Fish Endocrinology

© 2006 by Taylor & Francis Group, LLC

Fish Endocrinology

Editors Manfred Reinecke
Division of Neuroendocrinology, Institute of Anotomy University of Zürich, Zürich, Switzerland

Giacomo Zaccone
Department of Animal Biology and Marine Ecology University of Messina, Messina, Italy

B.G. Kapoor
Formerly Professer of Zoology, University of Jodhpur Jodhpur, India Present Address: Manohar Enclave, City Centre Gwalior, India

Science Publishers
Enfield (NH) Jersey Plymouth

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SCIENCE PUBLISHERS An Imprint of Edenbridge Ltd., British Isles. Post Office Box 699 Enfield, New Hampshire 03748 United States of America Website: (marketing department) (editorial department) (for all other enquiries)
Library of Congress Cataloging-in-Publication Data Fish endocrinology/editors, Manfred Reinecke, Giacomo Zaccone, B.G. Kapoor. Includes bibliographical references (p. ). ISBN 1-57808-318-4 (Set) 1. Fishes--Endocrinology. I. Reinecke, Manfred. II. Zaccone, Giacomo. III. Kapoor, B.G. QL639.F5535 2006 573.4’17--dc22 2006042325

ISBN (Set) 1-57808-318-4 [10 digits]/978-1-57808-318-3 [13 digits] ISBN (Vol. 1) 1-57808-414-8 [10 digits]/978-1-57808-414-2 [13 digits] ISBN (Vol. 2) 1-57808-415-6 [10 digits]/978-1-57808-415-9 [13 digits] © 2006, Copyright reserved All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, without the prior permission. This book is sold subject to the condition that it shall not. By way of trade or otherwise be lent, re-sold, hired out, or otherwise circulated without the publisher’s prior consent in any form of binding or cover other than that in which it is published and without a similar condition including this condition being imposed on the subsequent purchaser. Published by Science Publishers, Enfield, NH, USA An Imprint of Edenbridge Ltd. Printed in India

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During the past two decades, fish endocrinology has witnessed exciting developments due to our increased knowledge at all levels of biological organization, including molecular biology, cell biology, physiology and behaviour. New insights into development, neurobiology, immunology and molecular genetics closely correlate with classical aspects of endocrinology. The purpose of this book is to overview major advances in numerous research areas of fish endocrinology. Larvae of bony fish are the smallest independently functioning vertebrates and achieve dramatic rates of growth during early life. Thereby they undergo gradual morphological and physiological transformations in order to develop from the larval to the juvenile and finally to the adult stage. The endocrine systems that regulate the developmental and growth processes are thus of crucial importance. Section 1 deals with the growth hormone (GH)/insulin-like growth factor (IGF) system, a research field of rapidly growing importance and impact on future research. The section starts with a survey on the IGF system and the related insulin system in mammals (1). This chapter, thought to serve as a general introduction, is followed by chapters on insulin (2, 3) and on the IGF hormones (4) IGF and insulin receptors (5) and IGF- binding proteins (6) in fish. These chapters consider the roles of the different components of the insulin-IGF family in crucial physiological processes such as metabolism, development, growth, reproduction and osmoregulation. Particular sections of the book concentrate on classical fields of endocrine research. One is the gastro-entero-pancreatic (GEP) system, discussed in detail in Section 2. The histology and ultrastructure of the fish endocrine pancreas is dealt with for the African lungfish as a representative species (7). The functional aspects of glucagon and related hormones (8) and the development of the GEP endocrince system with its

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numerous hormones in fish (9) are described. The other classical challenge for endocrinologists, the hypothalamus-pituitary axis with its great importance as the central regulator of the hormone system, is covered in Section 3. This section includes general morphofunctional and developmental aspects of the bony fish anterior pituitary (10), the proopiomelanocortin (POMC) related peptides which interact in many physiological systems (11), the osmoregulatory actions of hypophyseal hormones in general (12), and prolactin (PRL) with its central role in freshwater adaptation in particular (13). Further topics explore the evolution, biology and function of the natriuretic hormone (NP) family, i.e., ANP BNP and CNP (14), cardiac , nitric oxide (NO) signalling (15) and the structure-activity relationships and myotropic actions of numerous peptides, such as tachykinins, bradykinins, and the neuropeptide Y (NPY) family, endothelin, vasoactive intestinal polypeptide (VIP) and galanin (16). Furthermore, the role of the fish pineal gland with its neuroendocrine messenger melatonin as a significant part of a complex time-measuring system is considered (17). These subjects are certainly challenges for future research. Numerous bony fish species such as salmon, seabream and tilapia have high commercial value and, thus, research results on the regulatory mechanisms involved in growth, development, reproduction and stress response have a major impact not only on the science of fish biology but also on the aquaculture industry. Accordingly, these aspects are not only considered in Section 1 but also in some chapters of Section 8 dealing with the neuroendocrine control of reproduction in fish and the synthesis and functions of estrogens (18, 19), stress response (20), the hypothamuspituitary-adrenal (HPA) axis and the roles of corticosteroids in ionic and osmotic balance, metabolism, and stress as well as in development, reproduction, and aging (21, 22). Furthermore, the influence of thyroid hormones on growth, development, parr-smolt transformation and reproduction is dealt with (23). Development, growth and reproduction are regulated by the integration of environmental influences such as food availability, temperature and season, with endogenous neuroendocrine and endocrine signals. For several years there has been severe concern that various hormonally active chemicals designated as endocrine disrupting compounds (EDCs) which are present in surface waters and aquatic sediments, may adversely affect the development, growth, reproduction

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and immune competence of fish. Several chapters (6, 19, 22, 23 and 24) are dedicated to the endocrine disruption of the growth, reproductive and immune systems. As one may expect, creating sections in a multi-author work will cause inevitable separations of related hormones and regulatory systems, e.g., insulin is covered in the insulin – IGF chapter (2, 3) and glucagon in the portion GEP system (8). Certain aspects of neuroendocrine regulation are guven not only in section 3 but also in the chapters 4, 18, 21, 22 and 24. The chapters in the book were peer-reviewed, besides the editors, by the following specialists: Augustine Arukwe (Ontario, Canada), Dianne Baker (Amherst, USA), Mercedes Blázquez (Barcelona, Spain), Lorenzo Colombo (Padua, Italy), Alex Eberle (Basel, Switzerland), Sture Falkmer (Trondheim, Norway), Luis Filgueira (Crawley, Australia), Olivier Kah (Rennes, France), Sakai Kikuyama (Tokyo, Japan), Werner Kloas (Berlin, Germany), Duncan MacKenzie (Texas, USA), Stephen McCormick (Turners Falls, USA), Tom Moon (Ottawa, Canada), Andreas Oksche (Gisesen, Germany), Brian C. Peterson (Stoneville, USA), Erika Plisetskaya (Seattle, USA), Gustavo Somoza (Buenos Aires, Argentina), Yoshio Takei (Tokyo, Japan), Mathilakath M. Vijayan (Waterloo, Canada) and James R. Wright (Nova Scotia, Canada). These experts contributed time and expertise and we would like to thank all of them. The subjects covered in the book reflect the newer areas of endocrinology as well as the traditional approach to the subject. An obvious trend is that shifts the earlier focus on central control mechanisms which lead to endocrine pathways of regulation towards greater considerations of peripheral paracrine/autocrine mechanisms. Most of the chapters not only review and discuss the state-of-the-art in the respective field, but also show perspectives of future research. Consequently, most chapters end with an epilogue that draws final conclusions and tries to anticipate future trends. We hope that the book will be of interest to a broad readership of scientists involved in basic fish research, comparative endocrinology, fisheries and aquaculture as well as for students of fish biology. Manfred Reinecke, Giacomo Zaccone and B.G. Kapoor

© 2006 by Taylor & Francis Group, LLC


Preface List of Contributors

v xiii

Section 1: Insulin and Insulin-like Growth Factors 1. A Survey on the Insulin and Insulin-like Growth Factor System Manfred Reinecke 2. Insulin Metabolic Effects in Fish Tissues I. Navarro, E. Capilla, J. Castillo, A. Albalat, M. Díaz, M.A. Gallardo, J. Blasco, J.V. Planas and J. Gutiérrez 3. Non-radioisotopic Immunoassay for Fish Insulin Tadashi Andoh 4. Insulin-like Growth Factor I and II in Fish Manfred Reinecke 5. Insulin and IGF Receptors in Fish J. Gutiérrez, I. Navarro, J.V. Planas, N. Montserrat, P Rojas, J. Castillo, O.V. Chystiakova, J.C. Gabillard, . A. Smith, S.J. Chan and N.B. Leibush 3


49 87 131

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x Contents 6. Insulin-like Growth Factor-Binding Proteins (IGFBPs) in Fish: Beacons for (Disrupted) Growth Endocrine Physiology Kevin M. Kelley, Tiffany D. Price, Maelanie M. Galima, Kathleen Sak, Jesus A. Reyes, Orlando Zepeda, Rebecca Hagstrom, Tuan A. Truong and Christopher G. Lowe 167

Section 2: Gastro-Entero-Pancreatic (GEP) System 7. The Endocrine Pancreas of African Lungfish: Light and Electron Microscopic Immunocytochemistry and Morphology Dirk Adriaensen and Jean-Pierre Timmermans 8. Glucagon and Friends Thomas P Mommsen and Ellen R. Busby . 9. The Development of the Gastro-Entero-Pancreatic (GEP) Endocrine System of Teleosts B. Agulleiro, M.P García Hernández, M.T. Elbal . and M.T. Lozano Section 3: Pituitary: Development, Hormones and Functions 10. Teleost Adenohypophysis: Morphofunctional and Developmental Aspects B. Agulleiro, M.P García Hernández and A. García Ayala . 289 199

223 257

11. Diverse Structures and Functions of Melanocortin, 325 Endorphin and Melanin-Concentrating Hormone in Fish Akiyoshi Takahashi and Hiroshi Kawauchi 12. Osmoregulatory Action of Hypophyseal Hormones in Teleosts Juan Miguel Mancera and Juan Fuentes 13. Osmoreception: A Fish Model for a Fundamental Sensory Modality A.P Seale, T. Hirano and E.G. Grau . 393


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Section 4: Natriuretic Peptides 14. The Natriuretic Peptide System of Fishes: Structure, Evolution and Function John A. Donald and Tes Toop Section 5: Cardiac NO Signaling 15. Nitric Oxide Modulation of Mechanical Performance in the Teleost Heart Bruno Tota, Sandra Imbrogno and Alfonsina Gattuso Section 6: Myotropic Hormones 16. Myotropic Neurohormonal Peptides in Fish J. Michael Conlon Section 7: Pineal Organ: Structure and Function 17. The Pineal Organ Horst-Werner Korf Section 8: Stress Response, Reproduction and Endocrine Disruptors 18. Morphofunctional Aspects of Reproduction from Synchronous to Asynchronous Fishes-An Overview Maria João Rocha and Eduardo Rocha 19. Current Perspectives on 17 -Estradiol (E2) Action and Nuclear Estrogen Receptors (ER) in Teleost Fish C.J. Martyniuk, N.S. Gallant, V.L. Marlatt, S.C. Wiens, A.J. Woodhouse and V.L. Trudeau 20. Stress Biomarkers and Reproduction in Fish Giulia Guerriero and Gaetano Ciarcia 571 541 507 487 443



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21. Neuroendocrine Mechanisms Regulating Stress Response in Cultured Teleost Species G. Mosconi, G. Cardinaletti, M. Carotti, F. Palermo, L. Soverchia and A.M. Polzonetti-Magni 22. The HPA Axis and Functions of Corticosteroids in Fishes David O. Norris and Steven L. Hobbs 23. Modes of Action and Physiological Effects of Thyroid Hormones in Fish J. Geoffrey Eales 24. The Impact of Environmental Hormonally Active Substances on the Endocrine and Immune Systems of Fish Helmut Segner, Elisabeth Eppler and Manfred Reinecke Index






Color Plate Section

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List of Contributors

Adriaensen, Dirk
Laboratory of Cell Biology and Histology, Department of Biomedical Sciences, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium. E-mail:

Agulleiro, B.
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain. E-mail:

Albalat, A.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Andoh, Tadashi
Hokkaido National Fisheries Research Institute, Fisheries Research Agency, 116 Katsurakoi, 085-0802 Kushiro, Japan. E-mail:

Blasco, J.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Busby, Ellen R.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C, Canada.

Capilla, E.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Cardinaletti, G.
Department of Comparative Morphology and Biochemistry, University of Camerino - v. F. Camerini, 2 - 62032 Camerino (MC), Italy.

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xiv List of Contributors Carotti, M.
Department of Comparative Morphology and Biochemistry, University of Camerino - v. F. Camerini, 2 - 62032 Camerino (MC), Italy.

Castillo, J.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Chan, S.J.
Departments of Biochemistry and Molecular Biology and Medicine and the Howard Hughes Medical Institute, University of Chicago, Chicago, Illinois 60637, U.S.A.

Chystiakova, O.V.
Sechenov Institute for Evolutionary Biochemistry and Physiology, Russian Academy of Sciences, St. Petersburg, Russia.

Ciarcia, Gaetano
Department of Biological Sciences, “Federico II” University, Napoli 80134, Italy.

Conlon, J. Michael
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates. E-mail:

Díaz, M.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Donald, John A.
School of Biological and Chemical Sciences, Deakin University, Geelong, 3217, Australia. E-mail:

Eales, J. Geoffrey
Department of Zoology, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. E-mail:

Elbal, M.T.
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

Eppler, Elisabeth
Division of Neuroendocrinology, Department of Anatomy, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

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List of Contributors


Fuentes, Juan
Centro de Ciências do Mar (CIMAR Laboratório Associado), Campus de Gambelas, 8005-139 Faro, Portugal. E-mail:

Gabillard, J.C.
Equipe Croissance et Qualié de la chair des poissons SCRIBE, INRA, Rennes, France.

Galima, Maelanie M.
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Gallant, N.S.
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Canada. K1N 6N5

Gallardo, M.A.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

García Ayala, A.
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

García Hernández, M.P.
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

Gattuso, Alfonsina
Department of Cellular Biology, University of Calabria, 87030, Arcavacata di Rende, CS, Italy.

Grau, E.G.
Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744, U.S.A.

Guerriero, Giulia
Department of Biological Sciences, “Federico II” University, Napoli 80134, Italy. E-mail:

Gutiérrez, J.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain. E-mail:

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xvi List of Contributors Hagstrom, Rebecca
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Hirano, T.
Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744, U.S.A.

Hobbs, Steven L.
Department of Integrative Physiology, University of Colorado, 354 UCB, Boulder CO 80309-0354, U.S.A.

Imbrogno, Sandra
Department of Cellular Biology, University of Calabria, 87030, Arcavacata di Rende, CS, Italy.

Kawauchi, Hiroshi
Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University, Sanriku, Ofunato, Iwate 022-0101, Japan.

Kelley, Kevin M.
Endocrine Laboratory/Dept. BIO-SCI, California State University, Long Beach, CA 90840, U.S.A. E-mail:

Korf, Horst-Werner
Dr. Senckenbergische Anatomie, Institut für Anatomie II, Fachbereich Medizin, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D60590 Frankfurt/Main, Germany. E-mail:

Leibush, N.B.
Sechenov Institute for Evolutionary Biochemistry and Physiology, Russian Acadeny of Sciences, St. Petersburg, Russia.

Lowe, Christopher G.
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Lozano, M.T.
Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain.

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List of Contributors


Mancera, Juan Miguel
Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain. E-mail:

Marlatt, V.L.
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Canada. K1N 6N5

Martyniuk, C.J.
Centre for Advanced Research in Environmental Genomics (CAREG), Department of Biology, University of Ottawa, Ottawa, Canada. K1N 6N5

Mommsen, T.P.
Departament of Biology, University of Victoria, PO Box. 3020, Stn. CSC, Victoria, B.C. Canada. V8P 3N5. E-mail:

Montserrat, N.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Mosconi, G.
Department of Comparative Morphology and Biochemistry, University of Camerino - v. F. Camerini, 2 - 62032 Camerino (MC), Italy.

Navarro, I.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Norris, David O.
Department of Integrative Physiology, University of Colorado, 354 UCB, Boulder CO 80309-0354, U.S.A. E-mail:

Palermo, F.
Department of Comparative Morphology and Biochemistry, University of Camerino - v. F. Camerini, 2 - 62032 Camerino (MC), Italy.

Planas, J.V.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Polzonetti-Magni, A.M.
Department of Comparative Morphology and Biochemistry, University of Camerino - v. F. Camerini, 2 - 62032 Camerino (MC), Italy. E-mail:

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List of Contributors

Price, Tiffany, D. Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA90840, U.S.A. Reinecke, Manfred
Division of Neuroendocrinology, Institute of Anatomy, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland. E-mail: reinecke@

Reyes, Jesus A.
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Rocha, Eduardo
Lab. Histology and Embryology, Institute of Biomedical Sciences, Abel Salazar - ICBAS, Lg. Prof. Abel Salazar no. 2, 4099-003, University of Porto, Portugal. E-mail:

Rocha, Maria João
Institute of Health Sciences (ISCS-North), Department of Pharmaceutical Sciences, Gandra, Portugal.

Rojas, P.
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645, E-08028 Barcelona, Spain.

Royal, Tiffany D.
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Sak, Kathleen
Marine Biology Program and Endocrine Laboratory, Department of Biological Sciences, California State University, Long Beach, CA 90840, U.S.A.

Seale, A.P.
Hawaii Institute of Marine Biology, University of Hawaii, Kaneohe, HI 96744, U.S.A. E-mail:

Segner, Helmut
Center for Fish and Wildlife Health, University of Berne, P Box 8466, CH.O. 3001 Bern, Switzerland. E-mail:

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Italy. Sanriku. Department of Biology. K1N 6N5 © 2006 by Taylor & Francis Group. Department of Biological Sciences. Japan. Soverchia.J. Centre for Advanced Research in Environmental Genomics (CAREG).A. 3217. Palmerston North. Deakin University. Centre for Advanced Research in Environmental Genomics (CAREG). Ofunato. University of Ottawa. V. Wiens. University of Truong. U. Private Bag 11222. University of Calabria.List of Contributors xix Smith. Massey University. Ottawa. Kitasato University. 87030. Department of Comparative Morphology and Biochemistry. School of Fisheries Sciences. A. University of Antwerp. Animal and Biomedical Sciences. Centre for Advanced Research in Environmental Genomics (CAREG). New Zealand. Geelong. Tuan A. Akiyoshi Laboratory of Molecular Endocrinology. Bruno Department of Cellular Biology. Department of Biomedical Sciences. Canada. Belgium. L. Ottawa. S. Takahashi. Italy. Department of Biology. K1N 6N5 Woodhouse. A. LLC .it Trudeau. Jean-Pierre Laboratory of Cell Biology and Histology. Camerini. Iwate 022-0101.62032 Camerino (MC). K1N 6N5 E-mail: Timmermans. Long Beach. F. Canada. University of Camerino .S.L. E-mail: tota@unical. CS. Department of Biology. Australia. Tota. Ottawa. E-mail: akiyoshi@kitasato-u. Tes School of Biological and Chemical Sciences. Arcavacata di Rende.C. Canada. B-2020 Antwerpen. University of Ottawa.v. Groenenborgelaan CA 90840. 2 . Marine Biology Program and Endocrine Laboratory. Toop. Institute of Veterinary. California State University.

Orlando Marine Biology Program and Endocrine Laboratory. CA 90840. Long Beach. California State University. © 2006 by Taylor & Francis Group. U.S. Department of Biological Sciences.xx List of Contributors Zepeda. LLC .A.

SECTION 1 Insulin and Insulin-like Growth Factors © 2006 by Taylor & Francis Group. LLC .

Production. E-mail: © 2006 by Taylor & Francis Group. Winterthurerstr. Regulation. University of Zürich. This chapter gives a short review on the well established insulin and IGF system in mammals with the intention to assist the readers to familiarize themselves with insulin and IGFs in fish.). IGF-I. Key Words: Insulin.. IGF-II. CH-8057 Zürich. Binding proteins.CHAPTER 1 A Survey on the Insulin and Insulin-like Growth Factor System Manfred Reinecke ABSTRACT Insulin and the related insulin-like growth factors (IGF) are essential hormones involved in fundamental physiological processes.unizh. insulin hormone (Navarro et al. Receptors.). Andoh). Author’s address: Division of Neuroendocrinology. Institute of Anatomy. Genes. Consequently. IGF hormones (Reinecke) and IGF binding proteins (Kelley et al. i. Growth hormone.e. insulin and IGF receptors (Gutiérrez et al. Among these are the maintenance of normal blood glucose levels (insulin) and the stimulation of development and growth as major functions. Switzerland. 190. LLC . the volume contains several articles dealing with the different aspects of the insulin/IGF family.

which mediate the effects of IGFI and IGF-II. Thus. the mature IGFs contain additional 6-8 residue C-terminal D-domains and the prohormones of both IGFs further C-terminal extensions. © 2006 by Taylor & Francis Group. the mature IGF molecules are single chain polypeptides. In further contrast to insulin. the IGF-I and IGF-II molecules contain B. the C-peptides of the IGFs are considerably shorter (Table 1.1) and.1) and both IGFs are 66% identical to each other. LLC . 3. Specific proteases of the IGFBPs. insulin and the IGFs also exhibit pronounced differences in their distribution patterns and partly in their physiological roles. The IGFs are structurally closely related to (pro-) insulin (Table 1.and A-domains of the IGFs are connected by a Cdomain (=peptide). As in proinsulin. Like the insulin molecule. 4. in contrast to insulin. They exert insulin-like effects on adipose and muscle tissue and mitogenic effects on various target cells. The prepropeptides of both mammalian insulin and IGF contain signal peptides.and A-domains of insulin. 2. they are not removed during prohormone processing. the B. the IGFs are highly conserved in the amino acid sequence. When compared to proinsulin. and to the insulin-like peptides in invertebrates. Six different binding proteins (IGFBPs) which transport the IGFs via the circulation towards specific tissues and target cells. The signal peptide of the prepro-IGF-II is generally shorter than those of the prepropeptides of IGF-I and insulin. These structures exhibit 50% sequence identity with the B.and A-domains.1) and exhibit lower levels of sequence similarity to other members of the insulin/IGF family such as relaxin. Within the mammals. It consists of several components: 1. the Epeptides. Type 1 and type 2 IGF receptors. Mature mammalian IGFI consists of 70 amino acid (aa) residues and IGF-II of 66-67 aa residues (Table 1. In addition. IGF-II and insulin exhibit remarkable similarities as also significant differences. Hormones IGF-I and IGF-II. which are generally cleaved during post-translational processing. However. IGF-I and IGF-II.4 Fish Endocrinology GENERAL Insulin-like growth factors (IGFs) constitute a family of polypeptide hormones with the two major forms. INSULIN AND IGF HORMONE STRUCTURES The structures of mammalian IGF-I. the mammalian IGF system is very complex when compared to other hormones such as insulin.


the expression of the IGF-II gene is regulated in a tissueand development-specific manner. the insulin gene is quite simple and compact in structure. encompasses less than 5 kb of DNA on chromosome 11. the insulin gene is transcribed into a single mRNA species from a single promoter. exon 1 transcripts are expressed in all organs while exon 2 transcripts occur predominantly in the liver. GH— which is the major regulator of liver IGF-I—increases the number of exon 2 transcripts more than that of exon 1 transcripts. 4 and 6. Furthermore. contains four different promoters (P1-P4) and spans approximately 30 kb of genomic DNA on the distal end of the short arm of chromosome 11. comprising three exons and two introns. In general. Mature IGF-II hormone is encoded by © 2006 by Taylor & Francis Group. The rat and mouse IGF-II genes contain six known exons and span approximately 12-15 kb of DNA. LLC . IGF-I gene is located to the long arm of chromosome 12 in human and in the central region of the homologous chromosome 10 in mouse. Prepropeptides with different Edomains. In rat liver.6 Fish Endocrinology INSULIN AND IGF GENES The human insulin gene. Hereby. called IGF-IEa and IGF-IEb. In contrast. the genes encoding the IGF prepropeptides are complex and big in structure and exhibit complex expression patterns. possesses two promoters and spans about 90 kb of chromosomal DNA. 4 and 5 while the IGF-IEb mRNA arises from the splicing of exons 1 or 2 with 3. evolve in the following manner: the IGF-IEa mRNA is derived from the alternate splicing of exons 1 or 2 with exons 3. the exons 1 and 2 are used in a mutually exclusive manner as are exons 5 and 6. The expression of exon 2 transcripts increases noticeably with the onset of growth hormone (GH) evoked growth. The human IGF-I gene contains at least six exons. the complex patterns of IGF-I and IGF-II mRNA species are all processed to produce the same IGF hormones. However. In rat. The human IGF-II gene comprises nine exons. The expression of the IGF genes is characterized by stage-specific promoters which act on numerous initiation sites and by differential RNA splicing that results in the generation of multiple prepropeptides. are differentially regulated and expressed. In humans. IGF-I mRNAs. exon 1 transcripts appear earlier in development than the transcripts of exon 2. The rat and mouse IGF-I genes exhibit an overall structure which is very similar to the human counterpart but the 3’ terminal exon structure and the resultant E-domain peptide sequences are different. Thus. which result from the activation of different promoters and transcription start sites.

IGFBP-3 ( subunit) and an acid-labile glycoprotein ( -subunit. IGFBP-1. The promoters P2. In the human genome. The expression patterns of the genes encoding the IGFBPs show marked changes beginning in embryonic and continuing through foetal. In contrast. Six different. These promoters remain active in non-hepatic tissues but P3 is shut-down postpartum in the liver and P1 is the predominant promoter active in the adult liver. IGF-II genes differ from the human gene in the distribution and usage of promoters. THE IGF BINDING PROTEINS AND THEIR PROTEASES After its release from the islet -cells insulin circulates in free form and directly interacts with its receptor. LLC . Promoter P1 is absent from the rat IGF-II gene. P3 and P4 are active in foetal nonhepatic tissues with P3 as the major foetal liver promoter. Its bioavailability is largely restricted by the capillary barrier. IGF in this complex has a half life of 20-30 min. The serum half-life of IGF bound to this complex is 12-16 hours. The mammalian IGF-II genes identified to date form a conserved linkage group with the insulin and tyrosine hydroxylase (TH) genes and are organized as a THinsulin-IGF-II genomic locus in the same transcriptional polarity. The regulation of IGFBP-3 and ALS. -3 and -4 bind IGF-I and IGF-II with similar affinity while IGFBP-2. the distance between the insulin and IGF-II genes is only 1.Manfred Reinecke 7 exons 7. is dependent on GH. About 20-30% of total serum IGF is associated with IGFBPs in a 40-50 kD complex which contains mainly IGFBP-2 and relatively little IGFBP-3. There is increasing evidence that the IGFBPs not only stabilize circulating IGF-I and IGF-II but may © 2006 by Taylor & Francis Group. early post-natal and adult life. The IGFBPs transport IGF-I and IGF-II with high specificity and affinity towards their target cells and do not bind insulin. -5 and -6 have higher affinity for IGF-II than for IGF-I.4 kb. and its IGF becomes available to tissue receptors. 8 and 9. the vast majority of the IGFs are linked to specific binding proteins (IGFBPs) and only less than 1% of IGF-I in serum is not bound to BPs. like that of IGF-I. The rodents. ALS). About 70-80% of total IGF (IGF I and II) is associated with a ternary 150 kD complex consisting of IGF ( -subunit). which probably accounts for the species-specific differences in IGF-II synthesis in the liver of rodents and humans. but structurally related binding proteins (IGFBP-1 to IGFBP-6) have been characterized in mammals and the potential existence of further yet not identified IGFBPs is under discussion. The 40-50 kD complex crosses the capillary barrier.

The type 2 IGF receptor is the mannose-6-phosphate (M6P) receptor which exhibits an additional binding site for IGF-II. Several target cell types secrete specific proteases which belong to several catagories such as serine proteases. THE INSULIN AND IGF RECEPTORS The insulin receptor is a tyrosine kinase that specifically binds insulin and displays a considerably low affinity for the IGFs. metalloproteinases and cathepsins. a transmembranal domain and an intracellular tyrosine kinase domain. Furthermore. have been identified. it is also called the IGF-2R/M6P-R and structurally different from the IGF-1R and the © 2006 by Taylor & Francis Group. and (2) activation of the mitogen protein kinase (MAPK) cascade through Grb2 and Ras activation leading to a mitogenic response. its transmembrane domains show 24% aa similarity. As the insulin receptor.8 Fish Endocrinology also modulate certain physiological activities of the IGFs. the type 1 IGF receptor (IGF-1R) is a heterotetrameric glycoprotein with two -subunits and two -subunits. Two different IGF receptors. reduce binding of the IGFs and increase their availability to the receptor. thus. The -subunits consist of a short extracellular domain. which further initiates binding of IRS-1 to the p85 regulatory subunit of phosphatidylinositol-3 kinase (PI3K) and Grb2. The -subunits are extracellular and their cysteine-rich domains bind the IGFs. Likewise. an IRS1 independent activation of the MAPK pathway by activation of another SH2-containing protein termed Shc is also likely. LLC . while the functionally important tyrosine kinase domains show 85% aa sequence similarity. Thus. Overall. One pathway of the IGF-1R is shared with the insulin receptor and involves phosphorylation of the 185 kDa insulin receptor substrate 1 protein (IRS-1). Binding of IRS-1 to these latter proteins leads to the initiation of two signalling pathways: (1) activation of PI3K and the subsequent formation of phosphatidylinositol-3-phosphate which serves as a signal for cell proliferation and increased glucose metabolism. the type 1 and type 2 IGF receptor. the affinity of the insulin receptor for the IGFs is considerably low. These cleave particular IGFBPs and. Ligand-binding initiates autophosphorylation on tyrosine and serine residues of the intracellular part of the -subunit of both the insulin receptor and IGF-1R. the IGF-1R has 56% aa sequence similarity to the insulin receptor. The IGF-1R has a high affinity for IGF-I. an about three times lower affinity for IGF-II and a particularly low affinity for insulin.

IGF-I and IGF-II are expressed in several organs and stimulate growth and development by selectively promoting mitogenesis and differentiation and inhibiting apoptosis via the IGF-1R. © 2006 by Taylor & Francis Group. The IGF-2R/M6P-R is a monomeric receptor (250 kDa) with a short intracellular domain (164 aa) and a large extracellular domain (2264 aa). The receptor shows a high affinity for IGF-II and virtually no affinity for IGF-I and insulin. The main source of IGF-I is the liver. In turn. However. the IGF-I serum levels are low at birth and increase progressively through puberty until the mid-adult life to decrease again thereafter. It exerts acute anabolic effects. In contrast. 1. From there. age is also significantly correlated to the amount of circulating IGF-I. The functional impact of the IGF-2R/M6PR is still enigmatic. the only established function of the IGF-2R/M6P-R is to remove IGF-II from the circulation. The main stimulus for insulin secretion is ingestion of a meal. resulting in maintenance of normal blood glucose levels. Therefore. GH released from the anterior pituitary stimulates synthesis of IGFI in the liver and its release into the circulation. The IGF-I serum levels decrease in humans suffering from chronic undernutrition but increase after improvement of nutrition. However. as yet. Thus.1).Manfred Reinecke 9 insulin receptor. the IGF-2R/M6P-R is discussed to have further physiological impact in mammals as well as to be involved in tumour genesis. fat and muscle cells—insulin acts as metabolic hormone. In addition to GH. which secrete insulin into the systemic circulation. In contrast to insulin. the physiological role of IGF-I is understood as endocrine (Fig. circulating IGF-I is a major feedback regulator of pituitary GH secretion because it specifically inhibits GH gene transcription and secretion. After binding to its receptor— which is essentially present in the liver. the serum IGF-II levels remain almost unchanged. According to the original somatomedin hypothesis put forward by Daughaday and co-workers in 1972. In humans. LLC . Another important regulator of liver IGF-I production is the nutritional status. IGF-I reaches its target cells where it interacts with the IGF-1R and exerts its effects. PRODUCTION SITES AND REGULATION OF INSULIN AND THE IGFS Insulin is exclusively produced in the -cells of the pancreatic islets. binding of IGF-II to the IGF-2R/M6P-R leads to internalization and degradation of IGF-II.

© 2006 by Taylor & Francis Group.10 Hypothalamus GH-RH Somatostatin Fish Endocrinology IGF-I Growth GH CMYK Pituitary GH Negative feedback IGF-I IGF-I Autocrine/ paracrine route IGF-I GH Endocrine route Liver IGF-I IGF-I Female and male gonad differentiation and function Fig.1 The mammalian GH/IGF-I system. LLC . 1.

In most—if not all—of these tissues.1). such as the granulosa and theca cells of the ovary. the IGF-II serum levels are low during development. liver exhibits the highest expression of IGF-II during early development. GH also stimulates IGF-I production (Fig. GH is an insignificant regulator of IGFII. increase after birth and persist on a high level throughout life. they seem to be independent of age and puberty. In contrast to IGF-I. it was detected that in sharp contrast to insulin IGF-I is expressed in numerous organs. Recently.Manfred Reinecke 11 Serum IGF-I is decreased by fasting over one week to about 40% of the normal value and restored after return to normal feeding. who was the first to identify the amino acid sequences of both IGF-I and IGF-II. In adult human and rat. 1. liver is also the main site of IGF-II production but. they also indicate that extrahepatic sites deliver a significant amount of serum IGF-I. In correspondence. the level of IGF-II in rat serum is higher during foetal than during post-natal life. Furthermore. In humans. growth plate chondrocytes and cells in the pancreatic islets. While the results confirm that liver is the principal source of circulating IGF-I. the IGF-II gene has for long been thought to be expressed only in liver and choroid plexus but some studies indicate further sites of IGF-II production. In rat.1). The results obtained in rat led to the wellaccepted thesis that IGF-II is an embryonic and foetal growth factor. the expression of IGF-II in mammals is speciesdependent. since studies in humans do not support this view. This marked decrease in serum IGF-I did not significantly affect postnatal body growth. Furthermore. 1. indicating that the level of endocrine IGF-I in all likelihood is not essential for postnatal growth and development. the hypothesis might apply only for rodents. Soon after its discovery. During early postnatal life. Little is known about the regulation of IGF-II in liver and extrahepatic sites because in contrast to IGF-I. autocrine and/or paracrine effects of local organ-specific IGF-I are probably more important than has been generally assumed (Fig. When considering the action of IGF-II. wrote in 1990: ‘Nature © 2006 by Taylor & Francis Group. in contrast to rats. it generally mimics the effects of IGF-I on numerous target cells. in contrast to IGF-I. it was shown that the complete and selective inactivation of the IGF-I gene in the livers of mice by the Cre/loxP recombination system reduced serum IGF-I by about 75%. However. The decrease of IGF-I serum level after reduced food intake correlates with reduced amounts of hepatic IGF-I mRNA. Humbel. the amount of liver IGF-II mRNA decreases significantly to reach a barely detectable level in the adult. LLC .

A. V.E. Prentki. W. W. Identification and molecular characterization of insulinlike growth factor binding proteins (IGFBP-1. The insulin-like growth factor system and cancer. Easom.H. Biol. Metabolic control of -cell function. Insulin and insulin-like growth factors. Biochem. Rev. Rev.L. J. C. S. Sara.C. S. The phylogeny of the insulin-like growth factors. Bondy. J. Biol.S.. Semin. K. Daughaday. The somatomedin hypothesis 2001. Vol. the physiological role of IGF-II is still far from being clarified. Endocr. Martini (Ed.-L. Jones. L. P 1991.L. Mannose 6-phosphate/insulin-like growth factor 2 receptor. 1990. R. and Hall.M. Eur. Int. Reinecke.D. expression and regulation of insulin-like growth . Firth. Insulin-like growth factors and their binding proteins: Biological actions. the readers are referred to the reviews given in the reference list. 22:53-74. Rev. A. These articles either cover the mammalian insulin and the IGF system in general or consider particular topics. S. D. and Ling. R. and Corkey. Insulin-like growth factors I and II. 11:253-266.).M. R.R.T. As the IGF-2R/M6P-R. factors I and II.J. M. and Fowlkes. L. -5 and -6). 14:176-181. J.. 2004. Schumaker. -granule transport and exocytosis. 1991. and Butler. M. Structure. 1995. Cellular actions of the insulin-like growth factor binding proteins.R. Insulin-like growth factor binding protein proteolysis. N. and Clemmons. Mammary Gland Biol. K. evolution. Nature (London). 16:3-34. -2. pp 23-31. and Ellis. and Collet. J. R.T. San Diego. In: The Encyclopedia of Endocrine Diseases.I. DaCosta. 2003. -3. the hormone IGF-II recently is also thought to be involved in cancer genesis and progression.. 235:107. and van Wyk. Yakar. 2002... 2002. S. Shimasaki.E. © 2006 by Taylor & Francis Group. 5:85-94. 1990. Rev. Rotwein. Rev. Somatomedin: proposed designation for sulphation factor. Cell Dev. Hall. Endocr. a bonafide tumor suppressor gene or just a promising candidate? J. -4. M. Academic Press. Cytol. and Roberts.J. J. 2000.A. 1972..C. 2003. 2000. C. The insulin-like growth factors and their binding proteins. Growth Factor Res. van den Brande. 3:243-266.. J. D.12 Fish Endocrinology is a sphinx presenting us with IGF-II as a riddle’. M. 23:824-854. D. Humbel. and Baxter. For further information. 195:127-137. 70:591-614. References Bunn. Semin. Raben. B. 2000.. Salmon. Trends Endocrinol. 183:1-94. Reinecke. Prog. Jr. Neoplasial. 1998. Liu. 3. Metabol. after thousands of publications have dealt with IGF-II. Cell Dev. Physiol. Evolution of. 190:445-462.. C. LeRoith. Cancer Lett. 11:267-275. Even now. J. Deeney. LLC . Endocr. M. LeRoith. Growth Factors 5:3-18.

Adamo. and Schmid. Physiological role of the insulin-like growth factor binding proteins.. 1995. 1994. Eur.).R. Metabolic effects of the IGFs. Roberts Jr (Eds. Zapf. J.. Molecular and cellular aspects of insulin-like growth factor action.E. LLC .. D.T. Zapf. Rosenfeld and C. 1994. insulin-like growth factors.Manfred Reinecke 13 Thissen. C. 577-616. In: Contemporary Endocrinology: The IGF System . 15:80-101. 132:645-654. J.. J. J.-M. and LeRoith. Endocr. M. © 2006 by Taylor & Francis Group. C. NJ.-P Ketelslegers. J. Werner.. Endocrinol. Nutrional regulation of the . Vitam. L. Jr. R . Froesch. 1999. Humana Press Totowa. Horm. 48:1-58. and Underwood. pp. E. Roberts. H. Rev.

muscle. and it occupies a key position in endocrine research. Castillo. Díaz. To begin with. as in other vertebrates. Universitat de Barcelona. Information on the effects of insulin on the main target tissues. has been examined. Spain. anabolic.V. J. E. Capilla. In muscle. biosynthesis and secretion of the hormone. from in vivo and in vitro studies. Gallardo. liver. Blasco. Although the effects of insulin in fish liver metabolism are.A. Molecular mechanisms involved in these processes are currently being studied in an intent to understand why fish are relatively intolerant to glucose in comparison with mammals. Diagonal 645. different studies reveal Authors’ address: Departament de Fisiologia. and adipose tissue. a brief overview is presented on the molecule and gene structure. Albalat. A. Insulin is hypoglycemic and acts as an anabolic hormone in teleost fishes. E-08028 Barcelona. Av. insulin promotes protein anabolism. J. Planas and J. M. in general. J.CHAPTER 2 Insulin Metabolic Effects in Fish Tissues I. *Author for Correspondence: E-mail: jgutierrez@.ub. LLC . glucose uptake and conversion into glycogen in most of the species © 2006 by Taylor & Francis Group. M. Facultat de Biologia. This chapter focuses on the effects of the insulin on metabolism in fish. Gutiérrez* ABSTRACT Insulin has attracted the attention of scientists for many years. Navarro.

1991. The primary amino acid sequence of insulin. GLUT. Fuirichi and Yone. information on hormone regulation of adipose tissue metabolism is scarce. 1968). 1972. Jensen et al. INTRODUCTION Since its discovery in 1922 insulin has become one of the most thoroughly studied molecules in scientific history.. Mesenteric fat is also a physiological target tissue of insulin in fish. as well as its tertiary structure. LLC . Shortly after its discovery in mammals. studies on crystalline insulin from fish islets described the tertiary structure of the molecule (Cutfield et al. Hepatocytes.. Carbohydrate. 1984. Nevertheless. Initial studies on the determination of insulin content in fish endocrine pancreas were made from Brockmann bodies of Thunnus thynnus (Planas. Gadus callarias (Reid et al. Furthermore. For example. certain invariant amino acids are conserved in all vertebrate insulins. 1976. Insulin is a polypeptide hormone secreted by the pancreatic -cells. insulin from different fish species was also isolated and characterized (McCormick and Noble. 1957). Gutiérrez et al. Teleost fish. Protein and lipid metabolism. which is consistent with the presence of insulin receptors. Its secretion was initially thought to be deficient in fish (Palmer and Ryman. Insulin appears to be anti-lipolytic and lipogenic. but it was later reported that plasma insulin concentrations are even higher in fish than in mammals (Thorpe and Ince. Insulin target tissues. the amino acid sequence of human insulin has a 72% identity with salmon insulin (Plisetskaya et al.. Glycogen. 2000. 2001). The first insulin amino acid sequence from a fish species was obtained in 1968 from the cod. 1991).. 1924. 1929). 1994). Conlon. Although glucose is the major stimulator of insulin © 2006 by Taylor & Francis Group. 1981). Myocytes. Mommsen and Plisetskaya. has been highly conserved among vertebrates (Conlon. 2001). as well as those that are important for maintaining the receptor-binding domain in its correct conformation (Mommsen and Plisetskaya.. Later. Key Words: Insulin. 1979). especially those residues that interact directly with the receptor. This chapter ends with a short overview of the actions of insulin on the brain related with food intake control. fish insulins have been extensively isolated and studied.16 Fish Endocrinology some peculiar characteristics of insulin action in various teleost species. Adipocytes. but control of lipid storage in muscle and liver needs further investigation. Due to the anatomical characteristics of the pancreas in many fish species with separation of exocrine and endocrine tissues.

Plisetskaya et al. the primary structure of insulin has been determined for at least one hundred vertebrate species. 2000a. Carneiro et al. appear to be more potent insulin stimulators than glucose (Ince and Thorpe.. 1988. 1995). especially arginine and lysine. including metabolism and growth. the structure of its receptor has been very well conserved during evolution (see Gutiérrez et al.. PROTEIN STRUCTURE AND GENES Protein To date. in fish the amino acids.. 1975. relaxin and other insulin-like peptides present in invertebrates (Blundell and Humbel. As anabolic hormone. 1988..I. 17 secretion in mammals. holocephalan (Conlon et al. 1994. Czech.600 Da. which possesses tyrosine kinase activity. insulin binds to its specific transmembrane receptor.. To exert its biological effects. and has an apparent molecular weight of 5. LLC . Berks et al. the most important functions of insulin are the stimulation of glucose uptake in liver and skeletal muscle and the increase of the influx of glucose and fatty acids in adipose tissue (Olefsky. Navarro et al. In its mature state. 1979. Smit et al. nerve growth factor (NGF). with the A and B chains linked by disulphide bridges. 1991). Conlon and Thim. like insulin. 1977. The presence of insulin receptors in fish has also been demonstrated and studied extensively and. insulin contains 51 to 58 amino acids distributed between the A and B chains. 1983. In addition. 1993. Cutfield et al.. The insulin © 2006 by Taylor & Francis Group. this volume). 1989.b). an important number of overlapping functions have been postulated between insulin and IGF-I in fish (Plisetskaya et al. Mommsen and Plisetskaya. 1988.. After autophosphorylation of the receptor. 1980. also see Reinecke. 1993). These include many fish species of elasmobranchs (Bajaj et al.. 1995a. Insulin belongs to a family of related peptidic hormones which also include insulin-like growth factors (IGFs). 1986). This chapter aims to review the literature concerning the characteristics of piscine insulin and focuses on the metabolic functions of insulin in fish.... 1989) and Agnatha (Peterson et al. In mammals.. Conlon et al. this volume). insulin plays an important role in the regulation of several physiological processes. an intracellular messenger signalling cascade is initiated. Planas et al. Fish insulins are structurally close to other vertebrate insulins.

two different forms of insulin have been isolated from the channel catfish (Ictalurus punctatus). two inter-chain linking A and B chains at positions A7-B7 and A20-B19. 2001). the invariant residues are always conserved (Conlon et al.. Gene The structure of the mammalian insulin gene consists of three expressed sequences (exons) and two intervening sequences or introns (Steiner et Fig.1. In addition. 1991).18 Fish Endocrinology molecule contains three disulphide bridges at invariable positions. © 2006 by Taylor & Francis Group.. Although some fish species possess amino acid extensions in the B chain. 1986. and some of them are thought to be important for the biologically active conformation of insulin (see review by Conlon. In addition to the invariant cysteines. Mommsen and Plisetskaya. The primary amino acid sequence of salmon insulin is shown in Figure 2. Most of the fish insulins sequenced contain—like their mammalian counterparts—21 amino acids in the A chain and 30 amino acids in the B chain. It has been further postulated that they are products of two different genes. 2. The thick lines between cysteine residues represent the three disulphide bridges of the molecule.1 Primary structure of salmon insulin. and one intra-chain within the A chain between cysteine residues at positions 6 and 11 (Mommsen and Plisetskaya. although the biological activity of the two forms is yet to be investigated (Mommsen et al. 2001a). 1991). 2000. a further ten amino acids have been fully conserved during vertebrate evolution. Adapted from Plisetskaya et al. Amino acids are presented using the threeletter code. LLC . (1994).

has been reported in mice and rats (Bunzli et al. The three exons (E1. while the second is located within the region encoding the C peptide (Fig. but their location within the gene has been very well conserved. a second gene. the insulin gene from tilapia was also sequenced and characterized. these two different genes were identified by Kavsan and co-workers in the salmon (1993). Not surprisingly. (1985). Navarro et al. 2. The length of the whole gene and the two introns is indicated. U: untranslated region.. LLC . containing two introns in similar positions. Some years later. 1985). is flanked by the typical eukaryotic transcription promoter and polyadenylation signal sequences. 1998). U TATAAA P BC I1 393 bp 1560 bp CAU I2 287 bp E1 E2 E3 Fig. In fish... which has lost the second intron. and in contrast to mice and rats. The first intron is in the 5’ untranslated region (5’UTR). The introns are quite variable between species in length and sequence.... and showed the same exon-intron distribution found in other organisms (Mansour et al.I. this event is thought to be the result of the recent genome duplication occurring in the salmonid lineage. Later. Lomedico et al. however. in the tilapia insulin gene these authors reported the presence of some potential control elements also found in mammalian insulin genes.b). the existence of a second gene coding for insulin in the genomes of fugu fish and zebrafish has been reported (Irwin. These two genes are most likely the product of fish genome duplication and indicate © 2006 by Taylor & Francis Group. C: C peptide. More recently. P: Signal peptide. 19 al. A: A chain. In addition. 1982. the first nucleotide sequence of the preproinsulin gene reported was from the chum salmon (Sorokin et al. Koval et al. The coding region. E2 and E3) and the two introns (I1 and I2) are represented. Adapted from Steiner et al. The structure of the gene was identical to the mammalian insulin gene. 1989a. The evidence suggests that these two genes have arisen through RNA-mediated transposition. 1972.2). located in exons 2 and 3. B: B chain. 2. 2004). the high frequency of polymorphic differences between the two reported sequences suggested the presence of more than one insulin gene. Although most mammals only contain one gene coding for insulin. 1979).2 Schematic representation of the salmon insulin gene.

especially in embryonic tissues. 1991. alternatively. as in mammals. 2004). insulin is primarily expressed in the -cells of the pancreas. thereby generating the mature form of insulin (Steiner et al. However. © 2006 by Taylor & Francis Group. INSULIN BIOSYNTHESIS AND SECRETION Biosynthesis Insulin is synthesized in the pancreatic -cells as a single-chain precursor called preproinsulin. 1999). 1986. When the -cell is appropriately stimulated.. suggesting either continuous processing during packaging in the secretory granules of the cell or the existence of an alternative cleavage site within the C peptide region of proinsulin (Conlon and Thim. However. In some fish species. The disulphide bridges of insulin are formed within the endoplasmic reticulum. 1997). Proinsulin is then exposed to several specific endopeptidases which excise the C peptide. a truncated form of the C peptide has also been isolated. Generally. observations made in this latter study concerning the presence of important conserved amino acids and potential proteolytic processing sites within the peptide sequence suggested that both insulins may be processed correctly in a molecule with biological activity (Irwin. Steiner. The C peptide is also secreted into the blood. the carboxyl-terminal A chain and a connecting peptide in the middle known as the C peptide. insulin is secreted from the cell by exocytosis and diffuses into the blood. either acquire a new function or sub-functionalize (Force et al. Removal of the signal peptide during insertion into the endoplasmic reticulum generates proinsulin. LLC . The molecule of proinsulin contains 86 amino acids distributed in three domains: the amino-terminal B chain. Insulin and the free C peptide are packaged in the Golgi into secretory granules.20 Fish Endocrinology that this dual-gene phenomenon is not restricted to polyploid fish species such as salmonids. which accumulate in the cytoplasm. however. the most duplicated genes are thought to degenerate into non-functional pseudogenes or. extrapancreatic sites of insulin gene expression have been reported. 1969. Hazelwood. but has no known biological activity (Steiner and Rubenstein. 1993). Conlon et al... Mommsen and Plisetskaya. 1984. suggesting that insulin may also have a role in early development (Muglia and Locker. 1986). In fish. information about distinct and/or specialized functions of these two insulin molecules in fish is yet to be gathered.

I. insulin is released into plasma. 1994). (1991) showed that glucose stimulates insulin secretion in anglerfish islets.. further studies are needed to fully understand the function of this peptide hormone secreted by its own target tissue. 1991. The concentration of plasma insulin in systemic circulation ranges from 1 to 30 ng ml –1. 1996).. proinsulin and C peptide (Chan et al. unexpectedly. Furthermore. 2001). In addition. 1993). other authors have found smaller responses in carp (Párrizas et al. Baños et al. The stimulators of insulin secretion in fish are the same as in mammals. 2004). although the effect was lower than that of arginine. LLC . However.. interferences in the measurement of plasma levels by radioimmunoassay can occur due to the presence in plasma of insulin-like activities (Urbinati et al. fish usually respond to glucose with an increase in circulating insulin (Plisetskaya and Duguay. (2004) showed that an intraperitoneal injection of glucose increased systemic insulin levels. © 2006 by Taylor & Francis Group. Secretion After -cell stimulation. called AdpInsl.. In addition.. Ronner (1991) found that the non-metabolizable glucose analogue 2-deoxyglucose (2-DG) induced an increase of insulin secretion in isolated endocrine catfish pancreas but not in mammals. the expression and secretion of insulin by carp adipocytes. although the role of carbohydrates is diminished in fish and there exist differences between fish species (reviewed by Navarro et al. 2002). More recently. However.. arginine is a good insulin secretagogue in catfish islets (Ronner and Scarpa. For example. 1987) and strongly stimulates insulin secretion in vivo in salmonids (Mommsen and Plisetskaya. Roy et al. Navarro et al. 1991). 1995).. plasma insulin levels in trout and eel (Legate et al. Among amino acids. 1997) and sea bream (Vega-Rubín de Celis et al. 1997). an intravenous glucose load increased. Although the sensibility of insulin to glucose is lower in fish than in mammals. Dumonteil and Phillippe. Milgram et al. showed a molecule exhibiting 98% homology with fish insulins. The cloning of this insulin. 1994. this insulin molecule was found to be biologically active and its secretion being stimulated by glucose. The efficiency of glucose as an insulin secretagogue in fish is still under discussion. suggesting that the metabolism of glucose might not be necessary for the stimulation of insulin secretion in fish. depending on the fish physiological situation (Mommsen and Plisetskaya. in sea bream. (2003) reported. Vega-Rubín de Celis et al. although not significantly. However. 21 1984. Navarro and Gutiérrez.

1998. The ability of peripheral tissues to uptake glucose in response to insulin—as a possible cause of this intolerance—remains a matter of debate (Moon. but the mechanisms by which insulin regulates plasma glucose levels are not fully understood. Capilla et al. 1991). In addition. Fish have been considered to be glucose intolerant in comparison to mammals. Baños et al. 2003).. both red and white skeletal muscle increase the rate of glucose utilization in the presence of high insulin levels after the injection of a glucose bolus (Blasco et al. insulin produced a lowering in blood glucose and an increase in the glycogen content of muscle in a freshwater © 2006 by Taylor & Francis Group.. Fish muscle accounts for 50% or more of the animal’s weight and should be important in the glucose clearance stimulated by insulin. 1987. in rainbow trout.. In one of the early studies on the anabolic actions of the hormone. 1996). trout adapted to diets with different percentages of carbohydrate from different sources. it appears that high levels of dietary carbohydrate are needed to stimulate insulin responses and the insulin receptor system.. In fact. muscle. Many studies have reported that plasma insulin levels decrease with natural or experimental deprivation of food (reviewed in Navarro and Gutiérrez. INSULIN EFFECTS IN SKELETAL MUSCLE Insulin Effects in Muscle in vivo In fish. It would again suggest the important role of amino acids (AAs) in insulin stimulation in accordance with the carnivorous habits of most of the species studied. liver or adipose tissue to the decrease in plasma glucose remains unclear. 2001). LLC . Many studies have focused on the effects of exogenous administration of mammalian insulin on plasma glucose levels and glycogen tissue reserves in different teleost species. 1995). the relative contribution of the different insulin sensitive tissues.. 2003).22 Fish Endocrinology The nutritional condition of the animal is the most important factor regulating insulin response. Although it is generally assumed that fish pancreas increases insulin secretion after adaptation to carbohydrate-enriched diets (Hilton et al. Although the hypoglycaemic effect of exogenous insulin is consistent in almost all teleost species. showed similar increases in post-feeding insulin levels to trout adapted to a carbohydrate-free diet (Capilla et al. the hypoglycaemic effects of insulin have been well described (reviewed by Mommsen and Plisetskaya.

(1988) showed that an administration of pharmacological doses of insulin to normally fed catfish did not affect glucose incorporation into muscle glycogen. with no effect on muscle glycogen. 1992..I. it was more evident in fed fish (Ottolenghi et al. mostly through the GLUT4 isoform. the existence of different members of the GLUT family has been recently reported in different fish species (Planas et al. although this effect was observed in both fed and fasted animals. Studies of labelled glucose administration in fish have shown that up to 10% of the 14C glucose is converted into glycogen. skeletal muscle and adipose tissue. 23 fish. Machado et al.. 2004a). In mammals. a South American teleost. In mammals. while more than 57% is partially or totally metabolized (Hemre et al. 2000. may be explained by seasonal components. Teerijoki et al. although. So far. 2001. 1982). Carneiro and Amaral (1983) injected insulin to Pimelodus maculatus. Krasnov et al. only a few studies have reported the effects of insulin on labelled glucose uptake in fish muscle in vivo. regardless of the nutritional state. Lates calcarifer. However. 1993). differences in doses and origin of the peptide used. Drakenberg et al. In contrast. Moreover. 2000b. In another study. Navarro et al.. Capilla et al. (1988) reported that insulin injections in catfish markedly increased the in vivo glucose incorporation into muscle protein. teleost insulins are more potent and hypoglycaemic effects are more rapid or prolonged in time (Duan et al. Although the mechanisms that mediate glucose uptake by insulin in fish are not well determined. some authors have failed to find any in vivo effect of insulin on muscle glycogen content. when homologous insulins were employed in fish experiments the main results were not very different from those obtained with mammalian hormones (Plisetskaya and Duguay. 2001. glucose transport across plasma membranes is mediated by a family of facilitative glucose transporters (GLUTs). (1997) subsequently proved that insulin did not stimulate the incorporation of 14C glucose carbon into muscle protein in the bony fish. 1993).. However. This hypoglycaemia was accompanied by an increase in the glycogen level in both white and red muscle. by © 2006 by Taylor & Francis Group.. at high and low ambient growth temperatures (Bhatt et al. in general. As such.. 1980). knowledge of the relative physiological importance of this hormone in muscle glucose metabolism in the whole fish remains limited. Navarro and Epple. The absence of mammalian-like insulin effects in some of these experiments.. insulin induced a decrease in blood glucose levels in catfish (Ictalurus melas). insulin stimulates glucose uptake in its target tissues. 2001). LLC .. Machado et al. for example. Clarias batrachus.

2001.3 shows the mRNA expression of GLUT4 in red and white skeletal muscle A 150 B 150 GLUT4/18S ratio GLUT4/18S ratio 100 50 0 100 50 0 * Control Fasted Control Fasted C 20 D 8 Insulin (ng/ml) Glucose (mM) 15 10 5 0 6 4 2 0 * * Control Fasted Control Fasted Fig. Adapted from Capilla et al. n = 5 for values of expression and n = 10 for plasma values. the expression of GLUT4 in red skeletal muscle has been shown to change in parallel with plasma insulin levels. 2002). *indicate differences at P<0. LLC . the expression of GLUT4 in trout white skeletal muscle does not appear to be affected by changes in plasma insulin levels. In fish. 2. The expression of GLUT4 mRNA has been normalised to the levels of 18S rRNA.. 2002). Interestingly. In particular. The circulating levels of insulin (C) and glucose (D) of control and fasted fish are also shown. Bryant et al.24 Fish Endocrinology promoting its translocation from intracellular storage compartments to the plasma membrane (Holman and Sandoval. high-carbohydrate diet (Capilla et al. Figure 2. Values are means ± SE.3 Insulin regulation of GLUT4 mRNA expression in red and white skeletal muscle in trout. (2002).05. although it is not yet known whether GLUT4 translocates to the plasma membrane in response to insulin in skeletal muscle. and to decrease when plasma insulin levels are reduced either by fasting or by feeding a low-protein.. © 2006 by Taylor & Francis Group. the expression of GLUT4 in trout red muscle has been shown to increase when plasma insulin levels are experimentally increased either by insulin or arginine treatment. Changes in GLUT4 mRNA expression in trout red (A) and white (B) skeletal muscle after fasting.

In the same study. 1999). Distribution and morphological characteristics of intramuscular adipocytes from salmon and trout have been studied (Zhou et al. Insulin Effects in Muscle in vitro The absence of established cell lines of fish muscle to perform in vitro assays of insulin actions has been a crucial limiting factor for many years. An anabolic role for insulin in lipid metabolism in agnatha has been indirectly demonstrated. 1995. Regarding insulin effects on lipid metabolism in fish muscle. 1997). It appears that the liver is the main site for lipogenesis in fish. since injection of antiserum to mammalian insulin elevated plasma FFA in adult lamprey (Plisetskaya.. 2001).. 25 of trout under fasting conditions. 1988). but most probably the incorporation of glucose carbons in muscle would involve lipogenic flux in hepatic tissue. insulin administration to lamprey larvae resulted in hypolipidemia accompanied by increased lipid content in muscle and liver. increasing the conversion of glucose carbons into skeletal muscle lipid in rainbow trout (Ablett et al. It appears that these cells would be used as an immediate source for supplying energy to the adjacent muscle fibres but their lipogenic capacity is unknown (Zhou et al. bovine insulin acts as a lipogenic hormone. LLC .I.. Although plasma concentration of free fatty acids (FFA) tends to decrease after insulin administration (Mommsen and Plisetskaya. and insulin-dependent uptake of AAs into muscle is usually accompanied by increases in protein synthetic rates (reviewed in Mommsen and Plisetskaya. Navarro et al.. decreased triacylglycerol lipase activity. while the lipogenic activity of muscle would be of minor importance (Hemre et al. In relation to protein metabolism.. anabolic actions of insulin in fish muscle have been demonstrated. Fauconneau et al. few studies have been reported. 1980). Fish skeletal muscle in vitro systems will be very useful for studying the insulin regulation of lipid metabolism such as FFA uptake and oxidation. In teleost studies. 1991). 1995). 1991). 1981) and catfish (Machado et al. caused opposite effects to those of insulin. and increased rates of lipogenesis in muscle and kidney (Kao et al. These results strongly suggest that insulin could be regulating the expression of GLUT4 in trout red muscle in vivo.. In a more recent study. administration of alloxan. © 2006 by Taylor & Francis Group. an insulin-secreting cell toxin. The route and fate of the glucose carbons was not determined. no information concerning possible direct insulin effects on the incorporation of these FFA into muscle is available..

26 Fish Endocrinology The recent development of myocyte primary cultures to test the biological effects of insulin in fish muscle has opened up numerous possibilities.. In this study. LLC . IGF-I and also IGF-II. 2004). © 2006 by Taylor & Francis Group. 1 μM insulin achieved the maximal stimulation of 2-DG uptake after 30 min of incubation (Fig. This is in agreement with the low presence of insulin receptors when compared to the number of IGF-I receptors in primary culture of rainbow trout muscle A 250 B L-alanine uptake (% above basal) 250 2-DG uptake (% above basal) b 200 150 100 50 0 200 b 150 100 50 0 a a a a Basal 100 Insulin (nM) 1000 Basal 100 Insulin (nM) 1000 Fig. Results are expressed as percentage of stimulation over basal levels and mean ± SE (n = 3 experiments). Preliminary experiments on the effects of insulin and IGF-I on 2-DG uptake in culture myocytes from sea bream showed similar tendencies (unpublished results). Different letters indicate significantly (P<0. respectively. have been demonstrated to be more potent than insulin in stimulating glucose uptake in these trout myocyte cultures (Castillo et al. However. where insulin provoked a maximal stimulation of glucose uptake at concentrations between 100 nM and 1 μM with similar incubation times. Codina et al. Similar effects have been described for mammals in a primary culture of human muscle cells (Ciaraldi et al. 2001). 2004). insulin-like growth factors.4A). (2004). 2.05) different values among groups. Stimulation of glucose (A) and alanine (B) uptake by insulin in trout myocytes at day 4 of development. Moreover.. primary cultures recapitulate muscle development more precisely than do immortal myogenic lines (Blanco-Bose et al. 2001). the effect of insulin on glucose uptake in rainbow trout skeletal muscle has been described using the myocyte primary culture as a model (Castillo et al. Cells were incubated with different concentrations of insulin for 1 h (A) or 2 h (B) and. Recently. 2.. Adapted from Castillo et al. 2004..4 Insulin effects on glucose and alanine uptake in trout myocytes. subsequently 2-DG or L-alanine were added and incubated for 30 or 20 min..

the presence of GLUT1 and GLUT4 has been described in trout heart (Planas et al. 2002).. Another caveat in fish is the signalling pathway by which insulin stimulates glucose uptake in muscle.. Preliminary experiments in trout myocytes strongly suggest that insulin stimulates the levels of GLUT4 mRNA and that this effect increases during in vitro myocyte differentiation (unpublished results). and in the previously reported skeletal muscle semi-purified preparations in several species of fish (Navarro et al. This finding would be in agreement with data on satellite cell cultures from rat foetuses where the expression of this glucose transporter increased in developed myotubes (Guillet-Deniau et al. 2001). Isolated cardiomyocyte preparations have been shown to be a good model for studying insulin-binding characteristics and metabolic effects in fish (Moon et al. Gallardo et al.5. Legate et al. 2000) as in mammalian heart tissue. Castillo et al. Although GLUT4 is considered to be mainly responsible for the stimulated insulin glucose uptake in rat cardiomyocytes (Fisher et al. LLC . These results confirm that IGF-I may also have a notable metabolic role in fish muscle. However. Additional information regarding this issue has been obtained from cardiomyocyte studies. Teerijoki et al. 2-DG uptake was partially inhibited by phloretin in a dose-dependent manner. In recent studies. 1994).. thus indicating a key role for intracellular proteins such © 2006 by Taylor & Francis Group. wortmanin reduced the basal glucose uptake and also the stimulatory effect of insulin in trout myocyte cultures. Navarro et al. Plisetskaya et al. 1996. this has yet to be demonstrated in fish..I. (1994) postulated an overlapping of functions between insulin and IGF-I in fish. these data indicate that insulindependent glucose uptake could be mediated by GLUT4 in fish myocytes. presented in Figure 2.. (2001) found no effect of cytochalasin B on glucose uptake in skeletal muscle membrane vesicles of rainbow trout. In fact. suggest the existence of at least one type of D-glucose transporter in trout heart tissue. Recent data from trout myocyte culture studies provide insights into the mechanisms that mediate glucose uptake in piscine systems. 27 cells (Castillo et al. (2004) reported that basal and insulin-stimulated 2-DG uptake in fish muscle may be mediated by specific transporters present in the cell plasma membrane since cytochalasin B inhibited glucose uptake.. and insulin stimulated 2-DG influx at physiological doses (unpublished results).. 2000b. 1999). In isolated trout cardiomyocytes. 1997). Together. These results.

1996) and fish cardiac muscle preparations (Gutiérrez et al. Symbols denote significant differences from control: (*)P<0. Similarly. Párrizas et al. as well as IGF-I and IGF-II.5 5 25 Phloretin (mM) Insulin (ng/mL) Fig. The inhibitor is added with the 2-DG..05. Regarding the effects on protein metabolism. 1983)... subsequently found a lack of stimulation effect of insulin on alanine uptake. it has been reported that insulin. it should be taken © 2006 by Taylor & Francis Group.001 0. increased both parameters.25 2. However. 1995. and even reported a decrease in protein synthesis. (2001). especially at developed stages of the culture (Codina et al. This observation is once again consistent with the higher number of IGF receptors in comparison with those of insulin.. increases the levels of the phosphorylated form of AKT (downstream element in the PI3K pathway). The 2-DG uptake was then followed for 30 min in a medium without D-glucose. Significant differences as in A (unpublished results). LLC . Cells were incubated for 30 min.. 1995). (B) Effect of different concentrations of insulin on 2-DG uptake. Cells were pre-incubated for 15 min using various concentrations of hormone.25 Basal 0. (2001) working with isolated trout cardiomyocytes. 2004). earlier studies showed that insulin stimulated amino acid uptake in fish opercular muscle explants in vitro (Inui and Ishioka.01. Gallardo et al.. a feature described not only in skeletal muscle but also in isolated trout cardiomyocytes (Moon et al.1 0. 2004) when high levels of stimulation of glucose uptake by insulin and IGF-I have been found (Castillo et al.28 A 2-DG uptake (% above basal) Fish Endocrinology B 2-DG uptake (% above basal) 120 100 80 60 40 20 0 150 125 100 75 50 25 0 ** ** ** * * * Basal 0. as phosphatydyl inositol-3-kinase(PI3K) in insulin-stimulated glucose transport in fish muscle. Values are means ± SD from 3 individual experiments. Values are mean ± SD of 6 individual experiments. although IGF-I. 2004). 2. (A) Effect of different concentrations of phloretin on 2-DG uptake by isolated trout cardiomyocytes.5 Phloretin and insulin effects on 2-DG uptake in trout cardiomyocytes. (**)P<0. The isolation cardiomyocyte procedure is as described in Gallardo et al. at low concentrations. as occurs in mammals (Castillo et al.

It is clear from all these in vivo and in vitro studies that muscle is also an important insulin target tissue in fish. However. Confirming this. Nevertheless. recent studies in trout muscle primary culture have found that none of the insulin treatments analyzed stimulated cell proliferation (measured by 3H thymidine incorporation into DNA). it seems that these studies are more conditioned by external factors. (2001) detected in ZF-4 cells from zebrafish embryos that IGFs were stimulators of cell proliferation and DNA synthesis. insulin is hypoglycaemic in almost all the fish species and acts as an anabolic hormone. the role of insulin in lipid storage or metabolization in these cell systems remains to be investigated. whose physiological relevance has to be interpreted with caution. In this latter work. LLC . which especially affect the actions of insulin on glucose metabolism. in vivo or in vitro. 2004). although insulin had a very low mitogenic activity. which may be related to differences in protein synthesis needs during muscular development. Most of the information from in vivo experiments related to insulin effects in muscle is based on exogenous insulin administration studies. Pozios et al. only a few studies have focused on the mitogenic effects of insulin on fish muscle. Navarro et al. are still scarce and further studies will be needed to obtain more information about the hypothetical proliferative effects of insulin in fish muscle. © 2006 by Taylor & Francis Group.I. it appears that studies using muscle in vitro models are consistent in demonstrating the anabolic role for insulin in fish carbohydrate and protein metabolism. however. while IGF-I was clearly mitogenic (Castillo et al. In general. and found that in this type of muscle cell the effects were more pronounced after IGF-I incubation. 2.. More recently. Recent studies have focused on the mechanisms involved in insulin-mediated glucose metabolism. Moreover.4B). In contrast to the available data regarding the metabolic effects of insulin in fish muscle. which is of great interest in terms of better understanding the role of insulin in fish carbohydrate utilisation. Available data on this issue. the capacity to stimulate amino acid uptake decreased with differentiation of muscle cells. (2004) described insulin-stimulated alanine uptake in primary culture of trout muscle cells (Fig. Castillo et al. 29 into account that the metabolic effects of insulin not depend only on the number of receptors available in the membrane. and many different elements in the signal transduction cascade interact to integrate the hormone’s physiological response.

1982). 1980). in the South American teleost Pimelodus maculatus (Carneiro and Amaral. this hypothesis has yet to be proved. GK has been characterized in © 2006 by Taylor & Francis Group. acts as a glycogen reservoir and glucose donor for other tissues and is also a key metabolic organ for turnover of AAs. Insulin depressed hepatic gluconeogenesis in the species studied when working with the intact fish. The latter authors sought to explain the different and even opposing effects of insulin observed in fish. However.30 Fish Endocrinology INSULIN EFFECTS IN LIVER Insulin Effects in Liver in vivo Piscine liver. LLC . arguing that the glycogen depletion observed in liver after insulin injection may not be due to a direct action of this hormone. In homeotherms. in Clarias batrachus (Bhatt et al. De novo glucose synthesis from alanine was diminished in both fed and fasted rainbow trout (Cowey et al.. especially gluconeogenic and glycolytic pathways and protein metabolism has been obtained in part from in vivo approaches. in fact. De la Higuera and Cardenas (1986) reported that insulin also decreased 14C glucose formation from glutamate carbons in rainbow trout. and the opposite actions or the absence of effects have been frequently observed in in vivo studies. like its mammalian counterpart. 1993).. is regulated by insulin in mammals. Insulin also stimulates AA uptake and protein synthesis in mammalian liver. 1978). such as epinephrine or glucagon. hypoglycaemia is accompanied by a slight increase in liver glycogen in animals adapted to a temperature of 6°C but at 20°C and 30°C. but depend on the stimulated production of other specific glycogenolytic hormones.. However. Other studies described a decrease in liver glycogen after insulin administration in hagfish (Inui et al. After insulin injection in carp. a very strong depletion of glycogen in liver was observed (Murat and Sefarty. insulin anabolic action on liver carbohydrate metabolism results in an increase in liver glycogen content and inhibition of gluconeogenesis.. Hepatic glucokinase (GK). the relationship between insulin and glucagon is not well established in piscine systems. although some data suggest that the interactions between the two pancreatic hormones are not the same as in mammals (Plisetskaya and Duguay. which plays a key role in glucose utilization in liver. 1977). 1975). Ictalurus melas (Ottolenghi et al. Information on how insulin can modulate the hepatic metabolic fluxes. these effects are not always observed in fish. Similarly. 1983) and in both fed and fasted catfish.

This study is one of the few to analyze insulin effects in lipid metabolism in vivo. Tools other than the exogenous administration of insulin have also been employed: the administration of large doses of arginine to stimulate endogenous insulin secretion. or in vivo neutralization of insulin by antiinsulin antibodies. as deduced from studies on the effects of diet adaptation on insulin and GK activity in trout and carp (Capilla et al. Minick and Chavin. and elevated liver triacylglycerol lipase activity compared to a control group of fish injected with non-specific rabbit serum. GK activity and gene expression in rainbow trout indicates that the induction of GK seems to be strictly dependent on dietary glucose. 2003. Castilla and Murat (1975) injected carp with insulin and showed that protein turnover was significantly reduced in liver. (1988) reported that an injection of pharmacological doses of insulin to normally fed catfish resulted in marked increases in the in vivo incorporation of 14C from glucose into protein in liver. insulin-deficient fish were hyperglycaemic. However. had diminished glycogen content in the liver... Few studies have analyzed the in vivo effects of insulin on hepatic amino acid uptake or protein metabolism.. In the model of administration of specific insulin antisera (Plisetskaya et al.. Navarro et al. Figure 2. being present concomitantly with high levels of insulin (Panserat et al. LLC . Machado et al. 31 various teleost species with respect to level of activity and gene expression.6 shows that diet-induced changes in trout liver GK activity and expression appear to be more dependent on plasma glucose than on insulin values. 2000). Further studies should focus on the direct effects of insulin on GK induction. 1972). bovine insulin did not affect 14C glycine incorporation into liver protein in the primitive hagfish Eptatretus stouti (Inui et al. 2001). Early studies regarding hormone effects in liver lipid metabolism are scarce and contradictory (Tashima and Cahill. 1992). 2004b). 1978). Although GK activity and mRNA in liver are induced by diet in various fish species (Panserat et al. Insulin may be necessary but not sufficient. and that there was an enhancement in protein synthesis. The relationship between post-prandrial insulin levels. 1989).. The importance of fish liver as a tissue with high lipogenic capacity has been well documented. and its findings are consistent with the observed reduction in triacylglycerol lipase activity in rainbow trout after insulin injection (Harmon and Sheridan. their possible regulation by insulin remains unclear.I. While Warman and Bottino (1978) reported hepatic lipogenesis to be unaffected by insulin in catfish (Pimelodus © 2006 by Taylor & Francis Group. 1968.

LLC . (1988) found that insulin administration in catfish induced an increase in the flux of glucose carbons into liver lipid. Values are means ± SE. C-free: carbohydrate free. Different letters indicate differences at P<0. Glucose (A) and insulin (B) plasma levels.32 Fish Endocrinology A 6h 24h 12 10 8 6 4 2 0 B 20 18 16 14 12 10 8 6 4 2 0 a a a Glucose (mmol/L) a b c c a Insulin (ng/ml) b b b c C-free HC LC C-free HC LC DIET DIET C GK activity (mU/mg protein) 70 60 50 40 30 20 10 0 d d C-free HC LC b b c a D 0.1 0 C-free HC LC DIET GK 16S Ratio GK/16S expression a b c C-free HC LC DIET DIET Fig. 2. maculatus).6 Diet effects on glucose and insulin plasma levels and glucokinase (GK) activity and expression in trout liver. HC: high carbohydrate. Adapted from Capilla et al.5 0. (2003).4 0.05. LC: low carbohydrate. and glucokinase activity (C) and expression (D) 6 and 24 h after feeding (only at 6 h for GK expression) in trout fed three different experimental diets. © 2006 by Taylor & Francis Group. Machado et al. The expression of GK mRNA has been normalised to the levels of 16S rRNA.2 0. n = 9 for plasma values and GK activity and n = 2 for values of GK expression.3 0.

but thereafter the hormone induced an increase in glycogen. particularly when glucose was added into the perfusate. 1998. 1975).. and the alanine flux to glucose and CO2 in sea raven hepatocytes. in vitro actions of insulin on fish liver have been analyzed using models of organ perfusion. Porcine or teleost insulin significantly increased the serine flux to glucose and glycogen. both in the absence and presence of glucose in the medium. 2000). (1985) working with isolated and perfused catfish liver. LLC . to increase hepatocyte glycogen content from amino acid carbons. (1981) who studied the effects of insulin on the carbohydrate metabolism of the catfish (Ictalurus melas). © 2006 by Taylor & Francis Group. insulin also increased glycogen content in hepatocytes.. Almost all the above experiments demonstrated the expected insulin effects characteristic of mammalian models. In earlier studies. showed that insulin did not influence liver glycogen decay during the first 2 h.I. Insulin decreased spontaneous glycogen lowering. 33 Although the regulation of hepatic lipogenic enzymes by dietary factors has been addressed in numerous studies (Likimani and Wilson. Ottolenghi et al. and produced a slight increase in the cell glucose level. Dias et al.. the latter having been demonstrated to be an excellent research model (Moon et al. or isolated hepatocytes. Insulin possesses anabolic effects. exhibiting some peculiar characteristics of insulin action in fish liver. Insulin Effects in Liver in vitro In addition to in vivo experiments. insulin has inhibited gluconeogenesis from lactate (Hayashi and Ooshiro. insulin must stimulate gluconeogenic flux. in agreement with a preferentially gluconeogenic role for the liver of this carnivorous species. In these experiments. Plisetskaya et al. 1982. in vivo regulation by insulin is yet to be explored in any detail. Shimeno et al. Thus. 1985). Navarro et al. which also corroborates insulin actions observed in intact fish. (1984) reported that insulin stimulated amino acid transport and 14C leucine incorporation into protein in hepatocytes isolated from coho salmon. Later.. One of the first in vitro experiments using isolated hepatocytes was performed by Ottolenghi et al. 1996. in the perfused liver of the eel. Foster and Moon (1987) studied in detail the effects of insulin on hepatocyte metabolism in sea raven (Hemitripterus americanus). Alvarez et al. in agreement with the effect observed in the entire fish. increasing glycogen stores but through increased amino acid utilisation and glucose production.

decreased total glucose production. glycogenesis and glucose oxidation by porcine insulin in isolated American eel hepatocytes. LLC . however. Porcine insulin (10 –8 M) maintained glycogen content. also increased Fru-2. increased lactate and alanine flux to glycogen in hepatocytes from summer and winter eels. is regulated by insulin in mammals. presence of glucagon. These studies once again show the role of insulin in stimulating the C3 precursor flux to glycogen. Regarding lipid metabolism. Mommsen et al. The researchers subsequently found. although the effect was strongly modified by seasonal changes.34 Fish Endocrinology Foster and Moon (1989) studied the regulation of glycogenolysis and alanine and lactate gluconeogenesis. Other authors have focused on the insulin regulation of the activity of key liver enzymes involved in carbohydrate metabolism. in the presence of glucagon. Foster et al.6-P2 concentration in hepatocytes. According to the authors. even when glucagon and GLP-1 concentrations were several-fold higher than in normal fish. a glucolytic enzyme that indirectly affects the gluconeogenic pathway. this effect is observed only at high insulin concentrations (Petersen et al. It is. Following this treatment. these effects suggest that PFK-1 is a potential regulatory point for insulin in the control of carbohydrate metabolism in the eel liver. It has been reported that insulin increases the activity of trout hepatic pyruvate kinase. 1975). complete immunoneutralization of insulin was obtained by intraperitoneal administration of anti-insulin serum. thus unclear which is the physiological role of insulin in carbohydrate enzyme regulation in fish liver. a characteristic feature in the liver of the fish species studied. For example. Another gluconeogenic enzyme. but no effects in piscine hepatocytes have been observed (Foster and Moon. PEPCK. early studies in liver slices of Notemigonus chrysoleucas showed contradictory effects of insulin. More recently. and had a small stimulatory effect on alanine gluconeogenesis in spring.6-P2) activation ratio and. The magnitudes of the insulin effects on metabolism were smaller in winter than in other seasons. 1987). glucagon and glucagon-like peptide (GLP) by arginine injection in rainbow trout. which stimulated lipogenesis and lipolysis at the same time (de Vlaming and Pardo. no alterations in key metabolites or carbohydrate metabolic pathways..6-diphosphate (Fru-2. (2001b) induced an increase in plasma insulin. © 2006 by Taylor & Francis Group. Insulin increased the fructose-2. 1990). in isolated hepatocytes from these fish. (1989) studied the kinetic characteristics of eel liver 6phosphofructo-1-kinase (PFK-1) in isolated hepatocytes and found that insulin decreased the sensitivity of PFK-1 to ATP an effect offset in the .

compared to mammals.. Furthermore. in vivo studies on the incorporation of radiolabelled substrates and their modulation by insulin have yet to be performed in fish. Navarro et al. fish are less able to clear a glucose load (Capilla et © 2006 by Taylor & Francis Group.. insulin actions on this metabolic pathway have not been reported in vitro in piscine liver systems. 1991. adipose tissue is known to be an important target for insulin. INSULIN EFFECTS IN ADIPOSE TISSUE In mammals. LLC . homologous to its mammalian counterpart. information regarding the direct effects of insulin on this tissue is scarce (Mommsen and Plisetskaya. from both in vivo and in vitro analyses. 1994).. the fish GLUT4 showed a lower affinity for glucose. this in vitro model has not been fully studied in fish. Mammalian insulin depressed glucagon-stimulated phosphorylation of TG lipase by 56% and inhibited glucagon-stimulated lipolysis.. In relation to carbohydrate metabolism. 1985). 1994). in contrast to the abundant literature on insulin control of adipocyte function in both isolated and cultured mammalian adipose cells. Although the presence of insulin receptors in fish adipose tissue has been described (Planas et al. Rates of de novo synthesis of FFA from 14C acetate were stimulated by insulin in acute and extended incubation periods in short-term culture of rainbow trout hepatocytes (Segner et al. 35 More recently. However. Insulin control of lipid mobilization and storage in this tissue requires further investigation. Harmon et al. (1993) investigated the regulation of trout hepatic triacylglycerol lipase (TG) by insulin in isolated hepatocytes of rainbow trout. The action of insulin in fish liver is similar to that described for other vertebrates. Insulin stimulates glucose and lipid uptake and inhibits the mobilization of stores. which could partly explain why. although specific effects are observed. 2002). in Oreochromis mossambicus (Sunny et al.. suggesting that insulin modulates lipolysis in trout liver by altering the phosphorylation of the TG lipase enzyme. 2000c). Sheridan and Harmon.I. Although hepatic lipogenic enzymes malicenzyme (ME) and glucose-6-phophate dehydrogenase(G6PDH) have been shown to be affected by other hormones such as cortisol and testorerone. A GLUT4 has been recently characterized from salmon adipose tissue (Capilla et al. in relation to specific characteristics of fish carbohydrate and protein metabolism. no effect of insulin on glucose uptake into adipocytes isolated from trout was reported (Christiansen et al. 2004a) and shown to be a functional glucose transporter in fish. In an early study.

(A) Insulin-stimulated salmon GLUT4 translocation in 3T3L1 adipocytes. 2004a).7B). is also able to accumulate lipids. However. Values are means ± SE of 6 independent experiments. In mammals. Moreover. 30 min. (B) Insulin-stimulated 2-DG uptake in isolated trout adipocytes. Values are presented as percentage of uptake over the basal value (100%). At the same time. The maintenance of lipid stores in adipose tissue is determined by a balance between lipid mobilization and deposition. insulin is the main regulator of lipid deposition. mostly as triacylglycerols. (2004a). 2. together with liver and muscle. In fish. Adipose tissue in fish. 2. *indicate differences at P<0. Insulin. and inhibited primarily by insulin. Values are presented as percentage of cells showing a plasma membrane ring obtained by counting 50 cells per condition as observed by immunofluorescence. analysed by confocal immunofluorescence microscopy (Fig. Although insulin has © 2006 by Taylor & Francis Group. lipid mobilization is known to be stimulated by catecholamines and glucagon. 1994).. 2. expression of the salmon GLUT4 in 3T3L1 adipocytes resulted in translocation of the transporter to the plasma membrane in response to insulin. Cells were pre-incubated for 30min with the hormone (100 nM) and uptake was measured after 2 h.7A).7 Insulin effects on GLUT4 translocation and glucose uptake in fish adipocytes. Values are means ± SE of 5 independent experiments.36 Fish Endocrinology A 100 90 80 70 60 50 40 30 20 10 0 B 2-DG uptake (% over basal) 160 140 120 100 80 60 40 20 0 * GLUT4 translocation (% positive cells) * Basal Insulin Basal Insulin Fig. This study also showed that insulin significantly stimulated glucose uptake in isolated trout adipocytes (Fig. LLC .05. the endocrine factors directly involved in their regulation are not well understood. Adapted from Capilla et al. the enzymatic machinery responsible for lipid mobilization and deposition in the adipose tissue is similar to that found in mammals (Sheridan and Harmon. al. 100 nM.

the anti-lipolytic effect was more evident when insulin reversed the lipolytic effect of glucagon in incubation. play a role in food intake regulation in fish through the inhibition of other orexigenic peptides. These data indicate that the adipose tissue’s accumulation of FFA coming from diet triglycerides. 1991). 1998). INSULIN AND FOOD INTAKE Food intake in fish is regulated by a complicated network of brain orexigenic and anorexigenic signal molecules (reviewed by Lin et al. in the presence of both hormones. central (icv) or peripheral (ip) treatment with insulin inhibited food intake 26 and © 2006 by Taylor & Francis Group. 37 been reported to have anti-lipolytic effects in vivo in fish adipose tissue (Harmon and Sheridan. 1994). insulin may. 2000. 2005). 1996) suggests that. 2001) and the increase of hypothalamic mRNA levels in fasted salmon (Silverstein et al. 1992). 2000). 2001).. Recent studies have demonstrated the inhibitory effect of insulin on lipolysis in isolated trout adipocytes. suggesting a minor role of this tissue in lipogenesis (Segner and Böhm. the activity of the enzymes controlling FFA biosynthesis in fish adipose tissue is lower than in liver. 1992).. direct effects of insulin on lipogenesis in fish adipocytes have yet to be demonstrated (Mommsen and Plisetskaya. Narnaware et al. or synthesized by other tissues. Soengas and Aldegunde (2004) showed that in rainbow trout. there have been few in vitro analyses of insulin action in lipid metabolism in this tissue in fish. More recently. may be regulated positively by insulin through this enzyme. However. directly or indirectly. 2000. Navarro et al. LLC . Insulin is thought to be one of the major signals informing the brain about body fatness and energy balance in mammals (Jéquier and Tappy.. Furthermore. However. 2006). 1999). There is some evidence that NPY is involved in controlling fish food intake. The presence of insulin receptors in semi-purified preparations of piscine brain (Leibush et al. Bernier and Peter. such as NPY (Silverstein and Plisetskaya. namely the stimulation of food intake in the goldfish (De Pedro et al. 2000). as in mammals (Albalat et al. as in mammals.. measured either by FFA or glycerol release (Harmon and Sheridan. Narnaware and Peter. intracerebroventricular (icv) injection of insulin had no effect on food intake in channel catfish (Ictalurus punctatus). However. 2000. Insulin was able to decrease the level of lipolysis in rainbow trout adipose tissue pieces. measured by glycerol release into the medium (Albalat et al. This factor notwithstanding. insulin administration increased lipoprotein lipase activity in trout adipose tissue.I... measured from 1 to 24 h after injection (Silverstein and Plisetskaya.

Protein metabolism is regulated by insulin in fish in a similar manner to that observed in other vertebrates. Muruzabal et al. opposite or unexpected effects are also reported from both in vivo and in vitro experiments. no clear physiological effects have been observed after leptin treatment in the salmon (Baker et al.38 Fish Endocrinology 52 h after administration. respectively. although gluconeogenic pathway presents specifically regulation in fish. In contrast.. 2000). As the information regarding the mechanisms of insulin action increases.. Some recent studies confirm the early findings on the preponderance of AAs over glucose in stimulating insulin secretion in fish systems. The presence of insulin peptide in the fish brain has been detected by immunocytochemistry. rather than via carbohydrate metabolism alteration. the possible mechanisms and peptides involved in the observed anorexic effect of insulin remain to be elucidated. 1993). While a leptin-like protein has been immunodetected using mammalian antibodies in various fish tissues (Johnson et al. suggesting that brain insulin comes from pancreatic secretion and is transported to the brain. there are numerous studies of insulin effects on fish hepatocytes.. In general terms. such as GLUT4 or GK are also present in fish. increasing glucose uptake and the accumulation of glycogen in muscle and liver. for many years. The existence of leptin in fish as a food intake inhibitor and antagonistic to insulin effects in brain as in mammals. and these cells have. LLC . is still a matter of controversy. This appears to be a direct action in the brain. The role of insulin in the control of lipid metabolism remains unclear. 2000. it is becoming increasingly clear that the key elements in glucose metabolism reported in mammals. whereas mRNA was undetectable (Plisetskaya et al. © 2006 by Taylor & Francis Group. and adipose tissue is one of the leastinvestigated insulin target tissues. but with specific functional characteristics. although the molecular basis of this characteristic is yet to be elucidated.. 1997). CONCLUSIONS In summary. offering new insights into the specific characteristics of piscine insulins. Although insulin can enter the brain across the blood–brain barrier in mammals (Banks et al. However. there is only indirect evidence that this may also be the case in piscine brains. To date. insulin acts—as in mammals—as an anabolic hormone in the control of carbohydrate metabolism. 2002).. the structure of insulin gene and peptide has been investigated in numerous teleost species over the last decade.

Endocrinol. H..E. I. the interactions between insulin and the complicated network of neuropeptides controlling food intake is a fascinating subject that has yet to be investigated in depth. M. and CIRIT (2001 SGR-00122 and CRA). Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): The role of insulin and glucagon.. The effect of . M. J. Diez. (In press). J.). as in mammals. Biochem. Regulation of lipoprotein lipase (LPL) activity in rainbow trout (Oncorhynchus mykiss) tissues. Navarro et al.M. Acknowledgements We thank Antonino Clemente and Rosa Marsol from the Piscifactoria de Bagà (Generalitat de Catalunya) and J. Endocrinol. Eur.. Gallego. R. Gen.O. Sinnhuber. Br. J. C. W..K. B. S. 2000. and Selivonchick. INRA. Rescan from SCRIBE. . J. Biochem Physiol. A. M. it appears that it could. Dogfish insulin.. also be a key signal informing the brain about the status of energy balance in fish. Schwabe. However. This study was supported by grants from the European Union (Q5RS2000-30068). 43:211-217.I. The English text was corrected by the Language Advisory Service of the University of Barcelona. C. Wood.A. Gen.. and Bautista. for their help . Rennes. Crow. © 2006 by Taylor & Francis Group. Gowan.. 1983. and for allowing us to use their sampling facilities and for their assistance. DGICYT Spain (AGL-2001-2903 ACU. Wollmer. Blundell. R. Comp.. Nutr. Weil.. Falkmer. A. Short-term modulation of lipogenesis by macronutrients in rainbow trout (Oncorhynchus mykiss) hepatocytes. L. The development of new in vitro systems such as myocyte cultures or isolated adipocytes will help to increase knowledge of insulin effects in fish tissues. Emdin. A.. Sánchez-Gurmaches. Pitts. S... Albalat.Y. A. and Strassburger. A 142: 347-354.P 1981. Bajaj.. References Ablett. C.P. Gutiérrez.. LLC . and Navarro. 2005. S. Primary structure.J.F. AGL 200203987).. M. Fauconneau and P Y. Gutiérrez. Holmes.. conformation and biological properties of an elasmobranchial insulin. D. prolonged administration of bovine insulin in rainbow trout (Salmo gairdneri R. J. and suggestions with myocyte culture. R. and Navarro. Tatnell. Albalat. 135:535-542.O. 84:619-628. Comp.M. Baró of the Piscifactoria Truites del Segre (Lleida) for providing fish. T. 39 been shown to be a good model of study of liver metabolism and its hormonal control. I. J. Comp. Alvarez..L. Given the important role played by insulin in metabolic control. We also thank P Le Bail. Lopez-Bote.

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LLC . RIA possesses problems relating to the handling of © 2006 by Taylor & Francis Group. We have developed competitive non-RI immunoassay systems including time-resolved fluoroimmunoassay and enzyme fluoroimmunoassay for flounder insulins. including potential health hazards. theories and techniques for development of non-RI immunoassay system for fish insulin have been reviewed along several examples. The most important requirements for the development of Author’s address: Hokkaido National Fisheries Research Institute. technical problems such as lower stability of labeled ligands.affrc. 116 Katsurakoi. In this chapter. Japan. E-mail: andoh@fra. and difficulties in the disposal of tubes and solutions used in the assay.go. Radioimmunoassay (RIA) systems for fish insulin have been developed in several species. Non-radioisotopic (Non-RI) immunoassay has the potential to solve such problems. but homologous RIA system for fish insulin is limited to salmon and catfish. Furthermore. Fisheries Research Agency.CHAPTER 3 Non-radioisotopic Immunoassay for Fish Insulin Tadashi Andoh ABSTRACT Development of exact quantification system is an essential step in solving recent problems related to insulin function in fish. 085-0802 Kushiro.

. Smith. INTRODUCTION Insulin (see: Navarro et al. © 2006 by Taylor & Francis Group. 1968. Conlon et al. this volume) is heterodimeric peptide hormone consisting of about 50 amino acid residues.. 2002. The first is a possibility that the insulinotropic effect of amino acids is stronger than that of glucose and the blood glucose level is relatively independent of insulin level in fish. and (2) biotinylation site of labeled ligand and a number of such sites. Andoh et al. Duan et al. Insulin promotes sulfation uptake in gill cartilage of fish at the physiological concentration (Duan and Hirano. LLC . Biotinylation. 1989. 1994. unlike mammals (Tashima and Cahill. Time-resolved fluoroimmunoassay. RP-HPLC—reversed phase high performance liquid chromatography. 1966. 1998a. including receptor binding process in target cell. The topics reviewed in the present study are also applicable to development of non-RI assay systems for other peptide hormones. 1977. Prospective projects in this field will be focused on: (1) secretion mechanism of insulin.. bfINS—barfin flounder insulin. 1960. 1992. 2002). Solutions to these problems are important to understand the physiological role and functional evolution of insulin in vertebrates and. Lanthanide. Ince and Thorpe.. Ligand labeling.. FIA—fluoroimmunoassay. EIA—enzyme immunoassay. Exact quantification of insulin is one of the most important and basic techniques to solve these problems. Andoh and Nagasawa. This hormone is secreted in fish from the different types of islets (see: Agulleiro et al. Abbreviations: TR-FIA—time-resolved fluoroimmunoassay. several differences between fish and mammals have been pointed out. Eu—europium. Kotaki et al. 1962. Andoh and Nagasawa. and (3) differential roles among molecular types of insulin and IGFs.50 Fish Endocrinology assays are: (1) assay design. The last distinction is the existence of plural molecular types of insulin in fish. Enzyme immunoassay. Key Words: Insulin.. although it is unclear whether there is any functional difference between them (Yamamoto et al. Competitive immunoassay. 1992.. Nguyen et al. However. 1998). Europium. c. consequently. The second difference is the promotive function of insulin in growth. Mommsen et al. to also understand the role of insulin in growth regulation system. this volume) corresponding to mammalian endocrine pancreas (islets of Langerhans) and regulates anabolism and growth in fish like in mammals. b... 1997). 2000. (2) signal transduction system. Plisetskaya.

Recently. 1994). Therefore... both of them being the first developed non-radioisotopic (nonRI) immunoassay systems for fish insulin. lamprey (Plisetskaya. 1986). 2002. We have reported enzyme fluoroimmunoassay (FIA. 1997) and TRFIA (Andoh and Nagasawa. 2002) systems for barfin flounder insulins (bfINSs). 2002). catfish (Plisetskaya et al. In the present chapter. They demonstrated simplicity. divergence in amino acid sequence among fish insulins is larger than that in mammals and crossreactivity against antiserum is different even between flounders (Andoh and Nagasawa. 1980). 1997. reliable RIA systems in fish are restricted to only certain fish species. © 2006 by Taylor & Francis Group. Andoh. Many EIA kits for hormones are commercially available. Unfortunately. 1971). Therefore. immunoassay systems used for mammalian insulins are for the most part not applicable in measuring blood insulin levels in fish. 1997). RIA is the most frequent method for quantification of hormones and growth factors. Therefore. non-RI immunoassay including enzyme immunoassay (EIA) and time-resolved fluoroimmunoassay (TR-FIA) have been used as substitutions of RIA for both peptide (Andoh and Nagasawa. trout (Tilzey et al. 1980. theories and techniques for development of non-RI immunoassay for fish insulin have been described and discussed in detail. Gutierrez et al. 1985. However. high specificity and sensitivity of this system. because crossreactivity between fish insulins and antibodies developed against mammalian insulins is usually very weak. In addition. Non-radioisotopic (Non-RI) immunoassay has the potential to solve such problems. 2002. 2005) and steroid hormones (Yamada et al. Furthermore. Plisetskaya et al. including potential health hazards.Tadashi Andoh 51 Radioimmunoassay (RIA) system was developed by Berson and Yalow (1959) using mammalian insulin. Yamada et al. RIA systems for fish insulin have been reported in hagfish (Emdin and Steiner. as yet. bonito (Furuichi et al. new immunoassay systems for the measurement of fish insulin had to be developed. LLC .. 1976). technical problems such as lower stability of labeled ligands. cod (Thorpe and Ince... molecular characterization of insulin has not been completely established in toadfish and bonito.. RIA also possesses problems relating to the handling of radioisotopes. toadfish (Patent and Foa. 1998c). Most of this information can be applicable to the development of non-RI immunoassay systems for not only fish insulin but also other peptide hormone and growth factors. 1984) and scorpion fish (Plisetskaya and Leibush. Andoh and Nagasawa.. 1974). and difficulties in the disposal of tubes and solutions used in the assay.

it is more specific. However. this volume). Taking all this into account. and (3) generally. Most commercial kits for insulins of humans and domestic animals use monoclonal antibodies. Therefore. highly specific and sensitive sandwich system sometimes depends on the use of IgG purified by strict specific affinity chromatography. Brockmann bodies correspond to Langerhans islets tissue of pancreas in mammals and consist of pure endocrine tissue including a lot of insulin-producing cells and is surrounded by only a thin tissue like membrane (Brockmann. production of monoclonal antibody is an important point of development of the sandwich system. (2) less quantity of antibody is needed. while the competitive method requires high purity of labeled ligand. 1991. LLC .. the sandwich method requires high purity of IgG. ANTISERUM Anti-insulin Serum Development Although molecular mass of insulin is about 6 kDa. this method requires a large quantity of specific antibody due to coating wells of microtiter plate and loss during labeling and purification. Therefore. This method requires at least two binding sites for IgG on the molecule. 1986). and insulin possesses several epitopes (Rathjen and Underwood. On the other hand. This point enables © 2006 by Taylor & Francis Group.52 Fish Endocrinology ANTISERUM DEVELOPMENT AND LIGAND LABELING Assay Design—Competitive or Sandwich? Although insulin is a small molecule (about 6 kDa). the sandwich method has been used in many assay systems including clinical test kits for human and domestic animals. 1986). see: Agulleiro et al. 1846. The advantages of sandwich method are: (1) high sensitivity. In addition. Mommsen and Plisetskaya. Most fish insulins are purified highly and easily by RP-HPLC from Pancreas or Brockmann bodies which contain insulin at high concentration. (3) linearity of standard curve. because specificity and sensitivity of this method depend highly on purity of IgG. insulin possesses several epitopes (Rathjen and Underwood. the advantages of competitive method are: (1) the whole antiserum can be used. and (4) measured value increases in proportion to hormone concentration increment. the sandwich system is more likely to be influenced by crossreactivity and non-specific binding of antibody. (2) wide dynamic range over three orders. we have adopted the competitive method for fish insulin immunoassay system from these backgrounds. Furthermore.

LLC . Titer elevation The relationship between titer elevation and applicability of antiserum to immunoassay sometimes appears to be unreliable. On the other hand. collectable serum is less than 10 ml/individual. The procedure of development of our antiserum (lot. reduction and affinity purification is generally considerably less than that of the purified IgG.5 mg/ml) (Lindmark et al. 951219-01) consisted of a primary injection of bfINS-II in Freund’s complete adjuvant followed by five booster injections of the insulin in Freund’s incomplete adjuvant. amino acid sequence of fish insulin has 10-14 amino acid residue differences from that of rabbit. Guinea pig produces high titer antiserum against mammalian insulin. The interval between injections was 2 weeks. 2002). Andoh and Nagasawa (1997.Tadashi Andoh 53 immunization without conjugation with other proteins unlike other peptide hormones. Furthermore. because guinea pig needs less antigen than rabbit due to its small body size. The peptide was conjugated with keyhole limpet hemocyanin using hetero- © 2006 by Taylor & Francis Group. especially in immunization of oligopeptide. because the amino acid sequence of guinea pig insulin has 14-18 substitutes in amino acid residues as compared to other mammals studied. However. suggesting that rabbit is an alternative for immunization against fish insulin. and the concentration of IgG is 2–5. 2002) used guinea pig for immunization with flounder insulin. Plisetskaya et al. (1986) first chose guinea pig rather than rabbit. We tried to immunize two guinea pigs with an oligodecapeptide consisting of B-chain N-terminus of bfINS-II. (1991) proved that chicken was a suitable animal for production of antibody against human insulin and also earn to purify the antibody from the egg.. Furthermore. Lee et al. Therefore.9 mg/ml is less than half in comparison to rabbit (12-14. (1985) used rabbit for immunization. 1983). Eighty mg of insulin was injected during first three times and thirty mg during the last two times. guinea pig is hardly suitable for antibody development for sandwich system for routine work. (1984) and Tilzey et al.. but used rabbits subsequently for higher antibodies (Plisetskaya et al. Guinea pig has been used for immunization against mammalian insulin. Gutierrez et al. the sandwich system needs fragmentation of IgG to Fab’ or F(ab’)2 and the final yield of fragments after digestion. Antiserum was obtained 2 weeks after the final injection.

3.2’-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) as a chromogen. No. Fig. This phenomenon can be explained by fine difference in antibody recognition sites between titer elevation checking and actual immunoassay. Two guinea pigs (GP1 and 2) were immunized and days after immunization were expressed as the upper figures. Titer elevation pattern was different between two animals (GP1 and GP2). Titer elevation was measured by colorimetric EIA using microtiter plate with immobilized bfINS-II and HRP-anti-guinea pig IgG system at intervals of two weeks. 3. 1981) before injection. LLC .. but did not recognize biotinylated bfINS-II at all. while that of GP2 maximized at 28 days and was approximately 1:100 at 70 days.54 Fish Endocrinology bifunctional cross-linking reagent (Kitagawa et al. The immunization was successful in both guinea pigs and the antisera recognized the peptide immobilized to microtiter plate with glutaraldehyde. Fig. © 2006 by Taylor & Francis Group. Titer of antiserum is expressed generally as visible dilution rate which is just relative value. Barfin flounder insulin-II was immobilized to microtiter plate at the concentration of 5 mg/ml and diluted antiserum from each animal was dispensed. we used an antiserum at 84 days from GP1 (Lot. Titer was detected using anti-guinea pig IgG labeled horseradish peroxidase and 2. According to these results. However.1 shows titer elevations of antibfINS-II in two guinea pigs. 951219-01) to develop FIA (Andoh and Nagasawa. Visible dilution rate of GP1 was less than 1:10000 after 70 days of the first immunization. 2002).1 Titer elevation of guinea pigs immunized barfin flounder insulin-II. 1997) and TR-FIA (Andoh and Nagasawa. although B-chain N-terminus was not labeled. titer elevation checked by the above method was the only easy indicator of antibody production in each animal.

1. carboxyl. Rockford. but measured values tend to be influenced easily by sample components. Biotin is a member of the vitamin B complex. (2) which site to biotinylate. It is sometimes more important than antibody production in a competitive assay system. Figure 3. These characteristics are suitable for labeling of ligand. and SH groups. This reaction progresses via primary amines containing both . Biotinylation has been used as the most popular non-RI labeling method. The other alternative is using a water-soluble type of reagents with a modified sulfo group at the active ester domain. biotinylation reagents are used after dissolving in organic solvent. LLC . The direct labeling allows to omit a step for binding of detection reagent. but excess labeling could decrease crossreactivity of labeled ligand with antibody.9 mM.and -amino groups under the condition of pH 7–9 and produces a stable amide bond between biotin and peptides. and (3) length of spacer arm chain. indirect labeling is preferred over direct labeling. Labeling via amino group is the most frequent in many immunoassay systems. The general advantages of the former are higher sensitivity and stability of the labeled ligand. and glycosylated sites.2 indicates the chemistry of biotinylation with SulfoNHS-biotin (Pierce. Biotinylation reagents for labeling via amino group have been shown in Table 3. IL). The number of labeled sites is decided by the balance of sensitivity and crossreactivity against the antiserum. The following three points used to be considered when biotinylating insulin: (1) number of biotinylated sites. Biotinylation is performed at room temperature or lower. because it is easier to control biotinylation efficiency at a lower temperature. Several kinds of reagents for biotinylation are commercially available and are used for labeling via amino. © 2006 by Taylor & Francis Group. Therefore. is chemically stable and its molecular weight is small (244 Da). Solubility of biotin in water is low—220 mg/l corresponding to 0. such as dimethyl sulfoxide. and the best number is different among each assay system. also called vitamin H.Tadashi Andoh 55 Labeling Technique of Ligand Labeling of ligand highly influences specificity and sensitivity in competitive immunoassay. It binds strongly with avidin and streptavidin (binding constant: 10 –15/M at 25°C). Excessively biotinylated ligand some times does not display crossreactivity. Generally. Number of biotinylated sites Multiple site labeling is effective for increased of sensitivity.

The number of biotinylated sites can be counted by mass spectrometry or photometric assay using avidin binding dye. LLC . ZipTip (Millipore.56 Fish Endocrinology Sulfo-NHS-biotin H 7 < pH < 9 Biotinylated protein Fig. Bedford. This product is a small column using octadecil silica resin for reversed phase chromatography and © 2006 by Taylor & Francis Group. This reaction progresses from pH 7 to 9. The former. In the case of biotinylation via amino group. The fraction containing salts and/or detergents requires additional purification for mass spectrometry. is more exact and sensitive as also simpler and faster for fractions of biotinylated ligands separated by RP-HPLC. MA) is fast and convenient for removing salts and detergents. each site reacts with biotinylation reagent influencing pH. with matrixassisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MASS). Bullesbach and Schwabe (1990) reported selective monobiotinylation of relaxin—which belongs to insulin superfamily—by pH regulation and combination with chromatographic separation. 3.2 Chemistry of biotinylation of protein via amino group using sulfo-NHS-biotin.

5 22.7 13.5 22.1 Commercially available reagents for biotinylation via amino group.5 Very low Very low Very low High High High Tadashi Andoh 57 © 2006 by Taylor & Francis Group. LLC .5 13.6 669. Commercial name NHS-Biotin NHS-LC-Biotin NHS-LC-LC-Biotin Sulfo-NHS-Biotin Sulfo-NHS-LC-Biotin Sulfo-NHS-LC-LC-Biotin Chemical name (+)–Biotin N-hydroxysuccinimide ester (+)–biotinamidohexanoic acid N-hydroxysuccinimide ester Biotinamidohexanoyl-6-aminohexanoic acid N-hydroxysuccinimide ester Biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt Biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt 6-((6-((Biotinoyl)amino)hexanoyl)amino)hexanoic acid.4 455.4 556.Table 3. sulfosuccinimidyl ester.7 443.4 30.5 567. sodium salt Molecular Arm length Solubility weight (Å) to water 341.4 30.

1990) is also useful and measurement with this dye is based on the same chemistry with HABA. Osaka.58 Fish Endocrinology the shape is like a pipette tip. 1996). 1973). the second method using avidin binding dye does not require special equipment and is inexpensive. binds to avidin in order to produce a complex that absorbs 500 nm (Green.6-ANS. A standard curve should be established using serial dilution of biotin. but it is impractical due to the need for a sample amount in microgram order. On the other hand. © 2006 by Taylor & Francis Group. This method is approximately more sensitive than HABA. There is no suitable method for determination of biotinylated site as yet. 1983). B-chain N-terminus is also reported as an important antigenic determinant (Geiger and Langner. Free biotin present in solution with this avidin-HABA complex will displace the HABA dye and result in decreased absorbance.. 1970. Biotinylation of these regions may possibly decrease the crossreactivity of labeled ligand. The determination is not so important in immunoassay except for a few cases. Colorimetric dye. The region A8-A10 is greatly variable in vertebrates including fish and important for antigenic determinant (Schroer et al. The buffer exchange based upon gel filtration is not suitable for mass spectrometry because of its dilution effect. (2) separation of each biotinylated ligand. Several fish insulins possess an additional -amino group at A9. 2-hydroxyazobenzen-4’-carboxylic acid (HABA). Fluorophotometric dye. Crossreactivity of labeled ligand with antiserum prevails over other considerations. Janolino et al.. LLC . The important points are: (1) biotinylation of ligand under the condition of low biotinylation efficiency. Identification of biotinylated site Mammalian insulin possesses three amino groups consisting of two Nterminal -amino groups of two amino acid chains and one -amino group of Lys at B29. Japan) providing custom-made service. Our recent TR-FIA system uses chemically synthesized B1 monobiotinylated bfINS-I which was purchased from an expert company (Peptide Institute. Protein sequencing is a simple procedure. 2-anilinonaphthalene-6-sulfonic acid (2. and (3) checking of immunoreactivity of each candidate. Chemical synthesis enables the introduction of biotin into any site specifically. but does necessitate special technique and is expensive. Mock and Horowitz.

Water soluble types modified sulfo group are also available (Table 3. lot. 22.5 Å.Tadashi Andoh 59 Arm length between biotin and ligand Three kinds of biotinylation reagents are commercially available. Thus. I-6136. 118F-4826). Insulin is a definitely small molecule compared both with avidin (MW: 60 kDa) and IgG (MW: 150 kDa) and the four binding sites of avidin for biotin are located deep inside avidin (Rosano et al.5 and 8. respectively. Japan) was diluted at the ratio of 1:3 with 50 mM Tris-HCl containing 150 mM NaCl. NHS-LC biotin and NHS-LC-LC biotin. Repurification of commercially available bovine insulin by RP-HPLC sometimes gives better results. but this product did not show crossreactivity with our anti-bovine insulin antiserum (Sigma.1). Block Ace solution (Dainippon Pharmaceutical. Cat. Note: EZ-Link Sulfo-NHS-LC-biotin is easily soluble in water. Reagents Bovine insulin (Cat. This solution is used for blocking and inhibition of degradation. Biotinylation Bovine insulin was mixed with the biotinylation reagent at the molar ratio of 1:5 and 1:100 in PBS and pH checked with pH test paper. respectively. I5500) and biotinylation reagent (EZ-Link SulfoNHS-LC-biotin) were purchased from Sigma and Pierce (Rockford. 1999).. This flexibility and length of the arm appear to be important for the crossreaction with antibody and binding with avidin. Bovine insulin biotinylated via amino groups without any extended arm is commercially available from Sigma. such as NHS-biotin. Biotinylation of bovine insulin The following example is a description of bovine insulin biotinylation using Sulfo-LC-NHS biotin. Phosphate buffered saline (PBS) was adjusted between pH 7. No.0 with NaOH. LLC . Osaka. In contrast.5 Å. Long chain arm with flexibility maintains binding for biotinylated insulin. Spacer arm lengths of those are 13. bovine insulin biotinylated with NHS-LC-biotin shows high crossreactivity against the above antiserum. 25 mM EDTA and 0. both with avidin and anti-insulin antibody.4 Å and 30.01% NaN3 (Tris-BA). After 2 hours © 2006 by Taylor & Francis Group. IL). it is important to store the reagent at below 0°C under the dry conditions. but might be hydrolyzed at room temperature.

Japan). Each fraction was diluted at the ratio 1: 1500 with Tris-BA and 10 l of each fraction was used for the measurement. LLC . Note: The molar ratio and pH are the most important factors which influence biotinylation efficiency.3B show typical results of separation of biotinylated bovine insulins.5 min on these profiles and mass spectrometric © 2006 by Taylor & Francis Group. biotinylation was terminated by addition of excess amount (over 100 times molar concentration of biotinylation reagent) of glycine. Kyoto.0 mm 150 mm.60 Fish Endocrinology at 25°C.1% TFA at the final concentration to the labeled ligand solution. Japan) with 2-(4-hydroxyphenylazo)benzoic acid as the matrix. Tosoh.1: 50: 50. because amine of tris reacts with biotinylation reagent and exhausts it. Those profiles were at a molar ratio of 1:5 and 1:100 for biotinylation reagent and insulin. Checking of immunoreactivities of biotinylated insulins Immunoreactivity of each fraction was assayed based on TR-FIA using microtiter plate coated with anti-guinea pig IgG and immobilized antibovine insulin (described below). Biotinylated insulin solution was injected onto RP-HPLC system using trifluoroacetic acid (TFA)/water (0. Biotinylated insulins are eluted after native insulin on RP-HPLC due to hydrophobicity of biotin in most cases.1: 100. v/v) and TFA/water/ acetonitrile (0. Trial and error is possibly needed. biotinylated insulin and unreacted insulin were separated by RP-HPLC after biotinylation. The flow rate was 100 l/min and the concentration of acetonitrile in the eluting solvent was increased from 0% to 25% over 5 min and from 25% to 40 % over 35 min at 30°C. pH checked under 5 and the mixture was left for one hour. We use an ODS-80Ts column (2. Tokyo. Fractionation was carried out peak by peak according to UV profile at 214 nm. A lot of absorbance peaks were observed after 29. v/v/v) after addition of 0. Results and discussion Figures 3. TrisHCl is not suitable for biotinylation reaction.3A and 3. Separation of biotinylated insulins Excess amount of biotinylation reagent. Note: Gel filtration does not give enough separation. Mass spectrometric analysis was carried out using Kompact MALDI-I time-of-flight mass spectrometer (Shimadzu. respectively.

The number of biotinylated sites varied from one to three and each biotinylated insulin was eluted depending on number of biotinylated sites. Insulin was reacted with sulfo-NHS-LC-biotin under the ratio of 1: 5 (A) and 1: 100 (B) and separated by RP-HPLC on an ODS-80Ts (2. © 2006 by Taylor & Francis Group. Frac. Two vertical arrows indicate retention times for native (bINS) and deamidated bovine insulins (da-bINS).3 Separation of biotinylated bovine insulin by RP-HPLC. Crossreactivity of each fraction was expressed as Count (cps) measured by TR-FIA system using anti-bovine insulin antiserum. Figures with horizontal arrow indicate number of biotinylated site established by massspectrometry. Tosoh) at 100 ml/min at 30 C. LLC . analysis indicated the number of biotinylated sites of each peak. No. 14 of A and 38 of B were used for development of TR-FIA systems (Fig.Tadashi Andoh 61 Fig.0 mm 150 mm. 3. 4).

frac.(Fig. These results indicate that fraction No. Competitive crossreaction was performed for 19 hours at 26°C. 3B.4 Binding inhibition curves for bovine insulin in two TR-FIA systems using monoor tribiotinylated bovine insulin and anti-bovine insulin antiserum. Concentration of biotinylated insulin in each assay was tuned to maximize the sensitivities by pre-experiments. No. No. 14 at 1:5 and No. Detection limit in No. 14) or tribiotinylated bovine insulins (Fig.4 corresponding to monoand tribiotinylated insulin. 38) and anti-bovine insulin antiserum (Sigma) were used. Microtiter plate was coated with goat anti-guinea pig IgG. The total count indicating total binding for tribiotinylated insulin was greater than that of monobiotinylated insulin. In fraction No. 14 it was 20 pg/well corresponding to 2 ng/ml. although nonspecific binding (at 100 ng/well) was the same. 3A. 38 at 1:100 were shown in Fig.62 Fish Endocrinology LC-tribiotinylated bovine insulin Count (cps) Bovine insulin (pg/well) Fig. 38 containing tribiotinylated bovine insulin is more suitable for the © 2006 by Taylor & Francis Group. Mono. 3. Fractions containing these peaks showed insulin immunoreactivity. 3. respectively. Eu-avidin was used for detection. frac. 38 was less than 10 pg/well corresponding to 1ng/ml. LLC . Binding inhibition curves for two TR-FIA systems using fraction No.

LLC . Andoh and Nagasawa. suggesting the absence of any functional divergence between bfINSs.Tadashi Andoh 63 development of TR-FIA for bovine insulin using antiserum employed in the present study. whereas LC-biotinylated bfINS-I was not recognized like other biotinylated fish insulin. Andoh and Nagasawa. 1972). 1997. Thus. There are two amino acid residues extension at the B-chain N-terminus of bfINSII and the N-terminal residue is pyroglutamic acid which does not react with the biotinylation reagent via an amino group even with an excess amount of biotinylation reagent. LC-biotinylated bfINS-II was recognized by the antiserum. 2002) to a system showing equal crossreactivities with both insulins (Fig. we developed FIA and TR-FIA systems using bfINS-II biotinylated with NHS-LC-biotin (LC-biotinylation. it is important to regulate biotinylation site. Binding activities of each bfINS with bfINS receptor are identical (Andoh and Matsubara. 1998a). On the other hand. Lindsay et al.5B). 951219-01) used in Andoh and Nagasawa (2002) recognizes B-chain N-terminus well. and our antiserum (Lot. 3. Furthermore. Bchain N-terminus of insulin is known to be an important epitope of insulin (Lindsay and Shall.. In contrast. Therefore. Critical points are the ratio of insulin to biotinylation reagent and separation of biotinylated insulin by RP-HPLC. Therefore. Biotinylation of barfin flounder insulin Several endocrinologists have tried to develop non-RI immunoassay for fish insulin. LCbiotinylated INS-II was substituted with chemically synthesized bfINS-I monobiotinylated with NHS-LC-biotin only at the B-chain N-terminus (Peptide Institute). No. Barfin flounder possesses two molecular forms of insulin. 2002).5A. Binding inhibition curves for both insulins in the improved system were identical.). Two molecular forms of insulin in barfin flounder originated from a single preproinsulin by proteolytic cleavage at different sites of the signal peptide region (Andoh and Nagasawa. all four amino groups of bfINS-I are not protected and can react with biotinylation reagent under the existence of excess amount of the reagent. we improved the above TR-FIA system for bfINSs (Fig. 1971. In the latter system. pers. but the results were unsuccessful due to the lack of crossreactivity between biotinylated ligand and antibodies (Plisetskaya. in order to produce crossreactive biotinylated insulin. 3. The latter system is more suitable for © 2006 by Taylor & Francis Group. comm. unpublished data).

6). 3. 951219-01) in both systems. However. © 2006 by Taylor & Francis Group. the total count in the improved system was lower than that in the former system (Fig. functional insulin quantification than the former system. Nakane and Kawaoi (1974) reported a highly efficient conjugation method for HRP and IgG via glycosilated sites using NaIO4. LLC . the bond between protein and DTTA is not stable under the coexistence with EDTA and metal ions. No. caused by less biotinylation sites and lower affinity for the antiserum.5 Binding inhibition curves for bfINS-I and -II in TR-FIA systems using LCbiotinylated bfINS-II (A) and B1 monobiotinylated bfINS-I (B). Direct HRP labeling of insulin There are two ways to conjugate HRP—via glycosilated sites of HRP and via amino groups. However. 3. Direct europium labeling of insulin Most proteins are labeled easily with europium via amino group using EuDTTA phenylisothionate (Wallac Oy). Therefore.64 Fish Endocrinology A bflNS-I B bflNS-I Counts (cps) bflNS-II bflNS-II Hormone (pg/well) Fig. direct labeling is not suitable for TR-FIA. The antiserum used was anti-bfINS-II (lot.

3. because crossreactivity with fish insulin was very low. In this assay. 1996). Conjugation efficiency of this method is several times higher than that via amino groups with glutaraldehyde (Nygren. such as hydroxysuccimidyl active ester and pyridyldisulfide groups. (1988) used this reagent to conjugate porcine insulin and horseradish peroxidase and separated A1 monoHRP-. this FIA system was not suitable for measuring plasma insulin level in fish. This reagent reacts selectively with amino groups and the introduces pyridyldisulfide group. We applied this method to bovine insulin and developed a highly sensitive and specific competitive FIA using anti-bovine insulin antiserum (Fig. B1 monoHRP- © 2006 by Taylor & Francis Group. 1982). However. A cross-linking reagent for protein conjugation is used for labeling via an amino group. 3. which is easily reduced to SH group. HRP-insulin is added to diluted antiserum after 6 hours of sample dispensation. This delayed dispensation of labeled ligand appeared to be effective for improvement of sensitivity. Zaitsu et al.Tadashi Andoh 65 Count (cps) Barfin flounder insulin-II (pg/well) Fig. LLC . Concentration of B1 monobiotinylated bfINS-I was changed from 90 pg/well to 360 pg/well. Andoh and Nagasawa. N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) possesses two reactive groups.7.6 Binding inhibition curves for bfINS-II in TR-FIA systems using LC-biotinylated bfINS-II and B1 monobiotinylated bfINS-I with anti-bfINS-II antiserum.

LLC . Crossreactivities of these biotinylated insulins were different from one another and monobiotinyaltion is suggested the most suitable (Zaitsu et al. bINSB: bovine insulin B-chain. hINSC: human insulin C-peptide. B1 diHRP-insulins by HPLC. hPP: human pancreatic polypeptide. namely: (1) coating the plate with anti-IgG (secondary antibody).7 Binding inhibition curves of FIA system using bovine insulin conjugated HRP. hGLU: human glucagon. bINSA: bovine insulin A-chain. (2) binding of anti-insulin antibody (primary © 2006 by Taylor & Francis Group.66 Fish Endocrinology Fluorescence intensity bINS hGLU SST hPP hlNSC bINSA bINSB Hormone (pg/well) Fig. SST: somatostatin-14. 3.. and A1. bINS: bovine insulin. ASSAY PROCEDURE The procedure of our solid-phase competitive non-RI immunoassay for bfINSs consists of five steps. 1991).

which are all rare earth metals. Furthermore. whereas those of natural biological sample are in 1-20 ns (Soini and Kojola. Sensitivity of TRFIA using europium appear to be almost the same or one digit lower than that of RIA. the enzyme immunoassay needs a further step for stopping enzyme reaction before measuring step. samarium. and (5) detection and measuring absorbance or fluorescent intensity (Fig.1% NaN3 (w/v). 3. 1983). such as avidin complex with lanthanide and enzyme. Competitive Time-resolved Fluoroimmunoassay (TR-FIA) For insulin Time-resolved fluorometric assay (Soini and Hemmila. Steps for detection are simpler than in EIA. this assay also needs some additional explanations. reaction time and components of blood. 25 mM EDTA. Assay buffer and Enhancement solution: Purchased from Wallac Oy.Streptavidin complex (Wallac Oy). Specific fluorescence of lanthanides can be measured after a selected decay time chosen to eliminate background influences. temperature. 0. such as europium (Eu). B1 monobiotinylated bfINS-I: Purchased from Peptide Institute (Osaka.Tadashi Andoh 67 antibody) with coated anti-IgG. (3) competition of hormone in sample and labeled hormone for anti-insulin antibody. Japan).9). LLC . However. The fluorescence decay time of lanthanide chelates is often in the order of 10-1000 s. Eu-avidin complex: Eu. Time-resolved fluorescence does not tend to be influenced by pH. Stages (2) and (3) can be performed in a single step. 3.8). terbium and dysprosium. The most important points of TR-FIA are a high degree of sensitivity and simplicity of the detection procedure. 1979) uses a long fluorescence decay time of lanthanide chelates in solution (Fig.8) containing 150 mM NaCl and 0. Dainippon Pharmaceutical) diluted with 50mM Tris-HCl containing 150 mM NaCl. Until now. (4) binding of molecules for detection. It is believed that TR-FIA is one of the most reliable systems for peptide hormone quantification. four elements have been used for TR-FIA. © 2006 by Taylor & Francis Group. Reagents Wash buffer: 50 mM Tris-HCl (pH 7.1% (v/v) of Tween 20. Those of other lanthanides are one or two digits lower compared with europium. Tris-BA: 25% Block Ace (v/v.

LLC . crossreactive competition of primary antibody for biotinylated insulin and insulins in sample (Step 2). binding of Eu-avidin (Step 3) and detection of Eu (Step 4). 3.8 Schematic procedure of TR-FIA for barfin flounder insulins.68 Fish Endocrinology Step 1 Step 2 Step 3 Step 4 Dispensation of Enhancement solution and detection of Eu Fig. © 2006 by Taylor & Francis Group. The procedure consists of four steps including coating of secondary antibody (Step 1).

this criterion is valid only for measurements © 2006 by Taylor & Francis Group. There are two criteria in microtiter plate selection for TR-FIA. 3. Researchers should check suitable plates for their own assay system. Rochester.9 Theory of time-resolved fluorometric assay. respectively. Most plastic plates show too high background for TR-FIA. which are excitation and emission wavelengths. Timeresolved fluorometric assay is influenced by transparency and fluorescence of material at 340 nm and 615 nm. Time-resolved fluorometric assay measures specific fluorescence of lanthanid after a selected decay time chosen to eliminate fluorescence of other substnaces. We use Maxisorp plate (Nalge Nunc International. Microtiter plate Many types of plates are commercially available. LLC . but suitable plates for TRFIA are limited to a few products. The first is the quality of plastic material. Lanthanide elements show a long fluorescence decay time unlike general substances. although those are good both for colorimetric and fluorometric assay. NY). However.Tadashi Andoh 69 Scattered light Fluorescence of coexisted substances (Background fluorescence) Fluorescence intensity Fluorescence of lanthanide Measuring time Time Excitation (about 1 ns light pulse) Fig.

70 Fish Endocrinology through the bottom of the plate.7). Shaking during washing is not needed in most cases. Furthermore. an antibody for competitive crossreaction. The first. The volume for each time of washing is adjusted to the maximum volume of each well. Mouse IgG was not suitable even after affinity purification.05% NaN3 (w/v) and 150 mM NaCl for several hours at room temperature and then stored in a refrigerator. Both whole IgG and Fc fragment IgG from guinea pig are suitable as antigens. Both rabbit and goat are suitable for immunization. Higher amount of anti-IgG bound to the plate gives higher counts.2 shows the suitability of secondary antibodies which are commercially available. Rabbit Fc fragment of IgG did not show enough crossreaction. is called primary antibody and corresponds to an anti-insulin antibody. The second is an antibody for immobilization of primary antibody to microtiter plate and is called secondary antibody. Concentration of anti-IgG for coating differs among assay systems. containing 0. Three or four times of dispensation and suction of the buffer is enough. The second criterion is the binding capacity of IgG. The starting concentration recommended is 10 g/ml. This is important to shorten time and elevate the sensitivity of the assay. Twenty-fold concentrated wash buffer is convenient for a stock solution. Table 3. Procedure Washing Microtiter plate is washed after every step with a wash buffer using a microtiter plate washer. IgG nonaffinity purified does not show enough binding. © 2006 by Taylor & Francis Group. Recently measurement equipment has been improved by allowing measurements from the upper angle of the plate. Any solid phase competitive immunoassay needs two kinds of antibodies. LLC . Coating of secondary antibody to microtiter plate A 96-well microtiter plate is coated with 200 l of 10 mg/ml of the affinitypurified rabbit anti-guinea pig IgG in 50 mM Tris-HCl buffer (pH 7. although the whole of IgG was suitable. solid-phase competitive immunoassay needs a highly purified secondary antibody. Anti-guinea pig IgG and anti-rabbit IgG correspond to this.

GP: guinea pig. Procedure: Goat affinity purified anti-guinea pig IgG was dispensed to each well and left in refrigerator overnight.3 Difference in binding capacity of microtiter plate coated with secondary antibody and stored in refrigerator. **: Antibody immnobilized to microtiter plate Gt: goat. Blocking was performed using Block Ace overnight and drying was performed by leaving the plate upside down on paper in refrigerator after coating and washing. Each value represents the mean of duplicate data. Fc: Fc frament of IgG. Rbt: rabbit. cps) 9050 8289 8173 1116 7184 3485 996 871 High High High Very low Middle Low Very low Very low Suitability OK OK OK No OK No No No *: Affinity purification was performed privately using rabbit whole IgG. 71 © 2006 by Taylor & Francis Group. Immunized animal Rbt Gt Gt Gt Gt Gt Gt Rbt Antigen GP IgG GP IgG GP Fc Rbt IgG Rbt IgG Rbt Fc M IgG M IgG Affinty purification Yes Yes Yes No Yes Yes Yes ? Company Rockland Chemicon Rockland ICN Chemicon Rockland Rockland Wallac Catalogue number 606-4102 AP108 606-1103 55622 AB21* 611-1123 610-1119 C120-105** Specific Crossreactivity binding (count. Table 3. IgG: whole IgG.Table 3. Treatment after coating Coating both without blocking and drying Blocked after coating Dried after coating Total binding 9780 10130 5737 Non-specific binding 1173 1007 885 Specific binding 8608 9123 4851 Tadashi Andoh Values indicate count per second (cps) measured by TR-FIA for barfin flounder insulins. LLC . M: mouse.2 Suitability of the secondary antibodies immobilizing guinea pig anti-barfin flounder insulin-II antibody in our TR-FIA system.

Eppendorf AG. Either just coated or a coated and blocked plate showed enough high-binding capacity. the microtiter plate is left for 24 hrs at room temperature. Table 3.72 Fish Endocrinology The microtiter plate coated with anti-IgG can be stored in a refrigerator for at least one week.3 shows the difference in the binding capacity of microtiter plate coated with secondary antibody and stored afterwards. coating efficiency of secondary antibody. temperature. but crossreaction at this temperature take a longer time for adequate binding than that at higher temperature. the order and speed of adding of the primary antibody is the most influential factor precluding intraplate variation even under equilibrated condition. This result indicates that wet condition is more suitable for preservation of the plate coated with secondary antibody. including sample and standards. depending on several fluctuating factors such as pipetting. The anti-bfINS-II antiserum is diluted with Tris-BA and 8 l of this solution is dispensed within 2 min to each well after two dispensations of the former. Table 3. The final dilution rate of the antiserum is 1:39200. LLC . Germany) after dispensations of other solutions. and speed of dispensation of each solution. Furthermore. In our TR-FIA system. It is well believed that antigen-antibody crossreaction stabilizes at 4°C. by using continuous pipette such as Multipette plus (4980 or 4981. Note: Measured values may vary. it is important to add primary antibody solution to 96 wells as fast as possible. Competition of biotinylated insulin and insulin in sample for primary antibodies After three washes. for reduction of the influence. 150 l of Tris-BA containing B1 monobiotinylated bfINS-I at the concentration of 390 pg/ml is dispensed to each well. The intraassay variation may be caused by high affinity which is called ‘functional affinity’ of the antibodies and not-theoretical crossreaction of polyclonal antiserum with the ligand. Hamburg. Extension of incubation time for crossreaction of primary antibody up to 24 hrs did not show enough reductive effect on intraplate assay variation. Either sample or standard insulin are added to this solution. Therefore. temperature change of microtiter plate during the assay by solution dispensation and © 2006 by Taylor & Francis Group. After shaking for several minutes.4 shows influence of dispensation order of primary antibody to each well. variation in plastic material of microtiter plate. while that of a dried plate was about half compared with the two former conditions.

19 Yes After sample adding** 3697±28 12.09±0. 2. Competitive crossreaction time Dispensation order of antiserum Before sample adding* 6hours 24hours Significant difference between concentrations Count (cps) Concentration(ng/ml) Count (cps) Concentration(ng/ml) 4273±19 7. sample adding. 4. sample adding.73±0. Tadashi Andoh 73 © 2006 by Taylor & Francis Group. 4. 1.4 Influence of dispensation oredr of solutions.32 4238±28 13. *: Procedure order at competitive crossreaction.Table 3. left for 20 min. dispensation of Tris-BA containing B1 monobitinylated bfINS-I. **:Procedure order at competitive crossreaction.26 No Yes Yes Significant difference Values were expressed as average±SEM caliculated from measured values of each 12 wells. 2. adding of diluted antiserum. LLC . dispensation of Tris-BA containing B1 monobitinylated bfINS-I.15 4589±24 10. adding of diluted antiserum. left for 20 min. 3.37±0. 1. 3.02±0.

Five minutes are enough for dissociation of Eu. each well receives 150 l of europium-streptavidin conjugate (Wallac Oy) diluted at 1: 4000 with Assay buffer (Wallac Oy).and interassay variation. However. it is important to perform all steps at room temperature and add preservatives to assay buffer. The plate is shaken for 5 min at a fixed speed at room temperature. To minimize this influence. A little bit of diffusion of spray and mist of solution containing europium causes unexpected high value of background. and is left for over 1-2 h at room temperature. and europium is dissociated from europium-streptavidin conjugate by adding 100 l of the enhancement solution (Wallac Oy). high sensitivity and inexpensiveness. HRP is used in numerous EIA systems because of its stability. FlA using HRP and its fluorogenic © 2006 by Taylor & Francis Group. The fluorescence of the europium is measured with a time-resolved fluorometer. alkaline phosphatase and glucose oxidase have been used for EIA. LLC . and revolving controller of the shaker for microtitre plate should be checked prior to this step and fixed at a suitable position. Especially. These enzymes are relatively stable at room temperature. Colorimetric assay using this enzyme is popular in the clinical test kit of mammalian insulin and their sensitivity and specificity are enough in most cases. Competitive Enzyme Fluoroimmunoassay (FIA) for Insulin Several enzymes. galactosidase. Detection with time-resolved fluorometer Excess europium-streptavidin conjugate is removed by 4 washes. a colorimetric system is sometimes unsuitable for measurement of plasma insulin level due to its low sensitivity. Binding of Eu-avidin complex After 3 washes.74 Fish Endocrinology washing appears to cause intraplate variation called ‘edge effect’. Solutions in each well should be shaken mildly but sufficiently. The tube keeping Eu-avidin should be spun down before use and pipetted carefully. This is important for reduction of both intra. Note: Europium remains a stable element even after many years under usual laboratory conditions. Note: This step enhances fluorescence of Eu to 105 times. including horseradish peroxidase (HRP). Avidin-HRP complex is also commercially available from several companies.

1980) which shows the highest sensitivity for HRP (Andoh and Nagasawa. In contrast. Fluorescence intensity Temperature (°C) Fig.Tadashi Andoh 75 chromogen improves this problem. © 2006 by Taylor & Francis Group. occurs at 15°C (Porstmann et al. 3. This protective function of HPPA has been reported in EIA systems using pHydroxyphenyl acetic acid (Porstmann et al. In addition. 1997).. 1977).. antibfINS-II antiserum. Figure 3. Incubation at over 25°C showed the highest degree of fluorescence intensity. the maximum activity of HRP using o-dianisidine. B1 monobiotinylated bfINS-I and HRP-avidin complex were used in this system. LLC . We use 3-(p-hydroxyphenyl)propionic acid (HPPA—Zaitsu and Ohkura.10 Relationship between fluorescence intensity and incubation temperature at the step for fluorogenic reaction in EIA using HRP and HPPA. 1985). this fluorogenic chromogen appears to possess protective function of HRP activity against high temperature. which is a colorimetric chromogen. Rabbit anti-guinea pig IgG. 1981).10 shows the relationship between fluorescence intensity and incubation temperature at the step for fluorogenic reaction in our FIA system using HPPA for bfINSs. phenol and 4-aminoantipyrin (Gallati.

Solubilize this reagent at 0. Incubation is for 60-150 min at 30°C or at room temperature. Switzerland) Fluorogenic chromogen: HPPA purchased from Sigma (Cat. Binding of HRP-avidin complex Solution of HRP-avidin complex is diluted with Tris-BA excluding NaN3 at the ratio 1: 50000. Note: Photometric interference among wells can be cut completely by using a black plate.025%.025% H2O2 solution.0). No. Procedure Competition of biotinylated insulin and insulin in sample for primary anti-insulin antibody The procedure and reagents are the same as those for TR-FIA. 1089153. Dispensation of fluorogenic reagents and measurement After 4 washes. 75 l of HPPA solution is dispensed followed by the addition of 15 l of 0. LLC . H6386) shows enough purity. Wash buffer: same with that of TR-FIA. Black plate (Greiner) or FluoroNunc. 150 l of this solution is dispensed to each well and the plate is incubated in crushed ice for 1-2 hours under moist conditions. Tris-BA: Same with that of TR-FIA. Rosch Diagnostics. The reaction is stopped by addition of 100 © 2006 by Taylor & Francis Group. No.5% (w/v) in 100mM phosphate buffer (pH 8.76 Fish Endocrinology Reagents Microtiter plate: Immuron 600. Note: NaN3 contained in Tris-BA buffer inactivates HRP-like activity of hemoglobin in blood sample. Maxisorp (Nunc). Capacity of IgG binding to a Black plate is greater than that of FluoroNunc. NaN3 also inactivates HRP activity.01% (w/w) for this step. Note: The plate is cooled in ice to reduce HRP inactivation by high temperature during dispensation and incubation. Thimerosal is a suitable preservative at the concentration of 0. H2O2: dilute 30% solution with DW to 0. HRP-avidin complex: Streptavidin-HRP complex (Cat. but NaN3 is excluded from the step of HRP-avidin binding. Basel. After three washes.

4 pg/well. Measurement time depends on the sensitivity of the equipment. 3. OTHER COMMENTS Specificity Crossreactivity with other peptides cannot be predicted in the newly developed assay system. Volume of sample or standard peptide in each well was ten ml of each. Non-specific binding was determined by subtraction at 10 g/ml of insulin-II. including insulin superfamily peptides. Concentration of H2O2 influences strongly to HRP activity.64 ng/ ml and the lowest detection limit was less than 5 pg/well corresponding 0.35 at 240 nm. Excitation and emission waves are 320 and 405 nm. respectively. In case of negative results. guinea pig anti-bfINS-II antiserum (1: 35. it is important to test it with other peptides and hormones. it appears to be effective to change to a ligand biotinylated at other site. Note: Speed difference of dispensation among the wells results in intraplate variation. ED50 in this curve was 56.3) and dispensed H2O2 solution at the same speed. Reaction stop solution (Gly-HCl buffer) enhances fluorescence of chromogen. While Na2SO4 (final concentration: 0. It has been shown in the above parts that difference of biotinylated site of labeled ligand resulted in crossreactive difference (Fig.Tadashi Andoh 77 l of 150mM Gly-NaOH (pH 10. LLC . corresponding to 5. © 2006 by Taylor & Francis Group. Conditions for HRPavidin binding and fluorogenic reaction were 120 min in crushed ice and 90 min at 26°C.5 ng/ml. B). Serially-diluted solutions (250 ng/ ml-488 pg/ml) of bfINS-I were used as standards. Absorbance of 0. 3.11. and the plate was left at room temperature overnight. respectively.5A.000) and 62 pg of biotinylated bfINS-II (final concentration: 413 pg/ml) were dispensed into each well.6%. w/v) is also used. this solution does not stop the reaction completely. The concentration can be measured by spectrophotometer. This sensitivity is slightly higher than that of TR-FIA system using the same antibody and labeled ligand. Example of measurement results Typical binding inhibition curve for bfINS-I using synthesized B1 monobiotinylated bfINS-I is shown in Fig. Therefore.025% H2O2 is 0. In this assay.

as shown in Fig. Increment of a biotinylated site of labeled ligand increases sensitivity. These studies suggest a possibility that sensitivity of fish insulin system can also be improved by a combination of a larger production of antibody by immunization of rabbits. the latter system is fast (less than 4 hrs) and easy to run. respectively. Andoh and Nagasawa.78 Fish Endocrinology Sensitivity In our TR-FIA system for bfINSs. In such a system. background also tends to increase after long incubation for fluorogenic reaction. the system is not suitable for micro-measurement of blood from the larvae and young. respectively.7. Delayed dispensation of the labeled ligand is effective in improvement of sensitivity in competitive system as described above (2. 3. Replacement of HRP and 3-HPPA for -galactosidase and 4- © 2006 by Taylor & Francis Group. The detection limits of two TR-FIA systems based on this combination for human (Storch et al. Ito et al. Especially. Fig.11). Combination of monoclonal antibodies and the sandwich system can improve sensitivity. A realistic possibility is improvement in detection system.3. but not realistic in most cases. To improve sensitivity is not easy both in TR-FIA and EIA. However.3. 3. although the system needs further development. 1996). (1997) improved fluorescent intensity approximately 2 Fold by using Eu-labeled streptavidin-biotinylated bovine serum albumin in competitive TR-FIA system for polyadenylate cyclase activating polypeptide 27 (PACAP27). LLC .4. These systems included two monoclonal antibodies for immobilization of insulin in sample to microtiter plate and for detection by labeled europium. Changing one or both of the above would be the most effective in solving the problem. Sensitivity of assay system mainly depends on antibody affinity for the labeled ligand and on sensitivity of the equipment. higher purification of IgG and changing to the sandwich system.2. 2002) insulins were 162 pg/ml and 18 pg/ml. However.. the sensitivity is not a problem.1993) and domestic animal (Lovendahl and Purup. In this method. because the detection limit is almost identical with the lowest insulin level of plasma in barfin flounders starved for three weeks. biotinylated bovine serum albumin was used as a bridge which enhanced the number of Eu-avidin molecules bound to biotinylated PACAP27.. FIA using HRP and 3-HPPA appears to show slightly higher sensitivity than TR-FIA (see: 3. the ratio of incubation times before and after adding the labeled ligand is important because of the difference in affinity between native and labeled ligands.

the value is defined 0. Avidin-HRP and HPPA were used for detection. Andoh and Nagasawa. 1998a. 1986. 1986. b). is 1. which contains four tyrosine residues. Insulin is extracted using acid-ethanol from Brockmann body and is purified by RP-HPLC. Mammalian insulin. The yield of purified fish insulin is estimated from the absorbance. therefore. -galactosidase-avidin is commercially available.. LLC . © 2006 by Taylor & Francis Group.Tadashi Andoh 79 Fluorescence intensity Barfin flounder insulin-I (pg/well) Fig.-D-galactosidase is an alternative which also gives better result (Kato et al. Cutfield et al. methylumbelliferyl. Standard for Measurements It is easy to purify insulin from Brockmann body which is a homologue of Langerhans islets in fish (Conlon et al.79 in fish insulin..11 Typical binding inhibition curve of FIA using B1 monobiotinylated bfINS-I and anti-bfINS-II antiserum for bfINS-I. 1975).05 at the concentration of 1 mg/ml water.. 3. This value should be defined based on the result of amino acid sequence analysis. Most of fish insulin contains three tyrosine residues.

(3) Eu contamination during pippetting.3). 1966). extended preservation under the acidic condition of mammalian insulin may induce deamidation at Asn residues in mammalian insulin (Sundby. Serially diluted insulin as a standard is prepared for each set of experiments. 1994). No. Unlike mammals. the immunological properties of mammalian insulins would remain stable. Xenopus insulin was immunologically unstable when stored in 0. (2) to use disposable wares.80 Fish Endocrinology Fish insulin is stable in fraction of RP-HPLC under the acidic condition about at least one week at 4°C like mammalian insulin. 1989). Salmon insulin standards can be stored frozen at –20°C for at least 3 months (Plisetskaya. 3. I would recommend the following steps: (1) to check the wash solution level in microtiter plate during washing. This problem appears to be caused by: (1) insufficient washing after dispensation of Euavidin. Berson and Yalow. which is a mixture of peptides and proteins from milk. Eu is chemically and physically stable even after several years under the laboratory condition. Block Ace (Dainippon pharmaceuticals). also appears to be suitable for storage. Under such storage conditions. Measured Value Spike Measured value spike is sometimes observed in TR-FIA.01 M HCl at –70°C even for 4 weeks. and (4) not to dry samples in microtiter plate either in the microtiter plate reader or in the laboratory after measurement. Neutralization and addition of BSA or suitable protein at the final concentration of 1% with preservatives (NaN3 or thimerosal) can extend this period. 1962.. © 2006 by Taylor & Francis Group. NaCl. LLC . although its two other properties—affinity for insulin receptors and biological activity measured as stimulation of glucose oxidation in rat adipocytes—remained intact (Shuldiner et al. (3) to open bottle of concentrated Eu solution after spinning down. However. Tris-HCl and phosphate buffer containing EDTA. A7030) and preservatives are suitable for dilution. and (4) Eu unforeseen diffusion in equipments and laboratory. Cat. This denaturation can be estimated by RP-HPLC. Insulin deamidated at A21 is eluted immediately after native insulin (Fig. To avoid this problem. (2) insufficient volume of wash solution. BSA (Sigma.

consequently. techniques for development of these assay systems and the differences between them using as an example assay systems for barfin flounder and bovine insulins. Although suitable concentration has not been established yet. The author also wishes to express gratitude to Prof. in my experiences. Plisetskaya. Therefore. it enables one to use less time. However. These techniques and suggestions will be applicable to assay systems for other peptides. 25 mM is high enough. HRP is inactivated by contact with metal ions. chelating by adding EDTA seems to be a simple and effective method. Its application to fish physiology is currently in great demand.Tadashi Andoh 81 Concentration of EDTA in Buffers Metal ions influence measured values both in FIA using HRP and TRFIA.. and transfer to an automated system. 2001). hormone measurements are limited in most cases by labor intensity. the © 2006 by Taylor & Francis Group. This assay technique has been applied to measurement for interleukin (Enomoto et al. LLC . Acknowledgments The author would like to thank Prof. Therefore. CONCLUSION The present study reviewed TR-FIA and FIA for insulin quantification. Quantification of hormone is one of the most basic and important techniques for physiological studies. University of Washington. This is an advanced system of TR-FIA and does not need the wash step. M. Immunoassay system using homogeneous time-resolved fluorescence (HTRF) based on fluorescence resonance energy transfer (FRET) is a candidate for the same. although quantification systems have been already developed. H. It is not easy to remove metal ions from solutions in both cases. Nagasawa. simplify the system. Most physiological phenomena progresses involve multiple changes in fish body. save labor. for her critical reading and helpful suggestions. to understand the physiological mechanisms. simplification of assay procedures is the next important step. and thus future researchers should pay attention to a number of indicators including hormones and bioactive substances. Emeritus E.. 2002) and insulin receptor tyrosine kinase activity (Biazzo-Ashnault et al. Dissociation of europium from labeled protein at detection step is also influenced by the existence of metal ions in TR-FIA.

Kareius bicoloratus.. De Pancreate Piscium. LLC . Inst.).S. and Schwabe. Peptides 21: 1785-1792. and Matsubara. Int. 1959. Berson. Hokkaido Natl. Monduzzi Editore. and Nagasawa. Detection of insulin receptor tyrosine kinase activity using time-resolved fluorescence energy transfer technology. Sci. Quantitative aspect of the reaction between insulin and insulin-binding antibody. Zool. T. C. Andoh.. and Nagasawa. M. and Nagasawa. Kojima. S. Bologna. T. Comp. 1149-1153. 22: 1023-1030. Purification and structural determination of insulins. Verasper moseri. and Dr T. H.A. 2001. Endocrinol. pp. Fish. 1998c. Zool. 208: 445-450. E. Development of non-radioisotopic immunoassay system for measuring flounder IGF-I. Hokkaido National Fisheries Research Institute.82 Fish Endocrinology University of Tokyo. H. E. D. 1997. Park. R. 38: 1996-2016. 2002. for their helpful suggestions and support for development of immunoassays. Brockmann. Andoh. Multiple molecular forms of glucagon and insulin in the kaluga sturgeon. Moller.S.. Competitive enzyme immunoassay using antiserum against mammalian insulin for monitoring the purification of fish insulin. 1986. 1998a. Verasper moseri. Homologous enzyme immunoassay for insulin and stimulation of insulin secretion by amino acids and glucose in the barfin flounder. and Nagasawa. J. Bull. H.. Cummings. 2000. S. University of Rostock. Fish. Biazzo-Ashnault. Res. Kijiya helped in sampling and measuring insulin. Y. Diabetes 15: 875-879. Biochem. Fisheries Research Agency.. Andoh. Sci. 291: 155-158. T. 1996. 35: 416-423. T. E. H. and Yalow. and Thim. Doctral dissertation. 1846. Development of a time-resolved fluoroimmunoassay for insulins and its application to monitoring of insulin secretion induced by feeding in the barfin flounder. Two molecular forms of insulin from barfin flounder. Sekiguchi and K.. D. S. References Andoh. Conlon. T. Huso dauricus. S. 1990. In: Advances of Comparative Endocrinology. Andoh. Andoh.A. B. Bull.. V. 61: 17-26. R. Peptide Protein Res. 1998b. Gen. Matsubara. 1966. The primary structure of ratfish insulin reveals an unusual mode of proinsulin processing. Monobiotinylated relaxins. 15: 939-943. 125: 365-374. Platichthys stellatus.S. (In Latin). T. E. Sci. Nagasawa. Zool. E. Kawashima and S. © 2006 by Taylor & Francis Group. Berson. W. R. Proceedings of 13th International Congress on Comparative Endocrinology. H. R. Sano. H. B. Hokkaido Natl. Anal. T. and Qureshi. 15: 931-937. Andoh. and Yalow. Verasper moseri (Pleuronectidae). Dafgard. glucagons and somatostatin from stone flounder. T. R. S. T. Deamidation of insulin during storage in frozen state. H. Res.. Invest. J. Inst. L. Andoh. T. Ding. Clin. FEBS Lett. and Nagasawa. H. Stimulation of insulin secretion by amino acids in starry flounder. and Nagasawa. are derived from a single gene. 62: 107-113. Falkmer. Kikuyama (Eds. A. 2005. E. Zhang. Bullesbach. J.

. Emdin. Sci. T.. 78: 235-237. 1996. H. H. R. Janolino.. 2002. LLC . D. Seguin. Goke.. Gen.. Clin. N. Moriyama. 1981. Nakajima.. Biochem. O. Nakamura. M. Cutfield. J. © 2006 by Taylor & Francis Group.. Biochem. Time-resolved fluoroimmunoassay for pituitary adenylate cyclase activating polypeptide 27 (PACAP27) using europium (III) ion chelate labeled streptavidin-biotin complex. Comp. P . V. 133: 221-230. Yoshida. Furuichi. 1992.. Japan. O. T. T.Tadashi Andoh 83 Conlon. J. and Nishimura. T. Green. Endocrinol. Gen. Hoppe-Seylers Z. App. Y. Endocrinol. Enomoto. K.. Eur. Shimozono.. 29: 1130-1135.3 cells using homogeneous time-resolved fluorescence. 42: 251-258. V. H. Tsuji. Comp. Preparation and characterization of hetero-bifunctional cross-linking reagents for protein modifications.. A.. B. Bull. Gen. 15: 1489-1495. Insulin-analoga mit N-terminal verkurzter B-Kette. A specific antiserum against insulin from the Atlantic hagfish Myxine glutinosa: characterization of the antiserum. Physiol.. insulin and glucagon-like peptide from the Pacific ratfish (Hydrolagus colliei). Y. Methods in Enzymol. Aikawa. Glucose and amino acid-stimulated insulin release in vivo in the European eel (Anguila anguilla L. E. Endocrinol. C. 1973. chracterization and stimulatory actions on [35S]sulphate and [3H]thymidine uptake in the branchial cartilage of the eel in vitro. Mathis. Anal.). S. M. W. Araki. 46: 1171-1181. and Ishikawa.. Duan. The isolation. T. Biotech. S. and Swaisgood. and Thim. T. Ince. J. Chem. R. K. Bull. A spectrophotometric assay for biotin-binding sites of immobilized avidin. T. Goto. Biomed. Hamaguchi. and Takemoto. 55: 393-397.. and Langner. 28: 73-79. Enzyme-linked immunoassay. and Maeda. 1977. purification and amino-acid sequence of insulin from the teleost fish Cottus scorpius. and Planas. Biochem. Fontecha. 1980. 1975. and Hirano. 1970. Kominami.. M. G. Biochem. Carrillo. K.. T. J. J. J.. Ohta. Y.. 158: 117-123. K. M. A radioimmunoassay method for determination of fish plasma insulin. D. Pharm. Endocrinol. J. C. Kato.. Fish. Endocrinol. Anal. 1989. R. H. Chem. A. its use in a homologous radioimmunoassay. and immunofluorescent microscopy. 1984. Gen. F. 133: 211-219. 73: 136-146.. E. Comp.. J. Pharm. Emdin. S. Spectrophotometric determination of avidin and biotin. 15: 699-703. Novel method for synthesis of the insulin-b-D-galactosidase conjugate and its applicapability for insulin assay. and Hirano. Endocrinol. Soc. I. H. Comp. (In German) Geiger. L. 31: 249-256. A. 1977. Fukui. 56: 1-7. Suzuki. M. T. Pharm. S. J. 1992. Multiple molecular forms of . Effects of insulin-like growth factor-I and insulin on the in vitro uptake of sulphate by eel branchial cartilage: evidence for the presence off independent hepatic and pancreatic sulphation factors.. Duan. Kitagawa. 1997.. P C. 1980. 18: 418-424. S. Dohi. J. Determination of the activity of peroxidase with the aid of the ‘Trinder reagent’. J. Gallati. H. S. Noso. Chem. A. Gutierrez. H. and Steiner. Kawauchi. High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3. J. 354: 1285-1290. Biomed. Ito. and Thorpe. Eel insulin: isolation.... G. M. Daily rhysms of insulin and glucose levels in the plasma of sea bass Dicentrarchus labrax after experimental feeding. F. Andrews. G. and Yone. Carne. 1986. M. Zanuy. Clin. Preaudat. Cutfield. and Falkmer.

Characterization of insulins and proglucagon-derived peptides from a phylogenetically ancient fish. Plisetskaya. 1994. T. B. P. Mommsen. In: Biochemistry and molecular biology of fishes. and Kawaoi. J. 4: 225-259. 1983. Physiol. P 1990. P 1971. S. Comp. Biochem. Nguyen.M. 1: 37-43. A. B 121: 3-11. Measurement of channel catfish (Ictalurus punctatus) plasma insulin in species-specific radioimmunoassay. H. 1962. 16: 41-46. 263: 658-665. D. W.M. T. and Silverstein. J. 55: 2141-2143.. J. L. Losert. (Tokyo) 51: 375-379. 1994. Fluorometric assay for avidin-biotin interaction. J. The acetylation of insulin.M. Fish Physiol. H. and Shall. Plisetskaya. experiments in . 22: 1084-1091. Lindsay. On the amino acid composition of the glycyl chain of bonito insulin-I. A. Mommsen. H. Two insulins from channel catfish: purification. Carbamyl. T. Plisetskaya.G. J. K. E. T. K. O. Mims. M. M. . J. and Kaminogawa. Agric. 30: 407-412. 300: 339-345.). M. S. Analytical Techniques. M. 1972. J. LLC . Plisetskaya. Plisetskaya. structures. P.. and . Aquat. 535-556. Conlon.. Bondareva. Mommsen... E. and Gorbman. Biochem.. P Mommsen (Eds. B. Conjugation of horseradish peroxidase to Fab fragments with different homobifunctional and heterobifunctional cross-linking reagents. J.and methylthiocarbamylinsulins. E. Peroxidase labeled antibody. Vol. T. J. Cytochem. K. M. Lindsay.. G. Biochem. 1971. Sci. 25: 61-70. Leonard. Methods 62: 1-13... J. Histochem. Fish Physiol. E. J. 1998. Kurioka. 10: 623-625. Ametani. the paddlefish (Polyodon spathula). Cytochem.. Lee.G. P W.. M. Mock. and Satake. 1982. A comparative study. D. 7180. 121: 737-745. Loge. Histochem. 2002.. M. Insulin in fishes and agnathans: history. E. and Foa. N. U. M. 1991. 1986. P. Amsterdam. Sci.. Dickhoff. 1991. and Sjoquist. Biochem.P Silverstein. J. structure. R. Chem. © 2006 by Taylor & Francis Group. Shimizu. Production and characterization of anti-human insulin antibodies in the hen’s egg. Thoren-Tolling. Biochem. Studies on insulin. Biol.. Nakane.. . IV. 1974. V. Biochim.. Endocrinol. Lindmark. and Horowitz. Fish Physiol. 80: 191-195. Biochem. M. T. Hochachka . J. A. Binding of immunoglobulins to protein A and immunoglobulin levels in mammalian sera. T. Biophys. Radioimmune assay of insulin in lower vertebrates. vivo and in vitro. K. Conlon. Development of radioimmunoassay for a model peptide hormone: insulin. J. Plisetskaya. J. and metabolic regulation. P.. M. P.P. Hatta.. Paquette. receptor-binding and cDNA sequences. W. Radioimmunoassay of insulin in fishes. Anim. Whittaker J. 2002. Biochem.. Some of not so favorite things about insulin and insulin-like growth factors in fish. M. Patent.. Physiol. and Purup. and Conlon. 2002. A. Immunol. Acta.T. E. E.84 Fish Endocrinology Kotaki. Mommsen. Comp. Biochem. Methods in Enzymol. Evol. and T. 1974. Rev. D. The assay of salmon insulin by homologous radioimmunoassay. J. 184: 234-240. A new method of conjugation. W. 3. Whittakaer L. S.. and Shall. J. Elsevier. T. Yamamoto. Lovendahl. and Plisetskaya.M. and Leibush. Technical note: time-resolved fluoroimmunometric assay for intact insulin in live stock species. 25. S. Nygren. Gen. pp. M. K.

T. and Evers. Yamada. Gen. Shuldiner. Thorpe. The X-ray three-dimentional structure of . Satoh. M.R. Immunol. F. J. 1993.. Acta 109: 175-181. Endocrinol. Waights. 29: 65-68. Schroer. Time-resolved fluorometer for lanthanoide chelates-a new generation of nonisotopic immunoassays. Mol. R. The development of a homologous teleost insulin radioimmunoassay and its use in the study of adrenaline on insulin secretion from isolated pancreatic islet tissue of the ranbow trout. and Hemmila. and Iwata. L. A. Clin. Yamashita. Immunol. Chem. alkaline phosphatase or b-galactosidase? J. 25: 353-361. Arosio. Biol. Comp. 1979. J. T. Kambegawa. J. H. Plasma insulin levels in teleostes determined by a charcoal-separation radioimmunoassay technique. Endocrinol. Biomol. Storch. E. Comp. Which of the commonly used marker enzymes gives the best results in colorimetric and fluorometric enzyme immunoassays: horseradish peroxidase. Studies on insulin. M. and Roth. Immnol. human insulin based on two monoclonal antibodies. W. Marbach. J. M. T. Tilzey. 1960. Engineering 16: 5-12. Method. Biochem. E. Comp. 1985. 237: 3406-3411. 126: 136-143. A. L. 1986. E. Porstmann. V. M.. B. A. Amano. Okuzawa. avidin. Two different insulins from langerhans islet of bonito fish. E. M. Comp.. Methods 79: 27-37. Clin. M. Sundby.. insulin recognized by monoclonal antibodies. 30: 332339. 23: 441-450. C. D. A. and Egger. J. Endocrinology 125: 469-477.. 1997. and Holmes. B. Chem. T. Gaede.. Clin. I.. Xenopus laevis. Chiba. Isolation and characterization of two different insulins from an Amphibian. P A. Okuyama. Porstmann. A. Med. 1985.. Porstmann. Indentification of antigenic determinants on . Gen. K. 40: 662-666. Gen. F. U. Mapping epitopes on the insulin molecule using monoclonal antibodies. 1989. H.. J. and Satake. A 81: 821-825. J. Bender. Chem. and Kojola. A time-resolved fluoroimmunoassay for . J. Bennet. Biochem. Endocrinol. Yamada. 1981. A.. Soini. Kotaki.. D. 11: 262-271. T. 157: 197201. Soini. Rosano. P and Kerp. J. Rathjen. Separation and characterization of acid-induced insulin transformation products by paper electrophoresis in 7 M urea. B. Species variation in the amino acid sequence of insulin.. F. C. Feldmann. Chimica. and Ince. 1983. 2002. and Underwood. Development of a time-resolved fluoroimmunoassay (TR-FIA) for testosterone: measurement of serum testosterone concentrations after testosterone treatment in the rainbow trout (Oncorhynchus mykiss). Opsanus tau. G. Endocrinol. Immunol. and Kim. Fluoroimmunoassay: present status and key problems. 1968.. 1976. and Iwata. Eur. Comp.. LLC .. 1999. L. H. Smith. E. Yamamoto.. R. Robinson. E. Maturational changes in brain contents of salmon GnRH in rainbow trout as measured by a newly developed time-resolved fluoroimmunoassay. 1962. 1966. 13: 693-700. Physiol. Effects of insulin in the toadfish. J. Salmo gairdneri (R). J.. Amer. K.. K. 106: 181-188. P and Bolognesi. and Cahill Jr. 1983. H. R. © 2006 by Taylor & Francis Group. Nugel.. (Tokyo) 48: 84-92. Tashima. I..Tadashi Andoh 85 Porstmann. A. Gen. Nugel. F. Temperature dependent rise in activity of horseradish peroxidase caused by non-ionic detergents and its use in enzyme-immunoassay.

Y. M. 15: 109-113. K.. (Tokyo) 39: 499-500. © 2006 by Taylor & Francis Group.. Nakayama. Nakayama.86 Fish Endocrinology Zaitsu. (Tokyo) 36: 1425-1430. New fluorogenic substrates for horseradish peroxidase: rapid and sensitive assays for hydrogen peroxide and peroxidase. M. Zaitsu. Y. and Ohkura. 1988. Bull. Nanami. 1980. M. K. Pharm. Y. K. and Ohkura. Anal. Pharm. Bull. Biochem. Zaitsu. Solid-phase enzyme immunoassay of anti-insulin antibodies: effect of labeling sites in insulin and of labeled number of horseradish peroxidase on the assay sensitivity.. and Ohkura. LLC . Chem. Chem. 1991. Preparation of [3-(2pyridyldithio)propionoyl]insulins and horseradish peroxidase-insulin conjugates by high-performance liquid chromatographic separation.

have been discussed here. their potential interactive regulation by different parameters such as other hormones. Switzerland. Paracrine. The IGF-I and IGF-II genes and their expression in liver and numerous other organs. E-mail: reinecke@anatom. Growth hormone. IGF-II.CHAPTER 4 Insulin-like Growth Factor I and II in Fish Manfred Reinecke ABSTRACT This chapter summarizes and interprets the present situation in research on insulin-like growth factor (IGF) I and II in fish. Growth. environmental temperature and salinity. cartilage.unizh. Development. University of Zürich. Finally. Endocrine. brain and gonads as well as their modes and potential interactions are considered in detail. Institute of Anatomy. LLC . The pituitary growth hormone (GH)/liver IGF-I axis as the main regulator of circulating (endocrine) IGF-I and the physiological impact of local (auto/paracrine) IGF-I in gills. maintenance. reproduction and osmoregulation. CH-8057 Zürich. Author’s address: Division of Neuroendocrinology. Reproduction. gastro-intestinal tract. Focus is also on the role of the IGFs in crucial physiological processes. 190. Key Words: IGF-I. endocrine pancreas. growth. kidney. and nutrition. an attempt is made to analyze current trends in IGF research and suggest future topics. © 2006 by Taylor & Francis Group. especially in development. season. GH/IGF axis. Osmoregulation.

Among the nonmammalian classes. It further discusses the expression and potential physiological roles of IGF-I and IGF-II in extrahepatic sites. As is the case in research on mammals. bony fish are the mostly studied with respect to the IGFs mainly due to their unique development from the larval to the adult life and to their high commercial value in aquaculture. the GH/IGF liver axis and parameters. seabream and tilapia in aquaculture industry. particularly human. The article deals with the IGF-I and IGF-II genes and sequences. but some recent studies also deal with the potential effects of the IGFs on gonadotropins.88 Fish Endocrinology INTRODUCTION The insulin-like growth factors IGF-I and IGF-II are major hormonal regulators of growth and development. osmotic pressure and other hormones. rat and (transgenic) mouse. kidney. most studies on the IGFs deal with mammals. there is also severe interest in the results obtained outside the science community. They selectively promote mitogenesis and differentiation and inhibit apoptosis (see Reinecke. on the IGF system. gastro-entero-pancreatic system. This is mainly true for localization and effects of the IGFs in the male and female gonads. brain. the IGFs and the type 1 IGF receptor (IGF-IR) from the unfertilized egg over the larval and the juvenile to the adult stage.) and receptors (Gutiérrez et al. This review focuses on the IGF hormones while the IGF-binding proteins (Kelley et al. such as gills. besides numerous scientific reasons to study the IGF system in fish. A second important role of the IGF system which is closely related to growth is its involvement in developmental processes. these species are the most investigated fish as the IGF system is concerned. such as endocrine-disrupting chemicals. Thus. this volume). which affect it. As to be expected. Third. the vast majority of investigations is on IGF-I. Several recent studies deal with the differential expression and action of GH. Because of the increasing importance of salmonids. male and female gonads. © 2006 by Taylor & Francis Group. the participation of the IGFs in reproduction has attracted great interest in fish research. temperature. seasons and environmental temperature and other parameters. The rising interest in the significance of the IGF system in fish growth and in potentially enhancing and impairing parameters is further expressed by the increasing number of studies dealing with the influence of nutrition. LLC .) have been considered in other chapters of this volume. such as nutrition.

such as chinook salmon (Wallis and Devlin. 1997b) and barramundi Lates calcarifer (Stahlbom et al. 1996.. The salmon IGF-I genes are composed of five exons and four introns. 47. 1999). 1989). 1998). with E domain encoded amino acids (aa) lengths of 35.. Consistent with the tetraploidy of the salmon genomes two IGF-I genes were detected. 1999). 1993).. 1994). The salmonids have been proposed to express alternatively spliced IGF-I mRNA transcripts encoding at least four different IGF-I transcripts. 2001) and turbot Psetta maxima (Duval et al. 1993). such as gilthead seabream Sparus aurata (Duguay et al. tilapia (Chen et al.. alternative splicing mechanisms of the E-domain and the transcripts produced in various bony fish species investigated. keta (Kavsan et al. 1992). 1998). Hashimoto et al.. zebrafish © 2006 by Taylor & Francis Group. tissue-specificity and differential regulation by GH (Duan et al.. chum salmon (Kavsan et al.Manfred Reinecke 89 THE IGF-I AND IGF-II SEQUENCES AND GENES Bony Fish CDNA sequences encoding IGF-I peptides have been characterized in several bony fish species. carp (Liang et al. LLC . zebrafish Danio rerio (Chen et al.. catfish (McRory and Sherwood. 1993.. Ea-1 to Ea-4.. 1998. Atlantic salmon Salmo salar (Duguay et al.. keta (Kavsan et al. 62. chum salmon O. More recently... 1997. early studies focused on salmonids such as Coho salmon Oncorhynchus kisutch (Cao et al... 1994). exon/intron arrangements. Schmid et al. 1997.. 2003). common carp Cyprinus carpio (Liang et al. Vong et al.. 1996.. Japanese flounder (Tanaka et al. tilapia Oreochromis mossambicus (Chen et al. 2002). Japanese flounder (Tanaka et al. 1992).. IGF-I sequences have been determined in several perciforms. The two IGF-I genes shared more than 90% similarity over their coding regions but differed in their splice donor sites. 1998). mykiss (Shamblott and Chen. Vong et al. tshawytschwa (Wallis and Devlin.. 1994). 1996).. Probably for the reasons stated in the Introduction. 1992). 2003). For detailed information on the organization of the IGF-I gene structures.. The transcripts showed different expression patterns during development. 1998).. Hashimoto et al. 1997. Reinecke et al. 1993) and chinook salmon O. 1993.. 1994). goldfish Carassius auratus (Kermouni et al. and 74 predicted residues (Shamblott and Chen. as well as in daddy sculpin Cottus scorpius (Loffing-Cueni et al. such as the two functional non-allelic IGF-I genes isolated from genomic DNA libraries of the chum salmon O. rainbow trout O..

1994).. Chen et al. nor between the TH and insulin genes. 2002). 1999). 2001) and turbot (Duval et al.7 kb in pufferfish and 2. 1996). In pufferfish (Chen et al. at least within approximately 20 kb of genomic sequence was detected. 2002). 2002). Thus...90 Fish Endocrinology (Chen et al. the readers are referred to the original publications. the overall organization of the IGF-II genes seems to be very similar in all bony fish species and resembles that established in mammals.. the chum salmon genome differs from that of the other teleosts investigated. In contrast to barramundi and pufferfish. in chum salmon neither a linkage between the insulin and IGF-II genes. 2002) only one IGF-II gene could be identified. and from pufferfish (Fugu rubripes. 1998) and barramundi (Collet et al. 2002). 1998. keta) (Palamarchuk et al. © 2006 by Taylor & Francis Group. In contrast.. 1998). 1997) and barramundi (Collet et al.. and common carp (Tse et al. from perciforms such as gilthead seabream (Duguay et al. as in other bony fish (Chen et al. 2002). 1998. 1993.. The chum salmon IGF-II gene consisted of four exons and activation of its only promoter gave rise to a single 4 kb transcript. Tse et al.. Although the chum salmon genome contains two non-allelic insulin and IGF-I genes (Kavsan et al. Recently.... 1998). In spite of some differences.. 1992) and chum salmon (Palamarchuk et al. All IGFII genes characterized in mammals are organized as TH-insulin-IGF-II genomic locus in the same transcriptional polarity. tilapia (Chen et al..5 kb in barramundi. Collet et al.. In general. the bony fish IGF-II genes are smaller and simpler organized than their mammalian counterparts. LLC . 2002). the complete nucleotide sequence and organization of the IGF-II gene has been described in chum salmon (O. insulin and IGF-II genes seems to be different in bony fish. the arrangement of the tyrosine hydroxylase (TH). This is probably due to the fact that the mammalian IGF-II genes are under the control of several tissue-specific and developmental stage-dependent promoters. the IGF-II and TH genes but not the insulin gene were adjacent to each other.... The TH and IGF-II genes were separated by only 1. and it has been concluded that an additional DNA fragment of unknown size was integrated between the TH and IGF-II genes as a consequence of the ancestral tetraploid origin of salmonids (Palamarchuk et al. Furthermore. possibly indicating that the insulin gene was inserted between the IGF-II and TH genes in higher vertebrates. daddy sculpin (Loffing-Cueni et al. cDNA sequences encoding IGF-II have been characterized only from the salmonids rainbow trout (Shamblott and Chen.. turbot (Duval et al. 1998).

1998). the daddy sculpin IGF-II prohormone exhibits 85-92% homology at the aa level to pro-IGF-II of rainbow trout. physiological studies on IGF-I indicate that the biological potency of this hormone is also remarkably conserved. 1993) and to bind to the bony fish IGF-IR (Leibush et al. does not seem to influence the biological activity as shown for IGF-I (Tanaka et al. The similarities between the fish prepro-IGF-I and –IGF-II molecules are rather weak in signal peptide and E-domain. experimental studies in bony fish can reasonably be performed by the use of human IGF-I. These broad-spectrum comparisons show that the aa sequences of both IGFs have been highly conserved throughout vertebrate evolution..and B-domains which amounted to 57% and 7%. they are in similar ranges. The aa sequence identities of the IGF-II hormones are in a similar range—65-95% among the different fish species and 62-78% to human. scorpius IGF-I A. Reinecke et al. For IGF-II.and E-domains and the six cysteine residues necessary for maintenance of tertiary structure.. however. fish prepro-IGF-I and IGF-II aa sequences exhibit a signal peptide. while the homologies with the human counterpart are usually a little lower (43-83%). the aa sequences of the bony fish IGF-I and IGF-II hormones share low homologies. Tsai et al. LLC . respectively. 1990. B-.and A-domains are remarkably high. human IGF-I has been shown to incorporate 35S-sulfate into fish branchial cartilage (Duan and Hirano. D. scorpius insulin (Loffing-Cueni et al.and D-domains are quite low (44-65% and 49-63%. ranging from 36% to 67%. In contrast. i.. As is also in mammals. 1998). 1991. © 2006 by Taylor & Francis Group.Manfred Reinecke 91 Generally.... 1997b. when compared to C. For instance.. Gray and Kelley. For IGF-I. The aa homologies of the IGF-II prohormones among the bony fish are somewhat higher than those of the IGF-I prohormones (Duguay et al. 1996). respectively). Depending on the fish species compared the mature IGF-I hormones show high aa sequence homologies which range between 66% and 97%. 91-100% and 72-90%.. 1994) and human and salmon IGF-I were equally potent in stimulating cartilage sulfation in salmon (Moriyama et al. 1996. For example. they amount to 91-100% when compared to other bony fish and still to 76-91% when compared to human. Duval et al. 2002). The C-domains further vary in length from 9 to 12 aa among the different bony fish species investigated which..e. gilthead seabream and tilapia. A-. but only 51% homology to human (LoffingCueni et al. Even lower homologies are between the IGF and insulin molecules as has been shown for the C. In addition to the structural conservation. The homologies of the fish C. the homologies of the fish B. 1999). Thus. C-..

are also present in bony fish. The mature shark IGF-I hormone has 70 aa and 66% and 62% sequence identity to human IGF-I and IGF-II. IGF-II-like peptides were also found in ray which differed from mammalian insulin and IGF-I but closely resembled IGF-II (Reinecke et al. 1998) and turbot (Duval et al.. 1997.and Bdomains corresponds to their key physiological impact as it has been established in mammals..and E-domains and the six cysteine residues necessary to maintain tertiary structure. Cartilaginous Fish Very few studies have dealt with the potential presence of the IGFs in cartilaginous fish. 1995). Later. Especially. which are involved in the recognition of the type 1 IGF receptor (IGF-IR) in mammals (Humbel. The physiological importance of the IGFs in © 2006 by Taylor & Francis Group. 1998). no alternative splicing occurs in shark. B-. Duval et al. Japanese flounder and turbot IGFI.. the existence of IGF-I and IGF-II in cartilaginous fish was undoubtedly established by the cloning of cDNA sequences encoding distinct and characteristic IGF-I and IGF-II preprohormones in a shark. PCR amplification of the E-domain region suggested that in contrast to most bony fish species. 1990). 1998) Japanese flounder (Tanaka et al. As it has been shown in tilapia. daddy sculpin. The particularly conservative phylogeny of the IGF A.92 Fish Endocrinology respectively. the IGF-II sequences also contain most of these crucial amino acids. The shark IGF-II hormone consists of 68 aa and shows 66% and 55% sequence homology with human IGF-II and IGF-I.. D. In the ray. C-... Raja clavata. respectively. 1994). IGF-I-like peptides were detected which exhibited immunoabsorption properties different from those of mammalian and non-mammalian insulin and IGFI (Reinecke et al. As in bony fish and higher vertebrates. The first indication that both IGF-I and IGF-II occur in cartilaginous fish was obtained by the use of gel-chromatographic and immunohistochemical techniques. GluB9. Loffing-Cueni et al.. and turbot (Chen et al. Likewise. LLC . Furthermore. A-.. the residues GluB3. goldfish (Kermouni et al. the B-domain residues ArgB21 and PheB23-TyrB24-PheB25. 2002) IGF-I. the spiny dogfish Squalus acanthias (Duguay et al. the elasmobranchian preproIGFs are composed of a signal peptide. respectively. 2002).. 1999. GlnB15 and PheB16 which in mammals participate in binding to the IGFBPs have been also identified in daddy sculpin.. such as daddy sculpin (Loffing-Cueni et al. 1992).

2 kb transcript but also in extrahepatic sites such as brain. 1995). C-. hagfish IGF exhibits characteristics for both IGF-I and IGF-II (Nagamatsu et al. 1991). D and E domains are relatively low when compared to human. IGF-Irelated peptides have been detected in islet and brain of M. the Atlantic hagfish Myxine glutinosa. D.and A-domains exhibit high (70%) homology to both human IGF-I and IGF-II. skeletal muscle and islet organ (Nagamatsu et al. IGF-I-immunoreactivity was present in all insulin © 2006 by Taylor & Francis Group. which clearly correspond to either IGF-I or IGF-II. thus. during evolution distinct IGF-I and IGF-II genes have first appeared in chondrichthyes.. all residues necessary to form the correct tertiary fold of an insulin/IGF-like molecule are conserved..e. Based on these findings. The sequence of a cDNA encoding a prepro-IGF peptide expressed in hagfish liver has been determined and Southern blot analysis has suggested that the hagfish IGF is a single copy gene (Nagamatsu et al. In contrast to the IGFs characterized in the other vertebrate classes. A-. however. has been investigated for the potential presence of the IGFs. 1993). The prepro-IGF consists of a signal peptide. 1991). which is considered to be the most primitive extant vertebrate species. clavata) liver and brain (Drakenberg et al. As also determined in bony and cartilaginous fish. 1991). hagfish IGF was also expressed predominantly in the liver as a 4. Thus. The homologies of the deduced aa sequences in the C. acanthias) testis and. and (2) Human IGF-I largely increased [3H] thymidine incorporation in cultured spermatocytes derived from shark (S. again indicating the particular physiological impact of these domains. whereas a single proto-IGF gene with equal sequence identity to and characteristics of mammalian IGF-I and IGF-II is present in the agnathan Myxine glutinosa. 1993).... LLC . Like it is characteristic for IGF-I in the other vertebrate classes.and E-domains as they are typical for the IGFs throughout evolution. Furthermore. the B. B-. glutinosa (Reinecke et al. i. In correlation. 1991). Cyclostomes Only one member of the cyclostomes.Manfred Reinecke 93 cartilaginous fish is indicated by the following results: (1) Ligand binding studies that have demonstrated that the IGF-IR is present in ray (R. enhanced DNA synthesis (Dubois and Callard.. the hypothesis has been raised that hagfish IGF may represent the (hypothetic) ancestral IGF hormone that duplicated and diverged into IGF-I and IGF-II (Duguay et al. heart..

LLC . skeletal muscle. Duguay et al. 1993) and in neurones throughout the brain (Reinecke et al. 1993).3 pg/ g DNA (Duan et al. 2004). As determined in tilapia (Caelers et al. 1996.. in gilthead seabream gills 75% of the amount of liver IGF-II mRNA and in kidney and muscle about 65% were detected (Duguay et al. 2003) and coho salmon (Pierce et al. intestine. heart. Similarly. the major site of IGF-I gene expression in bony fish is the liver (Duan et al. This result is consistent with those of other studies. 2003). In common carp.4. Recent studies in common carp (Tse et al.. Vong et al. Furthermore. 2004).04 pg/ g 18S rRNA and to 1. kidney.94 Fish Endocrinology and some somatostatin cells of the islet (Reinecke et al. brain 2. the level was as low as 0.. 2002.68 pg/ g 18S rRNA in liver while in kidney.. 1995). however. 1991). Pierce et al. It is reasonable to assume that © 2006 by Taylor & Francis Group.1 pg/ g 18S rRNA (Shamblott et al. gonad. An early investigation on coho salmon determined the IGF-I mRNA level in liver to 16 pg/ g DNA but markedly lower levels in extrahepatic tissues. glutinosa (Drakenberg et al. 1997). 1993. in rainbow trout. heart. spleen and testes. 2003. such as brain. muscle... respectively (Shamblott et al. and a significantly higher level of IGF-I mRNA in liver than in the other organs. Caelers et al. As found for IGF-I.. brain and spleen. 2004). gills 2. 2002. Vong et al. 2002.. 2004. IGF-II mRNA amounted to 2. kidney 3. hagfish IGF stimulated protein synthesis in rat myoblasts although with lower potency than human IGF-I or IGF-II (Upton et al... Matthews et al. kidney. kidney and gonads also expressed quite high absolute quantities of IGF-II mRNA. Vong et al. such as intestine 4. 1997.. gills. Tse et al. Evidence for a biological impact of hagfish IGF is given by the presence of the IGF-IR in the brain of M.4.. by the use of real-time RT-PCR the absolute amounts of both IGF-I and IGF-II mRNA were measured (Caelers et al.8 pg/ g 18S rRNA in kidney and gills. 1994..1.. and ovary 2. In rainbow trout liver. Thus. 1993)... other organs such as heart. Similarly.. such as gills.. Recently..8. intestine. 2004) also determined significantly higher relative IGF-I expression levels in liver than in the other tissues examined. 1996). in contrast to mammals. was revealed. THE IGFs IN THE LIVER AND EXTRAHEPATIC SITES Like in mammals.. the main site of IGF-II gene expression also is liver. 1995). IGF-II is expressed in high amounts not only in bony fish liver but also in numerous other organs throughout life.09 and 0. IGF-I mRNA accounted to 1.. the IGF-II mRNA levels in liver were about 2-8-times higher than in extrahepatic sites (Tse et al..

2000). 1998. 2002. Because in vivo results are often difficult to interpret due to the involvement of many physiological and partly interfering factors. there is good evidence that GH promotes IGF-I expression in fish liver and its secretion into the circulation. 2000). Shepherd et al. 1994. on primary cultured hepatocytes of salmonids (Duan et al. such as the IGFBPs. This is especially pronounced by a recent study in rainbow trout showing that the individual plasma levels of IGF-I and IGF-II were significantly correlated to the individual amounts of liver IGF-I and IGFII mRNA (Gabillard et al. tilapia (Ng et al. 1991. Thus.1 nM) are well within the range of circulating GH in fish. the liver of bony fish is the major source of both circulating IGF-I and IGF-II (Plisetskaya. These as well demonstrated the dosedependent promoting effect of GH on liver IGF-I mRNA expression.....e. 2004) and tilapia (Schmid et al.. Moriyama... 1993.. 1995. the IGF-I gene in fish liver seems to respond to physiological concentrations of GH. 1993.. 1995). 2003). 1995.. Vong et al. Duguay et al. 1999).. 1991. Biga et al. Gray and Kelley. 1998). Duan and Hirano. 1993. As it is also in mammals. in vitro studies have been performed. 1992) and barramundi (Stahlbom et al. 1992). 1996. Reinecke and Collet. The minimal effective GH concentrations in the in vitro experiments (0. 2003.. 1997. Moriyama et al. Shamblott et al. Injection or oral administration of GH significantly (3. Niu et al.. The GH-induced increase in liver IGF-I mRNA expression was associated to an increase in the level of circulating IGF-I (Funkenstein et al. Most studies on the potential role of GH in stimulating IGF-I production in fish liver have been performed in vivo. 1989... 1992. 1997.. Shamblott et al.. Tse et al. 1989.Manfred Reinecke 95 extrahepatic IGF-II may be of particular physiological impact in bony fish and that this role has been lost during phylogeny. Pierce et al. Shamblott et al.. The GH -liver IGF-I Axis in Growth Regulation GH action on liver IGF-I In accordance to the presence of high-affinity hepatic binding sites for GH as identified in the liver of salmonids Gray and Kelley. Hashimoto et al.. 2004).to 6fold) enhanced the IGF-I mRNA level in the liver of numerous species (Cao et al. i. Duan et al. 1995. LLC ... © 2006 by Taylor & Francis Group. Japanese eel (Mori et al..

1998.. Thus. 1995). Similarly... 1991). 1995). continuous infusion of bovine IGF-I via osmotic minipumps increased linear growth rate and weight gain (McCormick et al. 1998). IGF-I action on pituitary GH In trout pituitary. 1991. 1992) and tilapia (Shepherd et al. In adult Coho salmon. Kajimura et al. IGF-I bound with high affinity and specificity to the anterior pituitary of striped bass (Fruchtman et al. a strong relationship between the serum IGF-I level and GH synthesis and secretion from the anterior pituitary is suggested by in vivo and in vitro studies. LLC .. Both effects could be reversed by GH injections (Gray and Kelley. In agreement with these physiological studies.. indicating that the level of circulating IGF-I is correlated to growth. Kajimura et al. and IGF-I injected into trout induced a rapid inhibition of GH release (Perez-Sanchez et al. Shepherd et al. In agreement. 2002) and primary cultured pituitary cells from eel (Huang et al. 1998) IGF-I at concentrations within the physiological range (10-100 ng/ml) significantly inhibited GH release.. Gillichthys mirabilis (Gray and Kelley. GH and IGF-like material appeared pulsatile and their daily level profiles were significantly correlated with a 1.. 2002). In rainbow trout. Human IGF-I significantly reduced GH gene expression in cultured tilapia pituitary cells (Melamed et al.. © 2006 by Taylor & Francis Group.. 1992. 1997) with the outcome being dose related. a GH/liver IGF-I axis involved in the endocrine regulation of growth seems to exist in fish..96 Fish Endocrinology It is important to note that injections of GH not only increased liver and serum IGF-I but also significantly enhanced body growth with the effect being more pronounced in juvenile fish than in adults (Reinecke and Collet. Rousseau et al. hypophysectomy caused a significant decrease in the level of hepatic IGF-I mRNA in Japanese eel (Duan and Hirano... suggesting the presence of the IGF-IR (Blaise et al. 1997) and led to growth retardation in goby. Blaise et al. binding sites have been identified which exhibited a high affinity for both IGFs but a low affinity for insulin. IGF-I plays a predominant role in the regulation of pituitary GH synthesis and secretion in bony fish.. 1992). in tilapia and striped bass cultured pituitaries (Fruchtman et al. 1993). as in mammals. Thus. 2002) mainly in regions rich in prolactin (PRL) and GH cells. As in mammals. 1998.. Treatment with IGF-I inhibited GH release from trout pituitary cell cultures while GH treatment had no obvious negative feed back effect. thus. 2000.5 h delay (Niu et al.

2002). 2002). Melamed et al. 2002). 2001). Thus. IGF-I seems to influence disparately PRL and GH cells by activating separate as well as overlapping signalling pathways (Fruchtman et al. IGFI in physiological concentrations (10 nM) not only raised the GnRHstimulated release of GTH-I (=FSH) but also the intracellular content of FSH while the potential effect of IGF-I on LH cells was less pronounced (Baker et al. IGFI binding occurred at both cell types in striped bass (Fruchtman et al. GH release seems to be negatively regulated by both the hypothalamic neurohormone somatostatin and the levels of circulating IGF-I. 1998).. In striped bass and tilapia. 2000. 2000).. LLC . 1998.and time-dependent manner while insulin was about 100-times less potent and thyroid hormones (T4.. Whether the endocrine regulation of growth in fish may also involve GH-independent IGF-I gene expression in pancreatic islet cells remains to be clarified.. It has been further shown that the stimulation of PRL release by IGF-I is mediated via only mitogen-activated protein kinase (MAPK) while the inhibition of GH release is partially overcome by MAPK and phosphatidylinositol-3-kinase (PI-3-K) inhibitors.. IGF-I potently reduced GH release but markedly stimulated PRL release in a dose-dependent fashion from cultured pituitary (Fruchtman et al.. In primary cultures of eel pituitary cells. 1998). Kajimura et al. Thus. Rousseau et al.Manfred Reinecke 97 Several studies on primary cultured pituitary cells from goldfish. In Coho salmon primary pituitary cell culture.. In this experimental model. In agreement. supraphysiological © 2006 by Taylor & Francis Group. IGF-I and IGF-II with similar potencies increased gonadotropin (GTH)-II (=LH) cell content and release in a dose. T3) exerted no effect (Huang et al. particularly prolactin (PRL) and gonadotropins. In agreement with the observed effects of IGF-I on GH and PRL cells. underlining the evolutionary conservation and the impact of the somatostatin/GH/IGF-I neuroendocrine axis. feeding juvenile grass carp a somatostatin-inhibitor resulted in higher GH serum levels and growth acceleration (Xiao and Lin. as in mammals. Actions of the IGFs on other pituitary cells There is some evidence that IGF-I and IGF-II may regulate the expression of other pituitary hormones. IGF-I also significantly reduced GH gene expression. rainbow trout and eel have shown that somatotropin-release inhibiting hormone (=somatostatin-14) dose-dependently also inhibits GH release (Huang et al. while no significant effect of IGF-I was obtained on PRL mRNA (Kajimura et al.. 1998.. 2003)..

2000) and tilapia (Uchida et al.. liver IGF-I mRNA (Meton et al.. Larsen et al... Moriyama et al. © 2006 by Taylor & Francis Group. 1995) reaching a plateau at an intermediate ration size in coincidence with the best food conversion (Company et al. the IGFI plasma levels were significantly correlated to specific growth rate. coho salmon (Duan and Plisetskaya. 2003).. a clear correlation between individual serum IGF-I levels and individual growth rates was estimated in coho salmon (Pierce et al.. this was accompanied by a significant elevation of plasma GH concentrations (Duan and Plisetskaya.98 Fish Endocrinology concentrations of insulin were required to produce moderate effects on the FSH and LH cells.. 2001) and tilapia (Uchida et al... In tilapia. 1997). 1993). prolonged starvation led to growth retardation and a marked reduction in the condition factor. In coho salmon (Duan and Plisetskaya. The starvation-induced rise in coho salmon plasma GH levels was associated with a significant decrease in the hepatic GHbinding sites (Gray et al.. 1992).... 2000). Duan et al. 1993) and circulating IGF-I (Moriyama et al. In agreement. seabream (Meton et al. LLC .. The opposite effect has been obtained in gilthead seabream where increasing the feeding ration size caused a decrease in plasma GH concentration and an increase in hepatic GHbinding sites (Perez-Sanchez et al.. 2000. 1992).. 1997). In salmonids.. 2003) and of IGF-like peptide(s) in rainbow trout (Niu et al. in gilthead seabream (Sparus aurata). Starvation also markedly reduced the levels of circulating IGF-I in salmonids (Moriyama et al. rainbow trout (Chauvigné et al. 1993. 2001). 2000.. 2000). 2003) and barramundi (Matthews et al. Fasting for some weeks significantly decreased IGF-I gene expression in the liver of Japanese eel (Duan and Hirano. Nutritional regulation of IGF-I There is increasing evidence that the nutritional status of the individual also influences the amount of circulating GH and IGF-I in fish. plasma IGF-I levels and growth rate also increased (Perez-Sanchez et al. 1995).. Larsen et al. Refeeding of starved salmonids led to a rise in hepatic IGF-I mRNA (Duan and Plisetskaya. 2003).. the nutritional status most likely regulates the hepatic secretion of IGF-I by interacting with the GH/IGF-I liver axis. tilapia (Uchida et al.. 2003). 2003). Thus. Moreover. while there was no change in plasma GH levels after two weeks of starvation in tilapia (Uchida et al. barramundi (Matthews et al. 1994. 1993). 2001).. 2001). hepatosomatic index and condition factor (Uchida et al. with the increase of the feeding amount. 1993).

the potential role of diet composition is still a matter of controversy... intracerebroventricular or intraperitoneal injections of (g)ghrelin increased serum GH (Unniappan and Peter. 2000). 2003) as assumed in mammals (Korbonits et al. 2004) but also in brain. 2003). Therefore.. 2004). such as that fasting causes in increase in serum ghrelin which results in an enhanced release of GH from the anterior pituitary. In agreement. Furthermore.. Ghrelin In mammals. indicating no or little effect of diet composition on the GH/IGF-I axis (Beckman et al. the fish were fed either with high fat or low fat diet and no relationship was found between serum IGF-I levels and body fat. 2001). An early investigation on chinook salmon described that high body fat led to higher serum IGF-I levels (Shearer et al. 2003) and the serum ghrelin © 2006 by Taylor & Francis Group. starvation and refeeding likely affect the expression of IGF-I both at the endocrine (hepatic) and local (paracrine/autocrine) level. In another study. which would lead to a higher growth rate. 2003) ghrelin stimulated GH (and PRL) release in a dose-dependent manner from cultured whole tilapia pituitary. Numerous physiological roles of ghrelin have been described since its discovery in 1999 (see: Korbonits et al. Octanoylated goldfish (g)ghrelin peptides raised GH mRNA in cultured goldfish pituitary cells and promoted GH release which could be inhibited by somatostatin-14 (Unniappan and Peter. LLC .. ghrelin is mainly produced in the gastro-intestinal tract and in hypothalamus and serves as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R).. 1993).Manfred Reinecke 99 Furthermore. 2003). 2002) and tilapia (Kaiya et al.. Ghrelin mRNA was not only expressed in stomach as also found in rainbow trout (Sakata et al. changes in diet composition did not influence significantly hepatic IGF-I mRNA content in gilthead seabream (Meton et al. Both rat (Riley et al. a recent investigation showed that the levels of IGF-I mRNA in rainbow trout skeletal muscle increased dramatically after refeeding when compared to fasted individuals (Chauvigné et al. suggesting its additional role in the hypothalamic regulation of pituitary GH (Kaiya et al... 1997). While an earlier study in coho salmon indicated that the nutritional regulation of IGF-I expression seems to be restricted to the liver (Duan and Plisetskaya.. 2003). 2004).. Accumulating evidence indicates that ghrelin also occurs in bony fish and may exert similar functions. Ghrelin was identified from tilapia stomach and the amino acid sequence deduced from cDNA (Kaiya et al.

The above results are in line with the general opinion that plasma IGF-I is mainly derived from the liver under the control of GH and additionally indicate the influence of the season and the (linked) environmental temperature on the GH/liver IGF-I axis... In parallel. However.... both liver IGF-I mRNA and plasma IGF-I rapidly declined. as in other vertebrates. ghrelin has a potent effect on GH synthesis and release in bony fish. Duan et al. 1998). 2001).. when the feeding amount was restricted. liver IGF-I mRNA and plasma levels were about 2-times higher in fish reared at 16oC than in fish reared at 8oC after 6 weeks of incubation (Gabillard et al. Further regulators of liver IGF-I Seasons and environmental temperature There is severe indication that the levels of circulating IGF-I in bony fish change with the seasons.. 2003. Duguay et al. in gilthead seabream plasma GH increased in late spring and early summer. LLC . the highest growth rate and plasma IGF-I levels were measured in summer (Mingarro et al.. These results agree with those obtained in other salmonids where the plasma IGF-I level was also related to the environmental temperature (Beckman et al. 2003). 2002). increased again to intermediate amounts in winter and stayed at this height until March (Sakamoto and Hirano. 1998. whether ghrelin in fish is upregulated during fasting (Nieminen et al. 2004) and.. reached the minimum in October. There also seems to be a close relation © 2006 by Taylor & Francis Group. Thereafter. The levels of salmonids liver IGF-I mRNA and in parallel those of plasma IGF-I were elevated during late spring and early summer... 2002). Thus. Larsen et al. 1994. 1995. no influence of the environmental temperature on liver IGF-I mRNA and plasma IGF-I levels was found (Gabillard et al. 1993. 1992) remains to be clarified as also other potential effects of ghrelin on the fish IGF system. Interestingly enough. 2004). This may indicate that the environmental temperature does not directly regulate the plasma IGF-I level but promotes growth via IGF-I secreted from the liver after GH stimulation (Gabillard et al. In line with these seasonal changes of IGF-I in salmonids. Duan. remained low during autumn and winter to increase again in spring (Mingarro et al. plays a role in the starvation-induced increase in plasma GH as shown in salmon (Gray et al. thus.. In rainbow trout fed at libitum. 2003)..100 Fish Endocrinology levels were related to preprandial and postprandial status (Unniappan et al. Unniappan et al. 2003).

. the plasma GH levels rapidly increased in March and decreased in May without accompanying changes in the plasma IGF-I levels (McCormick et al. no significant inhibitory or stimulatory effects were evoked by salmon IGF-I (0. T3 (100 nM) significantly enhanced the amount of IGF-I mRNA in primary cultured hepatocytes. Receptors for thyroid hormone (3. Schmid et al. 1990). or extrahepatic sources of circulating IGF-I. the presence of the IGF-IR has been demonstrated in the liver of the barramundi (Drakenberg et al. 1998). such as thyroxin (McCormick et al. 1997. other hormones. LLC . The stimulating effect of T3 on hepatic IGF-I mRNA expression was time-dependent. Thus. no autocrine and/or paracrine IGF-I feedback mechanisms seem to be present in bony fish liver. Hormones In further analogy to mammals (Venkatesan and Davidson. Bres et al.. In addition. 1992. However. Agustsson et al. 1997).Manfred Reinecke 101 between the observed seasonal changes in serum GH and IGF-I and smoltification (Beckman et al. However.. 1998.. 1986. This may be attributed to effects of IGFBPs but further parameters may as well modulate the GH/IGF-I axis. at present is unclear. 1993.. 1990). There is some evidence that the mammalian hepatic IGF-I receptor is regulated not only by serum GH and but also by serum IGF-I (Venkatesan and Davidson.1-1000 nM) on the IGF-I mRNA level in primary cultured tilapia hepatocytes sensitive to GH (Schmid et al. 2002).1 M) was dose-dependent.3‘-triiodothyronine) T3. in Atlantic salmon reared under simulated conditions of normal seasonal photoperiod and an increase in temperature in February. the increase in IGF-I mRNA expression evoked by T3 (1 nM . In vitro. 2003) or cortisol. Recently. Plisetskaya. such as the endocrine pancreas (Reinecke et al...5. In agreement with the in vitro findings. Therefore. a significant increase (by 45%) of liver IGF-I mRNA occurred after repetitive intraperitoneal injections of T3.. Whether these include changes in liver GH-receptor densities (McCormick et al. 2001).. have also been identified in bony fish (rainbow trout) liver (Bres and Eales.. 2002). the influence of T3 on the expression of IGF-I in the liver of the tilapia has been investigated in vivo and in vitro (Schmid et al. 2002. 2003). 2000).. 1990). reaching about 150% of the starting amount after 4 h and 170% after 6 h. T3 directly stimulated the hepatic production of IGF-I in the tilapia in vitro and in vivo indicating © 2006 by Taylor & Francis Group.

as they are discussed for mammals (Ikeda et al. was significantly increased (Tse et al. In contrast to IGF-I that significantly inhibited GH release in vitro and in vivo (3. since in mammals.2). and in trout primary hepatocyte culture IGFII mRNA levels increased in a dose-dependent manner after administration of GH (Shamblott et al. On the other hand.. the amount of IGF-II mRNA in liver and several extrahepatic organs.. Similarly. in starved juvenile common carp (Cyprinus carpio) after injection of porcine GH.. LLC . intestine. The GH/liver IGF-II axis There is little information on the levels of circulating IGF-II in fish. 2005). and nothing is known about potential changes in serum IGF-II under different physiological conditions such as GH status. kidney and muscle. © 2006 by Taylor & Francis Group.. On the one hand.1. few studies have dealt with the potential GH dependence of IGF-II expression in bony fish liver. These results are supported by a recent study on cultured pituitaries of the tilapia (Kajimura et al.. the levels of IGF-II mRNA in liver and in several extrahepatic sites of seabream remained unchanged after GH treatment (Duguay et al. 2004). 2002). 2002) and cultured pituitary cells from striped bass (Fruchtman et al.. In these experimental models. 1996). the amount of IGFII mRNA in rainbow trout liver and pyloric caeca was significantly elevated after GH injection. where serum IGF-II ranged from 22 to 18 ng/ml. it is reasonable to assume that in bony fish both the IGF-I gene and the IGFII gene are under the control of GH both in liver and extrahepatic sites.. 1995). Data exist only for Atlantic salmon and rainbow trout (Gentil et al. 2003). because the earlier studies used RNase protection assay and the latter the highly sensitive real-time PCR to estimate changes in IGF-II mRNA expression. 1991). GH most likely regulates only the expression of the IGF-I gene but not that of the IGF-II gene. Vong et al. Therefore. The partly controversial results might well be due to species differences but more likely they are caused by different sensitivities of the detection methods. IGF-II only showed a tendency to suppress GH secretion in rainbow trout pituitary cell culture (Blaise et al. 1996. In agreement. 10-times higher concentrations of hIGF-II than of hIGF-I were needed to significantly inhibit GH-release. such as gills. Wilkinson et al.. This gives bony fish a unique state in phylogeny.102 Fish Endocrinology that in bony fish liver regulatory mechanisms seem to exist. smoltification and environmental temperature (Reinecke et al. 2002. 1995)....

Duan and Hirano... 1999) and carp (Fig. 1992. there is also an influence of GH on growth via local IGF-I.. gilthead seabream (Funkenstein et al. 1999) indicating that IGFI exerts direct effects on cartilage growth in bony and cartilaginous fish. Raja eglanteria (Gelsleichter and Musick. 1992). 4. 1997b)... Thus. 1991). Perrot et al. which has later been demonstrated also in other salmonids © 2006 by Taylor & Francis Group. 1985). LLC . 1997. 2000). Cartilage from salineinjected fish responded to IGF-I exposure in vitro with enhanced thymidine and sulphate uptake but these did not reach the basal uptakes obtained in the GH-injected fish (Tsai et al. 1990).. In agreement. 1991. such as tilapia (Reinecke et al. Gray et al. which has been shown to stimulate in vitro sulfate uptake into cartilage in several teleosts (Gray and Kelley. hypophysectomy and IGF-I injection suggested that the GH status of the animal is important for the sensitivity of cartilage to IGF-I (Gray and Kelley. 1994). An investigation on Gillichthyes mirabilis using GH-binding by the liver. 1992.. An early study determined that the stimulatory effect of GH on sulfate incorporation in eel cartilage was exerted indirectly and mediated by some IGF-like plasma factor (Duan and Inui. GH may have increased both uptakes via stimulation of local IGF-I because IGF-I peptide and mRNA are present in chondrocytes of juvenile and adult bony fish.1a).. However. in 1985. injection with GH over 2-3 weeks stimulated basal or IGF-I stimulated sulfate incorporation in coho salmon gill cartilage (McCormick et al. GH injection markedly increased thymidine and sulphate uptake into cartilage (Tsai et al. 1992) and in an elasmobranchian species. McCormick et al..Manfred Reinecke 103 THE IGFS IN EXTRAHEPATIC SITES Cartilage and Bone As outlined in the earlier section. as it has been shown in rat (Reinecke et al. Therefore. it has been shown that exposure of yearling coho salmon to seawater was accompanied by an increase of circulating GH (Sweeting et al.. In coho salmon. Gills Already. the observed GH-dependent growth-promoting effect of IGFI may not only be exerted via the endocrine route but also in an autocrine/ paracrine manner by IGF-I released from local chondrocytes. there is evidence that the GH/liver IGFI axis as endocrine system is involved in the regulation of fish growth. 1994). the clearnose skate.

1996. Mancera and McCormick.. McCormick. (b) Chloride cells in the gill epithelium of the tilapia show IGF-I-immunoreactivity. 1999).. 1999. (Sakamoto et al. 1991. Mancera and McCormick. Seidelin et al. © 2006 by Taylor & Francis Group. Seidelin and Madsen. 1997. 1992. (a) Several chondrocytes in the upper part of the carp gill cartilage exhibit IGF-I-immunoreactivity. 1999). 1998) and trouts (McCormick et al.. The osmoregulatory effects of IGF-I seem to be exerted directly because IGF-I stimulated Na+. Seidelin and Madsen. Yada et al. In mummichog (Fundulus heteroclitus) (Mancera and McCormick.1 IGF-I-immunoreactivity in gills.104 Fish Endocrinology Fig.. K+-ATPase in in vitro preparations of salmonid gill (Madsen and Bern. McLean et al.g... (c) In rainbow trout kidney. Agustsson et al. while insulin and IGF-II were without effects. numerous cells in the epithelium of the proximal tubule contain IGFI-immunoreactivity. Seidelin and Madsen. suggesting a participation of the GH/IGF-I liver axis in smoltification. 2001). LLC . K+-ATPase activity (McCormick et al. 1993). Transfer of euryhyaline fish from brackish to seawater increased plasma osmolality and gill Na+. 1998. 1991... (d) IGF-I-immunoreactive entero-endocrine cells are present in the upper small intestine of the turbot. 1998. kidney and intestine of bony fish. 4. 1991. 1999) which could be also achieved by GH application (e. IGF-I improved adaptation to seawater in a dose-dependent manner.

1997). the capacity of the GH/liver IGF-I axis to increase hypoosmoregulatory ability may be a common feature of euryhaline bony fish.. K+-ATPase compared with sham-operated controls (Shepherd et al.. seem to be involved in seawater adaptation interfering with IGFI and thus. Thus. However... Therefore. GH receptors were detected in tilapia gills (Fryer. 1991). not only GH but also cortisol and PRL. 1993). rendering the regulation of smoltification more complex. © 2006 by Taylor & Francis Group. K+-ATPase in coho salmon in vitro when preceded by in vivo treatment with GH (Madsen and Bern. 1993). 1996) have been identified (Reinecke et al. The injection of GH and IGF-I in mummichog caused significantly greater effects on plasma osmolality and gill Na+. LLC . during seawater adaptation IGF-I mRNA in coho salmon was not significantly altered in liver but increased markedly in gills (Sakamoto and Hirano. liver IGF-I mRNA was significantly higher in individuals reared in seawater than in those reared in freshwater (Inoue et al.. 2004). In the four-spine sculpin (Cottus kazika). Biga et al. The effects of IGF-I may depend on priming by GH because IGF-I raised the level of gill Na+. 1998). However. Thus. This assumption is supported by a study in tilapia where hypophysectomy lowered the levels of Na+. In contrast to the stimulatory effects of GH and IGF-I. 1999). 1997b). both liver-derived and local autocrine/paracrine IGF-I may participate in the regulation of plasma osmolality and gill Na+. K+-ATPase activity in Atlantic salmon and brown trout (McCormick.. 1999). 2003.. 4. K+-ATPase activity than that of either hormone alone (Mancera and McCormick. In accordance. other hormones seem to be also involved in these processes. prolactin (PRL) impaired osmoregulatory performance in brown trout and then completely abolished the stimulation of Na+.1b) which also express Na+. K+-ATPase activity in rainbow trout revealed that IGF-I was more effective than GH in reducing plasma osmolality after transfer from brackish to seawater (McCormick et al. K+-ATPase activity. 1979) and GH treatment increased IGF-I mRNA in gills of Coho and Chinook salmon and common carp (Vong et al. 2003) suggesting the potential impact of endocrine IGF-I in smoltification. 1996. Seidelin et al. K+-ATPase evoked by IGF-I (Seidelin and Madsen. K+-ATPase (McCormick.Manfred Reinecke 105 The comparison of the potencies of both hormones on plasma osmolality and gill Na+. IGF-I and cortisol had additive stimulatory effects on Na+. As potential production sites of IGF-I chloride cells of the filament epithelium of tilapia (Fig.

Thus. kidney is a major osmoregulatory organs in fish. In rainbow trout. 1997b. Because numerous gastro-intestinal hormones also did not coexist with IGF-I- © 2006 by Taylor & Francis Group. Shamblott and Chen.. Epithelial cells of the proximal tubules have been identified in tilapia (Reinecke et al. and also expresses high amounts of IGF-I mRNA. 1992).. Gastro-intestinal Tract IGF-I gene expression has been obtained in stomach (Loffing-Cueni et al. 1997a).1d. renal IGF-I may be involved in several other parameters of kidney function. 1991.. In addition to its possible role in osmoregulation.. 4. gastro-enteroendocrine IGF-I-immunoreactive cells have been detected in a cartilaginous fish. such as the stimulation of kidney growth and differentiation. 1995) and the renal IGF-I mRNA level in C.. tilapia (Reinecke et al. LLC . Berwert et al.106 Fish Endocrinology Kidney Besides gills. In contrast. renal blood flow and glomerular filtration rate and sodium absorption. 2003. 1996). Loffing-Cueni et al. and the cyclostome M.1c) as the potential sites of renal IGF-I production. presently we can only speculate about the physiological role of IGF-I in bony fish kidney... transfer to seawater increased the IGF-I mRNA level in kidney (Sakamoto and Hirano. Caelers et al... 1997b) and rainbow trout (Fig. Raja clavata. Reinecke et al. IGF-I-immunoreactivity coexisted with glucagon. 1998). In bony fish pancreatic islets. 1993. 1993). 2004) of several bony fish species. the ray. The evidence for a potential participation of renal IGF-I in osmoregulation is conflicting. turbot Scophthalmus maximus (Fig. In daddy sculpin. 2003a). 2003.. K+-ATPase gene level and activity in brown trout (Madsen et al. Atlantic salmon (Koppang et al. 1998) and shi drum Umbrina cirrosa (Radaelli et al. Reinecke et al.. somatostatin or Pancreatic Polypeptide (PP) but the IGF-Iimmunoreactive gastro-entero-endocrine cells did contain any islet hormone (Berwert et al. 1997b). 1995). 1995. as is likely in mammals (Hirschberg. as it has similarly described in mammals (see: Reinecke and Collet.. 2003). kazika (Inoue et al. Vong et al. the potential IGFI production sites have been identified as endocrine cells of the mucosal epithelium. glutinosa (Reinecke et al. Inoue et al. the same treatment did not affect Na+. 1997) and intestine (Duan et al.. 4... Below the phylogenetic level of bony fish... 1993. 1997.

1990).. The early appearance of IGF-I and its receptor may indicate a particular physiological impact of IGF-I in the developing fish gastrointestinal tract. 2003). Vong et al. Caelers et al.. Furthermore. the first IGFI-immunoreactive cells were found as early as in the first week post hatching in the intestinal epithelium of turbot (Berwert et al. 1995) and shi drum (Radaelli et al. 1999. LLC . Furthermore. 2003. In pig. daddy sculpin. 1998. barramundi. 2004). Their transient and early appearance during fish ontogeny suggests that IGF-I secreted from the IGF-I-immunoreactive epithelial cells may exert mitogenic functions in the developing gastro-intestinal tract by acting on neighboring epithelial cells. 1997. Because in addition to gill and kidney the intestinal tract is an important osmoregulatory organ in fish. nothing is known about its potential physiological impact or its synthesis sites.. This notion is supported by experiments in brown trout which suggest that IGF-I plays a role in the regulation of intestinal Na+. 2003a).. Reinecke and Collet. and (2) occur in greater numbers transiently during ontogeny (Reinecke and Maake.Manfred Reinecke 107 immunoreactivity (Reinecke et al. Collet et al.. 2003). K+ATPase activity (Seidelin and Madsen. and the IGF-IR appeared in the second week post hatching in shi drum gut. 1998). 1999). the amounts of intestinal IGF-I-immunoreactivity and of the IGF-IR coincided with villous growth and maturation (Schober et al. IGF-I potently stimulated crypt cell proliferation and villus cell density in adult rat (Steeb et al. 1998).. © 2006 by Taylor & Francis Group. 1999).. Loffing-Cueni et al. 1996. the presence of the IGF-II gene in gastro-intestinal tract has also been elaborated (Duguay et al.. 1995). In analogy. common carp and tilapia.... intestinal IGF-I may also be involved in osmoregulation (Koppang et al. Although the expression of intestinal IGF-II mRNA was raised by GH (Vong et al. In some bony fish species such as seabream. 1997a). 1994) and improved mucosal structure and function in transplanted rat small intestine (Zhang et al... This notion is supported by the observations that the IGF-I-immunoreactive cells: (1) in adults show pronounced inter-individual variations in frequency and distribution (Reinecke et al.. 1997b). The potential proliferative action of intestinal IGF-I may be regulated by GH that in juvenile common carp markedly increased the amount of intestinal IGFI mRNA (Vong et al.. IGF-I released from adult fish mucosal epithelial cells may exert paracrine effects in response to altering local demands. the IGF-I-immunoreactive cells very likely represent an individual class of gastro-entero-endocrine cells (Reinecke and Collet.

i. 1997a.e. Immunohistochemical studies in bony fish. glucagon and PP cells in turbot and common carp (Reinecke et al.108 Fish Endocrinology Endocrine Pancreas The IGF-I gene is expressed in the endocrine pancreas of different bony fish species (Plisetskaya et al. In the islets of the cartilaginous fish Raja clavata and Squalus acanthias. 1993. 1998).. the expression of IGF-I mRNA has been shown in principal islets (Brockmann bodies) of the salmon O. On the one hand. 1992. 1993). In several bony fish species IGF-I-immunoreactivity occurred in noninsulin cells. 1990).... islet-derived IGF-I may be involved in the paracrine regulation of insulin secretion from the -cells as it is assumed in mammals (Leahy and Vandekerkhove. These distribution patterns are in line with the restriction of IGF-Iimmunoreactivity to non-insulin islet cells above the phylogenetic level of bony fish (Maake and Reinecke. it is also conceivable that in addition to the © 2006 by Taylor & Francis Group.. 4. 1995). all insulin cells and some somatostatin cells contained IGF-Iimmunoreactivity (Reinecke et al. 1995). However. Hypophysectomy did not influence the amount of sulphation activity in eel pancreas (Duan and Hirano. 1993. 1992) which suggests that islet IGF-I is not regulated by GH.. in somatostatin cells in eel (Anguilla anguilla). On the other hand. 1995). gorbusha (Plisetskaya et al. 1995) which may suggest a significant physiological role for isletderived IGF-I not only in the adult but also during larval development. d) and goldfish. Thus. The observed effects may have well been raised by insulin (Plisetskaya. Berwert et al. 1993. some studies suggest that islet-derived IGF-I may also act as an endocrine hormone. 1997) which are not surrounded by exocrine parenchyma. 1997b. 1993). Reinecke et al.. 1993) and of Cottus scorpius (Loffing-Cueni et al. In agreement.. cartilaginous fish and cyclostomes indicate that IGF-I is not only present in cells of the exocrine parenchyma (Funkenstein et al.. and in somatostatin. Islet IGF-I appeared around day 10 post hatching in turbot (Berwert et al. Reinecke et al. Berwert et al. In goby.2b.. Richardson et al.. tilapia (Fig. b. 1997b).. ilectomy led to a decrease in 35SO4incorporation in cartilage but hepatic GH binding was unchanged (Kelley et al. in addition to somatostatin and glucagon cells a varying percentage of the insulin cells exhibited IGF-I-immunoreactivity. 1993. 1997) but also in islet cells (Reinecke et al.. the branching of IGF-I and insulin in the endocrine islets likely has occurred at the phylogenetic level of bony fish.. In the cyclostome Myxine glutinosa. LLC . 1995.

1991. IGF-II is present exclusively in -cells of the islets of mammals. IGF-II seems to coexist with insulin. 4. Thus. an exclusive coexistence of IGF-II and insulin was also detected in birds.. reptilians.c) and cartilaginous fish (Reinecke et al. As in mammals. b and c. 4. dog and human (Hill and Hogg.. bony (Fig.Manfred Reinecke 109 Fig. IGF-II occurs exclusively in insulin cells and IGFI in somatostatin cells. d) of tilapia pancreas. LLC . 1997). where it coexists with insulin within the secretory granules (Höög et al. 1993).2 Two consecutive sections (a. In contrast to IGF-I. 1995). indicating particular evolutionary conservation of the islet IGF-II system. GH-dependent liver IGF-I system islet-derived IGF-I constitutes a further endocrine GH-independent IGF-I system that is also involved in fish growth regulation. The first section is incubated with antisera against insulin (a) and somatostatin (b) and the second with antisera against IGF-II (c) and IGF-I (d). © 2006 by Taylor & Francis Group.2 a. amphibians (Reinecke et al.. Together with the observation that the release of insulin and IGF-II were strictly correlated in cultured human fetal islet cells (Bryson et al. Maake and Reinecke. 1989) this suggests a concomitant release of insulin and IGF-II. such as rat.. 1994). throughout phylogeny.

1996) and gilthead seabream (Funkenstein et al.. 1997b.. IGF-I-immunoreactive neurones were present in adult barramundi brainstem (Richardson et al. Torres-Aleman. McRory and Sherwood.... Loffing-Cueni et al. 1993). trout (Leibush et al.. 1999. 1993. 1997b) and hagfish (Reinecke et al.. carp. Tse et al. 1999. 1994. 1999). 1994. IGF-II mRNA is clearly expressed in the mesodermal portion of the choroid plexus and the meninges.. there is indication that IGF-II mRNA is transiently expressed in neurones © 2006 by Taylor & Francis Group. IGF-I in adult fish brain may support survival of neurones and glia cells as it is assumed for mammals (Hepler and Lund..110 Fish Endocrinology Whether this also implies that the IGF-II and insulin genes are activated simultaneously in vertebrates remains to be clarified. 1997. Reinecke et al. In adult mammalian brain. 1991) brain and IGF-I mRNA signals throughout the brain of developing sea bream (Funkenstein et al. Duguay et al.. while its presence in the cells of neuroepithelial origin is doubtful. Rather.. 2002) are expressed in the brain of several bony fish species. thus. 4. Together with the identification of the IGF-IR in the brain of daddy sculpin (Drakenberg et al. Perrot et al.. In general. Loffing-Cueni et al.. 1990. seabream and tilapia. 1997. there is no information on the exact distribution of IGF-I mRNA in fish brain and the IGF-I-immunoreactive neurones in barramundi.. the distribution patterns of IGF-Iimmunoreactive neurones in fish brain resemble the overall occurrence of IGF-I mRNA in rat brain (Bondy and Lee. tilapia and hagfish were sparsely scattered when compared to those containing IGF-II (see below). The potential sites of IGF-I synthesis in brain have been identified in the bony fish barramundi...3d) were constantly observed. while the frequency and distribution patterns of the IGF-Iimmunoreactive neurones in other regions showed marked variations among the individuals investigated (Reinecke et al. However. Perrot et al. 1995) and at all levels of adult tilapia (Reinecke et al. LLC . 1993). 1997b. Brain RT-PCR and Northern Blot studies have shown that both IGF-I mRNA (Duan et al. Funkenstein et al. glutinosa. and IGF-II mRNA (Collet et al. 1999).. 1998). 1997). 1997) the presence of the IGF genes indicates that they play a local physiological role in fish brain. and in the cyclostome M. However. The IGF-Iimmunoreactive Purkinje cells in cerebellum (Fig.. making a neurotransmitter or -modulator function of IGF-I unlikely. LoffingCueni et al. 1998.

LLC . (d) IGF-Iimmunoreactivity occurs in the ganglionic cell layer (gcl) of the cerebellum. (c) Section through the midbrain showing IGF-II mRNA in the optic tectum (TeO). particularly in the cell body layer of periventricular zone (cPZ). 4.3 IGF-II mRNA and IGF-I-immunoreactivity in the tilapia brain. © 2006 by Taylor & Francis Group. (a) High amounts of IGF-II are expressed in numerous cells of the choroid plexus.Manfred Reinecke 111 Fig. (b) A very selective in situ hybridization signal for IGF-II mRNA is present in the ganglionic cell layer (gcl) of the cerebellum.

4a) and Sertoli cells (Reinecke et al. IGF-II mRNA was strongly expressed in the epithelium of the choroid plexus (Fig. 1999). tilapia (Reinecke et al.. primary and secondary spermatocytes. suggesting an involvement of IGF-II in the regeneration of brain tissue (Walter et al. maintenance and regeneration of neurones (Caelers et al. the sustained neuronal IGF-II expression in adult fish brain that contrasts the situation in mammals may correlate with the continued postembryonic up to life-long brain growth as has been shown in many teleosts.. The discrete presence of IGF-II mRNA in neurones might indicate that IGFII acts as transmitter or modulator but the widespread occurrence of the IGF-II producing neurones more likely suggests that IGF-II plays a role in the differentiation.. The epithelial cells of human and rat choroid plexus seem to secrete IGF-II (Haselbacher and Humbel. 1982).. 4. the expression of IGF-II mRNA in the choroid plexus and its secretion into the cerebrospinal fluid were enhanced.112 Fish Endocrinology during early mammalian development. 1996. Caelers et al. A recent study has investigated the cellular distribution of IGF-II mRNA in the adult brain of the tilapia (Oreochromis mossambicus) (Caelers et al. 1997b. As in the mammalian brain (Walter et al. 1999). Reproductive Tract Male gonad The presence of the IGF-I and IGF-II genes in fish male gonads has been demonstrated in rainbow trout (Le Gac et al... IGF-II mRNA was also expressed in numerous distinctly distributed neurones at all levels of the tilapia brain (Fig. It is reasonable to assume that IGF-II from the choroid plexus of fish may serve similar needs. 1996) is consistent with the presence of IGF-I-immunoreactivity in trout and tilapia primary spermatocytes (Fig. 1999). c). 1999). 2003). Perrot and Funkenstein. and daddy sculpin (Loffing-Cueni et al. © 2006 by Taylor & Francis Group. shortly after the onset of an injury in rat brain.. However. 2003) the presumed function of IGF-II on neuron survival may be exerted independently from the physiologically varying GH levels. 4. 1997b).3c).3a) and in the meninges (Fig. The expression of IGF-I mRNA in isolated trout spermatogenic and Sertoli cells (Le Gac et al.. 2004).. 2003). Because in common carp brain the IGF-II mRNA levels in contrast to those of IGF-I mRNA were not significantly influenced by GH treatment (Vong et al.. Furthermore.. 4.3 b. Furthermore. IGF-II mRNA was found in numerous testicular cell types such as spermatogonia. 1998. LLC . 4.

Manfred Reinecke 113 spermatids and Sertoli cells (Perrot and Funkenstein. There is also evidence for a physiological role of IGF-I in the elasmobranchian male gonad.. in oocytes of ovarian fragments of striped bass.. IGF-I is likely to mediate the action of GH in the male gonad.. 1999). a marker for resumption of meiosis. Based on these data it can be assumed that IGF-I serves as paracrine/ autocrine regulator of fish spermatogenesis thereby interacting with steroid hormones. IGF-I stimulated all stages of spermatogenesis induced by 11-ketosterone (Nader et al. In Japanese eel cultured testes. which is compatible with the previous demonstration of GH receptors in trout testis (Le Gac et al. Furthermore. IGF-I stimulated thymidine incorporation into vitellogenic follicles of the goldfish (Srivastava and Van der Kraak. stimulated the incorporation of thymidine into spermatogonia and primary spermatocytes from cultured spermatogenetic rainbow trout testes (Loir and Le Gac. 1999). the amounts of testicular IGF-I and IGF-II mRNA were increased after GH-treatment (Le Gac et al. 1994. 1996). Morone saxatilis (Weber and Sullivan. In trout testis. IGF-I—and less pronounced IGF-II—increased germinal vesicle migration and breakdown. GH. both IGFs seem to stimulate DNA synthesis of bony fish male germ cells. in vitro. In rainbow trout. The density of gap junctions between granulosa cells as well as between granulosa cell and oocytes—which both are rare in incompetent follicles—was markedly increased by IGF-I in red seabream (Patino and © 2006 by Taylor & Francis Group. IGF-I and. 1996.. 2000). spermatids and Sertoli cells of gilthead seabream (Perrot et al. Thus.. 1994) and further induced final oocyte maturation in red seabream (Kagawa et al. Loir. Sertoli cell monolayers derived from premeiotic and meiotic regions displayed a doseresponse increase in DNA synthesis after addition of IGF-I. LLC . 1992). the IGF-IR has been identified where it was probably located at spermatogonia and Sertoli cells (Le Gac et al. 2000). with lower potency IGF-II. 1994).. Furthermore. 1999). 1999) which agrees with the localization of the IGF-IR in spermatogonia A. IGF-I markedly increased the thymidine uptake into cultures of premeiotic regions of spiny dogfish (Squalus acanthias) testis (Dubois and Callard. Female gonad Several in vitro studies indicate distinct physiological roles for IGF-I in the fish ovary. Perrot and Funkenstein. 1993).

Thus. Data on the presence of IGF-II in oocytes are conflicting.. estrogen and progesterone biosynthesis were differentially regulated by IGF-I in cultures of common carp ovarian follicles (Behl and Pandey. 1997b). 1999).. IGF-I immunoreactivity was detected in granulosa cells (Kagawa et al. follicular growth and oocyte maturation although further studies will have to verify this. the IGF-IR was localized at granulosa and theca cells of coho salmon (Maestro et al. In summary. LLC . 17 . 4d) and seabream (Radaelli et al. 2003b). Similarly. the intraovarian IGF-I production seems to switch from the young oocyte to the surrounding follicle cells during development (Schmid et al. Thus. IGF-II also induced final maturation of oocytes of red seabream in vitro (Kagawa et al. 1999. 1999). 1994). Perrot et al. in fish..c) IGF-I mRNA and peptide additionally occurred in theca cells. IGF-I binding in carp ovaries changed with the reproductive cycle (Maestro et al. but in contrast to IGF-I. 2000) and tilapia (Schmid et al. 2000). 4 b) but absent from oocytes in later stages of development (Schmid et al. © 2006 by Taylor & Francis Group. IGF-I was further present in young oocytes (Fig. IGF-II-immunoreactivity was described in oocytes at the perinucleolus stage (Schmid et al. an intraovarian IGF system does exist.. Radaelli et al. 1999. In red seabream. The above described actions of IGF-I under physiological conditions may well be exerted by circulating (liver-derived) IGF-I... Both IGF-II mRNA and peptide were located in granulosa cells and theca cells of later follicle stages in tilapia (Fig. the above studies indicate that IGF-I promotes proliferation and maturation in ovary. 1995) and in gilthead seabream (Perrot et al. 1997a) and in gilthead seabream additionally at previtellogenic oocytes (Perrot et al. 20 -dihydroxy-4-pregen-3-one) by the granulosa cells in the preovulatory coho salmon ovary (Maestro et al. 1999)... because both human and salmon recombinant IGF-I inhibited steroid production (testosterone.. but the IGFs are also produced in the ovary. While no IGF-II mRNA was detected in oocytes. 1999) (Fig.. 2000). The functional implication of ovarian IGF-I (and IGF-II) in fish seems to be an involvement in the autocrine/paracrine regulation of steroidogenesis. In agreement with the potential physiological role of IGF-I in the female gonad. 2003b)..114 Fish Endocrinology Kagawa. Furthermore.... As IGF-I. 1997a). 17 -hydroxyprogesterone) by the theca cells and stimulated steroid production (17 -estradiol (E2). they seem to be absent from earlier follicles. IGF-I also likely to play a crucial role in the regulation of steroidogenesis. 4 b.

(b) Immunohistochemical localization of IGF-I in young oocytes and in granulosa cells of a follicle at the third yolk globule stage of the tilapia. (c) Higher magnification showing IGFI-immunoreactive granulosa cells. In rainbow trout © 2006 by Taylor & Francis Group. 4. Both IGF hormones and the IGF-1R have been detected in fish embryos. thus. THE IGFs IN DEVELOPMENT Autocrine. coordinate the differentiation of tissues during critical periods of development.4 (a) Spermatocytes in rainbow trout testis exhibit IGF-I-immunoreactivity. suggesting a high organizational impact of the IGF system during ontogeny. at correct sites (in the case of para. steroid hormones and thyroxin. The regulation of organ differentiation thus involves a complex cascade and interaction of signals which have to be released at specific times. 1994). indicating that the important components of the IGF system exist during early development in teleosts and. the IGFs. LLC .and autocrine factors) and within a precise dose range (Segner et al. paracrine and endocrine signals such as GH..Manfred Reinecke 115 Fig. (d) IGF-II mRNA is present in tilapia granulosa cells of a follicle at the third yolk globule stage.

Ayson et al. rabbitfish and shi drum (Shamblott and Chen. 1997. In correspondence. a parallel age-related decline was found for tyrosine kinase activity of the IGF-1R.. such as during gastrulation and organogenesis. estrogens and aromatase activity are specific for ovarian differentiation. developmental and tissue-specific regulation of IGF-1R steady-state mRNA levels and polyadenylation were detected. In correspondence.. 2003 a. the number and binding capacity of the IGF-1R were highest at 5 weeks and both parameters decreased with age. 1999. Greene and Chen. The morphologically still undifferentiated gonad anlage already has the ability for gender-specific steroidogenesis.. Although both IGF-I and IGF-II mRNA were strongly expressed at all stages of development. 1997. because some evidence suggests that the steroids interact with other hormonal factors in gender differentiation (Piferrer 2001). Besides differentiation. 1999). seabream. 1999). 1999. Radaelli et al. and 11 -oxygenated androgens and 11 -hydroxylase activity for testicular differentiation. 2001). Ayson et al. 1993. Deane et al. IGF-I and IGF-II mRNA seem to be already present in unfertilized eggs (Green and Chen. the IGFs may be © 2006 by Taylor & Francis Group. Ayson et al. growth is an important parameter during development. IGF-II seems to be the predominant IGF as shown in rainbow trout and rabbitfish (Green and Chen. 2003). 2001).. 1997. However. Since sexual differentiation of the gonads is accompanied by pronounced cell proliferation and tissue growth (Nakamura et al. 1998). 2002).. The main inducers of gonadal sex differentiation in fish appear to be steroid hormones (Baroiller et al. Piferrer. Duguay et al. The distinct localization of IGF-I.. the distribution patterns of IGF-I and IGF-II mRNA and peptide were also tissue specific and age-dependent in trout. although in rainbow trout.. during yolk absorption.. 1999) and seabream (Perrot et al. at hatching. 1999. Perrot et al... b). In trout skeletal muscle (Méndez et al. 2002.. In this respect. It is still not clarified whether IGF expression in early development of fish is regulated by GH (Perrot et al.116 Fish Endocrinology (Greene and Chen. 2002)... LLC . low levels of GH mRNA were already detected during gastrulation and intermediate amounts of GH mRNA in early stage embryos (Yang et al. and at feeding. These results indicate a key role for IGF-I in growth and metabolism of trout muscle. in gonads of fish has been assumed to indicate a role of the IGF system in ontogenetic gonad differentiation (Reinecke and Collet. 1996. 1998). this does not necessarily imply that sex steroids are the only factors involved. and in part also IGF-II.

male and female gonads.. this © 2006 by Taylor & Francis Group. spleen. In turn. there can be no doubt that the so-called GH/liver IGF-I axis is present in fish as it is in other vertebrates. Therefore. skeletal muscle. 1999). Extrahepatic IGF-I probably is also controlled by stimuli different from GH. the expression of liver IGF-I and its secretion are influenced also by other parameters such as osmotic status. pancreatic islets. gastro-intestinal tract. To date.. LLC . in primary cultured rainbow trout pituitary cells (Weil et al. nutritional status. During puberty.e. gills. IGFI may promote pubertal development in fish. kidney. Incubation of the pituitary cells with physiological concentrations of IGF-I resulted in significantly higher GnRH-stimulated FSH release and remaining cell content of FSH and LH. environmental temperature and stress. the level of circulating IGF-I negatively regulates GH.. CONCLUSIONS AND SUGGESTIONS FOR FUTURE RESEARCH Both hormones IGF-I and IGF-II are present in bony fish liver as well as in numerous other organs such as brain. GH stimulates the expression of IGF-I in liver.. heart. eye. experimental data on a role of IGF-I (and IGF-II) in initial gonad differentiation are lacking to date. Apart from inducing growth. 2001)..Manfred Reinecke 117 mediators of steroid actions or act synergistically. The interaction between the different regulators of liver IGF-I which seem to constitute a network with many interacting partners will certainly be in focus of future research. The effect of IGF-I on synthesis and release of fish gonadotropic hormones was investigated using a primary culture of immature coho salmon (Oncorhynchus kisutch) pituitary cells (Baker et al. For fish. a promising field of research is to study the (organspecific?) interactions of other hormones like insulin (Navarro et al. 2000). However. GH also regulates the expression of the IGF-I gene in extrahepatic sites where it likely exerts auto/paracrine actions. from where it is secreted into the circulation to act as endocrine hormone. Thus. IGF-I appears to be involved in the regulation of the hypothalamus-pituitary-gonad axis. Similarly. A role of IGF-I as paracrine/autocrine and endocrine regulator of gonadal growth and differentiation has been recently demonstrated for the Japanese quail (Fu et al. the developmental period covering the transition from the immature to the mature reproductive system. the potentiating effects of IGF-I on GTH responses to GnRH were higher in early gametogenesis. i.

. As a first step. University of Zürich. the likely different physiological roles of the two (or more) IGF-IRs may be important. Acknowledgments The author wishes to thank Katarina Drakenberg. Lorenzo Berwert. Finally.. this volume) with the IGF-I system. Antje Caelers. Ivana David. gonadal steroids (Segner et al. In correlation. Giorgi Berishvili. this volume) or cortisol (Segner et al. attempts must be taken to clone the IGF-II receptor. Whether these results indicate a particular impact of IGF-II in fish remains to be clarified. Although some evidence has been presented for specific local actions of IGF-I. Werner Kloas (Institute of Freshwater © 2006 by Taylor & Francis Group. Elisabeth Eppler. ghrelin.118 Fish Endocrinology volume). much work has to be done before we can get an idea of the physiological impact of local vs systemic IGF-I in fish and the modes and potential interactions of its regulation (Reinecke et al. alterations in the expression of the IGF-II gene in different organs during development and with the physiological situation should be investigated. Sweden). this volume). the ‘hormone’ IGF-II will stay as enigmatic as it has been since its detection about 20 years ago. Recently. thyroid hormone (Eales. Whether this result implies that not only the IGF-1R but also a functionally significant IGF-II receptor exist in bony fish remains to be clarified as also the potential presence of the receptor in adults. such as in gills and gonad. Gunthild Krey. Schmid and Natallia Shved Institute of Anatomy. Thus. This gives bony fish a quite unique state because in mammals GH most likely regulates only the expression of the IGF-I gene. Caroline Maake.. it seems necessary to take a closer look on the IGFBPs (Kelley et al. 2005). Sture Falkmer and Vicki Sara (Karolinska Institute in Stockholm. the potential existence of an IGF type 2 receptor has been reported for trout embryos (Gutiérrez et al. In this respect. their production in liver and other organs and their regulative role in systemic circulation as well as in different tissues. Annette C.. an important topic to be dealt with is the determination of IGF-II serum levels in different fish species and their potential changes either during development or with different physiological conditions such as the nutritional status and the environmental temperature. LLC . The widespread presence of IGF-II in liver and numerous other organs of both juvenile and adult fish contrasts the situation in mammals. this volume). There is some evidence that in bony fish not only the IGF-I gene but also the IGF-II gene are controlled by GH in all organs.. Otherwise. Switzerland). this volume). Elisabeth Katz. Dominique Loffing-Cueni.

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T. Srivastava.M. Zool.J. Somatotropic actions of the homologous growth hormone and prolactins in the euryhaline teleost.. J.T. T. G. Physiol. Insulin-like growth factors as mediators of functional plasticity in the adult brain. Comp. S. Gen.T. Insulin-like growth factor mRNA in Barramundi (Lates calcarifer): Alternative splicing and nonresponsiveness to growth hormone. 2004.M.. W.T. and Chan. Appearance of insulin-like growth factor mRNA in the liver and pyloric ceca of a teleost in response to exogenous growth hormone. 113:331-342. and Peter. Steeb. S.. 270: 263-272. 1994..C. Vong. Identification of a second insulin-like growth factor in a fish species. Endocrinol. 94:2068-2072. 17:859-868.H. Regulation of DNA-synthesis in goldfish ovarian follicles by hormones and growth factors.. T. DNA Cell Biol. Integr. I. Nishioka. Shepherd. Physiol.B. Byrialsen. 1995.. and McKeown.P Cheng. R. McCormick.. Trahair. R. Regul. Prolonged administration of IGF peptides enhances growth of gastrointestinal tissues in normal rats. C. Metab.G. Mol. Endocrine control of .. K. and Chen. Acad. Physiol. D.. Biochim. S. Q. M. Madsen. S. L. Taniguchi. Wagner.S. B.. S. Acta 1575:63-74. Biochem. A. N. Kajimura. Mar. Acad.. In vitro and in vivo effects of ghrelin on luteinizing hormone and growth hormone release in goldfish. Am. M. 1994. Zool. Aquaculture 157: 311-323. Proc. Cheng.D. LLC . T. Bern. Proc. A. Biophys. Tse.A. T. Shamblott. Comp.M. Changes in plasma glucose. J. Hirano. 266:G1090-1098. Res. T. and Nakashima. Sakaguchi. Physiol. Dickhoff. © 2006 by Taylor & Francis Group. USA. H... Proc. Torres-Aleman I. Uchida. Sara. Sakamoto. K. 1993.G. Sci. Sci.J.C. and Hoeben. B. T. K+-ATPase expression in osmoregulatory tissues of brown trout (Salmo trutta). Comp. Natl.. L. R. the tilapia. Hirano.. Biotechnol. Sweeting.. 1998. 11:299-303. J. Aida. Shearer. V. and Chen. M. Chen.E.K.R. Horm. Riley. P.G. 31:114-119.. Acad. Richman. 1999.. 37:69-93. Shamblott.. 92:6943-6946. 1992. Aquaculture 45:185-197. T. USA. Genet. and Van der Kraak. and Grau.D. cartilage in coho salmon: GH influence in vivo on the response to IGF-I in vitro. T. Tsai.J. 1997. Effects of fasting on growth hormone/insulin-like growth factor I axis in the tilapia. H. 89:8913-8917.. 1997. M. P Madsen.. Natl. Gene and cDNA structures of flounder insulin-like growth factor-I (IGF-I): multiple mRNA species encode a single short mature IGF-I. Shamblott. Sci. 1999. K. H. Oncorhynchus kisutch. Oreochromis mossambicus. F.F. and Bern. Biochem.S. Control of growth and adiposity of juvenile chinook salmon (Oncorhynchus tshawytscha).. S. PCR-cloning and gene .A. 2:351361.T.F. R. I. Tanaka. G.S.A. Unniappan. J.. K. amino acid nitrogen and growth hormone during smoltification and sweawater adaptation in coho salmon. expression studies in common carp (Cyprinus carpio) insulin-like growth factor-II. A 134:429-439.I. Bolt. Age-related and tissue-specific levels of five forms of insulin-like growth factor mRNA in a teleost. 1994. and Chen.. and Read.. Oreochromis mossambicus.. M. Silverstein. 2002..M. M.S. 1985. Tomas. 2003. Biol.W. Effects of insulin-like growth factor-I and cortisol on Na+. K. E.H. Yamamoto.Manfred Reinecke 129 Seidelin.. and Kristiansen. C.. 286:R1093-1101. Stahlbom. Am. T. K. Mori. E. K. 1999. USA. Madsen. and Grau.S. C.K. Yoshizato. Sci. Natl. J. Ohkubo. Exp..

. W. D. N. and Chen... Reprod. © 2006 by Taylor & Francis Group. insulin and estradiol with GnRH-stimulated luteinizing hormone release from female rat gonadotrophs. Effects of insulin-like growth factor-I on in vitro final oocyte maturation and ovarian steroidogenesis in striped bass. Vong.. J. Neuroendocrinology 79:100-108. and Cheng. Morone saxatilis. Quantification of common carp (Cyprinus . 53:127-134. 193-201. 1999. Q.F. 1993. Steiner. 2000.E...L. Hohmann.P Chan.. T. Elliott. and Davidson. Berry.. Interactions of insulin-like growth factor-I.Y. Polack.J.L. 262:420-425. Biol. Weil. .Z. J.. G. Wallace. Wilkinson. Francis. 2003. Walter.R. Xiao.130 Fish Endocrinology Unniappan. Urano.M. Exp. Chan. 134:285-295.. Venkatesan. K. 1997. Endocrinology 140:2054-2062. 1995. and Devlin. Mol. Canosa. and Ballard. D. Mol.L. and Logan. A.Y. Xia. Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity. 144:73-79. Breton..M. Gen. Oncorhynchus mykiss. Frankel. R.. C.P Bain. Eur.T.J. 1992. Differential effect of insulin-like growth factor I on in vitro gonadotropin (I and II) and growth hormone secretions in rainbow trout (Oncorhynchus mykiss) at different stages of the reproductive cycle.. 2001. O. D. Endocrinol. A.C. A. W.V. Carre. S. Comp. S. B. M.. Changes in growth hormone and prolactin messenger ribonucleic acid levels during seawater adaptation of amago salmon (Oncorhynchus rhodurus). Kobayashi. Zhang. 105:79-90. 7:409-422.. J. Endocrinol.M. Development and characterization of a competitive polyclonal antibody enzymeimmunoassay for salmon insulin-like growth factor-II.. R. S. Am. T. and Le Bail. Gen.X. R. J. O. S.. Reprod.E. Early embryonic expression of the growth hormone family protein genes in the developing rainbow trout. J.A. Yang. G. Dev. Adamson. carpio) IGF-I and IGF-II mRNA by real-time PCR: Differential regulation of expression by GH.J.J. Endocrinol.J. 1999. Endocrinol. T. Yada. Smith. Francis.. Zool. Hill.. Endocrinol... Cwyfan-Hughes. and responses to central and peripheral injections. M. Insulin-like growth factor I receptors in adult rat liver: characterization and in vivo regulation. 2004. M.... 2004. LLC . Comp. and Lin. Endocrinology 140:520-532. 63:1049-1057. Orexigenic actions of ghrelin in goldfish: feeding-induced changes in brain and gut mRNA expression and serum levels. M. Comp. 258:E329-337.H. B. 1999. Wallis. 178:513-521. Greene.R. R. 2003. F. Blaise. and Carragher. C.. J. Mantell. F..H. and Rombeau.F... J. L. F. A.T. Duplicate insulin-like growth factor-I genes in salmon display alternative splicing pathways. Weiss. Weber. C. G. Ziegler. Holly. and Peter. Y. Roth.J. Evolution of the insulin-like growth factor (IGF) function—production and characterization of recombinant hagfish IGF.. H. T. Biochem. Diedrich K. Transplantation 5:755-761. and Hirano. H. and Sullivan. Insulin-like growth factor-I improves mucosal structure and function in transplanted rat small intestine. J. Physiol. Physiol.B. A.M. and Ortmann. W. P. J. Upton. P. J. Cysteamine—A somatostatin-inhibiting agent-induced growth hormone secretion and growth acceleration in juvenile grass carp (Ctenopharyngodon idellus). T. B 139. 1990.

U. Navarro1. Av. Palmerston North. New Zealand.B. Castillo1. Diagonal 2 Sechenov Institute for Evolutionary Biochemistry and Physiology. Rojas1. 3 Equipe Croissance et Qualié de la chair des poissons SCRIBE. Rennes. ** Author for Correspondence. I. J. Leibush2 ABSTRACT Insulin and IGFs belong to a superfamily of peptides with an important role in growth. 4 Institute of Veterinary.S. INRA. J. Authors’ address: 1Departament de Fisiologia.CHAPTER 5 Insulin and IGF Receptors in Fish* J.C. Our knowledge of the role of these peptides in fish has improved in recent years. E-08028 Barcelona. University of Chicago. Russian Acadeny of Sciences. N. differentiation and metabolism in all vertebrates. Facultat de Biologia. Smith4. P. Universitat de Barcelona. Chicago. Private Bag 11222. 5 Departments of Biochemistry and Molecular Biology and Medicine and the Howard Hughes Medical Institute. Illinois 60637 U. Animal and Biomedical Sciences.V.A. Gutiérrez1**.J. Massey University. France. Planas1. Russia. LLC . S. Chystiakova2. and who stimulated all of us to continue work in this field. Chan5 and N. Gabillard3. St. J. * This chapter is dedicated to Dr Erika M Plisetskaya (School of Fisheries. A. Spain. which are still active and productive. E-mail: jgutierrez@ub. Petersburg.V. Erika was also decisive in establishing our scientific collaborations. Leibush the very first experiments in fish insulin receptors in Sechenov Institute. O. who shared with Dr Boris N. © 2006 by Taylor & Francis Group. Montserrat1. Washington).

but the available data suggests that it is a very different molecule.. Insulin is probably one of the most commonly studied peptide receptors because of its structure. Emdin et al. IGF-I. The last part deals with signal transduction. Insulin and IGF-I receptors in fish have been widely studied by various authors. which begins with phosphorylation of the receptor and continues with a description of the main molecules known in the transduction cascade in fish. signal transduction. Localization. Both receptors share a similar structure: heterotetramers with tyrosine kinase activity (TKA). The description of receptor function begins with the binding properties. b). Párrizas et al.. Gutiérrez et al. This is followed by the analysis of binding regulation in various physiological situations such as ontogeny. like the number of receptors. focusing later on lamprey insulin receptors. 1994a. seasonal changes and nutritional influences. Fish. Capilla et al. Fish IGF-I receptors (IGF-IR) were initially found in carp ovaries (Maestro et al. The chapter ends with a description of the state of the art and some suggestions for future research in the field.132 Fish Endocrinology The hormone receptor is the first stage of endocrine action and its presence and activity determine the effect of the hormone on any given tissue. LLC . Signal transduction. 1991a. There is little information on the IGF-II receptor. Fish insulin receptor (IR) has been not studied so extensively as its mammalian counterpart. INTRODUCTION Hormone receptors represent the first level of hormone action and receptor presence determines the tissue capacity of response to a certain hormone. Receptors. This was followed by numerous studies of receptor changes during alimentary treatment (Gutiérrez and Plisetskaya. Structure. in Myxine and trout suggested that insulin receptor function was maintained throughout vertebrates. © 2006 by Taylor & Francis Group. Key Words: Insulin. The part dealing with the structure of receptors covers both protein and gene characteristics. b. 1998. A section has been included on the main methods and techniques used in receptor research. The first studies by Muggeo et al. Baños et al. Here we shall review what is known about these receptors in fish. (1980) studied the binding affinity and biological activity of Myxine insulin.... (1983) performed the first study of diet effects on insulin receptor. their affinity and specificity. 1991.. 1993) and in brain of cyclostomes. the presence and abundance of receptors in different tissues is also examined. 2003. Binding. IGF-II. and does not have TKA. Gutiérrez et al. some of whom have contributed to this chapter. (1981) compared the binding properties of scorpion fish and rat insulin receptors. 1991. Leibush et al. (1979a. Abblet et al. binding. etc. b. 2004a).

Recent studies (Chan et al. Párrizas et al. J. b. Moon et al. The binding and protein structure of IR and IGF-IR in fish are now fully understood (reviewed in “Large Complex” 150 kDa “Small Complex” 40-50 kDa Proteases BP3 BP1 BP2 BP4 BP5 BP6 INS IGF-I IGF-II INSR IGF-IR IGF-IR Fig. These studies encouraged research into the IGF-II receptor (IFG-IIR) and. 1999a.. 1997.. which is much more complicated than the receptors themselves. © 2006 by Taylor & Francis Group.. (1997a. Ayaso et al. Figure 5.. obtain the first data on this in fish (Méndez et al. in doing so. 133 elasmobranches and teleost (Drakenberg et al. (1995) described the abundant IGF-IR in fish skeletal muscle and heart. Greene et al.Gutiérrez. 2003c) demonstrate the existence of two IGF-IRs in different species although their respective function are still unknown.. (1996) studied IGF-I and insulin binding in trout cardiomyocytes while Castillo et al. Maestro et al. b. (1995) and Gutiérrez et al. Gabillard et al.1 summarizes the insulin and IGF-I system. 5. 1999) characterized IGF-IR structure and binding changes through the reproductive stage of carp and salmonids.1 Diagram of Insulin. IGF-I and IGF-II system that includes peptides. 2001a). 2002. Maestro et al. (2002) characterized them in cultured trout myocytes at different stages of development. and various binding proteins. et al. (1998) studied the changes of IGF-I receptor through trout larval development. LLC . heterotetramer receptors (insulin and IGF-I) and mannose phosphate IGF-II receptor. 1993)..

This protocol using the WGA as affinity chromatography has been used for semi-purification of receptors in skeletal muscle and heart of teleost fish (Párrizas et al. (1993) described the procedure to isolate and incubate hepatocytes for binding assays. Maestro et al. adipose. Affinity chromatography. The objective of this chapter is to review the information presently available on insulin. using mannose 6-phosphate M6-P bound to agarose to semi-purify IGF-II/M6-P receptors in trout. the procedure involves homogenization in cold buffer with enzyme inhibitors.134 Fish Endocrinology Leibush et al. (2001a). 1999). and with some modifications for semipurification of receptors from brain. subsequent centrifugation and finally re-suspension of the pellet in binding buffer. (1991) and Párrizas et al. b). As described by Leibush et al. b) for liver membrane preparation... (1996) described the © 2006 by Taylor & Francis Group. followed by filtration.2). 1998. Navarro et al.. Cell Isolation Plisetskaya et al. (Leibush et al. (1994a) (Fig.. LLC . METHODS AND TECHNIQUES USED Preparation of Receptors Plasma Membrane Preparation For most radioreceptor applications such as binding assays. 41% and 37% sucrose) and ultracentrifuged to obtain a higher level of membrane purification (Gutiérrez and Plisetskaya. etc. while less information is available on receptor physiological regulation and gene structure. IGF-II is still a relatively unknown molecule in poikilotherms in terms of its structure and function. 1997a.. 5. 1995). Semi-purification of receptors To prepare partially purified receptors. 2000a). The pellet can also be diluted in sucrose (upto 31% sucrose concentration) and subjected to a discontinuous sucrose gradient (45%. and Moon et al. ovary. (1981) and Gutiérrez and Plisestkaya (1991a. 1991a.. the proteins can be solubilized with triton and then purified using wheat germ agglutinin (WGA). 1994a. Gutiérrez et al. Planas et al.. 1996. membranes that are crudely or partially purified have proved satisfactory. as described by Gutiérrez et al. IGF-I and IGF-II receptors in fish. More studies are needed of insulin and IGFs receptors’ signal transduction pathways in fish. has been described by Méndez et al. b.

4°C) Affinity Chromatography 135 Mannose 6-Phosphate (M6-P)-Agarose Wheat Germ agglutinin (WGA)-Agarose Elution IR IGF-IR Elution IGF-IIR IGF-IIR Purified IFG-II/M6-P receptors Semipurified receptor preparations Fig. To check this. 20 min.. especially in isolated cells.2 Diagram of receptor purification of fish samples. do not interfere with results. et al. Wheat Germ Agglutinin (WGA) agarose leads to semi-purified receptor preparations. 4°C) Solubilization (Triton X-100) Centrifugation (150 000xg. which binds to the receptor but not to the binding proteins. 90min. J.Gutiérrez. 1994a. 5. It is important for any study on IGF binding. include slowspeed centrifugation steps and subsequent cell treatments prior to binding assays. ultracentrifugation provides a supernatant that is subjected to affinity chromatography. to be sure that contaminating IGF binding proteins. LLC . after mechanical and enzyme dispersion and tissue desegregation. all these procedures. 30-40 l (20 g glycoprotein) of semipurified receptors (Párrizas et al. Mannose 6-Phosphate (M6-P) agarose gives IGF-II receptors. was used (Maestro et al. In short. Whole Embryo Homogenization Centrifugation (40 000xg. which also bind IGF-I. b) from the eluted fraction of the WGA-column (the same as when using M6-P bound to agarose in IGF-II binding assays) are incubated at © 2006 by Taylor & Francis Group. After homogenization and solubilization with Triton.. Binding Assay The performance of the radioreceptor assay differs when it works on solubilized receptors and when it works on crude membrane preparations. 1998). des (1-3)-IGF-I. procedure for trout cardiomyocytes.

Binding assays with membrane preparations (Gutiérrez and Plisetskaya. 1991a. (c) hybridization of probe (which depends on the specificity of the probe designed). Incubation was terminated by removing the total volume of each well. Approximately 150 g/50 l of membrane are incubated in the presence of the radioactive and cold ligands. and radioactivity was counted. Binding to cells fixed on plates requires specific adaptation (Castillo et al.125I. various groups have studied the expression of mRNAs of insulin and insulin-like growth factor system by in situ hybridization. labeled probe is used (32P35S. Incubation was stopped by aspiration of medium. (b) fixation and pre-treatment of tissue (aldehyde fixatives are the most commonly used for both whole mounts and tissue sections).5 to 2 106 per well. If radioactivity. The data on the specific binding are analyzed according to Scatchard (1949). (e) incubation in antibody against the probe. In teleost fish.3% -globulin and 25% polyethylene glycol (45 minutes) and then centrifuged.3H). Binding in cardiomyocytes. The pellet is then washed and counted. To stop the reaction. © 2006 by Taylor & Francis Group. the process of in situ hybridization to whole embryos or sections involves several steps. the monolayers were washed. Binding studies are conducted with cells seeded at a density of 1. several authors used in situ hybridization for the detection of peptide and receptor mRNAs in insulin and insulin-like growth system. In the last decade. monolayers were washed and incubated for 12 h at 4°C with 125Ilabeled IGF-I or insulin in the presence of a range of increasing concentrations of cold peptides. 2002). In each well.136 Fish Endocrinology 4°C overnight in presence of cold insulin or IGF-I and with radioactive ligands (25pM). Unbound hormone is removed from a pellet obtained after slow-speed centrifugation and the radioactivity of the washed pellet is counted. and (g) visualization of the bound antibody under microscopy. “In Situ” Hybridization In general. cells were triturated by incubation with 1 ml 1 N NaOH for 30 min at 40°C. samples are precipitated by bovine 0. LLC . centrifuging and counting the radioactivity in the pellet.. (1996) was performed overnight at 4°C with shaking in 24-well plastic culture plates. b) are also performed overnight at 4°C. (f) washing of the unbound antibody. the treatment of the sample is the same. including: (a) synthesis of labeled anti-sense and sense RNA probes(s) (in recent years by non-radioactive labels such as digoxigenin). (d) washing to remove the unbound probe. described by Moon et al.

in vitro autoradiography of receptor-bound radiolabeled hormones has the advantage of both confirming receptor protein translation and allowing for various receptor pharmacology studies. autoradiography provides higher-resolution receptor localization. saturation. 1994). (2000) studied insulin-like growth factors and their ligands in gonads of gilthead seabream by random priming labeling of the probes with [32P]dCTP Schmid et al. (1997) studied the ontogeny of IGF-I mRNA during embryonic and larval development of the gilthead seabream. Likewise. the binding characteristics and reversibility of receptor binding (e. the expression of IGF-IR in the ontogeny of zebra fish was studied by Ayaso et al. and IGF2 in tilapia ovary using probes labeled with digoxigenin. and binding capacity. When studying fish larvae. Caelers et al. and finally (4) rapidly dried and exposed to film or photographic emulsion. et al. Hitchcock et al. Autoradiography In vitro autoradiography is a well-established technique for locating a great many receptor types. Kd. (2003) studied the expression of IGF-II in tilapia brain using similar procedures. affinity. 137 Funkenstein et al. the mRNA message for the receptor can be located by in situ hybridization.g. in vitro studies on tissue sections add significantly greater information on binding site © 2006 by Taylor & Francis Group. LLC .Gutiérrez. the treatment of the tissue such as the fixation and pre-treatment of the sample has been modified (Westerfield.. (1999) performed in situ hybridization for IGF1 . In this first approach. (2002). Sections are then (3) washed in cold buffer to remove any unbound ligand and to reduce dissociation of bound radioligand. Bmax) cannot be studied. but this fails to confirm either the presence of protein product or receptor binding characteristics. With the provision of appropriate controls. competitive inhibition and reversibility studies. et al. Although traditional ‘grind and bind’ receptor-binding studies can provide this information. A typical protocol for in vitro autoradiography on tissue sections involves (1) pre-incubation to remove endogenous bound ligand followed by (2) incubation in buffer containing radiolabeled ligand. In addition. This protocol can be variously adapted to include time-course. Although antibody staining can be used to the same effect. immunostaining is only semi-quantitative and lacks high resolution. (2001) performed in situ hybridization for insulin receptor in goldfish retina. Recently. without the various adaptors required for immunostaining. the probe was labeled with radioactivity [35S]UTP Perrot . Thus. J.

in reducing conditions and heating for 5 min at 95°C. Phosphorylation was started by adding 45 M ( -32P)ATP (12 Ci) and was stopped after 10 min at room temperature by addition of one-third of the total volume of LSBx with DDT. in presence of 100 M MgCl2 and 40 mM MnCl2 at 4°C overnight. semipurified receptors (8-12 g glycoprotein) were incubated with increasing concentrations of insulin and IGF-I (0.CMYK 138 Fish Endocrinology location. the versatility of in vitro autoradiography lends itself very well to studies of IGFRs in fish. such studies can even permit differences in binding characteristics of sub-populations of receptors to be examined. (1994a). blue = low binding. Further research in the future using this technique will undoubtedly add greatly to our knowledge of IGF action in teleosts (Fig. even down to the identification of specific cell types expressing particular receptors.6. in the manner described by Párrizas et al. 5. mu sk br cr ga Fig. skin (sk) and muscle (mu). all of which are very actively growing tissues. green = background. pH 7. Combined with various characterization experiments. 5. (1997b). 2005). very few studies have examined IGF-I receptor binding sites in the teleost using this approach (Boucher and Hitchcock. Red = highest binding.3). Tyrosine Kinase Activity (TKA) was determined. orange = intermediate binding. Briefly. Highest binding is seen in gill arches (ga). Smith et al..03 nM to 3 M) in Hepes. 5. brain (br). To date. 1998. Autophosphorylation and Tyrosine Kinase Activity The procedure has been described in Maestro et al. Scale bar = 500 m. Phosphorylated subunit of receptors was separated by SDS-PAGE and phosphorylated subunit was detected by autoradiography (Fig. However. Receptor glycoproteins (8-12 g) were © 2006 by Taylor & Francis Group. cranium (cr).4).3 Pseudocolor image of 1251-IGF-I binding sites in sagittal section of 85-days-old trout larva. LLC .

5. Receptor stimulation caused a dose dependent effect.6 518. air-dried.7 ± 37. 1997b). et al.4 106) cpm) for 10 minutes to allow autophosphorylation. (Modified from Maestro et al.1). Data are mean ± standard error (n = 4).1 Stimulation of tyrosine kinase activity by insulin and IGF-I in skeletal muscle from sea bream and rat (A) Results are expressed as radioactivity incorporated in the substrate: % of phosphorylation above the basal levels.4 Autophosphorylation of insulin and IGF-I semi-purified receptor from carp ovary under reducing conditions. pH 7. without adding hormone. the reaction was stopped by transferring samples to filter paper squares (Whatmann 3MM: Whatmann Clifton.01 Rat 937. and counted in a scintillation counter.3 ± 39.2nM Insulin 4nM 20nM 20nM 0 139 IGF-I 0.6 ± 24. A Insulin IGF-I B Insulin IGF-I Seabream 283. Results were expressed either as radioactivity incorporated into the substrate or as 32P transferred to the substrate per fmol of receptor (Table 5.023 ± 0.83 ± 2.4 305..3 Seabream 0.Gutiérrez. Carp ovary 0. J.02 0. after further incubation for 30 minutes.83 © 2006 by Taylor & Francis Group. pre-incubated for 16 h at 4°C with increasing insulin and IGF-I concentrations in Hepes buffer containing 100 mM MgCl2.25 mg/ ml and. Receptors were incubated with 50 M (32P)ATP (2. (B) Results are expressed in pmol of phosphorus incorporated to fmol of receptors.068 ± 0.2n M4nM 20nM 20nM 97.4 kD Fig. Fish preparations were tested for the presence of free phosphorus to assess whether differences in TKA were due to Table 5.4 (final volume 70 l). LLC . NJ) and shaking them in 10% trichloroacetic acid containing 10 mM sodium pyrophosphate. Paper squares were washed at least five times over a 2-3h period.1 ± 118 Rat 2733 ± 1 47. Synthetic substrate poly (Glu:Tyr 4:1) was added to a final concentration of 0.

LLC . Samples were heated at 95°C for 5 min and subjected to SDS-PAGE. 1994a).. Reaction was stopped by adding one third of total volume of Laemllli sample buffer at triple strength. (Modified from Maestro et al. Values of free phosphorus were very low and had no effects on TKA results. Wells were washed with cold PBS. T indicates total binding and NS. Western Blot of Signal Transduction Molecules Western Blot experiments on rainbow trout muscle cells in a primary culture followed Castillo (2006): the culture medium was replaced with DMEM + 0.5% BSA and peptides for 30 minutes. Cross-linking Cross-linking experiments for insulin and IGF-I receptors are described in Maestro et al.5). Samples were then incubated with 2 mM DSS (the cross-linker) in ice for 15 min.5% BSA for 2-3 hours and incubated with DMEM + 0. with or without insulin or IGF-I and labeled hormone (200.000 cpm) in 50mM Hepes. semi-purified receptors (60-100 g of glycoprotein) were incubated overnight at 4°C. (1997b). in presence of added unlabeled insulin or IGF-I. Labeled hormones linked to their receptors were detected by autoradiography (Fig. pH 7. and © 2006 by Taylor & Francis Group. to see the whole receptor molecule. or without it. Carp ovary Rat Ctrl Insulin T NS T IGF-I NS 205 kD 121 kD 86 kD Fig. non-specific.140 Fish Endocrinology adenosintriphosphatase (ATPase) activity (Párrizas et al..6. 1997b). 5. In short. Samples were run with reduction to visualize the subunit.5 Affinity cross-linking of 125 I-Insulin and 125 I-IGF-I to WGA receptor preparation of carp ovaries under reducing conditions. 5.

This finding was not corroborated subsequently.. Each receptor consists of two and two subunits linked by disulfide bonds to form an heterotetramer.5). 141 the cells were lysed and subjected to electrophoresis (SDS-PAGE). IR and IGF-IR subunits showed bands of 125 and 120 kDa. Lappova and Leibush. The subunits include ligand-binding sites. By means of cross-linking experiments in carp ovaries. 1995. respectively (Fig. 1997b). Stuart (1988) suggested that the subunit was not cleaved from the subunit in the elasmobranch sting ray. In an early study. Ligand binding causes tyrosine kinase stimulation. Samples were transferred to a PVDF membrane for 90 minutes. 1993. The membrane was incubated overnight at 4°C with the primary antibody (AKT and MAPK from Cell Signaling Tec. The subunit of the mammalian IR receptor has a molecular weight of 90-95 kDa. However. which is started by receptor autophosphorylation. Lapova and Leibush (1995) found in liver lamprey an subunit of 130 kDa. Inc. Immunoreactive bands were viewed by ECL. whereas the subunits contain tyrosine kinase activity.) diluted in a washing buffer. 1995) resulted in total molecular weight for both © 2006 by Taylor & Francis Group. which are members of the tyrosine kinase receptor super-family. 5.. The subunit of the insulin receptor (IR) has a MW of 130-135 kDa in birds and mammals and is located entirely extracellularly.. This lower molecular weight corroborates the findings of Drakenberg et al. STRUCTURE AND LOCALIZATION Structure of Insulin and IGF-I Receptors IR and IGF-IR are heterotetrameric glycoproteins of 350 kDa. (1993) in other fish species and may be due to differences in glycosylation.. Rather. et al. LLC .4) (Drakenberg et al. J. Membrane was washed for 30 minutes and incubated for one hour at room temperature with the corresponding secondary antibody and at indicated dilutions. and quantified with an image analyzer. It contains a transmembrane region and an intracellular domain with high homology to numerous protein kinases. 1985). autophosphorylation experiments revealed that the subunit in fish IR and IGF-IR had a similar molecular weight (97 kDa) and structure to that in mammals (Fig. 5. cross-linking experiments in non-reducing conditions in fish heart (Gutiérrez et al. Maestro et al. It is responsible for tyrosine kinase activity (Ullrich et al.Gutiérrez. Furthermore.

. identified as the cation-independent mannose-6-phosphate (M6-P) receptor. In addition. In several fish species.. Méndez et al. Ayaso et al. (Modified from Méndez et al. Structure of IGF-II Receptor The type-II IGF receptor (IGF-IIR). since IGF-II binding could not be completely displaced by either IGF-I or insulin in any of the receptor preparations tested (WGA semi-purified receptors or M6-P affinity column purified receptors). Through immunoprecipitation of cross-linked receptor with an antibody to rat IGF-II receptor. 1997. 5. This receptor binds IGF-II and IGF-I with high affinity (0. 2001a). Total bound to subunit (T) is indicated for trout and rat. Since IGFI and insulin were unable to displace the bound labeled IGF-II.6 Affinity cross-linking of 125I-IGF-II to semi-purified receptors (WGA) of 9 weekold trout embryos under reducing conditions. b.142 Fish Endocrinology receptors close to 350 kDa. Gabillard et al. (2001a) detected the presence of specific IGF-II binding in fish embryos. as it is a single-chain glycoprotein of approximately 250-270 kDa. © 2006 by Taylor & Francis Group. Non-specific binding was obtained by adding unlabeled IGF-II. 1999a. 5.. but does not bind insulin. but these differences are not detected by cross-linking and autophosphorylation techniques.6).. two types of IGF-IR(IGF-IRa and IGF-IRb) are described by the genetic approach (Chan et al. the cross-linking experiments demonstrated that labeled IGF-II bound to a protein of molecular weight~250 kDa (Fig. is structurally different to the IGF-I and IR receptor.. All these findings suggest that the protein structure of IR and IGF-IR has been very well conserved throughout vertebrate evolution. it suggests that this protein represents the IGF-II/M6-P receptor. 2003c).12-013nM). LLC . IGF-I and insulin (Ins). 2002. Greene et al. only one protein of molecular weight ~250 kDa was detected Trout larvae T IGF-II IGF-I Ins Rat T 209 kDa IGF-IIR 124 kDa a-sub IGF-IR Fig.

7 Immuno-precipitation of IGF-II receptors.7). Greene and Chen. turbot... Gabillard et al. is blank (immuno-precipitation of receptors without antibody). 1999. (Modified from Méndez et al. b.. such as rainbow trout. 1999a. 2003c). seabream and zebrafish (Elies et al. potential ATP and © 2006 by Taylor & Francis Group. Trout larvae T NS BI Rat T 143 209 kDa IGF-IIR 124 kDa 80 kDa Fig. 2001a).. 1996. 5. IR and IGF-I receptor genes have been fully or partially cloned from several teleost species. 1997. cross-linking and immuno-precipitation suggest that only a small percentage of the total IGF-II binding found in the WGA preparations. Chan et al.. coho salmon. the subunit contains N-glycosylation sites and cysteine-rich domains which are important for hormone binding. et al. 2002. Common to all IR and IGF-IRs. Labeled IGF-II was cross-linked to IGF-II receptor and immuno-precipitated with monoclonal antibody against IGF-II receptor. LLC . similar in size to its mammalian counterpart. 1997. The subunit contains a transmembrane domain. a highly conserved tyrosine kinase domain. 2002.. T is total 125I-IGF-II bound to the IGF-II receptors that could be immuno-precipitated. However. which constitutes further evidence of the presence of a type-II IGF receptor in fish that binds specifically IGF-II. corresponds to an IGF-II/M6-P receptor. Funkenstein et al..Gutiérrez. 5. flounder. This band could be completely displaced when an excess of IGF-II was added to the sample prior to immuno-precipitation. The deduced primary sequences of teleost IR and IGF-IRs correspond to proteins containing and subunits. Molecular Characterization of Insulin and IGF-I Receptors To date. (Fig. Ayaso et al. Maures et al.. Nakao et al. separated by a tetrabasic proteolytic cleavage site (R-X-R-R). 2002. data on binding. Bl. Most of this binding probably corresponds to the binding of IGF-II to the IGF-IR. NS is non specific binding (by adding unlabeled IGF-II before immuno-precipitation). J.

There appear to be several forms of IR and IGF-IRs in teleosts. 1997.... the primary sequences of teleost IR and IGF-IRs differ significantly from each other and form two clearly defined groups. and also differ in their mRNA sizes: 13 and 8 kb.. such as zebrafish and flounder. such as rainbow trout and coho salmon. Despite these common structural characteristics. which are the only two species in which the full sequence is known (Maures et al. 1999b). Greene and Chen. In flounder. Nakao et al. 1999b). and teleost IGF-IRs are more like mammalian IGF-IRs (63-88% similarity) than IRs (51-78% similarity) (Chan et al. These differences between the two IRs suggest that they may be encoded by different genes.. Non-salmonid teleosts.. Greene and Chen. In fact. Nakao et al. based on partial cDNA sequences (Chan et al. which in flounder have only 73% similarity to each other. If they do. The number of IR receptor isoforms found varies among the species studied. respectively. salmonids may have duplicated their IR genes. which correspond to three different genes.. comparison of the tyrosine kinase domain between the two IGF-IRs yields relatively high homology (approx. Maures et al. teleost IRs are more like mammalian IRs (65-85% similarity) than IGF-IRs (55-74% similarity). 85%) (Chan et al. Another interesting aspect is © 2006 by Taylor & Francis Group. c). the existence of two separate genes coding for IGF-IRs has been demonstrated in rainbow trout by Southern blotting (Greene and Chen. 2002). which share only 69% and 67% similarity to each other in zebrafish and flounder... 1999a). In contrast. 2002. have more than two isoforms. 1997. LLC . unlike in mammals. a fourth IR gene may have gone undetected in rainbow trout.. However. It will be important to confirm whether other diploid fish species contain two different IGF-IR genes. 2002. 1997. 1997). If this is so. For IGF-IR. 1997. In coho salmon. 1999a).144 Fish Endocrinology IRS-1 binding sites and an autophosphorylation site. 1999a. 2002). b. as the tetraploid salmonids do.. salmonids. 2002. Due to their tetraploid nature. Rainbow trout has three different forms of the IR. 2002). Funkenstein et al. have two different forms of IRs (Maures et al.. the two IGF-IR forms also differ in their mRNA sizes: 11 and 9 kb (Nakao et al.. it appears that all teleost species examined to date have two different forms. bringing their total number up to four. 2003b. 2002. as confirmed by Southern blotting (Greene and Chen.. 1999. the IGF-IR gene might have duplicated during teleost evolution. Greene and Chen. Gabillard et al. four different partial cDNAs corresponding to different isoforms of the IR were detected by RTPCR (Chan et al. Ayaso et al. since no complete sequence is available.. Elies et al. In salmonids. Nakao et al. 2002.

1979a. 1998). Myxine... 1995).. (1983) to the most recent ones by Capilla et al. although morphologically fish red muscle is a muscle. In this respect.. muscle. Leibush et al. because there is increasing evidence that the two genes are not redundant but may have distinct functions.. despite having a single insulin and IGF-I gene. 1996). as well as cardiac. where similar binding properties to those in higher vertebrates were found (Muggeo et al. From the initial studies of Ablett et al. 1995) suggests that the duplication of the receptor genes took place before the appearance of the chondrichthians.Gutiérrez. IRs were also described in fish ovary. it will be important to investigate the presence of separate IR and IGF-IR genes in the early vertebrates: cyclostomes and chondrichtians. even higher than white muscle does (Gutiérrez et al. metabolically it resembles the liver. b) confirmed that piscine liver possesses significant insulin binding.. quantitatively. 145 the different role of both IGF-IRs.. Ablett et al. The known presence of separate insulin and IGF-I genes in sharks (Duguay et al. Interestingly. Similarly. Braekkan (1956) and Wittenberger (1967) reported that. Planas et al. In evolutionary terms. To establish when the duplication of the ancestral gene took place. Gutiérrez and Plisetskaya 1991a. 1993.. is the most abundant tissue in fish. red muscle shows higher insulin binding than white skeletal muscle (Baños et al. LLC . showing in each case differences in binding characteristics. 1981. J. Furthermore. heart muscle displays significant IR binding.. 2000a). which are thought to evolve from the duplication of an ancestral common gene such as that found in protochordates (Pashmforoush et al. various studies (Leibush et al. which probably indicates the particular need of the heart to ensure a constant supply of metabolic fuels. though not in brain. Seasonal changes were also found in insulin binding in ovary and adipose tissue (see below). (2004a) IRs have been characterized in both red and white skeletal. Overall. with separate genes for IR and IGF-IRs. b). adipose tissue and brain (Gutiérrez et al. 1996. et al. the two different teleost IR and IGF-IR genes represent the first known genes in the vertebrate lineage. In mammals. the liver is a major target tissue for insulin. Localization of Insulin and IGF Receptors The earliest studies of insulin receptors in lower vertebrates began with red blood cells of the hagfish.. © 2006 by Taylor & Francis Group. Muscle is also a major target tissue for insulin and. it is fairly clear that all diploid fish have two distinct IR and IGFIR genes. 1983. it is worth mentioning that many years ago.

125I-IGF-II binding is localized in the granular layer. High level of specific binding of 125IIGF-I was also localized in gill arches and cranial cartilage of trout fry (Smith and Gutiérrez. Funkenstein et al. Boucher and Hitchcock (1998a) localized specific 125I-IGF-I binding sites in the goldfish retina. By means of autoradiographic techniques. Autoradiographic localization studies by Smith et al. swimbladder and the brain of seabream during ontogeny. cranial cartilage and gill arches of 16-day seabream larvae.. b) later described its abundance in muscles of various salmonid species. we found significantly higher IGFI binding than insulin binding. Both testes and ovary of adult seabream also show IGF-IR immunoreactivity (Perrot et al. (1999) localized IGF-IR immuno-reactivity in skin. and ear progenitors in early larval stages of zebrafish. from typical growth factor © 2006 by Taylor & Francis Group. Drakenberg et al. Párrizas et al. (1996) subsequently detected IGF-I binding in trout testis. (1993) demonstrated IGF-I binding in brain of cyclostomes. 2003). particularly in those areas that have the most active growth throughout life (such as the cerebellum and optic tectum).) and in different species. (2001a) found maximum levels of IGF-II binding in trout larvae at 5 weeks post fertilization during organogenesis. (1995a. Le Gac et al.. unpublished). (1991) in carp ovaries. Similar patterns of IGF-IR immuno-reactivity were also found in Shi drum (Umbrina cirrosa. while 125I-IGF-I binding is found in the molecular layer. kidney. In all tissues studied (including different muscle types.146 Fish Endocrinology IGF-I binding in fish was first reported by Maestro et al. 2000). elasmobranchs and teleosts. Perciformes) during early development (Radaelli et al. LLC . etc. Ayaso et al. where IGFI has been shown to stimulate proliferation of retinal progenitor cells (Boucher and Hitchcock. (2004) showed the presence of specific IGF-I binding sites and immuno-reactivity in various regions of the adult trout brain. ovaries. adipose tissue. brain. eye. (2002) localized IGF-IR mRNA expression in tail. 1998b). olfactory epithelium. with quite different binding sites. (1999) in turbot by in situ hybridization. while Cheng and Wu (2002) localized IGF-IR mRNA expression even earlier during zebrafish embryogenesis and Elies et al. Smith and Gutiérrez unpublished) found that in adult trout cerebellum. The presence of insulin and especially IGF-IRs in so many fish tissues appears to reflect a wide functional diversity. Studies of the piscine IGF-II receptor are scarce although Méndez et al. Perrot et al. intestine. (1997) demonstrated by in situ hybridization the presence of IGF-IR mRNA in chondrocytes. pancreas.

Insulin or IGF-I binding stimulates tyrosine autophosphorylation on subunits. Binding Studies The Schatchard curve has been the most common technique for characterizing binding properties (reviewed in Gutiérrez and Plisetskaya. MAPK. LLC . AKT etc (Le Roith et al. etc. 5.8 Signal trasduction cascade from the activated insulin and IGF-I receptor. the ligand binds to the subunit. et al. Their activation determines the final function of the receptor (Fig. After autophosphorylation..) and leads to the activation of pathways such as MAPKinase and PI3kinase. an important series of molecules are activated: IRSI. PI3K. osmoregulation. 1994). The specificity of this binding is also important.8). 2001). FUNCTION OF RECEPTORS To begin its action. Insulin IGF-I 147 INS-R PATP p85 IGF-IR RAS GTP RAF IRS m808 GRB1 RAS GDP PI3K p110 PDK AKT MEK MAPKK ERK MAPK Differentiation Metabolism Proliferation Fig. The characteristic of this binding is determined by the number of receptors and the affinity of the receptor to the ligand. the © 2006 by Taylor & Francis Group. J.Gutiérrez. actions to regulation of steroidogenesis. As binding determines receptor autophosphorylation. Schatchard calculation provides specific binding (Bsp). p85. its capacity varies and causes several grades of receptor response. 5. proliferation and differentiation. metabolism. which activates IRS molecules that recruit other proteins (GBRs.

The number of receptors can vary substantially as a function of the species. Within fish species. LLC . The specificity of the binding.10) (Párrizas et al. stage of development or physiological condition. (1998) described the same situation in © 2006 by Taylor & Francis Group. 2000a. 5. 1994a. a greater amount of cold insulin was needed to displace labeled bound IGF-I. Figs. The number and affinity of receptors determine the capacity of a certain tissue to respond to a specific ligand.5 nM (Kd). Another interesting feature of fish muscle is that IGF-IRs are more abundant.. is particularly important.000 fmol/mg. there are also differences in Ro. The greater presence of IGF-IRs suggests certain evolutionary changes in the role of IRs and IGF-IRs in muscle... 1997a. The highest binding value is obtained when the tissue possesses many binding sites with high affinity. tissue. Planas et al. 1995a). Maestro et al.. dissociation constant). 2002).20.. 1996. Study of IR in fish white skeletal muscle shows at once much lower binding than in mammal or bird muscle. in all fish species. brain (Gutiérrez et al. 2000b). b). and salmonids show the lowest levels (between 40 and 90 fmol/mg). Párrizas et al. 1991a. 1999). Leibush et al.148 Fish Endocrinology number of binding sites (Ro. 1995. heart. In fact. than IRs (between 150 and 770 fmol/mg. with insulin playing a more important role in regulation of muscle metabolism in birds and mammals than it seems to in poikiloterms (Planas et al. Leibush et al. It has already been mentioned that the binding specificity of IRs and IGF-IRs should be taken into account. Castillo et al.9.. while seabream and carp have higher levels (200-400 fmol/mg) (Navarro et al. which gives information about the selectivity of the receptor for its own ligand (see below). Changes of receptor affinity in the same tissue are not often observed. rather than cold IGF-I to displace labeled bound insulin. while in mammals the Ro are around 1. In an early study of salmonid skeletal muscle. This condition was later observed in various fish tissues: ovaries. Muscle in fish contains between 40-300 fmol/mg (Párrizas et al.. These differences between fish species were attributed partially to dietary habits (Párrizas et al. b). point where the slope line intercepts the x axis) and the affinity of the receptor (Kd. The same applies to heart muscle (Gutiérrez et al. see below).. Liver IRs in fish also have a lower binding capacity than in mammals (Gutiérrez and Plisetskaya. Affinity values for muscle IR are around 0. 1995). (1995) reported that the difference between the two receptors was that IGF-IR discriminated better between IGF-I and insulin than IR did.. 1995a... 1994b. 5. b.

IGF-IIR performs its function throughout IGF-IR. lamprey brain. while IGF-II has no TKA and its function. seems related to elimination of excess IGF-II... 5. Párrizas et al. (Modified from Planas et al. Insulin rise due to arginine injection or alimentary conditions (Gutiérrez et al.Gutiérrez. is subjected to large increases of a determined peptide. A possible pattern of these © 2006 by Taylor & Francis Group.. 2001a).9 Insulin and IGF-I binding in skeletal muscle of adult specimens of different fish species. 20 18 IGF-I Insulin 149 % Bsp/20 mg glycoprotein 16 14 12 10 8 6 4 2 0 Trout Sea bream Sea bass Carp Fig. under in vitro conditions. et al. (1998) pointed out that at the early stage of vertebrate evolution. while IR distinguished them poorly. (2000a) found the same up-regulatory response in adipose tissue of trout injected with arginine. IGF-IR was already highly specific in distinguishing the two related ligands (IGF-I and insulin). Later Planas et al.. A different situation appears for IGF-I and IGF-II binding. J. these terms are applied to experimental conditions in which the whole animal or a specific tissue. IGF-IR binds IGF-I and IGF-II with similar specificity (Méndez et al. 1991. In general. 1994a) provoked a rise in muscle IRs. LLC . 2000b). These authors suggested an asymmetric functional evolution of insulin and IGF-IR after their divergence from their common precursor.and down-regulation refers to increases and decreases in the number of receptors in the membrane. Leibush et al. Binding Regulation Up. In fact. at least in mammals.

1991b.. but to react by an increase in receptor numbers in response to a rise in blood insulin. These authors pointed out that in lamprey hepatocytes. treated with physiological insulin concentrations and at different temperatures (4-25°C). rapid down-regulation of IRs. 1994b). in vivo experiments in red muscles of carp and trout showed that hyperinsulinemia decreased insulin binding in both species by about 50% (Baños et al. but the © 2006 by Taylor & Francis Group. 1996).01) differences between groups. These results show the presence of regulatory mechanisms of IRs and IGF-IRs by their respective ligands. low temperatures did not constrain a process of internalization or down-regulation of IRs. as it did in homeotherms. tissues was to have few receptors in the membrane.. 1993). Although in vivo increases of insulin in trout and carp provoked upregulation of IR in liver (Gutiérrez and Plisetskaya. (Modified from Méndez et al. Similarly. In fact. This response seems to be characteristic of fish white skeletal muscle and adipose tissue. Párrizas et al... Comparison between larvae. Data are mean ± SE and different letters indicate significant (p<0.10 IGF-I binding in whole larvae or skeletal muscle of brown trout.. respectively (Moon et al.150 Fish Endocrinology IGF-I Binding 80 a % Bsp/20 mg glycoprotein Insulin Binding 6 5 4 3 2 1 0 ar ye 2 1/ 2 ks ks th s ar ye a a % Bsp/20 mg glycoprotein 70 60 50 40 30 20 10 0 ks ks th s ar ye ee ee ye ar on w w 1/ 2 1/ 2 m 5 12 b c c c c c c ee ee w w on 6 m 5 12 6 1 Fig. juvenile and adult fish sampled on the same day and under the same conditions. 1997). 2001b). This decrease was due to a decrease in the number of receptors while affinity did not change. Leibush and Lappova (1995) found in lamprey hepatocytes. in vitro studies of coho salmon hepatocytes showed IR downregulation when high levels of insulin were added to the culture medium (Plisetskaya et al. LLC 2 1 1/ 2 . specific binding of insulin or IGF-I in trout cardiomyocites decreased after incubation with high concentrations of insulin or IGF-I. 5.

Moreover. 2002). increasing rapidly to a maximum at the eyed egg stage (68. 1997.. In addition.10). IGF-IR binding showed a similar pattern to that found © 2006 by Taylor & Francis Group. Messenger for IGF-IR was found in all embryonic stages in rainbow trout (Greene et al. The whole picture confirms the importance of IGF-I in growth and development.. 1999) and turbot (Elies et al.. 1996. seabream (Funkenstein et al. 1999. 1999a. IGF-I binding decreased then to adult muscle levels (6. Gabillard et al. 1998.5% Bsp/20 g). and in vitro experiments may be artificial conditions. 1999) or trout (Greene et al. In situ hybridization revealed that the expression of IGF-IR was ubiquitous (Funkenstein et al. In rainbow trout. insulin binding was detectable 3 weeks after spawning.. 2003b. Changes during Ontogenesis As embryonic development is a period when growth factors play a very important role.. Perrot et al.. 2001b). b). in which administration of high concentrations of peptide to hepatoctyes or cardiomyocytes always provokes down-regulation. 1999a. 2002.76% Bsp/20 g). probably because of species specificity. 151 direction of the regulation seems to be dependent on the tissue and its respective functional needs... J. c). 5 weeks after spawning and coinciding with the organogenesis period. though with some differences of expression levels between tissues. Maures et al.. much higher than the peak found for insulin and also earlier than insulin (Fig. b. suggesting the presence of maternal mRNA. In the same study..2 g.. mRNA was also found in unfertilized egg. Maures et al. but not in seabream (Perrot et al. From this moment. 1997.. Mendez et al. 1999).9% Bsp/20mg). RT-PCR study revealed that both IGF-IRs (IGF-IRa and IGF-IRb) are expressed in embryos at similar levels (Gabillard et al. especially at the stage of eyed eggs. the experimental conditions should be taken into account. Variations in polyadenylate state were observed in turbot (Elies et al. zebrafish (Ayaso et al. followed by a decrease to characteristic values in adult trout muscle (1. in all these species. LLC .. 5. but at very low levels. their presence at various stages has been studied in several fish species. IGF-I was already detectable in newly laid eggs.. Elies et al.Gutiérrez. The presence of insulin and IGFs binding in whole eggs. larvae or fry has been studied throughout trout development. 1999).. Further experiments on binding regulation with cultured myocytes and adypocytes are needed.78% Bsp/20 g) (Maestro et al. 2003c). 2002). insulin increased through the eyed egg and yolk sac larvae stages to reach a maximum in fry of 0. Ayaso et al.. et al. (5. 2002..

The ratio between IR and IGF-IR established early in development (12 weeks) was found to be 0. subsequent drops to adult levels were observed (Méndez et al. after hatching. glucose and alanine uptake in cultured myocyte in trout more potently than insulin does.11 IGF-II binding during trout development: (1) new laid eggs.15 and 0.. Data are mean ± SE and different letters indicate significantly (p<0. 2001a). in a recent study Castillo et al. (3) 9th weeks of life. 2001a). (2001b) extended the studies by comparing 5-week and 12-week larvae with juveniles of 6 months.11).152 Fish Endocrinology 100 b 80 % Bsp IGF-II/20 mg glycoprotein 60 40 c 20 a 0 a gs gs Yo la lk s rv al ae k eg eg aw ed Fig.9% Bsp/20 g) (Fig.. Juveniles and adults were sampled simultaneously and under the same feeding conditions to minimize seasonal or nutritional effects. confirming its important role during this embryonic stage.17). Méndez et al. and (4) muscle of one-year-old juvenile.01) differences between groups. LLC Sp Ey Ju ve n nl le . but also in juvenile and adult fish. In fact. in IGF-IR with a peak at eyed eggs (77. 5. 5. This ratio was maintained throughout juvenile and adult fish growth (0. (Modified from Méndez et al. © 2006 by Taylor & Francis Group. (2004) demonstrated that IGF-I stimulates thymidine. and 1 and 2-year old adult fish. (2) embryos 5th week old. demonstrating that IGF-I binding in muscle is very important not only at development stages.18.

(1997a).. maximum insulin and IGF-I binding were found at the stages of primary oocyte growth and early vitellogenesis. 5. decreasing as vitellogenesis advanced. et al.. 2004b). 1997b) and in Salmo trutta (Maestro et al. maximum binding was observed in autumn and decreased in winter and spring. LLC .Gutiérrez. IGF-I 60 % Bsp/20 mg protein 153 Insulin 7 6 % Bsp/20 mg protein a a 50 40 30 20 cd 10 0 1 d 2 3 4 Maturation Stage 5 c b 5 4 3 2 c 1 0 1 2 3 4 Maturation Stage 5 b bc bc Fig. Maestro et al. © 2006 by Taylor & Francis Group. These results imply a role for both peptides in the maintenance of energy stores in adipose tissue. (2) early vitellogenesis. Later on. when insulin binding reached a nondetectable level... Different letters indicate significant differences between groups. All these seasonal binding variations depended on changes in receptor numbers. Seasonal Changes Important changes in fish ovary receptors occur during the gonad cycle (Fig.12). (Modified from Maestro et al. Capilla et al. 2000a). (4) late vitellognesis. Seasonal changes have also been observed in trout adipose tissue (Planas et al. In carp (Gutiérrez et al. J. while receptor affinity was not modified. concluded that IGF-I contributes to the regulation of ovarian steroid production.. 5. and (5) preovulatory follicles. 1993. 1997b). These changes suggested that both peptides were needed for regulation of ovarian function at different reproductive stages. Maestro et al.12 Changes in IGF-I and insulin binding throughout the various stages of carp ovary maturation: (1) primary oocyte growth. another peak in insulin and IGF-I binding occurred at the pre-ovulatory stage. 2002. 1999). Recent studies with fish isolated adipocytes confirm these hypotheses (Albalat et al. in a study of isolated theca and granulose layers from coho salmon... (3) vitellogenic follicles.

The authors point out that temperature would most likely affect the receptor turnover directly. Capilla et al. Ablett et al. the lamprey dies. (2003b) observed that in juvenile trout fed ad libitum and reared at different temperatures (8. (2003). differences that depended on their © 2006 by Taylor & Francis Group. insulin and muscle IRs on feeding trout with carbohydrateenriched diet. Capilla et al.154 Fish Endocrinology Fasting and Nutrition Experiments In early studies. there is less effect on circulating insulin and their receptors. The authors suggest that during this period of depletion of body tissues. (1983) and Gutiérrez et al. metabolism. This migration starts in October and is followed by 6-8 months of starvation. and 45 days’ fast in trout (Planas et al. hormone levels. (1994a) found in muscle IRs in various fish species. Insulin binding in muscle reaches a maximum in March. IGF-I may be inhibiting apoptosis to keep the lamprey alive until spawning. Chauvigné et al. 12. It has already been mentioned that Párrizas et al. IGF-I binding was inversely related to temperature. Gabillard et al. and then peptide binding to both receptors decreases until the end. (2003) found the same trend when studying the decrease of insulin levels and IGF-IRs in relation to trout growth at 18°C and 8°C. Leibush et al. Fifteen days’ fasting in carp and trout caused a decrease in muscle IRs (Párrizas et al. suggesting that temperature does not affect IGFIR expression. Temperature has multiple effects on food digestion. this effect was absent for muscle IGF-IR mRNA. 1994a). (1991) described the manner in which dietary treatment could modify IRs in fish liver and muscle in parallel with changes in plasma insulin. found no changes in either circulating glucose.. Baños et al. Nutritional conditions need to be profoundly modified to provoke changes in IRs and IGF-IRs. In addition. respectively. 16°C). insulin or their receptors.. (2000) studied insulin and IGFI binding in the muscle of lamprey during its pre-spawning migration. Thus. 2000a) caused a-decrease in adipose tissue IRs. (1998) found increases in plasma glucose. LLC . However. after spawning in May. all of which may affect receptor regulation. using high-quality diets with different carbohydrate content. IGF-I binding achieves a much higher peak in April. (2003) found that IGF-IRa but not IGF-IRb mRNA levels in muscle were maximal in fasted trout and declined after refeeding. When the experimental diet changes little. But this modification depends on the degree of change in diet composition.

155 feeding habits. It is noticeable that in fish heart.. 1994a). 1994b). so stimulating glucose entrance. 1993.. Using this assay. The TKA assay measures the phosphorylative capacity of the receptor (Párrizas et al. the TKA of IRs and IGF-IRs has been studied in several tissues and species. Changes in TKA were observed in some tissues of fish kept under special experimental conditions. closer to the numbers found in mammals. in trout muscle. which suggests more pronounced muscle response to insulin. 1995. high IRs and IGF-IRs were not accompanied by proportionally high TKA (Gutiérrez et al. 1995). respectively). 1994b.1). (1992) reported autophosphorylation of IRs in the liver of stingray and lamprey. while seabream and especially carp have more. 2004). Studies on regulation of binding to IGF-II receptors. Differences were found between species. Though Méndez et al. We found that trout and salmonids in general have few IRs in muscles. are very scarce. respectively. Signal Transduction The first stage in signal transduction is the autophosphorylation of the beta subunit as a consequence of the changes induced by the ligand bound to the alpha subunit (reviewed in Pirola et al. Maestro et al. However. 1997b). In rainbow trout adapted to high carbohydrate © 2006 by Taylor & Francis Group. in carp and trout fasted for 15 days. other than those referred to above. Receptor autophosphorylation determines phosphorylation of various molecules of the signal transduction cascade.. J. IR TKA is stimulated in a dose-dependent manner. heart and skeletal muscle has been described in different teleost species (Gutiérrez et al. IGF-II receptor in fish needs much more investigation. et al. The autophosphorylation of IRs and IGF-IRs has been studied in different fish species. in general. This is consistent with carp tolerance of high carbohydrate levels in diet. Párrizas et al. Stuart (1988) and Leibush et al. carp possessing high level and trout the lowest TKA (200% and 150% over basal level. Párrizas et al. Table 5.. They suggested that omnivorous species possessed more IRs.. (2001a) compared the regulation of IGF-I and IGF-II binding in EPC (carp epithelioma cell line) cultured cells. these values are clearly lower than those found in rat under the same experimental procedure (500-1000% over basal level. Autophosphorylation of IRs and IGF-IRs in ovaries. Thus. LLC ..Gutiérrez. (1994a) reported that.. muscle IR showed increased TKA (Párrizas et al.

1). After the events described above. Chystiakova et al. Perdue et al. We identified in cultured trout myocytes MAPK. When TKA is expressed as a percentage of the basal activity (phosphorylation without peptide stimulation). although Pozios et al. up-regulation of IR numbers paralleled an increase in TKA (Párrizas et al. suggesting that regulation of insulin and IGF-I function is dependent solely on the number and turn-over of receptors (Maestro et al. revealed that IGF-II was more potent than either IGF-I or insulin in stimulation of TKA. achieve a similar physiological effect because of their higher TKA. MAPK activation was transient (5-60 min) and needed a higher concentration of IGF-I than PI3K-AKT activation did. the increase in the number of IRs with a high TKA. results in an extremely efficient hormonal system. Recently. Méndez et al. 1994b).156 Fish Endocrinology diet. PI3K and AKT (Castillo © 2006 by Taylor & Francis Group. LLC . But when TKA is expressed as a pmol of phosphorus incorporated by fmol of receptor. IRS (insulin-receptor substrates) proteins are the first molecules phosphorylated in signal transduction. (2001a) using WGA preparations. However. This implies that IRs. although less abundant than IGF-IRs. (1991) observed that IGF-II binds—as does IGF-I—to the alpha subunit of the IGF-IR. stimulating IGF-IR’s TKA more efficiently than IGF-I. (2001b) found no changes in intrinsic TKA on comparing muscle IRs and IGF-IRs in larvae. During trout ontogeny. (2001) identified in zebrafish cultured cells the presence of MAPK and PI3K-AKT and their stimulation with IGF-I. juveniles and adult trout. Similarly. 1998).. as other vertebrate IGF-II receptors do. which responded to IGF-I stimulation. the threshold and duration of the IGF-I effect on the two pathways appeared to be different. the latter was more sustained (6h). Pozios et al. In addition. Méndez et al. During vertebrate evolution. IGF-IR and IRs seem to have similar activities in the various fish species analyzed. (2004) found the presence of MAPK and AKT in lamprey muscle lysates. the activity induced by insulin is much higher than that induced by IGF-I (Table 5. Interesting results were obtained when comparing the phosphorylation activity of insulin and IGF-I. no changes in TKA were observed. (2001) found a band with a molecular weight close to IRS in zebra fish.. two main pathways are followed: MAPK and PI3K-AKT. Not much information is available about IRS in fish. which suggests that the fish IGF-II/M6-P receptor lacks the tyrosine kinase domain. The M6-P preparations were devoid of TKA.

fasting is followed in some species by a decrease in IRs and IGF-IRs.) of fish. 157 et al. CONCLUSIONS AND FUTURE TRENDS IRs are present in insulin target tissues (muscle. IRs and IGF-IRs are regulated in in vitro assays. as it was once hypothesized that low tolerance to glucose in some fish species was due to receptor deficiency. This is important. It is clear now that this is not the case. and decrease later when they differentiate to myotubes. Many more studies of these receptors in fish are required. The structure of insulin and IGF receptors is well conserved: IRs and IGF-IRs are heterotetramers with TKA. etc. adipose tissue.Gutiérrez. Receptor changes as a result of nutritional treatments are observed only when hormone levels are profoundly modified. IGF-IRs in fish species have also been described and characterized. IGF-I and IGF-II stimulate these molecules. although salmonid species have low levels of IRs in muscle tissue. 2006). but in very low amounts. We also found that insulin. IGF-II receptors are also present. There is a remarkable abundance of these receptors. as occurs in some diabetic patients. et al. the presence and activity of MAPK are higher at early stages. even in tissues that in mammals are typical targets for insulin. 2006). it appears that during myocyte development. liver. This tendency has been observed in poikilotherms. This change is not so clear in AKT and its presence and response are maintained when myocytes are already differentiated (Castillo et al. up-regulation has been observed in fish muscle and adipose tissue.. LLC . whereas IGF-II receptors belong to the mannose-6-phosphate family of receptors without TKA.. Receptor regulation in vivo show different patterns: for example. Down-regulation of IRs has been observed when excess of peptide is administered to hepatocytes of different species. During the © 2006 by Taylor & Francis Group. For instance. J. Moreover. when satellite cells are proliferating. it is reversed in homeotherms when insulin seems to acquire a more significant role. The diversity of tissues that express insulin and especially IGF-IRs implies that these peptides have important functions. especially at embryonic stages. Recent findings suggest that metabolic functions of IGF-I in fish may even prevail over such functions of insulin.

suggesting the role for these receptors and their ligands during these processes. In myocytes in culture. Baró of the Piscifactoria Truites del Segre (Lleida) for providing. respectively. IR and IGF-IR TKA and their respective activity suggest that during vertebrate evolution. Signal transduction pathways have still not been widely studied. opens up new possibilities of studying the in vitro effects on metabolism and growth and of investigating the consequences of an excess of insulin and IGF-I on binding. embryos and larvae were revealed. The availability of piscine adipocyte and myocyte isolation and culture techniques. Further research in this direction using different fish models is required. variation of receptor numbers in gonads. while AKT is still active once myotube differentiation occurs. To find out which molecules and pathways were involved at each level of vertebrate evolution and how they were conserved is a very attractive research field awaiting new researchers. IGF-I and IGF-II. in order to understand better the regulation of receptors and to verify up-regulation in in vivo experiments. Fish IGF-II receptor is mostly unknown. markers of the proliferation stage could be indicators of better growth of muscle tissue. IR changed to a highly phosphorylated molecule. MAPK seems to be more active at proliferation stages. IGF-II receptor lacks TKA and its function in fish needs further exploration.158 Fish Endocrinology reproduction cycle and embryonic development. although it is known that in fish MAPK and AKT are stimulated by insulin. and for allowing us to use their sampling facilities and for their assistance. Its cloning would provide new tools for examining the role of IGF-II receptor in regulation of fish development and growth. and consequently overall fish growth. from its gene to its functions. LLC . For example. Acknowledgments We thank Antonino Clemente and Rosa Marsol from the Piscifactoria de Bagà (Generalitat de Catalunya) and J. brown trout and rainbow trout. To study which pathway is most activated under particular conditions (for instance: proliferation vs differentiation) could provide valuable data. Molecules involved in signal transduction are a very complicated and exciting area of research. © 2006 by Taylor & Francis Group. This research can also be applied in practice.

I. M. Zebrafish insulin-like growth factor-I receptor: Molecular cloning and developmental expression. C. Regul.. J. and Navarro. LLC . 1997. N. This study was supported by grants from the European Union (Q5RS2000-30068).C.. INRA. I. Comp. Endocrinol.F.. 2003.. Insulin-related growth factors stimulate . F. E. B.. 2005. R. Muscle insulin binding and plasma levels in relation to liver glucokinase activity.. Peptides 110:123-132. Insulin-like growth factor II mRNA is expressed in neurones of the brain of the bony fish Oreochromis mossambicus. Function of the red muscle in fish. the tilapia. P 1998b. J..P. J. Regulation of lipolysis in isolated adipocytes of rainbow trout (Oncorhynchus mykiss): The role of insulin and glucagon. T. Navarro. Insulin and insulin-like growth factor-I (IGF-I) binding in fish red muscle: Regulation by high insulin levels..Gutiérrez. hepatocytes and skeletal muscle plasma membranes of Rainbow trout. Fauconneau and P Y. E. plexiform layer and circumferential germinal zone in the retina of the goldfish. Neurol. C. . A. 50:129-139. Braekkan.-E. Hrusovsky. and CIRIT (2001 SGR-00122 and CRAqC). I. 159 We also thank P Le Bail.. S. Navarro.. © 2006 by Taylor & Francis Group. P 1998a.. Regul.. Eur. Peptides 68:181-187.Y. Rescan from .F. Albalat. 2002.. D. J. Boucher. 394:386-394. Neurol.-E.. Baró. Endocrinol. O. Nolan. N. Peptides 77:55-62. M. Nature (London) 178:747-748. 1998.J. Gen. The English text was corrected by the Language Advisory Service of the University of Barcelona.W. Influence of highcarbohidtate enriched diets on plasma insulin levels and insulin and IGF-I receptors in trout. Biochem. 1956. A 142:347-354. Cell. C. Taylor. S. for their help and suggestions with myocyte culture. Baños. A. Gutierrez. DGICYT Spain (AGL-2001-2903 ACU). Ayaso. 2003. and Gutiérrez. 394:394-401. 191:137-148. A. Physiol. 1983. J. Vachot. et al. Gutiérrez. C. Kaushik. Moon. I.M. C. and Byrnes.R.. S. and Hitchcock. NATO COLLABORATIVE RESEARCH GRANTS (CRG-921175) made it possible for most of us to contact each other and to collaborate in the study of insulin and IGF-I and IGF-II receptors. and Reinecke. Capilla.and Selivonchick. and Gutiérrez. Insulin-like growth factor-I binds in the inner . Comp. Panserat. Castejón. J. Schmid. Caelers. proliferation of retinal progenitors in the goldfish. Regul. Weil. S.F. Mol. The effect of high-protein and high carbohydrate diet on [ 125I ] iodoinsulin binding in erythrocytes. and Navarro. Castejón.. L. Baños. Médale. glucose metabolism and dietary carbohydrate in rainbow trout. Rennes. and Hitchcock. SCRIBE. Comp. J. J. J. A. Neurosci. Comp. 18:355–363. References Ablett. Boucher.

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CHAPTER 6 Insulin-like Growth FactorBinding Proteins (IGFBPs) in Fish: Beacons for (Disrupted) Growth Endocrine Physiology Kevin M. Kelley*. Price. Galima. California State University. Jesus A. LLC . insulin-like growth factor-binding proteins (IGFBPs) play important roles in the endocrine regulation of growth. BIO-SCI. Kathleen Sak. and specialized anabolic functions in cells and tissues. Truong and Christopher G. The expression and circulating levels of IGFBPs are highly sensitive to a variety of external and internal factors. development. Lowe ABSTRACT In fishes. Tiffany D.S. Tuan A. Reyes. a messenger of catabolism and stress. Rebecca Hagstrom. Understanding these relationships has practical Authors’ address: Endocrine Laboratory/Dept. Long Beach. This chapter considers the adaptive and maladaptive consequences of perturbing the expression and activity of © 2006 by Taylor & Francis Group. Maelanie M. and takes a particular look at their interplay with cortisol.A. Orlando Zepeda. CA 90840. U. as in all vertebrates. * Author for Correspondence: E-mail: kmkelley@csulb.

‘catch and release’. Stress. taking the view that IGFBPs are beacons for a disrupted endocrine physiology. Utilization of fish IGFBPs as indicators. Catabolism. LLC . the consequences of perturbing this system can be strongly maladaptive. they are much more. with the outcome that an ‘appropriate’ degree of IGF-mediated anabolic functions will proceed in a particular manner in a particular cell type. This chapter discusses the biology of IGFBPs in fishes. of a disrupted endocrine physiology is proposed. pollution) and perhaps improved fisheries management practices may be defined. yet there are also interesting differences from molecular. IGFBPs are centrally-positioned ‘integrators’ of the endocrine growthregulatory system. tissue. Key Words: Insulin-like growth factor (IGF-I.. EVOLUTIONARY EMERGENCE OF THE IGF AXIS AND ITS IGFBPs Across the invertebrate groups and to the chordates. By the time fishes had emerged. influence the expression and phenotype of the different IGFBPs. ‘insulin’ homologs are expressed and play important regulatory roles in cellular anabolism © 2006 by Taylor & Francis Group. IGF-binding protein (IGFBP). as they play a key role in regulating the tissue distributions of IGF peptides and profoundly influence IGF actions at target tissues. there were at least four of the six IGFBPs known in mammals. The magnitude and temporal aspects of these perturbations. both internal and external. Many of the biological roles of these fish IGFBPs appear to be well conserved with those seen in mammals. IGF-II). reflective of the severity of a stressor(s). IGFBPs. and/or organ system. Cortisol. Fisheries. As the IGF axis (IGF peptides.168 Fish Endocrinology relevance in fish and wildlife.g. and considers the potential consequences of ‘rocking the boat’ of fish IGFBPs. While IGFBPs fundamentally serve to carry IGF peptides in circulation and block the potential of IGFs to have insulin-like side effects. Growth. IGF receptors) is important for all cells and throughout the physiological system. whereby impacts of human activities (e. Many factors. or ‘beacons’. cellular and physiological perspectives. may have prolonged influences on the well-being of fishes. as a result of human interactions. as the effects of stressors—mediated initially via cortisol—can be broadcast into physiological impacts by IGFBPs. INTRODUCTION Insulin-like growth factor-binding proteins (IGFBPs) have a complicated evolutionary history that is unrelated to that of IGF receptors.

giving rise to IGF-I and IGF-II. therefore. 1997) or a hagfish (Myxine glutinosa. Separate IGFand insulin-specific receptor binding has been demonstrated in a wide © 2006 by Taylor & Francis Group.. and others. McRory and Sherwood. Nicoll et al. LLC . such as a tunicate (Chelyosoma productum. 1995). Chan et al. Moriyama et al. 2000. Duret et al. Pashmforoush et al. a solitary insulin-like receptor gene (e. Kelley et al. Bern et al. Fish IGF-I and IGF-II peptides exhibit ~80% homology with their mammalian counterparts and. mRNA slicing. Upton et al. LeRoith et al..g.... The divergence into ‘insulin’ and ‘IGF’. which possess the transmembrane and cytoplasmic domains involved in signaling. This duplication event appears to have occurred after the emergence of cephalochordates. In the chondrichthyean. 1995. in Amphioxus. both encoding separate . giving rise to two distinct regulatory peptides. 169 (e. 1998. 2000. this volume). and their divergence was critical to developing distinct anabolic roles of the two regulatory systems (next paragraph). 1998... since there appears to be only one insulin-like peptide (ILP) in Amphioxus californiensis (Chan et al. 1981. Kelley et al.. 2002). 1998. which is also the case for a wide variety of bony fish species (reviewed in Duan.(135 kDa) and . 1998. distinct IGF-I and IGF-II cDNAs have been identified (Duguay et al. Squalus acanthias. 1995).g.. 1998. 1991).. A prototypical IGF later duplicated in a gnathostome ancestor... see also Reinecke.. and they also link the -subunits to the -subunits.. with the respective mature receptor protein complexes consisting of 2 2-heterotetramers of around 350 kDa (reviews by Cheatham and Khan. this volume). As for the evolution of IGF. but two separate peptides in ‘later’ groups. The genes for mammalian insulin and IGF (‘type-I IGF’) receptors are homologous. Reinecke and Collet.g.. Reinecke and Collet. the ancestral insulin gene has duplicated. however..versus IGF-specific receptors (e. insulin and IGF (Reinecke. Within the chordate line. 1997.Kevin M. 1993). as we know them in today’s vertebrates. Comparison of the cloned vertebrate insulins with IGFs indicates an overall amino acid sequence identity of ~50% or less between the two peptides. Kelley et al.. Nagamatsu et al. 1991. Disulfide bridges link the two extracellular -subunits comprising the ligand-binding domain of the receptor.(90 kDa) subunits. 2000. in cyclostome. Cellular receptors specific for insulin and for IGFs also evolved early in vertebrate evolution. Drakenberg et al. 1996) duplicated early in Chordate evolution and diverged into insulin. is seen as differences in their nucleotide and amino acid sequences. prohormone processing. 1990). show strong conservation after an initial divergence.

with higher identities in the tyrosine kinase domains (>80%) and other conserved motifs involved in signaling (see: Gutiérrez et al.170 Fish Endocrinology variety of fishes (Leibush et al.. Maures et al. Perrot et al.. Reinecke and Collet.. exhibiting only 69% identity with each other. complete type-I IGF receptor cDNA clones have been sequenced for zebrafish (Ayaso et al. this volume). On the other hand. and it was found that fish possess two different IGF type-I receptor cDNAs (as well as two insulin receptor cDNAs). The fish type-I IGF receptors show ~60% overall sequence identity with the (single) human IGF type-I receptor. b). at the level of fishes. 1989). coho salmon (Oncorhynchus kisutch.. As an example.. rainbow trout (O. In addition to this physiologically relevant crossreactivity of peptides and receptors. Fig. Nakao et al. Elies et al. More recently. fish IGF typeI receptors are not more than 50% identical with insulin receptors. and turbot (Elies et al. Nicoll et al.. LLC . Using affinity cross-linking techniques. 1998. 1999a. 1996. and seabream (Sparus aurata. the two fish type-I IGF receptors demonstrate a significant degree of divergence between themselves. mykiss.. Chan et al. it has been demonstrated that IGF binds to a 110-130-kDa -subunit in fish. 1998. with the differences distributed throughout the molecules.. 2000. 2002. and it has ~1% the affinity for insulin binding as compared with that for IGF-I (data not shown). there are functionally distinct IGF receptors.. Therefore. Although studies are quite limited in fish. 1999. Navarro et al. Greene and Chen. Interestingly. Japanese flounder (Paralichthys olivaceus. with an affinity of ~1% of homologous ligand binding (Czech. Even in mammals. The entire receptor protein complex (under nonreducing conditions) has a molecular weight (~350 kDa) comparable to that of the mammalian type-I IGF receptor. this volume). 1999). Gillichthys mirabilis) muscle membrane IGF receptor -subunits in a specific manner. indicating obvious molecular divergence between the insulin and IGF receptors at the level of bony fishes. 2002).. 2002). Partial cDNA clones of insulin and IGF receptor tyrosine kinase domains (and proximal regions) were initially sequenced in turbot (Scophthalmus maximus. it has been shown that IGF receptor binding leads to © 2006 by Taylor & Francis Group.. 6. the two receptors show overlap in cell-signaling cascades and responses. see also Gutiérrez et al. 1997)... as in mammals.1 illustrates 125I-labeled recombinant barramundi (Lates calcarifer) IGF-I binding to goby (longjaw mudsucker. Kelley et al. 1999). insulin or IGF peptides are capable of binding their heterologous receptor. 1996).

. it should be noted that a second IGF receptor exists. yielding the widelyheld hypothesis that the IGF-II-binding property is a mammalian phenomenon.Kevin M.. b.. 2001. Finally. Nadimpalli and von Figura. This receptor. other vertebrates. but they do not bind insulin. LLC . the growth promoting actions of both IGF-II and IGF-I are mediated solely through binding to the type-I receptor. if any. possesses an MPR-300 receptor that specifically binds IGF-II (Mendez et al. originally identified in mammals (see Braulke et al.. Clairmont and Czech. © 2006 by Taylor & Francis Group. 1989. Their high affinities mean that IGFBPs effectively block IGF binding to the insulin receptor. Salmo trutta. Leibush et al. Instead. 1996. Yerramalla et al. Zhou et al.... see: Gutiérrez et al. The MPR-300 exists in fish. but it is nonetheless quite clear from many studies to date that the insulin receptor transduces a greater number of ‘metabolic’ functions in cells while IGF type-I receptor activates a greater number of functions related to ‘growth’ and/or specialized cell functions (described later). Where do the IGFBPs fit in? In the biological fluids and tissues of fishes. IGF peptides are bound to IGF-binding proteins (IGFBPs). It has no known growth signaling functions.. 2001. having no relation to the insulin/IGF type-I receptor family. 1995a. 1993. and invertebrates (Canfield and Kornfeld.. the MPR-300/type-II IGF receptor binds IGF-II with high affinity and translocates to lysosomes for degradation. However. The degree to which the two receptors induce distinct signaling has not been fully worked out.. in the sense that introduction of the IGFBP isolated IGFs from ‘cross-acting’ on insulin receptors (Kelley et al. 1995. Gutierrez et al. but it has not been possible to demonstrate binding to IGF-II.. and that this appears to be true from fish to mammals. and of all vertebrates. avoiding the otherwise strong potential for IGFs to exert insulin-like side effects. Kelley et al. Drakenberg et al. Pozios et al. more recently. is the cation-independent mannose-6-phosphate receptor (MPR-300).g. this volume). and PI3 kinase pathways (e. Parrizas et al. IGFBPs bind IGF peptides with affinities (Kd ~10 – 10 M) that are typically higher than that of type-I IGF receptor (Kd ~10 – 9 M). of the MPR-300/type-II IGF receptor are not clear. 1997.. receptor tyrosine kinase. 1994). 171 autophosphorylation. In mammals. even in mammals.. 2000. similar to that of mammalian insulin and IGF receptors. it was reported that embryonic brown trout. the type IGF receptor. 2001). 1995. MAP kinase. 1989. The ‘IGF axis’ roles.. Sorensen et al. for any vertebrate studied to date. This critical physiological role for IGFBPs is presumably fundamental to the evolutionary divergence of IGF axis. Nadimpalli et al. 2002).

IGFBP-2 of zebrafish (Duan et al. Funkenstein et al.. 2003). since the IGFBPs are rivals of the IGF receptor for IGF peptide binding. IGFBP-1 of zebrafish (Danio rerio. J. adaptable ‘status’. Lee and Cohen. Genbank accession #NM_205751). unpubl. 3.. and Duan. which is regulated through a myriad of alternative mechanisms. a host of post-translational modifications affecting IGFBP properties (e. Among the fish sequences reported to date (many are partial). 2003).Y. 6. 2002. zebrafish (Chen..L. tilapia (Oreochromis mossambicus. Le Roith. Furthermore.. they should be viewed as critical.g. see Fig. Maures and Duan. Cheng et al. 2002). 2002. 2002. glycosylation). 2002).2). LLC . in some experimental mammalian cell types. Thus..Y. Since no IGFBPs have been identified in non-vertebrate taxa. J. Kelley et al. unpublished. IGFBPs have a dynamic.172 Fish Endocrinology 2002). 2002. Gracey et al. Mohan and Baylink. and in mammals there are six distinct IGFBP genes. 2000. unpubl. Firth and Baxter. and most relevant to understanding the IGF axis. mykiss.. 1999) and seabream (Sparus aurata. © 2006 by Taylor & Francis Group. limited proteolysis.. Bunn and Fowlkes. the evolution of the IGFBP family presumably occurred entirely within the chordate line. alterations in expression of different IGFBP members.and extracellular matrix (ECM) binding activities affecting localization and/or IGF-binding affinities (reviewed by Clemmons et al. and different cell surface. 2002. at least two of the six IGFBPs also appear to have IGF-independent actions. Among fishes. and 5. 2001). Genbank accession #AY100478) all exhibit 40-50 % overall sequence identity with their mammalian counterparts. 2001) and goby (G... 2001. having identity with mammalian IGFBPs 1. It should also be noted that. centrally-positioned ‘integrators’ of the endocrine growth-regulatory system. and Wu. IGFBP-3 of round stingray (Urobatis halleri. rainbow trout (O.. Chen. four orthologs have been cloned and sequenced at the cDNA level. and IGFBP-5 of zebrafish (Ding. presumably through cell surface IGFBP receptors or via translocation of IGFBPs into the nucleus for direct involvement in gene regulation (see reviews by Duan. J. including cell-specific expression of IGFBPs. Baxter. phosphorylation. mirabilis. J. 2003. multimerization. 1998. 2.. WHERE DID FISH IGFBPs COME FROM? IGFBPs are unrelated to IGF receptors. a change in any of these aspects of IGFBP ‘status’ will alter how and where IGF peptides act at different cellular targets. Bauchat et al. C. Genbank accession number AJ299409).

corresponding to the 7-fold lower relative affinity for insulin). These functional motifs. Lee and Cohen.g. Kelley et al. IGFBPs 2-5 possess functional heparan-binding domains (allows cell surface association. since mammalian IGFBPs 3 and 5 (and their N-termini) are already recognized for such mechanisms of action (Duan. 2000. 2001). 1999. 2001. Interestingly. 2002). utilizing heparan-sulfate proteoglycans of the extracellular matrix). which corresponds to distinctive functional properties. 2001.. yet it is possible that this represents a pre-adaptation for an eventual IGFBP The fact that an IGFBP predecessor (similar to .. The only non-mammalian vertebrate for which an © 2006 by Taylor & Francis Group.g.Kevin M. with the most highly conserved sequences found in exons 1 and 4. Ross et al. Perlustrin. Chang et al.. Almeida et al. In contrast to the superfamily’s conserved N-termini. Thus. including twisted gastrulation (TSG. for example. cysteine-rich domains (see Vilmos et al. This family includes a variety of invertebrate member proteins. Mohan and Baylink....and C-terminal domains. see the following paragraph). 2001).. 2002... 2001). 2001) and a molluscan protein named perlustrin (Weiss et al. 173 The origins of the IGFBP family appear to be quite complex. Lamghari et al. 1999). Weiss et al. 2002. hypothetically. exon-1 of IGFBPs. This IGF-binding property is of course difficult to reconcile with the lack of a presence of an IGF in molluscans. perlustrin?) may have direct (IGF-independent) actions on cells is not surprising to the IGFBP research field. while IGFBPs 1 and 2 contain RGD motifs for binding to cell surface integrins. are thought to have been disseminated by a process of domain shuffling (Hwa et al. their C-termini can be more different between and even within sub-families. At least for part of their sequence. IGFBPs have relatedness to an ancient superfamily of regulatory proteins sharing homology in their N-terminal. In the IGFBP family. originally identified in shell matrix of abalone (Haliotis laevigata). LLC . Scott et al.. appears to have direct growth and differentiative actions in cells (e. which is encoded on a single exon of most member genes (e. Mammalian IGFBPs are encoded on four exons. leading to more functional distinctions within and among families. perlustrin is also capable of binding IGF-I with an affinity of Kd ~10 –7 M (recall Kd of IGFBPs is ~10 –9 M) and favors IGF binding over that of insulin (2. outside of the (superfamilyrelated) N-termini.. the IGFBPs may have emerged from a collective contribution of an ancestral exon from a cysteine-rich growth factor superfamily and accumulation of ‘functionally-useful’ domains from additional gene families.

and.. as mentioned above. O-linked glycosylation (IGFBPs 5 and 6). 1992.. A multiple sequence alignment for IGFBP-3 of different fish species is shown in Fig. IGFBP-1. 2003. and it shows the same organization. and limited proteolysis (IGFBPs 2-5) (reviewed in Conover. Limited IGFBP proteolysis. 1997). Kelley et al. 1998. mammalian IGFBP-3 has the greatest extent of glycosylation which results in a 45-50 kDa ‘doublet’ (as appears on a Western blot). 1995). Among the characterized fish IGFBPs. and the Cterminus of IGFBPs contains six additional conserved cysteine positions.and Ctermini is indicated by the red (100% identity) and blue amino acids. together. poor sequence identity with the midregion sequences (T. While all vertebrate IGFBPs are translated as 216-289 amino acid proteins. As referred to above.and C-termini maintain the structure of the high-affinity IGF-binding domain. Thus. Bunn and Fowlkes. given that fish IGFBPs bind mammalian IGF peptides with high affinity (discussed further below).M.and C-termini) showing the most variation across IGFBPs. Andress and Birnbaum. 6. and 5).174 Fish Endocrinology IGFBP gene has been characterized is that of chicken IGFBP-2 (Schoen et al... Price and K. The C-terminal half of IGFBPs includes domains conferring specialized functions. e. For example. phosphorylation appears to alter IGF-binding affinity in some IGFBPs (e. 3. with the mid-region (between N. However. Strong conservation in the N. 2002). these 18 cysteines are similarly conserved.g. their molecular weight at a given instant will depend on the status of these modifications. Kelley. 2002. 2002. 1996. which may be expected. the N-termini contain highly conserved cysteines (in 12 positions). Firth and Baxter. Lee et al. phosphorylation (IGFBPs 1. In the more ‘ancestral’ round stingray. in the circulation or in the microenvironment of a target © 2006 by Taylor & Francis Group. 2003). in the size range (24-33 kDa) of the other mammalian IGFBPs. a partial IGFBP-3 clone exhibits between 62-73% sequence identity in its C-terminus with that of the other species. on the other hand. the N. LLC . but when deglycosylated it is ~30 kDa. Clemmons et al. These include N-linked glycosylation (IGFBPs 3 and 4). which is widely believed to be a mechanism whereby IGF peptide availability (“bioactivity”) may be increased. Le Roith...2. as expected (see next paragraph). The influence of glycosylation on IGFBP properties is not yet understood. it is in the latter region where many IGFBP-specific post-translational modifications occur. data).g. Mohan and Baylink. reduces IGF-binding affinity in all known instances (with other possible effects. unpubl. however. including avian and mammal representatives.

3 and 5) are relatively well conserved with their mammalian counterparts. 1994). Garcia-Fernandez and Holland. 1994. 1999. 2004). it has been proposed that a major event in the evolution of the IGFBP family was the duplication of a cassette of DNA containing IGFBP 1 and 3 and other (HOX-A. More information from the fishes should help to shed light on the evolution of the IGFBPs subsequent to their origins. With this in mind. while the converse may also be said. EGF-R) genes. it would appear that to understand the origins of the IGFBP family.. Sequence analyses of the various cloned vertebrate IGFBPs indicates that IGFBPs 3.. 5 and 6 exhibit greater corresponding sequence identity than they do with IGFBPs. Also. and common chromosomal locations of IGFBP-1 with IGFBP-3 and IGFBP-2 with IGFBP-5.. 2004). Kelley et al. release of an IGFBP-specific protease can enhance IGF peptide access to its cell receptor. with the exception of a recent report by Shimizu et al. IGFBP-6 has up to 100-fold greater affinity for IGF-II over IGF-I. giving rise to the IGFBP 2 and 5 (and HOX-D.1. tshawytscha) IGFBP exhibited N-glycosylation. Allander et al.. 1989. 1994.. IGFBP proteolysis and the other post-translational IGFBP modifications seen in mammals have yet to be identified in fish (or other non-mammals). IGFBP-6 has not yet been identified in fishes. (2003b) that a Chinook salmon (O. 2000. from the presence of IGFBP proteins of molecular weights below the ‘standard’ size ranges (e.. Only one chondrichthyean sequence (Fig. 2) has been identified to date. LLC . while there are no sequences yet identified for IGFBPs 4 and 6 in any fish (or lower vertebrate). It has also been argued that a cassette of DNA containing IGFBP-6 (co-localized with HOX-C and ERBB3 genes) was the predecessor for the IGFBP gene family. 2. sequence data must now come from ‘earlier’ vertebrates and chordates (taxa with ancestors diverging from the main vertebrate line prior to chondricthyeans). Kou et al. Proteolysis of fish IGFBPs can only be inferred at this point in time. Headley et al. promoting anabolic actions. understanding the evolution of many of the functionally relevant properties (domains) of IGFBPs will very likely depend heavily on the data derived from fishes. 2000. Park et al. 175 tissue. where it serves as a potent inhibitor of IGF-II actions (Bach. However. Additionally. © 2006 by Taylor & Francis Group.. see Kelley et al. Peterson and Small. unlike the other IGFBPs. 2 and 4.g. Since the presently cloned and sequenced fish IGFBPs (1.Kevin M. ERBB-2) genes (Acampora et al. a notion supported by IGFBP-6 having the most divergent sequence of the six IGFBPs.

. Kelley et al.. and 20-25 kDa. as well as IGF-I.176 Fish Endocrinology GENERAL ASPECTS OF THE ENDOCRINE PHYSIOLOGY OF FISH IGFBPs The existence of IGFBPs in fishes was first recognized when a set of serum proteins were found to bind 125I-labeled human IGF peptides in a specific manner (Kelley et al. 1992). having molecular sizes of 40-50. In the teleosts tested. Noguchi.. 1992. Bauchat et al. diabetes). and it is mostly found bound into a ‘ternary’ complex with socalled ‘acid-labile subunit’ (ALS). 3 and 5. The fact that IGFBPs could be detected in the serum of the lamprey. mammals may be unique among vertebrate taxa in the sense that they circulate large concentrations of IGF-I bound to IGFBPs. suggesting their relationship to mammalian IGFBP-1. 2000. 1998. easily move to the tissue/cellular microenvironment to exert their © 2006 by Taylor & Francis Group. The identities of these proteins are yet to established. although additional putative IGFBPs have been periodically noted (e. 2. thereby maintaining a substantial circulating ‘reservoir’ of IGF (see Baxter. IGFBPs. 2001. indicated that the IGFBPs also emerged early in the vertebrate line. 2001. the other IGFBPs (32 kDa or smaller) will rapidly cross the capillary barrier and.g.. In both initial studies. along with the IGF peptides and receptors. The same authors demonstrated that the other two fish IGFBPs could be reduced by insulin treatment and were increased under catabolic circumstances (fasting. Firth and Baxter. 2002). which serves to lengthen the half-life of the complex in the serum. 2000. 1998. Reinecke and Collet.. and possibly IGFBP-2. 1992).. In mammals. Geotria australis (Upton et al. 2003). Upton et al. In contrast to IGFBP-3. Several studies since 1992 have confirmed the presence of three similar IGFBPs in the serum of a wider variety of fish species (reviews by Duan. 29-32. suggesting that this protein was the counterpart of mammalian IGFBP-3. circulate at much lower concentrations in fish as compared with mammals. Indeed. Park et al.. and that their serum levels were inversely correlated with growth. it was found that serum levels of the 40-50 kDa IGFBP could be increased by GH treatment and were positively correlated with growth rate (Kelley et al. but there is some confidence that they will be confirmed as IGFBPs. 2002). The collective molecular size [45-kDa IGFBP-3 + 7-kDa IGF + 90-kDa ALS] is large enough (~140 kDa ) to block its movement across the capillary barrier (~65-70 kDa). Nicoll et al. 1997. 1992). serum IGFBP-3 carries >90% of total IGF. LLC . in such manner. given the recent cloning of the cDNAs for fish IGFBPs 1. three major fish IGFBPs were identified... Kajimura et al..

. and jack mackerel. particularly to the rat’s 45 kDa IGFBP-3 band. Therefore. 2002. and then easier to detect. 2002). Galima et al.. itself has many important ‘local’ actions (e. It may also be notable that IGF-I circulates at levels comparable with that of insulin in fishes. 2002). by hormone injection or stress induction). Using size-exclusion chromatography under neutral conditions (which can be used to detect 140 kDa ternary complex in mammalian serum). Kelley et al. This holds true for all fish (and lower vertebrate) species tested to date (see Kelley et al. Dyer et al.. often rapidly. With the very low IGFBP levels are also very low concentrations of IGF-I in fish serum.3. In rat serum... Semicossyphus pulcher. so much so that IGFBPs may be difficult to detect by standard binding assays in fish under basal (‘normal’) physiological conditions. using Western-ligand blot analysis. without bound ALS. This is illustrated in Fig. circulate at much ... data). 2004. Larsen et al. This fish-tomammal difference has been consistently observed (see Baños et al. For example. 2000. 1999. LLC . Kajimura et al. Trachurus symmetricus). one or more of the IGFBPs will be elevated. whereas in the same two fish shown in Fig. as well as in other non-mammalian taxa. if the fish are provoked (e. IGF-I is present at 500-800 ng/ml. 2001. 1999. 6. With this apparent lack of a ternary complex in fishes.. lower concentrations in fish serum as compared with that seen in mammalian serum. in which 3 l of rat serum (‘R’ lanes in figure) are compared with the same volume of fish serum in terms of capacity to bind 125I-IGF-I. 2004. is dramatically higher in rat rat serum than in the two fish species shown (California sheephead. experiments in several different fish species demonstrate that levels of the © 2006 by Taylor & Francis Group. 2004). Beckman et al.g.). 6. 2003. a ternary IGFBP-3 complex does not appear to exist. In fish. Shimizu et al. Duan. 177 biological effects (regulation of bioactivity and distribution of IGFs. no IGF-binding activity reminiscent of the mammalian ternary complex can be detected in serum of several fish species (see Shimizu et al. etc.3. 2004 unpubl. 1999.. It should also be noted that IGFBP-3. IGFindependent actions. However. Kelley et al.. 2000. it is also clear that the 40-50 kDa IGFBP and all fish IGFBPs. Lee and Cohen. IGFBPs and IGFs are far less prominent features in the serum of fish as compared with mammals...Kevin M. serum IGF-I levels are between 20-30 ng/ml (Galima et al...g. Degger et al. 2002). in clear contrast to the mammals that may show 100-fold greater concentrations of IGF-I versus insulin. 2000. IGF binding.

most serum IGFBPs can rapidly partition into the tissue/cellular environment from the blood circulation. coho salmon. Although complicated. including liver. exhibited reduced .. (1995) corroborated the studies showing an inverse relationship between the serum level of these IGFBPs and insulin (and growth) status (e. bone. As stated earlier. cells almost always produce two or more IGFBPs (exception may be in some tumor cell lines). (1995) reported that release of the 30 kDa IGFBP from bass liver in vitro could be stimulated by GH. Firth and Baxter. Kelley et al. Mohan and Baylink. brain. since one IGFBP may be studied without interference from other expressed IGFBPs. and other species (Moriyama et al. All tissues. Fish tissues typically express one IGFBP but sometimes two. Morone saxatilis). 1997. cartilage. along with coordinate increases in serum IGF-I and growth rate (see Kelley et al. In contrast. yet there have been only a few attempts to date to assess their properties and regulatory roles in non-mammalian cells. gastrointestinal tract. IGF-I. As an aside. insulin’s direct inhibitory effect on release of the 30 and 24 kDa IGFBPs shown by Fukuzawa et al. IGFBPs are also directly produced by a variety of tissue and cell types of fish.. except the liver. or prolactin treatment. tilapia. Many questions need to be © 2006 by Taylor & Francis Group. expressed a 30 kDa IGFBP in vitro.g . 2002). while liver produced both 30 and 24 kDa IGFBPs. spleen. Fukuzawa et al. 2002). pituitary. IGF-I.. Siharath et al. mammalian . Roth. and triiodothyronine (T3). cellular levels. heart. since the ‘dynamics’ of IGFBPs in the microenvironment of mammalian cells are recognized as essential to a full understanding of the physiological roles of IGFBPs (Kelley et al. In addition to this route of entry of IGFBPs into tissues. muscle.. and thyroxine (T4). 1999. 1996. and gill filament. 2002. Shimizu et al. release with insulin.. fish cells may well present the better opportunities. epidermal growth factor (EGF). LLC . whereas estradiol was stimulatory. Two liver-secreted IGFBPs of similar size are also seen in goby.178 Fish Endocrinology 40-50 kDa IGFBP are significantly increased after GH injections. but was inhibited by insulin. there is also a small body of work beginning to address how they may act at the local. 2003a). 1993). This is a critical absence of data. The 24 kDa IGFBP on the other hand.. In terms of good experimental cell models. In addition to the interest in circulating IGFBPs in fish.. gonads. their results indicate that ‘local’ tissue IGFBP expression can be influenced by physiologically relevant factors. 2002. glucagon. (1995) provided the first demonstration of IGFBP production in fish tissues (of striped bass. Kelley et al. 2002. IGFBPs present at the cellular level have significant physiological relevance. kidney.

to begin to answer some of these functionally relevant questions. 6. as are mammalian IGFBPs 1-5? If so. while the 30 kDa band represents a membrane-localized 30 kDa IGFBP.. in a reptilian (iguana) heart cell (Schmidt. Kelley et al. 2002). 6. The upper 135-kDa band represents the á-subunit of the IGF type-I membrane receptor. at the molecular and cellular as well as physiological levels. Are fish IGFBPs capable of associating with the cell membranes.. (1999) could © 2006 by Taylor & Francis Group. Addition of excess unlabeled IGF-I (200 ng/ml) competitively inhibited 125 I-IGF-I to both proteins (‘nonspecific binding’). answered with respect to fish. 1998).1). and in goby muscle (shown in Fig. does this influence the ability of IGF-I to activate its cellular receptor? Do fish IGFBPs have any direct IGF-independent effects on cells. cell membrane IGFBP association has thus far only been demonstrated in a chicken hepatoma cell (Duclos et al.. Among non-mammals. in an affinity crosslinking procedure (as in Kelley et al. Duan et al. LLC . Kelley et al.1 Binding of 125 I-IGF-I to skeletal muscle membranes of the longjaw mudsucker. as do mammalian IGFBPs 3 and 5? Do any such IGFBP properties correspond with any post-translational modifications? Do IGFBP properties correspond with the presence or absence of particular functional domains in the protein sequences? Much work is needed. Gillichthys mirabilis.CMYK Kevin M. 179 IGF receptor asubunit Total I-IGF Binding 125I-IGF Nonspecific Binding 135 kDa MembraneMembraneMembranebound IGFBP bound IGFBP 30 kDa Longjaw mudsucker Fig. In zebrafish. 1999). 2001.

not demonstrate localization of recombinant IGFBP-2 to zebrafish embryonic (ZF-4) cell membranes. Gaps were introduced to maximize sequence homologies.2 Comparison of a partial IGFBP-3 primary sequence of round stingray (Urobatis halleri) with that of zebrafish.CMYK 180 srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus srIGFBP-3 zfIGFBP-3 tIGFBP-3 hIGFBP-3 chIGFBP-3 mIGFBP-3 zfIGFBP-2 Consensus Fish Endocrinology (1) (1) (1) (1) (1) (1) (1) (1) (1) (11) (1) (51) (1) (51) (10) (51) (1) (61) (30) (101) (1) (101) (60) (101) (11) (106) (73) (151) (1) (151) (102) (151) (51) (156) (114) (184) (3) (194) (137) (201) (88) (206) (153) (232) (40) (231) (177) (251) (138) (256) (203) (282) (90) (281) (227) (301) 1 50 -----------------------------------------------------------------------------------------MTGLCALCLT -------------------------------------------------GTRHSFAPCTVAPSLRAQPAKQRAPVAGVMQRARPTLWAAALTLLVLLRG -------------------------------------------------ASRSSAPSRRSKLASPLPSPPPSRPPGAMHPARPALWAAALTALTLLRGP -----------------------------------------MLSYVSCGL 51 100 -------------------------------------------------ALLAAFARLAESVSPVVRCEPCDDGAMVLCKPLPWDCDEPVKEPGCGCCL ---------------------VDVASVSQCKPVPLDCAEKLREPGCGCCV PPVARAGASSGGLGPVVRCEPCDARALAQCAPPPAVCAELVREPGCGCCL -------------------------------------------------PVAELAAGAVGGPVVRCEPCDARAVSQCAPPPTAPACTELVREPGCGCCL LLALVTFHGTARSEMVFRCPSCTAERQAACPMLTETCGEIVREPGCGCCP A C P C E VREPGCGCCL 101 150 ----------------------------------------CDSRALSQCN TCPLTEGQACGVYTGRCGTGLSCQHRPGESKPLQALLEGRG-----VCAK TCALSKGQSCGIYTSRCGSGLTCQPRPGETRPLLALLEGR-------GIC TCALSEGQPCGIYTERCGSGLRCQPSPDEARPLQALLDGRGLCVNASAVS -------------------------------------------------TCALREGDACGVYTERCGTGLRCQPRPAEQYPLRALLNGRGFCANASAAG VCARQEGEQCGVYTPRCSSGLRCYPKPDSELPLELLVQGLG--------R TCAL EG CGVYT RCGSGL CQPRP E PL ALL GRG 151 200 GTARARIFQNAAGNVNRSEEERN------ASNSGNEWSPNTHRMQE---APDKKQSGSPSHGHDNPETEGKEQNGTRTAGTGEAETVHHTTDISRDVQG SEHRNGISVPAHGEDNTTEEDG--------SPKGADTKDSRCGLTGLKARLRAYLLPAPPAPGNASESEE-----------------DRSAGSVESPSV -----------------------------------------------KFSLSTYLPSQPAPGNISESEEEHN------AGSVESQVVPSTHRVTDSKFCGR------KVDTEPTGSAEPRE--------VSGEVQDPLDIGLTEVPPPA G S EE T LTE 201 250 -----------SKTQPHRKLLKKEASRKSQLYKINNE--AENTETTNFSS GSRTPSEPSDPMMHSNKLEMIQKEQVKKSQVHKVVPFSGWIVQDIHNFSL -----------VLPHPKAEVIKNAEDNRSQRSKVKPLPGAVGTDGQNFSC SSTHRVSDPKFHPLHSKIIIIKKGHAKDSQRYKVDYE--SQSTDTQNFSS -----------HPIHTKMDVIKKGHAKDSQRYKVDYE--SQSTDTQNFSS -----------HPLHAKMDVIKKGHARDSQRYKVDYE--SQSTDTQNFSS ----------IRKPTKDSPWKESAVLQHRQQLKSKMKYHKVEDPKAPHAK H KMDVIKKG AK SQRYKV E S TDTQNFSS 251 300 EIKHDTEYGPCRREMEDILNNLKAVSILSPRGVYIPNCDKKGFYKKKQCR ESKRENEYGPCRREMESVMKQLKFTNVLNPRRFRIPNCDQKGFYKKKQCS GSKQETENGPCRREMESVLNSLKLTDIINPQGLHIPNCDKKGFYKKKQCR ESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKQCR ESKHETEYGPCRREMEDTLNDLKFLNTLSPRAIHIPNCDKKGFYKKKQCR ESKRETEYGPCRREMEDTLNHLKFLNVLSPRGVHIPNCDKKGFYKKKRCR QSQCQQELDQVLERISKITFKDNRTPLEDLYSLHIPNCDKRGQYNLKQCK ESK ETEYGPCRREMEDILN LKFLNVLSPRGVHIPNCDKKGFYKKKQCR 301 350 SSKGRRRGFCWCVDKYGTQLPSYDRNG--ELRCYSFESK----------PSKGRKRGHCWCVDKYGQPLPGYDGKEK--VHCYNMETK----------PSKGRQRGF----------------------------------------PSKGRKRGFCWCVDKYGQPLPGYTTKGKEDVHCYSMQSK----------PSKGRKRGFCWCVDKYGQPLPAFSVKGKGDV------------------PSKGRKQSFCWCVDKYGQRLPGYDTKGKDDVHCLSVQSQ----------MSVNGYRGECWCVNPHTGRPMPTSPLIRGDPNCNQYLDGQEMDPSVDPPN PSKGRKRGFCWCVDKYGQ LPGY KGK DV C S IGFBP-3 fish orthologs Round stingray sr = stingray zf = zebrafish t = tilapia h = human ch = chicken m = mouse Fig. LLC . Highest sequence similarities are seen in the N. Although zebrafish © 2006 by Taylor & Francis Group. and blue-colored residues high sequence identity (one absent). 6. Red-colored residues indicate 100% sequence identity among all IGFBP sequences. and zebrafish IGFBP-2. in a multiple sequence alignment using Vector NTI software. and human IGFBP-3. they hypothesized that this inability was due to the lack of a heparin-binding motif that plays a role in cell surface-ECM association in mammalian IGFBP-2. chicken. mouse. tilapia. with the bottom line representing the consensus sequence.and C-terminal regions of IGFBPs.

as described in text. and serum levels of IGFBP and cortisol were measured by western-ligand blot and radioimmunoassay. 2001). 1999). 181 Jack mackerel California sheephead kDa 45 30 24 R control stress stress control 200 150 100 R Cortisol (ng/ml) 50 Fig. inhibits IGF-I’s proliferative actions. Stress was induced by catching-associated experiences.CMYK Kevin M. LLC . IGFBP-2. In stressed fish.. rat serum exhibits a major 45 kDa IGFBP-3 band. serum IGFBPs are at low-to-undetectable levels in the fish. however. serum cortisol concentrations are increased by 50-100-fold (P<0.. When added to these cells. respectively (Kelley et al. IGFBP-2 possesses the integrin-binding motif. or IGFBP-1 (Bauchat et al. on the other hand.001. they represent only a fraction of the IGF-binding capacity of control rat serum. 6. -2 and/or -5). a result expected from a soluble IGFBP sequestering available ligand. n=11-45/bar) and levels of the 30 kDa IGFBP are dramatically increased. Kelley et al. RGD.3 Serum IGFBPs and cortisol concentrations in control and stressed jack mackerel (Trachurus symmetricus) and California sheephead (Semicossyphus pulcher). Even with these increases in the fish IGFBP. and a 24kDa IGFBP-4 band]. © 2006 by Taylor & Francis Group. Under control conditions. in strong contrast to the large quantities of IGFBPs present in control rat serum [see ‘R’ lanes. a 30 kDa IGFBP band (contains IGFBP-1. it was apparently not utilized in the ZF-4 cell system.

upon binding to IGF. Alternatively. 2002 and unpublished). allowing for such a release of peptide. Findings from the recently sequenced zebrafish IGFBP-1 cDNA indicate that an RGD motif is absent (Maures and Duan. IGFBPs can be growth promoting when they serve to localize IGF peptides at the cell membrane (in proximity of the IGF type-I receptors) and if they release the IGF peptide.4 Growth-promoting and growth-inhibiting mechanisms of action of IGFBPs at the cell membrane. when IGF-I is added to the cells.CMYK 182 Fish Endocrinology Growth promoting Growth inhibiting IGF IGF IGF BP IGF IG F IGF BP IGF Rec IGF Rec BP BP BP (+) (. This cell produces a single 30 kDa IGFBP that can be found both in soluble form and localized to cell membranes. LLC . in contrast to mammalian IGFBP-1. The presence of membrane-associated IGFBP appears to play an inhibitory role in IGF-I © 2006 by Taylor & Francis Group. 2002). 6. Interestingly. taking IGF-I with it. As shown in this diagram. rapidly leaves the membrane. membrane-bound IGFBP binds IGF-I and then rapidly falls off the membrane. the iguana IGFBP associates with heart cell membranes and. taking IGF away with it.. IgH-2 (Schmidt. Some mammalian IGFBPs exhibit reduced IGF binding affinities when they associate with the cell surface.) Fig. Kelley et al. IGFBPs can be growth inhibitory when they sequester IGF peptides. Schmidt et al. Studies on the regulatory roles of a cell-associating IGFBP from a lower vertebrate have been done in an iguana heart cell line. as relating to IGF action. making IGF unavailable to receptors. 2001.. 2002.

and a substantial—often dramatic— . and serum IGFBP-1 will increase rapidly in response to cortisol treatment or exposure to a stressor (e. These changes are most often closely associated with serum increases in the steroid. As in mammals. Lee et al.. cortisol is well recognized as a potent activator of IGFBP1 gene expression.. 2000.g. Therefore. 1997. which in all vertebrates is a critical ‘stress hormone’ (see Schreck. Figure 6. plays an important role in modulating IGF action— inhibition in this case. Cheng et al. both GH and insulin appear to be important in maintaining this anabolic endocrine status (Duan. Cheng et al. Wendelaar Bonga. cortisol. 1998.. Kelley et al... In mammals. 2002. 2001). Uchida et al.. An opposite endocrine status defines catabolism and growth inhibition: diminished levels of 4050 kDa IGFBP reduced IGF-I levels..Kevin M. there are examples of the alternative scenario. where membrane association of an IGFBP promotes IGF-I action. increased growth rate. Beckman et al. 2003. membrane association of the IGFBP reduces its binding affinity for IGF. 2002. This is © 2006 by Taylor & Francis Group..4 illlustrates these two types of mechanisms seen with IGFBP membrane association. Reinecke and Collet. Peterson and Small. 1999). this IGFBP. since the cells are significantly more responsive to the mitogenic actions of IGF-I under conditions in which IGFBP presence on the membranes has been reduced (this can be accomplished using IGF analogs capable of binding IGFBPs but not receptors). using membrane association to target its actions on a particular cell (cardiac) membrane. Underwood.. Shimizu et al. 2003. thereby localizing IGF-I in proximity to cell receptors but being ‘willing’ to give up the peptide (see above reviews on mammalian IGFBPs). FISH IGFBPs AS BEACONS OF DISRUPTED ENDOCRINE PHYSIOLOGY In fish. cartilage 35S-proteoglycan synthesis) is when the circulating levels of the 40-50 kDa IGFBP and IGF-I are elevated. 1998. Kelley et al. 1998. 1997. but levels of the 20-32 kDa IGFBPs are basal (often at the limit of detection). Kelley et al..g. 2004).. Ingenbleek and Bernstein. within hours of fasting in humans). in this case. 1993. 2004. elevated high-affinity IGFBP-1 may then sequester IGF peptides from activating target receptors (Ketelslegers et al. 183 action. 2004). From the mammalian literature. 2000. Noguchi. 1996. 1996. increase in levels of one or both of the 20-30 kDa IGFBPs (above references and Underwood. Katz et al. LLC .. 1996. an endocrine status indicative of anabolism (e.

.. these changes would be adaptive to dealing with a stressor(s). treated tilapia with cortisol and. Furthermore. indicating a comparable system in fish as that in mammals (Rodgers et al. Pankhurst and van der Kraak. on the other hand. Collectively.. 1995. 2000. The consistency with which increased cortisol and increased 20-30 kDa IGFBPs are correlated with the inhibition of growth in fish has prompted the proposal that IGFBP measurement can be an effective method to assess the anabolic/ catabolic status of fish (Kelley et al. 1997. so long as this altered endocrine status is not prolonged! It has been well established that human-derived stressors. Wendelaar Bonga. which implies that an IGFBP-1 protein of ~25. 1999..5 kDa (deduced size) might also be increased under these conditions. Cheng et al... LLC . Recent studies have corroborated this idea. leads to a reduction in energyexpensive anabolic processes. 1997.3). body growth rates were significantly inhibited. 2001. 1999). observed significant increases in 24-32 kDa serum IGFBPs (with no changes in higher molecular weight IGFBPs). 6. 30-60 minutes of moderate © 2006 by Taylor & Francis Group. cortisol increases levels of the 20-30 kDa IGFBPs in fishes (see Kelley et al. Maures and Duan (2002) found that expression of zebrafish IGFBP-1 mRNA could be stimulated by fasting as well as hypoxia. Our studies have also shown rapidly responsive alterations in serum levels of IGFBPs as compared with IGF-I levels. representing the presence of an important environmental stressor. 1993. 2003). Kelley et al. 1997). will induce significant surges in circulating cortisol in fish (see reviews by Schreck. Peterson and Small (2004) fasted channel catfish (Ictalurus punctatus) and found that serum cortisol levels significantly increased concomitantly with an increase in a 20 kDa IGFBP (with no changes in other IGFBPs) and that. plasma IGF-I concentrations in the tilapia were modestly reduced only after 24 hours. Lee et al. over 14–45day periods. 2002. these animals exhibit a >80-fold increase in serum cortisol concentrations and increased IGFBP levels from previously undetectable levels. In the jack mackerel (Fig. Mommsen et al. (2003).. while also exerting its well-known actions to mobilize metabolic fuels from stores (Ingenbleek and Bernstein. Kajimura et al. within two hours. Figure 6.184 Fish Endocrinology viewed as a mechanism whereby cortisol. Kajimura et al. from catching to handling to exposure to contaminants. indicating a more rapid response in serum IGFBPs than in IGF-I.3 shows a 30 kDa serum IGFBP in California sheephead that have experienced line-catching and a two-day confinement in experimental tanks.. 2004). 2001).

. McCormick. 2003. we also intended to make a case that IGFBPs serve as effective bio-indicators.. It is also a key regulatory system involved in development. repair of tissue after injury. Radaelli et al. Mendez et al. With respect to the latter. Allan et al. Perrot et al. 1991. 2002. Despite the still-normal IGF-I concentrations. but serum IGF-I levels are not reduced. Above. we considered cortisol as a messenger of stress that dramatically alters IGFBPs and the endocrine physiology of growth. the IGFBPs emerge as the most responsive to outside influences.. Kelley et al. 2002. and even more.. and a hedge against entropy. 2001. It mediates the growth-promoting actions of GH. Gabillard et al... the IGF/IGFBP/IGF-receptor system is involved in the function of virtually every cell. or ‘beacons’. then. Understanding this will have both scientific and practical relevance. Greene and Chen. Funkenstein et al. Madsen et al. 2000. which occurred quite rapidly. maintenance and remodeling of bone. 2003. 1996. 2002. osmoregulation (gill and kidney). Adashi. 1999. reproductive processes. Pera et al. This suggests that the alterations in IGFBP. However. the consequences of perturbing this system can be strongly maladaptive. Castillo et al. 1998. this volume). It is important to keep in mind that this is no ordinary endocrine system. 1998. 1999c. and Reinecke. cartilage and muscle. 1999. may have immediate inhibitory influences on tissue growth. Maures and Duan. a very common fisheries management strategy is to require ‘catch and release’ of wild fish (typically © 2006 by Taylor & Francis Group. 2001. of the status of the IGF/IGFBP/IGFreceptor system.. LLC . Understandably. Weber and Sullivan. it has not yet been defined whether such impacts of cortisol on IGFBPs and growth might be prolonged. Of all of the components of the growth endocrine system.. 185 handling (contact by touching their tails) leads to >50-fold elevations in serum cortisol concentrations and a dramatic increase in a 30 kDa IGFBP . when serum from these animals is added to cultures of cartilage explants (added at 5% volume). Beckman et al. even when IGF-I levels are not yet reduced. 2001. Seidelin et al. and many other physiological systems (for fish references on these subjects: McCormick et al. at all moments. It is ubiquitous.. 2002.. SUMMARY COMMENTS With the general aim of this chapter to discuss the present field of work on fish IGFBPs. Mohan and Baylink.Kevin M. central nervous system. 2004. 1995... IGF-I-mediated 35Sproteoglycan synthesis is substantially suppressed (compared with that in the presence of 5% control serum). Thus.

but they may also include xenobiotic compounds. where they may be repeatedly caught and stressed? What are the long-term effects on growth and health? Does catch and release make sense for some fisheries? Perhaps for some species. therefore.. The human HOX gene family.. is it really effective when one considers the impact of the catch-associated stress? As cortisol levels subside after a catch and release experience. References Acampora.S. how long does it take before IGFBPs return to normal? Further. Acknowledgments The authors thank the following persons from the Endocrine Lab at California State University at Long Beach. contributed to the content presented herein: James T. IGFBPs in particular. and Boncinelli. Haigwood. Julie Roth. R/F-192. Morelli. © 2006 by Taylor & Francis Group. Although the hypothalamo-pituitary-interrenal axis (when activated. but not others? Finally. Acids Res. and in part by the California State Resources Agency.S. project no.. D’Esposito. results in cortisol production and release into serum) is indeed influenced by a diversity of external and internal factors. Faicella. M. who in several different ways. Such factors may include other internal regulatory systems.. A. Stornaiuolo. A. Greg Nicholson. metals. E. Several of the reviews cited above discuss these aspects. Nucl.. Migliaccio. and recognition. what does this mean for animals in heavily-fished regions.. will further develop their utility... Nigro. through continuing research. This work was supported in part by the National Sea Grant College Program of the U. LLC . intended to protect marine and freshwater fisheries. It is. through the California Sea Grant College Program. Support was also received through N. V. Alex Gavrila. Mark Jamison. a wide variety of other factors can directly impact the IGF system. grants IBN-9600783 and IBN0115975.F. F. Pannese. more than cortisol can affect the IGF/IGFBP/IGF-receptor axis. Department of Commerce’s National Oceanic and Atmospheric Administration under NOAA Grant no. Simeone. and Shari Smolko. as effective ‘beacons’ for the status of growth endocrine systems in fish. D. or other environmental contaminants. 2002-2003 NA06RG0142. M. such as sex steroids or prostaglandins or growth factors. A. E. 17:10385-10402.186 Fish Endocrinology below a certain body size). our belief that IGFBPs. However. 1989.

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LLC .SECTION 2 Gastro-Entero-Pancreatic (GEP) System © 2006 by Taylor & Francis Group.

Authors’ address: Laboratory of Cell Biology and Histology.CHAPTER 7 The Endocrine Pancreas of African Lungfish: Light and Electron Microscopic Immunocytochemistry and Morphology Dirk Adriaensen* and Jean-Pierre Timmermans ABSTRACT Although the endocrine pancreas is well documented in many vertebrate species. but also the ultrastructure of the different endocrine cell types. and their expression of regulatory peptides in the endocrine pancreas and gut of the African lungfish Protopterus aethiopicus. data on lungfish are very limited. LLC . and to our knowledge no electron microscopic morphological or immunocytochemical data are available. *Author for Correspondence: E-mail: dirk. Belgium. Groenenborgerlaan © 2006 by Taylor & Francis Group. B-2020 Antwerpen.adriaensen@ua. Department of Biomedical Sciences. Light-microscopic (LM) and electron-microscopic (EM) immunohistochemistry and morphology were used to investigate the presence and University of Antwerp.

and pancreatic polypeptide (PP). whereas at least four different cell types have been described in most other vertebrate groups. and PP (F) © 2006 by Taylor & Francis Group. and the specific morphology of the endocrine granules in immuno-EM preparations. immunocytochemistry and EM strongly suggest that the entire islet population in Protopterus is represented by three endocrine cell types. SOM cells revealed DCVs of 150-400 nm diameter with an homogeneous core. The DCVs of GLU/PP cells were in the 100-250 nm diameter range. SOM-LI cells appeared to be randomly distributed throughout the islets. SOM-LI cells were far less numerous and scattered throughout the islets. pancreatic polypeptide (PP) and somatostatin (SOM). and cells containing GLU-LI were limited to the islet margins. INS-LI cells were mainly found in a palisade-like arrangement covering the central part of the islets.200 Fish Endocrinology The results demonstrate that the four regulatory peptides commonly detected in the mammalian endocrine pancreas. D. insulin (INS). i. staining of consecutive sections revealed that GLU and PP are expressed in the same cells. In addition. Endocrine pancreas. In conclusion. EM immunocytochemistry also revealed three clearly different endocrine cell types in the pancreatic islets of Protopterus. insulin (INS). B. facilitated the recognition of these three cell types in sections processed for EM morphology.. Besides in the islets. Islet organs. LLC . Electron microscopy.or PP-LI cells. glucagon (GLU). Immunocytochemically. Light microscopy. were surrounded by a small rim of GLU. while their processes were seen to contact the different endocrine pancreatic cell types. respectively A. this volume). were immunologically discernible in this dipnoan fish. The typical location of the cell types.e. Ultrastructurally. Protopterus. confirming the LM observations. INTRODUCTION At least four different populations of endocrine cells have been described in the endocrine pancreatic islets of many vertebrate species (see: Agulleiro et al.. endocrine epithelial cells immunoreactive for each of the four islet hormones could be detected in the epithelium of the midgut. these cell types have been shown to contain glucagon (GLU). LM immunocytochemistry showed that the large numbers of INS-like immunoreactive (LI) cells that populate the centre of the pancreatic islets. Lungfish. INS cells showed dense-cored vesicles (DCVs) with a maximal diameter of 100-250 nm and a flocculent core revealing a highly variable electron density. also revealing a highly variable electron density. Key Words: Immunocytochemistry. somatostatin (SOM). with a very homogeneous core that was mostly highly electron dense. although a minority was weakly contrasted. Hormones.

(2) the light microscopic immunocytochemical localization of the four typical islet hormones. LLC . Some light microscopic immunocytochemical information about different islet hormones has been reported for the lungfish Protopterus aethiopicus (Scheuermann et al. © 2006 by Taylor & Francis Group. 1979. 1979).. Bonner-Weir and Weir. including the pancreas in its wall (Fig. Youson and Al-Mahrouki. (3) the immuno-electron microscopic visualization of these hormones.1). 1991) and Protopterus annectens (Tagliafierro et al. Bonner-Weir and Weir. Despite the ever-increasing investigations in the field of comparative islet histology and the classification of lungfish islets as a separate evolutionary type (for review see Epple and Brinn. were dissected out and immersed in a cooled fixative dependent on further processing.Dirk Adriaensen and Jean-Pierre Timmermans 201 cells. They were fed thrice a week with pieces of heart muscle or liver and appeared to behave normally. The topographic location of these cell types varies from species to species (for reviews see Erlandsen et al. and to our knowledge no electron microscopic morphological or immunocytochemical data are presently available. MATERIALS AND METHODS Animals Adult specimens of the African lungfish Protopterus aethiopicus were kept separately in aerated low-water aquaria at approximately 22°C. 1976.. Anaesthesia and Dissection The animals were anaesthetized and sacrificed by injection of an overdose of Nembutal in the perivisceral cavity. The digestive tract was removed and tissue blocks containing the distal portion of the foregut and the proximal midgut. National and international principles of laboratory animal care were followed and the experiments were approved by the local ethics committee of the University of Antwerp. 1975. and (4) the ultrastructural characterisation of the different types of endocrine cells in the endocrine pancreas of the lungfish Protopterus aethiopicus.. 1999). The main aims of the present study were to provide a more complete report on: (1) the macroscopic and light microscopic features. 7. 1996). data concerning the endocrine pancreas of lungfish are very limited.

As a positive control identically processed rat pancreas as well as intestinal mucosa of Protopterus were used. Jackson 016-070-084) or a streptavidin-biotinylated horseradish peroxidase complex (diluted 1:200. or with rabbit antisera against porcine pancreatic GLU (diluted 1:200. Staining controls were performed by omitting the incubation step with the primary and/or secondary antibodies or by substituting non-immune sera for the antibodies. the sections were further processed using one of the following secondary antisera: AMCA-conjugated goat antiguinea-pig IgG (diluted 1:100. The Netherlands). Belgium. Consecutive 5 m sections were mounted on gelatine-coated glass slides. Jackson 106-065-003). Uden. Chemicon AB1976. © 2006 by Taylor & Francis Group. Light microscopic immunocytochemistry: Sections of the proximal midgut at the level of the pancreas were deparaffinized and incubated for 30 min with normal goat serum (diluted 1:5. dehydrated in ethanol. After rinsing in PBS. Dako A0619) and synthetic human SOM-14 (diluted 1:300. Jackson 111-095-144) or biotinylated goat anti-rabbit IgG (diluted 1:200.and FITC-labelled preparations were mounted. FITC-conjugated goat anti-rabbit IgG (diluted 1:100. ICN Biomedicals. Dako A0565 or Affiniti GA 1181. Dako A0564). ICN152890. biotinylated goat anti-guinea-pig IgG (diluted 1:200. Fluka 57595. Belgium). Sigma-Aldrich. Jackson 111-095-144). Heule. while the biotinylated preparations were incubated in Texas Red-conjugated streptavidin (diluted 1:100. After rinsing in PBS.202 Fish Endocrinology Light and Fluorescence Microscopy The intestinal segments were fixed (2h. or used for immunostaining. Belgium. deparaffinized. Biognost. Sigma-Aldrich). 4°C) in Bouin’s solution. Jackson 016-030-084).4). Fluka 72792. Bornem. Primary incubation was performed for 15-20 h at room temperature with a guinea-pig antiserum against porcine pancreatic INS (diluted 1:500. and either observed unstained. Belgium). pH 7.01M. human recombinant GLU. Sanbio. cleared in xylene. The specificity of the antisera was tested by preabsorption with an excess (50 g/ml diluted antiserum) of antigen (porcine pancreatic INS. Heverlee. followed by the use of DAB as a chromogen. Belgium). Sigma S9129. Dako X0907. stained by a routine trichrome staining. the AMCA. and embedded in paraffin. Jackson 106-155-003. After rinsing in phosphate buffered saline (PBS) (0. LLC . SOM-14. synthetic human PP (diluted 1:200. all sections were mounted and examined in a Zeiss Axiophot microscope equipped for light and fluorescence microscopy. AsseRelegem. TebuBio. Boechout. human PP.

The normal sera and primary antisera used. Ultrathin sections mounted on Pioloform-coated (single incubation) or uncoated nickel grids were floated face down on drops of solution placed on a paraffin film. TebuBio. whereas those labelled with anti-INS were incubated in biotinylated goat anti-guinea-pig IgG (diluted 1:200) after rinsing in TBS followed by a 2 h incubation in 15 nm goldlabelled streptavidin (diluted 1:50. washed in distilled water and successively incubated in normal goat serum and the primary antisera. Once the immunocytochemical procedure was finished. which are visible with the naked eye. contrasted with uranyl acetate and lead citrate. 1 h. Electron microscopic morphology The tissue specimens were fixed in 3% glutaraldehyde buffered with sodium cacodylate (overnight. © 2006 by Taylor & Francis Group. -GLU. the grids were air-dried. and embedded in Durcupan. divided into 1 mm³ pieces and immersed in a cooled fixative dependent on further processing. fixed with 2% glutaraldehyde for 5 min and rinsed again in distilled water. room temperature). Electron microscopic immunocytochemistry The tissue blocks were fixed in Zamboni’s solution containing 0.05 M. postfixed in 2% OsO4 buffered with veronal acetate (1 h.GAR10. and their dilutions and incubation times were the same as for light microscopic immunocytochemistry. contrasted en bloc in 0. were dissected out of adult specimens of Protopterus aethiopicus. room temperature).STP15. British Biocell EM. or –PP.5% uranyl acetate (veronal acetate buffer. dehydrated in ethanol and embedded in Durcupan. dehydrated in acetone.4). Belgium). rinsed in distilled water. They were washed in TBS.Dirk Adriaensen and Jean-Pierre Timmermans 203 Transmission Electron Microscopy Principal pancreatic islets. pH 7. were incubated for 2 h in 10 nm gold-labelled goat anti-rabbit IgG (diluted 1:50. the sections on grid labelled with anti-SOM. and viewed in a Philips CM10 transmission electron microscope. British Biocell EM. The sections were placed in 10% hydrogen peroxide for 5 min. For double incubation immunocytochemistry. the same procedure was then repeated with the opposite side of the grid face down on the series of drops. After thorough rinsing in TRIS-buffered saline (TBS) (0. 4°C). 4°C). Boechout. Ultra-thin sections were mounted on Formvar-coated copper grids. contrasted and viewed similarly as for electron microscopic morphology. TebuBio).05% glutaraldehyde (2 h. LLC .

In crosssections of this intestinal segment. © 2006 by Taylor & Francis Group. 7.204 Fish Endocrinology Preabsorption controls and staining controls were identical to those applied for light microscopic immunocytochemistry. The Fig. the pancreas appeared lunar. RESULTS General Aspects (Fig. Especially note the organization of exocrine and endocrine pancreas.1 Schematic representation of a transverse section through the proximal midgut of Protopterus aethiopicus. LLC . 7.1) The pancreas of Protopterus aethiopicus was located in the wall of the proximal midgut between the layers of the tunica muscularis.

were seen.4.2 Low-magnification light microscopic overview of an unstained paraffin section through the proximal midgut. the endocrine islets could easily be recognized. L: lumen of proximal midgut. M: mucosal layer. The principal endocrine islet was clearly outlined because it contained far less pigmented cells than the exocrine pancreatic tissue (Fig. x 6. blood cells or their precursors.2.1.3) Unstained sections (Fig. 7. Light and Fluorescence Microscopy Routine light microscopy (Figs. which were often grouped in islet-like structures close to or partly within the pancreas. 7. It measured up to 2 mm in diameter and was mostly visible with the naked eye as a round or oval yellowish spot at the surface of the fresh pancreas. which will not be discussed separately in the present study. since they were separated from the exocrine pancreas by a prominent connective tissue capsule. 7. The cytoplasm of endocrine cells was lightly stained compared to that of exocrine cells (Fig. 7. The imaged area is similar to the upper part of the scheme in Fig. © 2006 by Taylor & Francis Group. 7.2) of Protopterus gut and pancreas clearly revealed the high numbers of melanocytes and variably pigmented lymphoid cells. Sometimes a variable number of small accessory islets. Note the high numbers of melanocytes and variably pigmented lymphoid and blood-forming tissues (BFT). The principal endocrine islet (EN) is clearly outlined because it contains far less pigmented cells than the exocrine pancreatic tissue (EX). 7. LLC .3).Dirk Adriaensen and Jean-Pierre Timmermans 205 endocrine tissue of the pancreas was grouped in a large principal islet organ. The endocrine cells Fig.2). Also in trichrome stained sections. 7. often referred to as ‘Brockmann body’ in other fish groups.

4b).3 Light microscopic overview of a principal islet in a section of the pancreas of Protopterus. GLU-. PP. 7.206 Fish Endocrinology Fig. however.8) The pancreatic endocrine islets of Protopterus were found to harbour INS-. 7. The cytoplasm of endocrine cells is lightly stained compared to that of exocrine cells. intertwined with blood capillaries.and SOM-like immunoreactive (LI) cells.4-7. be made between different cell types. 7. separated from one another by blood capillaries (Figs.4a) showed that the large core of the islet was filled with INS-LI cells. The endocrine islet (EN) can easily be recognized. © 2006 by Taylor & Francis Group. Higher magnifications disclosed that the INS-LI cells were columnar or polygonal and appeared to be arranged in cords. Immunocytochemistry of the endocrine pancreas (Figs. especially at one or more poles contacting the blood capillaries (Fig. 7. 7. The INS-LI tended to be accumulated at the periphery of the cells. showing an apparently multilayered ciliomucous epithelium. as it is separated from the exocrine pancreas (EX) by a prominent connective tissue capsule (CT). Trichrome staining.4b. Overviews of the endocrine islets immunostained for INS (Fig. No clear distinction could. 7. appeared to be arranged in cords. LLC . x 35. especially in the central part of the islets.5a). The same sections as used for studying the endocrine pancreas invariably also contained a crosssection of the proximal midgut. leaving only a small peripheral layer with INS-negative cells.

while GLU-LI appears to be present in cells of the peripheral rim only. Sections through a principal islet organ immunostained (DAB) using an antiserum against INS.5a. No colocalization of the two peptides can be seen. 7. surrounding the core filled with INS-LI cells. No difference in immunoreaction was observed between the different GLU antibodies. the GLU cells appeared more irregularly shaped and smaller than the INS cells (Figs. in one section of the endocrine pancreas of Protopterus. 7. EX: exocrine pancreas. The number of intensely black pigmented cells is lower in the endocrine islet than in the exocrine pancreas (EX). (b) Higher magnifications reveal that the INS-LI cells are columnar or polygonal and arranged in cords. P: pigmented cells.4a. separated from one another by blood capillaries (C). and GLU (FITC-labelled in (b). In paraffin sections. © 2006 by Taylor & Francis Group. b: x 140. 7. 7. LLC .6b. (a) Low magnification overview showing a large number of INS-LI cells (stars) in the centre of the islet.5b. A B Fig. The INS-LI is accumulated at the periphery of the cells. 7. 7. INS-LI is limited to cell cords in the centre of the islet. surrounded by a rim of immunonegative cells (asterisks).5b) were exclusively found in the peripheral rim of the pancreatic islets of Protopterus. Simultaneous double fluorescent labelling for INS (AMCA-labelled in (a).b.b. especially at one or more poles contacting the blood capillaries (arrowheads). CT: connective tissue capsule.7) possessed a topographical distribution and morphology similar to the GLU cells. a: x 35.Dirk Adriaensen and Jean-Pierre Timmermans 207 A B Fig. x 100. Grouped endocrine cells revealing GLU-LI (Fig.6a). PP-LI pancreatic endocrine cells (Figs. 7.

7 Immunoreactivity (DAB) for PP in the endocrine pancreas of Protopterus aethiopicus. (a) GLU-LI. 7. SOM-LI cells seemed to be mostly solitary and intermingled with the other pancreatic endocrine cells. Further on.208 Fish Endocrinology A B Fig. Note the colocalization of both hormones in several endocrine cells (asterisks in a and b). 7.and PP-LI (Fig.b: x 360.b. P: pigmented cells.(Fig. a. Using simultaneous double immunofluorescence labelling.8a). they will mostly be referred to as GLU/PP-LI cells.6a. revealing the peripheral localization of PP-LI cells (stars). P: pigmented cells. 7. Light microscopic details of the same area in two consecutive immunostained (DAB) sections of an endocrine islet (EN). Asterisks: Central immunonegative cells. (b) PP-LI. © 2006 by Taylor & Francis Group. most of them revealed one or more processes— Fig. 7. INS-LI could be shown in a cell population clearly distinct from the one containing GLU.5a.6a. EX: exocrine pancreas.b) and PP-LI. 7. x 510. LLC .b). CT: connective tissue capsule. They were of variable size and shape and in paraffin sections. Comparison of the location of immunopositive cells in consecutive sections revealed that most of the peripheral islet cells appeared to colocalize GLU. with a slightly higher concentration at the periphery of the islets (Fig.

8b).b. L: lumen of the midgut. and/or A B Fig. a.b.b) were numerous and. the apical process of which seems to reach the luminal surface (arrows). (a) Light microscopic overview showing several INS-LI endocrine cells (arrowheads) in the multilayered cilio-mucous epithelium of the proximal midgut of Protopterus aethiopicus. P: pigmented cells. 7. INS-LI endocrine cells (Fig.8a. short and thick. LLC . (b) High magnification detail of an INS-LI epithelial endocrine cell. it was obvious that SOM.9a.Dirk Adriaensen and Jean-Pierre Timmermans 209 A B Fig. basally located in the epithelium. 7. a: x 280.9a.b: x 360. sometimes branching—that were seen in close proximity to other endocrine cell types (Fig. Details of pancreatic endocrine islets after immunostaining (DAB) for antiSOM. or slender.and GLU/PP-LI cells were largely confined to different cell populations. Many of the cells possessed a long apical process. © 2006 by Taylor & Francis Group. 7. The scattered immunoreactive cells have variable sizes and shapes and many of them reveal cell processes (arrows). Immunocytochemistry of enteroendocrine cells (Figs. 7. CT: connective tissue capsule of the islets. P: pigmented cells. 7.9-7. which often seemed to reach the luminal surface. endocrine epithelial cells immunoreactive for each of the four islet hormones could be detected in the cilio-mucous epithelium of the midgut.12) Besides in the endocrine pancreatic islets. EX: exocrine pancreas. for the major part. In consecutive sections. b: x 650. E: epithelial layer.

E: cilio-mucous epithelium.210 Fish Endocrinology Fig. and showed processes similar to the INS-LI cells.12) could be seen. Simultaneous double labelling yielded no colocalization between INS. mostly located between the basal epithelial cells. x 230. © 2006 by Taylor & Francis Group. P: pigmented cells. shorter basal processes.and PP-LI in the endocrine cells of the intestinal mucosa of Protopterus.11) and GLU-LI cells (Fig.10) were also abundant.12 GLU-LI mucosal endocrine cells (arrows). PP-LI endocrine cells (Fig. x 280. 7. x 390.(Fig. LLC . 7.10 PP-LI mucosal endocrine cells. L: lumen of the midgut. 7. Fig. Finally. 7. CT: connective tissue. 7. L: lumen of the midgut. some SOM.11 SOM-LI mucosal endocrine cell (arrow) in the cilio-mucous epithelium (E) of the midgut of Protopterus.and PP-LI in endocrine cells of the epithelial lining of the gut. L: lumen of the gut. P: pigmented cells. Fig. There was no evidence for colocalization of GLU. 7.

7.17) Transmission electron microscopic immunocytochemistry revealed three clearly different endocrine cell types in the pancreatic islets of Protopterus (Fig.13a.16.13-7.b. Transmission Electron Microscopy Immunocytochemistry (Figs. represented by 10 nm gold particles. The cells usually revealed a palisade-like arrangement and were in close proximity to blood capillaries with one or more poles (Figs. Low-magnification electron micrographs of a section of an endocrine islet in the pancreas of Protopterus aethiopicus treated for immunocytochemistry. 7. 7. At high magnifications.Dirk Adriaensen and Jean-Pierre Timmermans 211 Immunocytochemical control experiments Omission of the primary or secondary antibodies. LLC .14b. © 2006 by Taylor & Francis Group. 7. 7. 7. 7.13a. (b) The outer rim of the islet harbours mainly GLU/PP (A) and SOM cells (D). was found over the secretory granules of irregularly formed cells scattered throughout the A B Fig. b: x 820. represented by 15-nm gold particles. (a) Central part of the islet composed of strands of mainly INS cells (B) and capillaries (C). All controls performed supported the specificity of the reported immunocytochemical data. Note the connective tissue capsule (CT) of the islet.13a. the core of the endocrine granules appeared poorly delineated (Figs. as well as preabsorption of the primary antisera with an excess of antigen.17). with pigmented cells (P) and blood-forming tissue.b) INS-LI.14a). resulted in negative staining controls in all immunocytochemical procedures performed. was selectively located over the dense cores of the specific endocrine granules (dense-cored vesicles (DCVs)) of most of the endocrine cells covering the central part of the islet. 7. a. SOM-LI.

but their relationship to capillaries was often less obvious than for INS-LI cells (Fig. b: x 83.600. and more clearly delineated than those of INS cells (Figs. 7. © 2006 by Taylor & Francis Group. 7.15a.b. 7. more homogeneously electrondense. entire islets (Figs. 7. neighboured by capillaries (C).b.b. represented by 15-nm gold particles that are mainly located over the endocrine secretory granules (dense-cored vesicles (DCVs) a: x 2. SOM-LI cells were always in close contact to other endocrine cell types.500.14a. (b) High-magnification detail of the INS immunoreactivity. b: x 83. a: x 2.212 Fish Endocrinology A B Fig.15a).13b. LLC .17). (a) Overview of a SOM-LI cell (D) in the central part of an endocrine islet.15a. 7. (b) High-magnification detail showing the specific localisation of immunoreactivity (10-nm gold particles) over the DCVs. 7.500.and INS-LI cells in double-incubated sections showed that the cores of SOM-LI granules were larger.16a. surrounded by INS cells (B). 7.b) but clearly less numerous than the INSLI cells. because no limiting membranes of the granules were A B Fig. (a) Overview of a strand of INS-LI cells (B). High magnification of the contact zone between SOM.600. However.

© 2006 by Taylor & Francis Group. (b) High magnification detail. The electron micrograph shows parts of an INS. 7. b: x 83.100. 10-nm gold). neighbouring a SOM-LI cell (D) with 10-nm gold particles over the granules. Simultaneous double immunocytochemical staining of endocrine cells in electron microscopic sections of a pancreatic islet of Protopterus aethiopicus. (a) Overview. a seemingly homogeneous population of endocrine cells. 15-nm gold) and SOM-LI cell (D. Electron micrographs showing parts of an INS-LI cell (B) with 15-nm gold particles over the DCVs. contained. 7. LLC . All endocrine cells in the centre of the islets appeared to belong either to the INS.17. however.200. and of an immunonegative GLU/PP cell (A).(B.500.or to the SOM-LI population. The INS-negative rim of the islet. corresponding to the GLU/ PP-LI cells demonstrated with light microscopic immunocytochemistry. a: x 35. besides SOM-LI cells. it was impossible to measure the diameter of the granules. visible in these immunocytochemical sections. Fig. x 36.Dirk Adriaensen and Jean-Pierre Timmermans 213 A B Fig.b.16a.

7. the basement membranes and a fenestrated endothelium (Fig. Mostly columnar INS cells were always seen in the centre of the islets. containing mainly INS cells.214 Fish Endocrinology These cells harboured endocrine granules.21) The typical location of the different endocrine cell types. and the marginal rim.17). in contrast to the lightmicroscopic immunocytochemical sections and to the INS and SOM antisera. sometimes containing crystalline-like inclusions (Fig.19a-c). The DCVs. When seen in close contact to a blood capillary.18e).18–7. Nerve fibres. with a maximal diameter of 100-250 nm.19c). together with a clearly recognizable morphology of the endocrine granules.18b. the cores of which were homogeneously dense and well delineated. were frequently seen to contact INS cells (Fig.18d). SOM cells were readily recognizable by their content of numerous large DCVs with a maximal diameter of 150-400 nm. At the border between the centre of the islets. SOM-LI and unlabelled GLU/PP cells (Fig. and were separated from the lumen of the latter only by a narrow interstitial space composed of few collagen fibres. spots could be found showing closely apposed INS-LI. 7. 7. 7. identified by light microscopic immunocytochemistry and confirmed using electron microscopic immunostaining. enabled us to distinguish the three cell types in sections processed for electron microscopic morphology. Unfortunately.e).c. © 2006 by Taylor & Francis Group. DCVs contacting the surface membrane of INS cells neighbouring a capillary. The morphology of the endocrine granules served as an additional differentiation between the three different cell types. mostly containing many clear vesicles and some DCVs.18a).20a). This endocrine cell type appeared solitarily between INS cells in the centre of the islets (Fig. 7.19a) or between the cells of a third morphological type in the outer part of the islets (Fig. features of exocytosis of DCVs as well as a fenestrated endothelium could be noted (Fig. 7. 7. LLC . they displayed a palisade-like arrangement and close apposition to blood capillaries (Fig. Morphology (Figs. 7. the core of which was homogeneously fine-granular with a highly variable electron density (Fig. as well as omega-shaped membrane invaginations were indicative of the exocytosis of the content of the endocrine granules. 7. containing GLU/PP and SOM cells. 7. but markedly smaller than those of SOM cells. none of the GLU or PP antisera used has resulted in a positive immune reaction in the tissue sections used for electron microscopic immunocytochemistry. revealed a flocculent core of varying electron density.

GLU/PP cells were located at the periphery of the islets only (Fig. a: x 3. The DCVs of GLU/PP cells.700. LLC . however. b. revealed a homogeneous core that.21d). (c) High-magnification detail revealing typical crystalline-like inclusions in some of the DCVs.000. the SOM. d: x 10. High magnification of neighbouring cells allowed to differentiate between the three populations of endocrine cells in the pancreatic islets of Protopterus aethiopicus. 7. C: capillaries. (a) Low-magnification overview. or the GLU/PP cell population (Fig.500.18a-e. © 2006 by Taylor & Francis Group. (d. 7. Electron microscopic morphology of INS cells (B) in a principal endocrine islet of the pancreas of Protopterus. In sections processed for electron microscopic morphology. So far. c: x 59. in general showed the highest electron density of the three endocrine cell types (Figs. the maximal diameter of the DCVs was 100-250 nm. all of the endocrine cells observed seemed to belong either to the INS. 7. 7. and as such comparable to that of INS cells.Dirk Adriaensen and Jean-Pierre Timmermans 215 A B C D E Fig.700.21a-d).20b. e) Parts of INS cells (B) neighbouring a capillary (C). 7.20a). (b) Detail of the DCVs of INS cells. Note the fenestrated endothelium (arrowheads in d) and a nerve ending (N in d). arrow: nerve ending.e: x 24.c. although variable. with a maximal diameter of 100-250 nm and a flocculent core of varying electron density.

A B C Fig. Electron microscopic morphology of SOM cells (D) in a principal endocrine islet. (a) Low-magnification overview. A granule is extruded from the endocrine cell (arrow). 7. LLC . Note a neighbouring INS cell (B).c: x 24.300. b.700.19a-c. A granule is extruded from the endocrine cell (arrow). b: x 24. 7. (b) Detail of the DCVs of SOM cells. Electron microscopic morphology of GLU/PP cells (A) in a principal endocrine islet. (b) Detail of the DCVs of GLU/PP cells. (c) A capillary with fenestrated endothelium (C) close to a GLU/PP cell. © 2006 by Taylor & Francis Group. Note a neighbouring SOM cell (D).700. with a maximal diameter of 150-400 nm and a homogeneous core of highly variable electron density. c: x 33.216 Fish Endocrinology A B C Fig. (a) Low-magnification overview. a: x 3. a: x 3.300.000. (c) SOM cell near a capillary with fenestrated endothelium (C).20a-c. with a maximal diameter of 100-250 nm and a homogeneous core of highly variable electron density.

a. Comparison of the electron microscopic morphology of the DCVs in neighbouring endocrine cells of different types in a principal endocrine islet of the pancreas of Protopterus aethiopicus. It was confirmed that the exocrine pancreas lies in the intestinal wall and is marked by an especially strong and even distribution of true melanophores (Epple and Brinn.Dirk Adriaensen and Jean-Pierre Timmermans 217 A B C D Fig. but also the ultrastructure of the different endocrine cell types. The general organization and classical histological characteristics of the exocrine and endocrine pancreas described here appear to be largely similar to those reported for Protopterus annectens (Gabe. 1969. and their expression of regulatory peptides in the endocrine pancreas and midgut of the African lungfish Protopterus aethiopicus. LLC .500. Tagliafierro et al.700. 1996). 1975). 7. an observation that may © 2006 by Taylor & Francis Group. (a) INS (B) and SOM cells (D).. SOM (D) and GLU/PP cells (A) near a capillary (C). (d) Processes of INS (B). Discussion Light and electron microscopic immunohistochemistry and morphology were used in the present study to investigate the presence and distribution. (c) INS (B) and GLU/PP cells (A). (b) SOM (D) and GLU/PP cells (A).21a-d. b: x 10. c. d: x 24.

that in the rather primitive endocrine pancreatic organ of Protopterus aethiopicus. A first interesting finding was. Nevertheless. and GLU and PP mainly © 2006 by Taylor & Francis Group.. observed in the central part of the principal islets in the endocrine pancreatic organ of Protopterus aethiopicus. Using antisera against the mammalian peptides INS. SOM and PP the present study has demonstrated these four main pancreatic peptide . Negative staining in all immunocytochemical control experiments further indicated that the observed staining was very likely due to the presence of the different pancreatic hormones. In the Australian lungfish. Gabe. surrounded by a conspicuous connective tissue capsule (this study. the term ‘-like immunoreactivity (LI)’ has been used throughout the manuscript. no Brockmann bodies were present and the islet tissue was found throughout the dispersed exocrine tissue. SOM and PP apparently cross-react with the respective antibodies against mammalian hormones. The large number of INS-LI cells. The endocrine pancreatic tissue of Protopterus is mainly concentrated in one or just a few large (millimetre sized) islets. In a primitive actinopterygian fish. because they contain far less pigmented cells than the surrounding exocrine pancreas. with only limited distinct islet accumulation (Youson and Al-Mahrouki. therefore. correspond to a fair degree with the pattern described in many other vertebrates. even in unstained sections. The only other vertebrates known to have such encapsulated endocrine pancreatic islets are teleosts (Brockmann bodies). As already pointed out by Gabe (1969). Neoceratodus. the variably pigmented blood-forming and lymphatic islets seen around and partly within the pancreas are by no means restricted to the pancreatic area.and PP-LI cells. Since the latter have not been fully characterised yet for lungfish. with INS and SOM cells concentrated in the centre of the cell groups. 1999). INS cells were found in the islet centres and GLU cells peripherally. the general distribution of the endocrine pancreatic hormones seems rather similar to other fish. as was apparently also the case for SOM cells (Hansen et al. hormones in endocrine cells of the pancreas of a lungfish. 1987). GLU. and the surrounding small peripheral rim of GLU. clustered GLU and SOM cells were seen dispersed in the exocrine parenchyma in Neoceratodus.218 Fish Endocrinology be extended by the notion that the principle endocrine islets can easily be recognized. In addition. GLU. LLC . 1969). More particularly. the bowfin (Amia calva). a similar light-microscopic immunocytochemical distribution pattern of endocrine cells was described in large and medium sized islets in the pancreas of Protopterus annectens (Tagliafierro et al. 1996).. INS.

1988). 1999). 1982. such as amphibians (Kaung and Elde. teleosts (Abad et al. the biological importance of such local paracrine effects remains to be investigated in dipnoan fish. and PP-immunoreactive cells in the islet organ of Protopterus (see also Tagliafierro et al. Since SOM is known to be a potent inhibitor of secretion from many different endocrine cells. 1985) and in mammals (Larsson et al. and reptiles (Rhoten.. Tagliafierro and colleagues (1996) reported some overlap. we would like to refer to an excellent review paper on the phylogenetic development of the endocrine pancreas (islet organ) in fish (Youson and Al-Mahrouki. For other fish groups. The occurrence of cells having only PP. 1981. 1985). LLC . 1996) could have a functional significance and suggests a paracrine type of regulation on neighbouring cells. and revealed many processes that were seen in close proximity to the latter. Such a coexistence was found in foetal mammalian pancreatic A cells (Ali-Rachedi et al. El-Salhy et al. Except for the work of Tagliafierro et al.. Vaillant and Taylor. species differences exist regarding the coexistence of PP and GLU in the same pancreatic islet cells. the colocalisation of PP and GLU immunoreactivity within the same pancreatic endocrine cells appears to be a more common feature. 1987). 2001). 1980... (1996) the localization of endocrine cells in the gut epithelium of lungfish. 1979). the outlines of the GLUpositive peripheral rim match the PP positive as well as the INS negative rim exactly. Buchan..and PP-LI are unequivocally colocalized in numerous endocrine cells of the islet organ of Protopterus aethiopicus. 1981. In consecutive sections of the endocrine pancreas of Protopterus aethiopicus. viewed at a low magnification.. This close anatomical association of SOMimmunoreactive cells with INS-. but also that both the GLU. GLU-. In lower vertebrates. and more in particular © 2006 by Taylor & Francis Group.. High magnification revealed that GLU and PP cells not only have a similar topographic location at the periphery of the endocrine gland structure. 1984). and by a few authors in adults (Rombout et al. 1987).or GLU-LI in the islet organ of Protopterus aethiopicus cannot be excluded because of the lack of immuno-electron microscopic data. in addition to endocrine functions which could be similar to those found in cartilaginous fish (Tagliafierro et al. Within other vertebrate groups.. SOM-LI cells (D cells) appeared to be randomly distributed between the A/F and B cells.Dirk Adriaensen and Jean-Pierre Timmermans 219 colocalized in cells at the periphery (Youson et al. Kaung. but certainly not a complete co-expression of GLU and PP immunoreactivity in the islets of Protopterus annectens.

and Brinn. Varndell. Comp. GLU/PP).A. imaging and illustrations. 3:579-589. Van Noorden. Vermeiren for technical assistance. De Rijck for help with microscopy. Islet histophysiology: Evolutionary correlations. J. Wilander. Epple. (Teleostei). 70:9-19. Can. R.R. Bonner-Weir. and Abu-Sinna.E. 1982. R. 1979. three cell types with a typical granule morphology.C. R.M. Vindevogel for aid with the manuscript. Acknowledgements The authors wish to thank H.M. islet cell hormones. G. El-Salhy.L. Regulatory peptides in the amphibian pancreas.H. O. Zool.J. In conclusion. Res.P 1976. Ultrastructurally. and Rombout.A. Svensson..E. J. J. LLC . Endocrinol. SOM. Buchan. and Weir.M. the distribution of immunoreactivity for the four major pancreatic hormones in several species of lungfish appears to be relatively similar to that reported in several other fish groups. D. J. 1975.J. A. Taverne-Thiele.. 1985. S. and location within the islet organs.M. G. Comp.. and Polak. 1996). G. Hegre. © 2006 by Taylor & Francis Group. similar to those described in the present study. Erlandsen. S.. Endocrinol.. 27:320-349. 1988. Terloo and G. SOM. A. and Elde. References Abad. Van Daele and D. E. Jr. Gen. M. Spillemaeckers. F. Histochemistry 80:487-491.W.D. A. Histochem. 24:883-897. Endocrine cells showing INS. were also reported in Protopterus annectens (Tagliafierro et al. are now well characterised in Protopterus aethiopicus. De Pauw. Gen. and we would like to refer to the latter work for an elaborate discussion on the phylogenetic correlations of enteroendocrine cells.E... 63:2121-2124. Biomed. Gen.C. Cytochem. S. S. The organization of the endocrine pancreas: A hypothetical unifying view of the phylogenetic differences.. T. J.. Parsons. Comp. 38:28-37. Bloom. J. Ali-Rachedi. I. 1984. has received little attention so far. immunoreactivity (INS. M. Adrian. Distribution of cell types in the islet and evidence for the presence of somatostatin and gastrin within the D cell. The endocrine pancreas of anuran amphibians: A histological and immunocytochemical study. J. Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas.. All of the so far identified islet cells appeared to belong to one of the three identified populations. GLU and PP immunoreactivity.220 Fish Endocrinology Protopterus. D. Immunocytochemical and ultrastructural characterization of coexistence of pancreatic polypeptide and glucagon-like immunoreactivity in the pancreatic endocrine cells of Sparus auratus L. McEvoy. J. Endocrinol.. Pancreatic . Gapp.

Somatostatin cell processes as pathways for paracrine secretion. Immunohistochemical localization of polypeptide hormones in pancreatic endocrine cells of a dipnoan fish.M. Données histologiques sur le pancréas endocrine de Protopterus annectens Owen.-I. Biol. Rehfield. and Jorgensen. 2001. Abad. M.E. 1987.. H. G.. 1981. 1991.and glucagon-immunoreactivity in pancreatic endocrine cells of mouse. J. L. Cell. Hansen.. 1996. L. R. Gen. G. Cell Tissue Res.Dirk Adriaensen and Jean-Pierre Timmermans 221 Gabe. W. Rhoten. Endocrinol. 1980. A. Ontogenetic and phylogenetic development of the endocrine pancreas (islet organ) in fishes. Anat.. J. and Conlon.C.J. Co-localization of glucagon and pancreatic polypeptide in testudine pancreas. N.B. Microsc. Exp.H. J. Timmermans. 1969. 45:204-211. Kaung. Insulin-. M.H. Larsson. The endocrine cells in the gastroenteropancreatic system of the bowfin.-L.H..L. 196:173-181. A. 248:181-185. Carlini.. Morescalchi.. 102:288-298. B. and Mauceri. A..M. M. D. J.C.N.. Tagliafierro. 1981. M. 1987. Morphol. Arch. A. H..M. .: An immunohistochemical. J. Histochemistry 87:1-6. P. J. Rana pipiens.A. Experientia 43:428-430. Mol. Adriaensen. Neoceratodus forsteri. Hansen. F. 250:208-224. Endocrinol.. ultrastructural. Peptides 2 (Suppl. and Al-Mahrouki. Faraldi. A. De Magistris.A.. Amia calva L.. 1985. Anat. Peeze Binkhorst. and Schwartz. glucagon. 2):31-35.W. G. S. Rombout. J.. Comp. Endocrinol. Faraldi. Youson. G. and immunocytochemical analysis.H. Tagliafierro. Science 205:13931395. D. Morphol. 1979. 1999. C. Co-existence of pancreatic polypeptide (PP). Rec.P. 116:303335. Immunocytochemical localization of pancreatic endocrine cells in frog embryos and young larvae.F. Comp. and Elde. Putti. Naumovski. Scheuermann. and Taverne-Thiele. Gen. I.M. Interrelationships between somatostatin-like cells and other endocrine cells in the pancreas of some cartilaginous fish.W. Al-Mahrouki. Protopterus aethiopicus.A. J. Demonstration of carboxyl-terminal PP-like peptides in endocrine cells and nerves. Vaillant. Comp. Goltermann..-L. Distribution and morphometric quantitation of pancreatic endocrine cell types in the frog.-P and De Groodt-Lasseel. 91:185192. Immunocytochemical detection of islet hormones in the digestive system of Protopterus annectens.W. G. R. Della Rossa. 58:21-40. T. Kaung. and Pestinaro. Gen. Acta Histochem. M.and somatostatin-like immunoreactivity in the endocrine pancreas of the lungfish. 31:201-207. Youson. 1987. Fasulo. © 2006 by Taylor & Francis Group. and Taylor.. D.L.. LLC .N.

In particular. Stn. We limit our discussion of glucagon functions to fish. the differential expression of multiple genes in the three main tissues of production. glucagon-like peptide-1 (GLP-1) and GLP-2. As recent experimental advances have provided new and exciting insights into different aspects of proglucagon structure and function. 2 Department of Biochemistry and Microbiology. sites of transcription. Canada. receptor specificities. hormone interactions and functions. B.C. Victoria. University of Victoria. CSC. *Author for Correspondence: E-mail: tpmom@uvic. Canada. We review general topics regarding these related peptides such as gene numbers.CHAPTER 8 Glucagon and Friends Thomas P. especially teleosts. Victoria. Since much less is known about functions of GLP-1 and GLP-2 in Authors’ address: 1Department of Biology. where the hormone appears to be functionally more diverse than in mammalian glucose regulation. targets and not least mechanisms of action. New proglucagon gene sequences provide fascinating insights into evolutionary trends of proglucagon gene structure among fishes. endocrine pancreas. University of Victoria. Mommsen1* and Ellen R. LLC .ca © 2006 by Taylor & Francis Group. 3020. intestine and brain. Busby2 ABSTRACT The proglucagon gene encoding glucagon also contains two closely related hormones. B.C. sites of peptide processing. hints at complex modes of regulating peptide production and differential functions of the three individual peptides and their isoforms in fish. V8P 3N5. PO Box.

Receptors. Because of the narrowed scope. the ‘family’—often called the secretin-glucagon family—contains over a dozen similar peptides (Fig. Glucose regulation. At times. Intestine. but fascinating. 8. Challenges abound. we hesitated to call this selected review ‘glucagon-family peptides’ and ‘functional products of the proglucagon gene’ would have been too unwieldy. driven primarily by the huge potential especially of GLP-1 as a treatment for types I and II diabetes. At this point. LLC . we will restrict our discussion to the main functional products of the proglucagon gene. While overlap in structure. To this end. Key Words: Glucagon. Gene isoforms. This review is not meant to be comprehensive but rather as an introduction to the interesting world of glucagon and associated peptides in the fishes.224 Fish Endocrinology piscine models. receptors. peptides. the presence of half of these name-giving peptides (GIP and secretin) remains to be confirmed for the fishes. many of them associated with the gastrointestinal tract and/or associated with neuroendocrine cells. Teleost. or even where to start. © 2006 by Taylor & Francis Group. Recent evidence has been reviewed. mammalian models have to serve as suitable proxies. which to omit. GLP-1. INTRODUCTION The first stumbling block in writing a review on glucagon and related peptides in fishes is where to draw the line and which hormones to include or more importantly. the only questions are what to tackle first and how the fish models can be approached to best exemplify the power of comparative analyses.1). processing and function between the many members of the family is challenging and fascinating. Gene duplication. the writing will reflect the bias of the authors. Figure 8. GIP and VIP are included in the name of this superfamily. mostly Gsa.1 also presents the current state of knowledge with respect to specific hormone receptors. GLP-2. Brain. the authors are rather pleased about the recent revival of glucagon research. Proglucagon. Glucagon is now generally put into a family of peptide hormones with common structural features and all targeting specific membrane receptors with seven transmembrane domains. alas. intracellularly associated with G-proteins. Brockmann bodies. whose main interests lie in the metabolic actions and evolutionary relationships of these biochemically simple. namely glucagon and the two glucagon-like peptides (GLP-1 and GLP-2). indicating that functions of all three peptides go well beyond the currently accepted roles in metabolism. To a certain extent. Metabolic regulation. Evolution.

reptiles Fig. reptiles 35-mer.Peptide Glucagon GLP-1 GLP-2 GHRH PACAP VIP PTH related protein (PTHrP) Parathyroid hormone (PTH) Peptide His-Ile (PHI) GIP Secretin Exendin Helodermin Helospectin Present in teleosts yes yes yes yes yes yes yes no (?) ? no no no no no Receptor in mammals GR GLP-1R GLP-2R GHRHR VPACs. two genes in fish 31-mer. PTHrPR PTHrPR PHIR GIPR SecretinR GLP-1R VPACs VPACs Receptor in fish yes yes (*) yes VPACs. two genes in fish. coencoded with VIP in mammals 41-mer 27-mer 39-mer. (*) A GLP-2 receptor like sequence is annotated in the Tetraodon nigroviridis database and can be confirmed for Takifugu rubripes (data not shown). 225 © 2006 by Taylor & Francis Group. PAC1 VPACs.1 Members of the secretin-glucagon superfamily of hormones and their G-protein coupled receptors (family 2) in vertebrates. coencoded with PACAP in fish 27 & 38-mers 28-mer 125-mer Thomas P. GIP-glucose-dependent insulinotropic peptide (=gastric inhibitory peptide). Lengths of peptides are variable and are to be taken as guidelines only. PAC1 VPACs PTHrPR PTHrPR n/a no no n/a n/a n/a Comments on hormone 29-mer. multiple products 34-mer. 8. GHRH-growth hormone releasing hormone. single gene in fish 43-mer. VIP-vasointestinal polypeptide. LLC . but its identification as a true GLP-2 receptors remains an outstanding issue. Mommsen and Ellen R. Busby 84-mer 27-mer. PACAP-pituitary adenylyl cyclase activating peptide. reptiles 37 and 38-mers.

It exerted similar actions in all those vertebrates where researchers looked. In addition. encoded by a single gene and produced by the -cells of the endocrine pancreas of vertebrates. highly conserved in the mammals. leading not only to a new appreciation of glucagon per se. 1999). it all changed: glucagon now had two close relatives produced from the same gene with fascinating emerging functions. breaking the short scientific tradition of ‘peptide before gene’ – ‘glucagon’ was a relatively simple story: a short. within ten years. The hormone targeted the liver and generally opposed insulin’s overwhelming metabolic actions.7 billion years after their premiere in the vertebrates (Irwin et al. the proglucagon gene transcription was by no means restricted to the -cells and glucagon’s sphere of influence had extended to other tissues. see: Plisetskaya and Mommsen. thus. hormone interactions and function. Before their arrival—via the emerging molecular methods and. sites of gene transcription.. but every now and again have to mention mammalian models. receptor specificities. to gene transcription and to include different mechanisms of action. We will try to restrict ourselves to the piscine picture. © 2006 by Taylor & Francis Group. resulting in increased rates of hepatic gluconeogenesis and glycogenolysis and adipocyte lipolysis. mechanisms of actions and evolutionary trends alike. the glucagon-like peptides entered the scientific literature. processing sites. targets and not least mechanisms of action. to illustrate concepts or key differences between accepted mammalian dogma and fishy realities. targets. largely from studies on fishes which covered ancient and recent gene or genome duplications. The following is designed to give a brief glimpse of this picture that encompasses gene numbers. Then. some even opposing glucagon. single chain peptide of 29 amino acid residues. About a quarter of a century ago and thus some 50 years after glucagon (for historical aspects. but also to a relatively complex picture. 1996) and about 0. The accepted mechanism of message transduction was via adenylyl cyclase and cAMP resulting in Gsprotein activation and activation of protein kinase A. LLC .226 Fish Endocrinology Even if research on our piscine friends has started to lag behind—yet again—the comparative models will continue to provide key insights into hormone design. interesting and intriguing evolutionary aspects emerged.

1986) and with very small amounts of the N-terminally extended form of GLP-1 (Holst et al.. as pointed out elsewhere. GLP-1 and GLP-2 are released together with IP-2 in an uncleaved nonfunctional precursor fragment known as the ‘major proglucagon fragment’ (MPGF) (Orskov et al. At least theoretically. The teleostean glucagon gene contains six exons.4).3). processed from existing glucagon at a dibasic processing site. glicentin. are a 20 residue signal peptide. The different functional peptides are delineated by their expected dibasic cut sites. the primary protein product is glucagon.. 8. 8. LLC . Mommsen and Ellen R. The physiologically highly potent mini-glucagon (19-29). which encompasses a glicentin-related polypeptide (GRPP – 30 amino acids [aa]) and glucagon (29 aa). Interestingly. the proglucagon prohormone contains more than one biologically active peptide. © 2006 by Taylor & Francis Group. possibly representing a precursor yet to be processed.2). In spite of these low concentrations. 8. In mammalian -cells.4). containing GLP-1 (30 aa). 2002). 1994). are apparent for the fishes (Fig. while glucagon’s effectiveness is usually assessed in the low nanomolar range. Small amounts of oxyntomodulin are also found. Busby 227 PROGLUCAGON GENE STRUCTURE As in the case of the prohormone containing GHRH and PACAP (Parker et al. 8. Fish glucagons contain this key processing site (see below). large differences exist in the peptides produced from proglucagon in mammals between pancreatic -cells and intestinal L-cells (Fig. has been localized to -cells actively secreting glucagon and consists of a small percentage of the concentration of glucagon (Dalle et al. the main products in teleostean pancreatic -cells are glucagon and GLP-1 (see below). but much smaller differences between these tissues. separated by five introns (Fig. leading to the production of different products.. but the actual production of mini-glucagon or a physiological role for it in fishes remains to be demonstrated. an intervening peptide (IP1-6 aa) and a major proglucagon fragment. Coencoded in proglucagon.. 1996). miniglucagon is emerging as an important hormone.Thomas P. since its physiological effects are noted in the high femtomolar range. the intervening peptide-1 of the fishes shows no resemblance the mammalian N-terminal extension. intervening peptide-2 (12 aa) followed by GLP2 (34 aa) (Fig. Fish proglucagon lacks the equivalent of the N-terminally extended form of GLP-1 and only encodes the shorter form of GLP-1 and. neither at the nucleotide nor at the amino acid level (Plisetskaya and Mommsen. if any. starting from its N-terminal. 1993) and thus exemplifying a common theme for members of the secretin-glucagon family of peptides.

Oxytomodulin is a C-terminally extended glucagon that contains IP-1. but broken into its components. © 2006 by Taylor & Francis Group. SP: Signal peptide.2 Fish proglucagon-1 gene structure. For amino acid sequences of some of the corresponding peptides. UTR: Untranslated region. GRPP: Glicentin-related pancreatic polypeptide. where glucagon can be split to produce mini-glucagon. The figure indicates the dibasic site in glucagon (RR). IP-1: Intervening peptide-1. LLC .3. in fishes.228 Fish Endocrinology Intron 1 Intron 2 Intron 3 Intron 4 Intron 5 Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 5 -UTR SP GRPP Glucagon IP-1 RR GLP-1 IP-2 GLP -2 3 -UTR Glicentin Oxyntomodulin Mini-Glucagon Major Proglucagon Fragment Fig. 8. see Fig. 8. the Major Proglucagon Fragment (MPGF) is not produced as such. IP-2: Intervening peptide-2.

* * * * *. Conserved residues between gene products are indicated by asterisks. 2004).G lucagon and IP1 Te t r a o d o n 1 Ta k i f u g u 1 Ta k i f u g u 2 Te t r a o d o n 2 KR KR KR KR ** H S E G T F S N D Y SK Y L E D R K A Q D F VR W L M N N H S E G T F S N D Y SK Y L E D R K A Q D F VR W L M N N H S E G T F S N D Y SK Y L E T R R A Q D F VQ W L K N S H S E G T F S N D Y SK Y L E T R R A Q D F VQ W L K N S * * * * * * * * * * ** * * * ..* * .* * .RR RR RR RR ** E S E T H R R R . Data for Tetraodon nigroviridis were derived from the genomic DNA available at http:// www.* ..*. Amino acid differences in GLP-2s from the two proglucagon genes-1 are bolded. Busby G LP-2 Ta k i f u g u 1 RR H V D G T F T S D V NK V L D S M A A K E Y LL W V M A S K P S G E R Te t r a o d o n 1 RR H I D G T F T S D V NK V L D S M A A K E Y LL W V M T S K P S G E R Fig. proglucagon 2 on chromosome 2 (CHR2-CR642802).. The internal dibasic processing site potentially yielding mini-glucagon is bolded in the glucagon sequences..* . Proglucagon-1 is located on chromosome 3 (CHR3-CR644590).* * * .* *. Contiguous amino acid sequences starting from the processing site adjacent to the N-terminal region of glucagon to the end of the GLP-2 (gene 1) or GLP-1 (gene 2) are presented.3 Functional peptides in proglucagon sequences of two pufferfishes Tetraodon nigroviridis and Takifugu rubripes. LLC .A FS ESETNRRVEAFN E E * Thomas P. Data for Takifugu rubripes are from (Zhou and Irwin. 8.* * .* KR KR KR KR ** SG S A E SG T A E NG S L F NG S L F .fr/cgi-bin/ggb/ggb/tetraodon. 229 © 2006 by Taylor & Francis Group.- G LP-1 and IP2 Te t r a o d o n 1 Ta k i f u g u 1 Ta k i f u g u 2 Te t r a o d o n 2 TR KR KR RR -* H A D G T F T S D V SS Y L K D Q A I K D F VA R L K A G Q A H A D G T F T S D V SS Y L K D Q A I K D F VA R L K A G Q V H A D G T Y T S D V ST Y L Q D Q A A K E F VS W L K T G P G H A D G T Y T S D V SA Y L Q D Q A A R E F VS W L K T G R G * * * * * * * * * * *. Mommsen and Ellen R.genoscope.cns. Bioactive peptides are underlined. Other dibasic cut sites separating peptides are also indicated.

Like other prohormones in the glucagon superfamily such as secretin. 1997.. In Xenopus laevis. Processing of proglucagon into mature active peptides relies on action of prohormone convertases (PCs). respectively (Figs. VIP and PACAP (Sherwood et al. Considering specific gene structure and sequence similarity between glucagon and the two GLPs. 2000). amphibian proglucagons are altered from the depicted pufferfish structure in that they contain a duplication of the exon encoding GLP-1. including proinsulin.230 Fish Endocrinology Mammal Teleost Pancreatic -cells Glucagon GRPP Glicentin GLP-1/IP-2/GLP-2 (MPGF) N-terminally extended GLP-1 Mini-glucagon Intestinal L-cells Oxyntomodulin GLP-1 GLP-2 GLP-1/IP-2 N-terminally extended GLP-1 Oxyntomodulin GLP-1 GLP-2 see text no equivalent in fish Glucagon GLP-1 Oxyntomodulin GLP-2 (?) no equivalent in fish ? (see text) Fig. prosomatostatin © 2006 by Taylor & Francis Group.4 Peptide processing products of the proglucagon gene pancreas and intestine of mammals and teleostean fish. 8. GLP-1 and GLP-2. Irwin and Sivarajah. 4 and 5 completely accounting for glucagon..3). LLC . For instance. The proglucagon precursor structure is not completely consistent throughout all the vertebrates. proglucagons display functional units that are contained in single exons as clearly seen in our analysis of the pufferfish Tetraodon nigroviridis genome with exons 3. the latter peptides most likely originated through exon duplication of glucagon (Irwin et al. a hypothesis confirmed through an analysis of associated genes present in the vicinity of fish proglucagons (Zhou and Irwin. an additional exon duplication has resulted in three consecutive GLP-1 sequences (Irwin et al. 1999). 8. 2000). 2004). a family of enzymes responsible for maturation of most prohormones..2 and 8. resulting in the presence of at least two GLP-1 sequences occurring sequentially within the prohormone of many amphibians.

2004). GLP-1 and GLP-2 (Fig. 1995) and anglerfish (L. This arrangement is not restricted to the pufferfish but has also been described for rainbow trout (O. respectively... Busby 231 and proneuropeptide Y (Dhanvantari et al. 1983). 2000). most likely due to an ancient genome duplication (Jaillon et al. including IP-2 and GLP-2. LLC . this is only half (or even less) of the story for fishes.. transcription results in a shortened mRNA and prevents the expression and subsequent translation of GLP-2.R. 1995). can be alternatively spliced to eliminate the GLP-2 sequence from the mRNA by retaining the intron following GLP-1. In the context of proglucagon. 8.P Mommsen and E. PC1/3 and PC2 are of interest as they are primarily expressed in neural and endocrine tissue (Brakch et al. flanking glucagon.. we might expect between two and four proglucagons for those species—such as salmonids—that have incurred a more recent additional (partial?) genome duplication (Thorgaard et al. It involves the retention of intron 4 following GLP-1 (cf. Further supporting the idea of the existence of two non-allelic genes is our observation that the two proglucagon genes differ markedly in intron sizes and intronic sequences (T. Since this intron contains a polyA signal.3). unpublished). the two genes are encoded on chromosome 2 (proglucagon-2) and chromosome 3 (proglucagon-1). Teleostean fish definitely possess two independent proglucagon genes. In the pufferfish Tetraodon nigroviridis. but some can also cut at single basic amino acids.2. 8. mykiss) (Irwin and Wong. Uniquely for the fishes. 8. Taking this type of approach a little further. in contrast to the situation in mammals and most other diploid vertebrates that contain a single proglucagon gene (on chromosome 2). DUPLICATE PROGLUCAGON GENES However. 1996). 5).Thomas P. Irwin and Wong. Mommsen and Ellen R. americanus) (Lund et al. 5 -UTR 3-UTR ATAAA SP GRPP Glucagon IP-1 GLP-1 RR Fig. 2002) and are undergoing reduction in chromosomes © 2006 by Taylor & Francis Group.. the proglucagon-1 gene presented in Fig. Intron 4 includes a polyA signal and leads to truncation of the mRNA after GLP-1 (Fig. as seen in proglucagon.5 Alternative splicing of teleostean proglucagon-1. Alternative slicing of proglucagon-1 mRNA leads to truncated mRNA sequences missing portions of the precursor. 8. Busby. such as the arginine in position 6 of mammalian GLP-1. These enzymes cleave peptides primarily at dibasic cut sites. .

8. Proglucagon-2 is present in all teleostean genomes. therefore. tshawytscha). but the products are observed less frequently than those from proglucagon-1. the Brockmann bodies (Plisetskaya and Mommsen. Most relevant in the current context is the occurrence of two independent insulin genes in teleostean fishes (Irwin. 8.fishbase. Because of the persistence of the duplicate genes. the consistent differences in their respective products and active transcription into peptides. Oncorhynchus kisutch) or 68 (chinook salmon O. © 2006 by Taylor & Francis Group. hormone redundancy is more important to our finned friends than to other vertebrates. Of course. whose sequences differ from their counterparts derived from proglucagon-1. LLC . in subtle. respectively (http://www. The gene is composed of four exons and three introns. The second proglucagon gene is considerably shorter than proglucagon-1. resulting in potential production of glucagon and GLP-1 only. 2004) and 44 (Takifugu rubripes).org/). the pairs of proglucagons are not without other parallels in the fishes. only glucagon and GLP-1. lacking introns 4 and 5 and thus the entire coding region for GLP-2 (Fig. 8.. Both proglucagons are not only transcribed. The peptides most likely produced are.6 Structure of teleostean proglucagon gene-2. while representative salmonids are much higher at 60 (coho salmon.3). interesting questions arise about functional differences between these hormones pairs.6). 2004) and the storage of different insulins in endocrine pancreas Intron 1 Intron 2 Intron 3 5’-UTR SP GRPP Glucagon IP-1 RR GLP-1 3’-UTR Fig. 1996). accepted chromosome numbers (2n) for the pufferfishes are 42 (Tetraodon nigroviridis) (Jaillon et al. but also give rise to stored peptides as confirmed repeatedly by the isolation and characterization of duplicate glucagons from fish endocrine pancreas.232 Fish Endocrinology and genes. but consistent ways as exemplified by the amino acid sequences for two pufferfishes (Fig. Currently. respectively. whether the genes are processed differentially for paracrine actions or whether for some reason.

one GLP-2) are produced and their abundance in the producing tissues could be regulated by selective degradation. 1994). for the sake of this argument. all these treatments also lead to augmented secretion of insulin from Brockmann bodies. does not fulfill this function in the fishes (see below). For instance. Busby 233 (Mommsen et al. other amino acids and to a certain extent also by glucose. GLP-1. 1988). potentially setting up rather intricate and complex interactions between these two opposing hormone systems. PEPTIDE SECRETION Release of glucagon from the endocrine pancreas—the only tissue and only fish proglucagon-derived hormone analyzed in this respect—is increased especially by arginine. At any rate. since some of them show immunocytochemical staining for both pancreatic polypeptide and glucagon. in the case of the two insulins in catfish. 8. 2002). Of course. In species containing more than just a principal Brockmann body.Thomas P. The big question for such a multihormone/two-gene system is which hormones and what amounts of each are being produced. Most peptides derived from proglucagon have been purified from fish endocrine pancreas.7). the most powerful insulinotropin in mammals. In fact.. LLC . while others stain only for one of the two (Abad et al. including KCl are effective glucagonotropins. implying that endocrine pancreatic cells respond to similar stimuli and treatments are not targetting a specific hormone (Ronner. Mommsen and Ellen R. 2002). Recently. these multiple Brockmann bodies do not seem to be created equal. Curiously. it has been reported that the enigmatic miniglucagon may function as a powerful inhibitor of glucose-dependent insulin release in mammals (Dalle et al.. other species may have endocrine pancreatic cells infiltrating the liver. In vitro other compounds. an accumulation of endocrine cells with a small rim of exocrine tissue. that both genes are transcribed at similar rates and that their respective mRNA species have comparable stability. the purification from the Brockmann bodies does not mean that proglucagon transcription and peptide production are restricted to the pancreatic -cells. the list of tissues transcribing the proglucagon genes is constantly increasing in fishes (as in mammals) with gastrointestinal tract and brain attracting the main attention (Fig. stored © 2006 by Taylor & Francis Group. the Brockmann bodies of many species. Assuming. two GLP-1s. While most species possess distinct Brockmann bodies ranging from one to many.. five peptides (two glucagons.

. Data for fish are compiled from (Busby.7 Distribution of transcripts for proglucagon and glucagon receptors in mammalian and piscine tissues. 8. © 2006 by Taylor & Francis Group.234 Fish Endocrinology Proglucagon Mammal Adipose Brain Endocrine pancreas Gill Gonads Heart Intestine Kidney Liver Muscle Stomach no yes yes yes yes yes yes no yes no Glucagon receptors Mammal yes yes yes yes yes yes yes yes yes yes Fish yes yes Fish yes no yes yes yes yes yes yes yes yes Fig. Bold type indicates quantitatively important tissues. 2004). Some data for proglucagon were confirmed at the protein level with Western blots. 2002) and (Green et al. LLC . Data are based on results with PCR. Empty cells indicate lack of data. Northern blots or RNA protection assays.

. but substantial amounts of glucagon and GLP-1 are removed during passage of blood through the trout liver (Plisetskaya and Sullivan. Mommsen and Ellen R. Things become complicated by the fact that several tissues could potentially contribute to glucagon-related peptides in the circulation. mammalian models need to be consulted to allow comment on the removal of glucagon and GLPs from plasma. Busby 235 amounts were about 4:1 in favour of one peptide over the other (Mommsen et al. Alternatively. biologically inactive glucagon4-29 fragment is subsequently attacked at the peptide bond between Tyr13 and Leu14. 1989). Similar arguments apply also to GLP-1. With only a limited amount of data at hand for the fishes. The longer fragment may then be further degraded into miniglucagon by attack at a dibasic site. resulting in half-lives in mammals of 6-7 min for glucagon and GLP-2 and only about a minute for GLP-1 (Burrin et al. glucagon and the GLPs are degraded rapidly. but preponderance of glucagon-1 in data on Brockmann body peptides over glucagon-2 (Plisetskaya and Mommsen. Irwin. Glucagon is degraded in rat liver in several steps. unfortunately. it is quite possible that glucagon-1s are merely stored at higher concentrations. being able to differentiate between pairs of circulating hormones—which is yet to be achieved—will not supply the answer and in vitro perfusion systems for key tissues may be the technique of choice. a bond that is conserved also in teleosts (Fig. No turnover times are available for the fishes. The resulting. starting with removal of the N-terminal three amino acids.. Liver and kidney are the key removal sites in mammals. 2001). © 2006 by Taylor & Francis Group. PEPTIDE DEGRADATION One method to regulate the abundance of peptide hormones post secretion is to selectively remove one or more of the five hormones from the circulation. The dibasic site is conserved in the fishes either as RK (proglucagon-1) or RR (proglucagon-2). since no comparable mammalian data for an RK site exist.. but by analogy results for mammals can apply only to proglucagon-2. 2001) seems to hint at the prevalence of transcription of gene 1 and that indeed more glucagon-1s are produced than glucagon-2. Once secreted into the bloodstream.Thomas P. therefore.3). LLC . 2002). while residence times for glucagon-2 prior to secretion are short and glucagon-2 may account for the bulk of circulating glucagon. 2003). comparative data for glucagons are lacking. Cytosolic and endosomal enzymes are responsible for the degradation of glucagon after receptor/ligand internalization in the liver (Authier et al. 8. 1996.

This residue. Brubaker and Drucker. 1996) are for peptides. Representative amino acid sequences for GLP-2 from lamprey (Petromyzon marinus). pufferfish (Tetraodon nigroviridis). 8.. GLUCAGON Similar to other peptide hormones.. 2000. trout (Irwin and Wong. Gila monster (Heloderma suspectum) and rat (Rattus norvegicus). 8. The key to rapid GLP-1 degradation seems to lie with the alanine in position 2 at the N-terminal of the peptide (Pauly et al. In the fish genome. this Ala2 is not conserved and is replaced in all fish GLP-2s sequenced to date (Fig.8 GLP-2 sequences in vertebrates.R. specific focus has been on the removal of GLP-1 in the interest of creating longer-lasting analogues of GLP-1. making predictions about kinetics methods of removal of GLP-2 in fishes guesswork at this time.. 1996). unpubl. Lamprey Rainbow trout Pufferfish Amphiuma Gila monster Rat HSDGSFTNDM NVMLDRMSAK NFLEWLKQQG RGHVDGSFTSDV NKVLDSLAAK EYLLWVMTSK TSG HIDGTFTSDV NKVLDSMAAK EYLLWVMTSK PSGE HADGSFTSDI NKVLDTIAAK EFLNWLISTK VTE HADGTFTSDY NQLLDDIATQ EFLKWLINQK VTQ HADGSFSDEM NTILDNLATR DFINWLIQTK ITD Fig. Fish glucagon receptors belong to the superfamily of receptors with seven © 2006 by Taylor & Francis Group. However. DPP IV-like sequences are found directly adjacent to the proglucagon gene (Zhou and Irwin. Mommsen and E. glucagon’s main effects are mediated through binding to the extracellular portion of highly selective receptors. Not surprisingly because of an Ala in position 2. Data for lamprey (Irwin et al.8) by residues that do not lend themselves as readily as alanine as points of attack for DPP IV. adjacent to the N-terminal histidine. 2004).P.) are derived from the respective cDNAs. generating the inactive GLP-23-33. 1999). LLC . which is conserved in all vertebrate GLP-1s. Busby. 1996). amphiuma (Amphiuma tridactylum). removal of an Nterminal dipeptide by DPP IV also applies to GLP-2 in the mammals. 2004). are bolded. Data for amphiuma (Cavanaugh et al. is attacked by a somewhat selective dipeptidyl peptidase IV (DPP IV) to produce the biologically inactive GLP-13-31. largely in the context of treatment of diabetes (types I and II) (Drucker et al.. 1995) and pufferfish (T. rainbow trout (Oncorhynchus mykiss). Gila monster and rat (reviewed in (Plisetskaya and Mommsen. Residues in position 2.236 Fish Endocrinology Since therapy with GLP-1 is a distinct growth industry in humans.

it is selective for glucagon and does not bind GLPs. with small. It is somewhat surprising that only a single glucagon receptor sequence has been reported from fish to date. but systematic. extrahepatic actions for the hormone. Two different glucagon genes are actively transcribed in every species of teleost. differences between the two hormones conserved throughout a relatively long evolutionary time. Unfortunately. 2004). no shortage of different avenues of actions exists for the hormone. 2004) allows either glucagon to bind and perhaps fine-tuned distinctions between the two hormones do not become apparent until later in the post-receptor pathways. However.Thomas P. Chow and colleagues arrived at the conclusion that the fish receptor was less restrictive in terms of ligand specificities than its mammalian counterpart and could accommodate a broad range of glucagon structures (Chow et al. Generally. At the same time. by activating gluconeogenesis from carbohydrate intermediates such as pyruvate or lactate. Of all receptors for proglucagon-derived peptides. Even though its effects are best defined for the fish liver. Therefore. 8. regulating glucose. is spotty. 8. amino acid and lipid metabolism. although often implied. 8. LLC . Analyzing the binding pocket of the goldfish glucagon receptor. Unfortunately. Busby 237 transmembrane domains. perhaps fulfilling different physiological roles.7). glycogen synthesis is curtailed and the rate of glycolysis is decreased. The receptor shows between 50 and 56% sequence identity with glucagon receptors of other vertebrate groups. its presence in endocrine pancreas.. an important potential target for glucagon action. A whole array © 2006 by Taylor & Francis Group. 2004). the picture for the fishes is fairly limited even for the liver and evidence for extrahepatic actions. an extracellular loop containing the receptor Nterminal and an intracellular portion with the carboxyl terminal.9.10) and somewhat slower. As indicated in Fig. one would expect that different glucagon receptors would exist. 1994) or on recombinant receptors (Chow et al. often opposing the actions of insulin. hinting at active. glycerol and eligible amino acids. it is also possible that the comparatively reduced ligand specificity noticed for the fish glucagon receptor (Chow et al. liver is by no means the only target.. the hepatic glucagon receptor remains the only one for which binding data have been reported on hepatocytes (Navarro and Moon. at best. remains to be analyzed. Mommsen and Ellen R.. Glucagon functions as a powerful metabolic hormone. the hormone is instrumental in rapidly increasing blood glucose through the activation of hepatic glycogenolysis (Fig. The mRNA for the receptor is widely distributed in fish tissues (Fig.

6BPase Fig.6BPase-fructose 1. LLC . PFK2/F2. 8.6-bisphosphatase. ERK 1/2-extracellular signal-regulated protein kinases. The model is largely derived from data for fishes. Intracellular messengers are indicated by stippled lines.9 Message transduction and metabolic targets for glucagon in vertebrate liver.238 Ca2+ lyl eny Ad lase c Cy Fish Endocrinology ATP PDE 5’-AMP G R luc ec a ep go to n r Gsá C cAMP Ca2+ IP3 N cAMP Protein Kinase A Gq Calmodulin & ERK 1/2 Ca2+ Phos p Lipa hose C Phosphorylase Kinase CMYK Glycogen Synthase PIP2 Pyruvate Kinase TAG Lipase Acetyl-CoA Carboxylase cAMP IP3 CREB F1. PEPCK-phosphoenolpyruvate carboxykinase. TAG-triacylglycerol. DAG-diacylglycerol. IP 3inositoltrisphosphate. but also includes some mammalian mechanisms (Ca2+ influx & PDE inhibition). PIP 2-phosphatidylinositolbisphosphate.6BPase G6Pase PFK-2 / F2. G6Pase-glucose 6-phosphatase. F1. © 2006 by Taylor & Francis Group.6BPase Glycogen Phosphorylase DAG Transcription Protein Kinase C Ca2+ PEPCK F1. CREBcAMP-responsive element binding protein.6BPase-bimodal enzyme: phosphofructokinase 2/ fructose 2. G sa and Gq-G-proteins associated with the intracellular portion of the glucagon receptor. PDE-phosphodiesterase.6bisphosphatase.

8. IP3 (inositoltrisphosphate) and IP3-dependent release of Ca2+ © 2006 by Taylor & Francis Group.E-08 1. One of the substrates of protein kinase A is the cAMP-dependent response element-binding protein (CREB). all receptor mediated. glucagon ties into the inositoltrisphosphate (IP3) and Ca2+ systems of intracellular message transduction..10 Increase in cAMP and activation of glycogenolysis in response to hormones. Data are expressed as a percentage of maximum response in the presence of 1 M gf-glucagon (recalculated from Chow et al. Activation of glycogenolysis was assessed by release of glucose from endogenous glycogen after a 30-min exposure to goldfish (gf) glucagon or GLP-1.E-10 1. Intracellular cAMP concentrations after gf-glucagon exposure for 45 min of gf-glucagon receptor expressed in COS-7 cells.6-bisphosphatase.E-11 1. Busby 120 239 100 % of maximum response gfGlucagon gfGLP-1 cAMP 80 60 40 20 0 1. mediated via adenylyl cyclase.11) and subsequent increases in membrane diacylglycerol (DAG). glycogen phosphorylase. 2004). However. triglyceride lipase and fructose 1..9). a nuclear transcription factor that binds specifically to the cAMP response element and regulates gene expression.Thomas P. 8. Data are expressed as a percentage of the maximum amount of cAMP produced (redrawn from Chow et al. the influence of glucagon does not end there and metabolic regulation is achieved by a number of additional means. In addition to cAMP. 8. especially phosphorylation.E-05 Hormone concentration [M] Fig. phosphorylase kinase. Mommsen and Ellen R. involving activation of phospholipase C (Fig. of regulatory hepatic enzymes is subject to control by covalent modification.E-06 1. Included in this group are pyruvate kinase. cAMP and protein kinase A (Fig. 2004).E-07 1.E-09 1. LLC .

Figure 8. Cells were exposed to different concentrations of mammalian glucagon and phospholipase C activity was assessed after a 5-min incubation.E-11 1. In turn.240 Fish Endocrinology 160 PLC Activity [% of control] 150 PLC in Catfish Hepatocytes IP3 [% of control] 600 500 400 300 200 100 -1 0 1 2 3 4 5 6 140 130 IP3 Time course 120 Time [min] 110 100 1. Inset: Time course of catfish hepatocyte IP3 concentrations in the presence of 100 nM mammalian glucagon.. like the apparent induction of other hepatic enzymes in amino acid metabolism (alanine and aspartate aminotransferases). One target enzyme not included in Fig. (1997) and expressed as a percentage of control values without hormones. The physiological outcomes are detailed in Fig. with focus on mechanisms of intracellular message transduction and metabolic targets.9 presents a coarse summary of the events initiated when glucagon binds to its hepatic receptor. 1997). from intracellular stores. an enzyme tightly linked to general amino acid metabolism and also with urea synthesis in ureogenic teleosts. DAG and Ca2+ interact to activate protein kinase C. a © 2006 by Taylor & Francis Group. The enzyme is induced in toadfish (Opsanus beta) liver after glucagon injection (Mommsen et al. mostly by affecting the rate of acetyl Coenzyme A carboxylase. 8.E-10 1.E-06 1.E-07 1. it is possible that the observed increases are indirect and mediated via cortisol.E-05 Glucagon [M] Fig.E-09 1. 1993). Recalculated from Moon et al.E-08 1. 1992). (1997). largely the endoplasmic reticulum (Moon et al.11 Intracellular message transduction in catfish hepatocytes.E-13 1. another protein kinase that targets Ser and Thr residues in proteins. Data are redrawn from Moon et al. Activation of phospholipase C in black bullhead catfish (Ictalurus melas) hepatocytes.E-12 1. LLC .. Data are expressed as percentage over control values. Glucagon also inhibits the insulin-stimulated synthesis of triglycerides in rainbow trout liver (Cowley and Sheridan. 8. However. 8.12.9 is glutamine synthetase.

are noted (Moon. a few additional methods of glucagon action deserve to be mentioned. thus augmenting the effectiveness and duration of cAMP actions and another that the hormone increases influx of extracellular Ca2+ into parenchymal cells. One is that glucagon inhibits hepatic phosphodiesterase (PDE). is a sensitive and rapid indicator of changes in the protein kinase A status of liver (Moon et al. a prime target of glucagon action. 1998). as can be seen by a comparison of its N-terminal sequence (Fig. the key target for phosphorylation by phosphorylase kinase that leads to glucagondependent activation of the enzyme. 8. In contrast to mammalian models. 2003). Mommsen and Ellen R. The enzyme is highly conserved across vertebrates (and tissues). similarly. the alleged second messenger in this well-known activation cascade. even though one study reported increased rates of amino acid oxidation in response to glucagon (Foster and Moon. most of the differing residues either conserve the chemical character of the side chain and/or represent single (third) base changes. in spite of the fact that frequently few alterations in cAMP.13). Glycogen phosphorylase.Thomas P. Perhaps a search of the promoter regions of these enzymes for CREB or CREB-like sequences will establish a potential link between glucagon and transcription of hepatic genes in fishes..12 Metabolic targets and outcomes of glucagon action in teleostean liver. 8. thus potentially assisting IP3dependent routes of activation. the rat liver and zebrafish brain enzyme are identical in 661 of the total 793 amino acid residues of the enzyme monomer. known inducer of these enzymes (Mommsen et al. © 2006 by Taylor & Francis Group. no consistent increases in hepatocyte oxygen consumption rates are observed in fish hepatocytes. Overall. Busby Activation of pathways Glycogenolysis Gluconeogenesis Lipolysis Amino acid uptake Amino acid oxidation 241 Inhibition of pathways Glycogen synthesis Glycolysis Lipid synthesis Physiological outcome Glycogen depletion. glucagon does not increase the rate of urea synthesis in ureagenic fish species like the toadfish. 1999. Hyperglycemia Hyperglycemia Increase in plasma fatty acids Increase in plasma glycerol Hypoaminoacidemia Increase in ammonia excretion Fig. 1999).. 1987). LLC . Borrowed from mammalian models. with current research spotty about its applicability to fish systems. In addition. This includes Ser14. clone Fe:eBU804536 BU804536. mouse (Mus musculus.cns. pufferfish (Tetraodon nigroviridis.ensembl. LLC . © 2006 by Taylor & Francis Group.genoscope. Pufferfish sequences are not annotated in the databases. GenBank P09811). Species are: rat (Rattus norvegicus. http://www.13 N-terminal sequence of vertebrate glycogen phosphorylases. 8. GenBank Q8CI94).242 Fish Endocrinology Rat liver Zebrafish brain Tetraodon Takifugu muscle Mouse muscle 1 10 20 30 40 50 60 MAKPLTDQEKR RQISIRGIVG VENVAELKKG FNRHLHFTLV KDRNVATPRD YYFALAHTVR MSKPLTDHERR KQISVRGIAG LGDVAEIKKS FNRHLHFTLV KDRNVATPRD YYFALAHTVR MSKPLSDHERK KQISVRGLAG VENVTELKQN FNRHLHFTLV KDRNVATRRD YYFALAHTVR MSKPLSDHDRK KQISVRGLAG VENVTDLKQN FNRHLHFTLV KDRNVATRRD YYFALAHTVR MSRPLSDQDKR KQISVRGLAG VENVSELKKN FNRHLHFTLV KDRNVATPRD YYFALAHTVR *::**:*:::: :***:**:. chromosome 7.* : :*::: *: ********** ******* ** ********** pufferfish adult muscle (Takifugu rubripes. location 3907439:3909836:1. zebrafish (Danio rerio. Comparison of the 60 N-terminal amino acid residues of selected vertebrate glycogen phosphorylases. The Ser14 residue that is phosphorylated by glycogen phosphorylase kinase to activate the enzyme is bolded and boxed. GenBank AAS92629).

possess generally high plasma insulin titers and show relative peripheral insulin resistance (Mommsen and Plisetskaya. 2003). and it is quite possible that fish fall into this category. some powerful insulinotropins are also potent glucagonotropins. Therefore. Cell shrinkage alone—brought about by changing osmolarities of the bathing medium—also leads to activation of glycogen phosphorylase in fish hepatocytes. Further. 2001). in turn leading to shrinkage of hepatocytes and. changes in cell volume assist or exacerbate the initial hormonal stimulus. As usual. In this cascade. one wonders whether this argument applies to the fishes.Thomas P. Many of the physiological responses to glucagon depend on the nutritional status of the animal. incidentally fatty acid metabolism. counteracting and at times dampening the postprandial effects of insulin. Mommsen and Ellen R. the emphasis (real or revealing the researchers’ bias?) seems to be on plasma glucose where glucagon may act to prevent insulin-dependent hypoglycemia. including activation of glycogenolysis. another useful model for analyzing the functions of glucagon. based on a few contradictory observations: fish have comparatively slow insulinotropic responses to glucose. LLC . it is questionable whether glucose forms the centerpiece of their intermediary metabolism (Moon. opposes the actions of glucagon in mammals and vice versa. glucagon appears to be the main player in the regulation of blood glucose and.. leading to cell swelling by activation of key ion pumps. the metabolism of liver is readjusted to favour catabolic states. In birds. having earned glucagon the label as a ‘postprandial’ hormone. in birds. Busby 243 Glucagon affects membrane ion transport. 1991). Not all vertebrates are ‘insulin’ drive like the mammals. © 2006 by Taylor & Francis Group. with insulin playing only a subordinate role. However. After all. In mammals. but these phenomena remain to be confirmed for the fishes. the roles of glucagon that may provide a glimpse of the multifaceted potentials of this hormone in all vertebrates are yet to be defined. through resulting molecular crowding. insulin. but subtle differences exist in the timing and degree of activation between glucagon exposure and osmotic change alone (Hallgren et al. Unfortunately. possibly hints at important functions beyond the realm of the metabolism of simple carbohydrates. the strict conservation of allegedly glucose-centered hormones over half a billion years in a group of vertebrates that hardly ever encounter a jelly doughnut or equivalent in its diet. This is clearly reflected in the endocrine pancreas where glucagon (and GLP-1!)producing -cells predominate and an entire half is completely devoid of -cells.

. The first question should be whether these tissues secrete glucagon or whether glucagon acts in a paracrine fashion only. Extrahepatic Actions In agreement with the molecular evidence that proglucagon transcription is not restricted to the pancreatic -cells. Unfortunately. glucagon immunopositive cells occur throughout the principal Brockmann body. being outnumbered by insulin-positive cells by 4:1. For instance.7). Gómez-Visus et al. 1991). while glucagon cells were distributed throughout the largest and smaller islets of mullet (Mugil auratus and M.244 Fish Endocrinology In fishes. tilapia (Oreochromis niloticus) Brockmann bodies transplanted subcutaneously into nude. Responses to insulin are also glucose-dependent. the response of Brockmann bodies to glucagon in the activation of somatostatin release is glucose dependent. Further. 2000)—in spite of the fact that the donors themselves are severely glucose intolerant. Although glucagon receptors are distributed throughout the fish tissues (Fig. unencumbered by its counterpart in the systemic circulation. it is a recurring theme that key metabolic parameters of Brockmann bodies are glucoseresponsive. In the tilapia Brockmann body. the occurrence of glucagon and its local function remain to be defined. LLC . Even though the physiological role is still unclear. 1998) and in the brain. albeit required smaller glucose concentrations. 1999). 8. very little information is available on extrahepatic actions. 1986) and in Nile tilapia (Oreochromis niloticus) (Yang et al. glucagon immunopositive cells occur throughout the gastrointestinal tract of fishes (Rombout and Reinecke. 1984.. diabetic mice show ‘normal’ (by mammalian standards) glucose tolerance (Wright. 1993). Glucose Responsiveness Glycemia may fluctuate widely between species and within species of fishes. glucagon positive cells accounted for only 11. although insulin responses to glucose ingestion tend to be moderate and slow compared with many mammalian models (Navarro et al. but only at the periphery of the smaller islets of sea bass (Dicentrarchus labrax) (Lozano et al. Glucagon © 2006 by Taylor & Francis Group.. This puts the onus for differences in response on peripheral resistance to the glucostatic actions of insulin and less on the absence of glucose-sensitivity of insulin release. but relatively high concentrations of glucose (10 mM) are required to elicit the glucagon response.5% of the endocrine cells. saliens) (Lozano and Agulleiro..

resulting in enhanced release of glycerol and fatty acids. this effect is blocked by insulin. the list of metabolic targets © 2006 by Taylor & Francis Group. control of insulin production and release takes center stage.Thomas P. GLP-1 actions directly and indirectly oppose the actions of insulin and supersede or complement those of glucagon in that the hormone activates hepatic glycogenolysis.. 2000). there is still debate over the presence of GLP-1 receptors in mammalian liver. However. As such. fat and nutrients and thus controlling rates of nutrient intake and disposal.. 1998). 2000. As pointed out in Fig. at least in vitro (Harmon et al. just like in mammals. gluconeogenesis and lipolysis. the hormone functions as the foremost incretin. the model delineated above for hepatic actions of glucagon seems to apply (Fig. It is impossible to appreciate the role of GLP-1 in fishes outlined below without a brief discourse on its many roles in mammals. GLP-1 directly opposes glucagon at different levels. regulating pancreatic hormone output in response to enteral glucose. Here. most factors of the intestine/pancreas/liver complex delineated in Fig. GLP-1 GLP-1. Especially the activation of glycogenolysis is rapid. physiological effects have been elusive. GLP-1 has been termed ‘a better glucagon’ (Mommsen. Because of the different and intermeshed mechanisms of enzyme induction. Brubaker and Drucker. Clearly. except that the hormone activates O-methylglucose uptake in fish enterocytes. resulting in the release of large amounts of glucose from the liver that can be measured after a few minutes (Mommsen. adipose triacylglycerol lipase is activated by glucagon. However.7). 2000).14. Fundamental differences exist to the situation in fishes. governs numerous cellular tasks in different tissues and employs a variety of intracellular message transduction systems. a broadening of the hepatocentric view of glucagon is overdue. 8. assays to determine activation of gluconeogenesis require hours. the medical star of the proglucagon derived peptides (Drucker et al.14 do not seem to apply. albeit without (as in mammals) increasing rates of oxidation (Soengas and Moon. 8. Because in many species—goldfish being one of the few exceptions—GLP-1 is more powerful than glucagon in this respect (Fig. Focussing on intestine and endocrine pancreas in mammals. Not surprisingly.15).e. 2004). Unfortunately. 1993). the liver is an indirect target only and although a few direct hepatic actions have been reported. Busby 245 activated adenylyl cyclase in isolated fish brain membranes and in enterocytes. 8.. i. LLC . 8. Mommsen and Ellen R.



Fish Endocrinology


Liver Islet

GLP-1 in b-cells Insulin synthesis ­ Insulin secretion ­ Proliferation ­ Anti-apoptotic

GLP-1 in islet Islet neogenesis ­ Islet mass ­ Anti-apoptotic Precursor cells into b-cells ­

GLP-1 in d-cells Somatostatin secretion ­ GLP-1, GIP

GLP-1 in a-cells Glucagon synthesis ¯ Glucagon secretion ¯


Glucose Fat Nutrients
Vagus nerve





GLP-1 in intestine Gastric emptying ¯ Motility ¯


Fig. 8.14 The incretin concept: Interaction of intestinal hormones with pancreas and liver in mammals. Glucose and fatty acids in the intestine lead to increased output of GIP and GLP-1. Both are insulinotropins and especially GLP-1 also affects other endocrine cells in the pancreas. Insulin, in turn alters hepatic metabolism. Irregular shapes: intestinal cells producing GIP or GLP-1. Square boxes: effects of GLP-1. GLP-1RGLP-1 receptor; InsR-insulin receptor; GlucR-glucagon receptor.

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Thomas P. Mommsen and Ellen R. Busby


Glucose production [% of maximum]

100 90 80 70 60 50 40 30 20 10 0 1.E-10

hGLP-1 hGlucagon






Hormone concentration [M]
Fig. 8.15 Glycogenolysis in teleostean hepatocytes in response to glucagon and GLP1. Hepatocytes isolated from red Irish lord (Hemilepidotus hemilepidotus, Teleostei: Cottidae) were incubated with different concentrations of human glucagon and GLP-1. Glucose released by the cells from endogenous glycogen was measured after a 30 min incubation. Data are expressed as a percentage of the maximum response with 100 nM GLP-1 (T.P. Mommsen and E. Danulat, unpublished results).

is at present quite short, less reflecting physiological realities than limited research effort. To date, glycogen phosphorylase (and by implication phosphorylase kinase), pyruvate kinase, F1,6BPase, TAG lipase and PEPCK have been identified as targets, implying the involvement of differing routes of message transduction. Goldfish GLP-1 receptors expressed in COS-7n cells respond to GLP-1 with increasing cAMP levels and because of activation of glycogen phosphorylase and inactivation of pyruvate kinase, we assume adenylyl cyclase and cAMP to be involved. However, this is often not borne out in hepatocyte data, where cAMP levels go up significantly as GLP-1 concentrations exceed 0.1 M, while activities of GPase are noticeably increased with GLP-1 in the high picomolar range (Plisetskaya and Mommsen, 1996). Nevertheless, in some fishes, cAMP clearly serves as an intracellular messenger, especially at higher GLP-1 concentrations. In other experiments on fish liver, phospholipase C, IP3 and Ca2+ have been implicated (Moon et al., 1997)

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Fish Endocrinology

in GLP-1 actions. Further, the activation of PEPCK enzyme activity involves a transient peak in PEPCK mRNA (Fig. 8.16), alluding to a role of CREB or other transcription factors. The picture above is rounded out by the observation that GLP-1 is only a weak insulinotropin in fishes even in the presence of glucose. Although GLP-1 receptor transcripts are abundant in fish intestine (cf. Fig. 8.17), it is questionable whether the incretin concept based around glucose applies. It must be considered that fish are generally poor at dealing with carbohydrate diets (Hemre et al., 2001; Krogdahl et al., 2005), often glucose intolerant and insulin resistant (Wright, 2000) and amino acids are more powerful insulinotropins than glucose (Ronner and Scarpa, 1987). The differences in physiological roles between mammals and fish are even more astounding since the biologically active peptide is highly conserved. As such, fish hormones will activate mammalian receptors and


PEPCK mRNA, 5 10-9 GLP-1 PEPCK mRNA, 1 10 -8 GLP-1 PEPCK activity, 5 10 -9 GLP-1 PEPCK activity, 5 10 -8 GLP-1

[Percent of Control] ]





90 -4 0 4 8







Fig. 8.16 Phosphoenolpyruvate carboxykinase (PEPCK) in trout hepatocytes: mRNA and enzyme activity after exposure to GLP-1. Rainbow trout (Oncorhynchus mykiss) hepatocytes in primary cultures were incubated with zebrafish GLP-1 and sampled at the specified times. Soluble protein was extracted from one half of the cells for PEPCK activity measurements; polyadenylated mRNA was isolated from the other half and probed for the presence of PEPCK mRNA by quantitative PCR, using primer sets developed by Panserat et al. (2001). Data are from T.P. Mommsen, R. Sathiyaa and M.M. Vijayan (unpublished).

© 2006 by Taylor & Francis Group, LLC

Thomas P. Mommsen and Ellen R. Busby


Control Marker Brain Gall bladder Gill Male gonad Female gonad Lower intestine Upper intestine Kidney Liver Heart Pituitary Muscle Spleen

Fig. 8.17 Expression of brain-type GLP-1 receptor in goldfish (Carassius auratus) tissues. Southern blot. A. goldfish GLP-1R; B. -actin. Redrawn from Yeung et al. (2002).

mammalian hormones will bind to receptors present in fish tissues (Plisetskaya and Mommsen, 1996) or to fish receptors transfected into a mammalian cell line (Yeung et al., 2002). In direct comparison, the fish GLP-1 receptor is ‘structurally more accommodating’ than the mammalian receptor (Yeung et al., 2002), responding to a variety of GLP-1s and glucagons; however, EC50 for glucagons are about five times higher than for homologous GLP-1. Even though glucagon and GLP-1 share similar if not overlapping functions in fish liver, the two hormones seem to do so through different receptors. Extrahepatic Actions In stark contrast to the scenario developed above, extrahepatic actions of GLP-1 appear similar in mammals and fish. GLP-1 immunoreactivity has been detected in several parts of catfish (Clarias batrachus) brain, including the hypothalamus (Sarkar and Subhedar, 2001) and GLP-1 receptors have been located in the pituitary (Fig. 8.17) and the first piscine GLP-1 receptor was isolated from a fish brain mRNA preparation. Since centrally administered GLP-1 exerts a distinct inhibitory effect on channel catfish (Ictalurus punctatus) food intake (Silverstein et al., 2001) and GLP1 stimulates cAMP production by isolated rockfish (Sebastes caurinus) brain membranes (Mommsen and Mojsov, 1998), it is not far-fetched to predict that GLP-1 has a function in the teleostean brain. From the little

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Fish Endocrinology

available evidence, it appears that GLP-1 acts as an inhibitor of food intake due to signals of either satiety, the presence of nutrients in the gut, or interoceptive stress. In addition, GLP-1 leads to dose-dependent activation of adenylyl cyclase (Mommsen and Mojsov, 1998) and glucose transport by fish enterocytes and decreases the rate of glucose oxidation, without affecting the apparent affinity constant for glucose (Soengas and Moon, 1998). As a side-plot, included in Fig. 8.14, GIP affects GLP-1 release from intestinal L-cells and is a potent insulinotropin in its own right, but its presence in fish intestine or fish genomes, for that matter, remains to be tested. Unfortunately, no comparative fish data are available on the powerful cytotrophic and antiapoptotic actions GLP-1 exerts on mammalian endocrine pancreas (see Fig. 8.14), intestine and neuronal cells. One structural oddity really sets the fish GLP-1s apart. The ease of isolating and purifying biologically active GLP-1s from fish Brockmann bodies implies that fish GLP-1 do not require additional processing after biosynthesis. In contrast, the hormone of most other vertebrate groups requires removal of a six-residue peptide from the N-terminal as part of maturing GLP-1s. The fish proglucagon genes contain a rather short intervening peptide 1 and, hence, lack a similar N-terminal extension. Previously dismissed as an inactive precursor that does not interact with GLP-1 receptors, it appears now that the N-terminally extended mammalian GLP-1 can convert epithelial progenitor cells in mouse small intestine into insulin-producing cells in a glucose-dependent manner (Suzuki et al., 2003). This effect is unique to the extended form and not shared by mature GLP-1 and hence the fish seem to miss out on a powerful hormonal principle! GLP-2 This conserved peptide with about 33 residues remains enigmatic for the fishes, since to date, a role for GLP-2 has been identified in mammals only. GLP-2 acts more proximally than GLP-1 and just like GLP-1 it regulates blood glucose indirectly. However, its main contribution to nutrient assimilation is via trophic effects on the intestinal mucosa. The hormone also curtails apoptosis in the crypts and villi, reduces intestinal epithelial permeability and promotes intestinal glucose transport (Drucker, 2003). Finally, GLP-2 may play a role in regulating food intake (Tang-Christensen

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Thomas P. Mommsen and Ellen R. Busby


et al., 2000). GLP-2 exerts its different effects through binding to specific GLP-2 receptors. Unfortunately, little information on GLP-2 is available for the fishes, apart from the few published peptide sequences and numerous nucleotide sequences. Physiological roles remain to be identified. In principle, assignment of a physiological role should be easier than for the other coencoded peptides, since fish only express and produce a single GLP-2. Potential sites of production are intestinal cells, the brain and possibly the -cells, even though the current consensus is that pancreatic proglucagon-1 is alternatively spliced and no GLP-2 is encoded in the resultant mRNA. Furthermore, as pointed out above, proglucagon-2 lacks GLP-2 altogether, reducing the number of tissues that could contribute GLP-2. The peptide is less conserved than glucagon or GLP-1 (cf. Fig. 8) and curiously, all fish peptides lack Ala in position 2, rendering the peptide less susceptible to degradation by DPP IV, which is responsible for rapid removal of blood GLP-1 and GLP-2 in mammalian models. When we scanned the Tetraodon nigroviridis genome for glucagon-family peptide receptors, we located a receptor tentatively annotated as a GLP-2 receptor (GSTENT00013191001), but large similarities of the nucleotide sequence to fish glucagon and GLP-1 receptors require an in depth analysis before this seven transmembrane receptor can be classified properly. Epilogue The future looks bright for these peptides, with new functions, expanding areas of influence, broader mechanisms of message transduction and novel aspects of interactions with other hormones being constantly added to the existing repertoire. While the multitalented GLPs garner most of the attention, glucagon continues to fascinate in terms of processing, message transduction and a growing list of targets. The fish system provides some unique intricacies, as teleosts—due to an ancient genome duplication— retain two proglucagon genes giving rise to distinct, but seemingly closely related, sets of glucagon and GLP-1, whose biological functions have yet to be characterized in detail. For instance, the questions should be addressed whether these ‘isohormones’ are indeed functionally identical and simply represent system redundancy—an interesting topic in itself— or whether pairs of hormones are expressed and synthesized at differing rates and regulate different pathways to different physiological ends. Of

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Fish Endocrinology

course, these considerations are by no means specific to the proglucagonderived peptides. Fascinating questions abound, ranging from the minutiae of receptor dynamics, hormone processing turnover and message transduction, to the ‘bigger pictures’ of incretins, receptor evolution, hormone redundancies and the overall roles of glucagon-derived peptides in metabolism, control of appetite and intestinal dynamics. The complexity of the regulating the proglucagon ‘complex’ poses another interesting challenge, with regulatory points at multiple levels, including mRNA (alternative splicing), translation, peptide storage and secretion, tissue expression and peptide degradation, all occurring on two independent genes that are located on two different chromosomes. This, together with seemingly ‘sloppy’ specificities on the part of the receptors, amounts to an interesting witch’s brew of endless possibilities. In the fish model, GLP-1 appears to compete with glucagon for the same metabolic targets in the fish liver, while being functionally separated from glucagon in the brain and in the enterocytes. This overlap or seeming (additional) duplication in function needs to be addressed as does the apparent switch in function between fishes and other vertebrates. GLP-2 is still looking for a defined function in the fishes. However, considering abundance of its transcripts and its conservation over a long evolutionary history, without doubt some interesting physiological roles will be assigned to this peptide sooner rather than later. The post-genomic era has never looked more enticing. Acknowledgments We really appreciate the continuing fruitful discussions on ‘all things glucagon’ with Drs. Tom Moon, Matt Vijayan and Svetlana Mojsov, their feedback on this chapter and making available unpublished data. References
Abad, M.E., Taverne-Thiele, J.J. and Rombout, J.H.W.M. 1988. Immunocytochemical and ultrastructural characterization of coexistence of pancreatic polypeptide and glucagon-like immunoreactivity in the pancreatic endocrine cells of Sparus auratus L. (Teleostei). Gen. Comp. Endocrinol. 70:9-19. Authier, F., Cameron, P .H., Merlen, C., Kouach, M. and Briand, G. 2003. Endosomal proteolysis of glucagon at neutral pH generates the bioactive degradation product miniglucagon-(19-29). Endocrinology 144:5353-5364. Brakch, N., Rholam, M., Simonetti, M. and Cohen, P. 2000. Favourable side-chain orientation of cleavage site dibasic residues of prohormone in proteolytic processing by prohormone convertase 1/3. Eur. J. Biochem. 267:1626-1633.

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Thomas P. Mommsen and Ellen R. Busby


Brubaker, P and Drucker, D.J. 2004. Minireview: Glucagon-like peptides regulate cell .L. proliferation and apoptosis in the pancreas, gut and central nervous system. Endocrinology 145:2653-2659. Burrin, D.G., Petersen, Y., Stoll, B. and Sangild, P. 2001. Glucagon-like peptide 2: a nutrient-responsive gut growth factor. J. Nutr. 131:709-712. Busby, E. R. 2002. Expression, Regulation and Evolution of Proglucagon Genes in Vertebrates. Ph.D. Thesis. University of Victoria, Victoria, pp. 1-179. Cavanaugh, E.S., Nielsen, P and Conlon, J.M. 1996. Isolation and structural .F. characterization of proglucagon-derived peptides, pancreatic polypeptide and somatostatin from the urodele Amphiuma tridactylum. Gen. Comp. Endocrinol. 101:12-20. Chow, B.K.C., Moon, T.W., Hoo, R.L., Yeung, C.M., Muller, M., Christos, P and Mojsov, .J. S. 2004. Identification and characterization of a glucagon receptor from the goldfish Carassius auratus: Implications for the evolution of the ligand specificity of glucagon receptors in vertebrates. Endocrinology 145:3273-3288. Cowley, D.J. and Sheridan, M.A. 1993. Insulin stimulates hepatic lipogenesis in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 11:421-428. Dalle, S., Fontes, G., Lajoix, A.D., LeBrigand, L., Gross, R., Ribes, G., Dufour, M., Barry, L., LeNguyen, D. and Bataille, D. 2002. Miniglucagon (glucagon 19-29): A novel regulator of the pancreatic islet physiology. Diabetes 51:406-412. Dhanvantari, S., Seidah, N.G. and Brubaker, P 1996. Role of prohormone convertases .L. in the tissue-specific processing of proglucagon. Mol. Endocrinol. 10:342-355. Drucker, D.J. 2003. Glucagon-like peptides: regulators of cell proliferation, differentiation and apoptosis. Mol. Endocrinol. 17:161-171. Drucker, D.J., Lovshin, J., Baggio, L., Nian, M., Adatia, F., Boushey, R.P Liu, Y., Saleh, ., J., Yusta, B. and Scrocchi, L. 2000. New developments in the biology of the glucagon-like peptides GLP-1 and GLP-2. Ann. N. Y. Acad. Sci. 921:226-232. Foster, G.D. and Moon, T.W. 1987. Metabolism in sea raven (Hemitripterus americanus) hepatocytes: the effects of insulin and glucagon. Gen. Comp. Endocrinol. 66:102-115. Gómez-Visus, I., García Hernández, M.P., Lozano, M.T. and Agulleiro, B. 1998. Glucagon- and NPY-related peptide-immunoreactive cells in the gut of sea bass (Dicentrarchus labrax L.): A light and electron microscopic study. Gen. Comp. Endocrinol. 112:26-37. Green, B.D., Mooney, M.H., Gault, V.A., Irwin, N., Bailey, C.J., Harriott, P., Greer, B., O’Harte, F.P and Flatt, P 2004. N-terminal His(7)-modification of glucagon-like . .R. peptide-1(7-36) amide generates dipeptidyl peptidase IV-stable analogues with potent antihyperglycaemic activity. J. Endocrinol. 180:379-388. Hallgren, K., Busby, E.R. and Mommsen, T.P. 2003. Cell volume affects glycogen phosphorylase activity in fish hepatocytes. J. Comp. Physiol. B 173:591-599. Harmon, J.S., Rieniets, L.M. and Sheridan, M.A. 1993. Glucagon and insulin regulate lipolysis in trout liver by altering phosphorylation of triacylglycerol lipase. Am. J. Physiol. 265:R255-R260. Hemre, G.-I., Mommsen, T.P and Krogdahl, Å. 2001. Carbohydrates in fish nutrition: . Effects on growth, glucose metabolism and hepatic enzymes. Aquacult. Nutr. 7:1-20.

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Holst, J.J., Bersani, M., Johnsen, A.H., Kofod, H., Hartmann, B. and Orskov, C. 1994. Proglucagon processing in porcine and human pancreas. J. Biol. Chem. 269:1882718833. Irwin, D.M. 2001. Molecular evolution of proglucagon. Regul. Pept. 98:1-12. Irwin, D.M. 2004. A second insulin gene in fish genomes. Gen. Comp. Endocrinol. 135:150-158. Irwin, D.M. and Sivarajah, P 2000. Proglucagon cDNAs from the leopard frog, Rana . pipiens, encode two GLP-1-like peptides. Mol. Cell. Endocrinol. 162:17-24. Irwin, D.M. and Wong, J. 1995. Trout and chicken proglucagon: Alternative splicing generates mRNA transcripts encoding glucagon-like peptide 2. Mol. Endocrinol. 9:267-277. Irwin, D.M., Satkunarajah, M., Wen, Y., Brubaker, P .L., Pederson, R.A. and Wheeler, M.B. 1997. The Xenopus proglucagon gene encodes novel GLP-1-like peptides with insulinotropic properties. Proc. Natl. Acad. Sci. USA. 94:7915-7920. Irwin, D.M., Huner, O. and Youson, J.H. 1999. Lamprey proglucagon and the origin of glucagon-like peptides. Mol. Biol. Evol. 16:1548-1557. Jaillon, O., Aury, J.M., Brunet, F., Petit, J.L., Stange-Thomann, N., Mauceli, E., Bouneau, L., Fischer, C., Ozouf-Costaz, C., Bernot, A., Nicaud, S., Jaffe, D., Fisher, S., Lutfalla, G., Dossat, C., Segurens, B., Dasilva, C., Salanoubat, M., Levy, M., Boudet, N., Castellano, S., Anthouard, V., Jubin, C., Castelli, V., Katinka, M., Vacherie, B., Biemont, C., Skalli, Z., Cattolico, L., Poulain, J., De Berardinis, V., Cruaud, C., Duprat, S., Brottier, P Coutanceau, J.P Gouzy, J., Parra, G., Lardier, G., Chapple, ., ., C., McKernan, K.J., McEwan, P Bosak, S., Kellis, M., Volff, J.N., Guigo, R., Zody, ., M.C., Mesirov, J., Lindblad-Toh, K., Birren, B., Nusbaum, C., Kahn, D., RobinsonRechavi, M., Laudet, V., Schachter, V., Quetier, F., Saurin, W., Scarpelli, C., Wincker, P Lander, E.S., Weissenbach, J. and Roest Crollius, H. 2004. Genome ., duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate protokaryotype. Nature (London) 431:946-957. Krogdahl, Å., Hemre, G.-I. and Mommsen, T.P 2005. Carbohydrates in fish nutrition: . Digestion and absorption in post larval stages. Aquacult. Nutr. 11:103-122. Lozano, M.T. and Agulleiro, B. 1986. Immunocytochemical and ultrastructural study of the endocrine pancreas of Mugil auratus and Mugil saliens L. (Teleostei). J. Submicrosc. Cytol. Pathol. 18:85-98. Lozano, M.T., Garcia, A.A., Abad, M.E. and Agulleiro, B. 1991. Pancreatic endocrine cells in sea bass (Dicentrarchus labrax L.) I. Immunocytochemical characterization of glucagon- and PP-related peptides. Gen. Comp. Endocrinol. 81:187-197. Lund, P.K., Goodman, R.H., Montminy, M.R., Dee, P.C. and Habener, J.F. 1983. Anglerfish islet pre-proglucagon II. Nucleotide and corresponding amino acid sequence of the cDNA. J. Biol. Chem. 258:3280-3284. Mommsen, T.P 2000. Glucagon-like peptides in fishes: the liver and beyond. Amer. Zool. . 40:259-268. Mommsen, T.P and Mojsov, S. 1998. Glucagon-like peptide-1 activates the adenylyl . cyclase system in rockfish enterocytes and brain membranes. Comp. Biochem. Physiol. 121B:49-56.

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Thomas P. Mommsen and Ellen R. Busby


Mommsen, T.P. and Plisetskaya, E.M. 1991. Insulin in fishes and agnathans: history, structure and metabolic regulation. Rev. Aquat. Sci. 4:225-259. Mommsen, T.P Danulat, E. and Walsh, P 1992. Metabolic actions of glucagon and ., .J. dexamethasone in liver of the ureogenic teleost Opsanus beta. Gen. Comp. Endocrinol. 85:316-326. Mommsen, T.P Vijayan, M.M. and Moon, T.W. 1999. Cortisol in teleosts: Dynamics, ., mechanisms of action and metabolic regulation. Rev. Fish Biol. Fish. 9:211-268. Mommsen, T.P., Silverstein, J.T., Plisetskaya, E.M., Whittaker, L.J., Whittaker, J. and Conlon, J.M. 2002. Two insulins from channel catfish: Purification, structures, receptor binding and cDNA sequences. Fish Physiol. Biochem. 25:61-70. Mommsen, T.P Busby, E.R., von Schalburg, K.R., Evans, J.C., Osachoff, H.L. and Elliott, ., M.E. 2003. Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol. J. Comp. Physiol. B 173:419-427. Moon, T.W. 1998. Glucagon: From hepatic binding to metabolism in teleost fish. Comp. Biochem. Physiol. B 121:27-34. Moon, T.W. 2001. Glucose intolerance in teleost fish: Fact or fiction? Comp. Biochem. Physiol. B 129:243-249. Moon, T.W., Gambarotta, A., Capuzzo, A. and Fabbri, E. 1997. Glucagon and glucagonlike peptide signaling pathways in the liver of two fish species, the American eel and the black bullhead. J. Exp. Zool. 279:62-70. Moon, T.W., Busby, E.R., Cooper, G.A. and Mommsen, T.P. 1999. Fish hepatocyte glycogen phosphorylase - a sensitive indicator of hormonal activation. Fish Physiol. Biochem. 21:15-24. Navarro, I. and Moon, T.W. 1994. Glucagon binding to hepatocytes from two teleost fishes: The American eel and the brown bullhead. J. Endocrinol. 140:217-227. Navarro, I., Carneiro, N.M., Párrizas, M., Maestro, J.L., Planas, J. and Gutiérrez, J. 1993. Post-feeding levels of insulin and glucagon in trout (Salmo trutta). Comp. Biochem. Physiol. A 104:389-393. Orskov, C., Holst, J.J., Knuhtsen, S., Baldissera, F.G.A., Poulsen, S.S. and Nielsen, O.V. 1986. Glucagon-like peptides GLP-1 and GLP-2, predicted products of the glucagon gene, are secreted separately from pig small intestine but not pancreas. Endocrinology 119:1467-1475. Panserat, S., Plagnes-Juan, E., Breque, J. and Kaushik, S. 2001. Hepatic phosphoenolpyruvate carboxykinase gene expression is not repressed by dietary carbohydrates in rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 204:359-365. Parker, D.B., Coe, I.R., Dixon, G.H. and Sherwood, N.M. 1993. Two salmon neuropeptides encoded by one brain cDNA are structurally related to members of the glucagon superfamily. Eur. J. Biochem. 215:439-448. Pauly, R.P Rosche, F., Wermann, M., McIntosh, C.H.S., Pederson, R.A. and Demuth, ., H.U. 1996. Investigation of glucose-dependent insulinotropic polypeptide-(1- 42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization time of flight mass spectrometry— A novel kinetic approach. J. Biol. Chem. 271:23222-23229. Plisetskaya, E.M. and Mommsen, T.P. 1996. Glucagon and glucagon-like peptides in fishes. Int. Rev. Cytol. 168:187-257.

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Plisetskaya, E.M. and Sullivan, C.V. 1989. Pancreatic and thyroid hormones in rainbow trout (Salmo gairdneri): What concentration does the liver see? Gen. Comp. Endocrinol. 75:310-315. Rombout, J.H. and Reinecke, M. 1984. Immunohistochemical localization of (neuro)peptide hormones in endocrine cells and nerves of the gut of a stomachless teleost fish, Barbus conchonius (Cyprinidae). Cell Tissue Res. 237:57-65. Ronner, P 1994. Perfusion of pancreatic endocrine tissue of teleost fish. In: Biochemistry . and Molecular Biology of Fishes, P Hochachka and T.P. Mommsen (Eds.) Elsevier, .W. Amsterdam, Vol. 3. Analytical Techniques, pp. 179-189. Ronner, P and Scarpa, A. 1987. Secretagogues for pancreatic hormone release in the . channel catfish (Ictalurus punctatus). Gen. Comp. Endocrinol. 65:354-362. Sarkar, S. and Subhedar, N. 2001. Glucagon-like immunoreactivity in the forebrain and pituitary of the teleost, Clarias batrachus (L.). Gen. Comp. Endocrinol. 121:23-31. Sherwood, N.M., Krueckl, S.L. and McRory, J.E. 2000. The origin and function of the pituitary adenylate cyclase-activating polypeptide (PACAP)/glucagon superfamily. Endocr. Rev. 21:619-670. Silverstein, J.T., Bondareva, V.M., Leonard, J.B.K. and Plisetskaya, E.M. 2001. Neuropeptide regulation of feeding in catfish, Ictalurus punctatus: A role for glucagon-like peptide-1 (GLP-1)? Comp. Biochem. Physiol. B 129:623-631. Soengas, J.L. and Moon, T.W. 1998. Transport and metabolism of glucose in isolated enterocytes of the black bullhead, Ictalurus melas: Effects of diet and hormones. J. Exp. Biol. 201:3263-3273. Suzuki, A., Nakauchi, H. and Taniguchi, H. 2003. Glucagon-like peptide 1 (1-37) converts intestinal epithelial cells into insulin-producing cells. Proc. Natl. Acad. Sci. USA. 100:5034-5039. Tang-Christensen, M., Larsen, P Thulesen, J., Romer, J. and Vrang, N. 2000. The .J., proglucagon-derived peptide, glucagon-like peptide-2, is a neurotransmitter involved in the regulation of food intake. Nature Med. 6:802-807. Thorgaard, G.H., Bailey, G.S., Williams, D., Buhler, D.R., Kaattari, S.L., Ristow, S.S., Hansen J.D., Winton, J.R., Bartholomew, J.L., Nagler, J.J., Walsh, P Vijayan, M.M., .J., Devlin, R.H., Hardy, R.W., Overturf, K.E., Young, W.P Robison, B.D., Rexroad, C. ., and Palti, Y. 2002. Status and opportunities for genomics research with rainbow trout. Comp. Biochem. Physiol. B 133:609-646. Wright, J.R. 2000. Glucose homeostasis in the teleost fish tilapia: Insights from Brockmann body xenotransplantation studies. Amer. Zool. 40:234-245. Yang, H., Morrison, C.M., Conlon, J.M., Laybolt, K. and Wright, J.R. 1999. Immunocytochemical characterization of the pancreatic islet cells of the Nile tilapia (Oreochromis niloticus). Gen. Comp. Endocrinol. 114:47-56. Yeung, C.M., Mojsov, S., Mok, P and Chow, B.K.C. 2002. Isolation and structure.Y. function studies of a glucagon-like peptide 1 receptor from goldfish Carassius auratus: Identification of three charged residues in extracellular domains critical for receptor function. Endocrinology 143:4646-4654. Zhou, L. and Irwin, D.M. 2004. Fish proglucagon genes have differing coding potential. Comp. Biochem. Physiol. B 137:255-264.

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The Development of the Gastro-Entero-Pancreatic (GEP) Endocrine System of Teleosts
B. Agulleiro*, M.P. García Hernández, M.T. Elbal and M.T. Lozano

ABSTRACT This review attempts to integrate the limited data available on the development of the teleost gastro-entero-pancreatic (GEP) endocrine system, which consists of endocrine cells scattered among the epithelium of the digestive tract and the endocrine cells that make up the endocrine pancreas. The first appearance and subsequent distribution of endocrine cells in the teleost digestive tract correlate with significant stages in its development in relation with endogenous, endo-exogenous or exogenous feeding of larvae and the resulting changes in the gut morphology and histology. Serotonin (SER)-immunoreactive (ir) cells first appear in stomach and intestine before these regions are histologically differentiated and, therefore, prior to the onset of exogenous feeding in the sea bass, Dicentrarchus labrax. Gastrin (GAS)/cholecystokinin (CCK)-like peptides are found in the intestine
Authors’ address: Department of Cell Biology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain. *Author for Correspondence: E-mail:

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Fish Endocrinology

before the first feeding, after the opening of the mouth and before exhaustion of the yolk sac, or after this event in some species. Glucagon (GLU)-like immunoreactivity appears early in the intestine and later in the stomach. Immunoreactivity to peptide tyrosine tyrosine (PYY) and/or to neuropeptide tyrosine (NPY), or the expression of peptide tyrosine (PY), are detected in the cells of the intestinal epithelium of some larvae after the beginning of exogenous feeding and, in D. labrax, also in the stomach once the gastric glands have differentiated, these immunoreactivities coexisting with glucagon in this species but not in the turbot, Scophthalmus maximus. In D. labrax larvae, pancreatic polipeptide (PP) immunoreactivity is found in cells of intestine and stomach after the appearance of PYY and NPY immunoreactivities in the former but afterwards in the latter. Such immunoreaction is absent in the Japanese flounder, Paralichtys olivaceus. Both somatostatin 14 (SS 14) and somatostatin 25 (SS 25) are colocalized in the same cells of the stomach, before it is completely differentiated in D. labrax and S. maximus, and in the latter species also in the intestine. Met-enkephalin (met-ENK)-ir cells are present in the foregut of the leptocephalic larvae of the eel, Anguilla anguilla, its distribution extending to the whole gut in later stages, and in intestine and rectum of D. labrax from the onset of exogenous feeding. The occurrence of neurotensin (NT)- and substance P (SP)-ir cells during gut development has been studied only in S. maximus. Here they appear in the intestine after differentiation of the stomach anlage and as soon as the stomach has differentiated, respectively. Insulin (INS) immunoreactivity first appears in the stomach of D. labrax before the differentiation of the gastric glands and is present in more advanced developmental stages and in adults, while INSimmunoreactivity appears in both the stomach anlage and the upper intestine of S. maximus, disappearing altogether in later developmental stages. The endocrine pancreas of teleosts buds off from the digestive tract very early in development, making up a small islet that later becomes the principal islet. The first hormone to appear in the initial mass of endocrine cells is insulin (INS), followed by SS. In those species in which the occurrence of SS 14 and 25 has been tested, SS 25 is the first to appear, and is found at the same time as INS in D. labrax and gilthead sea bream, Sparus aurata. Glucagon appears some hours/days later, depending on the species, while PP and/or other peptide(s) belonging to the NPY family are the last hormone(s) to be located. Accessory, small or intermediate islets are found after the principal islet has formed, probably originating from the pancreatic ducts, at least in D. labrax. Accessory islets differ from the principal islet as regards the occurrence of cells containing peptide(s) of the NPY family in P olivaceus and S. maximus, or the . presence of GLU cells and of cells showing co-existence of GLU and some peptide(s) of the NPY family, in D. labrax.

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B. Agulleiro et al.


Pancreatic endocrine cells have only been ultrastructurally characterized during the development of D. labrax, where they display an ultrastructure that varies with the stage of their maturation process. However, right from their first appearance they have characteristic secretory granules that allow their correlation with similar endocrine cell types in advanced developmental stages and in adults, in which they have been immunogold labeled with the corresponding antisera. Key Words: Endocrine cells; Digestive tract; Islets; Teleost; Ontogeny.

INTRODUCTION The gastro-entero-pancreatic (GEP) system of teleosts comprises numerous types of hormone-containing cells scattered along the epithelial lining of the digestive tract or grouped in pancreatic islets. Immunocytochemical procedures, using both antisera raised against mammalian and teleost hormones, have allowed the localization of several hormones. Occasionally, in situ hybridization techniques have been applied. Ultrastructural studies have been carried out in some teleost species using either conventional or immunoelectron microscopic techniques. Some of the results on the endocrine pancreas obtained in these studies have recently been reviewed (Youson and Al-Mahrouki, 1999). The present chapter will focus on the endocrine cells which have been identified by the light- or electron-microscopic immunocytochemistry or by in situ hybridization. The ontogeny of the GEP endocrine system is studied in the context of the main developmental phases of the digestive system, which have been established in accordance with anatomical and histological features. In this respect, hatching, opening of the mouth (and, therefore, the onset of exogenous feeding), the complete absorption of the yolk sac and the appearance of the first gastric glands are important events that mark the development of the fish digestive tract. An undifferentiated gut is found in the larvae of some teleosts during the endogenous-feeding period (Boulhic and Gabaudan, 1992; Sarasquete et al., 1995; García Hernández et al., 2001), although in some species esophagus, presumptive stomach and intestine can be distinguished in this period (Segner et al., 1994; Ribeiro et al., 1999; Elbal et al., 2004). The opening of the mouth and the start of exogenous feeding frequently determine regional gut differentiation (Boulhic and Gabaudan, 1992; García Hernández et al., 2001). In the period of exclusive exogenous feeding, the differentiation of the stomach, in which the gastric glands appear, and of the intestine are

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Fish Endocrinology

completed and the pyloric caeca develop (Segner et al., 1994; García Hernández et al., 2001; Elbal et al., 2004). ONTOGENESIS OF THE ENDOCRINE CELLS OF THE TELEOST DIGESTIVE TRACT Little attention has been given to the endocrine cells of the developing gut in fish and very few studies have reported the occurrence of endocrine cells by electron microscopy (Rombout et al., 1978; Connes and Benhalima, 1984; Albertine-Berhaut, 1988). However, they have been studied in detail using conventional techniques during the ontogeny of the sea bass, Dicentrarchus labrax (García-Hernández et al., 1994b, c) where endocrine cells have been characterized from their appearance just after the opening of the mouth. The ultrastructure of these cells varies during stomach and intestine differentiation. In the earliest stages, all endocrine cells show ultrastructural features of undifferentiated cells, such as numerous free ribosomes, poorly developed organelles and scarce secretory granules. The ultrastructure of these cells is similar to that of the gut epithelial cells, to which they are attached by desmosomes. This seems to point towards a common origin for endocrine and epithelial cells in the digestive tract. As development progresses, the endocrine cells undergo a maturation process that involves a decrease in the number of free ribosomes, the development of organelles, and an increase in the number and size of the secretory granules, although these granules conserve their morphological characteristics (García Hernández et al., 1994b, c). Although gut endocrine cells have a replicating capacity in mammals (Fujimoto et al., 1980; Lehy, 1982; Kubben and Bosman, 1989), in D. labrax larvae only one intestinal cell type, considered as immature, is found in mitosis (García Hernández et al., 1994c). Dividing endocrine cells are not seen in the developing gut of Barbus conchonius, wich has been attributed to the long turnover time of endocrine cells (Rombout et al., 1984). Serotonin-Immunoreactive Cells Serotonin (SER) (5-hydroxytryptamine) is an important neurotransmitter in brain and also present in the enterochromaffin cells of the gut (El-Shaly et al., 1985). In teleosts, SER stimulates intestinal smooth muscle contraction (Jensen and Holmgren, 1985), controls acid and pepsine secretion (Holstein and Cederberg, 1984) and regulates the ionic

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B. Agulleiro et al.


selectivity of the intestinal epithelium (Kiliaan et al., 1989). In some vertebrates, SER-containing cells may be immature endocrine cells since they are frequently located in regions of cell proliferation in the gastric glands and intestinal mucosa (Inokuchi et al., 1984). It has been suggested that serotonin is the chemical inducer of muscle motility in the developing mammalian intestine (Totzauer, 1991). In adult teleosts, SER-immunoreactive (ir) cells have been demonstrated in the stomach of the gilthead sea bream, Sparus aurata (Abad et al., 1987), and a hybrid sparid fish, the pantex, Pagrus major x Dentex dentex (Radaelli et al., 2001), and in both stomach and intestine of the rainbow trout, Oncorhynchus mykiss (Beorlegui et al., 1992b; Barrenechea et al., 1994). SER-ir cells have been also found in stomachless teleosts: in the two anterior parts of the intestine of Poecillia reticulata and Leuciscus idus melanotus (Burkhardt-Holm and Holmgren, 1989) and in the entire gut tract of Zacco platypus (Ku et al., 2004). SER immunoreactivity co-exists with other hormonal peptides in the stomach of O. mykiss (Barrenechea et al., 1994). In teleosts, these cells seem to be characterized by their pleomorphic and highly electron-dense (NoaillacDepeyre and Gas, 1982) or small, round, dense (Kiliaan et al., 1997) secretory granules. SER-ir cells are not detected in the gut of leptocephalic larvae of the eel, Anguilla anguilla, where they appear after metamorphosis into glasseels and in adults (L´Hermite et al., 1985). They are numerous in the pyloric caeca and in the neck or the bottom of the gastric glands. In D. labrax, SER-ir cells are first identified in the last portion of the undifferentiated gut of the larvae before the opening of the mouth, and later in the gastric region before the differentiation of the gastric glands; they are present in the stomach and rectum of more developed larvae (García Hernández et al., 1994a). In adult specimens, they are localized in all regions of the digestive tract being most abundant in the pyloric stomach (Gómez-Visus, 1994). In the turbot, Scophthalmus maximus, SER cells first appear in the stomach anlage of young larvae and later in the differentiated stomach of larvae and juvenile specimens (Reinecke et al., 1997). Gastrin/Cholecystokinin-Immunoreactive Cells Gastrin (GAS), cholecystokinin (CCK) and caerulein make up a peptide family with a common C-terminal tetrapeptide sequence. The existence of

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Fish Endocrinology

multiple forms of GAS/CCK-like peptides in the gastro-intestinal tract of teleosts has been indicated by radioimmunological and physiological studies (Jönsson et al., 1987), although gastrin has not been identified in these fish (Johnsen, 1998). In some teleosts, cDNA encoding CCK (Peyon et al., 1998; Suzuki et al., 1999; Jensen et al., 2001; Kurokawa et al., 2003) or GAS (Kurokawa et al., 2003) has been cloned. Three forms of CCK, consisting of 7, 8 and 21 residues, respectively, have been identified in the pyloric caeca of O. mykiss (Jensen et al., 2001). In some teleosts, the administration of CCK increases gallbladder motility (Aldman et al., 1992) and the secretion of pancreatic enzymes (Einarsson et al., 1997), and delays gastric emptying (Olsson et al., 1999). Strong excitatory effects of GAS/CCK on gut muscle have also been demonstrated (Jönsson et al., 1987). In adult teleosts, the presence of GAS/CCK-containing cells has been demonstrated using antisera raised against several peptides or peptide fragments of the GAS/CCK family. In the stomachless fish B. conchonius, cells that immunoreact with antisera raised against the C-terminal portion or the C-terminal tetrapeptide of gastrin have been identified in the anterior part of the intestine, suggesting that they may contain gastrin-, CCK- or caerulein-like molecules (Rombout and Taverne-Thiele, 1982). Moreover, using similar antisera, GAS/CCK-like-containing cells have been demonstrated in the pyloric caeca and the proximal regions of the intestine of S. aurata (Abad et al., 1987) and of O. mykiss, in which they have also been found in the pyloric region of the stomach (Beorlegui et al., 1992b; Barrenechea et al., 1994). In flatyfish or Japanese flounder, Paralichtys olivaceus, endocrine cells that react with an antiserum against the middle fragment of human (h) GAS and with anti-CCK 28 have been located in the pyloric caeca but only CCK 28-ir cells have been found in the intestine (Yoshida et al., 1983), where strong in situ hybridization signals for CCK and GAS appear in typical endocrine-like cells (Kurokawa et al., 2003). In other teleosts, endocrine cells reacting with antisera against hGAS (Reifel et al., 1983) or against the specific C-terminal portion of GAS/CCK (Jönsson et al., 1987) have been detected in stomach and intestine. In the leaping mullet, Mugil saliens, and in S. aurata, hGAS 34-ir cells and CCK 8-ir cells have been observed, the former in stomach, pyloric caeca and anterior intestine and the latter only in pyloric caeca and anterior intestine (Elbal and Agulleiro, 1986; Elbal et al., 1988). No GAS/CCK-ir cells have been identified in the stomach of S. aurata (Abad et al., 1987). In Z. platypus, CCK 8-ir cells are found

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antisera against cholecystokinin octapeptide (CCK 8). one day before the first feeding and trypsinogen synthesis. CCK 8 and GAS 17 antisera react with identical cells throughout the development of S. in the anterior midgut and in the area adjacent © 2006 by Taylor & Francis Group. a synthetic fragment of Japanese flounder CCK 10 (anti-CCK) and in situ hybridization have been used (Kurokawa et al. Thus. On the other hand.. no CCK-ir cells have been detected in the stomach of larvae. LLC . These results suggest the existence of at least two different cell types that contain peptides belonging to the GAS/CCK family. 2001).and GAS 17-ir cells are seen in the intestine. 2004). In the tigerfish. However. Carassius auratus. 2003). CCK-ir cells are first found 45 days after hatching. labrax.. In the developing gut of D. juveniles or adults (Yoshida et al. In the tilapia. Agulleiro et al. the stimulative regulation of pancreatic enzyme secretion by CCK starts at first feeding. maximus (Reinecke et al. Oreochromis mossambicus. 1992). 1997). Hidrocynus forskahlii. The cells are more abundant in the proximal part of the intestine. Hippoglossus hipoglossus (Kamisaka et al. GAS 34.B. where they are more prominent in the upper intestine than in the stomach. 2000).. On the other hand... Using anti-flounder CCK 10 serum in the Atlantic halibut. 2000). The expression of CCK in the intestinal epithelium starts two days posthatching. These cells are detected at early developmental stages in stomach and intestine I. immunocytochemistry using an antiserum against . before the complete absorption of the yolk sac (García Hernández et al. In this species. and in the goldfish. 12 days after the onset of exogenous feeding and final yolk absorption. the CCK 8-ir cells being much more numerous than the other cells. some endocrine cells display immunoreactivity to GAS/ CCK/caerulein when antibodies to different fragments of this peptide family are used (Kiliaan et al.. After differentiation of the stomach. a peptide molecular form of which could include a sequence similar to the GAS and CCK C-terminal tetrapeptide-amide might be present in the stomach. only an antiserum specific for the common C-terminal tetrapeptide amide reacts with some cells of the stomach while CCK 8-.. In P olivaceus. h-gastrin 34 (GAS 34) and h-gastrin 17 (GAS 17) react with endocrine cells in the intestine at early developmental stages. 1983). CCK-ir cells are distributed from the pyloric caeca to the middle part of the intestine in larvae and juveniles (Kurokawa et al.. 1994a). GAS/ CCK-ir cells have been found in the stomach (Coetzee et al. the flounder gastrin mRNA starts to be expressed in the intestine of larvae during metamorphosis when the stomach differentiates (Kurokawa et al.. 263 throughout the intestinal tract except for the most proximal segment (Ku et al. 1991)..

saliens (Elbal et al. 1987). 1993. (this volume). Ku et al. Both GLU... Holmgren et al. L´Hermite et al. GLU-ir cells are observed in the stomach of M. to both GLU and glucagon-related peptides.and glycentin-like immunoreactivities are detected in endocrine cells of stomach and © 2006 by Taylor & Francis Group.. pancreas and gallbladder are already differentiated at hatching and the onset of exogenous feeding occurs one day after hatching. and to GLU and GLP in either the same (Nozaki et al. ten of the first thirteen amino acids being identical (Duguay and Mommsen. GLUir cells are present in the whole intestine of A.. Glucagon-Immunoreactive Cells Peptides containing a glucagon (GLU) sequence such as glycentin and oxintomodulin and a GLU-like peptide (GLP) seem to be products of the proglucagon processing in fish (Conlon. Plecoglossus altivelis.. possibly glycentin (Rombout and Reinecke. 1989). 2003). fish GLUs are peptides of 29 amino acids. to glycentin (Yoshida et al. The best-known and principal function of GLU in fish is its glycogenolytic action on the liver. In the ayu. As in mammals.. These cells might express a GLU-related peptide. 1983). Rombout et al. which results in hyperglycemia. 1985). Pan and Fang. since the immunoreaction is not abolished after preabsorption of the antiserum with GLU. the rate of amino acid uptake by the liver and ureagenesis (Mommsen and Moon. thus. 1979.. labrax larvae. For further information on the physiology of GLU in fish see Mommsen et al... 1994). 1985.. 2004). Cells immunoreactive to GLU (Langer et al. 1992b) or different (Al-Mahrouki and Youson. 1986 a. 1998) cells have been described in teleost intestine. anguilla glass-eels (L´Hermite et al. Beorlegui et al. high concentrations of GLU increase lipolysis.. Moreover. In D.264 Fish Endocrinology to the pyloric caeca but these cells are absent from the stomach. 1988). 1988. relatively variable in sequence although key amino acids are invariant in all species.. In this species. LLC . 1994a).. 1990). GLU-ir cells are first identified in the intestinal region and later in the gastric epithelium before differentiation of the gastric glands (García Hernández et al. 1982. b. in contrast to that observed in the pancreatic islets (García Hernández et al.. CCK may have a regulatory effect on the secretion of pancreatic enzymes and the contraction of the gallblader from early development. the same antiserum detects CCK-ir cells in the larval midgut from the day of hatching onwards (Kamisaka et al. Abad et al. 1984. 1994a).

. 1994b... and peptide tyrosine tyrosine (PYY) and pancreatic polypeptide (PP). and differ from the GLU-ir cells of the endocrine pancreas of the same species (Agulleiro et al. CerdáReverter and Larhammar. maximus... labrax (Gómez-Visus et al. neuropeptide tyrosine) family of peptides (see: Conlon. However. Neuropeptide Y-related Peptide-Immunoreactive Cells The neuropeptide Y (NPY. 1992. as reported for B. 1986. 1997). 1992. 1998). and salmon PYY. Kimmel et al. 1996. The ontogeny of GLU-ir cells has been studied only in S. In some teleosts. c). cDNAs encoding three different . 2002). Barton et al. 265 intestine of adult D... 1982). Agulleiro et al. 1991. 1993). labrax (Gómez-Visus et al. which are present in the endocrine cells of the gut and pancreas (Larhammar. PYY. 1985. or a homogeneous dense round core and a narrow clear halo. These cells are ultrastructurally similar to the cells correlated with GLU-ir cells of the differentiated stomach and intestine of larvae (García Hernández et al.. Nguyen et al. NPY has been isolated from the brain and PYY has been obtained from the stomach (Jensen and Conlon. 2000).. also in endocrine cells of intestine and pancreas. However. Conlon et al. an equatorial needle-shaped material can be seen. They have round secretory granules with a clotted content of variable electron density and an irregular clear halo. which is expressed exclusively in the central and peripheral nervous systems.B. we will use the term PYY in this review to avoid confusion. conchonius (Rombout and Taverne-Thiele. Conlon. occasionally. Here. GLU-ir cells appear early in the anterior intestine and afterwards in the stomach. 1998). investigations on the presence of peptides of the NPY family in the endocrine cells of the teleost gut and pancreas have been undertaken using antisera raised against mammalian PP PYY and NPY. © 2006 by Taylor & Francis Group. 1986. this volume) consists of NPY. 1995) and are named as PY by some authors. LLC . 1995). PYY or NPY in some papers. GLU cells have been identified and characterized using immunocytochemistry for electron microscopy in the stomach of D. . being found throughout the gastro-intestinal tract at more advanced stages (Reinecke et al.. In P olivaceus. These peptides are expressed in the brain and one of them. NPY-related peptides have been obtained (Kurokawa and Suzuki. The peptides of the NPY family found in the teleost endocrine pancreas are more similar to tetrapod NPY and PYY than to any tetrapod PP sequence (Andrews et al. the last referred to as PP.

1984) and P reticulata (Burkhardt-Holm and Holmgren.. 1984).. Danio rerio (Söderberg et al. or in the intestine and pyloric caeca of some teleosts (Stefan et al. 1998). Abad et al. 1981... 1979. On the other hand. 1994a). only the smaller granules are weakly labeled with anti-GLU (Gómez-Visus et al. mykiss (Beorlegui et al. 1986.and NPYir cells appear when the gastric glands differentiate. 1998). PYY and PP immunoreactivities seem to be co-localized © 2006 by Taylor & Francis Group. moreover. It has been suggested. 1978.. Rombout et al.266 Fish Endocrinology The physiological function of peptides of the NPY family in fish is not well known. PYY. Elbal et al. In D. 1990). When tested in mammalian systems.. only in the butterfly fish.. labrax. 1986. PP-. NPY and anglerfish polypeptide Y (aPY) immunoreact with endocrine cells of the intestine. 1992b) and D. 1979. labrax (Gómez-Visus et al. the co-existence of peptides of the NPY family and GLU has been reported in the digestive tract of A. Rombout and TaverneThiele. b). 1987. In the latter species. decrease heart rate in rats. Abad et al. anguilla (AliRachedi et al.. Whereas both granule types are immunogold labeled with anti-NPY and anti-salmo PYY.and NPY-ir cells appear in the intestine before PP immunoreactivity can be detected. 1989).. PP-ir cells are seen in the undifferentiated stomach. Ku et al. conchonius (Rombout et al. mykiss (Beorlegui et al.. LLC . an ancient teleostean group. Noaillac-Depeyre and Hollande. PYY-ir cells are also seen in the stomach (AlMahrouki and Youson. PP-ir cells have been found either in the intestine. 1987. the piscine PPs increase blood pressure. . 1986a). Pantodon buchholzi. They are also present in the gut of stomachless teleosts (Langer et al. O. Noaillac-Depeyre and Hollande. and NPY immunoreactivities have been detected in the endocrine cells of the pyloric caeca and intestine of O. Also. B... 2004). PYY-. saliens (Elbal et al. the former before and the latter after total absorption of the yolk sac (García Hernández et al. b. However.. while PYY. in situ hybridization has demonstrated that PYY is produced in the gut endocrine cells of the zebrafish. PYY-ir cells have been identified in the digestive tract of A.. Elbal and Agulleiro. antisera to PYY. In some Osteoglossomorpha. 1988) and in the stomach of M. anguilla (Ali-Rachedi et al. 1998). 1992a.. Langer et al. 1986a. being less frequent than PP-ir cells.. 1981... GLU/NPY-ir cells show two types of secretory granules differing in size and texture of the content. 1989). 1994). 1988).. that these peptides could direct or indirectly modulate SS and PP secretion in fish islets (Noe et al. 1982. and stimulate the appetite (Balasubramaniam et al.

. in the intestinal epithelium 3 days posthatching (as demonstrated by in situ hybridization). they are present in the entire intestinal tract. PY cells appear . 2000. © 2006 by Taylor & Francis Group..or 28-amino acids in length depending on the species. Thus. which contain a SS 14 sequence that generally varies by two amino acids in their C-terminal fragments.B. GLU and NPY-related peptides co-exist in some intestinal endocrine cells of D. 2000). Sheridan et al. LLC ..and PYY-like immunoreactivities are present in different endocrine cells at all stages of development (Reinecke et al. a unique 22-amino acid SS form has been reported in the catfish. Physiologically. In this species. Later. 1998). it is thought they may regulate pancreatic enzyme secretion (Kurokawa and Suzuki. several variants of SSs that come from PPSS II have been identified in teleosts (see Duguay and Mommsen. Somatostatin-Immunoreactive Cells Besides the SS 14 form that is identical in species of all classes of vertebrates and which results from the same precursor. 2002). both SS 14 and SS 25 depress plasma insulin and glucagon levels and stimulate glycogenolysis and lipolysis (Plisetskaya and Duguay. 2000). 1994.. PYY-like containing cells appear in the anterior intestine as soon as the stomach anlage is developed. Thus. 1994. Lin et al. 25. PPlike forms being more numerous in the stomach and PYY-like forms in the intestine (García Hernández et al. in this respect SS 25 reduces the plasma GLU levels more than SS 14 (Eilertson and Sheridan.. labrax larvae. maximus. at least two peptides of the NPY family seem to be present in the gut of D. These are large SSs. PPSS II could be processed into both an 14-amino acid peptide and 25or 28-aminoacid-peptide products (Lin et al. when the larvae start exogenous feeding. In O. No PP-like immunoreactivity has been found in the intestine of adult individuals (Yoshida et al. In addition. Agulleiro et al. Sheridan. 1997). 1994a). Sheridan and Kao.. showing their highest density in the upper anterior intestine. 1993). 1982). as has been demonstrated at light and electron microscopic levels in adult specimens.. 267 in some cells in both stomach and intestine. In S. mykiss. 1994a). In P olivaceus larvae. 1983). 1993.. GLU. labrax larvae (García Hernández et al. SSs in teleosts are involved in the modulation of pituitary GH secretion and the regulation of the pancreatic hormones associated with development and metabolism. Ictalurus punctatus (Magazin et al. preprosomatostatin (PPSS) I.

SS 14-ir cells are present in stomach and pyloric caeca of glass-eels (L´Hermite et al. anguilla. 1979. 2004). 1978. Elbal et al. and in cells of the stomach and intestine of O. 1988) and immunogold-electron microscopic techniques (NoaillacDepeyre.268 Fish Endocrinology The SS 14-ir cells are generally present only in the teleost stomach (Dubois et al. SS 14-like peptides seem to be located in granules with fibrillar content.. aurata. SS 25-ir cells first appear in the gastric epithelium of the undifferentiated stomach and later in the gastric glands. Yoshida et al.. mykiss (Beorlegui et al. In Osteoglossomorpha. SS-ir cells have been ultrastructurally characterized by conventional (Elbal and Agulleiro. SS 25 and SS 14 immunoreactivities co-exist in the stomach of both larvae (García Hernández et al.. 1998). secretory granules similar to those described © 2006 by Taylor & Francis Group. 1988). No SS-ir cells were found in the gut of some stomachless teleosts (Langer et al. 1997). Elbal et al. 1985.. 1983).. maximus. 1982. punctatus (Fletcher et al. whereas SS 25-like peptides have been identified in more scarce and denser granules (Elbal et al. The cells contain round or ovoid secretory granules of variable electron density and a fibrous or homogeneous content. L´Hermite et al. 1994b.. Reinecke et al. 1991. increasing in number in the former as the fish develop (Reinecke et al. Visus et al... Rombout and Taverne-Thiele... Langer et al. In A.. 1983.. 1991) and D. 1985. 1985). SS 22-ir cells have been detected in the intestine of I. In S. 1996).. 1986. LLC . but in some species they have also been demonstrated in pyloric caeca (Langer et al.... 1996). 1996). 1979) and intestine (Falkmer et al. 1996). Thus. 1982). 1994). Visus et al. 1979. aurata (Elbal et al.. 1986.. Both SS 25 and SS 14 immunoreactivities co-exist in cells of the stomach of S. platydus (Ku et al. SS 14/SS 25-ir cells are found in the stomach anlage and in the intestine.... Barrenechea et al.. SS 14 and SS 25 immunoreactivities in stomach and intestine vary greatly among the species (Al-Mahrouki and Youson. only SS 25-ir cells in the stomach or both SS 14and SS 25-ir cells in the stomach and intestine.. labrax (Visus et al.. only SS 14-ir cells are detected in the intestine. labrax. 1983). 1994a) and adults (Visus et al.. labrax have.. Abad et al. Elbal et al. throughout development and in adults (García Hernández et al. In the teleost stomach.. In D. which may indicate variations in the processing of prosomatostatin in this species. 1987. Elbal and Agulleiro. 1978. 1991). 1992a. although they are present in the proximal segments of the gut of Z. SS cells of D. Holmgren et al. In S. 1979.

labrax. Rombout and TaverneThiele. in the latter being more numerous in the anterior region. Met-ENK-ir cells are present in the foregut of leptocephalic larvae and in the whole gut of glass-eels. mykiss (Holmgren. 1988). Agulleiro et al. Elbal and Agulleiro. B. Elbal et al.... 1985). 1992). Kiliaan et al. 1991). auratus (Abad et al. 2001). Barrenechea et al.. Elbal et al. 1983) and the ion selectivity of the intestinal epithelium (Kiliaan et al. The physiological effects of NT in fish is not well known. Methionine-Enkephalin-Immunoreactive Cells Members of the enkephalin (ENK) family of pentapeptides are principally present in the brain but are also formed in some other tissues. 1984. Elbal et al. 1985.. 1988... the enkephalin-like peptides have an excitatory effect on the stomach of O. In D. 1992a.. © 2006 by Taylor & Francis Group. 1986.. 1985. Gadus morhua (Jensen and Holmgren. 1992). 1987. Radaelli et al. NT-containing cells have been detected with an antiserum raised against C-terminal NT in the stomach of S.. these cells are located in the epithelium of the intestine and rectum from the start (García Hernández et al.... 1992). 1980. 269 in adult specimens of other species (Noaillac-Depeyre. Abad et al. 1987) and the intestine of C. 1979.B. In vitro. 2004). LLC . 1979. 1983) and a variable effect on the intestine of the Atlantic cod. Burkhardt-Holm and Holmgren. 1986. 1979. Beorlegui et al. No immunoreactivity has been detected with antisera against the N-terminal fragment of NT (Rombout and Reinecke. Elbal and Agulleiro. Most attention has been focused on methionine (met)-ENK-ir cells. 1982.. 1984) and O. 1985). 1984) or against synthetic NT (Langer et al. Neurotensin-Immunoreactive Cells Neurotensin (NT) is a decapeptide that contains a well conserved Cterminal region. 1994) and in the intestine of stomachless and stomach-containing species (Langer et al.. mossambicus (Kiliaan et al.. auratus (Reinecke et al.. 1994a) or before of exogenous feeding (Mola et al. in the latter of which they are more numerous in the pyloric caeca (L´Hermite et al. conchonius (Rombout and Reinecke.. L´Hermite et al. 1989. which have been identified in stomach and intestine of some teleosts (Langer et al. Rombout and Reinecke. In vitro experiments seem to indicate that it affects the stomach muscle (Holmgren..

and after removal of the peptide signal proinsulin folds to form the correct tertiary structure (Steiner et al.. 2000). this volume).. Barrenechea et al. SP stimulates the contraction of the smooth muscle of the gut (Holmgren. Beorlegui et al... see: Conlon. 2002) or in both the stomach and intestine (Holmgren et al. maximus. these cells have also been described in juvenile specimens (Reinecke et al. Insulin-Immunoreactive Cells The Insulin (INS) structure consists of two polypeptide chains. a B-chain of approximately 30 residues and an A-chain of about 21 amino acids. 1987. Jensen and Holmgren.270 Fish Endocrinology The ontogeny of NT-ir cells has only been reported in S. In teleosts. 1992a. It stimulates the uptake of glucose and amino acids by skeletal muscle and liver. 1994).. 1989. A comparison of insulin sequences from representative teleosts has been performed (Mommsen and Plisetskaya.. 1997). Burkhardt-Holm and Holmgren. 1987.. 1992. SP immunoreactivity has been detected in endocrine cells of the gut of several teleosts. Substance P-Immunoreactive cells Substance P (SP) is an undecapeptide present in the intestinal tract of vertebrates.. 1991). for detailed information see: Navarro et al. increases protein synthesis in these tissues and fatty acid uptake into liver lipids (Mommsen and Plisetskaya.. but are absent from the stomach (Reinecke et al. Abad et al. they are found in all regions of the intestinal tract.. SP-ir cells are found in the intestine (Langer et al. 1993). Afterwards. Elbal et al. A total of 17 amino acid residues have been identified as invariants (Chan et al. INS is synthesized as a precursor (preproinsulin). INS acts primarily as a metabolic regulatory hormone in fish.. 1983. Elbal and Agulleiro. Rombout and Reinecke.. 1997). LLC . Liu et al. 1979. The ontogeny of SP-ir cells has been studied only in S.. maximus. Killian et al. 1988. Jensen et al. 1984. 1985. Chan and Steiner. 1991. 1982. The single chain proinsulin is then cleaved in the secretory granule to release insulin and the connecting C-peptide. this volume). cross-linked by one intra. They are first found throughout the intestinal tract when the stomach anlage appears. 1992.. © 2006 by Taylor & Francis Group.and two inter-chain disulfide bonds. 1986. They appear in the anterior intestine once the stomach has differentiated.

The antibodies used in these studies were anti-bonito INS. 1998. 1995. Guyot et al. In S. 1998).B. García Hernández and Agulleiro. Assouline et al. 2001.. Guyot et al. 1994. by conventional electron microscopy (Agulleiro et al.. 1998) or a cord-shaped mass of cells included in the dorsal wall of the anterior gut during very early developmental stages. 1997).. García Hernández et al.. 1996). maximus. a mammalian-INS antiserum revealed INS-ir cells in the stomach anlage and upper intestine at the onset of feeding while these cells were absent during the following developmental stages and in adults (Reinecke et al.. 2002.. 2000. on the other hand. Navarro et al.. 1974. 1982. 1992.. 1996).. Beccaria et al.. 1994d. Kurokawa et al. and by in situ hybridization (Argenton et al. Biemar et al. bicirrhosum and Gnathonemus petersii (Al-Mahrouki and Youson. 1990. Guyot el al. 271 INS immunoreactivity has been demonstrated using antisera against mammalian INS in endocrine cells of the stomach and intestine of the osteoglossomorpha P buccholzi and only in the intestine of Osteoglossum . Devos et al. In adult specimens. in the differentiated stomach (García Hernández et al. 1999. In D. Morrison et al. the immunostaining with fish INS antiserum being abolished after the preabsorption with bonito INS. Insulin-like Growth Factor-Immunoreactive Cells Insulin-like growth factor I (IGF-I) immunoreactive cells have been detected in fish gastro-intestinal tract and are described in detail by Reinecke (this volume). 1994a) and in adults (Visus et al. Kurokawa and Suzuki.. by immunocytochemistry (Rombout and Taverne-Thiele. LLC . these cells are characterized by round secretory granules of variable electron density that are immunogold labeled with bonito-INS antiserum (Visus et al. labrax. The teleost endocrine pancreas first appears as a cell cluster (Berwert el al.. fish...but not mammalian-INS immunoreactivity has been found in the stomach before differentiation of the gastric glands. 2002).. Agulleiro et al. 1979).... 2004). 2002. anti-salmon INS and anti-bovine/porcine INS. Rombout et al.. 1998). 2001. ONTOGENESIS OF THE TELEOST ENDOCRINE PANCREAS The developing endocrine pancreas of teleosts has been studied by conventional and histochemical techniques in early studies (Belsare. labrax (García Hernández and © 2006 by Taylor & Francis Group. such as newly hatched larvae of D.

1974). 1992).1) (García Hernández and Agulleiro. © 2006 by Taylor & Francis Group. 1992. In Clarias batrachus. (Navarro et al. 1995). The initial mass of endocrine cells detaches from the gut and migrates to the ventral zone to form a small islet (García Hernández and Agulleiro. aurata (Navarro et al. conchonius. GLU and SS are seen immediately after the appearance of the islet.. labrax (Fig. 2000) and S... while PP-ir cells appear one month after Fig. In B. 2000). 2001). 9.. 1992). 9.. 1998. 2001). maximus (Berwert et al.1 Pancreatic endocrine cells of the primordial cord of newly hatched Dicentrarchus labrax. maximus (Berwert et al. 2000). Note the secretory granules of a D1 cell (D1) with a content of varying shape and electron density. Kurokawa et al. cells immunoreactive to INS. Guyot et al.272 Fish Endocrinology Agulleiro. or one day after hatching in S. B. 1961) and is first seen 7 days after hatching (Belsare et al. the first islet comprises Gomori‘s fuchsin aldehyde-stained or insulin-containing cells (Falkmer.. (Kurokawa et al.. 1995) and P olivaceus . cells showing the co-existence of INS and SS 25 immunoreactivities (INS/SS 25 cells) in S. P olivaceus (Kurokawa et al. This first mass of pancreatic endocrine cells consists of INSand SS 25-ir cells in D. B cell. LLC . or only INS-ir cells in S. X 20000. aurata ...

Berwert et al. In P olivaceus. Agulleiro et al.and SS 14-ir and outer SS 25-ir cells are seen in the islet 2–5 days after hatching. in S. which becomes the principal one. small islets appear in areas surrounding the pancreatic ducts. whilst no immunoreactivity to any peptide of the NPY family occur in the islet of P olivaceus (Kurokawa et al. 1992). IGF-I immunoreactivity is detected in the principal and secondary islets from 11 days onwards.. 2004).and GLU. with SS 14-ir cells at the islet periphery. maximus... Afterwards. Oreochromis niloticus. this islet contained INSir cells. In S. In D. which appear 1–4 days after hatching... while a few NPY/PYY/PP/GLU-ir cells appear in a small peripheral area after a month (García Hernández and Agulleiro. most centrally located. INS-ir cells. labrax. are found (Berwert et al. SS 14. make up the islet (Bewert et al. see: Reinecke.. García Hernández and Agulleiro. Similarly. 1995). The cell © 2006 by Taylor & Francis Group. primordial islets consisting of INS. 1979.. which appear 6 days thereafter. one islet with INS immunopositive cells has been reported before hatching (Morrison et al... small and intermediate accessory islets appear 30 days .and GLU-ir cells throughout the islet. Kurokawa and Suzuki. maximus. 273 fertilization (Rombout et al. Rombout and Taverne-Thiele. persisting until juveniles (Berwert et al. small islets consisting of only INS-ir cells. 1982). which appear 5–6 days after hatching. Kurokawa and Suzuki. as do isolated and clustered endocrine cells of all cell types. ... 2000. These differ from the principal islet in the occurrence of peripheral PP-ir and PY mRNA expressing cells (Kurokawa et al. LLC . 1995. while a few cells were PYY-positive (Morrison et al. The subsequent stages of the developing endocrine pancreas are marked by: (1) an increase in size of the first islet. 1995).. while others are secondary islets. and (2) the appearance and increase in number and size of new islets (Belsare.. like the first islet at this stage. after hatching. SS25. 4 days after hatching. 1992). In D. Morsison et al. 1992. They are surrounded by GLU cells 4 days later.and SS 25-ir cells first appear close to the pancreatic ducts (García Hernández and Agulleiro. this volume). inner INS. and peripheral PP-ir cells. 2001) as well at hatching (Morrison et al. labrax. 1974.. In S. 1995). These small islets also contain SS 14-ir cells (5-6 days) and GLU-ir cells (8 days). while some of them contain PP-ir cells starting at 12 days (Berwert et al. maximus. In the Nile tilapia. 2002).ir cells. 1995. and SS14-. One day after hatching. 2002.B. Some small islets are of the principal type. 2000.. with SS 14-ir cells intermingled with the INS-ir cells. Kurokawa et al. 2004). 1998. Pack et al. 2004). Milewski et al. 2000). 1996.

. which allows its correlation with the same endocrine cell type in more advanced developmental stages and in adults. a gradual development of organelles and an increase in the number and size of secretory granules (García Hernández et al. medium electron-dense © 2006 by Taylor & Francis Group. increasing in frequency with age. LLC . 1991a). 1993. 1992). these islets increase in number and size. labrax. which stems from the first islet arising from the gut... Lozano et al. SS 25-ir cells appear in D. as has been demonstrated in adult salmonids (Plisetskaya et al. These cells are also found among the epithelial cells of the pancreatic ducts (García Hernández and Agulleiro. in which they have been immunogold labelled with antisera to the corresponding hormone (Agulleiro et al. 1988. 1992). 1994d). b). Thus. 1992).and GLU-ir cells and between SS 14... It has been studied in detail only in D. 1991b. The principal islet. Here. As development progresses. Thus. in adults. the principal islet contains endocrine cells arranged in wide cords surrounded by a narrow layer of exocrine tissue.274 Fish Endocrinology types and distributions are similar to those of the principal islet but the cells are surrounded by a thin layer of cells showing the coexistence of PP/ PYY/NPY and GLU immunoreactivities but no cells with GLU immunoreactivity only. labrax. labrax larvae before the GLU-ir cells can be identified. while the other islets are made up of homogenously distributed endocrine cells (Lozano et al. Sheridan et al. the cytoplasm differentiates progressively to show a decreasing number of free ribosomes. 1986. giving rise to the secondary islets (García Hernández and Agulleiro. 1994. Although some functional relationship has been suggested between SS 25... have been classified into type I (with a cell distribution similar to the principal islet) and II (with a cell distribution similar to the small islets described above) (Lozano et al.. each endocrine cell type has the characteristic secretory granules from the moment of their first appearance. In adult D. 1991a.. García Hernández et al. However. such as the inhibition of INS secretion. SS 25 cells have secretory granules with a loose. 1987). 1992). a rod-like content can be seen in some secretory granules of INS cells from early developmental stages to the adult. Little attention has been given to the ultrastructure of the developing endocrine pancreas. other functions may be attributed to SS 25 in early stages of development (García Hernández and Agulleiro.. 1992). 1994d).and INS-ir cells based on their arrangement in teleost islets (Nozaki et al. Abad et al. the ultrastructure of the pancreatic endocrine cells varies during development. which. seems to fuse with the small islets and/or isolated and clustered cells firstly found next to the pancreatic ducts in larvae (García Hernández and Agulleiro.

. although some have a polygonal core from their first appearance. © 2006 by Taylor & Francis Group. however. GLU cells have secretory granules. 9. a. which are smaller than those in the SS 25 cells in both larvae and adults (Fig. GLU. nucleus. NPY/PYY/PP/GLU cells show granules with a homogeneous medium or high electron-dense content and a clear halo in both larvae and adults. X 24000. N. 9.B. LLC . Agulleiro et al. 1998). coinciding with the end of the endotrophic period (Guyot et al.and NPY-like immunoreactivity being colocalized in the secretory granules (Fig. Secretory granules of a cell in which NPY-related peptides and glucagon co-exist immunogold-labeled with anti-glucagon (10 nm gold particles) and anti-PYY (30 nm gold particles). Fig. Note the absence of gold particles in the secretory granules of the adjacent SS 14 (D2) cell. the granule population being more heterogeneous in early developmental stages. aurata.2a). the cells forming the endocrine islet do not display any significant ultrastructural differences that would allow their classification into separate categories before 4 days after hatching.2 SS 25 (D 1) cell in the endocrine pancreas of adult Dicentrarchus labrax immunogold-labeled with anti-salmon SS 25. a x 40000. 275 content or a dark core. In S. 9. SS 14 cells have medium or high electrondense homogeneous secretory granules. with a round homogeneous core.2).

. Two bilateral rows of cells appear close to the midline within the dorsal endoderm during mid-somitogenesis (Biemar et al. García Hernández et al. undifferentiated. At 24 and 48 hpf. which may arise only from pancreatic ducts. Furthermore. a presumptive islet containing GLU-expressing cells which surround a central group of INS..... Agulleiro et al. the direct origin of pancreatic endocrine cells via the primitive gut has been confirmed by electron microscopy since the endocrine cells of the primordial cord are continuous with the gut lining cells. On the other hand. and (3) the presence of endocrine cells among the epithelial cells of the pancreatic ducts (García Hernández and Agulleiro. and there is no basal lamina between them (García Hernández et al. 1999. it has been demonstrated by in situ hybridization that INS is the first hormone gene to be expressed. Morrison et al.276 Fish Endocrinology In D.. These cells seem to accompany the endocrine cells during their migration from the gut to form the pancreas anlage. 1994d).. immunofluorescence double staining reveals the ocurrence of cells immunoreactive to both INS and SS 14 and cells © 2006 by Taylor & Francis Group. Later. The ductular origin of the endocrine cells which make up the islets that appear later is supported by: (1) the appearance of small islets in contact with the pancreatic ducts consisting of INS. mitotically active cells.. the cells aggregate into a cluster or prospective endocrine islet that also contains GLUexpressing cells (Biemar et al. except for the NPY/PYY/PP/GLU-ir cells. 2001). LLC .. since mitotic features are infrequent (García Hernández et al. they seem to originate from both gut and pancreatic ducts. the increasing number of endocrine cells within islets cannot be attributed to cell division alone. At 24 hours post fertilization (hpf). with no basal lamina separating them. rerio. 1994. 1994d. (2) the occurrence of clusters of different endocrine cell types adjacent to ductular cells. Agulleiro et al.. one-cell-thick domain that also contains SS-expressing cells (Argenton et al. García Hernández et al. Biemar et al.. 1994d). 1992. 1994d). 1992. 1994. 2001).and SS-expressing cells is formed (Biemar et al. flattened..and SS-25-ir cells with an ultrastructure similar to that of cells making up the primordial cord and also in contact with undifferentiated cells with occasional secretory granules. labrax. As regards the origin of the different endocrine cell types. 2004). 2001) but converge during late-somitogenesis to form a single. some migrating to join the principal islet originated from the gut (García Hernández and Agulleiro. 2001). In D. with no (or occasional) secretory granules have been found in the gut epithelium close to the primordial cord and surrounding the first islet after detachment from the gut.

. M.and SS-ir cells surrounded by GLU. Five to six days after fertilization. M.B. Gen. 1999). PPSS II is the predominant SS precursor.M. 276: 323-331. LLC . 2002). G. Gen.. References Abad. © 2006 by Taylor & Francis Group.. M. B. In addition. preprosomatostatin (PPSS) I. Agulleiro. Aspects ultrastructuraux et cytophysiologiques. this contains a group of INS-expressing cells surrounded by cells expressing GLU (the only pancreatic hormones tested) (Assouline et al. B. Appl.T.. Abad. one sole islet within the exocrine tissue is present. Gly 10). precursor of the conserved SS 14. Cell Tissue Res. most PPSS I-expressing cells also express PPSS II. 2001). Lozano. J. A. Aldman.T. 66: 123-136. 277 immunoreactive to a single hormone in the islet (Argenton et al. Oncorhynchus mykiss. J. Comp. Grove. Endocrinol. precursor of SS 14 variants (Tyr7. microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax). M. II. F. Agulleiro et al. 1992. Agulleiro. A comparative immunocytochemical study of the gastro-entero-pancreatic (GEP) endocrine system in a stomachless and stomach-containing teleost. it is detected ventrally and clearly separated from the gut (Assouline et al.T.W. Abad. Electron.E.. Somatostatin 14. and García Hernández M. the former being expressed later (24 hpf) than the latter (17 hpf) (Devos et al.P and Lozano. has been demonstrated during the early development of this species.E. 86: 20-25. D. 4: 65-78.E.. and PPSS II. J. Ontogeny of the endocrine . Comp. In young adult medaka. 1987.M. 2002).. Ayala.and PP-ir cells is found dorsally to the anterior gut and embedded in exocrine tissue (Biemar et al. Cell Tissue Res. B.. an islet made up of INS.and somatostatin 25-like peptides in pancreatic endocrine cells of Sparus aurata (Teleost): A light and electron microscopic immunocytochemical study.G. Endocrinol.. Comp.. afterwards they form a group that emigrate to the right side of the embryo. M. Elbal. and Agulleiro. II. Ichthyol. Albertini-Berhaut. S..P 1993.. The big and secondary islets.T. Gen. and Holmgren.H. Peeze Binkhorst. Duodenal acidification and intra-arterial injection of CCK-8 increase gallbladder motility in the rainbow trout. INS-expressing cells have first been located within the dorsal wall of the gut epithelium. García Hernández. L’intestin chez les Mugilidae (Poissons Téléostéens) à différentes étapes de leur croissance. Lozano. 1988. later. pancreas in sea bass (Dicentrarchus labrax): An ultrastructural study. Endocrinol. the expression of two different SS precursors. 2002). and Rombout. M. M.. 1994. 274: 303-314. 1992. In the medaka (Oryzias latipes) embryos. 86: 445-452. M.

Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas. M. J. Endocrinol. Van Noorden. Shively. Dev. A.. Hawke.. Balasubramanian. Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.. 1994. Tissue Cell 26: 309-321.. J. and Polak. Comp.. Belsare. 1974.A. S. A nonamidated peptide homologous to porcine peptide YY and neuropeptide YY. Argenton..F.W. Gen.. 1984. © 2006 by Taylor & Francis Group. Histological study of the organogenesis of the digestive system and swimbladder of the dover sole. Rigel. Adrian. Fisher. Forsch. M. E. Biol. L.A. 117: 299-303. and Reinecke. D.. S.. F. Epperlein. B. J. Endocrinol. Bourrat. C. Barton. and Scharfmann.E. C. M. Dev.P Gabrion. Ali-Rachedi. Scophthalmus maximus.M. Endocrinol. Mech. I. Morphogenesis of the endocrine pancreas in Clarias batrachus L. J.. S. 230: 189-203.E. Schmidteke. endocrine cells of the pyloric caeca and the intestine of Oncorhynchus mykiss (Teleostei). pancreas in the sea bass. Histochemistry 80: 487-491. M. Rainbow trout (Oncorhynchus mykiss) neuropeptide Y. and Bortolussi. T. Gen. 86: 483-495. L. 1990. W. Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet. P 1992b. Andrews.. 1992. Mikrosk... Dev. 2002. Beorlegui. Peptides 13: 1159-1163. Martínez. Díaz. D.L. 87: 217-221. LLC .M. Salmon pancreatic polypeptide exhibits neuropeptide Y-like activities in rats.. B. J.E. Gen. J. 110: 125-139.E. Beorlegui. and Dixon. 1992. Peptides 16: 113-122. Some peptide-like colocalizations in .. J.. Shaw. Development of the pancreas in medaka. and Gabaudan. H. Ontogeny of IGF-1 and the classical islet hormones in the turbot. 269: 353-357. 78: 80-92. 1998. A. J. Glucagon-producing cells.. A. Peptides 11: 673-677. F.R.... I. P ... 1999... R. Z. and Connes. Cell Tissue Res. W. Gapp. M. Comp. Segner. and Thim. Peers. Stein. D. Dicentrarchus labrax L. C. caeca and the intestine of Oncorhynchus mykiss (Teleostei).A. C. 1990. Zecchin. and Sesma. Beccaria. López. A.K. Maturation of the endocrine . Mech. F. A. and Martínez. Argenton. (Teleostei): An immunocytochemical and ultrastructural study. III. F. Studies on the development of endocrine glands in fishes.T. S. Assouline. Solea solea (Linnaeus 1758). Biemar. Boulhic. and Plisetskaya. Halton.. 2001. Aquaculture 102: 373-396. Comp. J. D. Bloom. King. an ancient teleostean group. C. A. and Driever. Leipzig 88: 981-986. Regulatory peptides in gastric endocrine cells of the rainbow trout Oncorhynchus mykiss: General distribution and colocalizations. R. R. Martínez.. Immunohistochemical studies of the endocrine cells within the gastro-entero-pancreatic system of Osteoglossomorpha. Endocrine cells and nerves in the pyloric . Berwert. D..278 Fish Endocrinology Al-Mahrouki. Anat. van Nguyen. Chance. 1985. Varndell. Barrenechea. D.M. Endocrinology 116: 26772681. and Youson. 1995.C. P 1992a.H. and Sesma.

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LLC . Hormones and Functions © 2006 by Taylor & Francis Group.SECTION 3 Pituitary: Development.

The duality of gonadotropins is practically confirmed in teleost fish. García Ayala ABSTRACT This chapter reviews recent literature on the teleost adenohypophysis. these hormones are differentially detected during ontogenic development and sexual cycle in distinct cells of the proximal pars distalis (PPD). García Hernández and A. Data on other teleosts such as perciforms which have Authors’ address: Department of Cell Biology.CHAPTER 10 Teleost Adenohypophysis: Morphofunctional and Developmental Aspects B. 30100 Murcia. Faculty of Biology. with special emphasis on the identification and characterization of the hormoneproducing cells in adults and during ontogeny. Spain. *Author for Correspondence: E-mail: aguleiro@um. Both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) have been shown to be present in most species studied either by isolation or by cloning of the encoding cDNA. Agulleiro*. Attention is given to the different molecular forms of the hormones and their expression in the pituitary © 2006 by Taylor & Francis Group. LLC . P. In salmonids. M. University of Murcia. they also have different physiological roles in the control of reproduction.

and their ultrastructure are well established in adult teleosts. their possible differential distribution or physiology not having been established to date. except in the gilthead sea bream. SL1 and SL2. The location of these cells in the PPD. their levels being differently regulated during development and in response to alterations in environmental salinity. At the ultrastructual level. the occurrence of two variants. Teleost thyrotropic hormone (TSH) is usually detected using antisera against the human TSH -subunit. PRL and SL are synthesized in distinct cells. Sparus aurata. appear during early development. both hormones are found in different cells. the time of their first identification depending on the species and even on individual studies. Two variants of the hormones that make up the growth hormone (GH)prolactin (PRL)-somatolactin (SL) family have also been found in a number of teleost species. adrenocorticotropic hormone (ACTH) and -melanotropic hormone ( MSH). in which cells containing both GH and PRL have been identified at an early developmental © 2006 by Taylor & Francis Group. and their ultrastructual features are common. have been less studied than the other teleost pituitary hormones and the cells synthesizing them are usually detected by use of antisera raised against human or synthetic molecules.e. the two PRL forms. GH. The members of the proopiomelanocortin (POMC) family. differ in their osmoregulatory and somatotropic actions. has been suggested in some species. although both coexist in the same cells. the distribution and the ultrastructural characteristics of these cells are well established in teleosts. the occurrence of LH and FSH in the same cells and/or in a synchronous manner being found in some species. As regards the ontogeny of the different pituitary cell types. The two GH forms are structurally very similar. i. However.. However.290 Fish Endocrinology not been so extensively studied. depending on the species. depending on the species. FSH and LH cells are not distinguishable from each other but both have distinct features that vary greatly through the sexual cycle which allow to distinguish them from other adenohypophyseal cells. are not so conclusive but point to patterns of gene expression different from that seen in salmonids. In addition. From their first appearance. Proteolytic conversion of ACTH to -MSH seems to occur either only in some immature secretory granules or in all secretory granules of MSH cells. except for LH. PRL and GH cells appear at the same time or separately. Cells containing TSH are similarly distributed in the dorsal PPD among all teleosts studied so far. depending on the species. respectively. PRL being found before or after GH cells. glycosylated and/or non-glycosylated SL forms occur in SL cells. PRL177 and PRL188. As regards SL. LLC . the rostral pars distalis (RPD) and the pars intermedia (PI). all of them. Pituitary cells appear in salmonids earlier (before hatching) than in other fish with a shorter pre-hatching development.

and by the direct innervation of the endocrine cells. depending on the species. by in situ hybridization techniques. but appearing before gonad differentiation or during gonadal development. the AH is divided into three parts: rostral pars distalis (RPD). Cell ultrastructure. proximal pars distalis (PPD) and pars intermedia (PI) (Scheme 1). by an almost complete interdigitation of NH and AH. Key Words: Adenohypophysis. is an endocrine gland that secretes a number of hormones and appears to be under the influence of specialized nerve cells within the brain. Teleost. Ontogeny. LLC . Identification of the hormone-secreting adenohypophyseal cells has been a long process involving several methods including staining techniques such as polychrome staining. either by identification of the hormone itself in the cells using immunocytochemical techniques for light and/or electron microscope or by detection of the expression of the genes encoding them. NH) and epithelial (adenohypophysis. As regards the two variants of PRL. which has allowed precise identification and characterization of the synthesis sites. 291 stage and in juvenile specimens. INTRODUCTION The pituitary. or to their © 2006 by Taylor & Francis Group. Hormone localization. a high expression of SL1 and a low or null expression of SL2 have been observed during the earliest developmental stages of S. In teleosts. The last hormone to be detected during ontogenic development is LH. which is present in all vertebrates. although both variants later co-exist in pituitary SL cells. which is not present in the larval stages. Moreover. Regarding the SLs. the tilapia. PRL177 is found before PRL188 in the only species studied to date. Eight different hormones have been found to be secreted by the teleostean adenohypophyseal cells and these have been grouped into families according to similarities in structure and function. several teleost pituitary hormones have been isolated or their encoding cDNA cloned. aurata. according to the distribution of the endocrine cell types. Agulleiro et al. In recent years.B. Oreochromis mossambicus. AH) tissues. ultrastructural studies and immunocytochemical procedures which initially used antisera raised against mammalian pituitary hormones. the pituitary is characterized by a lack of a neurohypophyseal median eminence and of a portal-hypophyseal vascular system (both normally present in vertebrates). It is a composite of neural (neurohypophysis.

1981). whereas the -subunit. GTH I and GTH II. 2000). which confers the biological activity. and the thyrotropin (TSH). the hormones have been grouped into three families: the gonadotropin-thyrotropin family. neurohypophysis.. All three consist of two non-covalently bound subunits. and . GTH I and GTH II are designated as FSH and LH. TELEOST ADENOHYPOPHYSEAL HORMONE-PRODUCING CELLS Cells Producing Hormones of the Gonadotropin-thyrotropin Family This family of hormone comprises three glycoproteins. LLC . © 2006 by Taylor & Francis Group. At present. rostral pars distalis. In this way. Interposed between the endocrine cells. origin from a common precursor molecule. PPD. respectively. stellate or non-secretory cells can be seen in the three areas of the AH.292 Fish Endocrinology RPD PPD NH PI NH GTH cells TSH cells GH cells PRL cells SL cells MSH cells ACTH cells Scheme 1 Schematic drawing of a sagittal view of the pituitary of S. The subunit is common to the three hormones. the growth hormone-prolactin-somatolactin family and the proopiomelanocortin family. proximal pars distalis. due to their structural and functional similarities with tetrapod gonadotropins (Quérat et al. NH. PI. two distinct gonadotropins (GTH). although there are some species-dependent variations. is unique to each one (Pierce and Parsons. The distribution of the different cell types containing distinct hormones within the AH (Scheme 1) is quite regular in teleosts. dumerilii showing the distribution of the different endocrine cell types. RPD. pars intermedia.

Van der Kraak et al. the pattern of production and secretion of FSH and LH in these fish seems to be different from salmonids.. 2002.. Odontesthes bonariensis (Miranda et al. 1992.. including a primitive species. 1996). 1993. and a significant degree of colocalization of both hormones in the platyfish. Elizur et al.. 1996. It is not clear whether a single gonadotropin cell type producing both FSH and LH.. Liu et al. 1989. 2003b). Suzuki et al. 1990. These differences might reflect differential reproductive patterns between annual and multiple spawners (Kajimura et al. Hassin et al. 1991) and the pejerrey. 2000. Yoshiura et al... Gen et al.. 1994. Koide et al. Swanson et al.. 1997... Kato et al. Swanson et al. 1992. 1996.. Vissio et al. 1989. Tanaka et al. 1988a. The physiological roles of these hormones have been established mainly in salmonids. Jackson et al. Prat et al. 1996. or two distinct cell types synthesizing either FSH or LH. The scarce available data on non-salmonid teleosts suggest that their roles might vary to some extent. 1994) has been revealed... the results depend on the antisera used. 1992). 1988. the same antisera do not distinguish distinct FSH and LH cells in some species (Mukai and Oota.. 1999) seem to confirm the duality of gonadotropins in fish. 1995... the extensive data on cloning of the cDNAs which encode for the two subunits of FSH and LH in a variety of teleosts (Sekine et al. Although a FSH-like molecule could not be found using chemical procedures in some species (Banerjee et al... Mateos et al... Mateos et al.. occur in teleosts. 1997a. 1999. FSH is involved in vitellogenesis and spermatogenesis and LH in final oocyte maturation.. Most studies have used antisera to the -subunits of salmonid FSH and LH. Weltzien et al. b. Agulleiro et al.. To a great extent.. Naito et al. 2003.B.1991.... García Hernández et al... 2001. 2001). 1999. 2003).. the Japanese eel.. 1989. 1990a. Weltzien et al. Xiphophorus maculatus (Magliulo-Cepriano et al. Okada et al. Koide et al.. However. 2003a). 1988c. By the use of these antisera the occurrence of FSH and LH cells in salmonids (Nozaki et al. 1989. ovulation and spermiation (Suzuki et al. Tyler et al. Shimizu and Yamashita. Anguilla japonica (Yoshiura et al. LLC . 1993. 1993. © 2006 by Taylor & Francis Group. Kawauchi et al.. García Hernández et al. Lin et al. 2001).. 1989... Kajimura et al. 1993. since both FSH and LH are synthesized synchronously during gametogenesis in some species (Sohn et al. 1995. 293 Gonadotropins and gonadotropic cells FSH and LH have been isolated and characterized in a number of teleosts (Itoh et al. 1991.. Copeland and Thomas.

. 2003b. A gradual variation in the relative number of FSH and LH cells has been found in salmonids during the sexual cycle. b). 1998. spawning period and increase at spawning. The use of homologous antisera to the -subunit of FSH and LH demonstrates that both gonadotropins are produced in different cells in some non-salmonids (Kagawa et al.. 2003). c). On the other hand. 2001. but does not label some GTH-like cells... c). Clarias gariepinus. in immature fish (Zandbergen et al. dumerilii (García Hernández et al.. In situ hybridization also shows the presence of FSH. However. Oncorhynchus mykiss (Naito et al... 1991). and the number of LH cells exceeds the number of FSH cells in the immature bluefin tuna. © 2006 by Taylor & Francis Group. García Hernández et al.. 1990b... Pandolfi et al.. 2000. 2003) and in H. 2003). cells in which FSH and LH immunoreactivities co-exist also occur (García Hernández et al. 1991).mRNA and LH. Calman et al. Miranda et al. However..mRNA in distinct cells of the rainbow trout. hippoglossus (Weltzien et al.. and the Atlantic halibut. reacts with all GTH cells in mature specimens (Koide et al. In addition.. Kagawa et al. as well as in related species (Shimizu et al. although in the Mediterranean yellowtail.. 2002b). 1990a. they are equally distributed in the PPD and in the periphery of the PI in S.. Sparus aurata (García Ayala et al. aurata (García Ayala et al. 1998) and O. that is homologous to the known LHs. Hippoglossus hippoglossus. an antiserum to the -subunit of the only GTH characterized in the African catfish. bonariensis (Miranda et al. (Kagawa et al. Antisera to S. b). Thunnus thynnus. Seriola dumerilii. 2003). 1998.. allow the identification of FSH and LH cells in this species.. 1992). Fundulus heteroclitus.. dumerilii gonadotropins also allow the detection of FSH and LH cells in the gilthead seabream. antisera against some synthetic fragments of the FSH and LH -subunits of the mummichog. in F heteroclitus.. probably FSH cells. although in the latter co-existence of both subunits in a single gonadotrope cannot be ruled out (Weltzien et al. Calman et al. FSH cells are numerous during both periods (Shimizu et al. 2003b. 1993). in S. ranging from the sole presence of FSH cells in early vitellogenic fish to a predominance of LH cells in mature fish collected prior to spawning (Nozaki et al. although LH cells are scarce in the pre. 2002a.294 Fish Endocrinology Segura-Noguera et al. 2001a) or react with GTH cells in some sciaenids (Yan and Thomas.. LLC . 1991). 2002a... Naito et al.. FSH and LH cells show distinct distribution patterns in the PPD of some salmonid and non-salmonid species (Nozaki et al. 2001). 2001. 2001).

point to a single. They are characterized by the variable number and size of the secretory granules. 1987. 1991. Marchelidon et al. it is impossible to establish a correlation between the production of either FSH or LH and the ultrastructure of GTH cells. Paralichthys olivaceus (Hiroi et al... García Hernández et al. 295 GTH cells are very heterogeneous as regards their ultrastructural features. 1988). 1991. Bandyopadhyay and Bhattacharya.... Teleost TSH -subunits have a high degree of amino acid sequence homology with the human (h) TSH -subunit. TSH is the principal regulator of thyroid hormone synthesis and secretion (Ng et al. Moriyama et al. Most investigations. 1989. 1997... 1999). 1984. 1995).. in the case of the latter species. Sohn et al.. Chatterjee et al. Borg et al. Thyrotropin and thyrotropic cells TSH has been isolated and characterized (Kühn et al. which may become large vacuoles.. Salmon et al. Larsen et al. both hormones.. depending on the functional stage of the fish. 1995. polymorphic GTH cell type with varying ultrastructure. the differently dilated cisternae of rough endoplasmic reticulum (RER). 1997. 1997.. 2002b) has demonstrated that the secretory granules of GTH cells of varying ultrastructural features contain indistinctly FSH or LH or. 1993a) and S. as in mammals. 1997). 2001) in some teleosts.. 1982) and may be differentially regulated by negative feedback from the thyroid hormones (Larsen et al. Banerjee et al. In teleosts.. Byamungu et al. 1987). Thus. Moriyama et al.B. LLC .. 1997) and the cDNA encoding the TSH -subunit has been cloned and sequenced (Ito et al. 1993. 1993.. 1986.. Yoshiura et al. in some cells. 1998. Electron microscopic immunocytochemistry in O.. while a highly developed and dilated RER has been related to high synthesis activity (Cook et al.. 2002b). dumerilii (García Hernández et al. Agulleiro et al. Thus. The varying features have been attributed to the functional stages of these cells... globules and IMs seem to be crinophagic structures that develop by fusion of the secretory granules (Sharp-Baker et al. Antisera raised against the © 2006 by Taylor & Francis Group. mykiss (Naito et al.. and the occurrence of globules and large membrane-bound irregular masses (IM) (Peute et al.. García Ayala et al. and to play a role in low-salinity adaptation during the metamorphosis and shore emigration of the Japanese flounder. including those carried out with conventional electron microscopy (see: García Hernández et al. 2002b).. 1993. 1994. TSH seems to be involved in metamorphosis (Miwa and Inui.

.. 1986. Quesada et al. its amino acid sequence being deduced by cDNA cloning (see: Funkenstein et al. Their genes seem to have evolved from a common ancestral gene (Ono and Kawauchi. Although presumptive TSH cells have been described ultrastructurally in some teleosts (see: García Ayala et al. 1992. b. 1994. In some species.. and H.. © 2006 by Taylor & Francis Group. hippoglossus have been detected using an antiserum against the -subunit of the pituitary glycoproteins (Weltzien et al... Anathy et al. 1988. 1994a. 2001.. LLC . Yan and Thomas. 1991. 1996.. 1998). round secretory granules with a homogeneous content of medium or high electron density and dispersed or parallel-arranged cisternae of RER. Nagler et al. 2002.. japonica (Ueda et al. Mahmoud et al.. Cavari et al. Segura-Noguera et al. TSH cells have only been identified with immunoelectron microscopy in O. García Hernández et al. Mukai and Oota.. Pendón et al. TSH cells of the coho salmon. 1983. 1996. 1986) and S... 1991a. Poecilia latipinna (Batten... Vissio et al.. prolactin (PRL) and somatolactin (SL)—the latter hormone recently described only in teleosts—are structurally and functionally related hormones.. Ayson et al. 2003c)... Einarsdottir et al.. 1991. the molly. 2002... Growth hormone and growth hormone cells GH has been isolated and characterized from several teleosts (Kawauchi et al. Two variants of GH have been found in some species (RandWeaver et al. 2003. Cambré et al. 1997. Toubeau et al. 2001). Chang et al.. Cells Producing Hormones of the Growth hormone-prolactin-somatolactin Family Growth hormone (GH). Pandolfi et al... TSH cells are located in the PPD of most teleosts studied (Ueda et al... Law et al. 2000. 1983). 2001a. 1994.. 1986. 1996. 1998).. 2003c). mykiss (Van Putten et al. Sánchez Cala et al. 1995.. 1983). C. gariepinus (Peute et al. 1996. Hong and Schartl. Venkatesh and Brenner. 1997. Oncorhynchus kisutch.. Ohkubo et al. 2000.296 Fish Endocrinology latter crossreact with fish TSH cells and have been used for their immunocytochemical identification. Lemaire et al. as in A. 1991c. 1991. 1984). 1994).. Weltzien et al. Inoue et al. Rendón et al.... 1993.. dumerilii (García Ayala et al. They have tiny. TSH cells appear only in the RPD. The TSH cells are small and ultrastructurally homogeneous. 1992. Doliana et al. since anti-teleost sera are still not available. 1994. Chen et al. Xu et al. 2003). 1996. Venugopal et al.

.. Vissio et al. Villaplana et al. Sarasquete et al. in the PI (García Hernández et al. 1996. © 2006 by Taylor & Francis Group.1986.. Weltzien et al... except in the barbel. Sánchez Cala et al. 1994. Borski et al... Calduch-Giner et al.. 1991. GH cells are characterized by large homogeneous secretory granules (Ueda et al. although they are structurally very similar and no differential distribution or physiology of the two forms has been established by now. 1996 a. 1997. shape and size of the secretory granules (Fig.. 2000. 1995... an ultrastructural heterogeneity that might be related to varying functional cell stages (Villaplana et al. Quesada et al. reproduction (Le Gac et al. 2002). 1994). 2000. Huang and Specker. Rendón et al. GH cells generally surround the neurohypophyseal processes that invade the PPD (Cambré et al. aurata (Herrero-Turrión et al. Yada et al. Batten.. Carassius auratus. 297 Jackson et al..1) vary greatly from cell to cell. 1994. 1996. García Hernández et al. the number. 2003c). 1993). some GH cells are also found in the RPD (Huang and Specker. 1985.. participates in metabolic regulation (Sheridan. 1983).. Most if not all of these functions are exerted via the IGF system (see: Reinecke. Mukai and Oota.. and S. 10. 1997. 1993. At the ultrastructural level. 1997. Prolactin and prolactin cells The amino acid sequence of the PRL of a number of teleost fish has been established by chemical procedures or by cDNA cloning (Santos et al. 1993) and the immune function (Sakai et al. this volume) Teleost GH cells have generally been demonstrated with antisera raised against fish GH sequences. 1979).B. or in both the RPD and PI (Villaplana et al. Yan and Thomas. Pandolfi et al. 1997. Anti-mammalian GH sera do not react with teleost pituitary cells. 2000). 2000). aurata. 1996.. 2003a) by in situ hybridization.. Agulleiro et al. 1986)... 1997).. GH gene expression has also been demonstrated in the RPD of the tilapia. although in the goldfish. McKeown and Álvarez. Barbus barbus (Toubeau et al. 2001a... LLC . 1991). 1994). they are small. such as osmoregulation (Sakamoto et al. as revealed by immunogold labeling (Cook et al. 1994) and in other functions. b. 1986.. 2003). García Ayala et al.. GH plays an essential role in the regulation of growth and development (Donaldson et al.. In S. 1988. Besides the PPD. 2003).. but also may be scattered through this area (Batten. Segura-Noguera et al. Oreochromis mossambicus (Nishioka et al...

. In some species.. Yada et al. PRL 177) prolactin. G. In fish. 1991).. Rentier-Delrue et al. Yamaguchi et al. 2002). Yasuda et al. 2002). 1994. Golgi complex.. two PRL forms have been demonstrated (Specker et al. 1988. their levels being differently regulated during development (Ayson et al. 1988. X 25000.298 Fish Endocrinology Fig. 1994) and in response to © 2006 by Taylor & Francis Group. 1988). 1988.. PRL plays an important role in freshwater osmoregulation by preventing both the loss of ions and the uptake of water (Manzon. Chao et al. Song et al... Although the two PRL forms mostly are highly homologous.. Jackson et al. 1988. Suzuki et al. 1986.. 10. 1999.. tilapia PRLs share less amino acid identity and are classified as long (188 amino acids.. b. PRL 177 and PRL 188 differ in their osmoregulatory and somatotropic actions (Auperin et al. 1989. Nucleus.. N.. 1997).. As regards the PRL variants. PRL 188) and short (177 amino acids. 1987. 2000). LLC .. Kuwana et al. Shepherd et al. but its involvement in immunoregulation has also been proposed (Sakai et al. 1996a.1 Immunogold labeled growth hormone (GH) cell of a 60-day-old larva of Sparus aurata. 1985. the former showing the greatest similarity with other fish PRLs (Yamaguchi et al.

These cells have also been detected by in situ hybridization in S. the hybridization signals are significantly greater in freshwater. 1991. generally scarce. 1996. barbus in which an anti-ovine PRL serum was used (Toubeau et al. 2003c). Segura-Noguera et al.. The secretory granules are round. 2000. García Ayala et al. 1991. except in B. Sánchez Cala et al... variably sized and very electron dense (García Ayala et al. 2000). more numerous and more highly immunolabeled secretory granules than the PRL cells of their seawateradapted counterparts (Hwang and Wang. 299 alterations in environmental salinity (Auperin et al.. or thick strands (García Hernández et al. © 2006 by Taylor & Francis Group. mossambicus (Nishioka et al. In the latter species. PRL cells are located mainly in the RPD... Specker et al... mossambicus (Nishioka et al. 1997... 1981. or small.. As regards the distribution of PRL 177 and PRL 188. which reflects the osmoregulatory role of PRL. Weltzien et al. 1991.. Yada et al. Vissio et al.rather than in seawater-adapted fish. Ayson et al.. LLC . Hwang and Wang.. isolated or clustered PRL cells were also seen in the PPD (Cambré et al.. Cambré et al. 2001a)... 1983. follicles (Quesada et al. or in the PPD and PI (Toubeau et al. 2003). suggesting that their biological significance is not a function of differential release of one particular type of secretory granule (Specker et al. 1995. both compact masses and follicles (Nagahama et al. Specker et al. Villaplana et al. 2003).... Sánchez Cala et al. aurata (Herrero-Turrión et al. 1994).... 1994.. 1993. both hormone variants co-exist even in the same secretory granules. round or elongated and high or medium electron dense (Villaplana et al. Vissio et al.. Moreover. Agulleiro et al. PRL cells have been characterized ultrastructurally with immunoelectron microscopy in some species (Batten. 1994. 1996. heterologous in most studies. 1994. 1993. they co-exist in the same cells of the RPD in O. where they are arranged in compact masses (Batten.. Villaplana et al. PRL cells have mainly been demonstrated by immunocytochemistry using anti-teleost PRL sera... Pandolfi et al. 1993)... 1991). the PRL cells of freshwater-adapted tilapia show a more developed RER and larger. 1997). 2003). 1986. Yan and Thomas. As might be expected. Uchida et al. 2003). 1993).B. Rendón et al. 2003b) and O. Toubeau et al. Mukai and Oota. 2004).. Specker et al... Naito et al. 1994. 1997. 1986. In the latter.. 1986. 1993. 1993). García Hernández et al. Huang and Specker. 1996. 1988. 1993. 1986. 1996). Huang and Specker. 1993.

Villaplana et al. Astola et al. 1996. No definitive function of SL has been determined. Zhu and Thomas... 1997. 1990... Also two variants of SL—SL1 and SL2— seem to occur in teleosts. In addition.. Pendón et al. where they are mainly organized in discontinuous cords lying against the NH (Rand-Weaver et al. chum salmon. the cDNA encoding the SL has been cloned and the amino acid sequence of SL elucidated in many teleosts (Ono et al. 1995. 1995). 1995.. Sciaenops ocellatus (Zhu et al... 1991. 1997.. 2003c). aurata (Cavari et al.. Cheng et al. Mousa and Mousa. although various roles have been suggested. 2000). Johnson et al.... 1997. SL. 1995).. 1993.. 1995. 1996) and S.. phosphate and fat metabolism (Lu et al. 1994b. Glycosylated and non-glycosylated forms of SL co-exist in the pituitary of S. 1997). Ayson et al. applying antisera against cod SL. García Hernández et al. no SL-immunoreactive cells have been found in the white mouth moray eel. 1991a) and later from other species (Rand-Weaver et al.. gilthead seabream or sole. 1993. 2000. Moreover. aurata (Villaplana et al. 1991b. Johnson et al. Cavari et al. 2000). 1993. 1997). although they may also be isolated or clustered in the inner areas of this zone and in the PPD of S. Oncorhynchus keta. In © 2006 by Taylor & Francis Group. SL might be involved in the control of some steps of reproduction (Rand-Weaver and Swanson.. 1992. 1997. Dores et al.. Kaneko et al.. including response to stress (Rand-Weaver et al. Rand-Weaver et al. dumerilii (García Hernández et al.. 1994a. Gadhus morhua (Rand-Weaver et al. Takayama et al. as suggested by the obtention of two deduced amino acid sequences differing in seven amino acids in S. Iraqui et al. 1993b). SL cells have been identified by immunocytochemistry.. aurata (Cavari et al. 1997). LLC . 1999).. Solea senegalensis. SL-immunoreactive cells have been demonstrated in the PI.. 1997. although non-glycosylated SLs are present in salmonids (Rand-Weaver et al.. 1996). 1993..300 Fish Endocrinology Somatolactin and somatolactin cells SL was first isolated and characterized from the pituitary of the cod. 1996) and the presence of two different SL transcripts in the pituitary of the red drum. and response to low illumination or to darkness (Zhu and Thomas..1996. Yang et al. Company et al. 1999. Weltzien et al... the synthesis and secretion from the SL cells is increased during sexual maturation and spawning (Olivereau and Rand-Weaver.. 1995. Pandolfi et al.. 2001a. Rendón et al. calcium and acid-base regulation (Kakizawa et al. 2003. b).. 1996). 1991b. Most SLs characterized to date are glycosylated forms. 1999. In accordance... 1996). May et al. Exceptionally.. Sánchez Cala et al. Mousa and Mousa. Gymothorax meleagris (Dores et al.

Fig. 1993b). 1991b. 2003). © 2006 by Taylor & Francis Group. polymorphic (round. a). SL cells are periodic acid-Schiff (PAS) positive. oval. and varying cell activity correlates with organelle development (Kaneko et al. 2002). dumerilii (García Ayala et al. 1997) and S. 301 salmonids (Rand-Weaver et al. 1993b). Note the labeled irregular mass resulting probably from fused secretory granules (asterisk. 10. x 12600. LLC .. 2000. the secretory granules are round and quite homogeneous in size. Using in situ hybridization techniques. 1994a. In O.. In S. mykiss (Kaneko et al.2).. Olivereau and Rand-Weaver. The different outcomes of the PAS reaction depend on the presence of glycosylated or non-glycosylated SL forms.. Nucleus. and the co-localization of SL1 and SL2 mRNAs has been demonstrated in S. N. 2001). aurata (Villaplana et al. Agulleiro et al.2 Immunogold labeled somatostatin (SL) cells of juvenile Sparus aurata. polygonal and pear-shaped) or irregular secretory granules and irregular immunogold-labeled masses (Fig.. aurata (Herrero-Turrión et al. mykiss. SL cells have been characterized by electron-microscopic immunocytochemistry with anti-cod SL serum. Sánchez Cala et al. 10...B. a x 32000. b) and a few non-salmonid species (Segura-Noguera et al.. the gene expression of SL has been demonstrated in cells bordering the NH in the PI of O.

In teleost fish. -MSH. 1982. In teleosts. perhaps reflecting different stages of hormone synthesis and release. where it exhibits complete biological activity. Segura-Noguera et al. In the two latter species.. known as the immunological tail because they can originate immunological differences (Bentley. 1996. 1991. 1998... 1986... they have been identified in the RPD of most teleosts studied by anti-human (h) ACTH (1-24) serum. 1991). The secretory granules of ACTH cells have been immunogold labeled with anti-ACTH (11-24) in P latipinna (Batten. since antibodies to teleost ACTH are not available (Benjamin. 1993. García Hernández et al. aurata (García Hernández et al. 1999. Lee et al.. barbus (Toubeau et al. Although little attention has been paid to ACTH cells... The amino acid sequence of the portion 1-24.. 1996. 1997b. 1992. and -MSH (Nakanishi et al. 1991. 1995) and in adaptation to hyposmotic environments (Mancera et al.. appears to be constant in mammals. Okuta et al.. Adrenocorticotropin and adrenocorticotropic cells ACTH is a polypeptide composed of 39 amino acid residues. 1996. Infrequent substitutions occur in this region in non-mammalians. this volume). Variations in the ACTH molecule occur in the C-terminal region. 1997. 1998). they are round © 2006 by Taylor & Francis Group.. Villaplana et al. 2000. 2003). Cambré et al. Yan and Thomas. Vissio et al.. 2002). Rendón et al. LLC . 1986) and with anti.. the adrenocorticotropin (ACTH) and three melanocortin sequences: -melanotropin ( -MSH). Immunoreactivity to h ACTH (1-24) is also present in the MSH cells of all species studied with the exception of B.. Alrubaian et al.. 1979). Cells producing hormones of the proopiomelanocortin family In tetrapods. ACTH (1-24) in S. 2002) (See: Mancera and Fuentes. the nucleotide sequence of a cloned cDNA for the POMC lacking the -MSH sequence has been determined in some species (Salbert et al. ACTH (1-24).302 Fish Endocrinology which seem to arise from the fusion of various secretory granules. Arends et al. occur in different SL cell types.. ACTH regulates cortisol release and is involved in the regulation of the stress response (Balm and Pottinger. dumerilii and S. the proopiomelanocortin (POMC) gene encodes the sequence of the opioid -endorfin. Sánchez Cala et al. Toubeau et al. 2003).

1988). 2003c). as well as differences in antisera properties and in the technical approaches used. the morphological features of the larvae at the time of hatching differ greatly (Balon et al. 2002. García Hernández et al.. Monoacetylated -MSH is the principal molecular form in teleosts. some being scattered in the PPD (Toubeau et al.. 1986. Villaplana et al. 1986). Furthermore.. 1997b. -MSH is responsible for changes in coloration and seem to control background adaptation in teleost fish (Baker et al... In a few species. 1991. dumerilii (García-Hernández et al. © 2006 by Taylor & Francis Group. 1984). Rendón et al. ACTH immunoreactivity is also found in secretory granules with a homogeneous and electron-dense core.. developmental differences due to rearing conditions which strongly affect the larva development (Blaxter. 2000) or surround the NH branches in this area. Mukai and Oota.. 2000. ONTOGENY OF THE TELEOST ADENOHYPOPHYSIS The pattern of larval development and the length of the embryonic period varies considerably between different species. LLC .. Cambré et al. In S. Sánchez Cala et al.-endorfin sera (Weltzien et al. MSH cells have been generally demonstrated by immunocytochemistry with anti-mammalian -MSH sera. a varying electron-dense content and a clear halo. 1986). -MSH and ACTH . that could be immature granules in which the proteolitic conversion of ACTH to -MSH and CLIP takes place.. In contrast.-MSH serum labels the numerous secretory granules that contain a fine granular material of variable electron density and compactness.. (11-24) immunogold labeling co-exist in all secretory granules (Batten. MSH cells are also immunostained with anti-h ACTH (1-24) and anti. 2002). Agulleiro et al. 1996. 1997. García Hernández et al.. Segura-Noguera et al. MSH cells have been characterized ultrastructurally by immunocytochemistry (Batten. in melanotropic cells of P latipinna. mostly located in the Golgi area. thus.. since anti-teleost MSH sera are not available. 1988).. with . its amino acid sequences being remarkably well conserved. Ferrandino et al. aurata (Villaplana et al. 303 and evenly sized although smaller than in P latipinna (Batten.. 1986. 2002). 1995. Villaplana et al. MSH cells occupy most of the PI (Batten.. 2003). 1986. 1981).B. anti. Melanotropin and melanotropic cells -MSH originates from the direct cleavage of ACTH in MSH cells (Smith and Funder. 1997b) and S.

which appear only in the PPD (García Ayala et al. Afterwards. most endocrine cell types may migrate from the adenohypophyseal region of their first appearance to other regions during ontogeny. whereas all types of involutive endocrine cells have been found in adults. 1996. Shen and Leatherland. 1989.. it has been suggested that as a result of local factors or intercellular recognition signals.. 2003). the increasing number of which cannot be attributed simply to cell division. Villaplana et al. some undifferentiated cells remain grouped in the most cephalic region of the AH or dispersed in other regions and can even be seen in the oldest larvae. 2002... 1996). which supports a possible fundamental role for the NH in the differentiation and development of the AH (Kaneko et al. 2003). Little attention has been given to the early organization of the AH at the electron microscopic level. their secretory granules can be immunogold labeled.304 Fish Endocrinology can result in variations as regards the time at which the different endocrine cell types are first detected. Synapticlike structures between axon terminals of the NH and the differentiating endocrine cells are observed from the first appearance of the NH (Villaplana et al. 1996. 1996). No migration occurs in the case of FSH and LH cells. aurata this aspect has been studied in detail (Villaplana et al. 1978.. On the other hand... since mitotic figures are rarely observed during development. While the endocrine cell types gradually become more differentiated on development.. Saga et al. Small evaginations of the NH into the AH are evident shortly after hatching (Leatherland and Lin 1975. Only one anterior and one posterior region can be distinguished at the beginning (Hwang and Sun. The number of endocrine cells. The appearance of NH precedes the occurrence of the definitive zonation in the AH. 1993. Villaplana et al.. 1993b. 2001. 1996). These cells could be the source of the adenohypophyseal cells. 2001. 2002. 2003). which appear when the pituitary already shows the zonation characteristic of adults. Villaplana et al. LLC . Only in S. even within the same species. The pituitary first appears as a small mass of undifferentiated cells embedded in the ventral floor of the diencephalon (Naito et al. 1996). 1993a). allow their identification as soon as the cells can be detected immunocytochemically. which together with their distribution patterns. the quantity and size of their secretory granules and the development of their organelles increase as the fish develop. or TSH cells. The different endocrine cell types show differential ultrastructural features.. A variation in © 2006 by Taylor & Francis Group. and become more numerous and larger with development (Villaplana et al. No involutive cells appear in the developing pituitary.

The appearance of LH cells. 305 the pattern of localization throughout development has also been reported for PRL and ACTH cells of the annual cyprinodont Cynolebias whitei (Ruijter. aurata (García Ayala et al. coincides with the onset of vitellogenesis and spermatogenesis in salmonids (Nozaki et al. On the other hand. Plecoglossus altivelis (Saga et al. LH might play this role in non-salmonids (Saga et al.... studies on the ultrastructural identification of endocrine cells are scarce. 1987) and FSH and LH cells of X. maculatus (Magliulo-Cepriano et al. bonariensis (Miranda et al... García Ayala et al. 2001). although a role in the regulation of initial gonadal growth and development has been suggested for FSH in salmonids (Naito et al. 1987). Most attention has been paid to FSH and LH cells. aurata (García Ayala et al. 2003). sea bass. Saga et al. Thus. 1994). which are important regulators of teleost development (Power et al. however. 2003). just after hatching in S. 1990b. TSH cells have been © 2006 by Taylor & Francis Group. LLC . mykiss (Ito et al. Saga et al. maculatus (Magliulo-Cepriano et al. while LH but not FSH cells are found in newly hatched larvae of the ayu... 2003) and shortly after hatching in P olivaceus (Miwa and Inui. Cottus kazika.. 1993). bonariensis (Miranda et al. 1999.B. 1993) and S.. 1999). Although a number of immunocytochemical studies deal with the chronological appearance of some endocrine cell types during development.. 1991. LH seems to regulate gonadal development during downstream migration (Mukai and Oota... 1993). 1993. The expression of TSH. their ontogeny has been studied using antisera against the -subunits... and X.. both FSH and LH have been related to sex differentiation or to the temperature-dependent sex determination of O. 1994). The number of LH cells exceeds the number of FSH cells in immature T. Agulleiro et al. maculatus (Magliulo-Cepriano et al. 1990).... and precedes gonad differentiation in S. mykiss (Saga et al. the . 1993). TSH cells have been identified very early.. aurata (García Ayala et al. Exceptionally. 1 to 2 weeks before hatching in salmonids (Leatherland and Barret. 2001). FSH cells are identified for the first time in neonatal (2 days) X. 1994). 1995).gene is significantly higher in immature than in mature O. with the start of gonadal development in X. 2001)... 1993).. 2003).. Saga et al. maculatus (Magliulo-Cepriano et al. Dicentrarchus labrax (Cambré et al. Naito et al. 1994) and 2 and 3 weeks after hatching. 1998) and O. In the fourspine sculpin. 1993b. thynnus (Kagawa et al. These data agree with the role of TSH as stimulant of the secretion of thyroid hormones. in O. respectively.

1999). 1988). keta (Naito et al.. in C. From their first appearance. which suggests a continuous involvement of the pituitarythyroid axis in metamorphosis and in the physiological adaptation to freshwater of these fish (Grandi et al. mossambicus (Hwang. as suggested by the differential PRL 177 and PRL 188 gene expression and the intensity of immunoreaction with antisera to both hormone forms in larvae.. japonica (Arakawa et al. 1999). however.. around hatching (Ayson et al. In O.. Grandi et al. aurata (Villaplana et al. since no significant differences in GH gene expression are observed between freshwater and seawater larvae (Ayson et al. 2003) PRL cells. 1999). b). Immunoelectron microscopy has allowed the characterization of PRL cells in prehatching larvae of C. GH cells appearing before (Cambré et al. does not seem to play a critical role in osmoregulation during the early developmental stages of this fish. where they gradually increase in number and size during development. LLC . It may well be that PRL 177 and PRL 188 are involved in the freshwater adaptation of O.. 1990) and in 2-day-old larvae of S. in newly hatched larvae of O. aurata (Villaplana et al.306 Fish Endocrinology demonstrated as late as 50 days after hatching. 1990). GH. immunocytochemistry and hybridization signals show that PRL 177 appears before GH and PRL 188. aurata. although GH and PRL mRNAs have been detected from 2 days after hatching onwards.. 2003). 1993b). 2000) and the cichlid fish Cichlasoma dimerus (Pandolfi et al. 2 days after hatching in S. 1994). Laiz-Carrión et al... and 12 days after hatching in A. 2003). they are not revealed in pituitary cells until 4 days after hatching (Herrero-Turrión et al. In S.. Moreover. 1994). 1993. However. © 2006 by Taylor & Francis Group. in other studies. 1990.. 2003a. Power and Canario. PRL cells show abundant and strongly labeled secretory granules and an extensive RER and Golgi complex (Hwang. or undergo a differentiation process involving organelle development and an increase in size and number of the secretory granules. 2003).. GH and PRL cells have been detected simultaneously by immunocytochemistry: 2 weeks before hatching in O.... TSH cells have been detected from the leptocephalus stage onwards in some species of Anguilla (Ozaki et al... 1 day before hatching in P altivelis (Saga et al. whitei (Ruijter and Creuwels. 2003). mossambicus. in P altivelis (Saga et al. 2000. as the GH cells also do (Villaplana et al. 1992) or after (Saga et al. . at hatching .. reared either in freshwater or seawater (Ayson et al. they have been identified at different times. mossambicus.. 1992). gariepinus (Volkaert et al. 2001b). 1994).

. Moreover.. Agulleiro et al. 2001) and P altivelis (Saga et al. 2000). although ACTH cells have also been reported to appear 8 days after hatching in the latter species (Power and Canario. suggesting the presence of a non-glycosylated form of SL in the early stages of development.. 2003). which may co-exist with a glycosylated form in adults (Villaplana et al. in old larvae and juveniles. 1999). 2003c). hatching in C. ACTH and MSH cells appear 3 and 1 days before hatching. although high levels of SL1 transcripts and low or absent levels of SL2 transcripts have been found in the embryonic stage and in newly hatched larvae of S.. and 2 days after . 1993). LLC . before the first appearance of GH and PRL cells. 1992). respectively.. MS cells of S.. aurata (Villaplana et al. Alosa sapidissima (Laiz-Carrión et al. SL cells are PASnegative in larvae and juvenile S. and just after hatching in P altivelis (Saga et al.. No immunogold labeling © 2006 by Taylor & Francis Group. 1997). .. 2003). These cells have only been described in mammals (Asa et al. cells that are immunoreactive to both GH and PRL antisera have been demonstrated in the central region of the AH of newly hatched S.. Pandolfi et al. in A.. ACTH cells have also been detected in D. sapidissima (Laiz-Carrión et al. However. the size and heterogeneity of their secretory granules increasing during development (Villaplana et al. SL cells appear 3 days before hatching in the American shad.... 1990). 1988. 2001b). aurata show ultrastructural features similar to those of the PRL cells but their secretory granules are labeled with one or both antisera to GH and PRL (Villaplana et al. in O. aurata and C. and 1 week before hatching and 3 weeks later. and have been referred to as mammosomatotrops (MS). keta (Naito et al. 1991) and chicken (Ramesh et al. 1999) and S.. dimerus.. 1997. respectively. dimerus (Pandolfi et al.. Kineman et al.. 2000). 2003). 1993b). labrax 1 day after hatching (Cambré et al. 307 In addition to GH and PRL cells. and in the RPD of old larvae and juveniles (Villaplana et al. at hatching in S. SL cells have been characterized by immunogold labeling from 4-day-old larvae onwards in S. aurata larvae... aurata.B. 2002). MS cells might give rise to both GH and PRL cells and. aurata (Villaplana et al. 1998). they could be related to a temporary increase in the production of GH when this hormone is required for vigorous growth (Villaplana et al. aurata. 2001b). 1997. MSH and ACTH cells have been demonstrated simultaneously with immunocytochemical techniques several weeks before hatching in O. mykiss (Saga et al.. Contrary to findings observed in adults. hybridization signals for both SL transcripts have first been detected co-localized in SL cells of 4-day-old larvae (Herrero-Turrión et al.

. C.... E. T. G. G.. S. Endocrinol. Moriyama. and Flik. Cloning and expression of two proopiomelanocortin mRNA in the common carp (Cyprinus carpio L. Wilson. E. F. Gen. 1992. Kaneko. H. 95:143-152.. and Valdivia. Corticotrope and melanotrope POMC-derived . 12:13-24. J. T. A. H. T. W. J. Leunissen. C. Endocrinol. © 2006 by Taylor & Francis Group. H.. Evidence that two . Martens. References Alrubaian. and Halliday.. B.. N. J. 26:315-324. Neuroendocrinology 48:423-431. and Pottinger. Sollars. J. Losinski. Changes in pituitary and plasma levels of MSH in teleosts during physiological colour change. Martial. E. Mol. Moriyama.. R. T. 1984. Comp. F. Kovacs.). 2003. J. Comp.. M.. S.308 Fish Endocrinology has been obtained with ACTH antisera in the developing pituitary. 132:384-390. a pituitary hormone related to growth hormone and prolactin from gilthead seabream. 9:1061-1066. Comp. Anathy. Vermeer. and Kawauchi. S. Ayson. E. I. J. and Hirano. Gen. P. Endocrinol. G. Y.. Pendón. P H. 98:279-288. de Jesús. Hirano. peptides in relation to interrenal function during stress in rainbow trout (Oncorhynchus mykiss). and Kawauchi. M. Astola. Arakawa. L. K. 1994. Differential expression of two prolactin and growth hormone genes during early development of tilapia (Oreochromis mossambicus) in fresh water and seawater: Implications for possible involvement in osmoregulation during early life stages. and Hirano. E. J. G. Mol. G.. J. Endocrinol. cDNA cloning. A.. M.. Gen. while secretory granules of MSH cells are labeled from 2 days onwards in S. 104:330-336. 117:251-259. Hasegawa. Auperin. 1999.. Cloning and expression of somatolactin. Gen. 115:292-300. where they resemble those found in adults (Villaplana et al. 2001. tilapia (Oreochromis niloticus) prolactins have different osmoregulatory functions during adaptation to a hyperosmotic environment.. Cell. and Bowley. Amemiya. Endocrinol. G. T. T. Ayson. T. Comp... Kaneko. A. Pandian. Biosc. Ortiz. A. S. aurata. Baker. 2000. Wendelaar Bonga. F. T. Cloning. 2002). J. S. J. Danielson. Y. Anguilla japonica. 1988. F.. and Dores. 1996. Endocrinol. I.. Rentier-Delrue.. Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC. and Mathavan. Comp. 55:142-149. Isolation. E. G. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus). V.. K. Endocrinol. Domokos. 1998. R.. B.. Endocrinol. T. M. Hirano. R. and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone. Electron microscopic and ultrastructural immunocytochemical analysis. LLC . T.. Ayson. Human fetal adenohypophysis. Endocrinol.. de Jesús. F. Immunocytochemical detection of prolactin and growth hormone cells in the pituitary during early development of the Japanese eel. P 1994. Gen. Comp. G. Comp.. sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis). F. Tsukamoto. C. Sci.. Arends. Venugopal.. T. Sparus aurata. Gen.. Zool. and Prunet. T. Horvath. Koteeswaran. 143:23-31. Balm. Amemiya. Laszlo. 1995.. S.. Gen. Asa. B.

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E. and prolactin-cells from the gilthead sea bream (Sparus aurata L. 2000. Endocrinol. Early . 2001.. T. García Ayala. Gen. and LH . Embryol... Hirano. (Teleostei). Anat. J.. A. Andersson. E. Mugoyo.. M. Vissio.. D. M. Comp.. Identification of mammosomatotropes. C. M.. 203:449-460. . García Hernández. and Agulleiro. A.. Immunocytochemical and ultrastructural characterization of mammosomatotrope-. B. P. F. and LH from pituitary glands of Atlantic halibut (Hippoglossus hippoglossus L. Teleostei): An ontogenic study (from newly hatched to adults).. A. Moriyama. T. Comp. Weltzien. P Miao. and Swanson. and Agulleiro. M. Villaplana. Teleostei). growth hormone-. 1997. Anat. 2003b. A 134:315-327. and Andersen. M. LLC .). 125:410-425. García Hernández. and Ollevier. B. J. García Ayala.... Comp. B. 2003c. Lescroart. Teleostei): An ontogenic study.. Swanson. Volckaert. P and Agulleiro. 93:214-233. P. Molecular characterization and expression of FSH . P. Endocrinol. 2001. G. Anat. P 2003a. W.. 255:347-357. Paz. 1999. García Ayala. Identification and localization of eight distinct hormone-producing cell types in the pituitary of male Atlantic halibut (Hippoglossus hippoglossus L. and Agulleiro. B. Li. Immunocytochemical demonstration of melanotropic and adrenocorticotropic cells from the gilthead sea bream (Sparus aurata L.). B. Norberg. P G. The complete . organization of the pituitary gland in Sparus aurata L. The adenohypophysis of the swamp eel. García Ayala.322 Fish Endocrinology Villaplana.. Biochem. . B. and Maggese. Gen. Yada. L. García Hernández. B. T. Grisez.. M. Embryol. Comp. An ultrastructural study. M.. Andersen. M. Zhang. and Kawauchi.. © 2006 by Taylor & Francis Group. A. Aquaculture 201:117-136.. 131:97-105. Endocrinol. D. Villaplana. B. García Hernández. O. Weltzien. Synbranchus marmoratus.-A. M. Isolation and characterization of FSH . Embryol. J. Anat. and Grau. O. 1996.-A.. Ontogeny of immunoreactive somatolactin cells in the pituitary of gilthead sea bream (Sparus aurata L. Gen. 193:441-452.. Weltzien. Physiol. and Agulleiro. M. A. B 122:423-431. Kobayashi. M. García Ayala. Immunocytochemical and ultrastructural characterization of somatolactin cells from the gilthead sea bream (Sparus aurata L. 1994. 2003.. F.. E. A. Chaves Pozo. A. F. and Agulleiro. S. Biocell 20:155-161. Biochem.. O. V. Villaplana. Norberg.... Immunohistochemically detected ontogeny of prolactin and growth hormone cells in the African catfish Clarias gariepinus. 131:87-96. H. Morphol... growth hormone cells and prolactin cells in the pituitary gland of the gilthead sea bream (Sparus aurata L. Teleostei) using light immunocytochemical methods: An ontogenetic study. M. Comp. Endocrinol. amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus). Changes in plasma levels of the two prolactins and growth hormone during adaptation to different salinities in the euryhaline tilapia. Physiol. H. Helvik. García Ayala. Xu. E. Norberg. an immunocytochemical analysis.. 1996. Teleostei) by light and electron microscopy: an ontogenic study. 2002. Villaplana. 196:227-234. and common subunit in male Atlantic halibut (Hippoglossus hippoglossus).-A. P and Andersson. Gen. F. B. Comp. 202:421-429. Villaplana. B. A. Embryol. Oreochromis mossambicus... F.

1987. 1997. and Sano. Y. Yasuda. 84:389-400. Janssen-Dommerholt. Specker. 99:275-288. Yoshiura. 1986. R. Yang. Biochem. and Thomas. Chem. Gen. 244:528-541. Y. Comp. Immunomodulatory effects of prolactin and growth hormone in the tilapia.. E. Complete amino acid sequences of a pair of fish (tilapia) prolactins. Cloning and . H. 66:280-290. 113:69-79. J.. and Aida. 1988. 2002. and Thomas. M. A. Aida. Y. P 1995.. and Kawauchi. Y. P 1999... Kikuchi. spotted seatrout (Cynoscion nebulosus).. H. H. W. Lin. Yokoo. Yamaguchi. Endocrinol.. Zhu. M.. 101:21-31. of the pituitary cell types in three sciaenid fishes: Atlantic croaker (Micropogonias undulatus). K. Agulleiro et al. H. Y. Cloning and characterization of rainbow trout (Oncorhynchus mykiss) somatolactin cDNA and its expression in pituitary and nonpituitary tissues. S. Arab.... Peter. Comp. H. Kawauchi. King. K. LLC . Yoshiura. 1995. Y. H. GTH-cells in the pituitary of the African catfish. Goos. and Thomas... J. Kikuchi. K. Suetake. Endocrinol. Nishioka. Japanese eel (Anguilla japonica). Biophys. Ruijter. 173:483-492. Soc. A.. Yan. Elevations of somatolactin in plasma and pituitaries and increased -MSH cell activity in red drum exposed to black background and decreased illumination. T. K. K. Gen.. Kobayashi. Yamaguchi. 323 Yada. K. Biochem. M. radioimmunoassay and plasma levels after exposure to stressors or various backgrounds. J. B. 1997. Gen. Clarias gariepinus. 11:255-263.. Schulz. phylogenetic relationship of red drum somatolactin cDNA and effects of light on pituitary somatolactin mRNA expression. A. Comp. Azuma. and Aida. Fish Physiol. Zhu. Duality of gonadotropin in a primitive teleost. Carassius auratus. Comp. E. 1999. Comp.-Y. Kobayashi. and red drum (Sciaenops ocellatus). Th. H. and Grau.. J.. K. © 2006 by Taylor & Francis Group. T. Endocrinol. Red drum somatolactin: Development of a homologous . and Bern. H. and Peute.. Oreochromis mossambicus. L.. 1993. Comp. during gonadal maturation: An immuno-electron microscopical study. Primary structure of common carp prolactins. R. Yasuda. 114:121-131.. P. Kato. Aida. M. Hirano. P 1991. Kajimura. Y. T. 106:271-280. Proc. J. Y. S. Zandbergen. 1996. Histochemical and immunocytochemical identification . Gen. 263:9113-9121. Comp. Endocrinol. T. Gen. Gen. Itoh. Yoshiura. R. Gen. C..-I. Biol. H. J. Arch.. 10:47 Yoshiura. T. Miyazima. Endocrinol. tPRL177 and tPRL188. G. V.. D. K. Endocrinol. Endocrinol.. Molecular cloning of cDNA that encodes the subunit of teleost thyrotropin form the goldfish. T.. Comp.. Primary structure of chum salmon prolactins: Occurrence of highly conserved regions. Endocrinol.B. and Chen. 105:379-389. Jpn. A.. K.. Y.. Hirano. Zhu. Endocrinol. R. S. M. van den Branden. A. J. Endocrinol. Y. Molecular cloning of the cDNAs encoding two gonadotropin subunits (GTH-I and -II ) from the goldfish. and Kato. K. Comp. and Thomas. Y. Uchida. Gen.

MCH. Kitasato MCs and MCH are responsible for a wide spectrum of biological roles. including immunoregulation and food intake. *Author for Correspondence: E-mail: akiyoshi@kitasato-u. Endorphin and Melanin-Concentrating Hormone in Fish Akiyoshi Takahashi* and Hiroshi Kawauchi ABSTRACT Melanocortins (MCs) such as ACTH and MSH and endorphin are encoded by the proopiomelanocortin (POMC) gene. Japan. POMC is hypothesized to have evolved due to a recombination of the MC segment. In addition to classical functions. Melanin-concentrating hormone (MCH) gene is expressed mainly in the hypothalamus. which is primarily expressed in the pituitary © 2006 by Taylor & Francis Group. Iwate 022-0101. Thus. has been characterized as a melanotropic peptide inducing aggregation of melanosomes of the skin. the peptides derived from POMC Authors’ address: Laboratory of Molecular Endocrinology. Ofunato. such as body color change and stress response. the structure of which is well conserved throughout vertebrates. LLC . Sanriku. School of Fisheries Sciences.CHAPTER 11 Diverse Structures and Functions of Melanocortin.

these peptides also act cooperatively. MCH Receptor. Skin pigmentation. proMCH. a peptide derived from the N-terminal region of POMC potentiates the interrenal effect of ACTH. Thus. these peptides not only exhibit a variety of functions and antagonism during body color changes. Pituitary-interrenal axis. and -endorphin ( END). but they are also antagonistic during other responses such as immunity and feeding. Release of POMC-related peptides. These POMC-related peptides interact in many physiological systems. MCH inhibits the release of ACTH from the pituitary. MCH genes-related peptide. INTRODUCTION Body color change in fish in response to background coloration is an important process for environmental adaptation. while -END stimulates it. On the other hand. Energy metabolism. Increasing evidence has shown the diverse actions of these hormones in both the central nervous system and peripheral tissues.326 Fish Endocrinology and MCH genes are multifunctional due to the occurrence of multiple receptors in a wide variety of tissues. Proopiomelanocortin. the peptide hormones encoded by the POMC and MCH genes exhibit multiple effects that maintain a discreet homeostatic status in © 2006 by Taylor & Francis Group. Immunomodulation. Melanophorestimulating hormone. -MSH inhibits feeding. LLC . During feeding. MCH is derived from a different precursor protein. Surprisingly. adrenocorticotropin (ACTH). Adrenal mitogenic hormone. In addition to their antagonistic effects. -MSH. These also interact mutually in many biological processes. The -melanophore-stimulating hormone ( -MSH) produced in the pituitary gland turns the body a dark color by stimulating melanin synthesis. Key Words: Molecular evolution. which is produced in the hypothalamus area of the brain. Endorphin. while melanin-concentrating hormone (MCH). while the effect of Mgrp is stimulatory. which also contains a functional peptide called the MCH generelated peptide (Mgrp). in which peptide hormones having opposite effects play roles as mutual physiological antagonists. -MSH is synthesized as a part of a large precursor protein called proopiomelanocoritn (POMC) together with other peptides including -MSH. including antagonistically in body color change and food intake. Adrenocorticotropin. Food intake. Melanocortin receptor. turns the body a pale color by aggregating melanin granules. Melanin-concentrating hormone. For example.

1976). the gene products of AtT20/D16v cells (a mouse pituitary cell line derived from ACTH cells) were subjected to double immunoprecipitation using antisera against ACTH and -LPH or -END (Mains et al. ACTH. LLC . -MSH. The precursor. a single protein that contained ACTH and -LPH was discovered. First. 1977a. © 2006 by Taylor & Francis Group. and -END. STRUCTURE OF PROOPIOMELANOCORTIN Identification of POMC Elucidation of the structure of POMC as a precursor of multiple peptide hormones has occurred over three major periods. named proopiocortin. and corticotropin-like intermediate lobe peptide (CLIP). 1973). 11. The second group consists of -lipotropin ( -LPH). One group consists of ACTH. in the latter half of the 1970s. During the second epoch.1). each part contains one MSH sequence— -MSH in N-POMC. This chapter deals with the structural characteristics of POMC-related peptides and MCH and their precursors. The latter two peptides are identical to the middle and C-terminal portions of -LPH. Characteristically. the complete cDNA nucleotide sequence for the common precursor of ACTH and -LPH was derived from the bovine pituitary neurointermediate lobe by Nakanishi et al. These interrelationships suggest that ACTH is a precursor of small peptides (Scott et al. His-Phe-Arg-Trp. -MSH. along with the functional diversity and interrelationships of these peptides in fish. That study showed that POMC consists of three segments. 1979). and are collectively called melanocortin.. MSH and ACTH exhibit a core sequence. During the third epoch. was purified from camel pituitary (Kimura et al. and LPH (Fig.. the name ‘proopiomelanocortin’ for the common precursor was proposed by Chrétien et al. b). respectively. Thus. (1979).. The amino acid sequences of the latter two peptides are identical to the N-terminal half and C-terminal half of ACTH. As a result. (1979). Of the three MSHs. a protein that reacted to both antisera was identified. the presence of -MSH was originally demonstrated by a cDNA cloning study. N-POMC. -MSH in ACTH and -MSH in LPH. respectively. 1977. two groups of peptides were identified during the 1950s to 1970s. indicating that -LPH is a precursor for -MSH and -END (Li and Chung. Roberts and Herbert. In these studies.Akiyoshi Takahashi and Hiroshi Kawauchi 327 many aspects of the fish endocrine system.

Human prePOMC is composed of 241 amino acids (aa) and contains a signal peptide. The primary structure of POMC has been delineated in African lungfish. 1999). 1999b. 11.1 Outline of POMC based on African lungfish and bovine studies (Nakanishi et al. It is present as a single gene per haploid genome on chromosome 2 at p23. Lee et al. B = Arg in lungfish or Lys in bovine. Structure of Fish POMC POMC in sarcopterygians According to Nelson (1994). However. 11.. Exon 2 consists of 152 bp and encodes a 26 aa sequence for the signal peptide and the first 18 aa of N-POMC. -MSH of the Australian lungfish POMC has one substitution in the melanocortin core sequence (from HisPhe-Arg-Trp to His-Phe-His-Trp). extending from the N-terminal to Cterminal.328 Fish Endocrinology RK RR KR BR KKR ACTH JP a-MSH CLIP BR KK b-LPH g-LPH b-END KR KK N-POMC Pro-g-MSH AMH g3-MSH g-MSH N-b-LPH b-MSH Fig. including lungfish. Amemiya et al. Peptide terminology modified from Eberle (2000).2). Neoceratodus forsteri (Dore et al. LLC .. as is observed in tetrapods (Fig.. and an unnamed group consisting of Porolepimorpha and Dipnoi. Lungfish POMC contains three MSHs.. Exon 1 contains an untranslated nucleotide sequence consisting of 86 bp. Lee et al.. the Class Sarcopterygii (lobe-finned fish) is composed of three subclasses.. NPOMC. Tetrapoda.. Coelacanthimorpha including coelacanths. ACTH. 1999a) and Australian lungfish. 1979. N-POMC is not divided into subsegments because of the absence of a -MSH segment and pairs of basic amino acids. respectively. Protopterus annectens. ACTH. © 2006 by Taylor & Francis Group. 1999b. Relative positions of consecutive basic amino acids are shown. In teleost POMC. The 7665 base pair (bp) human POMC gene is composed of three exons and two introns (Takahashi et al. (Amemiya et al. 1999a). Exon 3 consists of 833 bp and encodes the remaining segment of POMC including the rest of NPOMC. and -LPH. 1983). and -LPH. Introns A and B are 3708 and 2886 bp.

whereas -MSH and -END of coelacanths are less closely related to their tetrapod counterparts than those of lungfish (Takahashi et © 2006 by Taylor & Francis Group. -MSH. 11. and chondrostean has a mutation in the melanocortin core sequence. and tetrapods shows that -MSH and CLIP of coelacanths share greater identity with their tetrapod counterparts than lungfish.. basal neopterygian. 2003b). lungfish. ACTH segment (stippled).Akiyoshi Takahashi and Hiroshi Kawauchi 329 Fig. Ac.-MSH.2 Molecular architecture of fish POMC. Desacetyl(Ac). The occurrence of three MSHs with different amino acid sequences and one -END indicates that the structural organization of the coelacanth POMC is identical to that of lungfish. and primitive ray-finned fish. The primary structure of coelacanth POMC was partially delineated by amino acid sequence determination of several POMC-related peptides from Latimeria chalumnae (Takahashi et al. N-POMC containing -MSH. The MSH segment (black).-END were identified in an extract from the rostral pars distalis. LLC . CLIP and N. -MSH. and -END segment (hatched) are shown. Comparison of the amino acid sequence of each segment in coelacanths. -MSH of Australian lungfish. tetrapods.

11. Teleost POMCs consistently lack the -MSH segment.. 2003). Oreochromis mossambicus (Lee et al.. Oncorhynchus mykiss (Salbert et al. Acipenser transmontanus (Amemiya et al. and salmonids (Salbert et al. 1988). POMC cDNA has been identified in chondrosteans.. sturgeon.. Carassius auratus (Cerdá-Reverter et al. Polyodon spathula (Danielson et al.. tilapia.. including gar. Oncorhynchus nerka (Okuta et al. rainbow trout.. Lepisosteus osseus (Dores et al. zebrafish. goldfish. 1996. Oncorhynchus keta (Kitahara et al.. 2000). sockeye salmon. 1997). paddlefish. Takahashi et al. Anguilla rostrata (Alrubaian.. 11. Dicentrarchus labrax (Varsamos et al. 2003). paddlefish and gar POMCs possess three MSH segments. Arends et al.. 1999. 1999b). and the insertion of consecutive gaps into the region corresponding to the -MSH segment is needed to align them with POMCs of the primitive group (Fig. and that the sequence was eventually deleted in the teleost lineage. as is seen in Australian lungfish. 1992).. 1997. 1999. Two different POMC cDNAs have been cloned in sturgeon. 2003).. These structural features of rayfinned fish POMCs suggest that the -MSH segment has progressively accumulated mutations in the lineage of early-evolved ray-finned fish. 1992.. 2003b). © 2006 by Taylor & Francis Group... Okuta et al. LLC . Danio rerio (Hansen et al.. 1998. 1996). including bichir. POMC in actinopterygians In Class Actinopterygii (ray-finned fish). 1999) and paddlefish. Takahashi et al. 2002) and seabass. and in teleosts... Cyprinus carpio (Arends et al. Both -MSH and CLIP may have evolved at different rates than -MSH and -END in both coelacanths and lungfish..330 Fish Endocrinology al.2). chum salmon. carp. Alrubaian et al. Although the primitive ray-finned fish exhibit a -MSH segment. including eel. Thunnus obesus (Takahashi et al. In contrast to the consistent occurrence of three MSH segments in POMCs of lobe-finned fish.3). carp. while bichir.. Polypterus senegalus (Bagrosky et al. 2003). 2003b). the melanocortin core sequence of the -MSH segment exhibits an amino acid substitution. sturgeon. The occurrence of two POMC subtypes suggests that duplication of the POMC gene might have frequently occurred during the evolution of ray-finned fish.. 1998. the number of MSH segments in ray-finned fish is inconsistent (Fig. Teleost POMCs lack the -MSH segment. 1999). in basal neopterygians. tuna. Alrubaian et al. 2000). Danielson et al..


2).4): (i) duplication of a consecutive segment from -MSH to -END. and holocephalans. The segment between -MSH and -MSH. This segment shows relatively high nucleotide sequence identity with the -END segment. 2000). (ii) insertion of the duplicated segment between -MSH and an original -MSH segment. 1999). called the Cterminal extension of -MSH (CTED). located between ACTH and -MSH. and the other encodes MSH-A. 2000. and N-Ac. is another unique structure of cartilaginous fish POMCs. One encodes ACTH and -END.2). -MSH. 2004a).. 1999a). a member of the primitive cartilaginous fish group (Takahashi et al. 1999a. -MSH is also present in POMC of ratfish. suggesting that -MSH acts as a functional peptide. -MSH and -MSH.. 11. POMC cDNAs of stingray.. Petromyzon marinus (Fig. and (iii) subsequent differentiation of the duplicated -MSH to -MSH and mutation of duplicated -END to CTED (Amemiya et al. spiny dogfish. These classifications imply that the immediate evolutionary antecedent of the former two MSHs is different from that of the latter two. Thus. Based on these structural characteristics. and is called proopiocortin (POC). is called -MSH (Amemiya et al. MSH-B.332 Fish Endocrinology POMC in chondrichthyans The Class Chondricthyes (cartilaginous fish) is divided into two subclasses. 2003) encode four MSH segments (Fig. -MSH has been identified from dogfish pituitary extract in addition to -... 11. 1995a). and is called proopiomelanotropin (POM) (Heinig et al. 1999a). 1995. Dasyatis akajei (Amemiya et al. the lamprey differs from © 2006 by Taylor & Francis Group. and a different -END. Thus. Takahashi et al. 11.... although no homology is observed in the amino acid sequence.. On the basis of sequence identity and the number of amino acid residues in MSH segments. including ratfish.. Chimaera phantasma. elasmobranchs including sharks and rays. In elasmobranchs. and galeoid shark. POMC in agnathans Two different types of POMC cDNAs have been cloned in sea lamprey. the occurrence of four MSHs is a common feature of cartilaginous fish.-END (Takahashi et al. Squalus acanthias (Amemiya et al. -. Heterodontus portusjacksoni (Dores et al.. Takahashi et al. -MSH might have appeared in the lineage of cartilaginous fish as follows (Fig. The fourth MSH. 2004a). LLC . the four MSHs of cartilaginous fish POMC are classified into two groups: one comprises -MSH and -MSH and the other.

4 Occurrence of -MSH in cartilaginous fish. Processing and Tissue Distribution of POMC General features of processing Each segment of POMC (N-POMC. 1980. and -END are encoded by a common POMC gene. . and MSH-B and MSH-A correspond topologically to -MSH and -MSH of gnathostome POMC. Taken from Takahashi et al. and Arg-Lys. 1997). 1980.. -END. 2004a. Smith and Funder. Castro and Morrison. 11. C-terminal extension of -MSH (CTED). LLC . -MSH. These segments are flanked by pairs of basic amino acids such as Lys-Lys-. Chrétien and Seidah. 1988. -Arg-Arg-. 1981. Posttranslational processing has been extensively investigated in mammals (Eipper and Mains. . gnathostomes. C. the final © 2006 by Taylor & Francis Group. The amino acid sequence identity between POC and POM is only 30%.. -MSH. respectively. . Although the POMC gene is expressed in both ACTH cells of the pars distalis and MSH cells in the pars intermedia of the pituitary. Lys-Arg. -MSH. ACTH. END. ACTH contains -MSH and CLIP and -LPH contains -MSH and . -L. MSH. and the pituitary gland has been found to express high levels of the POMC gene. which act as cleavage sites to generate peptide hormones. E. Krieger et al. where ACTH. . The locations of -END sequences in the POC and POM are conserved.Akiyoshi Takahashi and Hiroshi Kawauchi 333 g a d C b E occurrence of d–MSH and CTED intermediate g a b E b E insertion b g E duplication a b E Fig. and -LPH) itself comprises small segments: N-POMC contains -MSH and joining peptide (JP). -MSH-like sequence. -MSH.

.-MSH in goldfish (Tran et al. In addition to intact N-Ac. 1974)... In the MSH cells. is cleaved to form the N-terminal peptide. -MSH. Those identified in the pars intermedia are Des-Ac. POMC is cleaved to N-POMC.. 2001).-END. phosphorylation. and END were present in the pars intermedia and all the ACTH was present in the pars distalis. these carp peptides probably originate in the pars intermedia. Rodrigues et al. and Di-Ac. Proprotein convertase (PC) 1 is a major processing enzyme in ACTH cells and both PC1 and PC2 act in MSH cells. and JP ACTH is cleaved to form -MSH and CLIP and -LPH . including N-Ac. and -LPH. -MSH. Biosynthesis and post-translational processing in fish Rodrigues and Sumpter (1983) showed by radioimmunoassay that the majority of -MSH. and acetylation.- © 2006 by Taylor & Francis Group. The Ser residue in rodent CLIP is partially phosphorylated and the -END generated in MSH cells is acetylated at the N-terminus and truncated at a pair of basic amino acids in the C-terminal region. CLIP.-END1-29 were detected in an extract of the whole carp pituitary (Takahashi et al. (1983) found newly synthesized -MSH.334 Fish Endocrinology gene products are different in each cell type. and -END.-END. The main POMC-related peptides identified biochemically in the pituitary pars distalis of fish are ACTH in spiny dogfish (Lowry et al. 1974) and C-terminally truncated forms of N-Ac.... 2001). -MSH in spiny dogfish (Bennett et al. In the ACTH cells. POMC is a glycoprotein that possesses carbohydrate moieties in the C-terminal regions of 3-MSH and CLIP and . Several of the generated peptides and POMC itself undergo modifications such as glycosylation. 1974) and -END in carp (van den Burg et al. Considering the large population of MSH cells compared to that of ACTH cells in carp pituitary (van den Burg et al. and END in the pars intermedia of the pituitary of rainbow trout by pulsechase analysis. -MSH. 2001).-END in carp (van den Burg et al. ACTH. .. -MSH. Cterminally truncated multiple forms of N-Ac. A significant part of -LPH is further cleaved to -LPH and -END.. N-POMC is further cleaved to form an extreme N-terminal peptide (here called adrenal mitogenic hormone (AMH) according to Lowry et al. three forms of -MSH with different degrees of acetylation. 1991). -MSH. 2000). -MSH. and N-Ac. -MSH is an N-terminally acetylated and C-terminally amidated form of ACTH1-13. CLIP in spiny dogfish (Lowry et al. For example. LLC .-MSH. amidation. 1983). Moreover. N-POMC. 1989) and tilapia (Lamers et al..

5). 1-15. Therefore. and 1-10 are released from the pars intermedia of carp pituitary (van den Burg et al. 1-18. 1995. Pars distalis POMC N-POMC ACTH b . it can be concluded in general that POMC is processed differentially in the two pituitary lobes and the processed peptides undergo similar modifications to those observed in mammals (Fig.. which are contained in POC.POMC a-MSH CLIP N-b-LPH b..5 Major products of POMC in fish (gnathostome) pituitary based on peptide characterization and immunological distribution.Akiyoshi Takahashi and Hiroshi Kawauchi 335 END1-29. 1995b).END Pars intermedia POMC N . Tissue distribution Although the major tissue expressing the POMC gene is the pituitary gland.MSH products in cartilaginous fish Fig.MSH N . which are contained in POM. the central nervous system and other peripheral tissues have also © 2006 by Taylor & Francis Group. In sea lamprey. 11. As the POC and POM genes are expressed specifically in the pars distalis and pars intermedia. nasohypophysial factor and ACTH. 2001). have been isolated from pituitary extract (Sower et al. 11. LLC .. respectively. -MSH and -MSH may be produced in the pars intermedia of cartilaginous fish pituitary. Takahashi et al.Ac -b-END g -MSH d . preferential production of ACTH in the pars distalis and that of MSH in the pars intermedia is shown to be conserved among vertebrates. and MSH-A and MSH-B.

is highly conserved among fish ACTH. In fish. The N-terminal 22 aa sequence. The production of POMC-related peptide has also been observed in carp brain (van den Burg et al. 1987. ACTH is generally composed of 39 aa as is seen in tetrapods (Fig.. 1992). but not by antiserum to N-Ac-END-II. 11. 1995). LLC . Salbert et al. the topographic distributions of ACTH and MSHs in the lamprey pituitary are similar to those in gnathostome vertebrates. and tilapia. Thus. (1992) revealed that two POMC genes. on the other hand. excluding agnathans. are expressed in cells of the rostral pars distalis and pars intermedia in triploid female trout. 1987). the distribution of POMC-producing cells has been studied mainly in the pituitary and brain. 1988.336 Fish Endocrinology been shown to express the gene in mammals (Smith and Funder. Bird et al. It has been established in tetrapods that ACTH4-10 (Met-Glu-His-Phe-Arg-Trp) is the active core for steroid synthesis and ACTH15-18 is important for binding with receptors (Inouye and Otuska. In sea lamprey pituitary. ACTH cells were stained by antiserum to N-POMC.. Sower et al.6). POMC neurons hybridized with the POMC-A probe in triploid female trout (Salbert et al. (1989) demonstrated the distribution of POMC in trout brain—parvocellular neurons located in the medial basal hypothalamus were stained by antisera against -MSH. Naito et al. and N-POMC. POMC-related Peptides in Fish ACTH In fish. (1984) showed the distribution in chum salmon pituitary: MSH cells in the pars intermedia were stained by antisera to N-POMC and N-Ac-END-II. Axons from these neurons extend into various regions of the brain but do not appear to project into the pituitary gland. whereas MSH-A and MSH-B derived from POM are present in the pars intermedia (Nozaki et al. including these functionally important segments. Castro and Morrison. 1995a)... In the brain. © 2006 by Taylor & Francis Group. In the pars distalis. 2001).. They suggested that the difference in stainability might be due to tissue-specific processing and modification of POMC. including tuna. immunocytochemistry has shown that ACTH and nasohypophysial factor derived from POC are present in the pars distalis. ACTH is composed of 40 aa. Ramachandran. seabass. ACTH. 1995. POMC-A and -B. even though these peptides are encoded on different genes (Takahashi et al. in which an additional amino acid is found in the CLIP segment. 1997). In Perciformes.


. While the ACTH segment flanked by basic amino acids (Lys and Arg) is composed of 39 aa in salmonid POMCA.. 1980). which is acetylated at the N-terminal residue and contains an amide at the C-terminal (Fig. This -MSH is referred to herein as common -MSH.. Alrubaian et al. 1992. 1999a. The latter is a form of the former that is truncated by one-residue at the C-terminus. and is amidated at the C-terminus (Takahashi et al. Most fish -MSH are composed of 13 aa and are identical to mammalian -MSH (Takahashi et al. -MSH -MSH is derived from the middle portion of -LPH. Amino acid sequencing and cDNA cloning studies revealed that -MSH of lobe- © 2006 by Taylor & Francis Group. In chum salmon. 1998.. Arends et al. Sea lamprey ACTH is composed of 60 or 59 aa.. 2006). a different type of -MSH consisting of 15 aa has been isolated from pituitary extract and termed -MSH-II (Kawauchi et al. paddlefish. Dores et al.. Several lamprey ACTH molecules are phosphorylated (Takahashi et al. derived from a precursor POM. 1995b). Okuta et al. in addition to the common -MSH... Moreover. 11.. 1999.. In elasmobranchs. carp. In agnathans. Another sequence corresponding to -MSH is the N-terminal 22 aa sequence of ACTH derived from a precursor POC. which has not yet been identified in pituitary extract. Danielson et al. 2000). is the peptide corresponding to -MSH (Figs. The amino acid sequence of -MSH-II differs from common -MSH at the C-terminus where Val-NH2 has been replaced by Ile-Gly-His-OH.. 1999. the segment is composed of 42 aa in POMC-B.. A rather significant diversity was exhibited between the two salmonid ACTHs. 2000. -MSH -MSH refers to ACTH1-13. 2003). 2 and 6). MSH-B. MSH-B is composed of 20 aa. These results indicate that the ACTH-A may have evolved as an invariant copy. LLC . has a free N-terminal. the C-terminal residue of -MSH (Val in common MSH) is replaced by Met in dogfish and galeoid shark and Lys in stingray (Amemiya et al. salmonid ACTH-A shows higher sequence identity with other ACTHs than salmonid ACTH-B.6).338 Fish Endocrinology Two different POMC cDNAs have been cloned in sturgeon. Takahashi et al. and salmonid fish (Salbert et al. while ACTH-B may have accumulated mutations after the duplication of POMC in the salmonid lineage. 2001). 1996.

MSH-A. the amino acid sequence of fish 3-MSH is inconsistent (20 to 25 aa) owing to diversification of the C-terminal region (Fig. therefore.2 and 11. which is flanked by pairs of basic amino acids.. Moreover. -MSH has been identified in spiny dogfish pituitary extract (Takahashi et al. -MSH of lobe-finned fish and cartilaginous fish is predicted to be composed of 12 aa as in mammals. 1995a. -MSH of primitive ray-finned fish—including sturgeon. Scyliorhinus canicula. 1981.Akiyoshi Takahashi and Hiroshi Kawauchi 339 finned fish and ray-finned fish is composed of the same 17 aa as amphibian -MSH (Fig. In spotted dogfish. However. 11. Bichir POMC has an intact melanocortin core sequence. a -MSH of 18 aa residues has been isolated (Love and Pickering. in which the N-terminal is Lys and C-terminal is Phe-NH2. 1982).3-MSH or 3-MSH. in sturgeon and paddlefish.. The12 aa form. and His-Phe-Arg-Ser in gar). 1998. is composed of 16 aa and its C-terminus is amidated as predicted by the presence of the amide donor Gly next to the C-terminal Ala on POMC-II (Arend et al. 1974). 1999).7) (Takahashi et al.b). 11.7).7). although the -MSH sequence lacks both N-terminal and C- © 2006 by Taylor & Francis Group. In fish. which was isolated from sea lamprey pituitary extract.. 2000). -MSH Two forms of -MSH have been isolated from bovine pituitary pars intermedia (Browne et al. Hammond et al.. In agnathans. According to the criteria in mammals. identified in pituitary extract (Takahashi et al. consists of 18 aa as is seen in mammalian and avian -MSH (Fig. Takahashi et al. is called [Lys]1-MSH or simply -MSH. 2004a).. the elasmobranch -MSH segment. 2000).. It is. was found to correspond to -MSH (Figs.. 11. while in spiny dogfish a C-terminally truncated form of -MSH consisting of 16 aa has been isolated (Bennett et al. The ratfish -MSH segment of POMC is flanked by pairs of basic amino acids and is composed of 16 aa due to a deletion in the Nterminal region (Takahashi et al. paddlefish and gar—is probably nonfunctional because of a single mutation in an essential residue in the melanocortin core sequence (His-Phe-His-Trp in sturgeon and paddlefish... unlikely that -MSH is generated from the precursor in these fish. LLC . The other is a C-terminally extended form of -MSH and is called [Lys]. Carp -MSH-II. 1974. 11..3). In cartilaginous fish. Takahashi et al. One exception is observed in carp. a cleavage site on the N-terminal side of -MSH (Arg-Lys in tetrapods and Arg-Asn in dogfish) has been substituted with Gln-Asn. 1999).

© 2006 by Taylor & Francis Group. A. Italics show MSH-A isolated from lamprey pituitary extract.340 Fish Endocrinology Fig. LLC . B. Basic amino acids in the C-terminal region of -END acting as a processing signal are in bold. Basic amino acids flanking hormone segments are shaded. Underlines in the C-terminal region of trout A-type -END are dodecapeptides derived from POMC-A. Lamprey MSH-A and -MSH compared with bovine. Structure of fish (gnathostomes) -MSH and -END compared with bovine. 11.7 Structure of -MSH and -END. Trout means rainbow trout.

LLC . The -MSH isolated from dogfish pituitary extract is composed of 12 aa with a free N-terminus and amidated C-terminus. Given that a pair of basic amino acids. -END is consistently located in the C-terminal region of POMC.. Takahashi et al. and the number of amino acids in the segment differs among fish species. Galeoid shark and stingray POMCs possess similar amino acid sequences. In sturgeon POMCs.8). 1999a). In ratfish. The tetrapod -END segment is consistently composed of 31 aa (Fig. the melanocortin core sequence of -MSH in POMC-A is mutated as described above. 1999a. 2004a) (Fig. whereas lungfish -END is composed of 34 aa (Amemiya et al... Dores et al.. On the POMC. 11.. the location of basic amino acids suggests that the ratfish -MSH may be composed of 16 aa and possess no modification at the C-terminal. 1999b. Dores et al. the dogfish -MSH segment is flanked by consecutive basic amino acids at the N-terminus and a monobasic amino acid at the C-terminus.. Basic amino acids flanking hormone segments are shaded.. However.8 Structure of -MSH. 11. 1999). 11. 2003). 2003.. Lee et al. © 2006 by Taylor & Francis Group. 2003b). the coelacanth peptide may be a C-terminally truncated form. -Lys-Lys-.Akiyoshi Takahashi and Hiroshi Kawauchi 341 terminal cleavage sites (Bagrosky et al.. no analgesic effects have been reported in fish.7). Coelacanth -END isolated from the rostral lobe of the pituitary is composed of 30 aa (Takahashi et al. -MSH -MSH has only been observed in cartilaginous fish including elasmobranchs and holocephalans (Amemiya et al. -END -END is an endogenous opioid peptide in which the five N-terminal residues are essential for activity. 2000. is present in the C-terminal region of the -END segment on its putative precursor. stingray spiny dogfish galeoid shark ratfish KKDEKNYKMTHFRWGRGPK KKKDGKIYKMTHFRWGRGPK KKKDGKVYKMAHFRWGRGPK KKDGKLYKMTHFRWSEGSR Fig. while that in POMC-B retains an intact core sequence. One possible explanation is that the ‘intact’ core sequence arose as a result of a secondary His to Arg back mutation (Danielson et al. 1999. Underline shows -MSH identified in dogfish pituitary extract. as can be seen in lungfish -END.

2003). 1999b. the two salmonid -ENDs are distinct (Salbert et al.. Alrubaian et al. This extension might have occurred via a point mutation that changed the stop codon to a Trp codon. Danielson et al. the structure of -END is known in carp. In contrast to the relatively high sequence identity in these fish.342 Fish Endocrinology -END of Perciformes including tuna and tilapia is composed of 32 aa (Lee.. 1996. two peptides derived from the C-terminal extension have been isolated from rainbow trout pituitary extract (Tollemer et al. 1999. ratfish POMC is composed of 33 aa and is characterized by a deletion of three residues corresponding to elasmobranch -END10-12 (Takahashi et al.. 1998. 2003.. 2003). carp. Moreover. Takahashi et al. In ray-finned fish. 2000.. A 34 aa -END is found in teleosts including Salmoniformes ( -END-A in sockeye salmon). Acipenseriformes (sturgeon and paddlefish).. The sequence identity between the two -ENDs in sturgeon and paddlefish is 97% and 88%. Hansen et al. 1999. Bagrosky et al. while the galeoid shark -END is 35 aa long and lacks a C-terminal residue found in spiny dogfish and stingray POMC (Amemiya et al. In Cypriniformes. Danielson et al. and all exhibit a 33 aa sequence. 2003). Two carp POMCs encode an identical sequence of -END (Arends et al... 1999)... two POMC subtypes have been identified in salmonids.. Okuta et al. the spiny dogfish and stingray -ENDs are composed of 36 aa.. respectively (Alrubaian et al. Amemiya et al. 1997... the sequence identity of -END-A to -ENDs of other species is higher than to salmon -END-B. and Polypteriformes (bichir) (Okuta et al. 2004a). and sturgeon. Dores et al. In rainbow trout. from Tyr-Gly-Gly-Phe-Met to Tyr-Ser-Gly-Phe-Met. 1998. 2002).. 1997. In elasmobranchs. 2000). Cerdá-Reverter et al.. and the sequence identity between the two forms is only 47%. 1992. 2000. paddlefish. in Anguiliformes (eel).. The 29 aa -END-B encoded on POMC-B is five residues shorter than the -END-A on POMC-A.. LLC . 1997). 2003b. and in primitive ray-finned fish including Semionogiformes (gar). Takahashi et al. 1999. These features of -END together with those observed in ACTH (see section on ACTH) suggest that POMC-B of Salmoniformes appeared as a result of a paralogous event and then evolved as a variant copy. Sockeye salmon END-B exhibits a substitution in the five essential N-terminal amino acids. 1999a. 1996). Dores et al. Takahashi et al. In holocephalans. Sea lamprey POC and POM contain a -END consisting of © 2006 by Taylor & Francis Group.. 1992)... Indeed.. goldfish and zebrafish (Arends et al. POMC-A possesses a C-terminal extension beyond the -END segment (Salbert et al.

and -MSH) and one END ( -END) (Takahashi et al. POMC has also been identified in invertebrates (Salzet et al. A single END segment is consistently found in all gnathostome classes (Fig. the variation in MSHs in the POMCs of extant vertebrates indicates that the POMC has diverged by duplication. The class-specific number of MSH segments provides important insight into the molecular evolution and diversity of POMC in gnathostomes (Fig. during the early stages of cartilaginous fish evolution. 11. and deletion of the MSH segment. The lack of the fourth Cys may support the hypothesis that salmonid POMCB is a variant copy formed after duplication in a paralogous event (see sections on ACTH. 1995a).3).. ray-finned fish. This segment is characterized by the presence of four Cys residues clustered in the first half of the segment. respectively (Takahashi et al. two or three in ray-finned fish. and one or two in agnathans. 11. AMH AMH is the term for the N-POMC region excluding -MSH and the JP segment proposed by Lowry et al. 2001. . -MSH may have been derived from - © 2006 by Taylor & Francis Group. Salmonid AMH in POMC-B has only three Cys residues. A sequence comparison of -END throughout vertebrates shows that -END6-9. Evolution of POMC From ancestor to extant fish A sequence comparison of POMC throughout vertebrates shows that the number of MSH segments is more or less class-specific. which may have been inherited by early vertebrates. 2003a). The ancestral form may have possessed three MSHs ( . and cartilaginous fish. However. 1999) and thus it may have originated in the ancestral invertebrate.. 11. LLC . (1983).9). which exhibit 31% sequence identity with humam -END and 37% mutually. First. Sequence comparison shows the N-terminal 38 aa sequence is highly conserved among gnathostomes (Fig. -END and from Ancestor to Extant Fish).2). Lys-Ser-Trp-Asp.. four in cartilaginous fish. 1997. The presence of the consensus sequence clearly classifies -END into the fishtype and tetrapod-type. insertion.. three in lobefinned fish. is a consensus sequence of orthologous END of lobe-finned fish. while other gnathostome POMCs including salmonid POMC-A have four Cys residues in the AMH segment. Stefano et al.Akiyoshi Takahashi and Hiroshi Kawauchi 343 32 and 35 aa.

no changes occurred in the number of MSH segments. Second. These results suggest that mutations have accumulated in the -MSH segment of the basal group and that the segment has been completely deleted in teleosts. each copy evolved in concert with the specialization of tissue function during the course of evolution (Fig. after duplication of the ancestral POMC gene. Although POMCs of the basal group of ray-finned fish possess three MSHs. and finally. Sea lamprey has two different POMCs. The tissue-specific expression of the POC and POM genes in the lamprey shows that.4. 1995a. © 2006 by Taylor & Francis Group. most -MSHs exhibit mutations in their melanocortin core sequence. (2004a).. MSH. Abbreviations are the same as in Fig.. probably before the appearance of holocephalans and elasmobranchs. and each precursor has a different number of MSH. the POC and POM genes are expressed in the pars distalis and the pars intermedia.344 Fish Endocrinology Chondrichthians E Actinopterygians E teleosts -L basal group E Substitution mutation in -MSH-core sequence deletion of -MSH occurrence of -MSH Early Vertebrates E inheritance of three MSHs and one END Sarcopterygians E duplication and specialization Agnathans E E Early Invertebrates E Fig.9 Schematic diagram of the molecular evolution of POMC in vertebrates : taken from Takahashi et al. Ficele et al. POC and POM. As a result. 11. 11. in lobe-finned fish. 1998). 11. respectively (Takahashi et al. in the lamprey. In contrast to the expression of a common POMC gene in both the pars distalis and the pars intermedia of the gnathostome pituitary (see section on biosynthesis and posttranslational processing in fish). the number of MSH has increased to four.9). LLC . the presence of only two MSHs ( -MSH and MSH) in teleosts demonstrates a decrease in the number of MSHs in rayfinned fish.

For example. Unlike in the lamprey. There has been much controversy over the relationship between coelacanths. One of the two POMCs in salmoniformes seems to be a variant copy accumulating more mutations than its counterpart (see section on POMC-related peptides in fish).. and can provide insight into the evolution of fish into land animals from both an anatomical and molecular perspective. Takahashi et al. while the lungfish -MSH and -MSH exhibit tetrapod-specific structure.and tetrapod-specific structures in POMC of lobe-finned fish is evidence that these fish are the closest living relatives of tetrapods. 1991). 1992. Okuta et al. Lobe-finned fish POMC shows interesting characteristics (Amemiya et al. LLC .. The higher sequence identity between the two than that observed between lamprey POC and POM suggests that they may be paralogous genes.. 1999. Danielson et al.. (2003b) compared coelacanth and African lungfish POMC-related peptides with tetrapod homologues to identify which of the two POMCs is more closely related to that of tetrapods. and tetrapods. However. lungfish. to tetrapods than the lungfish. The co-existence of fish. Based on sequence comparison.. 1995.. 1999). In the case of -MSH and CLIP the coelacanth was found to be more closely related . Alrubaian et al. this hypothesis is not consistent with results of analyses of mitochondrial and nuclear data (Zardoya et al. whereas in the case of -MSH and -END. 1999b). 2001).. African lungfish ACTH and -END exhibit fish-specific structure.. 1996). Anatomical evidence suggests that lungfish are more similar to tetrapods than the coelacanth (Janvier. Arends et al.. 1998. while the primary structure of coelacanth hemoglobin indicated that the coelacanth was the closest living relative of tetrapods (Gorr et al. Transition from fish to tetrapod Lobe-finned fish are considered the closest living relative of tetrapods (Meyer. A similar characteristic is observed in coelacanth POMC-related peptides (see section on POMC in sarcopterygians). 1996. the relationships between the three groups based on protein and nucleotide sequence data are inconsistent. indicating that the lungfish POMC is a mosaic structure of fish and tetrapod types. Venkatesh et al. the two POMCs in these species exhibit the same number of MSHs. the coelacanth was found to be less closely related to tetrapods than the © 2006 by Taylor & Francis Group.Akiyoshi Takahashi and Hiroshi Kawauchi 345 Two POMC subtypes have also been observed in ray-finned fish (Salbert et al. 1998).

. Thereafter... chinook salmon.. MSH.. carp (Bird et al. 1995). molly. rainbow trout (Naito et al. Verasper moseri (Amano et al. including chum salmon. 1989). 1988). The putative hormone was termed ‘melanophore-contracting hormone’. when Baker and Ball (1975) reinvoked the dual hormone theory to control teleost melanophores. whereas the -MSH and -END segments in coelacanth POMC have evolved faster than those in lungfish POMC. They suggested that MCH is a neurohypophysial peptide that is synthesized as a prohormone in the hypothalamus and processed to an active form. and then transported to the pars nervosa of neurointemediate lobe for storage and release. Oncorhynchus tshawytscha (Minth et al. © 2006 by Taylor & Francis Group. However. the development of this bioassay facilitated the discovery of MCH in chum salmon (Kawauchi et al. 1983). difficult to infer which of the two fish POMCs is more similar to tetrapods based on the segments of POMC. barfin flounder. and its hypothalamic origin was first suggested by Enami in 1955. Sparus auratus (Mancera and Fernández-Llebrez. Consequently. It was known in the early 1940s that pituitary extract from some fish induced pigment aggregation. 1989). gilthead seabream. Tissue Distribution The localization of MCH-immunoreactive neuronal somata and fibers has been examined by immunocytochemistry in several species of teleosts. LLC . therefore. is responsible for pigment dispersion in the integumentary melanophores of lower vertebrates. and cichlid fish. STRUCTURE OF MELANIN-CONCENTRATING HORMONE Identification of MCH The dual hormonal control of color change in response to background coloration by two antagonistic pituitary melanotropic hormones was first postulated by Hogben and Slome in 1931. no advances were made in the understanding of MCH until 1975. 2003). It is. Rance and Baker (1979) subsequently found the highest activities in the neurointermediate lobe of rainbow trout pituitary using an in vitro trout scale bioassay and they called the putative hormone ‘melanin-concentrating hormone (MCH)’. Poecilia latipinna (Batten and Baker.346 Fish Endocrinology lungfish. the sequence comparison showed that the -MSH and CLIP segments in coelacanth POMC have evolved more slowly than those in lungfish POMC. then known as intermedin. 1985).

1995). 2004b)..Akiyoshi Takahashi and Hiroshi Kawauchi 347 Cichlasoma dimerus (Pandolfi et al.. MCH-ir fibers have also been detected in extrahypothalamic areas. Structures of Fish MCH and Related Peptides cDNA and gene structure In Salmoniformes. the preproMCHs are composed of 136 and 135 aa residues.. and that their synthetic activity was altered in response to the color of the environment (Bird and Baker. have been cloned from the hypothalamus of chum salmon (Ono et al... Breton et al. 2003). 1985.. 1991). the mammalian MCH gene is composed of three exons and two introns (Thompson and Watson. In tilapia and barfin flounder. optic tectum and midbrain (Naito et al.. two types of MCH cDNAs. In teleost fish. while a single MCH cDNA. 1995). It was originally proposed that MCH-ir neuronal somata were located only in the nucleus lateralis tuberis from which axons projected to the pituitary.. such as the olfactory bulb. and an approximately 160 bp 3'-untranslated region. 1995)... Gröneveld et al. which project to the pituitary gland. Hypothalamus areas expressing the MCH gene have been identified in rainbow trout (Suzuki et al. Batten and Baker. 1989).. Two MCH genes have been cloned from a chum salmon liver DNA library (Takayama et al. MCH transcripts are most prevalent in the magnocellular neurones of the nucleus lateralis tuberis. Suzuki et al. MCH1 and MCH2. 1995a. pretectal thalamus.. The two genes are 86% identical at the nucleotide sequence level. 1993b). Takahashi et al. 1990. LLC . The second group of neuronal somata do not project into the pituitary but project centrally (Baker et al. However. They are intronless genes. The two preproMCHs are composed of 132 aa and show 80% sequence identity with each other. a second group of parvocellular MCH neuronal somata has also been identified in the dorsal hypothalamus. located in the area above the lateral ventricular recess of the third ventricle in the rainbow trout.. and rainbow trout (Baker et al.. MCH2 has been cloned in coho salmon. 1995b. MCH is located at the C-terminus of preproMCH. respectively (Gröneveld et al. 1988). 1988. a preproMCH region. © 2006 by Taylor & Francis Group.. The TATA box is located about 20 bp upstream from the putative cap site. Batten et al. Oncorhynchus kisutch (Nahon et al. 1990). In contrast to the salmon MCH gene. 1995). 1989). chinook salmon (Minth et al. each of which commonly consists of an 80 bp 5'-untranslated region. 1989.

1995. The importance of the Trp15 residue for interaction with the MCH receptor is also suggested by © 2006 by Taylor & Francis Group. tilapia scale assay (Kawazoe et al... 1988. Biosynthesis of MCH from a larger protein has been demonstrated in rainbow trout (Bird et al. 1986..348 Fish Endocrinology Expression. Breton et al. 1988). 1995.. Fish MCH is two amino acid residues shorter than mammalian MCH... rainbow trout. 11. 1983). whereas in rainbow trout. There is one replacement in the amino acid sequence of barfin flounder MCH compared to other teleost MCHs in the N-terminal outside the ring structure formed by a disulfide bond (Takahashi et al.. MCH MCH isolated from chum salmon pituitary extract is composed of 17 aa and possesses one disulfide bond (Kawauchi et al. Minth et al. 1990).9 K product (MCH) was scarcely detectable. the 15 K product (proMCH) was more prevalent.. and carp scale assay (Baker et al. the expression level of MCH2 is higher than that of MCH1 (Baker et al. 1987). over time. 1993a).. Baker et al. Presse et al. Nahon et al. which consists of 19 amino acids (Zamir et al. Suzuki et al.. Gröneveld et al. whereas MCH increases.... LLC . cDNA cloning studies have revealed that the amino acid sequence of chinook salmon. 1990). Structure-activity studies using fragments and analogues of salmonid MCH have revealed that most of the residues in the disulfide bridge are essential for the melanin-concentrating activity in an eel skin assay (Lebl et al. The other three replacements are present outside the ring. coho salmon. The two additional amino acids in mammals are present in the N-terminal outside the ring structure. 1991. 1988.10). Fish MCH possesses four replacements relative to mammalian MCH... 2004b).. In human MCH.. 1995. and tilapia MCH are identical to the chum salmon peptide (Ono et al. the replacement of this Leu with Ala has a negligible effect on binding with the receptor (Audinot et al. 1989. biosynthesis and processing The two MCH genes are expressed to nearly the same degree in mature female chum salmon hypothalamus (Ono et al... proMCH decreases progressively in abundance.. 2001). 1997). 1990. Matsunaga et al. However. 1989). Nahon et al. 1995a) (Fig. One of the replacements in the ring—Val7 in fish and Leu9 in mammals—is a physicochemically conservative replacement. 1989. One hour after the injection of radiolabeled amino acid into the basal hypothalamus of anesthetized white-adapted fish. while the 2.

. 2003). Takeuchi et al.and C-termini. and is referred to as Mgrp (it is also called neuropeptide EI. The segment flanked by the processing signals is composed of 13 aa in salmoniformes and 22 aa in tilapia and barfin flounder. the Arg6 residue (Arg4 in fish) in the N-terminal exocyclic sequence and Trp17 residue (Trp15 in fish) in the C-terminal exocyclic sequence are essential for binding to the receptor (Audinot et al. rat. RECEPTORS FOR MELANOCORTINS AND MCH Melanocortin (MC) Receptors in Mammals Identification of MC receptor in tetrapods Two PCR subclones resulting from the amplification of human melanoma cDNA were determined by DNA sequencing to encode novel G-proteincoupled proteins (GPCRs) that are highly related to one another.flanking the Nterminal side of NEI in one of the proMCHs is mutated to -Arg-Gln(Minth et al. respectively). 2003. in salmoniformes or NAL in barfin flounder having Ala and Leu at the N.. 1989). Leukocytes and the brain are also known to express the gene. respectively. Gantz and Fong. and chicken (Mountjoy. 1992). and their pharmacological characteristics.and C-termini. 1995c). mouse.. In chinook salmon. fish proMCH possesses another processing signal (Fig. 2000. The presence of naturally occurring Mgrp has also been demonstrated in tilapia (Gröneveld et al. -Arg-Arg. In contrast to the highly conserved nature of the MCH segment among fish. 11. LLC .Akiyoshi Takahashi and Hiroshi Kawauchi 349 an eel skin assay (Matsunaga et al. the amino acid sequence of the Mgrp segment is variable. The MC-R family. MC1-R shows nearly the same degree of affinity for -MSH and ACTH. although the MC1-R gene is mainly expressed in melanocytes and melanoma where it participates in pigmentation. Even the pair of basic amino acids is not conserved. which consists of five subtypes. Northern hybridization analysis demonstrated expression specifically in melanocytes and adrenal cortex (Mountjoy et al. 1989). In human MCH... 2001). and biological significance have been characterized in mammals.. has been cloned from human.10). MC2-R is a specific receptor for © 2006 by Taylor & Francis Group. Mgrp In addition to the processing signal generating MCH. tissue distribution. NEI having Glu and Ile at the N.

10 Structure of preproMCH compared with that of human. Basic amino acids flanking the peptide segments are shaded. LLC .350 Fish Endocrinology Fig. © 2006 by Taylor & Francis Group. Functional peptides are shown by underlines. 11.

bone marrow. MC4-R has also been cloned in spiny dogfish (Ringholm et al.. 69% for MC4-R. and thyroid gland. Later. MC4-R shows nearly the same degree of affinity for -MSH and ACTH.1). LLC . 1994. and stomach. and is expressed in brain and several peripheral tissues including placenta. 2003a). 64% for MC3-R. The MC2-R gene is preferentially expressed in adrenal cortex and in adipose tissue. Shimizu et al. spleen. 2003) and goldfish (Cerdá-Reverter et al. Fugu rubripes and confirmed the assembled DNA sequences by PCR and resequencing. possesses roughly the same degree of affinity for ACTH and -. which lacks MC3-R. pufferfish MC5-R gene contains three introns in the coding region (Logan et al. especially in the hypothalamic area.. 46% for MC2-R. Cammas et al. duodenum. Pufferfish MC2R gene contains only one intron. the entire coding region of all MC receptors is contained in a single exon. The sequence identity of zebrafish MC receptors with human homologues is 54% for MC1-R. The human and mouse MC2-R genes are separated into two and four exons. They also demonstrated that zebrafish has six MC-Rs.Akiyoshi Takahashi and Hiroshi Kawauchi 351 ACTH. and is involved in energy homeostasis. or -MSH distinguishes this receptor from the other MC receptor subtypes. The MC5-R gene is expressed in a variety of tissues including skeletal muscle. 1997. Logan et al. and MC5-R has been cloned in goldfish (Cerdá-Reverter et al. pituitary. has only four (Table 11. (2003) screened genomic databases of zebrafish and pufferfish.. while the coding region is located in a single exon (Naville et al. particularly in sebaceous glands. -. while pufferfish. and 70% for MC5-R-B.. which is in the identical position relative to the coding sequence as intron 3 of the MC5-R gene.. adrenal glands. 70% for MC5-R-A. 2003). ovary. lung. including two MC5-R subtypes. and MSH. leukocytes. adipocytes. 1997). which is thought to be associated with energy metabolism. uterus. 2003c). (2002) determined the nucleotide sequences of one MC4R and two MC5-Rs obtained from a zebrafish genomic library. testis. In tetrapod species. -. MC5-R shows the highest affinity for -MSH. Unlike tetrapod species. © 2006 by Taylor & Francis Group. but not for -. and is expressed predominantly in brain. thymus. and its affinity for ACTH. MC3-R. MC5R is involved in the regulation of exocrine functions.. pancreas. respectively. MC receptor in fish Structure Ringholm et al..

In situ hybridization studies show that goldfish MC5-R transcripts are widely distributed in the goldfish brain. 2003a).1 Identification and pharmacological characterization of fish melanocortin receptors (MC-R) and melanin-concentrating hormone receptors (MCH-R) Receptor MC1-R MC1-R MC2-R MC2-R MC3-R MC4-R MC4-R MC4-R MC4-R MC5-RA MC5-RB MC5-R MC5-R MCH-R1A MCH-R1B MCH-R1 MCH-R2 MCH-R2 Species Zebrafisha Pufferfisha Zebrafisha Pufferfisha Zebrafisha Zebrafisha. infundibular hypothalamus. Low levels are also detected in the cerebellum. dRingholm et al. ND: not determined. tuberal hypothalamus.352 Fish Endocrinology Table 11. The goldfish MC5-R mRNA is found in peripheral tissues including kidney. including the ventral telencephalon. muscle. dorsal and ventral thalamus.b Pufferfisha Goldfishe Zebrafisha Pufferfisha Pufferfisha Zebrafisha Pufferfisha Affinity order of ligands ND ND ND ND ND -MSH > ND -MSH > -MSH > -MSH > -MSH = ND -MSH = ND ND ND ND ND NDP-MSH >> NDP-MSH >> NDP-MSH >> NDP-MSH >> NDP-MSH >> NDP-MSH >> -MSH >> 1-MSH -MSH >> 1-MSH -MSH > 1-MSH -MSH = 1-MSH -MSH > 1-MSH -MSH >> 1-MSH An overview of affinity is shown by the displacement of [125I]NDP-MSH from different melanocortin receptors by MSH-related peptides. LLC . spleen. The spiny dogfish MC4-R gene © 2006 by Taylor & Francis Group. fat. tectum and tegmentum mesencephali.. (2003c). pituitary and ovary. skin and retina. (2003). high expression levels are found in the telencephalon. (2002). Reference: aLogan et al. (2003a). cCerdáReverter et al. In the brain. ventral thalamus. preoptic area. and spleen (Cerdá-Reverter et al. (2003).b Zebrafisha. and hypothalamic inferior lobe.. pre-optic area. gill. medulla. gill. bRingholm et al. Tissue distribution In goldfish.b Pufferfisha Goldfishc Dogfishd Zebrafisha. reticular formation. eCerdá-Reverter et al. 2003c). with low expression levels in the intestine. and spinal cord. posterior tuberculum. vagal and facial lobes and spinal cord (Cerdá-Reverter et al. MC4-R mRNA is found in brain and some peripheral tissues including the ovary.

2002). while rodents. guinea pig... Part of the nucleotide sequence of a second MCH receptor. Mori et al.. 1999. followed by -MSH and 1-MSH (Ringholm et al. 1999. MCH Receptors Identification of MCH receptor in mammals The MCH receptor. 2001). 2003). Hill et al. and show 38% sequence identity to each other.. MCH- © 2006 by Taylor & Francis Group. CerdáReverter et al. 2003c). Full-length DNA for MCH-R2 was determined using PCR with a primer set based on the partial sequence (An et al. whereas the MCH-R2 gene has five coding exons and one noncoding exon (Kolakowski et al. -MSH exhibits the highest affinity for goldfish and zebrafish MC4-R expressed in the same human cells.. Lembo et al. and slightly weaker signals in the optic tectum. Saito et al. The MCHR1 gene has two exons. 2001.. 2003a). 1999. 2001.Akiyoshi Takahashi and Hiroshi Kawauchi 353 is expressed in several brain regions but not in any of the peripheral tissues (Ringholm et al.. These ligands bind with the same potency to the zebrafish and goldfish MC4-R as to human MC4-R. and telencephalon.. was identified using the reverse pharmacological approach wherein a human orphan GPCR (SLC1) having 40% amino acid sequence identity to human somatostatin receptor specifically bound to MCH (Bachner et al.1) (Ringholm et al. 2002. followed by -MSH and 1MSH (Table 11... Rodriguez et al. was initially obtained by searching the human genomic database for the MCHR1 amino acid sequence. 1996. 1999). excluding those chemically synthesized. olfactory bulb. mouse. Strong signals are observed in the brain stem and hypothalamus. 2001. later named MCH-R1.. 1999. rhesus monkey. do not have a functional MCH-R2 gene or encode a nonfunctional MCH-R2 pseudogene (Tan et al.. 2001) Human. hamster. Human MCH-R1 and R2 are composed of 344 and 339 aa. MSH exhibits the highest affinity for spiny dogfish MC4-R expressed in human embryonic kidney 293-EBNA cells... and rabbit. dog... An et al. respectively. 2001. and ferret have been shown to express the two MCH receptor genes... Chamber et al. 2003). Pharmacological characteristics Among the several ligands. Sailer et al. Goldfish MC5-R expressed in the human cells binds to -MSH and -MSH with similar affinity (Cerdá-Reverter et al. Shimomura et al. including rat. MCH-R2. LLC .

.. 1999. Hill et al. 2003). because no transcripts are detected in adult tissues. 2001. Hervieu et al. In zebrafish. Hoogduijn et al. Pufferfish contains two MCH receptors corresponding to MCH-R1 or -R2. pharmacological characteristics. 1983). 1999). Mori et al. 2001). Verlaet et al.. two of which are subtypes of MCH-R1.. 2002). and the third is MCH-R2. Hill et al. and physiological functions (Lovejoy and Balment.. The addition of cortisol to the superfusion buffer results in a decrease in basal release of ACTH from the pituitary cells and a diminution in the ACTH-releasing activities of © 2006 by Taylor & Francis Group. (2003) for zebrafish and pufferfish using the whole genome shotgun datasets as described in Section on Structure.. MCH-R1-A and -B. LLC . MCH receptor in fish The structure of the MCH receptor in fish was provided by Logan et al. although MCH-R1 is more abundant than MCH-2R (Hill et al. BIOLOGICAL FUNCTIONS OF POMC-RELATED PEPTIDE AND MCH Hypothalamic Control of the Release of POMC-related Peptides Regulation of the release from the pars distalis Corticotropin-releasing factor (CRF) and urotensin I (UI) belong to a family of neuropeptides related in structure.354 Fish Endocrinology R1 is expressed in many regions of the central nervous system (Chambers et al. 2002. the expression of MCHR1-A is embryo specific. and can be detected in adults and young embryos. The expression of the MCH-R1 gene is also observed in extracentral nervous tissues (Saito et al.. 2000. 2001. 2001. The expression of MCH-R2 parallels that of MCH-R1-B during embryonic development. Mori et al. Saito et al. An et al. MCH-R2 is expressed at higher levels in the adult torso than the head. 1999. 1999. MCH-R2 distributes similarly in the human central nervous system to MCH-R1. and only weak expression can be found in embryos younger than five days (Logan et al. 1999.... and UI has greater potency than CRF (Fryer et al.. They have been shown to elicit ACTH release in goldfish... Mori et al.. 2001). Expression of MCH-R1-B appears stronger than MCH-R1-A. Zebrafish has three MCH receptor genes. 2001. 2001... Lembo et al.

Thus..11).. The removal of cortisol from the superfusion buffer results in a recovery of basal ACTH release and a recovery of the ACTHreleasing activities of urotensin I and CRF (Fryer et al. 1996).. Regulation of the release from pars intermedia The regulation of the release of POMC-related peptides from the pars intermedia is mainly under inhibitory control by the hypothalamus. These results show that CRF and UI are members of the hypothalamus-pituitary-interrenal (HPI) axis and stimulate ACTH release while their production is suppressed by cortisol in a negative feedback system. Penny et al. which is derived from a precursor that is also common to Mgrp. as was shown by ectopic transplantation of the pars intermedia.Akiyoshi Takahashi and Hiroshi Kawauchi 355 urotensin I and CRF. In rainbow trout.. In carp. the release of ACTH is under rather complicated hypothalamic control (Fig. 2000). while injection of the glucocorticoid antagonist increases CRF mRNA levels (Bernier et al. Tran et al. stimulation of the release of POMC-related peptide from the pars intermedia by CRF has been demonstrated by enhanced in vitro release of -END (van den Burg. arginine vasotocin not only simulates ACTH release but also exhibits synergistic effects on CRF (Baker et al. A variety of neurotransmitters mediate neural stimuli associated with the regulation of POMC-related peptides from the pars intermedia. Among © 2006 by Taylor & Francis Group. The involvement of CRF in ACTH release has also been shown in other fish such as rainbow trout (Baker et al. 1986). which caused uncontrolled secretion of MSH (Iturriza. 2001). Baker et al. They also showed that chronic treatment of rainbow trout with MCH resulted in less in vitro release of ACTH from the pars distalis (Baker et al. The level of CRF mRNA in the telencephalon-preoptic region in goldfish brain decreases in response to intraperitoneal implants of cortisol whose release from interrenal tissue is stimulated by ACTH.. LLC . Thyrotropin-releasing hormone (TRH) and isotocin have also been shown to stimulate ACTH release in goldfish (Fryer. 1979)... (1985) showed that MCH reduced the in vitro release of ACTH by the pituitary pars distalis taken from stressed trout. The effects of MCH. 1969. 1996) and tilapia (van Enckevort. 1989). are in opposition to those of Mgrp in rainbow trout. 1984).. 1989. Mgrp stimulates ACTH release in vitro (Gröneveld et al. 1996). In tilapia. 1999). 11.

2004).11). and red porgy (Van der Salm et al.. it also stimulates the release of MSH from the pars intermedia of gilthead sea bream (Rotllant et al. tilapia (Lamers et al. TRH also affects the relative abundance of the three forms of -MSH. Although CRH is the major hypothalamic hormone stimulating the release of ACTH from the pituitary pars distalis. © 2006 by Taylor & Francis Group. MCH inhibits -MSH release by the pituitary gland at picomolar concentrations. and red porgy. which would be consistent with the concentrations of this hormone in plasma of normal fish (Van der Salm et al. carp (van den Burg et al. In tilapia. tilapia (Lamers et al. 11. NPY: neuropeptide Y.. 1994). 2004). 2000b).. Brain (hypothalamus) Mgrp/AVT/IT/UI CRF/TRH MCH NPY DA + Pars dustalis distalis (ACTH cells) POMC + – + – + – neurointermediate lobe Pars intermedia (MSH cells) (MSH cells) POMC ACTH b-END a-MSH Des-Ac-a-MSH Di-Ac-a-MSH N-Ac-b-END Fig.... 1989). AVT: arginine vasotosin. tilapia (van Enckevort et al. 1987). 1991. 1991). The release of -MSH is significantly enhanced when endogenous MCH is immunoabsorbed by the addition of MCH antiserum to the culture medium (Barber et al. 2004).356 Fish Endocrinology them.11 Hypothalamic stimulatory (+) and inhibitory (–) control for the release of POMC-related peptide from fish pituitary. 1989). Pagrus pagrus (Van der Salm et al.. Neuropeptide Y stimulates the release of MSH in goldfish (Fryer.. A peptide hormone that is responsible for inhibition of the release of MSH is MCH (Fig. 1989). See text for other abbreviations. LLC . IT: isotocin. 2000).. In red porgy. and red porgy (Van der Salm et al. DA: dopamine.. concentrations of MCH below 10 M inhibit -MSH release from the pituitary gland (Gröneveld et al. 1995d). 2004). 11. 2003). and TRH stimulates the release of -MSH in fish including goldfish (Tran et al.. the inhibitory effect of dopamine on MSH release has been reported in goldfish (Omeljaniuk et al...

1988. Baker. and increases plasma levels of MCH (Kishida et al. In vitro bioassays have demonstrated the direct antagonism between -MSH and © 2006 by Taylor & Francis Group. Administration of -MSH disperses melanin granules in melanophores of killifish. Anguilla anguilla. while plasma levels of -MSH do not change concomitantly with background adaptation in flounder.. Adaptation to black background activates the MSH cells in tilapia (van Eys and Peters. 1986). and CRF also affect the relative amount of Di-Ac.. dopamine. 2004). 2004b). Adaptation of rainbow trout to white background activates the expression of MCH gene in hypothalamus (Suzuki et al.MSH more than the release of -MSH in tilapia (Lamers et al.. TRH enhances the in vitro release of Di-Ac. Pleuronectes flesus (Baker et al. LLC . 1981). 1995). Rodrigues and Sumpter (1984) concluded that MSH is not involved in the physiological color change. 1957).. 1991. The role of MSH in melanophore function seems to vary.-MSH. 1984). 1981) and increases plasma levels of -MSH in salmonids and eel. Long-term administration of MCH reduces melanin content of skin and plasma -MSH levels in rainbow trout (Baker et al. Based on the studies in rainbow trout. depending on the species. MCH. but may be involved in morphological color change. 1991... In vivo and in vitro treatment with MCH aggregates melanin granules in melanophores of various fish (Kawauchi et al. In addition to TRH. -MSH and Des-Ac.-MSH released from pituitary cells in red porgy (Van der Salm et al. 1983. 1993). These results suggest differential hypothalamic control of the release of different forms of -MSH.. 2000). 1994. On the other hand.. 1986. Fundulus heteroclitus (Pickford and Kosto. Rodrigues and Sumpter. Long-term administration of -MSH stimulated melanophore development in tilapia (van Eys and Peters.. Takahashi et al. 1991).Akiyoshi Takahashi and Hiroshi Kawauchi 357 which exhibit different degrees of acetylation. 1989). the in vitro studies have shown that effects of -MSH on melanophores are mediated through the activation of an intracellular cAMP/protein kinase A signaling pathway (Fujii. 1984. elevates tissue concentrations of MCH in both hypothalamus and pituitary (Green et al. Baker. 1991). and influences their release from the pars intermedia. Antagonism between -MSH and MCH on Melanophores The biological mechanisms by which -MSH and MCH regulate skin pigmentation have been studied extensively in teleosts (Fujii and Oshima. The effects of MCH are opposite those of -MSH. Eberle.

© 2006 by Taylor & Francis Group. Hypophysectomy results in a marked atrophy of interrenal cells and a decrease in plasma cortisol levels (Donaldson and McBride. while MCH secreted from the neurohypophysis induces the opposite response through MCH receptor-dependent activation of inhibitory GTP-binding protein. Burton and Vokey. Sugimoto (2002) explained the regulatory system for melanophore responses to -MSH and MCH in fish as follows.12). 11. -MSH secreted from the pituitary activates adenylate cyclase to increase intracellular cAMP levels through the melanocortin receptor-dependent activation of stimulatory GTP-binding protein. 1967. Ball and Hawkins. 1993). 1976. Effects on Interrenal Function Effects of ACTH on interrenal function Pituitary-interrenal axis The involvement of ACTH in interrenal function has been well documented in teleost fish (Fig. melanin granules aggregated initially with MCH and then dispersed with increasing concentrations of -MSH added to the MCH vehicle. 1993. These changes are prevented by treatment with ACTH. 11. Young.12 Stimulatory (+) effects of POMC-related peptides on fish interrenal tissue. melanin granules dispersed initially with -MSH and then aggregated with increasing concentrations of MCH added to -MSH vehicle. On a black background. In contrast.-MSH + + -MSH + + cortisol release + ACTH N-POMC interrenal growth + Fig. Pseudopleuronectes americanus (Baker. In vitro studies have also confirmed the cortisol-releasing activity of ACTH by incubation Di-Ac.-MSH Des-Ac. In these experiments. Augmentation or diminution of cAMP signaling induces dispersion or aggregation of melanin granules. LLC .358 Fish Endocrinology MCH on the melanophores by titrating the two hormones against one another in rainbow trout and winter flounder. 2000).

and agnathans. these data suggest that the HPI axis was established at an early stage in vertebrate evolution. Balm and Pottinger.Akiyoshi Takahashi and Hiroshi Kawauchi 359 of diced head kidneys from rainbow trout (Rance and Baker. O’Toole et al. a corticosteroid unique to elasmobranchs. 1997). Effects -MSH on interrenal function In fish.. Sumpter et al. Armour et al.. Rance and Baker (1981) demonstrated for the first time the in vitro corticotropic activity of -MSH using diced interrenal tissue from rainbow trout. 2000a. Dexamethasone-treated fish do not respond to this stressor with any increase in either ACTH or cortisol levels.. 1999) and in non-salmonid fish such as tilapia (Balm et al.. one of which was confirmed as 11-deoxycorticosterone. 1999). 1995. 1985.. 1986.. Hazon and Henderson. Des-Ac. Young. the in vitro corticotropic activity of ACTH isolated from sea lamprey pituitary was examined using pronephric tissue (Takahashi et al. 1975.. As is the case in teleost fish. Similar plasma profiles of ACTH due to artificial stress have been observed in other salmonid fish such as rainbow trout and brown trout (Pickering et al. 1986. 1999. 1995b). 1974).-MSH has full intrinsic activity: its molar potency is 0. In elasmobranchs. 1981. gilthead seabream (Arends et al. Takahashi et al.. 1994). cartilaginous fish. and plasma cortisol levels 50-fold compared to unstressed fish.9 © 2006 by Taylor & Francis Group. ACTH and stress It has been well documented that ACTH is released from the pituitary gland in response to stress (Wendelaar Bonga.. Therefore. LLC . increased the plasma ACTH levels approximately 10-fold.. indicating that the dexamethasone exhibited a potent negative feedback effect. and ACTH enhances in vitro synthesis of the steroid in rays and dogfish (Klesch and Sage. hypophysectomy reduces the plasma levels of 1 hydroxycorticos-terone. Sumpter and Donaldson (1986) showed that in coho salmon. ACTH commonly stimulates corticosteroid production in ray-finned fish. produced by crowding and confinement. In short. Rotllant et al. and sea bass (Rotllant et al.. Nunez and Trant. b). 1984. Ruane et al. an acute stress. 1993) or by in vitro superfusion using diced head kidneys from tilapia (Balm et al. 1993.. 1994) and gilthead sea bream (Rotllant et al.. ACTH has been isolated from the pituitary of spiny dogfish (Lowry et al. ACTH converted 11-deoxycortisol to at least two steroid products.. In agnathans. b). 1990. 2000a. 1973. 2003).

caused a rapid and pronounced elevation in the plasma -MSH level.9% NaCl. Rotllant et al. Salmo trutta. -MSH and Di-Ac. the HPI axis exhibits a different response to stress in accordance with background color.360 Fish Endocrinology 10 –2-fold less than ACTH1-24 but nearly 100-fold greater than -MSH. Thus. The involvement of -MSH in the HPI axis has also been indicated by analysis of the stress effects on the plasma -MSH level. 1985). 2000b). Sumpter et al.-MSH and -MSH. The plasma -MSH level changes primarily in response to background color. (1986) also showed in rainbow trout that although handling and confinement caused no increase in the plasma -MSH level. Sumpter et al.12). Injection stress. 1986).MSH is about 120-fold less potent than ACTH1-39. (1992) compared the corticotropic activities of DesAc. increased plasma -MSH levels in black-adapted rainbow trout. Interestingly.-MSH. a more severe stress. Rodrigues and Sumpter. In brown trout.. confinement. 1984).-MSH in a superfusion experiment with tilapia interrenal tissue. a superfusate from pars intermedia is only 2. The difference in potency among the three type of MSH with different degrees of acetylation suggests that the N-terminal Ser residue affects binding to MC2-R. which consisted of exposure to air plus restrainment..and long-term confinement of the fish caused an increase in plasma -MSH levels (Arends et al. © 2006 by Taylor & Francis Group. LLC . -MSH-related peptides released from the pars intermedia may have a significant effect on interrenal function (Fig. but not in white adapted fish (Gilham and Baker. Di-Ac.. in which the corticotrophic activity is caused by ACTH. 1984. 1999. The rearing of trout with a black background results in significantly higher -MSH levels than that with a white background (Baker et al..7-fold less active than that from the pituitary pars distalis. followed by confinement. Similar plasma profiles of -MSH in response to stress were observed in gilthead seabream: both short. 11.-MSH showed the highest corticotropic activity followed by Des-Ac. When Lamers et al. Although Di-Ac. elicited by administration of 0. the plasma -MSH level in black-adapted fish did not change significantly in response to thermal shock. whereas -MSH in white-adapted fish showed a significant elevation (Pickering et al. (1985) showed that a combination of three different types of stress (handling. and thermal shock) caused a sustained rise in -MSH levels in brown trout.

. the adrenal cortex may be under the dual control of two distinct peptides—N-POMC-related peptides and ACTH—derived from a common precursor. which suggests that the proliferation of adrenal cells may be under the control of factors other than ACTH. immunoneutralization with antisera against N-POMC51-74 corresponding to a part of 3-MSH that shows an absence of adrenal mitogenic activity (Estivariz et al. 2003). 1984). although intact N-POMC1-76 showed no effects. is unknown.. evidence indicates that ACTH inhibits the proliferation of adrenal cells in vitro (Ramachandran and Suyama.. while N-POMC1-76 had no effect (Estivariz et al. 1997). Furthermore. 1979. Moreover.. An in vitro study showed that N-POMC1-28 and N-POMC2-59 enhanced 3 H-thymidine incorporation in adrenal cells. In human pituitary. the peptide induces adrenal tumor cell proliferation. the origin of the shorter peptides. 1996). 1988).Akiyoshi Takahashi and Hiroshi Kawauchi 361 Effects of N-POMC on interrenal function Mitogenic activity ACTH is the major corticotropic stimulus for the adrenal cortex (Wendelaar Bonga.. The involvement of N-POMC in adrenocortical mitogenic activity was demonstrated both in mammals and teleosts in the 1980s.. Dunlap and Grizzle. 1975. Estivariz et al. called AMH (see section on General features of processing).. 1982). 1977. while it inhibits adrenal steroidogenesis (Fassnacht et al. and intact N-POMC antiserum diminished adrenal mitotic activity after bilateral adrenal enucleation (Estivariz et al. Walsh et al. and administration of N-POMC1-76 after treatment with trypsin enhanced DNA synthesis in adrenal cortex. POMC. Di Blasio et al. In spite of the accumulating evidence for mitogenic activity of the Nterminal non. 1982) completely inhibits compensatory adrenal growth.. ACTH stimulates the proliferation of adrenal cortical cells in vivo (Kolanowski et al. Although. Kahri et al. N-POMC1-76 is generated from POMC. antiserum against N-POMC1-28 inhibited compensatory adrenal growth following an unilateral adrenalectomy (Lowry et al. Using human peptides. (1982) showed that in vivo administration of N-POMC1-28 stimulated adrenal growth and mitosis in intact rat. According to an in vitro study using synthetic N-POMC1-28..-MSH region of N-POMC. although it is unlikely that a smaller peptide is generated in the pituitary because of the absence of the pars intermedia. LLC . 1990. 1983). as has been observed in response to antiserum against N-POMC1-28 © 2006 by Taylor & Francis Group. Taken together.

LLC . 1982). 2003). 1983). Prior treatment of the mouse peptide with trypsin enhances the activity (Pedersen and Brownie. cleaves pro.3).. Human NPOMC1-76 exhibits a similar effect on isolated perfused rat adrenal cells (Al-Dujaile et al. salmon N-POMC may function in its intact form and may induce effective interrenal growth (Fig. postulated that N-POMC was specifically cleaved postsecretionally by a protease expressed by the adrenal to generate AMH. 1980. and synthetic bovine 3-MSH also potentiates the corticosteroid release in response to ACTH (Pedersen et al.. The identification of a serine protease that is upregulated during growth of the adrenal cortex. 2001). is expressed exclusively in the outer adrenal cortex.-MSH region in mammals and relatively good conservation of the N-terminal 37 aa containing four Cys residues between salmon and human N-POMC (Fig.. The importance of N-POMC to adrenal mitogenesis has also been observed in fish (Takahashi et al. 1981. and provides evidence for the hypothesis (Bicknell et al. Considering the importance of the N-terminal non. a basic amino acid is not present at the corresponding position in salmon NPOMC.. whereas human N-POMC1-76 had no effect when given as an intact molecule (Estivariz et al. N-POMC potentiates the steroidogenic effects of ACTH. Mouse N-POMC potentiates the effects of ACTH1-24. the peptide does not potentiate the effects of ACTH1-24 on adrenocortical cells when given simultaneously with ACTH in a bolus dose.12). however.. 1980). 1983).. Potentiation of ACTH activity In addition to the potential adrenal mitogenic activity. It should be noted. 11. N-POMC may be cleaved at a pair of basic amino acids at N-POMC49... Farese et al. 1985).362 Fish Endocrinology (Lowry et al. indicating that AMH is not generated from NPOMC1-76 in circulation. causing corticosteroid release from rat adrenal gland in vitro under static conditions (Pedersen and Brownie. 1982). Considering the inhibitory effect of NPOMC1-28 on adrenal steroidogenesis in vitro (Fassnacht et al. 50. Interrenal cells showed a significant increase in nuclear size over 6 days following the injection of chum salmon with a 75 aa N-POMC fragment that did not contain MSH.-MSH. the potentiating effect of N-POMC may originate from 3-MSH. the N-terminal half of the salmon N-POMC may be responsible for the increase in nuclear size. therefore. 11. The potentiating effect is only © 2006 by Taylor & Francis Group. It is. In humans. Therefore. 1980). that salmon NPOMC affected trout interrenal function when an intact molecule was injected. However. However.

11. b). In addition to the indirect functions via cortisol. salmon N-POMC also potentiates ACTH-induced cortisol release from interrenal tissue in vitro (Fig. ACTH itself modifies the immune system by decreasing the number of circulating leukocytes in coho salmon (McLeay. and increasing phagocyte respiratory burst activity in rainbow trout (Bayne and Levy. mast cell degradation. 2000a). In short. ACTH has numerous effects on immune function... In mammals. 1991) (Fig. which performs a homeostatic function in mammals and fish (Weyts et al. In fish. 1999. In the fish immune system. although results from in vitro studies are conflicting (Holm and Majzoub. 1999. antibody production by antibodyproducing cells. 2000a. IL-2. and interferon.. inhibiting lymphocyte mitogenesis in carp (Weyts et al.. -MSH has been recognized as a potent immunomodulatory agent (Lipton and Catania.13).. 2002). although it increases the rate of apoptosis (Weyts et al. Engelsma et al. 1973a.12) (Takahashi et al. MSH In mammals. 1999). but in general its tends to suppress immune responses. Harris and Bird. as in mammals. natural killer cell activity and cytokines. LLC . 1997. lymphocyte proliferation. It inhibits the production and activity of immunoregulatory and pro-inflammatory cytokines such as interleukin (IL)-1. suppressing the number of circulating lymphocytes. In rainbow trout. Lugar et al. 1985). ACTH is a main stimulator of cortisol release from the interrenal gland. Immunomodulation ACTH In the previous two decades. it has been recognized that the neuroendocrine and immune systems are intimately linked and involved in bi-directional communications.and down regulates the expression of co-stimulatory molecules on antigenpresenting cells. 1995). 11. Harris and Bird.Akiyoshi Takahashi and Hiroshi Kawauchi 363 apparent when the cells are exposed to a priming infusion or to a simultaneous continuous infusion. 1998). -MSH has suppressive effects on a number of immune responses in mammals. cortisol exhibits inhibitory effects.. © 2006 by Taylor & Francis Group. and phagocytosis. the stress response induces activation of the HPI-axis and the sympathetic nervous system. -MSH also exhibits an immunostimulatory effect by increasing the production of immunoglobulin E.

364 Fish Endocrinology leukocyte number lymphocyte mitogenesis – ACTH + lymphocyte proliferation MCH a-MSH a-MSH -MSH + + + + Des-Ac.-MSH Des-Ac-a-MSH respiratory burst of phagocytic cells + + ACTH + b-MSH -MSH + Di-Ac. LLC + N-POMC + MCH .13 Stimulatory (+) and inhibitory (–) effects of POMC-related peptides and MCH on fish leukocytes.-MSH Di-Ac-a-MSH + b-END -END N-Ac. 11.-END N-Ac-b-END a-MSH plus MCH -MSH no effect b-END -END phgocytosis + a-MSH -MSH + MCH Fig. © 2006 by Taylor & Francis Group.

All these results indicate that MSHs have a stimulatory effect on the immune response of trout and carp in vitro. bone marrow macrophage differentiation. Sibinga and Goldstein. 1984.. MSH stimulates the proliferation of leukocytes prepared from head kidney of rainbow trout and carp (Harris and Bird. -MSH1-16. Watanuki et al.-MSH have also been shown to stimulate the phagocytic and respiratory burst activity of trout head kidney phagocytes in vitro (Harris and Bird. peripheral blood mononuclear cell interferon. the effect of -END on the function of leukocytes has been well documented. carp phagocytic cells treated with DesAc. -MSH and Des-Ac.-MSH have also been shown to increase the phagocytosis of phagocytic cells prepared from head kidney (Watanuki et al.. T-cell proliferation. Moreover. 2003).-MSH and DiAc. including monocyte chemotaxis. although the mode of action opposes that in mammals (Fig. and natural killer cell activity. Heijnen et al. neutrophil and macrophage superoxide production. macrophage phagocytosis and cytotoxicity. Watanuki et al. although the effects are mostly inhibitory in mammals. phagocytosis and chemotaxis were also significantly increased in kidney phagocytic cells taken from rainbow © 2006 by Taylor & Francis Group.production. In carp. Watanuki et al.Akiyoshi Takahashi and Hiroshi Kawauchi 365 -MSH also plays a role as a modulator of immune responses in fish. (2002) showed that both Des-Ac. This inhibitory effect is comparable to that observed in mammals.-MSH inhibited the production of IL-1 by carp macrophages that had been stimulated by treatment with lipopolysaccharide. In carp. and -MSH (Takahashi et al. Harris and Bird (2000a) have suggested that cells other than phagocytes releasing one or more macrophage-activating factors were required for -MSH to exert its stimulatory effects on trout phagocytic cells. 2003).13).-MSH. 2000b). Des-Ac. 1997. 1991).-MSH exhibit increased production of superoxide anion. In fish. LLC ..-MSH and di-Ac. 1998. increased production of superoxide anion has also been observed in response to other MSHs including carp -MSH and spiny dogfish MSHs such as Des-Ac. 11. 2000). (1999) showed that the production of superoxide anion in phagocytic cells increased significantly in rainbow trout injected with -END. indicating that the peptides augment the activity of the cells. In addition to the phagocytic activity.-MSH or Di-Ac. -END -END modulates many aspects of the immune system in mammals (Fisher and Falke. On the other hand.. -MSH. 1988. 1999.

Even treatment with naloxone.13). indicating their mutually antagonistic nature. Rainbow trout and carp macrophages incubated with N-POMC significantly enhanced the production of superoxide anion. Thus. 2000). (2001) who investigated the function of the peptide isolated from chum salmon (Fig. N-POMC The first evidence demonstrating the association of N-POMC with the immune system in vertebrates was reported by Sakai et al. However. LLC . 11. Both rainbow trout and carp macrophages showed increased phagocytosis when treated with N-POMC. both -END and N-Ac. because acetylation of the Tyr results in a significant loss of analgesic activity (Smyth et al. 2000).. © 2006 by Taylor & Francis Group. However. Namely. 2000. MCH significantly increased both the percentage of phagocytosis and the phagocytic index of rainbow trout head kidney phagocytes.366 Fish Endocrinology trout injected with the hormone. did not completely suppress the production of superoxide anion induced by -END in rainbow trout phagocytic cells (Watanuki et al. These functions are similar to those of -MSH. 1979)..-END increase the production of superoxide anion by head kidney phagocytic cells in carp (Takahashi et al. the mode of action of -END on fish phagocytic cells is unclear. 11. 11.. In addition to an immunomodulating effect in mammals.. 2000). and treatment of rainbow trout head kidney leukocytes has been shown to increase bactericidal activity. The N-terminal Tyr residue is crucial for the binding with the opioid receptor. All these results clearly show that -END activates the function of phagocytic cells in fish (Fig. a potent opioid receptor antagonist. Watanuki et al. Direct in vitro studies have confirmed the effect of -END (Watanuki et al.. MCH Harris and Bird (1998) provided the first evidence that MCH can directly influence immune responses in vertebrates (Fig. -END exhibits potent opiate-like analgesic activity. the stimulatory effects of both hormones were diminished. when MCH and -MSH were added to cells together.13). Similar results were obtained in in vivo studies in which rainbow trout and carp injected with N-POMC showed significantly increased superoxide anion production as well as phagocytosis by macrophages.13).

Although the major lipotropic hormone in mammals is ACTH.. while only ACTH has potent lipolytic activity in rats (Ramachandran and Lee. Guinea pig adipocytes are sensitive to both -MSH and ACTH. 1983). cells other than phagocytes are required for MCH to exert its stimulatory effects on trout phagocytic cells through the release of one or more macrophage-activating factors (Harris et al. 2002). while MCH alone has no effect on the proliferation (Harris and Bird. Des-Ac. It has been named after its typical ‘cobalt-blue’ body color (Oguri. which contains MSH cells (Kaneko et al. Yamazaki.14). Another © 2006 by Taylor & Francis Group.-MSH shows the highest lipolytic activity in isolated rat adipocytes.Akiyoshi Takahashi and Hiroshi Kawauchi 367 MCH is also antagonistic of the stimulatory effects of -MSH on leukocyte proliferation. It has also been suggested that as in the case of -MSH. 11. This phenomenon seems to be related to a lack of most of the pars intermedia in the pituitary. Effects on Energy Metabolism and Food Intake Effects of MSH on lipolysis POMC-related peptides have been shown to stimulate lipolysis in mammalian adipocytes (Boston. significant species differences have been shown with regard to the relative potency of the peptides. 1990). integumental melanophores are considerably reduced in number (Oguri. Richter. 1993). 1997). 1974. Intra-arterial administration of Des-Ac.-MSH increases circulating levels of free fatty acids.-MSH stimulates hepatic triacylglycerol lipase both in vivo and in vitro (Fig..-MSH in rainbow trout. administration of Des-Ac. whereas -MSH has no effect... and -MSH (Kawauchi et al. LLC . 1972). In the cobalt variant. Both -MSH and ACTH exhibit potent lipolytic activity against rabbit adipocytes. the hormone shows no significant effect on lipid mobilization in rainbow trout (Takashima et al. Kaneko et al. Among the chum salmon MSH-related peptides. 1993). 1987). However. 1999). Moreover. 2000b). followed by -MSH. The finding of lipolytic activity of MSH in rainbow trout led Yada and co-workers to investigate the role of MSH in the ‘cobalt’ variant of rainbow trout (Yada et al. The cobalt variant is found on rare occasions at trout experimental stations and commercial farms. 1974. 1976. (2000) demonstrated the lipolytic activity of Des-Ac. ACTH1-24. 1984). Yada et al.. while adipocytes from hamsters only respond to ACTH (Ng..

Taking into account the lipolytic activity of MSH.-MSH as in the normal trout in vitro.-MSH. characteristic of this variant is obesity: enlarged liver and fat accumulation in the abdominal cavity have been reported (Yamazaki. LLC . whereas its activity in vivo was lower than that in the normal trout (Yada et al..14 Stimulatory (+) and inhibitory (–) effects of MSH-related peptides and MCH on fish energy metabolism and food intake. 1993)..368 Fish Endocrinology lipolysis + Des-Ac. The hepatic lipase activity of the cobalt responded to Des-Ac. MC3-R and MC4-R are preferentially expressed in hypothalamus. No significant difference has been observed in the basal activity of triacylglycerol lipase of cultured liver slices between the cobalt and normal trout.-MSH MCH Food intake _ MSH -END + Fig. 2002). Thus. the lower levels of lipase activity in vivo in the cobalt variant of rainbow trout are due to a lack of stimulatory regulation by lipolytic hormones such as Des-Ac. Kaneko et al. Oguri. the scarceness of MSH cells caused by the lack of most of the pars intermedia in the pituitary seems to be correlated with the abnormal fat accumulation in the cobalt variant. 1974. a central nervous system region © 2006 by Taylor & Francis Group. 11. 1976. Effects of POMC-related peptides on food intake MSH Among the five different MC-receptors.

1986. Fasting upregulates AGRP mRNA levels in the nucleus lateral tuberis.14)..-MSH4-10 amide (SHU9119) completely blocks the inhibition caused by MT-II. 1996). 1986.. Food deprivation decreases the expression of hypothalmic POMC mRNA (Brady et al. Cys11)-cyclo. and (3) administration of SHU9119 significantly enhances food intake... Fan et al. LLC . has been discovered (Ollmann et al. D-2-Nal7.. Tsujii and Bray. 2003a. (Ac-Nle4. (1997) showed that melanocortinergic neurons exert a tonic inhibition of feeding behavior using melanocortin analogues as follows: (1) intracerebroventricular administration of the agonist. Furthermore. (2) co-administration of the specific melanocortin antagonist. In goldfish brain.. both POMC and MC4-R are expressed in the hypothalamic area appropriate for involvement in food intake (CerdáReverter et al.Akiyoshi Takahashi and Hiroshi Kawauchi 369 that controls many physiological functions. the goldfish genome has a gene homologous to the mammalian AGRP gene. D-Phe 7. The first evidence for an inhibitory role of MSH in the regulation of food intake was provided by studies in rats in which intracerebroventricular administration of the peptide resulted in suppression of food intake (Panskepp et al. D-2-Nal7. Kim et al. whereas (Ac-Cys3. including feeding behavior. which is exclusively expressed in the ventrobasal hypothalamic nucleus lateral tuberis. 11. b). progressive fasting did not affect POMC mRNA expression levels in the brain (Cerdá-Reverter et al.. an endogenous selective antagonist of MC3-R and MC4-R. 2003a. Asp5. © 2006 by Taylor & Francis Group. b). Intracerebroventricular administration of (Nle4. 1989).-MSH (NDP. In contrast to mammals. 2003a.-MSH) and MT-II inhibits food intake in fasted fish. Lys10. suggesting that melanocortins exert a tonic inhibitory effect on food intake (CerdáReverter et al. 7 D-Phe ). Huszar et al.-MSH4-10 amide (MT-II).. inhibits feeding in mice. Several lines of evidence have hitherto demonstrated the association of MC-receptors with food intake. 1990. b). Asp5.MSH3-11 (HSO24) stimulates food intake in fed fish. 2003). (1997) reported that a targeted disruption of MC4-R resulted in obesity associated with hyperphagia. whose gene is normally expressed in the hypothalamus and whose levels are increased in obese mice. Vergoni et al. 1997). 1976. Lys10)-cyclo. On the other hand. All these results suggest that melanocortin is involved in the regulation of food intake and its activity is inhibited by an endogenous antagonist AGRP in goldfish (Fig. Poggioli et al. Lys10)-cyclo. agouti-related protein (AGRP).. Arg5. the teleostean homolog of the arcuate nucleus (Cerdá-Reverter and Peter. Nle4. (AcNle4.

Grossman et al. LLC .. Hanada et al.. circumstantial evidence for the association of MCH with food intake was obtained in barfin flounder (Takahashi et al. In fish. 1997). which are and 1 opioid receptor antagonists. The first evidence that opioid receptors participate in this feeding response was obtained in goldfish (de Pedro et al. which inhibits food intake.. Effects of MCH on food intake In rodents. (v) Mice carrying a deletion of the entire coding region of the MCH gene are hypophagic and lean (Shimada et al. respectively. 2003). Rossi et al.. Moreover. Mediation of -END effects on increasing food intake via the opioid receptor has also been confirmed in rodents (Silva et al. 1996.. (vi) MCH receptor antagonists reduce food intake in rats (Borowsky et al. The feeding stimulation by -END is antagonized by -funaltrexamine and naloxonazine.. while no increase is observed upon intraperitoneal injection.. 2002). 1986). but not by the receptor antagonist nor-binaltorphamine.. 2002.370 Fish Endocrinology -END In contrast to -MSH. 2003).. 1998).. and (vii) Disruption of MCH-1R in mice results in lean and hyperactive animals in which hyperphagia may be a compensatory response to the increased lean mass and hypermetabolic rate (Chen et al. The © 2006 by Taylor & Francis Group. 2002). Marsh et al. hypothalamic -END is increased by feeding (McLaughlin et al. 1995. Takekawa et al. 2001.14). 1996. High levels of pituitary and plasma -END are observed in genetically obese mice and rats (Margules et al. Intracerebroventricular injection of -END induces an increase in food intake. 1 receptor antagonist 7-benzidilidenenaltrexone. 2002. Hervé and Fellman. 1985). 1977) and paraventricular nuclei (Leibowitz and Hor. 1996) (Fig. (ii) Transgenic mice overexpressing the MCH gene tend to overeat and are obese (Ludwig et al.. 2001). 1996. (iii) Fasting (24 or 48 hr) in rodents results in increased MCH gene expression (Qu et al.. 2004b). -END stimulates food intake in rodents upon microinjection into the hypothalamic ventromedial (Grandison and Guidotti. 1982) and nucleus accumbens (Majeed et al. 2000). Gomori et al... (iv) Increased MCH gene expression is observed in genetically obese rodents (Qu et al.. several recent investigations have shown an association between MCH and enhanced feeding behavior: (i) intracerebroventricular injection of MCH stimulates feeding (Qu et al. 11.. 1978).. or 2 receptor antagonist naltriben. 1997...

both total length and body weight were greater in the fish reared with a white as opposed to black background.14). in turn.. LLC . © 2006 by Taylor & Francis Group. The activities of leukocytes are modulated by POMC-related peptides in fish. 1999). resulting in a diverse number of MSH segments on POMC. These results indicate that a white background stimulated production of MCH in the brain and. (b) Phylogenetic Origin of POMC POMC has also been found in platyhelminths (leech. 1998). there are missing links.. 1998). Furthermore.. 11.Akiyoshi Takahashi and Hiroshi Kawauchi 371 MCH gene expression level in the brain was higher in fasted fish than controls. However. Rearing of barfin flounder for 6 months in a tank with a white background resulted in increased MCH gene expression in the brain compared to those reared with a black background. Ciona intestinalis (Dehal et al. MCH enhanced body growth. 2002). SUMMARY (a) Diversity of POMC Structure The class specific number of MSH in fish and the occurrence of the two POMC subtypes in some fish species suggest that POMC has evolved by internal gene duplication and deletion of the MSH segment. Gene duplication has also increased the number of POMC copies. 1997. The number of MCH-immunoreactive (ir) cell bodies in the hypothalamus was also greater in the fasted fish. probably by stimulating food intake (Fig. and nematode. POMC are preferentially found in immunocytes (Salzet et al. which showed a correlation between fasting and increased MCH gene expression. Caenorhabditis elegans (Bargmann. Theromyzon tessulatum) and mollusks (Mussel. immunoregulation may be a common effect of POMC among vertebrates and invertebrates. Moreover. because POMC and the melanocortin receptor family have not been found in the invertebrate genome. Mytilus edulis). suggesting that it appeared at an early stage of invertebrate evolution. Stefano et al. These results are comparable to those in rodents. Taken together. including the urochordate. In these invertebrates. some immunocytes have been shown to produce POMC-related peptides and express the melanocortin receptor in mammals (Lugar et al..

372 (c)

Fish Endocrinology

Physiological Interaction Between POMC Gene Products and MCH Gene Products in Fish

(i) Regulation of body color change is a typical mutual antagonistic interaction between MSH and MCH. Moreover, MCH inhibits the release of MSH from the pituitary pars intermedia. Thus, MCH substantially antagonizes the melanin-dispersing effects of MSH at both pituitary and integumental levels. (ii) Although both -MSH and MCH enhance the activity of phagocytic cells, when -MSH and MCH are added to cells together, the stimulatory effects of both hormones are diminished. MCH also inhibits the proliferation of lymphocytes induced by -MSH. (iii) The inhibitory effect of MSH on food intake has been demonstrated in goldfish. In addition, there is indirect evidence of a stimulatory effect of MCH on food intake in flounder. (d) Physiological Interaction Among POMC-related Peptides in Fish

Melanocortins stimulate cortisol release from interrenal tissue, although MSH is less potent than ACTH. N-POMC enhances the effects of ACTH, while N-POMC itself does not stimulate cortisol release. Both -MSH and -END may also interact in cortisol release (Balm et al., 1995), although they exert opposing effects on food intake. (e) Physiological Interaction Among MCH-related Peptides in Fish

MCH and Mgrp exhibit opposing effects on ACTH secretion from the pituitary. MCH inhibits the release, while Mgrp stimulates it. (f) Versatility of POMC and MCH Genes

In addition to the change in skin color, POMC-related peptides and MCH-related peptides play important roles in many physiological processes including the HPI axis, immunoregulation, and energy metabolism. Indeed, MC5-R is expressed in several goldfish tissues. Furthermore, MC4-R and MC5-R are distributed in several regions of the brain. The distribution of MC receptors is comparable to that observed in

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mammals, indicating that the versatility of melanocortins might have been established at an early stage in the evolution of vertebrates. On the other hand, although MCH is present in the hypothalamus of all vertebrates that have been examined, only neopterygians employ MCH as a circulating hormone (Sherbrooke and Hadley, 1988; Baker, 1991; Bird et al., 2001). Given the fact that MCH mainly regulates brain functions such as food intake and energy metabolism, its ability to affect color change might have been evolutionary ramified in the ray-finned fish lineage. In conclusion, both the POMC and MCH genes are multifunctional, because they code for two or more functional peptides. Their versatility originates primarily from the occurrence of multiple hormone segments in the genes. Moreover, their effects are diversified by the occurrence of multiple receptors distributed in a wide variety of tissues. References
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Akiyoshi Takahashi and Hiroshi Kawauchi


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Osmoregulatory Action of Hypophyseal Hormones in Teleosts
Juan Miguel Mancera1 and Juan Fuentes2

ABSTRACT The osmoregulatory system in fish is under control of several hormones. Adenohypophyseal and neurohypophyseal hormones play an important role in osmoregulation, through their direct or indirect action in target organs. The neurohypophysis secretes two main factors—arginine-vasotocin (AVT) and isotocin (IST). AVT may be related to changes in perfusion patterns through osmoregulatory organs, such as gill and kidney or involved in changes of ion and water transport. At present, the role of AVT in adaptation to hyperosmotic/hypoosmotic environments remains contradictory. The
Authors’ address: 1Departamento de Biología, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain. E-mail: 2 Centro de Ciências do Mar (CIMAR Laboratório Associado), Campus de Gambelas, 8005139 Faro, Portugal. E-mail:

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Fish Endocrinology

osmoregulatory action of IST in teleosts is also unclear. At the adenohypophyseal level, prolactin (PRL) is involved in freshwater adaptation and presents a clear hyperosmoregulatory role. In contrast, the growth hormone (GH)/insulin growth factor (IGF) axis seems to play a role in seawater adaptation in salmonids. In some non-salmonid fish, there is substantial evidence that the GH/IGF axis is active in ion regulation, while in other species very little work has been done to clarify this issue. The physiological role of somatolactin (SL) remains unclear. Although attempts have been made to relate this hormone with osmoregulation, the evidence for such a function is not compelling at present. Hormones derived of proopiomelanocortin (POMC) (adrenocorticotropin -ACTH- and melanotropin -MSH-) control the interrenal tissue and amongst the factors produced by this tissue, cortisol is the primary one with ionoregulatory action. A hypoosmoregulatory role of this hormone is well established in many species. In addition, several lines of evidence indicate a role for cortisol either in ion uptake in freshwater or brackishwater-adapted fish. Therefore, a ‘dual osmoregulatory’ role for cortisol has been proposed, with a stimulatory action on ion secretion in hyperosmotic media, and an increase of ion uptake in cooperation with PRL in hypoosmotic environments. The osmoregulatory actions of thyroid hormones are ambiguous, but cooperation with GH and cortisol has been demonstrated. Gonadotropins (GTH I and II) induce synthesis and release of sexual steroid in gonads. Several lines of evidence point to a role of sexual steroids, mainly 17 -estradiol, on calcium balance and seawater adaptability in some teleosts. Key Words: Teleost; Hypophysis; Osmoregulation; Arginine-vasotocin; Prolactin; Growth hormone; Somatolactin; Adrenocorticotropin/ melanotropin; Thyroid-stimulating hormone; Gonadotropins.

INTRODUCTION Adaptation of fish to constant or changed environmental salinity involves the action of several endocrine factors that may change the rate of active sodium and chloride transport and alter the rates of secretion or uptake of other ions and/or water. A diverse array of endocrine factors often interacts or has synergic action in the control of ion balance. Thus, the anatomical source of hormones involved in the control of osmoregulation is heterogeneous and includes several tissues (see Bentley, 2002, for a detailed description). The heart (atrial and ventricular natriuretic peptides), the urophysis (urotensis I and II), the corpuscles of Stannius (stanniocalcin), the liver (insuline-like growth factors, also produced by other tissues such as gill or kidney), the gonads (sex steroids), the kidney

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Juan Miguel Mancera and Juan Fuentes


(cortisol and angiotensinogen) and the pituitary gland contribute to ion balance. However, the pituitary gland may be considered the ‘master’ gland in the control of osmoregulation across the vertebrates. The pituitary (hypophysis) is an important endocrine gland that controls through its different hormones, several endocrine functions in animals such as osmoregulation, reproduction, growth, etc. Anatomically, the association of two tissues with different embryological origin constitutes the pituitary gland: the neurohypophysis derived of neural tissue and the adenohypophysis derived of endodermal tissue (Gorbman et al., 1983; Norris, 1997; Bentley, 2002). The neurohypophysis connects to the hypothalamus through the infundibular stalk and releases hormones synthesized in different hypothalamic nuclei, mainly in the supraoptic (SON) and the paraventricular (PVN) nuclei. The neurohypophysis secretes two structurally related hormones: AVT a member of the vasopressinergic family and IST a member of the oxytocinergic family. The teleostean adenohypophysis comprises three subdivisions: the rostral pars distalis (RPD) containing prolactin and adrenocorticotropic cells; the proximal pars distalis (PPD) containing somatotropic, gonadotropic and thyrotropic cells; and the pars intermedia (PI) including somatolactin and melanotropic cells. Adenohypophyseal hormones group into three families in teleosts based on their molecular biochemistry: (a) GH/PRL family including PRL, GH and SL hormones; (b) glycoprotein hormones containing GTHs (GTHI and GTHII) and TSH; and (c) proopiomelanocortin-derived hormones, such as ACTH and MSH.
Table 12.1 references)

Main actions of pituitary factors in ion balance processes (text for
Administration i.c.v. in vitroa in vitrob i.p. i.p. in vitroc Species eel trout sea bass several salmon several Action drinking glomerular filtration rate gill Cl – secretion gill ATPase gill ATPase cortisol secretion

a: in situ kidney preparation; b: gill cell culture in Ussing chamber; c: perfussed head kidney.

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Fish Endocrinology

Several hypophysial and extrahypophysial hormones are involved, directly or through other endocrine glands, in the control of the osmoregulatory system of teleosts. Previously timely and excellent reviews on this subject have been published (Foskett et al., 1983; Mayer-Gostan et al., 1987; Bern and Madsen, 1992; McCormick, 1995, 2001; Sakamoto et al., 2001). Here, we will focus at neurohypophyseal levels on AVT actions, while at adenohypophyseal levels we will discuss some of the effects of different hormones released into the bloodstream with direct action or whose osmoregulatory action is mediated by other endocrine factors. Arginine-vasotocin (AVT) The two primary circulating neurohypophysial peptides in teleosts are AVT and IST and their plasma concentrations are similar. These nonapeptides are synthesized in the form of prohormones in the perikarya of magnocellular neurons at hypothalamic levels. Both peptides are translated and stored in the nerve terminal of these neurons at the pars nervosa of the hypophysis. After an appropriate stimulus, the precursors are cleaved and hormones released into the blood stream (Norris, 1997; Bentley, 2002). AVT is the fish counterpart of the arginine vasopressin (AVP) of mammals. An osmoregulatory role of AVT in teleosts has been proposed, with several effects described on osmoregulatory organs, while the osmoregulatory role of IST remains unclear. In 2002, reviews on the osmoregulatory role of neurohypophyseal peptides have appeared (Warne, 2002; Warne et al., 2002). However, the osmoregulatory action and targets of AVT in fish is far from well established. The number of species and experimental approaches described to date are limited and often provide ambiguous, if not contradictory, results. In this section, we will give an overview of the proposed osmoregulatory actions of these hormones. At neurohypophyseal levels, content of AVT exhibits a short-lived response to osmotic challenge. Although, this response may be related with an acute stress response as suggested for trout (Gilchriest et al., 2000). In contrast, in fish adapted long-term to different environmental salinities (freshwater -FW- versus seawater -SW-), comparative levels of pituitary AVT content provide often contradictory results. Thus, the use of different techniques for evaluating pituitary AVT content such as

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Juan Miguel Mancera and Juan Fuentes


histology, immunocytochemistry and radioimmunoassay or the use of different species, with different degrees of euryhalinity or osmoregulatory characteristics (eel, flounder, rainbow trout and medaka) could explain these conflicting results (Balment et al., 1993; Warne, 2002; Warne et al., 2002). Nevertheless, experimental data from several teleost show that pituitary AVT content is sensitive to osmotic challenge. Hence, a depletion of AVT during the initial period after transfer from FW to SW takes place, while a small increase takes place after the inverse transfer. These pituitary changes correlate inversely with variations in circulating plasma AVT levels observed during acute osmotic challenge (Haruta et al., 1991; Balment et al., 1993; Bond et al., 2002). All this data suggest that the osmoregulatory role of AVT may be related to hyperosmotic adaptation. AVT receptors are present in branchial tissue of different species and appear in higher numbers in the gills of SW-adapted fish than FW-adapted fish (Guibbolini et al., 1988; Warne et al., 2002). According to pharmacological criteria, the receptors identified belong to a new receptor type NHF functionally similar to the mammalian neurohypophysial V1a type (Guibboline and Lahlou, 1990). However, at present, there is not data on the specific localization of the receptor/s in the different cell types present in the gill tissue (i.e., chloride cells, respiratory, pillar or mucous cells) an issue that once clarified will probably have deep implications in the functional characterisation of AVT. Recently, Evans (2002) pointed out there is not experimental evidence at present to support a direct role of AVT in the control of ion transport in fish gills, a statement that still stands. Hence, the osmoregulatory role of AVT in the gills could be only associated to changes and maintenance of blood perfusion pattern and flow through this osmoregulatory organ (see Olson, 2002). However, a recent study using a primary culture of sea bass (Dicentrarchus labrax) gill respiratory cells, has shown that AVT (an also IST) are able to stimulate Cl – secretion in SW-adapted fish. These peptides could act by means of two distinct V1-type receptors located in gill respiratory cells. Whether these receptors are present in gill chloride cells and the exact role of AVT on chloride cell function remains unclear (Guibbolini and Avella, 2003). AVT receptors have been described in the kidney of teleost fish. Initial studies attributed a diuretic effect to AVT, however later studies suggested the existence of an antidiuretic effect using physiological doses

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Fish Endocrinology

and contrasting with the diuretic effect obtained at supra-physiological doses used formerly. The mechanisms by which AVT brings about the reduction in urine production are not well understood, but the existence of both a decrease in glomerular filtration rates and an increase in tubular water reabsorption have been postulated (Amer and Brown, 1995; Brown and Balment, 1997; Warne, 2002; Warne et al., 2002). In keeping with its water handling effect, AVT applied intracranially also results in a decreased water intake in eels (Kozaka et al., 2003). In addition, angiotensin I (one of the metabolites of the renin angiotensin system with dipsogenic effects) is able to reduce circulating plasma levels of AVP (Balment et al., 2003). In line with all these evidences, it would be possible to speculate that the physiological action of AVT in fish osmoregulation is probably directed to water handling mechanisms and not to ion transport itself. Prolactin (PRL) PRL is a pleiotropic hormone with several endocrine actions across the vertebrates. In a recent publication, Bole-Feysot et al. (1998) refer to over 300 separate functions of PRL in vertebrates some of them related to the osmoregulatory process. This hormone is a member of the GH/PRL family, which also includes growth hormone, mammalian placental lactogens and teleostean somatolactin. This peptide hormone contains between 175-200 amino acids and three disulfide loops and it is synthesized in the form of pro-hormone. Most piscine species described so far seem to present a single PRL form. However, in some teleost, it has been reported the existence of two different PRL forms (Oncorhynchus keta, Cyprinus carpio, Anguilla anguilla, Oreochromis niloticus and Oreochromis mossambicus) (see Manzon, 2002). In species of the genus Oreochromis (tilapia) both PRL forms present differences not only at the structural level but also in their biological roles, such as responses to changes in environmental salinity (Auperin et al., 1994; Yada et al., 1994) and regulation during development (Ayson et al., 1994). The first evidence of the osmoregulatory action of PRL in teleosts emerged from research by Pickford’s group. This group demonstrated that hypophysectomized FW-adapted killifish Fundulus heteroclitus could survive only if treated with PRL (Pickford and Phillips, 1959). After this first evidence, several experimental evidences have shown that PRL is involved in FW adaptation and supported the hyperosmoregulatory role of PRL (Hirano, 1986; McCormick, 1995; Manzon, 2002).

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Juan Miguel Mancera and Juan Fuentes


Synthesis and release of hypophyseal PRL increased in FW- or brackish water (BW)-adapted teleost (Nishioka et al., 1988). In the euryhaline teleost gilthead seabream (Sparus aurata), adaptation to BW also activated PRL cells (Mancera et al., 1993). Accordingly, expression of PRL mRNA also increased in hypoosmotic media (Martin et al., 1999). Circulating plasma levels of PRL decline after transfer from FW to SW or increase after transfer from SW to FW (for references see Manzon, 2002). PRL receptor (PRLR) cDNAs have been isolated and characterized in several teleosts (Prunet and Auperin, 1995), showing the highest expression levels in osmoregulatory organs (O. niloticus: Sandra et al., 2000; Carassius auratus: Tse et al., 2000; S. aurata: Santos et al., 2001; Paralichthys olivaceus: Higashimoto et al., 2001). In addition, it has been demonstrated the changes depend on environmental salinity, with the lowest PRLR expression in the higher environmental salinity (Shiraishi et al., 1999; Sandra et al., 2000). The effects of PRL on osmoregulatory system relate to the prevention of ion loss to the external milieu and to prevention of water influx, typical osmoregulatory problems faced by fish living in hypoosmotic environments. Thus, effects of PRL on different osmoregulatory organs (gill, kidney, intestine, urinary bladder and skin) and on water permeability and ion balance have been established (see Hirano, 1986; McCormick, 1995; Manzon, 2002). At branchial levels, PRL treatment decreases osmotic permeability and increased mucus secretion (Clarke and Bern, 1980; Brown and Brown, 1987), both processes contributing to difficult the transit of ions and water thought the gills. It has been demonstrated that PRL treatment reduced gill Na+,K+-ATPase activity in SW- or FW- adapted teleost (F. heteroclitus: Pickford et al., 1970; O. mykiss: Madsen and Bern, 1992; O. mossambicus: Sakamoto et al., 1997; S. aurata: Mancera et al., 2002). In the sparid silver seabream Sparus sarba, a reduction of gill Na+,K+ATPase activity and of Na+,K+-ATPase mRNA expression have been observed in SW- or BW-adapted fish after PRL treatment (Deane et al., 1999; Kelly et al., 1999). In addition, PRL antagonizes the hypoosmoregulatory action of both cortisol and GH, two seawateradapting hormones, in salmonids (O. mykiss: Madsen and Bern, 1992; Salmo salar: Boeuf et al., 1994; Salmo trutta: Seidelin and Madsen, 1997). The existence of two types of chloride cells has been proposed in the gills of teleosts: -cells typical of SW and -cells typical of FW (Pisam and

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Fish Endocrinology

Rambourg. 1991; Pisam et al., 1995). The existence of PRLR has been described in chloride cells (Weng et al., 1997; Santos et al., 2001). In addition, PRL has strong effects on morphology, distribution and density of these cells and PRL treatment stimulated development of -cells and dedifferentiation of -chloride cells (see McCormick 1995; Sakamoto et al., 2001; Manzon, 2002). At renal levels, PRL increases Na+ reabsorption and water excretion, by stimulation of glomerular size and urine output (see Clarke and Bern, 1980; Braun and Dantzler, 1987). Similarly to the gills, the existence of chloride cells has been reported in the kidney (see above). The effects of PRL treatment on renal Na+,K+-ATPase activity are contradictory. Pickford et al. (1970) reported a stimulation of this activity in F heteroclitus . after PRL treatment. However, in salmonids (S. salar: McCormick et al., 1989; S. trutta: Madsen et al., 1995; Seidelin and Madsen, 1997) and in the silver seabream (Kelly et al., 1999), this activity remains unaffected by hormonal treatment. Manzon (2002) suggest that renal role of PRL on kidney function is related to water balance and not ion balance. If this idea is correct, this could explain the failure of PRL treatment to change renal Na+,K+-ATPase activity. In addition, the nephron is a heterogeneous structure that presents different segments with different physiological roles and biochemical characteristics respect to ion and water physiology. It is possible to speculate that negative results on the effects of PRL treatment on renal Na+,K+-ATPase activity are related to the heterogeneous nature of the kidney structure and often treatments seem ineffective. An experimental in vitro model of kidney function similar to that developed by Renfro’s lab (Duda and Renfro, 2001) may shed new light into the hormonal control of renal ion transport. The information on the endocrine effect of PRL in the intestinal tract is scarce and generalization of PRL effects in the fish intestine is somehow difficult. The presence of PRLR in the intestinal tract (Prunet and Auperin, 1995) points to an action of PRL in the gut, likely related with the osmoregulatory process. At intestinal levels, PRL seems to decrease permeability to water and ions and Na+,K+-ATPase activity an action consistent with a decrease in the absorption of Na+, Cl– and water (see Collie and Hirano, 1987). However, recent reports show that ovine PRL increases intestinal Na+,K+-ATPase activity in S. sarba (Kelly et al., 1999), but not in S. trutta (Seidelin and Madsen, 1999). In addition, the existence of regional differences in the intestinal tract difficult studies on PRL effects in intestine. Definitely further research will be necessary in

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which present a single-chain polypeptide of about 22kDa and a high sequence similarity between different teleostean GHs (Rand-Weaver and Kawauchi. later other papers confirmed this role in salmonids (see McCormick. 1988). in non-salmonid teleosts. Manzon. two different forms of GH (GH I and II) have been isolated. which carries out some or all of the physiological actions of GH. 1991). Björnson. 1997). 1980. the osmoregulatory role of this axis is less clear (see Mancera and McCormick. LLC . 1997). Growth Hormone (GH) GH is other member of the GH/PRL family. mykiss (Sakamoto and Hirano. but there is no © 2006 by Taylor & Francis Group. there is no functional difference established for both types of GH at present (see Björnsson.. According to the somatomedin hypothesis. trutta. plasma levels of GH have been reported to rise during smoltification or after transference from FW to SW in salmonids (see Sakamoto et al. Brown and Brown. GH mRNA expression increased after exposure to SW. Therefore. the physiological actions are often referred as those of the GH/IGF axis (Holly and Wass. In salmonids. 1993. 1995).. McCormick. Smith (1956) reported a positive influence of GH treatment on SW adaptability of S.Juan Miguel Mancera and Juan Fuentes 401 order to clarify the intestinal response to PRL in the scenario of osmoregulation. instead of isolated effects of GH. kisutch (Gray et al. PRL induces an increase in mucus production by stimulation of differentiation and proliferation of mucous cells. 1989). FW-adapted fish (where higher PRL levels are expected) always have a more slippery skin than SW-adapted fish (see Clarke and Bern. 1987. Björnsson. This increase in the mucus layer reduces permeability of skin to ions and water. Histological and immunocytochemical studies have shown that the synthesis and release of hypophyseal GH increased in SW-adapted fish compared with FW-adapted fish (Nishioka et al. 1995. 1998b).. 1993). 1997). 1990) and O. In this way also. For this reason. 1993). the GH/IGF axis seems to play a clear role in SW adaptation in salmonid teleosts (see Sakamoto et al. However. while.. In addition to its role for the promotion of growth and differentiation (McLean and Donaldson. In the skin. The GH receptor has been characterized in gills of O. 1993. GH stimulates the production and release of IGF-I from the liver and/or other organs. 2002). Accordingly.

In the air-breathing climbing perch Anabas testudineus. also improved salinity tolerance and gill Na+. in the silver seabream S. 1995). In addition to gills. However. 1988. In the gilthead seabream and other teleosts. salar previous to smoltification (Nonnotte et al. Pituitary GH cells and plasma GH levels behave differently depending.. 2001).K+-ATPase activity (Mancera and McCormick. Madsen et al. In addition.K+-ATPase activity (Flik et al. Xu et al. mossambicus).. The role of GH and/or IGF-I as osmoregulatory hormones has been studied in only a few non-salmonid species. increase of gill Na+. on the species studied and the environmental salinity (Nishioka et al. 1998b). immunocytochemical and morphological data suggested an activation of GH cells in fish acclimatized to brackish water (see Mancera et al. injection of ovine GH improves the drinking response and. sarba adapted to SW or BW. McCormick. treatment with GH hormone before SW entry increases salinity tolerance.. 1998a). 1995. GH receptors have been located in other osmoregulatory organs such as intestine and posterior body kidney (Sakamoto and Hirano.cotransporter in gill chloride cells (Boeuf et al. Mancera and McCormick. The improved SW tolerance is reflected by proliferation of -chloride cells (involved in salt secretion) and diminution of -cells (involved in ion uptake). Nevertheless. Pelis and McCormick. GH treatment increased salinity tolerance. However.. In tilapia (O. LLC . 1995). administration of GH increased gill Na+.. 1998). little is known on the influence of GH treatment on these osmoregulatory organs.K+-ATPase activity and Na+. 1999. Kelly and co-workers (1999) describe no © 2006 by Taylor & Francis Group. Seidelin and Madsen. consequently the water handling in Atlantic salmon pre-smolts after transfer to SW but not in FW fish (Fuentes and Eddy. In the euryhaline mummichog.. treatment with GH and/or IGF-I .K+-ATPase subunit expression and abundance of Na+-K+-2Cl.. 2000). F heteroclitus. while induces changes in intestine of S. However. opercular chloride cell number and gill Na+.K+-ATPase activity (Leena and Oommen. 1993. GH treatment has not effect on kidney Na+-K+-ATPase activity (Madsen et al. it has been demonstrated that in different salmonid species. 1995.. the osmoregulatory role of this axis is less clear. suggesting that GH may interact also with the renin angiotensin system to produce an integrated response to SW entry in salmonids. trutta. to our knowledge.402 Fish Endocrinology clear evidence of the existence of this receptor in chloride cells. 1994. 1991). In S. 1997). in non-salmonid teleosts. 1995).

IGF-I carries out some or all of the physiological actions of GH (Reinecke. 1999). 1999. In S. gill and renal IGF-I mRNA expression increased after GH treatment and transfer to SW in salmonid species (Sakamoto and Hirano. 1999). Pelis and McCormick. 2001). heteroclitus. 1993). LLC ... little is known on the influence of GH treatment in other osmoregulatory organs in nonsalmonid species. Similar to salmonids. 1995. auratus. it has been reported in different species of salmonids a strong synergy of GH. IGF and cortisol to improve salinity tolerance. In the euryhaline teleost S. According to the somatomedin hypothesis.K+-ATPase activity and subunit expression. Somatolactin (SL) SL is a pituitary hormone of teleosts that was first isolated in cod by RandWeaver et al. gill Na+.K+-ATPase . sarba. 2001).K+ATPase activity in the kidney of SW. Mancera and McCormick. 1998a. Similarly.and -subunit mRNA expression (Deane et al. plasma IGF-I increased during smoltification and SW adaptation. 1999).K+-ATPase activity and development of chloride cells (McCormick. mossambicus (Weng et al. GH treatment did not cause any significant changes in Na+. Accordingly. all actions consistent with improved hypoosmoregulation. In addition. In salmonids.and BW-adapted fish (Kelly et al. 2000). no differences in gill Na+. 1993) and in O.and saline-treated fish either adapted to SW or BW (Mancera et al.. 1999. The name of this hormone derives from the structural similarity of somatolactin and both growth hormone © 2006 by Taylor & Francis Group. GH reduced Na+. Seidelin and Madsen.. 2001. abundance of Na+K+-2Cl– cotransporter in gill chloride cells (Seidelin et al. The osmoregulatory role of GH and/or IGF-I also is also played through its interaction with other osmoregulatory hormones. In salmonids and non-salmonid species several studies have shown that IGFI treatment increases salinity tolerance. cooperation between GH and cortisol for increasing hypoosmotic capacity has been reported (Mancera and McCormick.K+-ATPase activity were observed between GH. In the same species. 2001)..Juan Miguel Mancera and Juan Fuentes 403 alterations in this activity in GH-treated fish compared to saline-treated animals. in the non-salmonid F. The underlying mechanisms for this interaction relates to the capacity of GH to increase the abundance of cortisol receptors (see McCormick. this volume). (1991) and belongs to the GH/PRL family (Rand-Weaver and Kawauchi. Sakamoto et al. mediated by increased gill Na+.. 2002).

1999).. However.2 Effects of some extra-pituitary endocrine factors related to ion balance in the control of release of pituitary hormones.. 2003) PRL secretion (Frutchman et al. SL has been associated to calcium regulation based on activation of somatolactin cells by exposure to low external calcium (Kaneko and Hirano...p. intra-arterial. 2003) PRL secretion (Eckert et al.. intra-peritoneal. the physiological actions of SL in fish remain elusive and despite several attempts the evidence to support an involvement of SL in osmoregulation is not compelling (see Kaneko.404 Fish Endocrinology Table 12. 2003) GH secretion (Eckert et al. Hormone/factor Ang II Ang II Ang II VNP CNP Low osmolarity IGF’s IGF’s Administration i. in vitro in vitro in vitro in vitro in vitro Species tilapia eel flounder tilapia tilapia tilapia tilapia tilapia Action PRL plasma levels (Leedom et al.. 1996). but not salmonids that lack PIPAS cells. (1991) demonstrated that SL cells correspond to periodic-acid-Schiff (PAS)-positive cells of the pars intermedia (PIPAS cells) in several teleosts. apart from the effect of salinity on the levels of expression.. Thus. in vitro i. On the other hand. 2003) GH secretion (Eckert et al. anguilla).p. Rand-Weaver et al. 1993).. some studies have shown variations in the expression of SL mRNA in the pituitary gland in response to changed environmental salinity. Even before the isolation of SL. For the most part. (somatotropin) and prolactin. several studies showed that PIPAS cells are activated under conditions of low environmental osmolality or calcium in goldfish (Carassius auratus) and eel (A. LLC . Such instances have been shown in catfish (Tang et al. 2003) AVT plasma levels (Balment et al.a. a hypercalcaemic action of SL has been indicated and plasma © 2006 by Taylor & Francis Group. i.. 2000) i. there is no convincing evidence to attribute specific roles to SL in the process of salinity adaptation. However. 2000) GH secretion (Frutchman et al.a. 2003) PRL secretion (Seale et al. 2001) and chum salmon (Taniyama et al..

expression of Na+.. ACTH is the key factor for releasing of cortisol from interrenal tissue. Madsen et al. 2001. and two lipotropins. two or four melanotropins (MSH) depending on the organism. 2002) and in this section. such as adrenocorticotropin (ACTH). 2001. 1996). Mancera et al. Cortisol enhances salinity tolerance. In fish. Evans.. Seidelin et al. 1999. 2003).. In most fish species a single gene is present. © 2006 by Taylor & Francis Group. The use of homologous protein available for experimental bioassays should help to provide evidence for physiological roles of a peptide that although present at high levels lacks a clear physiological function. but duplications of the POMC have been also described (Gonzalez-Nunez et al.K+-ATPase activity. Therefore. 2001. Laiz-Carrión et al. Mommsen et al. a stimulatory role of MSH has been also described.. The hypoosmoregulatory role of cortisol is well established in many teleosts. 1997). POMC Family (ACTH and MSH) Proopiomelanocortin (POMC) is a precursor for several pituitary hormones. stress and immune function (Wendelaar Bonga.. gill Na+. 1995. 2003).Juan Miguel Mancera and Juan Fuentes 405 SL levels are elevated in stressed and exercised fish with higher plasma Ca2+ levels (Kakizawa et al.. LLC . 1990. The physiological roles of SL in the control of osmoregulation and other physiological processes are far from being clarified. Sakamoto et al. The POMC gene encodes for -endorphin and for other nonopioid peptides. There are excellent reviews about osmoregulatory action of cortisol in fish (McCormick 1995. 1995. Pelis and McCormick.K+-ATPase -subunit. a hypothalamic-pituitaryinterrenal axis has been described with cortisol as the main product of this axis. we will give only a general overview of the osmoregulatory actions of this hormone. while in some species. 1995.. to a certain extent.. but at adenohypophyseal levels. 2002. MSH in some species indirectly affects teleost osmoregulation as a results of its action on cortisol release (Wendelaar Bonga. development and proliferation of gill chloride cells. 1999). ionic and osmotic regulation. similarly to other vertebrates. and abundance of Na+-K+-2Cl– cotransporter in gill chloride cells (McCormick. ACTH and. 1997. growth. Endocrine control of cortisol secretion involves different factors. Cortisol directly and/or indirectly plays an important role in several aspects of fish physiology including intermediary metabolism.

Laurent and Perry. Eckert et al. 1996). 2000). Na+ and Cl–). cortisol treatment increased plasma osmolality. plasma osmolality and ion levels in BW-adapted fish (Mancera et al. and 3.. gill chloride cell abundance. Yada and Ito. catfish and medaka maintained in hypoosmotic conditions (Flik and Perry. 1990.. thyroxine. 1995) and an improved drinking response after transfer to SW in rainbow trout and Atlantic salmon (Fuentes et al. 2000. These ‘contradictory’ evidences for cortisol action in ion regulation have been integrated by McCormick (2001) who proposed a ‘dual osmoregulatory’ role for cortisol: (i) a stimulatory action on ion secretion in hyperosmotic environments. In S..K+-ATPase activity. 2002). 2001). it has been reported an activation of PRL and ACTH cells in BW-adapted fish with respect to SW-adapted fish and it has been also suggested a cooperation of PRL and cortisol in the osmoregulatory process at low salinities (Mancera et al. 1997. tilapia. mossambicus adapted to FW. eel. a true thyroid gland typical of higher vertebrates is lacking and thyroid tissue appears scattered in the lower jaw (Power et al. and (ii) an increase of ion uptake in cooperation with PRL in hypoosmotic environments. 1985. 2001). Indeed. 1993b. Eckert. aurata. In fish... Dang et al. 1994) and this treatment increased gill Na+..T4. In O..406 Fish Endocrinology Several lines of evidence indicate a role of cortisol either in ion uptake in FW. 2002).K+-ATPase density in gill chloride cells of several species such as rainbow trout. Marshall. In S. 2001). and that the activity of the proton pump in the gills is fundamental for sodium uptake in hypoosmotic environments (Lin and Randall. Thyroid Stimulating Hormone (TSH) TSH induces the synthesis and release of thyroid hormones (THs. some evidences suggest a positive interaction of cortisol with PRL for maintenance of ion balance in FW fish (Parwez and Goswami. 1989. surface area and Na+. Certain other evidences point to an intestinal effect of cortisol in fluid movement in salmonids during smoltification (Veillette et al.or BW-adapted fish. Thyroid © 2006 by Taylor & Francis Group. Perry. 1999. It is necessary to remark that cortisol also increased H+-ATPase activity in gills of salmonids. treatment with cortisol increased ion regulatory capacity after transfer to low salinity environments (Mancera et al. 1995.T3) from thyroid tissue. aurata. ion levels (Ca2+.. LLC . In addition.5’-triiodo-L-thyronine.. cortisol treatment increased chloride cell density in gill and operculum (Dang et al. 2002).

K+-ATPase activity has been shown (Dange. an interaction between T3 and GH or cortisol has been reported in SW acclimation of S. treatment with T3 alone failed to increase gill Na+. (1982) using treatment with thiourea (a classic antagonist of the thyroid system) found that a functional thyroid is essential for maintenance of sodium and osmotic balance and for survival of F. Receptors for thyroid hormones have been found using ligand binding studies and molecular techniques (see Power et al.K+-ATPase activity in this same species at physiological concentrations of T3 and T4. 1993. However. In addition. salar (Madsen and Korsgaard. Such results are probably the consequence of the dose of treatment. This interaction has been related to the capacity of T3 to up regulate the number of gill cortisol receptors (Shrimpton and McCormick. However. 1995). LLC . Knoeppel et al. a role in the development of hypoosmoregulatory capacity of has been demonstrated in the summer flounder Paralichthys dentatus (Schreiber and Specker. 1990) but not in other species (see McCormick. trutta and O.K+-ATPase activity and chloride cell number in S. In non-salmonid teleosts. kidney and intestine and this could suggest a role of these hormones in osmoregulation. Shrimpton and McCormick.K+-ATPase activity and salinity tolerance in this species (Mancera and McCormick. repeated T4 injections increase gill Na+. 1998). In O. Gonadotropin (GTHs) Gonadotropins (GTH I and II) control reproductive cycle of teleost and induce the synthesis and release of sexual steroid in gonads. 1999). 2000)...K+-ATPase activity. although the mechanism for this interaction is not currently known. mossambicus T4 treatment has no effect on osmoregulatory system while a cooperation between T4 and cortisol to increase gill Na+. 1989) and Salmo gairdneri (Madsen. 1987. Thus. the information linking osmoregulation with the thyroid system is also unclear. chloride cell morphometric (area and immunoreactivity intensity). the thyroid system has been mainly studied by its association with the process of smoltification and it is believed that the thyroid system plays an important role in the migratory process. McCormick. A recent report (Subash Peter et al. 2000) describes increased hypoosmoregulatory capacity of FWadapted fish characterised by an increase of gill Na+. To our © 2006 by Taylor & Francis Group. 1995). 2001) in gills. In salmonids.Juan Miguel Mancera and Juan Fuentes 407 hormones regulate several physiological processes but the osmoregulatory role in teleost is equivocal (Grau. 1986). mykiss (Leloup and Lebel. heteroclitus in SW. 1998). and decrease of renal Na+.

In non-salmonid species. Madsen and Korsgaard. Williams and Wigham. is reduced by E2 treatment in O. However.K+-ATPase activity. However. LLC . 1979).408 Fish Endocrinology knowledge. (2001) have reported that E2 treatment impairs hypoosmoregulatory capacity in O. The pathway used by E2 to interact with the osmoregulatory system is not known. treatment with several . Le François et al. mykiss (Schlenk et al. These results substantiate a chronic stimulatory action of E2 on gill Na+.. mossambicus by decreasing gill Na+. hepatosomatic index. In F heteroclitus adapted to BW and SW. there is no published information on related direct effect of GTHs on osmoregulatory organs in teleosts. several lines of evidence indicate a negative relationship between sexual maturation and SW adaptability (McCormick and Naiman.K+-ATPase activity in this species (Guzman et al.. 1997). a direct effect of E2 on osmoregulatory organs can also be possible. in vitro studies have shown that treatment with this hormone also stimulates PRL release from the pituitary gland of O. in the euryhaline teleost S. eel and long-jawed mudsucker (Nagahama et al. it is possible that E2 exerts a negative effect on the osmoregulatory system by increasing plasma PRL levels. 1994).. 2004). mossambicus and O. 1995). Accordingly. 1986.K+ATPase activity. an implant of E2 (10 g/g body weight) for upto 15 days significantly increased plasma calcium.. hepatic flavin-containing monooxygenase activity..K+-ATPase activity in different salmonid species (Miwa and Inui. an enzyme related to adaptation to hypersaline environments. 1989). This negative effect on hypoosmoregulatory capacity has also been shown after administration of exogenous gonadal steroids (17 methyltestosterone and 17 -estradiolE2-) (see McCormick. Olivereau and Olivereau. In addition. 1986. In addition. E2 treatment reduced gill chloride cell density and gill Na+. Vijayan et al. 2000). E2 receptors have been © 2006 by Taylor & Francis Group. injections of E2 decreased gill Na+. 1975. 1985. plasma metabolic parameters and gill Na+. auratus maintained in SW. Accordingly. In salmonids. Thus. the negative effects of E2 on adaptation to hyperosmotic environment have been reported.K+-ATPase activity as well as metabolic capacity in liver and gill. several evidences suggest an indirect role of sexual steroids on osmoregulatory system in teleosts. Histological and ultrastructural studies have demonstrated that E2 stimulated PRL cells of tilapia. Recently. It is well established the negative effect of PRL on gill Na+.K+-ATPase activity and increased plasma osmolality and ion levels (Mancera et al. 2004). mykiss (Barry and Grau.

Am. N. Estradiol-17 and thyrotropin-releasing hormone stimulate . tilapia (Oreochromis niloticus) prolactins have different osmoregulatory funtions during adaptation to a hyperosmotic environment..J. V. synthesis. Fish Physiol. Mol. Gen. A selective survey of the endocrine system of the rainbow trout (Oncorhynchus mykiss) with emphasis on the hormonal regulation of ion balance.F. 62: 306-314. and Prunet. 1992. . Kaneko. Endocrinol.G.K+-ATPase activity in the sea bream. Isolation. M. Endocrinol. Comp. Warne. M. Endocrinol.J.A.A.. P 2002. J.. 95: 143-52.Juan Miguel Mancera and Juan Fuentes 409 demonstrated in both intestine and kidney of salmonids (Persson et al.Ile5.Thr9] angiotensin I. T.D. S. and biological activity of flounder [Asn1. A Comparative Account in Vertebrates. S. Comp. Bentley. F..P and Grau. LLC .M. Evidence that two . M. B. Springer-Verlag. Flik. Tierney.. R. Hasegawa. 1995. G. prolactin receptors in tilapia (Oreochromis mossambicus) after a change in salinity or hypophysectomy.. 1994. and Prunet. F. J. J. P 1994. Rentier-Delrue. 2000) although in the later estrogen receptors have not been detected in the gill. Martial. Regulation of gill ..M. Auperin. A. 11: 189-194. Biochem. 130: 92-8. and Hirano. The fact that E2 down regulates pituitary expression of PRL (Cavaco et al.A. Endocrines and Osmoregulation. and Hazon. Acknowledgments The present publication has been made possible by the funding for travelling from DGES HP1999-0098 and HP2001-0061 (Ministerio de Ciencia y Tecnología) from integrated actions to J.. 269: R775-R780. J. Bern. B. Differential expression of two prolactin and growth hormone genes during early development of tilapia (Oreochromis mossambicus) in freshwater and seawater: Implications for possible involvement in osmoregulation during early life stages.M. The authors would like to express their gratitude to Drs S. T.. T.J. Ayson. Aquaculture 100: 237-262. References Amer. Barry. Endocrinol.M. Canario and D. © 2006 by Taylor & Francis Group. Rentier-Delrue. S. Gen. Power for constant support and encouragement. 145: 213-220. H. prolactin release from the pituitary gland of a teleost fish in vitro. and Madsen.. Comp. Glomerular actions of arginine vasotocin in the in situ perfused trout kidney. J. and Brown.S. Physiol. McCormick. Gen. F.. 1993. R. 2000) and gilthead seabream (Socorro et al. Berlin.A. 2003. Warne J. 1986. Balment. Endocrinol. Martial. 2003) could explain the fact that E2 treatment increases gill Na+. J. Auperin. J. and Takei. Balment.. Arginine vasotocin and fish osmoregulation. G. P 1995. and E 82/00 and E65/02 from Conselho de Reitores das Universidades Portuguesas to J. Y. 12: 13-24.

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