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Walter BorG, M.D.
Selected Publications
"" ~
Hospital of
Saint Raphael A4!1j or teaching D:tJiliare of
A member of the Saint Raphael Healthcare System Yale University School ofMedicin e

1450 Ch apel Streer interna l Medicine

New Haven. Connecticut 0651 1 Training Program
Phone: (203) 789 -3358
Fax: (203) 789-3222
November 10, 1998

Re: Walter Borg, M.D.

To Whom It May Concern:

It is, indeed, a pleasure for me to be able to recommend Walter Borg. Currently, I am in a

teaching position at the Hospital of Saint Raphael where I run the Endocrinology Clinic and
prepare and deliver most of the Endocrinology lectures. I am also, currently, the President of the
Connecticut Endocrine Society and a long-term member of the Endocrine Society. I was in active
subspecialty Endocrine practice for the past 32 years. I am a Clinical Professor of Medicine at
Yale and actively teach at Yale University on a weekly basis. I spend one month directing the
Endocrine section consultative service at Y ale-New Haven Hospital. I have also served as
Chairman of the Hospital of Saint Raphael, Department of Medicine on two separate occasions in
the past.

I had the privilege of observing Walter Borg as an Endocrinology fellow at Yale-New Haven
Hospital with one month's intensive observation as his preceptor. I also watched him weekly
during Endocrinology rounds. He is, indeed, a superb physician. He is able to obtain complete
histories and perform complete physical examinations on all of his patients. His clinical judgement
is excellent and is based on his avid reading ofthe Endocrine literature. His case presentations are
excellent and he is extremely well organized in his record keeping . He has a superb general
medicine knowledge to go along with his expertise in Endocrinology. He is more than capable to
teach medical students, residents, and fellows early in their training. His lectures and
presentations are extremely well organized and contain all of the pertinent literature data needed
to substantiate his views. He is extremely professional. I believe that Dr. Borg has shown a great
grasp of medicine and he performs at the highest level of competence.


, ~
Norman J. Marieb, M.D.


GEltr.RI) N. 13uR~ow" MD

November 4, 1998

To Whom It May Concern:

I have been asked to write a letter of recommendation Dr. Walter Borg. I have related to him in my role
as a Professor of Medicine and a member of the section of endocrinology.

I have had close interaction with Dr. Borg seeing patients. He is an intelligent, committed physician with
good clinical judgment and an excellent ability to obtain history and perform physical examinations. He is
scrupulous in his case presentation and record keeping and has a broad knowledge in both general internal
-~<nedicine and endocrinology.

I have not seen him in a teaching situation but the quality of his lectures is such that they have been carefully
prepared and are inforrnative< His professional attimdes and behavior have been of high standard. Dr. Borg
is a clinically competent endocrinologist.


~~~<-d?~ ~3~~
Gerard N. Burrow, MD

Special Advisor to the President for Health Affairs

Yale University School of Medicine

333 CEDAR STf<..EET, NEW HAVEN CT 0652{)-80,s5 L220 SHM 2\iJ.7HS J7HS PHONE 20J.7HSA693 FAX


P.O. BOX 208064
Sonia Caprio, M.D.
TELEPHONE: 203-785-4648 Thomas O. Carpenter, M.D.
FAX; 203-737-1998 Scott Rivkees, IvLD.
Myron Genel, M.D., Associate Dearl
JoAnn Ahern, M.S_N., R,N., cnE.
Apri14,2000 Nancy A. Held, i'vLS., R.n, c.nE.
Mary Savoye, R.D., CD/[\;
Patricia Gatcomb, R.N., cnE.
Elizabeth Boland, A.P.R.N.
Miranda Vincent, A.P.RN.

To Whom It May Concern

Dear Sirs:

It's a great pleasure to write this letter of recommendation for Dr. Walter Borg, as I have known
Walter ever since he arrived at Yale 10 years ago. It has been particularly gratifYing to observe
Dr. Borg's growth and development from a young person new to the US with only one year of
post-medical schoo] experience to a well-trained endocrinologist with a special knack for
research and scholarly activities.

--. Walter came to us on a special Juvenile Diabetes Foundation International fellowship, having
impressed the JDFI leadership by his interest, enthusiasm and intelligence at a JDFI workshop
for European medical students. Coming sight unseen, we didn't know what to expect and frankly
we did not expect that much. It quickly became apparent, however, that Walter had the potential
to be one of our most productive trainees. He was incredibly well organized, researched the
literature extensively and was able to prioritize! esearch questions in a focused and goal-directed

Having been placed initially in a basic science laboratory, Walter recognized that this area of
research did not hold his interest. Instead, he shifted his area of study to the problem of
hypoglycemia counterregulation. This was a wise and productive move for Walter and for our
group. Dr. Borg has made important contributions to our clinical research projects and he has
had a leadership role in studies of the role of the VtvlH in regulating counterregulatory hormone
response to hypoglycemia. The results of Dr. Borg's studies have been published in top quality
journals. Walter's abilities to comprehensively search and organize published literature has
served him well in writing a variety of chapters and reviews, also.

Despite his many accomplishments, Dr. Borg had to suspend his research activities in order to
become Board certified in endocrinology. Most of the last 6 years have been spent in clinical
training in internal medicine and adult endocrinology. Although I have not had an opportunity to
observe Dr. Borg's performance in the clinical arena, I'm much impressed by the letters that he
has received from his clinical mentors They indicate that Walter has developed strong c1ihlcal
skills Those skills in combined with his abilities as an independent investigator make Walter an
outstanding candidate for any position that he

Sincerely, ; ; )

Ir~ 1/ Uv&'-­
William V. Tamborlane, M.D.
Professor of Pediatrics
Yale University
wolofMedicil1L' Rohert S. Sherwin, M.D.
Section of Endocrinology CN.H. Long Professor
333 Cedar Street, JIFlvfP of internal Medicine
P.o. Box 208020 Director, Di"betcs
New HlIVt:lI, Conl/celiell! 06520-H020 Elldocrinology Research
Telephollu: 2()3~785-·4! 83
Fax: 203-737-5558
E-mail: robcn.shcrwill@yale.cdu
May 8,2000

Walter Borg, M.D.

Yale University School of Medicine

333 Cedar SI. TMP 532

New Haven, CT 06510

Dear Walter,
I would like to thank you very much for your active participation in our project. I should
emphasize that if not for your relentless effort and unique skills we would not be able to
successfully accomplish our goals. We are indeed fortunate, to have you as a member of
our team. since there are very few young physician-scientists with such a well-balanced
training in both research and clinical fields as yourself Due to this unique training you
are the great asset to the American Medical Community.
I am committed to be supponive in your future endeavors in any way I can. I
strongly believe that you have a bright future in the field of diabetes and endocrinology,
and your service in those areas will be of great value for the U S. Healthcare.

llook forward to our continuous future cooperation.

, /

C,9rdiaP(l / / .
Robert S Sherwin, M.D.

C N.H Long Professor of Medicine

Director, Endocrinology Fellowship Program

Director, Diabetes Endocrinology Research Center


Medical Center

555 Willard Avenue

Newington CT 06111

In Reply Refer To:

November 30, 1995

To whom it may concern,

I am writing in strong support of Dr. Walter Borgls application for a position in your fellowship
program. I have known Dr. Borg for more than one year and have observed him many times in the
general medicine clinic. I have also developed a research proposal with Dr. Borg evaluating the
effects of smoking cessation on diabetes control in diabetic smokers. Thus I feel qualified to
comment on his clinical skills and character, as weIl as his dedication to research.

Dr. Borg has outstanding clinical skills. Compared to other residents he is definitely in the top
10%. He accurately diagnoses conditions and devises appropriate treatment and follow-up plans
for his patients. He also has good common sense in his approach to diagnosing medical

Dr. Borg's personal skills have also made him well liked by other residents, patients, and staff
~, members. He is extremely hard working, meticulous, and well organized. He devised an outpatient
history and physical exam checklist to better monitor his patients in' the general medicine clinic at
the Newington VA,

Dr. Borg is also a very dedicated scientist. He is very interested in diabetes and has published
many original articles on glycemic control. He has also shown great initiative in planning of a
project evaluating glycemic control after smoking cessation in subjects with NIDDM.

I enthusiastically support for Dr. Borg's application to your fellowship program. I hope you give
his application serious consideration.

soc' A---~
Dir~e~en~ Medical Clinic

VA Medical Center, Newington

Assistant Professor of Medicine

University of Connecticut School of Medicine


150 Sargent Drive Fax (203) 781-4383

New Haven, CT 06511 Physician Access Line (203) 781-4331

December 14, 1998

RE: Walter Borg, M.D.

To Whom It May Concern:

Dr. Walter Borg has asked me to write concerning my opinions about him as a physician that I
formed during a one month period when I was the endocrinology attending on the Yale inpatient
endocrine service. Dr. Borg was the fellow on service responsible for seeing all of the inpatient
endocrine consults and responsible for teaching both residents and medical students. Dr. Borg
overall is an excellent physician with solid clinical skills. His histories were thorough and his
physical exams complete and I found no deficiencies in these areas when cO!llpared to my own
history and physical examination of the same patients. His clinical judgment was excellent I
consider his clinical judgment to be excellent both in the area of endocrinology and in general
internal medicine. His case presentations were crisp and his notes in the medical record easily
read and logical in their order. He has sound knowledge of general internal medicine and far
greater knowledge in the field of endocrinology than I would have expected for a fellow at his
level. He excels in teaching medical students and residents and was well prepared with graphics
and pertinent photographs when he did didactic teaching, several sessions of which I observed in
person and learned a great deal myself. The area where I felt that he could be slightly faulted was
in the area of test ordering and I believe he tended to order a full panel of tests without regard to
cost. However, he was only led in this direction because of his intense intellectual curiosity. His
professional attitude and behavior toward patients is without fault and he behaved in an entirely
ethical manner. I would recommend him without reservation.

Gordon Reid, M.D.
Assistant Professor of Medicine
Yale School of Medicine

Jtmerican J'ederationfor Cfinica{!R...esearcfi

6900 Grove Road· Thorofare, NJ 08086-9447 • (609) 848-7072 • FAX (609) 384-6504
r~ell UnlVCr5l!y MedICal College
New York University
University 01 lowa
Iowa CIty VAMe
rneu University Medical College
June 17, 1994
ltVerslty 01 Texas~Southwestern

Walter P. Borg, MD
SLACK Incorporated
Yale University
Dept. of Medicine/Endocrinology
NA TIONAL COUNCIL 333 Cedar street LMP1
New Haven, CT 06511
"sny ot Alabama at B,rmingham
BirmIngham VAMC
Dear Dr. Borg:
"ver5Ity 01 Texas-Southwestern

~lve~slty 01
California-San Diego
Congratulations on having been chosen to receive a
San Diego V AMC Trainee Investigator Award for your abstract
u;w..,en,.ly of Calilornla·trvlne
presentation at the Clinical Research Meeting in
HOWARD J. EISEN Baltimore, Maryland, April 29 - May 2, 1994.
" Temple University
Ptliladelphia VAMC
MONICA M. FARLEY In addition to your prize the AFCR also wishes to
Emory UniversIty
Atlanta VAMe
present to you a one year complimentary associate
membership in the Federation. Your membership will
Stanford University
Palo Alto VAMC
begin on July 1, 1994. Please complete the enclosed
form for our records and return it to the AFCR National
Indiana University
Inolanapoli~ VAMC
headquarters in the envelope provided. Please do not
send a check.
Nortnwestern Universl'ty
VA LakeSIde
Your membership includes a subscription to the Journal
UnIverSity 01 Nebraska of Investigative Medicine and its supplements and the
r.. a!lor,Cj; Inslttutes ot HeatUl
AFCR newsletter. We hope that you will enjoy these and
MARK S. PALLER all benefits that membership in the AFCR provides.
JfW.i;'~5,ity 01 Minnesota
Er.;;iCor.O Medical Center
r,,€w We look forward to your participation in the activities
KEITH M. RAMSEY of the AFCR and if you have any questions please feel
free to contact the National Office.
ot Colorado

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E:19i.Jn'J MedlCal CU:1lef

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AFCR President

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May 5-8. 1995
San Diego. CA Official Publication.· Jo:.,·rr,al of Investigative Medicine
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~p:;df~ ~~~ President ASCI .

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Baltimore, MD

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Page 1 of 1

SUbj: Nomination of Walter Borg for election to AACE BOD

Date: 10/8/20073:28:24 P.M. Central Daylight Time

From: MHARRI;:J.I,.@OIolIV!ardhe.!aJth.Qrg

To: oqa!y@a?

Dear Betty et al:

I would like to nominate Dr. Walter Borg for election to the AACE BOD for 2008. Dr. Borg is an active and influential member of my
Socioeconomic and Coding Committee and have devoted countless hours to AACE affairs over the past 2 years. He will be a leader in our
organizations for years to come. Sincerely, R. Mack Harrell, MD, FACP ,FACE

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Monday, October 08,2007 America Online: Guest

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• (Professional Medical Corporation)



Telephone (337) 235-1200

September 10} 2007

Dr. Walter Borg

501 W. St. Mary} Suite 120
Lafayette, LA 70506


Dear Dr. Borg,

Mr. is a wonderful man who came to see me two years ago about a hernia
and some ED. In his follow up visits we found that a co-worker of his, who had similar
problems, was found to be hypogonadat with a very low testosterone. $0, we studied him
and found a similar situation with a 121 testosterone, and then we did a CT scan of his head
and found suspicious pituitary area. We did the MRI, and Dr. Wojak thought it did go along
with the pituitary adenoma, and then we sent him to Dr. Todd Baquet. At the time I did not
know that you were available, and the patient wants a second opinion, and I have heard
such good things about you that I am looking forward to his seeing you.
Endosed are his records and I sure appreciate your help.

Respectfully yours,

Frank R. Bacque, M.D.


Page 10f1

Subj: RE: Baton Rouge Meeting

Date: 11/2/20067:52:15 AM. Central Daylight Time
- -~rom:
0: DrWBo(

Dr. Borg,
Thcank you very much for your service to the society and for your kind words. We are also lucky to have members
like you that are willing to dedicate themselves to the improvement of healthcare in the state of Louisiana. If you are
interested, I would like you to consider serving on the Executive Committee for the Lafayette Parish Medical Society­
which also carries with it an automatic position on the LSMS delegation.

Thank you again for your service and interest.

Andy Blalock

From: [mailto:]

sent: Sunday, October 29, 2006 5:01 PM
Subject: Baton Rouge Meeting

Dear Dr. Blalock,

It was a real pleasure to meet you in Baton Rouge. Lafayette Parish Medical Society is very fortunate to have you

as a leader.

I am looking forward to serve our Society for many years to come.

--" !Viii keep in touch. Please let me know if I can be of any assistance in the meantime.


Walter Borg, M.D.

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Friday, August 10,2007 America Online: DrWBorg

Dear Dr. Borg,

I am writing to,"Thank You". You are the finest doctor I have ever known. As far
as I am concerned, you are the best in several states for sure. I have been to
many doctors in different states, in the last several years and had never found one
who was interested or cared about my health, like you did.l appreciate the time,
interest and care you gave to me and my health issues. With you as my doctor I
felt safe. I wonder if I will ever find another doctor like you.
I don't mind telling you, that I am so disappointed with my new doctor and will
be looking for another one.
I had signed all the necessary papers with your office and his office to have my
medical records sent to him. I waited over two months for an appointment with him
and had hoped I would find him to be the kind of doctor you are. On the day of my
appointment, when he walked into the room, he didn't even give me the time of
day. He dropped into his chair and said, what can I do for you?He did not have my
medical records from your office, his nurse had not given him the copy of my
medical history and medications that I had given to her. I gave him my copy and
went over what had been going on. He appeared not to know anything about my
condition and really didn't seem to be interested. He asked very few questions
and said he wanted some test run and walked out of the room. This visit was
approximately ten minutes. When he walked out I didn't see him again. He hacJn't
said, nice to meet you, see you later or even good-bye. I'm not anyone special,
_.l!~ a little common courtesy would have gone a long way. His nurse came in and
said for .me to go to the lab and call them in ten days for the results. On the tenth
and eleventh days I called and left messages and sent an email. On the third day I
received the results in the mail. I didn't understand them and had questions, so I
called again and sent another email, with two phone numbers where they could
reach me and asked to schedule an appointment with the doctor because I have
not been feeling well. Also that I had questions about the test and any orders he
had that I should follow and that J n~ new prescriptions. The nurse finally
called me late on the third day and said, the doctor doesn't know why you're not
feeling well and there is nothing on the results that concerns him. She'said, you
don't need to cOme back until the end of October. Oh, and your thyroid level is too
high, so reduce your Cytomel to one a day. I told her one of my other doctors had
alreacJy Iower~ my Synthroid by 25 mg. the week before. She just brushed that
aside and said, he wants you to take one Cytomel. So for a couple of weeks now I
have been on .075 mg and one Cytomel daily. I really don't feel well, I have
gained around thirty pounds in the last three months, my back has been hurting a
lot and I have broken a toe. My Pulmonary doctor has put me on fluid pills,

because I have swelled up so much. I'm always bloated and move very slowly. My
stomach is always hurting and J just feel like a wreck. I feel like and look like, I did
the first time I went to see you. It's really discouraging for me.
Anyway, t don't even know if you remember me or if my records are still with
you. I thought I'd take the chance that you would. I wanted to let you know how
things were going and to ask if you would possibly recommend a doctor that I
could see, that would live up to your standards. After having the best, it's hard to
settle for less. Of c;ourse I can only go to a doctor that will accept and
PPO PLUS. 00 you think you will ever accept Medicare again? If you did, I would
certainly be back in your office.
Dr. Borg, I would really appreciate any help you could give me in selecting another
Again I want to thank you, for three years of the best health care I have ever
had from a doctor. You are a good human being and a nice person. Oh and did I
mention the best of doctors.
Thank you Dr. Borg.
I also hope all is well with you. Have a blessed week!

PS My email address
PaseI of I

Subj: Letter of Recommendation

Date: 111112009 8:00:33A.M.CentralStandard
To: DrWBorg

To Whomit mayConcern,

I haveknownDr Borgas a colleague for severalyears.He and I havebothbeenon a nationalcommitee for

American Association (AACE).Hiscontribution
of Endocrinologists io the committeehasalwaysbeenpositiveand
On a personalnote,Dr. Borghasbeenexceeding
subtantial. generous withhistimein assisting mewithlearning
the processof publication, in my groupsuccessfully
resulting obtaining two publications
in the InsulinJournal.
I am a generalinternistanda diabetologist who provides criticalservicesin an underserved
ruralTennessee. I
providecriticalserviceto chronicallyill patients,one thirdwho do not haveinsurance.My practicepatternsreflect
what is medicallynecessary,in the bestinterests,and affordableto my patients.I am not a fellowhiptrained
endocrinologist.My patientsrountinely havediabetes, thecomplications of diabetes,depression,
and rheumatoidarthritis.My practicepatternsare shapedby the povertyof thoseI serve.I am currentlytrainingin
psychiatryto betterservemy chronicallyill patients.

Dr. Borgaskedto reviewchartslastyear underinvestigation by the statemedicalboardof Lousiana.I invested

severalhoursin the reviewof thoseiharts. Hischartsclearlvstatethatthe scoDeof his oracticeis timrteO-'O--
Hischarts,as wellhiswritingin theendocrine community, clearlystatethatDr. Borgdoesnot manage
or psychosocialaspectsof chronicillness..

Dr. Borq'schartsclearlvreflecta practicepatternwhlchis consistentwithconsultantendocrinoloqists who have

beentrainedin elitelnstituions.Histhinkingpatternis consistent withconsultativeendocrinologiists
who havebeen
trbinedin eliteinstututions.
Manyof theopinionsexpressed by Dr,Borgare heldfellowendocrinologists on our
AACEcommitee,lf Dr. Borgwas in my area,and if my patients couldaffordDr. Borg,I woulduseDr. Borgas a
consultan.. Or. Borg'scharts,in my opinion,do not fall belowthe levelof carerequiredfor actionby a state

Dr.,Borgis b!'illiant,
to makean academiccontributjonto endocrinologv
andan ongoing,substantial contribution
to theworkof AACE. I respectfully
requeston the behalfof endocrinology
thatDr.Borgbe continued to be licensed.


WlliamF. Conway,MD,FACP

A Good Credit Score is 700 or Above. Seeyours in just 2 easy steps!

Pase1 of I


Date: 1 1 1 2 1 2 0 0192 : 3 9 : 1 3
A . M . C e n t r aS
l t a n d a r dT i m e
From: DrWBorg

From:tbailey@amcri nstitute. com

S e n t .1 1 1 1 1 2 0 079: 5 8 : 5 1P . M .C e n t r aS
l t a n d a r dT i m e
Subj:The Conundrumof StateMedicalBoards

To Whom lt May Concern:

I have reviewedthe medicalrecordsin questionof Dr Borg.

evaluationsthat I have ever encounteredin my past 20 yearsas a practicingspecialist

- in endocrinology
aTd rnelab-oiism Ev€try ie6t"o rAeied vibi mdileul-ously Aocurnenied-and-justmtd.


TimothyS. Bailey.MD. CPI

AMCR InstituteInc.
700 West El Norte ParkwaySuite201
Escondido.CA 92026
\ .\ '\ /

New year...newnews.Be the firstto knowwhat is makingheadlines.

Monday, January12,2009 America Online

Walter P. Borg, M.D.

List of selected publications


Borg, W.P.: Case Against Bioidentical Hormones. JPANDS, 2008. 13(2): 47-51.

Borg, W.P.: Need for Objective Approach. The First Messenger. American
Association of Clinical Endocrinologists, 2006. 15(5): 1,7.

Borg, W.P.: AMA Calls for Reality Check on Bioidentical Hormone. The First
Messenger. American Association of Clinical Endocrinologists., 2007.

Borg, W.P.: Reproductive Endocrinopathies and Psychopathology

Common misconceptions. The First Messenger. American Association of Clinical
Endocrinologists., 2007. 16(6): 9-10.

R. Sherwin, W.P. Borg, Boulware, S.D:: Metabolic effects of IGF-1: Implication for
the therapy of Diabetes Mellitus. In: Blackman, M.R., Harman, S.M., Roth J.,
Shapiro J.R. (Ed.): GH and IGF-1: Basic and Clinical Advances.1993 Raven Press.

W.P. Borg, M.J. During, R.S. Sherwin, M.A. Borg, M.L. Brines, G.I. Shulman:
Ventromedial hypothalamic lesions in rats suppress counterregulatory responses
to hypoglycemia. J Clin Invest. 93:1677-1682; 1994.

WP Borg, and WV Tamborlane, Summary and Commentary: Mechanism of

Awareness of Hypoglycemia: Perception of Neurogenic (Predominantly
Cholinergic) Rather Than Neuroglycopenic Symptoms by Dwight A. Towler, et al.
From Research to Practice. Diabetes Spectrum 7:249-250; 1994.

R. Sherwin, W.P. Borg, S.D. Boulware: Metabolic effects of insulin like growth
factor 1 (IGF-1) in normal humans. Horm Res 41(Supp.2):97-102; 1994.
Walter Borg, M.D. -2-

WP Borg, MJ During, RS Sherwin, MA Borg, GI Shulman: Ventromedial

hypothalamus plays a critical role in triggering counterregulatory responses to
hypoglycemia. Abstract. Clinical Research 42:213A; 1994.

W.P. Borg, MA Borg, GI Shulman: Local Ventromedial Hypothalamus Glucopenia

Triggers Counterregulatory Hormones Release. Abstract. Diabetes
43(Suppl.:1):446; 1994.

MA Borg, RS Sherwin, MJ During, WP Borg, GI Shulman: Local Ventromedial

hypothalamus glucose perfusion blocks counterregulation during systemic
hypoglycemia. Abstract. Diabetes 44(Suppl.:1):2; 1994.

T. W. Jones, WP Borg, S. D. Boulware, G. McCarthy, R. S. Sherwin, and W. V.

Tamborlane: Enhanced adrenomedullary responses and increased susceptibility to
neuroglycopenia: mechanisms underlying adverse effects of sugar ingestion in
healthy children. J Pediatr 126:171-177; 1995.

Borg W.P. and Sherwin R.S.: Metabolic effects and potential clinical applications of
insulin-like growth factor 1. Diabetes Annuals. 1995 Elsevier Science B.V.

R. Sherwin, W.V. Tamborlane, and W.P Borg: Carbohydrate, Lipid, and Amino
Acid Metabolism in the Nonpregnant Patient. In Diabetes Mellitus in Pregnancy.
Principles and Practice. Second Edition. E.A. Reece, D.R. Coustan. Eds New
York, Churchill Livingstone 1995.

W.P. Borg, R.S. Sherwin, M. During, M.A. Borg, and G.I. Shulman: Local
Ventromedial Hypothalamus glucopenia triggers counterregulatory hormone
release. Diabetes 44:180-184, 1995.
Walter Borg, M.D. -3-

W.P. Borg, CHL Shackleton, SL Pahuja, and RB Hochberg. Long lived

testosterone esters in the rat. Proc. Natl. Acad. Sci. USA 92:1545-1549; 1995.

D.G. Maggs, Borg W.P., During M.J., Sherwin R.S. Microdialysis techniques in
the study of brain and skeletal muscle. JDFI/EASD Meeting 1995.

D.G. Maggs, Borg W.P., Sherwin R.S. Microdialysis techniques in the study of
brain and skeletal muscle. Diabetologia 40:S75-82; 1997.

MA Borg, RS Sherwin, MJ During, WP Borg, GI Shulman: Local Ventromedial

hypothalamus glucose perfusion blocks counterregulation during systemic
hypoglycemia. Diabetes 44(Suppl.:1):2; 1995.

MA Borg, RS Sherwin, WP Borg, WV Tamborlane, GI Shulman: Lactate perfused

locally into the ventromedial hypothalamus suppresses counterregulation during
systemic hypoglycemia. Diabetes 45(Suppl.:1):195; 1996.

MA Borg, RS Sherwin, WP Borg, WV Tamborlane, and GI Shulman: Local

Ventromedial hypothalamus glucose perfusion blocks counterregulation during
systemic hypoglycemia in awake rats. J Clin Invest. 99:361-365; 1997.

MA Borg, GI Shulman, WP Borg, WV Tamborlane, RS Sherwin: Recurrent

iatrogenic hypoglycemia and IDDM suppress counterregulation caused by
localized 2-DG perfusion of the ventromedial hypothalamus. Diabetes
46(Suppl.:1):159; 1997.

TW Jones, WP Borg, MA Borg, SD Boulware, G McCarthy, D Silver, WV

Tamborlane, and RS Sherwin: Resistance to neuroglycopenia: An adaptative
response during intensive insulin treatment of diabetes. J Clin Endocrin & Metab.
82:1713-1718; 1997.
Walter Borg, M.D. -4-

WP Borg, MA Borg, and WV Tamborlane. The brain and hypoglycemic

counterregulation: Insights from hypoglycemic clamp studies. Diabetes Spectrum
10:33-38; 1997.

MA Borg, WP Borg, WV Tamborlane, ML Brines, GI Shulman, RS Sherwin:

Recurrent hypoglycemia and diabetes mellitus impair counterregulation induced by
localized 2-deoxy-glucose perfusion of the ventromedial hypothalamus in rats.
Diabetes 48:584-587, 1999.

WP Borg, MA Borg, WV Tamborlane, RS Sherwin: Local VMH Alpha-Adrenergic

Blockade Impairs the Epinephrine Response to VMH Glucopenia. Diabetes
48(Suppl.:1):239; 1999.

WP Borg, RS Sherwin: Classification of Diabetes Mellitus Including MODY. In

Advances in Internal Medicine, Volume 45. Mosby 2000.


p C P ES f


Anthony S. Fauci, MD Joseph B. Martin, MD, PhD,

Chief, Laboratory of Immunoregulation; Director, PRep (c), MA (Bon)
National Institute of Allergy and Infectious Dean of the Faculty of Medicine;

Diseases, National Institutes of Health, Bethesda Caroline Shields Walker Professor of

Neurobiology and Clinical Neuroscience,

Eugene Braunwald, AB, MD, Harvard Medical School, Boston

MA (Hon), MD (Hon), ScD (Hon)

Distinguished Hersey Professor of Medicine. Dennis L. Kasper, MD, MA ( Hon)

Faculty Dean for Academic Programs at Brigham William Ellery Channing Professor of

and Women's Hospital and Massachusetts General Medicine, Harvard Medical School;

Hospital, Harvard Medical School; Vice-President Director, Channing Laboratory; Co-Director,

for Academic Programs, Partners HealthCare Division of Infectious Diseases; Executive

System. Boston Vice-Chainnan, Department of Medicine,

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Mallinckrodt Professor of Medicine, Harvard Stephen L. Hauser, MD
Medical School ; Physician and Director, Chainnan and Betty Anker Fife Professor,
Massachusetts General Hospital Cancer Center, Department of Neurology, University of
Boston California San Francisco, San Francisco

Jean D. Wilson, MD Dan L. Longo, AB, MD, FACP

Charles Cameron Sprague Distinguished Chair Scientific Director, National Institute on

and Clinical Professor of Internal Medicine, The Aging, National Institutes of Health,

University of Texas Southwestern Medical Center, Gerontology Research Center.

Dallas Bethesda and Baltimore


New York St. Louis San Francisco Auckland Bogota Caracas Lisbon London Madrid

Mexico City Milan Montreal New Delhi San Juan Singapore Sydney Tokyo Toronto

2080 the psychiatric aspects of diabetes are not discussed here, most prob­
Endocrinology and Metabolism
lems can be anticipated and handled if common sense is coupled with
sympathy and finnness. It is also appropriate to offer cautious hope
The -insulin r~sistance is severe, and ketoacidosis may occur despite that the disease will be handled better in the future than is possible now.
high endogenous insl}lin levels. The Alstrom syndrome is a rare autoso­
mal recessive disease"cfiaracterized by childhood blindness due to Please refer to Chap. 60, Insulin, oral hypoglycemic agents, and
retinal degeneration, nerve deafness, vasopressin-resistant diabetes in­ the pharmacology of the endocrine pancreas, in Goodman &
sipidus, and, in males, hypogonadism with high plasma gonadotropin Gilman's The Pharmacological Basis of Therapeutics, 9th ed. , New
levels. The patients thus appear to have end organ resistance to multiple York, McGraw-Hill.
hormones. Other features include baldness, hyperuricemia, hypertri­
glyceridemia, and aminoaciduria. Superficially, the patients may re­ BfBLIOGRAPHY
semble subjects with the Laurence-Moon-Biedl syndrome but can be
differentiated on initial examination by the absence of polydactyly G ENERAL REVIEW
and · mental deficiency. Insulin resistance in the Alstrom syndrome
UNGER RH, FOSTER DW: Diabetes mellitus, in Williams ' Textbook ofEndocri­
is mild. Ataxia-telangiectasia is characterized by cerebellar ataxia, nology, 9th ed, JD Wilson, DW Foster (eds). Philadelphia, Saunders, 1997
telangieclasia;apredispositicmto breast cancer, and a variety.ofabnor­ (in press)
malities in the immune system in addition to insulin resistance. The
Rabson-Mendenhall syndrome consists of dental dysplasia, dystrophic GENETICS AND PATHOGENESIS
nails, premature. puberty,. and acantbosis. nigticans. The insulin resis­ ATKINSON MA, MACLAREN NK: The pathogenesis of insulin-dependent diabe­
tance is probably due to an insulin receptor abnormality. Lepre­ tes mellitus. N Engl J Med 331: 1428, 1994
chaunism is characterized by an elfin appearance of the face, hirsutism, BINGLY PJ et al: Can we really predict IDDM? Diabetes 42:213, 1993
absence of subcutaneous fat, thickened skin, and insulin resistance. COUSTAN DR et al: Gestational diabetes: Predictnrs of subsequent disordered
glucose metabolism. Am J Obstet Gynecol 168: 1139, 1993
Defects are found in both the 0. and the f3 subunits of the insulin GHOSH S, SCHORK NJ: Genetic analysis of NIDDM. The study of quantitative
receptor, causing expression in plasma membranes to be markedly traits. Diabetes 45: I, 1996
diminished. Not listed in Table 334-12 is insulin resistance due to HAGOPIAN WA et al : Glutamate decarboxylase-, insulin-, and islet cell-antibod­
hormone excess (acromegaly, Cushing's syndrome), myotonic dystro­ ies and HLA typing to detect diabetes in a general population-based study
phy, and thalassemia major. The insulin resistance in these conditions of Swedish children. J Clin Invest 95:1505, 1995
usually is not clinically significant. HARRISON LC et al: MHC molecules and f3-cell destructive immune and nonim­
mune mechanisms. Diabetes 38:815, 1989
·INSULIN ALLERGY Insulin allergy is due to 19E antibodies HYOTY H et ill: A prospective smdy of the role of coxsackie B and other
to insulin. Manifestations include immediate reactions with local sting­ enterovirus infections in the pathogenesis ofIDDM. Diabetes 44:652, 1995
ing or itching, delayed local reactions with brawny swelling lasting PALMER JP, McCuLLOCH DK: Prediction and prevention of IDDM-1991.
up to 30 h, and generalized urticaria or frank anaphylaxis. Systemic Diabetes 40:943, 1991
reactions are usually seen in patients who have stopped insulin therapy I'ERMUTT MA et al: Glucokinase and NIDDM: A candidate gene that paid off.
Diabetes 41 :1367, 1992
for one reason or another and have then resumed treatment. The allergic
POLONSKY KS et al: Non-insulin-dependent diabetes mellitus-a genetically
reaction may occur as early as the second injection on resumption of programmed failure of the beta cell to compensate for insulin resistance.
therapy. Mild reactions can be treated with antihistamines. If the N Engl J Med 334:777, 1996
problem is severe, desensitization procedures are required. A I-day PuGLIESE A et al: HLA-DQBl*0602 is associated with dorrtinant protection
insulin desensitization procedure is shown in Table 334-13. Once the from diabetes even among islet cell antibody-positive first-degree relatives
patient is desensitized, insulin therapy should not be interrupted . of patients with lODM. Diabetes 44:608, 1995
THAI AC, EISENBARTH GS: Natural history of lODM. Diabetes Rev 1:1 , 1993
UMPIERREZ GE et al: Diabetic ketoacidosis in obese African-Americans. Diabe­
cult to accept that one has a chronic disease that requires a change in tes 44:790, 1995
life-style. This is particularly true in the case of diabetes, since patients
are usually aware that they are vulnerable to late complications and DIABETIC COMPLlCATIONS
that their life expectancy is shortened. It is not surprising that the AIELLO LP et ai: Vascular endothelial growth factor in ocular fluid of patients
emotional response to diabetes often hampers treatment. The primary with diabetic retinopathy and other retinal disorders. N Engl J Med
reaction can range from denial with an accompanying refusal to cooper­ 331:1480, 1994
ate to excessive preoccupation with the illness. The physician should BEISSWENGER PJ et al: Formation of immunochemical advanced glycosylation
end products precedes and correlates with early manifestations of renal
make every effort to bring the patient to a middle ground of acknowl­
and retinal disease in diabetes. Diabetes 44:824, 1994
edging the disease and responding prudently without becoming ob­ BORG WP et ai: Local ventromedial hypothalamus glucopenia triggers counter­
sessed. The goal is to live with diabetes, not for it. Patients with regulatory hormone release. Diabetes 44: 180, 1995
diabetes are no different from other patients, in that they may use BOYLE PJ et ai: Brain glucose uptake and unawareness of hypoglycerrtia in
their disease manipulatively with both family and physician. These patients with insulin-dependent diabetes mellitus. N Engl J Med
problems are particularly acute with children and adolescents. While 333:1726, 1995
BROWNLEE M: Glycation products and the pathogenesis of diabetic complica­
tions. Diabetes Cart: 15: 1835, J 992
'fable 334-13 FOSTER DW, McGARRY JD: The metabolic derangements and treatment of
diabetic ketoacidesis.N Engl J Med 309:159; 1983
Insulin J)cscnsitization ':' KROLEWSKl AS et al: Glycosylated hemoglobin and the risk of microalbumin­
Time, h Dose, U Route Time, h Dose, U Route uria in patients with insulin·dependent diabetes mellims. N Engl J Med
332:1251, 1995
0 0.001 Intradennal 3.5 0.2 Subcut. LEURS PB et al: Tissue factor pathway inhibiror activilY in patients with lODM.
0.5 0.002 Intradermal 4 0.5 Subcut. Diabetes 44:80, 1995
I 0.004 Subcut. 4.5 I Subcut. PARTANEN J et"al: Natural history of peripheral neuropathy in patients with
1.5 0.01 Subcut. 5 2 Subcut. non-insulin-dependent diabetes mellitus. N Engl J Med 333:89, 1995
2 0.02 Subcut. 5.5 4 Subcut. SEQUIST ER et al: Familial clustering of diabetic renal disease: Evidence for
2.5 0.04 Subcut. 6 8 Subcut. genetic susceptibility and diabetic nephropathy. N Engl J Med
3 0.1 Subcut. 320:1161 , 1989
SIPERSTEIN MD: Diabetic ketoacidosis and hyperosmolar coma. Endocrinol
* Following desensitization, use 2 to 10 U of regular insulin every 4 to 6 h for 24 to 36 h Metab Clin North Am 21 :915,1992 . .
after the 6-h injection before switching to intermediate-acting insulin. TOWLER' DA et ai: Mechanism of awareness of hypoglycemia. Perception
SOURCE: Schedule ofJA Galloway. For detailed information see JA Galloway, R Bressler, of neurogenic (predominantly cholinergic) rather than neuroglycopenic
Med Clin North Am 62:663 . 1978. symptoms. Diabetes 42: 1791, 1993



9th Edition

Jean D. Wilson, M.D.

1 Charles Cameron Sprague Distinguished Chair
j and Clinical Professor of Internal Medicine
The University of Texas Southwestern Medical Center
Dallas, Texas
j Daniel W. Foster, M.D.
Donald W. Seldin Distinguished Chair
and Chairman of Internal Medicine
The University of Texas Southwestern Medical Center
Dallas, Texas
j Henry M. Kronenberg, M.D.
Professor of Medicine
Harvard Medical School
Chief, Endocrine Unit
Massachusetts General Hospital
Boston, Massachusetts

1 P. Reed Larsen, M.D.

Professor of Medicine

I Harvard Medical School

Chief, Thyroid Division
and Senior Physician, Brigham and Women's Hospital
Boston, Massachusetts


1 A Harcourt Health Sciences Company
Philadelphia London New York St. Louis Sydney Toronto


10'12. Roy H. Chou MeV. Field .lB. Tilne-acrion characteristics of regular and 1052. Ro~nsto,k J. Vega GL, Ra$kin P. Effect uf intensive diahetf'.' treatment
NI)H insulin in inSl1irtHfe;:{lt:ri diabetics. j Clin Endocrinol Metab 1980~ on low-density lipoprotein apoHpoprotdn P kinetrcs in type I d iah{< tics.
50:475-47'1. Oi.het~s 1988; 37:393-397.
1O~:1. 1\oltc MS, Poon V, (;rod,k; C \1. d al. Reduced solubility of short-acting J()5$. Ruhinstein ,,,,- Pierce CE Jr II, Bloomgankn Z. Rapid healing of diahelic
solubk insuliuK wht~n mixed \<ilth Iongf'r~ac(ing insulin. Dlabeles 1983~ foot ulcers with continuous snlx:utaneolls in~ulin lnrusinn. Am J Med
:1:0>:1 177-1 I Ill. 1983: 75:161-165.
JO:!4. Skykr.JS. Insulin pharmacology. M('d Clin :-iorth Am 1988; 72:1337-1354. 10;,4. Lauriuen T. Frost-Larsen K. Larsen H-W. et at The effcct of nt>ar·norrllal
iO~S Zinman B. The physjologic replacement of in~Hlin: an elusIve goal. N blood glucose levels upon rctinopathy: two-year follow-up. DiabelUlogta
f.nglJ Med 1989; 321:363-:>70. 1983.2:;:174 (abstract).
iO~6 Sonnenberg GEt Chanteiau E. Sundermann S. et at Human and porcine 1055. Lauritzen T. Frost-Larsen K. Larsen HW, et a1. Two-year experienCt· with
regular insulins art: c<ruaUy effective in !'ulx:utaneous replacement ther­ continuous subcutaneous insuHn infusion in relation to retinopathy and
apy: resultS of a double-blind crQ.'i-'iover .study in type 1 diabetic patients neuropathy. Diabetes 1985; 34(SuppI3}:74-79.
with continuous subcutaneous insulin luftLSioll. Diabetes 1982; 31:600­ 1056. Holman RR, Mayon-White V, Orde-Peackar C. et al. Prevention of de(~rio­
tiM. ration of renal and sensory-nerve function by mort: inleosj<\."e manage­
1O~7. Home PD, Malisi-Bened~"i M. Shepherd f;.AA. et aL A comparison of the ment of insuhn-dependent diabetic palit:llts: a two-year randomrsed pro­
activity and disposal of S(':rni·s~lHhetic hum;tn insulIn and pordne insulin spective studv. Lancet 1983; 1:204-208.
in normal man by 'he glutose clamp technique. Diabetologia 1982; 1057. Engerman RL, Kern TS_ Progression of incipient diabetic rerinopathy
22:11-45.' _. during good glycemic control. Diabetes 1987: 36:808-~12.
JO;!i\. Heding LG, Marshall Persson B. c, a!. Immunogenici,,, of monocom· 1058. RosellStockJ, Friberg T, Raskin P. Effect uf glycemic control Ull microvas­
pone-nt human and porcine insulin in newly djagnosed type I (insuhn­ cular complications in patients with type I diabetes mellillls. Am J Med
dependent) diabetic chil<ln'n. Diabetologia 1984; 27 (Suppl) :96-98. 1986,81:1012-10IR
1029. Skyler JS, Pfdffer EF, Raptis S. et aL Biosvnthetic human insulin: progress 1059. Teutsch SM, Herman WH, Dwyer DM, et al. Mortality among diabetic
amI prospects. Diabetes Care 1981; 4:140-143. patients using continuous subcutaneous insuhn-·infusion pumps. ~! Engl J
1030. Zuppinger K. Acbi C, Fankhauser S, et aL Comparison of human and Med 1984: 310:361-368.
porcine insulin therapies in children with newly diagnosed diabetes melli­ 1060. Mecklenburg RS, BensonJW JI; Blumenstein BA, et al. Long-term meta­
ttls. Diabetologia 1987; 30:912-915. bolic control with insulin pump ulerapy: Report of experience "ith 1~7
1031. Teuscher A, Berger WG. Hypoglycemia unawareness in diabetics tranrr patients. N Engl.J Med 1985; 313:464-468.
ferred from beef/pork insulin to human insulin. Laocet 1987; 2:382-385, 1061. Jorgens V, Gruber M, Bott U, et aL EfIective and safe translation of
1032. Schade DS. Brittle diabetes: strategies, diagnosis, and treaunent. Diabetes intensified insulin therapy to general internal medicine deparunenlS.
Metab Rev 1988; 4:371-390. Diahetologia 1993; 36:99-105.
1033. Schade DS, Santiago]V. Skyler JS. et aJ. Unstable diabetes and insulin 1062. Perriello G, De Feo P, Torlone E, et aL The dawn phenomenon ,n type I
resistance. In: Schade DS, Santiago ]V, Skyler JS, et aI., eds. Intensive (insulin-<lependent) diabetes mellitus: magnitude, frequency, variability,
Insulin Therapy. Princeton: Excerpta Medica, 1983: 264-283. and dependency on glucose counterregulation and insulin sensitivity.
10'14. Fukuda M, Tanaka A, Tahara y, et at Correlation between minimal Diabetologia 1991; 34:21-28.
secretory capacity of pancreatic beta<ells and stability of diabetic control. 1063. Boden G, Soriano M, Hoeldlke RD, et aI. Counterregulatory hormone
Diabetes 1988; 37:81-88. release and glucose recovery after hypoglycemia in noninsulin-<lependent
1035. Nathan DM, Singer DE. Hurxthal K. et al. The clinical information value diabetic patients. Diabetes 1983; 32:1055-1059.
of the glycosylated hemoglobin assay. N EnglJ Med 1984; 310:341-346. 1064. White NH, Skor DA, Cryer PE, et at Identification of type I diabetic
1036. Goldstein DE, Is glycosytated hemoglobin clinically useful? N EnglJ Med patients at increased risk for hypoglycemia during intensive therapy_ N
1984; 310:384-385. Engl J Med 1983; 308:485-491.
1037. Ramsay RC, Goetz Fe. Sutherland DER. et a1. Progression of diabetic 1065. Cryer PE. Glucose counterregulation in man. Diabetes 1981; 30:261-264.
retinopathy after pancreas transplantation for inslllin-<lependent diabetes 1066. ('..erich J, Davis J, Lorenzi M, et al. Hormonal mechanisms of recovery
mellitus. N EnglJ Med 1988: 318:208-214. from insulin-induced hypoglycemia in man. AmJ Physiol 1979;)236:E380­
1038. Schiffrin A, Belmonte MM. Comparison between continuous subeutane~ E38".
ous insulin infusion and multiple injections of insulin: a one-year prospec­ 1067. Bolli G, De Feo P, Compagnucd P, et a!. Important role of adn~nergic·
tive study. Diabetes 1982: 31:255-264. mechanisms in acute glucose counterregulation following insulin-induced
1039. Skyler JS, Skyler D1., Seigler DE, et at Algorithms for adjusunent of hypoglycemia in type I diabetes: evidence for an effect mediated by beta­
insulin dosage by patients ",ho monitor blood glucose. Diabetes Care adrenoreceptors. Diabetes 1982; 31:641-647_ '.
1981; 4:311-3I8. 1068. De Feo P, Bolli G, Perriello G, et aL The adrenergic contribution to
1040. Rizza RA, Gerich JE, Haymond MW, et al. Control of blood sugar in glucose counterregnlation in type I diabetes mellitus: dependency on A­
insulin-<lependent diabetes: comparison of an artificial endocrine pan­ cell function and mediation through beta2-adrenergic receptors. Diabetes
creas, continuous subcutaneous insulin infusion. and intensified conven­ 1983; 32:887-893.
tional insulin therapy. N Engl J Med 1980: 303:1313-131R 1069. Boden G, Reichard GA Jr, Hoeldlke RD, el aL Severe insulin-induced
104 L Raskin P. Open and dosed insulin infusion s)istems: newer methods of hypoglycemia associated wiuI deficiencies in the release of counter-regula.
insulin delivery. In: Ellenberg M, Rifkin H. eds. Diabetes Mellitus: Theory tory hormones. N EnglJ Med 1981; 305:1200-1205. .
and Practice. 3rd ed. New Hyde Park. NY: Medical Examination Publish­ 1070. Cryer PE. Decreased sympalhochromaffin activity in IDDM. Diabetes
ing. 1983: 94 1-957. 1989; 38:405-409.
1042. Slama G, Garrel D. Tchobroutsky G. MUltiple daily insulin injections 1071. Dagogo-Jack S, Rattera;;arn C, Cryer PE, et aL Reversal of h}poglycemia
through subcutaneously implanted needle. Lanret 1980; 1:1078, unawareness, but not defective glucose counterregulation, in IDDM. Dia­
1043. Reeves ML, Seigler j)E, Ryan EA, et al. Glycemic control in insulin­ betes 1994; 43:1426-1434.
dependent diabetes mellitus: comparison of outpatient intensified con­ 1072. Boyle PJ, Kempers SF, O'Conn"r 'AM, et al. Brain glucose uptake and
ventional therapy with continuous subcutaneous insulin infusion. Am J unawareness of hypoglycemia in 'patients with insulin-<lependent diabetes
Med 1982; 72:673-680. mellitus. N En I Med 1995; 333:1726-173L
1041. Pickup JC, Keen H. Parsons JA. et aL Continuous subcutaneous insulin Borg ViP, Sherwin RS, During MJ, et at Local ventromedial hypothalamus
infusion: improved blood-glucose and intermediary-tnetabolite control '0 glucopenia triggers counterregulatory hormone release. Diabetes 1995;
diabetics. Lancet 1979; I:1255--1~58.
1045_ Tamborlane wv. Sherwin RS, Gene! M. et al. Reduction to normal of nawareness 0 ypog ycem.a.
plasma glucose in juvenile diabetes by subeutaneous administration of
insulin wilh a portable infusion pump. N EnglJ Med 1979; 300;573-578. l075. Somog); M. Exacerbation of diabetes by excess insulin action. Am J Men
1046. Felig P, Bergman M. Imens,ve ambulatory treallnen t of insulin-<lependent 1959; 26:169-191.
diabetes. Ann Intern Med 1982; 97:225--230. 1076. Wilson DE. Excessive insulin therapy: biochemical effects and clinical
1047. Kitabelli AE, Fisher IN, Matteri R. et al. The use of continuous insulin repercussions. Current concepts of counterregulation in type I diabetes.
delivery systems in treatment of diabetes mellitus. Adv Intern Med 1983; Ann Intern Med 1983; 98:219-227.
28:449-490. 1077. Tordjnan Klvl, Havlin CE, Levandoski LA, et a!. Failure of nocturnal
1048. Mecklenburg RS, Benson JW Jr, Becker NM. et at Dinical use of the hypoglycemia to cause fasting hyperglycemia in patients with insulin­
insulin infusiun pump In 100 patients ,,~th type I diabetes. N Engl J Med dependent diabetes mellitus. N EnglJ Med 1987; 317:1552-1559.
1982: 307:513-518. 1078. Winter RJ Profiles of metabolic contrnl in db.hetlc chtldren- fre-quency
1049. Schade DS. Santiago ]V, Skyler JS, et aL Hazards of intensive insulin of asymptomatic nocturnal hypoglycemia. Metabolism 1981; 30:66&-672.
therapy. In: SchacJe DS. Santiago lV, Skyler JS. et al .. eds. Intensive Insulin 1079. Kahn CR. Rosenthal AS. Immunologic reactions to insulin: insulin ai!('rgy,
Therapy. Princeton: Excerpta Medica, 1983: 287-301. insulin resiSlanCt\ and the autoinlmune insulin syndrome. Diabetes Care
I ()50. Broussolle C, Jeandinler N, Hanaire-Broutin H, et at French multicentre 1979; 2:283-295.
experience of implantable insulin pumps. Lancet 1994; 343:514-515_ 1080. Kahn CR, Mann 0, Rosenthal AS, et aL The immune response to illSulin
1051. Sehade DS, Santiago]V, Skyler JS, et aL E!fects of intensive treaunent on in mall: interaction of HLA alloantigens and the development of the
substrate and hormonal abnormalities. In: Schade DS, SantiagoJV, Skyler immune response. Diabetes 1982; 31:716-723.
JS, et aI., cds. Intensive Insulin Therapy. Princeton: Excerpta Medica, 1081. Galloway .lA, Bressler R, Insulin treaunent in diabetes. Med Clio North
1983: 71-87. Am 1978; 62:663-680.

sO ~ffi\JY ~©

ocrlno ogy
Mark A. Sperling, M.D.
1 Vrra I. Heinz Professor and Chainnan

Department of Pediatrics

University of Pittsburgh School of Medicine


Children's Hospital of Pittsburgh

Pittsburgh, Pennsylvania

w.s. Saunders Company

A Divisioll of Hmcmut Brace &: CO"'JlO1I1
Philadelphia London Thronto Montreal Sydney 1bkyo
Hypoglycemia in the Child 271

hyperinsulinemia is present and demands further evaluation metabolism in children with maple syrup urine disease. J Clin Invest
62:398, 1978.
and treatment. In children, an adenoma is more likely than
10. Pagliara AS. Karl IE, DeVivo DC, et al: Hypoalaninemia: a
f)-cell hyperplasia. Treatment shou~d .~e best directed toward concomitant of ketotic hypog.lycemia. J Clin Invest 51:1440. 1972.
the likelihood of surgery. If the inItial sample reveals an [I. Pagliara AS, Karl IE. Haymond M, Kipnis DM: Hypoglycemia in
insulin concentration of more than 100 J.LU/mL, a tumor is infancy and childhood. J Pediatr 82:365 (pt n, 558 (pt 2).
possible, but factitious h.yperinsuli~emia must .be. e~clu~ed.
[2. Stanley CA. Baker L: Hypcrinsulinemia in infants and children:
At this stage, a C-pepude level IS helpful; If It IS high, diagnosis and therapy. Ad,' Pediatr 23:315, 1976.
indicating endogenous hyperinsulinemia, a tumor or ade­ 13. Arnie! SA. Simonson DC, Sherwin RS, et al: Exaggerated epinephrine
noma secreting insulin is most likely. If it is low, factitious responses to hypoglycemia in normal and insulin·dependent diabetic
hyperinsulinemia with exogenous administration of insulin children. J Pediatr 110:832, 1987.
must be suspected, and further eva1l!ation is indicated. If the 14. Jones TW. Boulware SD, Kraemer DT. et a[: Independent effects of
youth and poor diabetes control on responses to hypoglycemia in
initial cortisol concentration is less ~han 10 J.LgldL and/or the chi ia
"fowth hormone concentration'iS less than 5 nglmL at the 5. Jones TW, Borg WP. Boulware SD, et al: Enhanced adrenomedullary
'"time of documented hypoglycemia, adrenal insufficiency response and increased susceptibility to neuroglycopenia: mechanisms
and/or pituitary insufficiency must be suspected, and a full underlying the adverse effects of sugar ingestion in healthy children. J
Pedialr 126: 171 f 995.
endocrine evaluation, including appropriate imaging studies,
DeFeo P, Gallai V, Mazzotta G, et al: Modest decrements in plasma
should be performed. glucose concentration cause early impairment in cognitive function
'If the history is suggestive of hypoglycemia but the and later activation of glucose countelTegu[ation in the absence of
physician is not present at the time of acute symptoms, a hypoglycemic symptoms in normal man. J Clin Invest 82:436,
careful history and physical examination may provide im­
17. Schwartz NS, Clutter WE, Shah SD. Cryer PE: Glycemic thresholds

portant diagnostic clues. A history of salt craving, a change for activation of glucose counterregulatory systems are higher than

in growth velocity, access to insulin, and the relationship of the threshold for symptoms. J elin Invest 79:777, 1987.

symptoms to time elapsed since the last meal are clearly 18. KelT D, Diamond MP. Tamborlane WV, et al: Influence of

relevant. During physical examination, the presence of hepa­ counterregulatory hormones. independently of hypoglycemia on

cognitive function, warning symptoms and glucose kinetics. Clin Sci

tomegaly, hyperpigmentation, or neurological findings of 85:197, 1993.

raised intracranial pressure provides clear direction for fur­ 19. Bloch CA. Clemons P, Sperling MA: PubertY;decreases insulin

ther investigation. Ultimately, however, suggestive history sensitivity. J Pedialr [10:481. 1987, .'

even in the absence of findings requires a 24-hour fast 20. Amiel SA. Caprio S, Sherwin RS, et aJ: Insulin resistance of puberty:
under careful observation. If symptoms of hypoglycemia are a defect restricted to peripheral glucose metabolism, J Clin
Endocrinol Merab 72:277, [991.
provoked, the steps are those described in Figure 11-5. An 21. Thornton PS, Alter CA, Katz LE, et a1: Short- and long-term use of
algorithmic approach to hypoglycemia has been described98 octreotide in the treatment of congenital hyperinsulinism, 1- ~e.diatr
and is detailed in Chapter 5. Rarely, an oral glucose tolerance 123:637, 1993.
test may be helpful in suspected reactive hypoglycemia. 22. Thomas PM, Cote GJ, Hallman DM, Mathew PM: Homozygo'Sity
mapping, to chromosome I I p, of the gene for familial persistent
hyperinsulinemic hypoglycemia of infancy. Am J Hum'Genet 56:416,
23. Glaser B. Chui KC, Anker R, et al: Familial hyperinsulinism maps to
Treatment chromosome II p 14-15.1. 30cM centromeric to the insulin gene.
Nature Genet 7:185, 1994.
24. Augilar-Bryan L, Nichols CG, WechSler SW. et a1: Cloning of the ~
The treatment of each major cause of hypoglycemia has cell high-affinity sulfonylurea receptor: a regulator of insulin
been discussed in its relevant section. secretion. Science 268:423, 1995.
25. Thomas PM, Cote GJ, Wohlik N, et al: Mutations in the sulfonylurea
receptor gene in familial persistent hyperinsulinemic hypoglycemia of
infancy. Science 268:426, 1995.
References 26. Steiner DF, Philipson LH: Pas de deux or more: the sulfonylurea
receptor and K' channels. Science 268:372, 1995.
27. Thornton PS. Glaser B, Herold K, et al: Familial hyperinsulinism (HI)
I. Cryer PE: Glucose homeostasis and hypoglycemia. In Wilson lD,
inherited in an autosomal dominant (AD) form differs clinically and
Foster DW (eds): Williams Textbook of Endocrinology, 8th cd.
genetically from the more common autosomal recessive form. Pediatr
Philadelphia, W.E. Saunders. 1992, pp. 1233-1253.
Res 37:587, 1995. Abstract
2. Gerich JE: Glucose counterregulation and its impact on diabetes
28. Stanley CA. Baker L: Hyperinsulinism in infancy: diagnosis by
mellitus. Diabetes 37:1608, 1988.
demonstration of abnormal response to fasting hypoglycemia.
3. Dinneen S, Gerich 1, Rizza R: Carbohydrate metabolism in non·
Pediatrics 57:702, 1976.
insulin dependent diabetes mellitus. N Engl J Med 327:707, 1992.
29. Finegold DN, Stanley CA. Baker L: Glycemic response to glucagon
4. Haymond MW: Hypoglycemia in infants and children. Endocrinol
during fasting hypoglycemia: an aid in the diagnosis of
Metab Clin North Am 18:211, 1989.
hyperinsulinism. J Pedialr 96:257, 1980.
5. Bier DM. Leake RD. Haymond MW, et al: Measurement of "true"
30. Levitt-Katz LE. Satin-Smith MS, Stanley CA, Cohen P: Insulin-like
glucose production rates in infancy and childhood with 6,6~
growth factor binding protein-I levels in the diagnosis of
dideuteroglucose. Diabetes 26:1016. 1977.
hypoglycemia due to hyperinsulinism. Pedlatr Res 37:538. 1995.
6. Chaussain JL: Glycemic response to 24 hour fast in nonnal children
and children with ketotic hypoglycemia. J Pedialr 82:438. 1973.
31. Gottschalk ME. Geffner ME. Yasuda PM, Shields WD: Reversal of
7. Chaussain JL, Georges P, Olive G, Job JC: Glycemic response to 24­ microcephaly and developmental delay after cure of hyperinsulinemic
hour fast in normal children and children with ketotic hypoglycemia, hypoglycemia. J Pedialr 117:432, 1990.
II: hormonal and metabolic changes. J Pediatr 85:776. 1974. 32. Price W A. Zimmer B. Conway R, Szekely B: Insulin-induced
8. Seltergren G, Lindblad BS, Persson B: Cerebral blood flow and
factitious hypoglycemic coma. Gen Hosp Psych 8:291, 1986.
exchange of oxygen, glucose, ketone bodies, lactate, pyruvate and
33, Editorial: Meadow's and Munchausen. Lancel 1:456, 1983.
amino acids in infants. Acta Pediatr Scand 65:343. 1976.
34: Sperling MA: Insulin biosynthesis and C-peptide: practical application
9. Haymond MW, Ben·Galim E. Strobel KE: Glucose and alanine from basic research. Am J Dis Child 134:1119, 1980. "

The ~el\' En nd Jotllll.lI "f .\!cdicinc

~r_hE~~i J.lhJ 75 j.Lg of gestoden~ gin:n 011 tby~ 1 [() 21 of dH:­

men'trlul Llde ,'-ttcr one month of medll'lprcdni\"lol1c therapy,
pttlHun: m.lgneric resonance iJl1Jging showed compkrc IT\olu­
nun of .,11 the intiltr;w.:s except the one in the !l\'poth"I,lJllm, and
H rcmJHH.:d unchanged thereafter.
Dunn\! the next two vcars, the do,\~ of Jw.:tl1\·I{)n:dni~(Jlonc \\".1.s.
,"I ,khl.lll,- reduced to 6 ~lg per d"y "nd then repl~ce:d bv (orti,one

J":CLitl" t 37.5 Jng per d~}r). Shorr Iv thcrt:;'lfrcr~ the p~1tit.:fH h(:g.'Ul to
reF0rt erl5udes of [lintnc55 thJ.t la>ted one to [\\0 hours- ~lnd 0(­
dlrn:d .lbout once a week. These cpisoJC5 wert' unn:brc-d to eat­
!n~ or and were nor accompanied by other symp[ool.\. -Jl1C
p~ttt,:nl',,, other syrnptorns were freqLlcnt cpi\odl",\ of hypcr­
FRAN(,:OISE FERY, MD" PH.D., LAURENCE PLAT, M,D., l,,,trC[l\l,l r",ulting from i11lp"ired thirst perception, the cle\~dop'
PHILIPPE VAN DE BORNE, M.D., PH.D., fI1Lnr (;f cushingoid teatures, and progressive \\Tight lo:.~ of 10 kg.
Sh!..' \\"a' hO$pitalized for asseSSJ11t:nt of t:lintne~s. ()n adrnls~,on,
her bluod pressure and heart rate were normal but she still had
h'pothermi,L Laboratory tests were norm;:" except te'r ;1 plasma
glucos,' "uncentration (measured while she was LIsting) of ,,5 mg
pcr deciliter (3J mIllol per liter), which prompted tllrrher e,'alu·
YPOGLYCEMIA stimulates rapid increases

H in the secn:tion of several hormones, in­

cluding cJteclmLmlines, glucagon, cortisol,
and growth hormone, th,lt act in concert to increase
,Ition uf ,e,eral aspects of glucose homeostasis, Dttring the c,·alna·
tion dl\.' pJ[ient continued to receive her usual tn:~urnent ti)[ hV'­
popituttJ.ri~ln, except that her n10rning dose of 25 n-lg of corti\)ol~e
.>Cerate "<.1$ ddayed until the end of testing on each studv li:ly,
the plasma glucose concentration. The chief role of
the central nervous system in triggering the release
The ,tudies were approved by the ethics committee of the Fac·
of such coumerregulatory hormones during hypo­
ulty of \Iedicine of the University of Brussels, and the p"tient
glycemia is well recognized. The specific region of gaw oral int()rmed consent,
the hypothabmus responsible for this process is l'bsnu glucose was measured by " glucose oxichse method
probably the ventromedial region, because bilateral ,Bochri'lger Mannheim, Mannheim, German),), and glycosvlated
lesions or perfusion of f)-glucose into this region re­ hemoglobin 'Y:'S measured by affinity high· pressure liquid chroma­
tograph,' (Bio· Rad LaboratOries, Hercules, Calif.); the normal
duces the increases in plasma glucagon and catechol­ r'.mge is ,LO to 6,5 percent. Plasma instllin, growth hormone, cor·
amines in response to h\'pogJycemia in rats.1.2 vVe rislll, giuc"son, and pancreatic polypeptide were measured by
describe a patient \\ith a Iwpothalamic sarcoid infIl­ fadioll1u11unoa5sJ.y>i-5 Plasma and urinary c;]techobrnines \ven:
trate who had complete loss of the counterregula­ mc,lsurcd by high-pressure liquid chrom~tograph)' with dectro·
chen1icl! detection. 6
tory response to hvpogln:emia.
A 31-year-old \\'onlJll in whom ,arcoidosis had been diagnosed Base-line Hormonal Status
ar the age of 27 ye,lfs Iud an IS-month history of secondary Plasma growth hormone concentrations were unde­
amenorrhea, a I2-1l1()l1th hisron' ofm:ight gain orabout 20 kg,
and polydipsia and polyuri." ApJ.rt from. the presence: of obesitr
tectab!.::, and plasma cortisol concentrations, measured
(weight, 86 kg; height, 171 em) ~nd J tendency toward hvporher· apprminutdy 14 hours after the evening dose ofl2.5
ntia) the resnIts of the ph~'sicJl cxanlin<ltion were norn1aL .:\ W'lter~ mg of cortisone acetate, averaged 1.5 f-Lg per deciliter
deprintion test confirmed the ctL.1gnosis of central diabetes insip­ HI I1mol per liter), whereas plasma glucagon .md
idus. Measurement' of pl.1Sma allLi uriDar), hormones disclosed
catecholamine concentrations and urinarv catechol­
partial pituitar), insufiicienc,', with low pbsn1.l concentrations of
free thyroxine and corti"o! .md hvperprolactinemia. Pbsma lute­ amine excretion were within the normal ~ange,
inizing hormone .lnd ti,liide'"rill1uiating honl1one concentrations
were l1ornlai, but the rc~ponSC$ to gonadotropin-releasing hor­ Glucose Homeostasis
fil0ne were: snpranonn.1L ;\l.lgnctic n:sonance 1:Hlaging ren::;.1kd ',1 Blood glucose was measured at least eight times
nonnal pirnitltv gland bur multiple intiltrates in the brain,
inclnding one in tht: hypoth~tblIl1il,..- reglon. The diagnosis of sar­
per d;l\' throughom the 33-day hospitalization, and no
coidosis was contirmed h)' the: findings of a high pbsma ;111­ hypoglrcemia was detected. The lowest values oc­
giotemin~con\Trting (nZ,'m\! cOBeemration, bilateral hll~r lvm­ cnrred on waking; the mean (±SD) of these "alnes
phadenopathy with r( interstitial intiirrates on computed \\'as 51->::'::8 mg per deciliter (3,2::'::0.5 m11101 per li­
tOlllography of the el1'''t, ;lnd typical tindings on transbrunchiJl
ter). The mean blood glucose concentrations twO
biopsy, Treatment was inmated with 32 mg of methvlpredn;so­
lone per day, 100 p.g of len ,thYroxine per cta\', 30 JLg of dcsmo­ hOllr~ and four hours after a meal were 11 0::'::45 and
pressin acetate p~r day~ .Hltl :1 dHnbin~ltion of 30 j1.g of ethin~:l t':S~ 90::'::3;1 mg per deciliter (6.1::'::2.5 and 5.0::'::1.9 mmol
per liler), respectively. The mean value at 3 a.m. was
85:!:28 mg per deciliter (4]::'::1.6 mmo! per liter).
From the Dcpanrnenb of Elldncrinolog.y (FE. LP,~ I.i\!J :.lnd Inrl:!'rnJ.j
The glycosylated hemoglobin value was 5.3 percent.
Medicine (E.c.) and 11K Ttn'C[[CnSil)f1 Cliilic (PIl.t Hopit,l ErJ5n1c, Uni· During a three-day fast, the patient'S plasma glu­
versite Lion: de Rntxdk.,>" Hru:>~\:b. Hdgimn. Addrc:>~ reprim n:qucs{!), to cose concentration fell from 66 mg per deciliter (3]
Dr. FerY;.l[ tht: DcpJIu)1(,;l1t u-t' En,-iocrinolog:r~ H()pit~ll ErJSnh:-, 80~ routt:
de Lellnik~ B-l070 Brussels, Hdgium, or;u ftery@mt'd.ulb 1111nol per liter) to 50 mg per deciliter (2.8 111mol per
© 1999, Nbss~1ChI!$t:tts. l\-lcdtl';l! Sonety. liter), and the plasma insulin concentration fell from

852 March 18, 1999


Insulin injection Insulin injection

t t

80 c
160 o::=::
u'" g'E.
:::l 120 urn
a a
:20 40

15 2000
o::t, 1000

5 500

l]) 800 l])
0 40 c:
§ 30
600 ..r:::­
0 l]) E
::c c-'"

•••••••• •


a 0 -~
~30 0 30 60 90 120 -30 0 30 60 90 120
Minutes Minutes
Figure 1. Changes in Plasma Glucose and Counterregulatory Hormone Concentrations Induced by the
Intravenous Administration of Insulin in the Patient (Solid Circles) and in 16 Patients with Panhypopi­
tuitarism (Open Circles).
The values in the patients with hypopituitarism are means (:!eSE), and the stippled areas are the mean
(:!eSE) values in normal subjectsJ The patient received 10 9 of glucose intravenously at minute 30. To
convert values for glucose to millimoles per liter, multiply by 0.0556; to convert values for cortisol to
nanomoles per liter, multiply by 27.59; 10 convert values for epinephrine to picomoles per liter, mUltiply
by 5.458; and to convert values for norepinephrine to nanomoles per liter, multiply by 0.0059.

12 fLU per milliliter (72 pmol per liter) to 5 fLU Effect of Acute Hypoglycemia on the Release
of Counterregulatory Hormones
millilirer (30 pmol per liter). No ketones were
recred in urine during the fast. After the intravenous injection ofO.IS U of human
The response to the ingestion of 100 g of glucose insulin per kilogram of body weight, the plasma glu­
was abnormal and delayed, with peak plasma glucose cose concentration decreased from 54 mg per deci­
and insulin concentrations of 181 mg per deciliter liter (3.0 mmol per liter) to 29 mg per deciliter (1.6
(10.1 m11101 per liter) and 120 fLU per milliliter (720 mmol per liter) in a 3D-minute period. The patient
pmo! per liter), respectively, at 120 minutes. The was then given 10 g of glucose intravenously. Despite
plasma glucose concentration was 42 mg per decili­ this injection, the plasm;} glucose concentration re­
ter (2.3 mmol per liter) at 240 minutes and 38 mg mained low for the next 60 minutes, when she re­
per deciliter (2.1 mmol per liter) at 300 minutes; the ceived another lO-g injection of glucose and drank a
corresponding plasma insulin concemrariorlS were cola. This experimentally indw.:ed hypoglycemia did
16 and 11 fLU per milliliter (96 and 66 plUol per li­ not activate any hormonal counterregulatory response.
ter). The patient had no sympathoadrenal symptoms To be able to distinguish between deficiencies of
during the hypoglycemia. growth hormone and cortisol and a deficiency ofoth-

Volume 340 Number 11 853

The New and Journal of .vledicine

--- Insulin infusion-­ -- Insulin infusion -­


, ,
. , 60


o(/) 4 ,,
,, 600


0.6 ~--~--~~--L---+T300
0.4 250 ro
200 ~

• • , • • • • • •,,

0 30 60 90 -30 0 30 60 90
Minutes Minutes
Figure 2. Plasma Glucose, Counter regulatory Hormone, and Pancreatic Polypeptide Concentrations
during a Hypoglycemic-Clamp Study in the Patient.

To convert values for glucose to millimoles per liter, multiply by 0.0556; to convert values for cortisol

to nanornoles per liter, mUltiply by 27.59; to convert values for epinephrine to picomoles per liter, mul­

tiply by 5.458; to convert values for norepinephrine to nanomoles per liter, multiply by 0.0059; and to

convert values for pancreatic polypeptide to picomoles per liter, multiply by 0239.

er cOlillterregulaton' hormones, we compared the pa­ liliter (l.05 nmol per liter), respectively, during the
tient's responses to hypoglycemia with those ob­ last 30 minutes of the clamp study. These values 'are
served under similar experimental conditions in 16 60 to 80 percent lower than those reported for nor­
patients \vith p~mh\'popitllitarism (mean age, 42±10 mal subjects in whom hypoglycemia of a similar
years; body-mass index [the weight in kilograms magnitude was induced. 9 · 12 The plasma pancreatic
divided the square of the height in meters), polypeptide concentration rose, reaching a maximal
(Fig. l). In these patients the hypoglyce­ value of 281 pg per milliliter (67 pmol per liter) at
mia was corrected JS dl1ciently as in normal subjects, 90 minutes. Throughout the test, the patient had
despite deticiencies of cortisol and growth hormone moderate symptoms ofneuroglycopeni::t (slow speech
biu presum::tblv normal secretion of glucagon and and drowsiness) but no sympathoadrenal symptoms.
Changes in Muscle Sympathetic-Nerve Activity
Effect of a Hypoglycemic Clamp on the Release in Response to Hypoglycemia, Chemoreceptor-Reflex
of Counterregulatory Hormones Activation, and Baroreflex Deactivation
We performed ;1 In:poglycemic-damp study in the The patient's muscle sympathetic-nerve activity was
patient to assess the response of counterregulatory recorded continuously from the peroneal nave pos­
hormones. During a primed constant intravenous in­ terior to the fibular head with a nerve-tramc analyzer
fusion ofimulil1, ,lLiministcn.:d at a rate of 4.5 mU per (Bioengineering Department, University of Iowa,
kilogram per milllHt: tt)[ 90 minutes, the plasma glu­ Iowa City) during the hypoglycemic-clamp study and
cose concentration decreJsed ti'om 59 per decili­ several other experimental conditions. Sympathetic
ter (3.3 I11mol per liter) to a mean of ±2 mg per bursts were identified by inspection of the neurogram.
deciliter (l.7±O.1 rmnol pn liter) between minutes The sympathetic-nerve activity and heart rate tend­
30 and 90 (Fig. 2). There were no changes in plasma ed to decrease during hypoglycemia (from 42 bursts
cortisol, growth hormone, or glucagon concentra­ per minute to 30 bursts per minute and from 68 beats
tions at any rime. Plasma epinephrine and norepineph­ per minllte to 63 beats per minute, respectively) (Fig.
rine concentrations did not change during the first 3). In contrast, sympathetic-nerve activity increased
hour and then increased slightly, averaging 185 pg per (to 50 bmsts per minute) during activation of the
milliliter (1009 pmol per liter) and 178 pg per mil­ chemoreceptor reflex by maximal end-expiratory ap­

854 . March 10, 1999


Electrocardiographic Activity

Respiratory Activity

10 seconds

Sympathetic-Nerve Activity

Base Line Hypoglycemia Apnea Nitroprusside Infusion

Figure 3. Electrocardiographic, Respiratory, and Muscle Sympathetic-Nerve Activity in the Patient at Base Une and during Hypo­
glycemia, Maximal End-Expiratory Apnea, and an Infusion of Sodium Nitroprusside.
The plasma glucose concentration was 30 mg per deciliter 0.7 mmol per liter! during hypoglycemia. Sodium nitroprusside (0.4 f.L9 '
per kllogram per minute) was infused intravenously for 10 minutes.

nea, especially at the end of the period of apnea, Resting energy expenditure, determined by indirect
when stimulation of the chemoreceptor renex is calorimetry, was 65 percent of the predicted value
maximaL Barordlex deactivation bv an intravenous adjusted tor sex, age) and lean body mass. I3
infusion of SOdilll1 nitroprusside (0.4 /-Lg per kilogram
per minute) for 10 minutes also markedlv increased DISCUSSION
the heart rate, to 94 beats per minute, and muscle
Sarcoid granulomas have a predilection for the hy­
svmpathetic-nerve activity, to 84 bursts per minute.
pothaLm1lls, and patients with neurosarcoidosis fre­
Release of Counterregulatory Hormones quentlv present with panhypopituitarism resulting
in Response to Other Stimuli from the loss of the ability of the hypothalamus to
To determine whether the defect in the release of release certain hormones. H These patients also com­
counterregulatory hormone was specific tor hypo­ monly have central diabetes insipidus, impairment of
glvcemia, we evaluated the secretory capacity of the thirst, and alterations in body temperature associated
pancreatic alpha cells and the response of the auto­ with a low metabolic rate_lS In addition to these symp­
nomic nervous system to other stimuli_ A 30-minLite toms, our patient had selective loss of the counter­
intravenous intusion of arginine induced a 7.5-told regulatory response to hypoglycemia.
rise in tht: plasma glucagon concentration and a 10­ The tact that our patient did not have hypoglyce­
f(Jld rise in the plasma insulin concentration, The plas­ mia during a three-day [;1st is not surprising, because
ma norepinephrine concentration increased sixtold, in normal subjects a selective, drug-induced deficien­
and the heart rate increased trom 60 beats per min­ cy of any counterregulatory hormone lowers plasma
ute to 92 beats per minute once the patient stood glucose concentrations aher three days of fasting but
up after having been Iving down for 10 minutes. does not calise hypoglycemiaY· On the other hand,
the patient'S late, persistent hypoglycemia after the
Other Tests .ingestion of glucose is similar to that in patients with
The rectal temperature, monitored contiilllously a combined deficiency of epinephrine and g1ucagonF
with a calibrated themnc probe, averaged 36.0±0.5°C. Glucagon and epinephrine have a crucial role in

Volume 340 Number 11 855

The New England Journ;ll of Medicine

the rccon:ry Irom imulin-induccd hypoglyccmi.l, at' an indchlt'd £/) Dr. J:

lh·ters for the "'amm:menU off/fIrma
<,,,d pn"rrmry: po/ypfptide. til I};: EO. fttrLass, "",d Dr S.
whereas cortisol and growth homlone Me of minor
RL"fetnjfji>r YI'l'iewmg the mn.ntucript all4 far hdp};,' fUMtstWns,
importance.. I'.I~ The normal pattern of glucose re­ ~nd to r.
Stokes arid M. Drqp'$ for editarial assistance.
covery that we found in the patients with pituitary
insufficiency but presumably normal responses of REFERENCES
other countcrregulatory hormones8 further illustrates
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this concept .md points to ddcctivc cltcchohmine \i::-n(rnlin:~h;lf h~'p()thalarnic fesi(ms ~n rats- ~urp"'e.s.-' omnt!O':f'l"Cgufatory re­
and g!uc.lgon responscs as rhe !"3.crors responsible for s[l0rti<.'S to h\"poglvccmi,. J elin InWst 1994;93:1677·81.
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vt.nrr(tlncdiai hyp·orhaTamus glucos.e perfusIon bfocks cownCf'l"e~ULHion
tion of insulin in our patient. durin SV;'lCmlC h· Ivccmia in awake mrs. Gin InvCst 199 7 '99:3615.
Besides stimulating the release of pituitary hor­ . l C rt ~ AU " ott t ell: lef, ,o:ate: an;na lmmu·
nO,J...'i...<;")'Y ofinsulm. I ('lin EndocnnoJ l\1ct'.lb J965;25:1375-M.
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sympathetic retlexes involving brain-stem neurons sp<>nS< til ilJ>ulin-induceu hypoglycCUll;, in man. J Clin In,·c.t 1976;58:7­
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ularory n(}rmonc: rc-sponscs to insulln-jndllc~d acute hypngly..:cmiJ. in
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in plasma norepinephrine and the heart rate during I nomlt:: tailure in iIl.~ll1in -dependent dhlbete5 mellitus: recent anre.("cdcnt
hypogly'(cmia reduces <luronomic R"Sponscs to.. symptoms of; .mu defense
the postural tcst provide further evidence of the in­ against subse'luent hvpogl!"cnria. J Clm In,"",,! 1993;91:819·28.
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of h\,><:>gIyccm.ia in plticnts with insulino=. .N Engl J Med 1993;329,
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hypoglycemia in humans is cOllIToversial,11,22 the com­ pl.l$nu (ortisoI during hypoglycemia preserves subsequent countcr.n.::gub· ~

plete abrogation of glucagon release in our patient "'{" ["'1',,0"'_'. ) Clin In,,"'t 1997;100:429-38.

12_ Vcnc:mJ:n T~ Mitrak.ou .0\" Mobn 1\1, Cryer P, Gcri~h J. rnuuc(lon of

during hypoglycemia, rogether with the large increase hypoglycemia UIld,,'McncSS by asymptomatic nocturnal hypoglyccmi•.

induced by arginine, supports the concept that the Dialx"" 1993;42:1233··7.

13. R.:tVlliSin E, Lillioj>. S, Anderson T£, Christin L, Rog.mJus C. Deter·

autonomic nervous system has a major role in the minan[s of 14-h{)ur t"flCrgy !!xpcndirurc in nlMI: mcrn<kh and. n:su)u; using
alpha-cell response to hypoglycemia. a rcspimorv chamlxr. j Clin Invest 1986;78:1568·78.
14. $rum CA, Ncdon FA, Lebovitz HE. Hypothalamic insufliLicru::y: the
The glycemic threshold tor the ;lctivation of coun­ cal1"C ofhYl)npirnirarism in sar~oidn'ris. Ann Intern Mcd 1978)t8:589~94_
terregulation is about 65 mg per deciliter (3.6 mmol 15_ Br.mnsrcin GO. The hypothalamus. In: Mclmcd S, cd. The piwitary.
per liter) in nonl1al subjecrs,"·12 but it is lower in C;llllbritigc. Mlss.: Buekwdl Science, 1995:309·40.
16. Royle Pl, Shah SD) Cryer i>K Insulin, gh.f{:agon.,. and catechol.amincs
patients with intensively rreated diabetes mellitus in po:vcnrion of hypoglycemia during fasting. Am J Phys>o11989;156:
who have recurrent hypogiycemia,13 in patients with Eb51·E66L
insulinomas,lO and in normal subjects in whom hypo­ 17. Tsc TI', ClutTer WE, $hah SD, Cryer PI'. Mechanisms of postprandial
glucos, counrc'!"gu!:uion in man: rili)-S;ologic roles of glucagon llld cpi·
glycemia is induced 12 ro 24 hours before test­ nl.':phnnc VtS-ol-\"is io.''iUnn in rhe prevention of hyp()gly(emia L)tc aha glu­
ing.t I ,l1 Therefore, the possibility that the low plasm;l msc ing.-scion. I Clin Inn:st 1983;72:2711·86.
18. G.:rit:h JE. Glucose Ct.>untcrrrgulu:ioll ;md irs i01p.ll.:t on l.UIDctt::s rud­
glucose concentrations in our patient lowered the litus. Di"""[c, 1988;37:1608,lZ
drreshold for sympathOJdrenal activation CMillot be 19. Cryer PI::. Glucose COUDtcrregulmon: prevention and com..'Crion of
mled out. However, this possibility is lullikely, be­ hypog!\,«lI\i. in hum.",,,. Am J Phy.,ioI1993;264:EI49·E1S5.
20. Fag. uS: L N;ld;).sson J.=~ Reme C. Sympathetic outtlow in human mlL~k
cause in normal subjects, after the plasma glucose nerves iOCR""" durin!,- hrpoglyccmi,. DUhctL'S1986;35:1124·9.
concenrration has been maintained at 52 mg per dec­ 21. Havel PI. :\lurn B. A.:rivarlon of IDccmomiL' nerves and the atif"cn:al
iliter (2.9 mmol per liter) fOl: 56 hours, stepwise low­ medulla ~'()n(nbutC5 to tncn:ascd glucagon secretion during moderare
in..';.ulin-itlliucco h~1X)g1yt:cmi.a in women. Diabetes 1997~46:801-7.
ering of the plasma glucose conccntr.lbOn to 45 mg 22. Hil~ted J~ r:r.wJseo H, Holst H", ChrisI:C:nsen NJ, ~idscn SL. Pl.t<:,ma
per deciliter (2.5 mnlol per liter) induces an increase glUClgOQ ,:md glucose recovery mer hypoglyL"Cmia: the effect of tool ';1U­
t<~l"mi<:: bl<x:k.ldc. ,\era Eudocriuol (Cnpcnh) 1991;125:460·9,
in the secretion of catecholamines and glucagon.24 23. "'-mid $.\. Sherwin RS, Simonson DC, Tambo,l.n.: W\'. Effi:cr of
A defect in rhe defense against hypoglycemia like inrensive insulin thenpy on glycemic thresholds for counn.:rrcgubwry hor~
that fOlmd in our patient 111;1\' not be rare, but it is UltmC rdea:...<'. Di.lMn::.'i 19XR;37:901-7.
24. Bayk Pl. o.:.lgy Rl, O'Connor AM, Kempers SI'. Yeo it.>" QU.llls C
ditlicult to diagnose because of the absence of sym­ fu.Upurion in br:llu glucose Hptake following n.:",-urn:n( hrp()gl~·ccm.ia. Pmc
parhoadrenal symptoms of hypoglycemia. Natl ACdd $,; l! <; A 1994;91:9352·6.

856 March 18, 1999

OOZI·972XJO II$O:UIOlO Vol H6. No.2
Tht: .Jnumal ofCIiOltaJ Endoa1mlliJg}- & Metabolism Prilitt'd til U,S.A
CnpynJ?:ht © 20m by Thl" EndocnnE.> Soc!ety

Oral Glucose Augments the Counterregulatory

Hormone Response during Insulin-Induced
Hypoglycemia in Humans*
Department of Pediatrics, Internal Medicine and the General Clinical Research Center, Yale University
Ekhool of Medicine, New Haven, Connecticut 06520; and Department of Pediatrics Baystate Medical
Center, Springfielt.i. MaEsachusetts 01199

ABSTRACT glucose levels between the two hypoglycemic clamp studies, and basal
It has been suggested that the counterregulatory hormone (eRH) CRH concentrations were alS() similar. As expected, there was a brisk
response to acute hypoglycemia is triggered via glucose sensors sit­ rise in all CRH during the control (hypoglycemia+noncarbohydrate
uated in either the hypothalamus or the portohepatic area. If the drink) study. ill the experimental study, administration of OG
latter were critical during hypoglycemia, one would anticipate that (hypoglycemia +OG), to raise intraportal glucose levels during systemic
ingestion ofglucose, by rdising glucose levels in the portal circulation, hypoglycemia, did not attenuate CRH responses. illdeed, OG enhanced
should attenuate CRH responses previously described in animal stud­ the rise in epinephrine, glucagon, and GIl.
ies. To evaluate the effect ofraising portal, but not peripheral, glucose Increases in cortisol and norepinephrine did not differ between the
levels during insulin-induced hypoglycemia, we performed hypogly­ two studies.
cemic clamp studies in five healthy adult males on two occasions. On Therefore, our data suggest that increasing the level of glucose in
one occasion, subjects received oral glucose lOG) (25 g) during hypo· the portal vein above that in the systemic circulation, during hypo­
glycemia; and on one occasion, noncarbohydrate-containing drink of glycemia, enhances (rather than suppresses) eRH responses. Thus,
equal volume, while maintaining plasma glucose at 55 :!: 2 mgldL ingestion of glucose may reverse hypoglycemia directly by provision
(3.08 mmolfL). of substrate, as well as indirectly by stimulating countereguiatory
As a result, there were no sigoificant differences in systemic plasma mechanisms. (J Clin Endocrinol Metab B6: 645-648, 2001)


prompt and coordinated counterregulatory hormone
(CRH) response resulting from hormonal and neural acti­
fed state and serve to limit postprandial hyperglycemia (13).
However, during hypoglycemia, it might have the adverse
effect of reducing the capacity of oral glucose (OG} feeding
vation (1). Although CRH responses to hypoglycemia have to promote glucose recovery.
been extensively studied (2), the putative mechanisms of In comparison with animal models, there is paucity of data
hypoglycemia detection have not been fully elucidated. In­ available regarding the locus of hypoglycemia detection in
deed, studies in animal models have provided conflicting human subjects. In an experiment in nature, it has been
results as to whether the preeminent locus of hypoglycemia recently shown that the counterregulatory response to hy­
detection resides in the brain (3-5) or in the portohepatic poglycemia was markedly attenuated in a patient with hy­
area (6-8). pothalamic sarcoidosis (14). Experiments examining the in­
A variety of experiments in rats suggest that the ventro­ fluence ofportal vein glucose sensors in human subjects have
medial hypothalamtLs (VMH) plays the central role in stim­ been impeded by practical difficulties in trying to prevent
ulating catecholamine and glucagon responses to hypo­ reductions in glucose in the portal system while lowering
glycemia (9-12). On the other hand, epinephrine and plasma glucose systemically. In the current study, we have
norepinephrine responses were reduced in studies in dogs in attempted to overcome this obstacle by introducing glucose
which systemic hypoglycemia was induced by insulin while into the portal vein via the oral route, while a stable and
euglycemia was maintained within the portal vein by direct reproducible hypoglycemic stimulus was induced systemi­
intraportal infusion of exogenous glucose (6). The suppres­ cally using the hypoglycemic damp technique (15). Surpris­
sion of antiinsulin hormones by entry of glucose into the ingly, CRH to hypoglycemia tended to be increased rather
portal system might facilitate the switch from the fasted to than diminished by ex; administration, in compariSon with
control hypoglycemia studies without OG.

Received July 20, 2000. Revision received October 16, 2000. Accepted
October 25, 2000. Materials and Methods
Address all correspondence and requests for reprints to: Rubina A. Subjects
Heptulla, M.D., Department of Pediatrics, Division of Endocrinology,
Baystate Medical Center, 3300 Main Street, Springfield, Massachusetts We studied five healthy, nondiabetic, lean (body mass index, 24 + 1
011'1'1. E-mail: rheptul! kg/m') altult males (age, 26 + 1 yr) on two occasions. All subjects were
~Supported by NIH Grants DK-20495, HD-30671. RR-06022, and seen in the outpatient department of Yale General Oinical Research
RR-125. Center, in the morning, after a 10- to 12-h overnight fast. A detailed

646 HEPrULLA ET AL. .JCF: & M • 2001
Vol 86. ~o. 2

9-J c ·200
.-.-- Hypo - non-<:arbohydralB drink E0.. :000
8') --0- Hypo + oral glucose

70 ­
0..___ 800

:!2 wO>
OJ &J !1!~

E 400
SG 200
~ C1l
0 40
a:: a
CJ 30
25 ~ Hypo + non-carbohydrate drink
a> - 0 - Hypo .. oral glucose
10 c 20
0 r- --,-----[ I r--,-----r--~-,---
E ·.5
o 20 40 60 80 100 120 140 160 180 0::1
Time (min) .c::l '0
FIt;. 1. Data are expressed as means::':: SE for plasma glucose con­ o
centrations. Shaded circles represent the hypoglycemia study along ~ o
",-jth noncarbohydrate drink, and open circles represent data of the
hypoglycemia and OG study where patients received 25 g OG at 80 t25 g of oral glucose To convert plasma glucose values to millimoles per liter, multiply
by 0.056.
medical history and physical examination were obtained for each sub­ 160 *
ject. All subjects were in good health and taking no medications. The c 140
nature and purpose of the study was explained to them, and written CJLJ 120 *
voluntary consent to participate in the study was obtained. The Human 00> 100
Investigation Committee of Yale University School of Medicine ap­ ~.3
C) 80
proved the study protocols.
To produce a standardized hypoglycemic stimulus, we performed a 0
one-step hypoglycemic clamp procedure, as described by Simonson IT 20 40 60 80 100 120 140 160 180
af. (16), in all the subjects. Two cannulas were inserted, one in an an­ Time (min)
tecubital vein for infusion of insulin and glucose and the other in the
dorsal hand vein for blood sampling. The hand was placed in a heated FIG. 2. Data are expressed as means::':: SE for plasma epinephrine,
(65 degree C) box to arterialize the venous blood sampling (17). The GR, and glucagon concentrations. Shaded circles represent the hy­
subjects were given continuous iv infusions of insulin (120 mU per poglycemia study along with noncarbohydrate drink, and open circles
square meter of body-surface area per nUnute), and target plasma glu­ represent data of the hypoglycemia and, OG study where patients
cose values were achieved by varying the rate of infusion of 20% glumse received 25 g OG at 80 min. To convert plasma epinephrine values to
in water. Plasma glucose was measured, at the bedside, at 5-min inter­ picomoles per liter, multiply by 5.458.
vals ( Coulter, Inc. glucose analyzer, Beckman Coulter, Inc.,
Fullerton, CAl. In all the subjects, plasma glucose concentrations were 4.2% (at 11.1 ng/mL); glucagon, 8.3%; and cortisol, 8.6%. For epineph­
stabilized between 90 and 108 mg/ dL (5.0 and 6.0 mmol/L) before the rine and norepinephrine, the interassay variability levels were 17% and
induction of hypoglycemia. The length of the euglycemic phase was 60 16%, respectively. The intraassay variability values were 6% for epi­
min in all subjects, the plasma glucose concentration was reduced to nephrine and 4% for norepinephrine.
approximately SO-55 mg/dL (2.8-3.1 mmol/L), over a period of ap­
proximately 10 min, by reducing the rate of glucose infusion. Plasma
Statistical analysis
glucose concentrations were then maintained at that level for a further
100 min. The plasma glucose concentra tions and hormone responses in the two
During one hypoglycemic clamp study, a load of 25 g glucose studies were compared by ANOVA with repeated-measures design.
was given orally as a cola-flavored drink at 80 min, the time when When there was a statistically significant group-time effect, two-tailed
plasma glucose concentration had reached approximately 50 mg/dL paired t tests were used to localize the effects. Differences were regarded
(2.8 mmol/L). During the other clamp, patients were given a caffeine­ as statistically significant if P values were less than 0.05.
free diet cola equal in volume and color to the OGdrink at 80 min
of the study (0-60 min euglycemia, 60-180 min hypoglycemia, 80 min Results
administration of the oral drink)
Plasma insulin concentrations in the basal state 18 ::':: 1 vs.
M eas urements 7::':: I/LU/mL (57 ::':: 7 vs. 50 ::':: 7 pmol/L)] and during the
Blood samples were taken at 10-to 20-min intervals for measurements insulin infusion [178::':: 8 vs. 190::':: 10 /LU/mL (1277::':: 57 vs.
of plasma insulin, epinephrine, norepinephrine, cortisol, glucagon, 1363 ::':: 70 pmol/L)] did not differ between the studies, re­
and GH. gardless of whether or not OG was given during hypogly­
Plasma catecholamines were measured by high-performance liquid cemia. As shown in Fig. 1, there were also no significant
chromatography using an electrochemical detector; and plasma insulin,
differences (P < 0.8) between the two studies, with respect
GH, cortisol, and glucagon were measured by double-antibody RlAs. All
the samples from each subject were analyzed in a single assay, and all to basal glucose concentrations or in the fall in plasma glu­
tests were run in duplicate. The intraassay variations for the GH were cose levels during both hypoglycemic clamps. Thus, virtu­

ally an identical hvpoglycemic stimulus was achieved, nisms underlying alterations in CRH responses induced by
whether or not the subject ingested OG. ex:;. However, with respect to GH, it is noteworthy that
As shown in Fig. 2, basal concentrations and the early ghrelin (a GH-releasing acetylated peptide from the stom­
(60-80 min) rise in plasma epinephrine, GH, and glucagon ach) has been shown to stimulate GH release independent of
levels were similar in both studies. However, during the last regulation by hypothalamic GHRl-I (21). Previous studies by
60 min of the hypoglycemic clamp with OG, the plasma Donovan et al. (7, 8) did not examine changes in GH levels
concentrations of epinephrine 11212 ::':: 145 pg/mL (6605 ::':: during their hypoglycemia and portal euglycemia experi­
790 pmol/L)J, GIl (23 ::':: 3 Jl.g/L), and glucagon (134 ::':: 23 ments. The increased glucagon responses with OG could be
ng/L) rose to values that were .signifiGmtly higher than mediated by neural connections linking the portal venous
plasma epinephrine [824::':: 181 pg/mL (4490::':: 986 pmol/L), system with the YMH (22). There may be other mediators of
P < 0.015], GH (14 ::':: 3 fJ.g/L,.P < 0.015), and glucagon (86 glucagon secretion (like endothelin-1) that may account for
20 pg/mL, P < 0.(5) levels observed during the hypoglyce­ the increased glucagon response (23). In addition, the study
mic clamp without ex:; (Table 1 ).In contrast, as shown in Fig. design with hyperinsulinemic euglycemia before hypogly­
3, administration of OG did not significantly affect plasma
cortisol (P < 0.08) or plasma norepinephrine (P < 0.16) re­ 40
sponses to hypoglycemia.
Discussion - - - Hypo + non-carbohydrate alnk
30 -0-- Hypo + oral glucose
The present investigation was undertaken to examine the
role of glucose sensors within the portal venous system in
regulating CRH responses to hypoglycemia in human sub­
tEc 25

jects. Animal experiments from our own laboratory have '0 20

suggested the YMH is responsible for detection of hypogly­ Jl
(5 15
cemia (9-12); whereas other studies have shown that main­ 0
tenance of euglycemia in the portal vein suppresses the CRH 10
response to systemic hypoglycemia (6). In this study, we
attempted to produce similar experimental conditions in hu­ 5
man subjects by using the glucose clamp teclmique to cause
an identical reduction in systemic plasma glucose concen­
trations during two studies: one with and one without in­ 600
gestion of OG. Relatively large iv doses of insulin were used
in both studies so that precise control of systemic glucose =E 500
- - - Hypo + non-carbohydrate drink
concentrations could be achieved in the face of ingestion of
OG in amounts (25 g) within the range that might be used
~ -0-- Hypo + oral glucose
c:: 400
clinically to treat hypoglycemia. ~
Oral glucose wa<; ingested during the experimental study 0.
after mild hypoglycemia was induced and CRH responses c:: 300
were initiated. Practical considerations precluded measure­ e0
ments of glucose concentrations in the portal vein in these z 200
subjects. However, in animal studies, ingestion of 40g OG
leads to a rapid rise in glucose concentration and appearance
en 100
of approximately 27 g glucose intraportally within 15--30 min 0::
of glucose ingestion (18-20). Under these conditions, one
might estimate that we would have observed a detectable 0
o 20 40 60 so 100 120 140 160 180
reduction in plaSJIla epinephrine concentration vs. control
TIme (min)
experiments if a portal glucose sensor was operative in hu­
mans. However, ingestion of OG enhanced, rather than re­ fIG. 3. Data are expressed as means ± SE for plasma cortisol and
duced, the adrenomedullary responses to hypoglycemia. norepinephrine concentrations. Shaded circles represent the hypo­
glycemia study along with noncarbohydrate drink, and open circles
Moreover, the ingestion of ex:; also increased GH and glu­ represent data of the hypoglycemia and OG study where patients
cagon responses to hypoglycemia. received 25 g OG at 80 min. To convert plasma norepinephrine values
This study was not designed to investigate the mecha­ to picomoles per liter, multiply by 5.458.

TABLE 1. Counterregulatory honnone response to hypoglycemia with and without oral glucose

Basal Peak Basal Peak

Hypo + 824 :!:: 181 0.7 0.1 14.5 ± 3.
Hypo + OG 27 ± 7 12.12± 14.5 0.7 ± 0.1 22.6:!:: 3.
Pvalue NS 0.015 NS 0.015
NS. Not significant.
648 HEPI'ULLA ET AL. JCE& M. 2001
VoL 86. No.2

cemia induction may have also contributed to the reduction 6. Hevener AL, Bergman RN, Donovan CM. 1997 Novel gJucosenror ior hy­
poglycemic detection localized to the portal vein. DiabeteS. 46:1521-1525.
in glucagon response (24). 7. Hamilton-Wessler M, Bergtffiln RN, Haller JB, Watanabe RM, Donovan eM.
Regardless of the mechanisms involved, the data support 1994 The role of liver glucosensors in the integrated sympathetic response
the contention that the increased CRH responses were the induced by deep hypoglycemia in dogs, Diabetes. 43:1052-1060.
8. Donovan CM. Hamilton-Wessler M, Halter JB, Bergman RN. 1994 Primacy
result of absorption of glucose into the portal system We olli\'er glu~osensors in the sympathetic response to progrl?SSive hypoglyce­
controlled for nonmetabolic effects of volume and taste by
having the subjects, during the control study, ingest a de­ 9. Borg MA, Borg WP, Tam""rlane WV, Brines ML, Shulman GI, Sherwin RS.
1999 Chronic hypoglycemia and diabetes impair coun terregulatioo induced by
caffeinated drink of the same taste and volume as they in­ localized 2-<ieoxy-glucose perfusion of the ventromedial hypothalamus in rats.
gested during theexperiroental'stu<;ly. Moreover, the pattern Diabetes. 48:584587.
10. Borg MA, Sherwin RS, Borg Wl', Tamborlane WV' Shulman GI. 1997 Local
of a 4O-min delay in response for all·three hormones is more ventromedial hypothalamus glucose perfusion blocks counterregulation dur­
consistent with the time requi:r~l:to absorb ing~'Sted glucose ing systemic hypoglycemia in awake rals. , Oin Invest. 99:361-365.
into the portal circulation than what might be expected from 1 L Borg Wl', Sherwin RS, During MJ, Borg MA, Shulman GI. 1995 Local ven­
tromedial hypothalamus glucopenia triggers coWlterreguiatory hormooe re­
a more immediate neural response. It is also noteworthy that lease. Diabetes. 44:180-·184.
these rt.'Sponses were very consistent and selective, so that 12 B org WP, During MJ. Sherwin KS, Borg MA, Brines ML, Shulman GJ. 1994
statistical significance was readily observed with a relatively Ventromedial hypothalamic lesions in rats suppress counterregulatory re­
small number of subjects. Ingestion of ex:; did not seem to mg
alter plasma cortisol and norepinephrine responses to hy­
poglycemia, but further studies in larger numbers of subjects
are needed to evaluate these responses more definitively.
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ment of hypoglycemia are potentially important. Further 16. Simonson Dc.. TamborJane WV, DeFronzo RA. Sherwin RS.1985 Intensive
studies are needed to determine whether similar effects are insulin therapy reduces rounterregulatory hormone responses to hypoglyce­
mia in patients with type I diabetes. Ann Intern Med. 103:184-190.
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18. Abumrad NN, Cherrington AD, Williams FE, Lacy WW, Rabin D. 1982
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.Diabetes. Vol 48. Issue 3 584-587. CopHight
. t !999 by American Diabetes Association
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l'ubMcd Citation
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BorlkM./\ II Sherwin. R. S
Chronic hypoglycemia and diabetes impair Alert me when:
'counterregulation induced by localized
2-deoxy-glucose perfusion of ~he ventromedial
hypothalamus in rats
MA Borg, WP Borg, WV Tamborfane, ML Brines, GI Shubnan and RS Sherwin

Department oflntemal Medicine, Howard Hughes Mcdicallnstitule, Yale University School of Medicine, New Haven, Connecticut

06520-8020, USA

Previous studies have demonstrated that the ventromedial hypothalamus (VMH) plays a critical role in sensing and
responding to systemic hypoglycemia. To evaluate the mechanisms of defective counterregulation caused by
.iatrogenic hypoglycemia and diabetes per se, we delivered 2-deoxy-glucose (2-DG) via microdialysis into the VMH to
produce localized cellular glucopenia in the absence ofsystemic hypoglycemia. Three groups of awake chronically
catheterized rats were studied: I) nondiabetic (with a mean daily glucose [MDG] of 6.9 mmolll) BB control rats (n =
5); 2) chronically hypoglycemic nondiabetic (3-4 weeks, with an MDG of2.7 mmol/I) BB rats (n = 5); and 3)
moderately hyperglycemic insulin-treated diabetic (with an MDG of 12.4 mmolll) BB rats (n 8). In hypoglycemic
'rats, both glucagon and catecholamine responses to VMH glucopenia were markedly (77-93%) suppressed. In
diabetic rats, VMH 2-DG perfusion was totally ineffective in stimulating glucagon release. The epinephrine response,
'but not the norepinephrine response, was also diminished by 38% in the diabetic group. We conclude that impaired
:counterregulation after chronic hypoglycemia may result from alterations ofthe VMH or its efferent pathways. In
diabetes, the capacity ofVMH glucopenia to activate the sympathoadrenal system is only modestly diminished;

however, the communication between the VMH and the alpha-cell is totally interrupted.

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• f)ownlnad t() (itatlQl1:Vl;1!l;tg<";[
Monica A. Borg·, Robert S_ Sherwin·, Walter P. 8?rg*, William V.

Tamborlanet , and C.erald I. Shulman *

* +
Yale Univenity School of Medicine, Department of Internal Medicine and - Department of Pediatrics., New Haven,
Connecticut 06520

The ventromedial hypothalamic nucleus (VMH} is necessary for the integrated hormonal response to hypoglycemia. To
determine the role of the VMH as a glucose sensor, we performed experiments designed to specifically prevent glucopenia in
the VMH, while producing hypoglycemia elsewhere. We used awake chronically catheterized rats, in which local VMH
glucose perfusion (100 mM or IS mM ofD-glucose) was combined with a sequential euglycemic- hypoglycemic clamp. In
twO control groups the VMH was perfused either with (a) an iso-osmotic solution lacking glucose, or with (b)
.nonmetabolizable L-glucose (100 mM). During systemic hypoglycemia glucagon and catecholamine concentrations
promptly increased in the control animals perfused with either 100 mM L-glucose or the iso-osmotic solution lacking
glucose. In contrast, glucagon, epinephrine and norepinephrine release was inhibited in the animals in which the VMH was
'perfused with D-glucose; hormonal secretion was partially suppressed by the VMH perfusion with 15 mM D-glucose and
'suppressed by - 85% when the VMH was perfused with 100 rnM D-glucose, as compared with the control groups. We
'conclude that the VMH must sense hypoglycemia for full activation of catecholamine and glucagon seeretion and that it is a
key glucose sensor for hypoglycemic counterregulation. (J Clin. Invest. 1997.99:361-365.)

~Key words: VMH; microdialysis; hypog~vcemia; glucagon; epinephrine

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J Counterregulatory Hormone Response during Insulin-Induced Hypoglycemia in Humans. J Clin Endocrinol Metab
l 86: 645-648 [1\~st@~J ffiJll Tt!tl
• Beverly, 1. L, De Vries, M. G., Bouman, S. D., Arseneau, L M. (2001). Noradrenergic and GABAergic systems in
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1 doundy, V. A., Cincotta, A. H. (2000). Hypothalamic adrenergic receptor changes in the metabolic syndrome of
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• Levin, B. E., Dunn-MeynelJ, A. A., Routh, V. H. (1999). Brain glucose sensing and body energy homeostasis: role in
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;Dial'>etes. Vol 44, Issuc 2 180·1 R4, Copyright © 1995 by American Diabetes Association t. Similar articles found in:
Dlal'>etcs Onln",


JLocal ventromedial hypothalamus glucopenia triggers

" Pup\kd.CJtalion
,. Search Medline for articles by:
ElorL\£.J'~ II £!.\@liUl. G. L
,. Alert me when:
[1-:" articles cite thIS artIe\..:
,counterregulatory hormone release ,. Download to (i!"tilmM;lI1ager

;WP Bo.-g, RS Sherwin, MJ During, MA Borg and GI Shulman

[)epanmcnt of Inlemal Medicine, Yale University School 0f,Medicine, New Haven, Connecticut 06520·8020.

To lest the hypothesis that nuclei of the ventromedial hypothalamus (VMH) playa key role in the detection of
20unterregulatory responses to hypoglycemia, we'delivered the glucopenic agent 2-deoxyglucose via bilaterally placed
microdialysis probes into the VMH of conscious, chronically catheterized rats. The goal was to produce cellular glucopenia
localized to the VMH. The volume of brain tissue exposed to 2-deoxygJucose was determined by adding
[3HJ2-deoxyglucose to the dialysate; its distribution in cerebral tissue was almost exclusively limited to the VMH. Rats with
microdialysis probes placed into the frontal lobes served as a control group. Local perfusion of2-deoxyglucose (but not
glucose) into the. VMH caused a prompt twofold increase in plasma glucose in association with a striking elevation of
plasma glucagon (3.5-fold), epinephrine (30-fold), and norepinephrine (3.5-fold), No effect was seen when 2-deoxyglucose
,was delivered into the frontal Jobes. We conclude that glucopenia localized to the VMH triggers the release of
counterregulatory hormones that defend against hypoglycemia. Thus, the neurons that sense glucopenia may be simated in

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u\l>strac:tl LE.lllLJ ext]

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hypoglycemia, hyperinsulinemia, and excess corticosterone on hypoglycemic counterregulation. Am. J. Physio!. 281:
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• Hepmlla, R. A., Tamborlane, W. V" Ma, T. Y.-z.., Rife, F., Sherwin., R. S. (200 I). Oral Glucose Augments the
Counterregulatory Hormone Response during Insulin-Induced Hypoglycemia in Humans. J Clin Endocrinol Metab
86: 645-6481Abstract] [Full Textl
• Beverly, 1. L., De Vries, M. G., Bouman, S. D., Arseneau, L. M. (2001). Noradrenergic and GABAergic systems in
the medial hypothalamus are activated during hypoglycemia. Am. J. PhysiOl. 280: 563R-569 [Abstract) [Full Text)
.• Evans, M. L., Matyka, K., Lomas, J., Pemet, A., Cranston, L C P., Macdonald, L, AmieI, S. A (1998). Reduced

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\),tract] U'-ull Tt;xt}

• Jones, T. W., Borg, W. P., Borg, M. A, Boulware, S. D., McCarthy, G., Silver, D., Tamborlane, W. V., Sherwin, R. S.
(1997). Resistance to NeurogJycopenia: An Adapralive Response during Intensive Insulin Treatment of Diabetes. J
Clin Endocrinol Metab 82: 1713·1718 filllstraq1 f£t!UTelli
• DaHman, M. F., Akana, S. F., Bhatnagar, S., Bell, M. E., Choi, S., Chu, A., Horsley, C, Levin, N., Meijer, 0.,
Soriano, L. R., Strack, A. M., Viall, V. (1999). Starvation: Early Signals, Sensors, and Sequelae. Endocrinology 140:
4015-40231Abstractl [Full Tt.:·xt]
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Oetenninant of the Impaired (beta}-Cell Secretol)' Response after Protein-Energy Restriction in the Rat.
Endocrinology 139: 3382-3389 L-il?ma~tl U'll[j Jex,!}
• Beverly, J. L, de Vries, M. G., Beverly, M. F., Arseneau, L M. (2000). Norepinephrine mediates glucoprivic-induced
increase in GABA in the ventromedial hypothalamus of rats. Am. J. Physiol. 279: 990R-996 It\bst@f!l [Full Text)
• Boundy, V. A., Cincotta, A. H. (2000). Hypothalamic adrenergic receptor changes in the metabolic syndrome of
genetically obese (ob/ob) mice. Am. J. Physiol. 279: 505R-514 [Abstract] [Ful! Text]
• Levin, B. E., Dmm-Meynell, A. A., Routh, V. H. (1999). Brain glucose sensing and body energy homeostasis: role in
obesity and diabetes. Am. J. Physiol. 276: 1223"&-1231 filll§tract] [Fuil Text}
• Jackson, P. A., Pagliassotti, M. J., Shiota, M., Neal, D. W., Cardin, S., Cherringtol4 A. D. {I 997). Effects of vagal
blockade on the counterregulatol)' response to insulin-induced hypoglycemia in the dog. Am. J. Physiol. 273:
1 178E-1 188 filllstract] [Full Text}
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Hypothalamus Glucose Perfusion Blocks Counterregulation during Systemic Hypoglycemia in Awake Rats. J. Clin.
Invest. 99: 361-365 [Abst@ill [Full Text]

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sion). In case of complex problems with manuscript handling, dite the overall speed of review (the current average time for
the matter is referred to the Managing Editor or the Assistant review is 40 days), they must do so without compromising the
Managing Editor. quality and fairness of the process. Fast track publishing may
The Associate Editors do not take calls from the authors be appropriate for certain types of papers; however, this can be
concerning decisions or other matters directly related to manu­ achieved by using the "Rapid" category, which is available to
scripts. All such enquiries should be addressed to the Editor in all authors who wish to compete on a level playing field, with
the form of a written communication, which will be forwarded defined rules. Finally, it is noteworthy that important and
with the complete file to the appropriate Associate Editor. The novel work often experiences a "spontaneous acceleration"
Associate Editor and/or the editorial staff will then communi­ during review. This is because the reviewers are usually excited
cate with the authors by letter or by teJephone, as appropriate. about reviewing such work and tend to do so more quickly.
The following are the major reasons [or the policy of not ac­ Legal status ofsubmitted manuscripts. Recent attempts by
cepting phone calls. . corporate entities to gain access to confidential work under
review have made it necessary to define the legal status of man­
1. The Journal is a written medium, and all communica­
uscripts submitted to The Journal. All such manuscripts are
tions concerning its activities must be recorded in written form. deemed to be the property of the submitting author(s) until
This is important not only for uniformity and fairness, but also
such time as a decision is made and the copyright is officially
because it provides a written record for other editors and/or assigned by the authors to the Society. However, by submitting
office'staffwho may handle the manuscript in the future. the manuscript to The Journal, the authors agree to su bject it to
2. The Journal receives manuscripts from all over the world. the confidential peer review process outlined in the foregoing
At present approximately 40% of submissions are from foreign pages.
countries, The Editorial Board feels that it would be unfair if Scientific integrity. Duplicate publication and outright sci­
only authors in certain time zones and with low costs ofcalling entific fraud are rare events that nevertheless have a very seri­
have direct access to the editors to discuss their manuscripts.
ous impact on the integrity of the scientific community. If the
3. For fair and proper handling of enquiries the Associate
editorial board uncovers possible evidence for such problems
Editor must have access to the entire manuscript file at the time
in submitted manuscripts. it will first confront the correspond­
of making the assessment. However, the individual editors do
ing author to complete confidence, to allow an adequate clarifi­
not keep set office hours at the main Jeloffice, where the files cation ofthe situation. Ifthe results ofsuch interactions are not
are kept. A written enquiry allows the entire file to be pulled
satisfactory, the board will contact the appropriate official in
and sent to the Associate Editor for a proper evaluation. the institution from which the manuscript originated. It wili
4. Manuscript decisions are made during the weekly meet­
then be up to the institution in question to pursue the matter
ing of the Editorial Board. Enquiries and rebuttals are also appropriately. Depending upon the circumstances, The Jour­
discussed at the meeting. Thus, communication with the au­
nal may also opt to publish errata, corrigenda, or retractiGns,
thors is most useful after an initial evaluation of the written
which will be linked to the original article in the Index
material has been made by the Associate Editor, and the matter Medicl/s.
has been discussed by the Editorial Board.
A lesser problem arises when a member of the scientific
"Preapproval" ofpapers and selective 'rast-track pllblish­ community disagrees strongly with the methodology and/or
ing. .. With increasing competitiveness in science, some jour­ conclusions of an article that has been published in The Jour­
nals have begun to foster the notion that "first is best." It has nal, but does not allege actual fraud. The Journal does not have
now become commonplace for authors to contact journal edi­ a "Letters to the Editor" section to publish a communication
tors ahead of time to see if their work can be given special regarding this. Therefore, the concerned individual should ei­
consideration because of its perceived importance or popular ther contact the authors directly to discuss the disagreement. or
appeal. As a matter ofgeneral policy, the leI does not provide allow the natural corrective mechanisms ofscience to settle the
such advance determinations. This is because a preliminary issue with time.
judgment based upon a phone call or an abstract is prone to In the final analysis. the peer review process has its inherent
significant error. Also, the assessment of "importance" and flaws, and in the eyes of the author the outcome of a manu­
"interest" usually requires opinions from experts in the particu­ script review may not always be equitable. However, in the
lar field, i.e., peer review. The Journal also does not promise absence of a better system, expert refereeing by peers remains a
expedited review of selected manuscripts. Selecti vely accelerat­ tried and true method for the scientific community to maintain
ing peer review fora specific manuscript by prearrangement its own standards ofexcellence. The editors of The Journal are
increases the possibility of inadequate evaluation of the work com mitted to promoting the quality and fairness ofthis process
and imparts to the reviewers and editors an implicit bias in in every way possible, as well as enhancing its speed and effi­
favor of acceptance. Also, other authors whose papers were ciency. The board remains open to suggestions from the scien­
accepted earlier following conventional review may have their tific community of ways to improve this process and to sustain
publication dates unfairly delayed. Furthermore, the authors the excellence of The Journal.
seeking a fast track may be motivated by the realization that
other similar work b already under review elsewhere. If they Ajit P. Varki
gained this information by being confidential reviewers of the for the Editorial Board
other work, The Journal may then be aiding and abetting an
The peer review system followed by the JeI attempts to References
treat all manuscript submissions in as fair and uniform a fash­ I. Varki. A. P. 1992. The times they are a·changin.Changing wilh the times.
ion as possible. While the editors make every attempt to expe­ J. Clin Invesl. 89:721-722.

EdilOria! vii
Res Ipsa Loquitur. Positive and Negative Trends Affecting the Jet Editorial

1 2000


.,a.. ;It
« o

i V)



1 ~

::s "'"Z
1000 Z

1910 1980 1990

Figure 4. National Institutes of Health research funding trends from

1970 to 1992. Annual NIH budget as a percentage of total federal or
national spending on health care.
1!1113 lfit '985 ,m 1987 1988 19119 '990 1991 liKl2 , _

Figure J. JCI manuscript submission and publication rates. Figures e

for 1993 are projected.
% 60
w 50



tn 300 w ~,
40 ::i
0:: Figure 5. Veterans Ad­
ii: "c:

o ministration research '
r- 30 0 funding trends fr,om
200 '/I.
y (5 1980 to 1992. Annual


t 1970 1980 1990

VA research budget as
a percentage of total VA
spending on medical
0 a
1981 1989 1991 1993
Table I. JCl Manuscript Submissions by COlJrttryo/Origin
Figure 2. Days from submission to first decision (all manuscripts)
and to final publication (accepted manuscripts). Accurate data not Percent of lotal 5ubmissioll$
available before 1987. Figures for 1993 are based on data from man­
uscripts received through October 1993. 1987 1992

Australia 1.6 1.3

Canada 2.9 3.5
II) 80 France 2.5 4.5

Germany 2 3.4
Israel 1.3 1.1
:::> 60 Italy
z 1.8 2.4
~ 40
1:::= us
:::> Switzerland 2.0 2.0
United Kingdom 2.0 3.4
~ 20
USA 71.2 60.8
Figure 3. leI manuscript
submissions: US 11'5. nOll- All others 5.6 5.1
0 Us. Data not available be­
1915 1985 1995 tween 1983 and 1986. Accurate cJata not available before 1987.

The Journal of Qinical Investigation, Inc. Ajit Varki. Editor .

Volume 93. January 1994. I Michael Held. Managing 'Editor

Res Ips(1 LoqllilUr: P(Jsilil'e (111(:/ Negatil'e Trends Ajfrciing ,he JCI 1
Diabetes, Diabetes, and the American Diabetes
Philip E. Cryer

of talented and committed associate editors, who, with their

individual areas of scientific expertise, have taught me a
great deal and, with their concern for science and the

ow at the end of my tenure as editor of Diabetes,
journal, have made my job easy. The associate editors are
I wish to offer some personal thoughts about our
journal, the disease we oppose, and our Associa­ Drs. David D. Chaplin, Michael L. McDaniel, Mike M. Mueck­
tion. I am pleased, but not satisfied, with our ler, M. Alan Permutt, Julio V. Santiago, and Josepli R.
journal. I am disappointed, but not surprised, that the disease Williamson in St. Louis and Dr. George S. Eisenbarth in
remains recalcitrant. I am concerned, but optimistic, about Denver. Special thanks are due Mary Weis, who as editorial
our Association. The challenges are great, but so are the assistant continues to lead the St Louis editorial office so
opportunities. capably, Karen Muehlhauser and Lisa Chandler in that office
and the professional publications staff at the Association'~
National Center, including, but not limited to, Susan Lau,
Established in 1952, Diabetes has become a leading, arguably
Peter Banks, Matt Petersen, Stacey Wages, and Jennifer
the leading, diabetes-oriented research journal in the world.
Gross. Finally, I am grateful to Dr. R. Paul Robertson, the
The history of the journal was summarized by my predeces­
previOUS editor, for his advice and support and for· handling
sor (1).
submissions to the journal from the editors and their ass0­
ciates. . .
Diabetes will remain at Washington University in S1. Louis
through 1996, its second 5-year tour at this institution. It is appropriate to mention some measures. of journal
However, I will step aside as editor at the end of 1995. Dr. performance. Perhaps the most informative descriptive sta­
Julio V.Santiago. the hardest working of the hard-working tistic is the time between manuscript receipt and the initial
associate editors, will serve as editor through 1996. The editorial decision, a combined measure of the performance
rationale for this change included the perception of a poten­ of the editors, the reviewers, and the editorial office (as well
tial conflict between my role as editor of Diabetes and my as the various telecommunication and mail services). For
role as president-elect and then president of the American regular publication manuscripts, the mean :t SD (range)
Diabetes Association, as well as the increasingly time-con­ receipt to initial decision interval decreased from 56 :t 24
suming extra-university demands of the latter. After all, I still days (1-154 days) in 1992, our first year, to 44 :t'22 days
have a rewarding day job that I hope to retain! The Associ­ (1-173 days) in 1993 (P < 0.001). It was unchanged at 44 :t
ation has selected Dr. Gordon C_ Weir as the new editor for 23 days (1-125 days) in 1994. Although it is too early to
the term starting in 1997. I wish Dr. Weir and his colleagues calculate the final 1995 figure, it does not appear to have
well. changed substantially. These are similar to the 1989-1991
I am grateful to many individuals for making my term as intervals (1). Thus, this measure'seems to have been reduced
editor both enriching and pleasurable. They include our to its minimum using the current editorial methods. Perhaps
authors. The quality of a scientific journal cannot exceed the new methods can reduce it further.
quality of the science submitted to it for publication. They The interval from receipt to initial (often final) decision for
also include our reviewers, including previous and current rapid publication manuscripts also decreased from 15 :t 10
members of the editorial board. Those invaluable volunteers days (1-66 days) in 1992 to 12 :t 7 days (1-33 days) in 1993
provide critical expert advice to both authors and editors. It (p < 0.05), was unchanged at 12 :t 8 days (1-30 days) in
has been a particular pleasure to work with a diverse group 1994, and has not changed substantially in 1995. Thus, these
receive substantially expedited review. The trade-off is that
there is usually only one outside reviewer and written
From the Division of Endocrinology, Diabetes and Metabolism and the General
critiques are not provided routinely.
Clinical Re5e'arch ('A!llter and Diabeles Research and Training ~nter, Washington The acceptance-to-publication interval is one quantifiable
University &hool of Medicine, St. Louis, Missouri. measure of th.e activity of the National Center publications
.Address correspondence to Dr. Philip E. CJ:yer, Division of Endocrinology,
DIabetes and Metabolism, Washington University School of Medicine (Box 8121), office. These intervals were 132 :t 33 days (84-270 days) in
660 S. Euclid Ave" St. Louis, MO 63110. 1992, 130:t 17 days (95-266 days) in 1993, and 120 ::!:: 17 days
Received for publication 16 June 1995 and accepted in revised form 30 June (86-212 days) in 1994 for regular publication ~anuscripts.
IDDM, insulin-dependent diabetes mellitus; NlDDM, non-insulio-<lependent dia­ Surprisingly, to me at least, diskette submission has not
betes mellitus; SI, Systente International. shOltened this interval substantially. It has, however, re­

duced the cost of publication of the journal considerably. Science Citation Index "impact factor" (basically a measure
Parenthetically, Diabetes is income-neutral to the Associa­ of the relationship between the number of citations of
tiOR Its costs are covered by revenue (including subscription articles in a journal and the nunlber of articles publiShed in
fees) not public support income. The intervals for rapid that journal) was 5.861 in the most recent year for which data

1 publication manuscripts have been somewhat shorter. Ef­

forts to further shorten the interval for the latter have been
initiated, but I am chagrined to acknowledge a similar
are available (1992). That was higher than that of any other
diabetes-oriented journal (range 5.261-0,111). Diabetes is a
successful venture of the American Diabetes Association.
promissOry note concerning the acceptance to publication
interval for both regular and rapid publications 4 years ago
Aside from understandable disagre.ements about negative Were it not for hypoglycemia, diabetes would be easy to
editorial decisions, there has ~~n rather little conflict be­ treat. One would simply give enough insulin (or, in those
tween authors and the editors over the past 4 years. We have individuals with enough residual endogenous insulin secre­
stayed the course with respect to Syslkme International (SI) tion, sulfonylurea) to lower plasma glucose levels to or
units in principle, although we have probably missed some below the nondiabetic range. However, hypoglycemia is a
violations in practice. The SI unit table was u{Xlated and reality. Glucose is a critical metabolic fuel, particularly for
modifie.d to include suggested numbers of significant digits. the brain. With current treatment methods, hyperinsuline­
Indeed, my irritation with use of excessive significant digits mia., whether exogenous or endogenous, sufficient to lower
has been a source of amusement to some of the 'a5Sociate plasma glucose levels carries the risk of lowering them too
editors. Perhaps the issue that continues to raise the most far. Hypoglycemia is, indeed, the limiting factor in the
discussion is our use of the figure of 6.0 to convert insulin management of diabetes (4). Because of that reality, the
concentrations from tLU/ml (mUll) to pmolfl. The difficulty, management of diabetes is currently much more complex
of course, is in converting biological activity to molar units. than lowering plasma glucose concentrations.
V0lund et al. (2) found 1.0 unit to correspond to 6.0 nmol In theory, we could eliminate iatrogenic hypoglycemia if
human insulin by quantitative amino acid analysis. This is we were to learn to replace insulin in a truly physiological
very close to that of 5.95 nrnol/U determined by bioassay (3). fashion, to prevent, correct, or compensate for compromised
The change to the 6.0 conversion figure in the SI unit table in defenses against developing hypoglycemia, or both. The
Diabetes was made in 1989 by the previous editors. We have latter notion is based on the premise that it is the integrity of
continued to use that figure. the glucose counterregulatory systems--in their broadest
Changes in journal practices have included applying the sense, including both phYSiological defenses against falling
Association's duality of interest policy to authors as well as plasma glucose concentrations (primarily decrements in
editors, accepting manuscripts on diskette, increasing page insulin and increments in glucagon and epinephrine) and
charges (to $50), and refonnatting the journal. The latter symptom-initiated behavioral defenses (e.g., food ingestion)
made Diabetes more readable in my opinion. Readers seem -that determines whether or not relative or absolute ther­
not to have noticed; we received only one (positive) com­ apeutic insulin excess results in an episode of hypoglycemia
ment from a reader. (4). This concept likely explains the frequency and sever­
Diabetes has strict editorial page limitations, 1,680 pages ity of iatrogenic hypoglycemia Those people with the most
per volume (year). The average was 1,686 pages published compromised glucose counterregulation, e.g., most people
per year in 1992-1994. This necessitates rather high rejection with fully established insulin-dependent diabetes mellitus
rates: 68-71% for regular publications and 72-88% for rapid (IDDM), suffer iatrogenic hypoglycemia, including severe
publications in 199'2-1994. Nonetheless, editorial decisions hypoglycemia, most frequently. However, it seems unlikely
have been and con,tinue to be baSed on the editors' scientific that this concept explains all iatrogenic hypoglycemia. The
judgment. Indeed, page counts have not been made known to former notion, physiological insulin replacement, is attrac­
the associate editors, wh.Q With my concurrence have made tive because of its conceptual simplicity despite its current
the vast majority of the editorial decisions, until after a given impracticality.
volume is completed. Insufficient insulin secretion is the proximate cause of
Particularly in view of limited editorial space, the editors diabetes. Relative resistance to insulin action is a feature of
believe that Diabetes should focus on publication of original the pathophysiology of both non-insulin-dependent diabetes
research. Thus, the bulk of the editorial pages have been mellitus (NIDDM) and IDDM. It may well playa role in the
committed to regular and rapid publications. There were 203, pathogenesis of NIDDM (5-8) and is relevant to the manage­
218, and 195 regular publications and 15, 14, and 6 rapid ment of NIDDM to the extent that measures that reduce
publications in 1992, 1993, and 1994, respectively. One, insulin resistance (e.g., caloric restriction and exercise)
usually invited, Perspective in Diabetes has been published result in improved glycemic control. Nonetheless, regardless
each month. We do not have a regular letter to the editor of the degree of insulin resistance, diagnosable diabetes does
section, although some (five in 1992-1994) letters that clarify not develop until insulin secretion declines and becomes
articles published in the journal or speak to controversial insuJficient to meet the metabolic demand (5-8). Thus,
issues stimulated by a published paper have been published. relative or absolute insulin defiCiency is a prerequisite of
We have published very few editorials. clinical diabetes.
Clearly, the journal can be improved. Overall, however, I Ideally, we would like to prevent a given disease. If we
Jelieve D'iabetes is not broken and does not need to be fixed cannot prevent it, we would like to cure it If we can neither
in a major way. It will be interesting to learn the views of the prevent nor cure the disease, we would like to manage it
new editors. Over its 44-year history, Diabetes has become a effectively. If the disease is, like diabetes, the. result of
widely respected and influential subspecialty jOlUTlal. Its insufficient secretion of a hormone, effective management

implies physiological replacement of that hormone. Few disease and the leading cause of blindness in working-age
chronic homlone deficiency diseases can as yet be prevented adults, of nontraumatic lower extremity amputations, and of
'or cured, but several can be managed effectively. For exam­ end-stage renal disease requiring dialysis and transplanta­
ple, in appropriate doses, thyrorine ingestion in hypothyroid­ tion. The cost of all health care for people with diabetes was
ism mimics normal thyroid hormone secretion and produces estimated to be $105 billion, 15% of all health care expendi­
physiological hormone replacement. That is not the case for tures, in the U.S. in 1992 (10). Notably, less than one-half of
insulin replacement in diabetes. All current insulin replace­ one percent of that sum was spent on diabetes research.
ment regimens are imperfect compared with normal insulin Founded in 1940 as a professional society, the American
secretion. Insulin-treated (or sulfonylurea-treated) people Diabetes Association is now a remarkable amalgamation of a
with diabetes have periodic hypoinsuI!nemia with its result­ leading professional society and a leading voluntary health
ant hyperglycemia and, not infrequently, periodic hyperinsu­ organization. Our Association has grown substantially and in
linemia with its risk of hypoglycemia Parenthetically, even if many ways matured over the past few years. Our volunteers
drugs that enhance sensitivity to endogenous insulin (in a have been highly successful in fund raising; our annual
generic sense) are shown to be safe and effective, it is most expenditures, about two-thirds derived from public support
unlikely that they would produce euglycemia that is sus­ and the remainder from revenue, are now about $90 miJlion.
tained over a lifetime of NIDDM, since NIDDM is typically a The mission of the American Diabetes Association is a
progressive disease (9). Insulin replacement would ulti­ noble one: to prevent and cure diabetes and to improve the
mately be necessary in most, if not all, patients who survived lives of all peopJe affected by diabetes. The Association
to that point in the course of their diabetes. provides organizational and fund raising support and an
Perfect insulin replacement would correct all of the met­ array of programs, organized around the themes of research,
abolic abnormalities of diabetes that are known to be information, and advocacy, to the diabetes community.
clinically relevant, perhaps with no risk of hypoglycemia. It Among these programs, only research will prevent and cure
would alleviate all symptoms and prevent acute metabolic diabetes. Research has inlproved the lives of all people
complications, almost assuredly prevent the specific chronic affected by diabetes, and it will continue to do so.
complications (retinopathy, nephropathy, and neuropathy) Although our research awards and grants budget is small
(10), and likely reduce atherosclerotic risk Aside from the relative to federal research expenditures, our Nationwide
need for therapeutic intervention, it would approximate cure Research Program is unique and makes a difference. It
of the disease just as thyrorine replacement does in hypo­ supports the training of the next generation of diabetes
thyroidism. FurthemlOre, a truly safe and effective method of investigators through Career Development Awards, Mentor­
- ..Derfected insulin replacement would undoubtedly be appli­ Based Postdoctoral Fellowships, and Medical Student Fel­
~le to people with NIDDM as well as those with lDDM. lowships. Furthennore, it supports the testing of innovative
At a minimum, perfected insulin replacement would re­ ideas through investigator-initiated Research Awards, Clini­
quire rninute-to-rninute plasma glucose-regulated insulin de­ cal Research Grants, and Uons SightFirst Research Awards.
livery into the circulation. (To the extent that factors in Finally, it supports selected areas of special research inter­
addition to glucose are relevant to physiological regulation est, including the Association's Genetics of Non-Insulin­
of insulin secretion, even that might not provide perfect Dependent Diabetes (GENNID) study, designed to facilitate
insulin replacement.) Several approaches are on the horizon identification of the gene abnormalities responsible for sus­
(some have been there for a rather long time). These include, ceptibility to NlDDM, and the Diabetes Prevention Trial I,

but are not necessarily limited to, development of a dosed­ designed to determine if lDDM can be prevented. Thus, to a
loop insulin delivery system (sensor, ~omputer, pump), large extent, the Association nurtures the human and con­
transplantation of normal ll-1Sulin-secretirig tissues (pancre­ ceptual infrastructure of future diabetes research.
as, islets), and implantation of cells converted to the task of The American Diabetes Association's research awards and
glucose-regulated insulin secretion by gene manipulation. grants expenditures were $8.6 million in fiscal year 1995. i

Because we do not know how diabetes will be prevented These were dollars actually spent by investigators. Associa­
and cured, we must continue to support a broad range of tion overhead costs are not included in this figure, nor are
fundanlental research potentially relevant to those goals. other expenses conventionally allocated to research by not­
Diabetes will be prevented and cured, but we do not know for-profit charitable organizations. In fiscal year 1995, train­
when. Pending that time, perfected insulin replacement is a ing awards (n 73), investigator-initiated awards and grants
reasonable short-term goal for the diabetes research COITUllU­ (n = 63), and support of our areas of special research
nity. Pending that time, there is also a pressing need to interest constituted 35, 43, and 22%, respectively, of our
continue to communicate the outcomes of research in peer­ research awards and grants expenditures.
reviewed journals such as Diahetes and for even greater Over the past decade, our research awards and grants
efforts by the volunteers and staff of the American Diabetes expenditures have not grown in parallel with the approxi­
Association. mately threefold increase in our public support. Measures to
increa.')e our research expenditures are being pursued ac­
tively. Policies enacted recently will ultimately increase our
THE AMERICAN DIABETES ASSOCIATION research awards and grants expenditures in parallel with our
--"s the readers of Diabetes know all too well, diabetes is a public support. Among other direct inputs, affiliate voluntary
.Dmmon, currently uncurable but treatable, potentially dev­ contributions to research are critically inlportant and are
astating chronic disease that is extraordinarily expensive in being solicited actively. Although it will invite the charge of
both human and dollar costs. An estimated 14 million Amer­ parochialism, I must point out that tlle Missouri Affiliate
icans, more than 5% of the U.S. population, have diabetes. It continues to lead the way in this category. In fiscal year 1995,
is a m~or cause of premature death from ?-therosclerotic its voluntary contribution to research was $405,000. If the I~
other 51 affiliates were to average that sum, our research ation volunteers, we can make a difference. We can acceler­
• awards and grants budget would triple! Finally, the newly ate the prevention and cure of diabetes and, pending that,
esta,blished American Diabetes Association Research Foun­ improve the lives of all people alfected by diabetes through
dation is expected to boost the awards and grants budget in research as well as through our other programs and services.
the future, hopefully the near future.

1 The American Diabetes Association Strategic Plan for

1995-1998 reflects widespread support for increased support
of research. It envisions a near doubling of our awards and
1. Robertson RP: HIstory of Diabeles, the journal (1952-11191). DUlbele.~
41:1-5. 1992
2. V_lund A, Br.mge J, ~ K, Jensen I, Markussen J, Rikel U, Sorensen
grants budget over 3 years, increased support of the training AR, Schlichtkrull J: In vitro and in vi\'o potency of insulin analogues

1 component of our research program, and increased Associ­

ation-wide ownership of our Nationwide Research Program.
It seems clear to me thai the{~arch support gene is
designed for cllnical·use. Diabetic Med 8:839-847. 11191
3. Pingel M, Velund A, Seremen E. Serensen AR: Assessment of insulin
potency by chemical and biological methods. In Hornume D''Ugs. Gueri·
guian JL, Bransome ED. Outschoom AS. Eds. Rockville, MD. U.S.
expressed, albeit at diverse ~,'in all American Diabetes Pharmacopeia! Convention, 1982, p. 200-207
Association volunteers and staff and that the rate of its 4. CIyer PE: Hypoglycemia: the limiting factor in the management of lDDM.
Diabetes 43:1378-1389,1994
transcription is increasing. Nonetheless, there is a critical 5. TllYlor SI, Acdli D, Imai Y: Insulin resistance or insulin defkiency: which
need for positive transcription factors. The medical and is the primary cause ot NIDDM? Diabetes 43:735-740. 1994
6.. Bogardus C: The case for insulin resistance as a necessary and suftlcient
scientific community can serve as those transcription fac­ cause of !we n diabetes mellitus.. J lAb ain Med 125:556-559, 1995
tors. .! would hope that many more physicians, other health 7. ROOertson RP: Diabetes and insulin resistance: philosophy, science and
care providers, and biomedical scientists would become the multiplier bypothesis. J Lab ain Med 125:560-565, 19Q5
8. Pimenta W, KoQltkowski M. Mitrakou A, Jenssen T. Yki.,.Jarvinen H,
actively involved in their American Diabetes Association Evron W, Dalley G, Gerich J: Pancreatic beta-reU dysfunction as the
chapters and affiliates, as well as the national organization. primary genetic lesion In NIDDM. J Am Med Assoc 273:1855-1861, 1996
While that involvement might be focused, ideally it would 9. V.I. Prospective Diabetes Study Group: U.I. Prospective Diabetes Study
16: overview of 6 years' therapy of type n diabetes: a progressive disease.
include participation in the full range of .Association pro­ Diabetes 44:1249-1258, 1995
grammatic themes-research, information, and advocacy-and 10. Diabetes Control and Complications Trial Research Group: The relation­
ship of glycemic exposure (HbA 1,,) to the risk of development and
the critical area of income development. With the many progression or retinopathy In the Diabetes Control and Complications
already engaged and the legion of other committed Associ­ Trial. Diabetes 44:968-983, 1995


Proc. Natl. Acad. Sci. USA
Vol. 92, pp. 1545-1549, February 1995

Long-lived testosterone esters in the rat

*Yale University School of Medicine, Department of Obstetrics and Gynecology, and the Comprehensive Cancer Center, New Haven, CT 06510; and tChildren's
Hospital-Oakland Research Institute, Oakland, CA 94609
Communicated by Seymour Lieberman, St. Luke's-Roosevelt Institute for Health Sciences, New YorkA NY, November 7, 1994

ABSTRACT Over the past decade it has become increas- There are now numerous studies showing that fatty acid
ingly clear that steroid hormones are enzymatically esterified esters of almost all of the families of steroid hormones are
with fatty acids. These steroidal esters are the natural analogs synthesized in vitro (12, 13). However, there are only a few
of synthetic esters that are used therapeutically. One such which demonstrate that steroid esters exist endogenously.
family of pharmacological steroids is the synthetic alkyl esters After the esters of the A5-33-hydroxysteroids were discovered
of testosterone, androgens with great hormonal potency. We in the adrenal gland (1, 2), esters of dehydroisoandrosterone
have investigated whether testosterone esters exist naturally and pregnenolone were found in blood (14) and pregnenolone
by using the rat as a model. Most tissues of male rats, includ- esters were found in human ovarian follicular fluid (15). Fatty
ing blood, have very little if any ester (quantified by immu- acid esters of reduced metabolites of progesterone and tes-
noassay as a nonpolar saponifiable metabolite), but fat and tosterone have been isolated and characterized by MS, reveal-
testes have sizable quantities, -3 ng of testosterone equiva- ing androsterone esters in human breast cyst fluid (16) and
lents per g of tissue. Testosterone in fat averages 9 ng/g. The allopregnanolone (as well as pregnenolone) in bovine corpora
fat from female rats and long-term (>2 weeks) castrated luteum (17). There are very few studies of endogenous esters
males has no detectable testosterone ester. The presence of of biologically active steroids. Esters of E2 are present in
testosterone esters was confirmed by GC/MS, which clearly limited concentration in blood (17), with much greater
showed the presence of testosterone in the hydrolyzed ester amounts in fat (18). Relatively large amounts of E2 fatty acid
fraction of fat from intact males but not long-term castrates. esters have been found in human ovarian follicular fluid,
Upon castration, testosterone levels in the fat completely enabling their complete characterization (18). Other reports of
disappear within 6 hr. To the contrary, it is not until 48 hr esters of active steroids are more tenuous. Esters of testoster-
after castration that a measurable fall in the testosterone ester one (19,20) and the corticoids, cortisol and corticosterone (21,
fraction was observed; even after 10 days a small amount of 22), have been reported to circulate in sizable amounts in
ester is still present in the fat. These experiments demonstrate human blood. Although the report of corticoid esters in blood
the existence of a previously unknown androgen with a po- was first published in 1960, there has been no independent
tentially important physiological impact; testosterone esters, confirmation. The evidence for testosterone esters in blood is
natural analogs of potent therapeutic agents, occur in the fat uncertain since neither long-term inhibition of steroidogenesis
where they can serve as a reservoir of preformed androgen to nor castration reduced the concentration of the putative com-
stimulate neighboring target tissues. pound (20).
The existence of natural esters of androgens is of impor-
Although fatty acid esters of sterols, such as cholesterol, have tance because, like estrogen esters, synthetic alkyl esters of
been known for decades, the existence of naturally occurring androgens have been used therapeutically for decades due to
fatty acid esters of steroids is a much more recent discovery. their high potency and prolonged action (23). Thus, biological
In 1979, putative steroidal esters of the A5-3(3-hydroxysteroids, esterification of androgens, such as testosterone, would be
pregnenolone (1), dehydroisoandrosterone, and 17a-hy- expected to have a dramatic effect on both the potency and the
droxypregnenolone (2), were discovered in the adrenal gland. life of the male hormone. This paper reports a study in which
They were named lipoidal derivatives to convey their nonpolar the existence of fatty acid esters of testosterone (TL) in tissues
nature and their ability to be converted back to the parent steroid of the male rat was examined and the hypothesis was tested that
as a result of mild hydrolytic procedures. They were subsequently
TL is long-lived when compared to testosterone.
identified as fatty acid esters (3). These findings raised the pos-
sibility of the existence of similar esters of biologicaLly active MATERIALS AND METHODS
steroids. Such compounds would be the natural analogs of syn- [1,2,6,7,16,17-3H]Testosterone (100 Ci/mmol; 1 Ci = 37 GBq)
thetic steroidal esters that have been used pharmacologically as purchased from New England Nuclear was refluxed with alkali
extremely potent and long-lived hormones. The well known ther- to remove labile 3H (24) and then purified by HPLC (25); final
apeutic use of one such family of pharmacological steroid hor- specific activity was 92 Ci/mmol. Testosterone stearate and
mone esters, the estrogens (4), led to experiments which showed [3H]testosterone stearate were synthesized by esterification
that estradiol (E2) is biosynthetically converted into a lipoidal with stearyl chloride and purified exactly as described (25).
derivative, LE2 (5), a nonpolar metabolite, which was identified Sprague-Dawley rats (Charles River Breeding Laboratories)
as a family of C-17 fatty acid esters of E2 (6). Although LE2 is not between 3 and 5 months old were castrated under methoxyflu-
estrogenic when esterified (7)-i.e., it does not bind to the es- rane (Metofane; Pitman-Moore, Washington Crossing, NJ) an-
trogen receptor directly (8)-it is converted to E2 by esterase esthesia (day 0). At the stated times, animals were decapitated
action. The fatty acid esters comprising LE2 are extremely long- while under Metofane anesthesia and the tissues were removed
lived (9) and, thus, act as a reservoir of E2. They represent the and immediately frozen. Blood was obtained by cardiac puncture,
most potent of the naturally occurring steroidal estrogens (10, placed on ice, and centrifuged at 4°C, and the serum was re-
11). moved. Fat from various areas was combinded and mixed. Ap-
The publication costs of this article were defrayed in part by page charge Abbreviations: TL, testosterone ester; E2, estradiol; LE2, E2 lipoidal
payment. This article must therefore be hereby marked "advertisement" in derivative; THF, tetrahydrofuran.
accordance with 18 U.S.C. §1734 solely to indicate this fact. VTo whom reprint requests should be addressed.
1546 Biochemistry: Borg et al. Proc. Natl. Acad. Sci USA 92 (1995)

proximately 250 mg of each tissue was weighed, transferred to a displaces '20% of the bound tracer. The blank (no tissue)
test tube (16 x 125 mm) containing 2 ml of methanol and carried through the entire procedure was generally 7-10 pg.
homogenized with two 10-sec bursts of a Polytron homogenizer The results of the RIA were corrected for the blank and for
(Brinkmann). To correct for experimental losses, a representative recovery of the internal standard and normalized for the size
fatty acid ester, 5000 cpm of [3H]testosterone stearate (13 pg; of the aliquot and the weight of the tissue. They are reported
testosterone molar equivalent), was added as an internal standard as pg, molar equivalents, of testosterone (pg T equiv) per g of
in 50 ,ul of ethanol. When testosterone was measured, an internal tissue.
standard of [3H]testosterone, 5000 cpm, was also added. The To verify that the alumina chromatography eliminates tes-
suspension was mixed, 4 ml of chloroform was added, and it was tosterone from the nonpolar TL fraction, two experiments
Vortex mixed again. Two milliliters of water was added and, after were performed. In one, 100,000 cpm of [3H]testosterone was
thorough mixing, the suspension was clarified by centrifugation at added to 250 mg of fat and the amount of radioactivity con-
1600 x g and the bottom organic layer was removed with a taminating the TL fraction was measured: it was - 100 cpm, or
Pasteur pipette. The aqueous layer with the tissue residue was 0.1%. In another experiment, 10 ng of testosterone was added
extracted again with 4 ml of the organic layer obtained by par- to 250 mg of female fat and the TL was analyzed. There was
titioning chloroform/methanol/water in the ratio 2:1:1. The or- no measurable testosterone in the TL fraction (see Table 1).
ganic extracts were combined, evaporated under N2, and then put When the fat samples from female rats or long-term castrated
under vacuum at 50°C for 10 min to remove all traces of alcohol. males (>2 weeks) were analyzed by this protocol, the amount
With serum, a slightly different extraction procedure was of TL measured was not significantly different than the blank
used to increase the extraction yield. Four milliliters of freshly (see Fig. 2) and in most experiments it was the same as the
distilled tetrahydrofuran (THF) was added to 1 ml of serum, blank. When TL in fat samples from male rats was analyzed,
followed by the 3H internal standards and then 1 ml of brine. significant amounts were found (see Table 1 and Fig. 2). If the
The solution was vigorously mixed and then centrifuged. The TL fraction was not saponified (mock procedure) no testos-
THF was removed and the aqueous residue was extracted terone was found (see Table 1). We assayed 10 ng of testos-
again with an additional 2 ml of THF. The THF layers were terone stearate directly by the RIA, and none was detected
combined and evaporated under N2. The residues from the
extracts of the various tissues and serum were dissolved in 1 ml (data not shown). The testosterone ester does not cross-react
of benzene/hexane (3:1) and transferred to a column (6 x 0.5 in the assay unless it is hydrolyzed. The accuracy with which
cm) of alumina (3% H20) equilibrated in the same solvent. this assay measures TL was determined by adding various
The column was washed with 10 ml of benzene and the TL amounts (100-1500 pg T equiv) of exogenous testosterone
fraction (which contains the 3H internal standard) was eluted stearate to -250 mg of female rat fat. The TL was measured
with 10 ml of ethyl acetate/benzene (1:20). The column was as described and the results including the 95% confidence
washed with an additional 10 ml of ethyl acetate/benzene limits are shown in Fig. 1.
(1:20) and the testosterone fraction was obtained with 10 ml To confirm the RIA identification of testosterone in the TL
of ethyl acetate/benzene (2:3). The TL fraction was trans- fraction, fat samples from male and long-term (>2 weeks)
ferred to a screw cap test tube (16 x 100 mm) equipped with castrated male rats were analyzed by GC/MS. One gram of
a Teflon liner and evaporated under N2, and the residue was each (divided into four separate 250-mg samples) was ex-
dissolved in 100 ,ul of benzene, 900 ,lI of methanol, 100 ,lI of tracted and saponified as described above. After the addition
10% aqueous potassium carbonate, and heated overnight at of acetic acid and extraction with isooctane, the methanolic
50°C. Samples undergoing mock saponification were treated in solutions were applied to C18 SPICE columns (Analtech). The
the same manner but the potassium carbonate was omitted. columns were washed with 5 ml of methanol/water (1:1), after
Afterward, 100 ,ul of 9% aqueous acetic acid was added and which the testosterone was eluted with 5 ml of methanol/water
any residual ester was removed by extraction with 2 ml of (7:3). The four samples were combined, the methanol was
isooctane. Testosterone esters partition in the isooctane, and removed under N2, and the aqueous residue was extracted
testosterone partitions in the aqueous methanol. The hydro- twice with 2 vol of ether. The ether layers were evaporated
carbon layer was discarded and 900 ,ul of water was added to under N2 and the residues were dissolved in benzene.
the aqueous methanol layer. The alcohol was removed under The testosterone fractions were derivatized before analysis
N2 and the aqueous residue was extracted twice with 5 ml of by conversion of the C-3 carbonyl group to a methyloxime and
ethyl ether. The ether extracts were combined and evaporated the 1713-hydroxyl group to a trimethylsilyl ether (27) as follows.
under N2, and the residue was transferred to a test tube (12 x The samples were dried under N2, dissolved in 50 IlI of a 2%
75 mm) with several washings (total < 0.5 ml) of acetonitrile. solution of methoxyamine hydrochloride in pyridine (Pierce),
After the organic solvent was evaporated, the residue was and heated for 1 hr at 60°C. After removal of pyridine under
dissolved with vigorous Vortex mixing in 150 p,l of human N2, 50 ,ul of trimethylsilylimidazole (Pierce) was added and
serum that had been stripped of endogenous steroid with derivatization was allowed to proceed for 1 hr at 100°C. Excess
dextran-coated charcoal (26). We have found that dissolving reagent was removed by diluting the sample with 1 ml of
the residue in steroid-free serum and then analyzing for the cyclohexane and passing it through a short column (in a
steroid by using a nonextraction RIA decreases the blank. Pasteur pipette) of Lipidex 5000 (Packard) equilibrated with
However, this step is not absolutely necessary. An aliquot of 30 cyclohexane. The testosterone derivative was eluted with 2.5
Al of serum was assayed to determine the recovery of the 3H ml of cyclohexane. The solvent was evaporated under N2, the
internal standard, usually '40%. Samples in which the recov- resulting residue was dissolved in 20 Al of cyclohexane, and 2
ery was <25% were not used. The unesterified testosterone ,ul was injected into the GC/MS.
fraction, obtained from the alumina column with ethyl ace- The GC/MS analysis was carried out with a DB 1 column (15
tate/benzene (2:3), was evaporated under N2 and then dis- m) (J & W Scientific, Rancho Cordova, CA) housed in a
solved in steroid-free serum (recovery 70%). Both the TL Hewlett-Packard 5790 gas chromatograph attached to a 5970
(hydrolyzed) and testosterone fractions were analyzed by RIA: mass spectrometer (Hewlett-Packard). The sample was in-
two aliquots of the serum (50 ,u each) were analyzed directly jected by splitless injection with the oven temperature remain-
by using a nonextraction 1251 RIA for testosterone CAC-TKTT ing cool (50°C). After 3 min, the oven temperature was in-
(Diagnostics Products, Los Angeles). The commercial RIA is creased to 230°C at the rate of 27°C/min. The oven temper-
described by the manufacturer as specific for testosterone- ature was then raised by 2°C/min to a final temperature of
that 5a-dihydrotestosterone cross-reacts only negligibly 300°C. The eluent was analyzed by selected ion-monitoring
(which we confirmed). In the assay, 10 pg of testosterone using the parent ion (M+) of testosterone methyloxine tri-
Biochemistry: Borg et aL Proc. NatL Acad Sci USA 92 (1995) 1547
methylsilyl ether (m/z 389) and fragments m/z 358 (M-OCH3) 2000
and m/z 125 (A-ring fragmentation).

The procedure designed to measure TL in various tissues
quantifies the testosterone released by saponification from the
nonpolar, TL, fraction. An internal standard of [3H]testoster-
one stearate is added as a representative ester of TL to correct .-
for procedural losses. The tissue is extracted and the extract is
chromatographed on a column of alumina in order to obtain
the nonpolar TL fraction free of testosterone (see above). The
TL fraction is then saponified with potassium carbonate, par-
titioned between aqueous methanol and hexane to remove
unhydrolyzed steroid, and analyzed with a RIA for testoster-
one. The results are corrected for the recovery of the 3H
internal standard. The validity of the procedure was tested in
several ways. We showed that this procedure eliminates tes-
tosterone from the TL fraction. When [3H]testosterone was
added to fat, negligible radioactivity was found in the TL
fraction. Furthermore, when 10 ng of testosterone was added 0 200 400 600 800 1000 1200 1400 1600 1800 2000
to female fat (equivalent to 40 ng/g, an amount far in excess T-St added (pg T equiv.)
of endogenous testosterone) none of the added testosterone
was measured in the TL fraction (Table 1). When fat from FIG. 1. Quantification of testosterone esters in fat. Various con-
male rats was analyzed by this procedure, with the exception centrations of testosterone 17-stearate (T-St) were added to 250-mg
that a mock saponification was carried out in which potassium portions of female rat fat. The fat was extracted and the TL fraction
carbonate was omitted from the incubation, no assayable tes- was isolated and saponified; testosterone was measured by RIA as
tosterone was found in the TL fraction (Table 1). This was described in the text. Individual samples are shown. Dotted lines
expected since testosterone esters are not measured in the represent 95% confidence limits.
RIA unless first hydrolyzed to testosterone. When various not in the fat of the female or the long-term castrate. While there
amounts of testosterone stearate were added to fat and then may be some Th in both of the latter groups, the amount is below
carried through the entire procedure, including saponification, the sensitivity of the assay (see Materials and Methods). For
the exogenous ester was accurately detected (Fig. 1). A variety comparison, in the experiments shown in Fig. 4, testosterone in
of tissues from male rats was analyzed for TL by this assay. As male fat was also measured, averaging 9700 ± 1100 pg/g.
shown in Table 1, most tissues contain only negligible amounts TL was also detected in the testes (Table 1), where it is 4000
of saponifiable testosterone in the Th fraction. This includes pg T equiv/g. Since the testes have very high levels of testoster-
serum in which a larger sample volume, 1 ml, was analyzed in one, we again showed in this tissue that the TL was not caused by
order to increase sensitivity. However, male fat contains signif- contamination with the endogenous testosterone. The TL frac-
icant amounts of TL: the TL in fat was assayed in intact males, in tion was put through a mock hydrolysis (without potassium
males that had been castrated for periods of >2 weeks, and in carbonate) and then analyzed. There was no testosterone in the
females. The results of this experiment are presented in Fig. 2. nonhydrolyzed TL fraction from testes (Table 1).
The amount of TL measured in these experiments (in T equiv per The hydrolyzed TL fraction isolated from male rat fat was
g of tissue ± SEM) is as follows: males, 2400 + 174; females, 54 also analyzed by GC/MS in order to confirm that the immu-
+ 14; long-term castrated males, 106 ± 45. In the experiments
shown in Fig. 4 (the normal male is at time 0), the TL in male fat 3000 -
averaged 2700 ± 199. In all experiments on intact male rats, the
TL in fat ranged from 1100 to 4100 pg T equiv per g. Thus, there male
is a relatively large amount of TL in the fat of the intact male but 2500 - 1 female
Table 1. TL in tissues of rat 1-1
T L > 2 week castrate male

TL, pg T 0 2000 -
Tissue equiv/g 0

Male fat* 2400 ± 174 0

Male fat (nonsaponified)t ND St0 1500 -
Female fat ND 0.)
Female fat + testosterone (40 ng/g)t ND
Testes§ 4025 ± 537 0O 1000 -

Testes (nonsaponified)t ND IJ
Male serum, female serum, male brain,
male liver, male muscle, male spleen ND 500 -
ND, nondetectable values ranging from 0 to 150 pg/g. Each tissue
was analyzed in at least three different assays. Data for TL are means
± SEM expressed as pg T equiv per g of tissue. 0 -
*From data in Fig. 2.
tThe TL fraction was isolated as usual and then subjected to a mock FIG. 2. TL in adipose tissue of rat. Samples of fat (-250 mg) were
saponification from which potassium carbonate was omitted. analyzed for TL by RIA of the saponified TL fraction. Results are
ITestosterone, 10 ng per sample, was added to -250 mg of fat. means from -10 separate experiments. Total number of fat samples
§Mean of four experiments on a total of eight different samples: range, analyzed are as follows: male, n = 29; female, n = 22; castrate, n =
1626-5728. 16. Error bars are SEM.
1548 Biochemistry: Borg et aL Proc. Natl. Acad. Sci USA 92 (1995)

2800A 2500B
2600 male 2000 castrate male
1800 500
16.7 mm 17.2 16.7 mi 17.2 16.7 17.2
mm 7.
FIG. 3. GC/MS of saponified TL fraction from rat fat. Partial chromatograms of the [M]+ (m/z 389) of testosterone methoxime trimethylsilyl
ether. Samples of fat (1 g) from intact (A) and long-term castrated (B) male rats were extracted, saponified, and converted to the methyloxime
trimethylsilyl ether as described in the text. The chromatogram (C) is testosterone run simultaneously as a reference. Analysis was performed on
samples in A and B with aliquots that were representative of identical portions of tissue (as determined by recovery of the 3H internal standard).
In each panel the ordinate is the selected ion, m/z 389. The two incompletely resolved peaks at 16.9 and 17.0 min represent the syn and anti forms
of the methyloxime derivative.
noassayable material is testosterone. The TL fraction from a at those times (Fig. 4A) and even after an entire day (Fig. 4B),
normal male and a long-term male castrate was converted to TL levels did not decline appreciably. Only after 48 hr of cas-
testosterone by saponification, purified by chromatography on tration is a decrease in the concentration of TL noticeable. The
a C18 column, derivatized to form the methyloxime trimeth- TL levels in fat declined slowly thereafter, until 10 days when
ylsilyl ether derivative, and then analyzed by single ion mon- only very low levels were found.
itoring GC/MS: at m/z 389 parent [M]+, at m/z 358 [M-
OCH3]+, and at m/z 125 (A-ring fragmentation). The saponified
TL fraction from the intact male rat gave a response for all three DISCUSSION
ions at the correct retention time for the methoxylamine tri-
methylsilyl ether derivative of testosterone. The partial ion chro- These experiments show that TL exists in the fat and testes of
matogram (m/z 389) of a standard of testosterone derivatized in the male rat. Although the structure of TL is not definitively
the same manner and the two fat extracts are shown in Fig. 3. The proven, it is highly likely that TL is a heterogeneous family of
two unresolved peaks at 16.9 and 17.0 min, observed with au- fatty acid esters of testosterone: its physicochemical charac-
thentic testosterone and the male fat extract, represent the syn teristics are the same as the fatty acid esters, and similar
and anti forms of the methyloxime derivative (27). These peaks lipoidal derivatives of other steroids have been shown to be
are not present in the saponified TL fraction of fat from the fatty acid esters (3, 6, 15-17, 28). The evidence that testoster-
long-term castrated male rat. one is released by hydrolysis of the nonpolar TL fraction from
Since steroidal esters are long-lived, presumably because male rat fat rests on strong evidence, both immunological and
they are protected from metabolism (9), we performed an spectral. The immunological (RIA) data show little if any TL
experiment to compare the kinetics of the disappearance of TL in the long-term castrated male or in the female rat (Fig. 2).
and testosterone from fat after castration of male rats. As This is consistent with a metabolite of testosterone. Most
shown in Fig. 4A, almost all of the testosterone in the fat compelling is the GC/MS analysis, which shows testosterone
disappeared by 3 hr after castration and by 6 hr there was no in the hydrolyzed TL fraction of male fat but not the long-term
longer any measurable testosterone in the fat. To the contrary, castrated male rat.
10000 - 5000
9000 - 4500
8000 - 4000

0 7000 - ) 3500
co --TL
- 6000 - ., 3000

QL 5000 - ' 2500

4000 - $ 2000
: 3000 - I~ ~ ~~~~--4
F 1500
2000 - 1000
1000 - 500
0 -
0 1 2 3 4 5 6 0 48 98 144 192 240
hours after castration hours after castation
FIG. 4. Effect of duration of castration on concentration of testosterone (T) and TL in male rat fat. Each point is the mean of at least three
separate determinations. Total number of different fat samples analyzed for each point is .5. Error bars are SEM.
Biochemistry: Borg et aL Proc. NatL Acad Sci USA 92 (1995) 1549
There is no TL in the other tissues tested (Table 1), including terone through the action of esterase(s). Hormonal communi-
muscle and blood. There is also no TL in the brain. This is of cation from the target tissue to the fat could signal adipose cells
interest because the first demonstration of the fatty acid es- to synthesize or activate the esterase, leading to the release of
terification of a steroid hormone was the esterification of androgen at times when testicular production or blood levels of
testosterone by a brain preparation (29). In this study, while we testosterone are low. Since, in this view, the androgenic stimu-
did not find TL, we did find relatively large amounts of lation would be only through local fat and not the circulation,
testosterone in the brain (5900 ± 1000 pg/g; mean ± SEM). specificity of the target tissues is possible. Regardless of how TL
Thus, although both the substrate and the enzymatic capacity may act, the existence of this highly unusual, long-lived metabolite
to esterify testosterone is in the brain, there is no TL. Although of testosterone undoubtedly has important ramifications for our
this might appear to be contradictory, there are several reasons understanding of the physiology of the sex steroids.
why this might occur, including rapid hydrolysis of TL; the
acyltransferase enzyme found in the in vitro experiments with We gratefully acknowledge Donna Raucci and Esther Roitman for
brain has a high Km (low affinity) for testosterone, insufficient their skillful technical assistance. This work was supported by the
for the in vivo concentration; or the enzyme and substrate are National Institutes of Health (Grants DK-34400 to C.H.L.S. and
present in different regions of the brain. Regardless of the CA-25951 to R.B.H.).
reason, the synthesis or accumulation of TL is obviously more
complex than might be otherwise apparent. The tissue distri- 1. Hochberg, R. B., Bandy, L., Ponticorvo, L. & Lieberman, S.
bution of TL is consistent with our studies of the E2 esters in (1977) Proc. Natl. Acad. Sci. USA 74, 941-945.
humans in which we found that there is very little in muscle or 2. Hochberg, R. B., Bandy, L., Ponticorvo, L., Welch, M. & Lieber-
blood and that most was present in fat (18). It does not agree man, S. (1979) J. Steroid Biochem. 11, 1333-1340.
with the findings of a substance with the properties of TL in 3. Mellon-Nussbaum, S., Ponticorvo, L. & Lieberman, S. (1979) J.
Biol. Chem. 254, 12500-12505.
the blood of men (19, 20). The finding of high levels of a 4. Deghenghi, R. & Givner, M. L. (1979) in Burger's Medicinal
compound with the properties of testosterone esters in blood Chemistry, ed. Wolf, M. E. (Wiley, New York), pp. 917-939.
is unusual since the hydrophobic esters of steroids do not 5. Schatz, F. & Hochberg, R. B. (1981) Endocrinology 109,697-703.
appear to be secreted into blood (18, 30). Although fairly high 6. Mellon-Nussbaum, S., Ponticorvo, L., Schatz, F. & Hochberg,
concentrations of some steroid esters circulate in blood, they R. B. (1982) J. Biol. Chem. 257, 5678-5684.
are the esters of the A5-3f-hydroxysteroids pregnenolone and 7. Littlefield, B. A., Gurpide, E., Markiewicz, L., McKinley, B. &
dehydroisoandrosterone (14). They are formed in blood by Hochberg, R. B. (1990) Endocrinology 127, 2757-2762.
lecithin:cholesterol acyltransferase (LCAT) for which they are 8. Janocko, L., Lamer, J. M. & Hochberg, R. B. (1984) Endocri-
good substrates (14, 31). E2 esters are in also blood, but E2 is nology 114, 1180-1186.
9. Lamer, J. M. & Hochberg, R. B. (1985) Endocrinology 117,
a poor substrate for the enzyme, and this is probably the major 1209-1214.
reason why only very low levels are present. However, testos- 10. Lamer, J. M., MacLusky, N. J. & Hochberg, R. B. (1985) J.
terone is not esterified at all by LCAT (14). As pointed out Steroid Biochem. 22, 407-413.
above, the compound measured in human male blood (19, 20) 11. Zielinski, J. E., Pahuja, S. L., Lamer, J. M. & Hochberg, R. B.
did not disappear after long-term castration (surgical or phar- (1991) J. Steroid Biochem. Mol. Biol. 38, 399-405.
macological) or even complete inhibition of steroidogenesis 12. Hochberg, R. B., Pahuja, S. L., Zielinski, J. E. & Lamer, J. M.
with aminoglutethimide, and its level in blood was inversely (1991) J. Steroid Biochem. Mol. Biol. 40, 577-585.
proportional to that of testosterone (20). There are obvious 13. Poulin, R., Poirier, D., Theriault, C., Couture, J., Belanger, A. &
differences between those studies and this one, especially the Labrie, F. (1990) J. Steroid Biochem. 35, 237-247.
14. Jones, D. L. & James, V. H. T. (1985) J. Steroid Biochem. 22,
species used and many of the techniques, and so it is not 243-247.
possible to conclude that TL is not in human blood. Obviously, 15. Roy, R. & Belanger, A. (1989) Steroids 54, 385-400.
the confirmation of the presence of TL in human blood re- 16. Raju, U., Levitz, M., Banerjee, S., Bencsath, F. A. & Field, F. H.
quires direct experimentation. However, whatever that sub- (1985) J. Clin. Endocrinol. Metab. 60, 940-946.
stance is, its biological properties are very different than those 17. Albert, D. H., Ponticorvo, L. & Lieberman, S. (1980) J. Biol.
that we found for TL in rat fat (see Fig. 4). Chem. 255, 10618-10623.
These studies demonstrate the presence of a nonpolar me- 18. Larner, J. M., Shackleton, C. H. L., Roitman, E., Schwartz, P. E.
tabolite of testosterone, TL, in the fat and testes of the male & Hochberg, R. B. (1992)J. Clin. Endocrinol. Metab. 75,195-200.
rat. This metabolite is extremely long-lived when compared to 19. Addo, S. B., Diamond, E. & Hollander, V. P. (1989) Steroids 54,
the parent steroid, testosterone (Fig. 4). Prolonged biological 20. Addo, S. B., Holland, J. F., Kirschenbaum, A., Mandeli, J. &
half-life (ti12) is linked to increased androgenic potency, as Hollander, V. P. (1990) Steroids 55, 492-494.
evidenced by the synthetic androgenic esters that are used 21. Weichselbaum, T. E. & Margraf, H. W. (1960) J. Clin. Endocri-
therapeutically (23). This relationship of biological tlq2 and nol. Metab. 20, 1341-1350.
potency has been confirmed with the naturally occurring E2 22. Margraf, H. W., Margraf, C. 0. & Weichselbaum, T. E. (1963)
17-fatty acid esters, LE2, which because they are protected Steroids 2, 155-165.
from metabolism have a considerably extended biological life 23. Wilson, J. D. (1990) in The Pharmacological Basis of Therapeutics,
(9) and are therefore remarkably potent estrogens (10, 11). eds. Goodman-Gilman, A., Rall, T. W., Nies, A. S. & Taylor, P.
Likewise, it would be expected that because the endogenous (Pergamon, New York), p. 1421.
24. Osawa, Y. & Spaeth, D. G. (1971) Biochemistry 10, 66-71.
testosterone esters, TL, are long-lived, they are highly potent 25. Lamer, J. M., Pahuja, S. L., Brown, V. M. & Hochberg, R. B.
androgens. It is very interesting that in the relatively long (1992) Steroids 57, 475-479.
period after castration, when the levels of testosterone are 26. Holinka, C. F., Hata, H., Kuramoto, H. & Gurpide, E. (1986)
undetectable, sizable quantities of TL are still present in the fat Cancer Res. 46, 2771-2774.
(Fig. 4). At these times, although testosterone is not being 27. Shackleton, C. H., Merdinck, J. & Lawson, A. M. (1990) in Mass
synthesized by de novo steroidogenesis, the androgen can still Spectrometry in Biological Materials, ed. McEwen, C. (Marcel,
be produced, not by the steroidogenic enzyme that synthesizes New York), pp. 297-377.
C19 steroids, the 17-hydroxylase, but by enzymatic hydrolysis of 28. Lamer, J. M., Pahuja, S. L., Shackleton, C. H., McMurray, W. J.,
TL. While the amount of testosterone that is produced may be Giordano, G. & Hochberg, R. B. (1993) J. Biol. Chem. 268,
small if diluted into the entire body pool, locally sizable 29. Kishimoto, Y. (1973) Arch. Biochem. Biophys. 159, 528-542.
amounts may be secreted. We hypothesize that stimulation of 30. Belanger, B., Caron, S., Belanger, A. & Dupont, A. (1990) J.
specific androgen target tissues can occur by a paracrine Endocrinol. 127, 505-511.
mechanism through which TL in neighboring fat supplies testos- 31. Roy, R. & Belanger, A. (1989) J. Steroid Biochem. 33, 257-262.
Local Ventromedial Hypothalamus Glucose Perfusion Blocks Counterregulation
during Systemic Hypoglycemia in Awake Rats
Monica A. Borg,* Robert S. Sherwin,* Walter P. Borg,* William V. Tamborlane,‡ and Gerald I. Shulman*
Yale University School of Medicine, *Department of Internal Medicine and ‡Department of Pediatrics, New Haven, Connecticut 06520

Abstract signals. This view is best supported by data showing that pre-
vention of intercerebral glucopenia by infusion of glucose into
The ventromedial hypothalamic nucleus (VMH) is neces- both the carotid and vertebral arteries nearly abolishes coun-
sary for the integrated hormonal response to hypoglycemia. terregulatory hormone responses to systemic hypoglycemia (2,
To determine the role of the VMH as a glucose sensor, we 5). Recent studies suggest that the ventromedial hypothalamus
performed experiments designed to specifically prevent glu- (VMH) may be the specific region in the CNS that is responsi-
copenia in the VMH, while producing hypoglycemia else- ble for activation of counterregulatory mechanisms, since focal
where. We used awake chronically catheterized rats, in lesioning in this area abolishes the hormonal response to sys-
which local VMH glucose perfusion (100 mM or 15 mM of temic hypoglycemia (6). Moreover, production of local neuro-
D-glucose) was combined with a sequential euglycemic– glycopenia by perfusion of 2-deoxy-glucose into the VMH is
hypoglycemic clamp. In two control groups the VMH was able to trigger the release of counterregulatory hormones, de-
perfused either with (a) an iso-osmotic solution lacking glu- spite systemic normoglycemia (7).
cose, or with (b) nonmetabolizable L-glucose (100 mM). The present study was undertaken to examine whether the
During systemic hypoglycemia glucagon and catecholamine converse is also true; namely, can hormonal responses to sys-
concentrations promptly increased in the control animals temic hypoglycemia be blocked by preventing neuroglycope-
perfused with either 100 mM L-glucose or the iso-osmotic nia in the VMH, alone? For this purpose, the hypoglycemic in-
solution lacking glucose. In contrast, glucagon, epinephrine sulin clamp technique was combined with bilateral VMH
and norepinephrine release was inhibited in the animals in microdialysis in Sprague-Dawley rats; the microdialysis perfu-
which the VMH was perfused with D-glucose; hormonal se- sate contained extracellular fluid (ECF) with added glucose.
cretion was partially suppressed by the VMH perfusion with This approach allowed us to selectively prevent hypoglycemia
15 mM D-glucose and suppressed by z 85% when the VMH within the chosen brain region, and to test its effect under stan-
was perfused with 100 mM D-glucose, as compared with the dardized hypoglycemic conditions.
control groups. We conclude that the VMH must sense hy-
poglycemia for full activation of catecholamine and gluca- Methods
gon secretion and that it is a key glucose sensor for hypogly-
cemic counterregulation. (J. Clin. Invest. 1997. 99:361–365.) Animals. Male Sprague-Dawley rats were purchased from Charles
Key words: VMH • microdialysis • hypoglycemia • glucagon • River Laboratories (Raleigh, NC). Animals were housed in an envi-
epinephrine ronmentally controlled room with a 12-h light/dark cycle, and were
maintained on standard ad libitum rat diet (AGWAY Prolab 3000,
Waverly, NY) comprising of 22% protein, 5% fat, and 51% carbohy-
Introduction drate (the remaining 22% consists of ash, crude fiber, and moisture).
VMH microdialysis canulas placement. Rats (mean body weight
The central nervous system (CNS)1 plays an important role in
of z 28065 g, range 250–315 g) were anesthetized by intraperitoneal
sensing glucopenia and triggering counterregulatory hormone injection (1 ml/kg) of a mixture of Xylazine (AnaSed 20 mg/ml; Lloyd
release during hypoglycemia. (1–3). Although a region within Laboratories, Shenadonoah, IA) and Ketamine (Ketaset 100 mg/ml;
or in close proximity to the liver may activate the sympatho- Aveco Co., Fort Dodge, IA) in a ratio of 1:2 (vol/vol) and placed on a
adrenal system (4), the CNS appears to be the dominant center stereotaxic frame. Thereafter, the skull was exposed, and holes were
responsible for the sensing and integration of hypoglycemic drilled bilaterally in chosen coordinates, through which the guide can-
ulas were lowered slowly into the brain. The stereotaxic coordinates
were determined from the atlas of Paxinos and Watson (8). Specifi-
cally, VMH canulas were placed by using the coordinates 2.6 mm pos-
Address correspondence to Gerald I. Shulman, M.D., Ph.D., Yale terior and 3.8 mm lateral in relation to bregma, and the angle of 208 in
University School of Medicine, Department of Internal Medicine/En- relation to horizontal plane passing through bregma and lambda. The
docrinology, Box 208020, FMP 104, New Haven, CT 06520-8020. canulas were then secured to the skull with stainless steel screws and
Phone: 203-785-5447; FAX: 203-785-6015; E-mail: gerald_shulman@ dental acrylic. Animals were then allowed to recover from the stereo- taxic procedure for 12–16 d before study. One day before each exper-
Received for publication 19 July 1996 and accepted in revised form iment, microdialysis probes of side-by-side design (9) were inserted
5 November 1996. into the guide canulas. The length of the probes was 10.5 mm, as mea-
sured from the bregma-lambda plane. The exposed microdialysis
1. Abbreviations used in this paper: CNS, central nervous system; membrane was 1.0–1.5 mm (approximately the size of the VMH), so
ECF, extracellular fluid; VMH, ventromedial hypothalamic nucleus. that we could selectively perfuse this brain region. On the morning of
the experiment, the perfusion medium was loaded into 1-ml syringes
J. Clin. Invest. and delivered at a flow rate of 2.5 ml/min using a Harvard perfusion
© The American Society for Clinical Investigation, Inc. pump (model 22; Harvard Bioscience). Four groups of rats (two ex-
0021-9738/97/01/361/05 $2.00 perimental and two controls) were studied. In each the VMH was
Volume 99, Number 2, January 1997, 361–365 perfused with a sterile, ascorbate-free, artificial, extracellular fluid so-

Ventromedial Hypothalamic Nucleus as a Glucose Sensor 361

lution (ECF: 135 mmol/liter NaCl, 3 mmol/l KCl, 1 mmol/liter MgCl2, 35 mg/kg body wt; Abbott Laboratories, North Chicago, IL). The
1.2 mmol/CaCl2, and 2 mmol/liter sodium phosphate buffer to pH polyethylene carotid artery catheter was extended to the level of the
7.4). The VMH probe was perfused in the first group with 100 mM aortic arch, and the silicone internal jugular vein catheter was ad-
D-glucose (metabolically active isomer) and in the second group with vanced to the level of the right atrium. At the end of the procedure,
15 mM D-glucose. The third and the fourth groups of animals served both catheters were flushed and filled with heparin (42 U/ml) and
as controls: The third group had no D-glucose added to the perfusate polyvinylpyrrolidone (1.7 g/ml) solution, plugged, tunneled subcuta-
(i.e., ECF only was perfused into the VMH) and the fourth group of neously around the side of the neck, and externalized behind the
rats had 100 mM of the non-metabolizable, L-glucose isomer added head through a skin incision. Catheters remained sealed until the day
to the perfusate. The last group of animals served as a control for the of the study. Only those animals that had completely recovered were
high osmolarity of the perfusate used in groups perfused with D-glu- used in the studies.
cose. High concentrations of glucose were used in the perfusate to en- Specifically, the recovery of the animals after both stereotaxic
sure that hypoglycemia was prevented throughout the VMH, because and catheter surgery was assessed according to a standard protocol
the efficiency of glucose delivery through the VMH dialysis system is approved by the Yale Animal Care and Use Committee. Briefly, for a
very low, (, 5%). To estimate the efficiency of glucose delivery, we period of one week after each surgery, animals, housed in the Yale
performed three in vitro experiments, using the same microdialysis Animal Facility, were monitored for body temperature, feeding activ-
system, where only ECF (without glucose added) was perfused in a ity and body weight on a daily basis. We defined recovery as: (1)
100 mM glucose solution via the VMH microdialysis probe. Under maintaining normal rat body temperature (z 37.58C); (2) attitude —
these conditions the effluent from the probe contained only z 4.2 acting normally; and (3) weight gain z 7 g/day. Any sign of infection
mM glucose (or z 4% of glucose) in the solution. Calculations were (focal, localized to the surgerized areas, or generalized) was noted,
made, by extrapolating these data to our animal experiments, consid- and sick animals were excluded from participating in the experiments
ering that in vivo (using identical microdialysis probes, solutions, and (z 10%).
flow rates), the efficiency of the microdialysis system would be much Euglycemic/hypoglycemic clamp. The hyperinsulinemic clamp tech-
lower, and the delivery of glucose to be closer to 1–2% of the infused nique, as adapted for the rat (11), was used to provide a standardized
glucose, or values that approximate brain ECF glucose levels during hypoglycemic stimulus. Each of the animals was fasted for z 24 h be-
euglycemia in the rat which are about 1.5–2.0 mM (10). Given these fore the study and then received a sequential euglycemic–hypoglyce-
considerations we estimate that the doses of glucose delivered to the mic clamp study. On the morning of the experiment, the catheters
VMH did not produce hyperglycemia in brain ECF. were flushed and maintained patent by a slow infusion of saline (20
At the end of each experiment, the accuracy of probe placement ml/min) that contained a small amount of heparin (1–2 U/ml). Ani-
was confirmed histologically by cresyl violet staining. Only those ani- mals were fully awake and freely moving about in their cages, unteth-
mals that showed bilateral probe placement into the desired brain re- ered. After a 60-min rest period, blood samples were withdrawn for
gion were included (z 25% of rats failed the histological criteria). measurement of baseline concentrations of glucose, insulin, glucagon,
Fig. 1 presents a photograph of rat brain with microdialysis guide can- epinephrine, and norepinephrine. Thereafter, a primed (2,160 pmol
ulas tracks placed bilaterally in the VMH. over 1.5 min) continuous infusion (120 pmol/kg21 ? min21) of porcine
The ability of the microdialysis system to deliver glucose selec- insulin (Eli Lilly & Co., Indianapolis, IN) was initiated and main-
tively to the VMH, has been validated previously in our laboratory, tained for 150 min. A variable infusion of exogenous glucose was ad-
using radiolabeled 2-deoxy-glucose (7). Based on these data, we as- justed based on plasma glucose measurements obtained at 5-min in-
sume that the VMH was specifically affected by dialysis, and that no tervals to achieve the desired glucose level. During the first 60 min,
other adjacent area could be significantly influenced by this manipu- the rats were maintained at euglycemia (mean plasma glucose 6.4
lation. mM). Thereafter, plasma glucose was allowed to fall over z 40 min to
Surgical procedures. At 6–8 d before study (i.e., z 7 d after ste- hypoglycemic levels (z 2.5 mM) and then clamped at this level for
reotaxic surgery), animals underwent an additional aseptic surgical z 4565 min using a variable infusion of exogenous glucose. Experi-
procedure for placement of internal jugular vein and carotid artery ments were terminated if the plasma glucose (a) fell below 4.5 mM
catheters under intraperitoneal pentobarbital anesthesia (Nembutal, during the last 45 min of the euglycemic phase and (b) rose above 3.9

Figure 1. Photograph of the rat

brain with the microdialysis
guide cannulas tracks placed
bilaterally in the VMH. The
exposed dialysis membrane
was approximately the size of
the VMH, that is 1.0–1.5 mm.
Three different concentrations
of D-glucose (0, 15, and 100
mM) and 100 mM L-glucose
were used in the experiment.

362 Borg et al.

mM (secondary to glucose infusion), or inadvertently fell below 2.0 cine standards, respectively. Plasma catecholamines were measured
mM during the hypoglycemic phase. Only the studies during which with a radioenzymatic method (Amersham Corp., Arlington Heights,
the mean blood glucose level achieved during hypoglycemia was in IL), as previously described (6, 7).
the range of 2.3–2.7 mM were prospectively analyzed. In the first ex- Data are expressed as mean6SE. Comparison between the ex-
perimental group of rats, where the VMH was perfused with 100 mM perimental groups was made by ANOVA with a repeated measure
D-glucose, 14% of animals with proper canula placement (as verified design, followed by the Student’s t test to localize effects. Glucose in-
histologically) did not meet these criteria. In the second group (per- fusion rates during the hypoglycemic steps represent average values
fused with 15 mM D-glucose) 12% of animals were excluded. Two for the designated time interval. Values for percent suppression of
control groups (where ECF only or L-glucose perfusion were intro- hormonal responses reflect changes from the mean of the peak coun-
duced) had 25% and 20% animals excluded, respectively. Histologi- terregulatory response (i.e., the mean of the 135 and 150 min data
cal results and the accuracy of the clamps were the only criteria for points) results in the control studies.
excluding or including the clamp studies for the data analysis.
Blood samples for measurement of plasma insulin, glucagon and
catecholamines were taken during the euglycemic (30- and 60-min) Results
and hypoglycemic (120-, 135-, and 150-min) phases of the experi-
ments. During blood sampling, dilution by fluid in the dead space of At the outset of the study, neither the basal concentrations of
the catheter was avoided by withdrawal of 0.5 ml of blood before glucoregulatory hormones nor the weight of the animals (Ta-
sample collection. This volume exceeded the dead space volume by ble I), were significantly different in the four experimental
about five fold. A new syringe was then used for the sample collec- groups. During the insulin infusion, plasma insulin rose to
tion. Subsequently, the initial syringe was reattached, and the con- nearly identical levels, and plasma glucose was comparable
tents reinfused. Blood recently obtained from littermates was trans- during each phase of the study in all groups (Table II and Fig.
fused during the clamp to quantitatively replace the blood withdrawn 2). In the hypoglycemic phase of the study, the desired level of
during the experiment. The protocol was reviewed and approved by
glycemia (i.e., z 2.5 mM) was achieved after z 45 min.
the Yale Animal Care and Use Committee.
During euglycemia the concentrations of epinephrine, nor-
Analytical methods and calculations. Plasma glucose was mea-
sured in duplicate using a Beckman Glucose Analyzer II (Beckman, epinephrine, and glucagon were not significantly different
Fullerton, CA). Plasma insulin (Binax, South Portland, MA), and among the four groups of animals. The effects of the different
glucagon (ICN Biomedicals, Inc., Carson, CA) were determined by a VMH microdialysis perfusions on counterregulatory hor-
double antibody radioimmunoassay. Insulin measurements during mones are summarized in Fig. 3. In both experimental groups
the basal and insulin infusion periods were made using rat and por- (VMH perfused either with 100 mM or 15 mM D-glucose) hor-

Figure 2. Plasma glucose concentrations during euglycemic-hypoglycemic clamps plus VMH microdialysis perfusions.

Ventromedial Hypothalamic Nucleus as a Glucose Sensor 363

Table I. Basal Hormonal and Weight Data of the
Animals Studied
VMH perfusions with

100 mM 15 mM 0 mM 100 mM
D-glucose D-glucose D-glucose L-glucose

(n 5 6) (n 5 7) (n 5 9) (n 5 8)

Insulin (nM) 0.1160.04 0.1260.03 0.1360.02 0.1260.02

Glucagon (ng/liter) 146613 152617 180615 212643
Epinephrine (nM) 0.760.1 0.660.1 1.260.4 1.160.3
Norepinephrine (nM) 1.760.3 1.660.2 2.260.5 2.160.5
Weight (g) 295620 270620 280630 285625

monal responses were diminished, despite a similar degree of

systemic hypoglycemia. Specifically, 100 mM D-glucose perfu-
sion into the VMH suppressed the release of epinephrine and
norepinephrine by 90% and glucagon by 75% (P , 0.05 as
compared with both control groups). The VMH perfusion with
15 mM D-glucose had a less pronounced effect, but also sup-
pressed epinephrine and norepinephrine by z 75% (P , 0.05
vs. both control groups) and glucagon by z 45% (P , 0.05
only as compared with the VMH perfusion with ECF). In con-
trast, there was a sharp increase in the concentrations of
plasma catecholamines and glucagon in the two control groups,
when hypoglycemia was induced.
The diminished counterregulatory hormone responses in
Figure 3. Effect of D-glucose VMH perfusion on the hypoglycemia-
the animals receiving VMH D-glucose perfusion was associ-
induced increment in plasma counterregulatory hormones.
ated with a 3–4-fold higher rate of exogenous glucose infusion
than was required in the control studies to maintain the plasma
glucose plateau at 2.5 mM (Table II).
the glucagon response, is reduced in the face of systemic hy-
Discussion poglycemia. No inhibition of counterregulatory hormone re-
lease occurred when the VMH was perfused with ECF only
This study demonstrates that when hypoglycemia is selectively (no D-glucose added) or with 100 mM L-glucose or when re-
prevented in the VMH, but not in the remainder of the CNS or sults from those studies are compared with those of naive rats
elsewhere, the catecholamine response, and to a lesser degree previously reported (6). In keeping with these findings, the two
experimental groups receiving the VMH D-glucose perfusion
Table II. Mean Plasma Glucose and Insulin Concentrations required more exogenous glucose to maintain the hypoglyce-
during Euglycemia and Hypoglycemia mic plateau than did control animals which had intact counter-
regulatory responses.
VMH perfusions with
While the characteristics of counterregulatory responses
100 mM 15 mM 0 mM 100 mM during hypoglycemia are well established (6, 7), the physio-
D-glucose D-glucose D-glucose L-glucose logic events that occur to link glucopenia with counterregula-
tory hormone secretion remain obscure. Although the bulk of
(n 5 6) (n 5 7) (n 5 9) (n 5 8)
data suggest that the brain plays a critical role in this process,
Euglycemia (T 5 0–60 min) the specific anatomical location of the center for sensing and
Glucose (mM) 6.460.1 6.460.2 6.560.1 6.460.1 integration of hypoglycemic signals is uncertain (5, 12). In pre-
Insulin (nM) 4.960.5 4.760.4 4.960.5 5.060.6 vious studies we performed hypoglycemic clamp studies in bi-
Hypoglycemia (T 5 105–150 min) laterally VMH lesioned rats as well as in rats with lesions in
Glucose (mM) 2.560.2 2.560.1 2.460.1 2.460.1 different brain areas (6). A marked inhibition of both the glu-
Insulin (nM) 5.060.6 4.960.4 5.060.4 5.260.7 cagon and catecholamine response to hypoglycemia was evi-
Exogenous glucose dent only in the VMH-lesioned animals, consistent with earlier
infusion rate studies implicating the hypothalamus in the activation of hy-
(mg·Kg21·min21) 5.261.2* 4.461.1* 1.560.5 1.360.4 poglycemic counterregulation (13–15).
In this study we establish in a more specific and physiologi-
* P , 0.05. Glucose values were obtained by averaging measurements cal way the critical role that the VMH plays in the counterreg-
during the 0–60 and 105–150 min of the study. Insulin data for the eugly- ulatory response to hypoglycemia. Specifically, these data
cemic phase represent the average insulin level of the sample obtained demonstrate that the catecholamine response to systemic hy-
at the end of this phase. Insulin values during hypoglycemia represent poglycemia is blocked, and the glucagon response is attenu-
the average of measurements obtained at 120 and 150 min. ated by direct glucose delivery to the VMH. By using the se-

364 Borg et al.

quential euglycemic-hypoglycemic clamp technique, we avoided Acknowledgments
the confounding effects of variable glycemic levels among the
study groups. Additionally, the bilateral intracranial microdial- We are indebted to Dr. David Maggs for his comments and sugges-
ysis technique allowed us to minimize or to abolish cellular tions. We appreciate the assistance of Aida Groszmann and Andrea
glucopenia in an area of brain restricted to the VMH. Probe Belous for their help in the measurements of plasma hormones. We
also are grateful to Jenny Sherwin and Kimberly Tishler for their ex-
placement, while obviously invasive, did not appear to signifi-
pert technical assistance.
cantly alter the hormonal response to hypoglycemia, since the This research was supported by Public Health Service grants R01-
magnitude of the hormonal response in the control rats re- DK 20495, R01-DK 40936, and P01-DK 45735. Drs. M.A. Borg and
ported here was very similar to that reported by our group in W.P. Borg are the recipients of fellowship grants from The Juvenile
naive animals during comparable hypoglycemic clamp studies Diabetes Foundation International.
(6). It is noteworthy that VMH perfusion with 100 mM D-glu-
cose was sufficient to block the sympatho-adrenal and to atten-
uate pancreatic a-cell response to systemic hypoglycemia, References
whereas only partial inhibition of both glucagon and cate-
cholamine response was seen, when 15 mM glucose was per- 1. Bernard, C. 1858. Lecons sur la Physiologie et la Pathologie en Systeme
Nerveus. J.B. Bailliere, Paris. 549–555.
fused through the probes. While the reason for this discrep- 2. Biggers, D., S. Myers, D. Neal, R. Stinson, N. Cooper, J. Jaspan, P. Will-
ancy is uncertain, it is likely that the low efficiency of glucose iams, A. Cherrington, and R. Frizzel. 1989. Role of brain in counterregulation
delivery via the probe may have prevented complete abolition of insulin–induced hypoglycemia in dogs. Diabetes. 37:7–16.
3. Lautt, W. 1980. Hepatic nerves: a review of their functions and effects.
of hypoglycemia throughout all neurons in the VMH during Can. J. Physiol. Pharmacol. 58:105–123.
the 15 mM but not the 100 mM glucose perfusion, especially 4. Donovan, C., J. Halter, and R. Bergman. 1991. Importance of hepatic
VMH neurons located at greater distances from the probe. It is glucoreceptors in sympathoadrenal response to hypoglycemia. Diabetes. 40:
unlikely that the inhibition of counterregulatory release in- 5. Frizzel, R., E. Jones, S. Davis, D. Biggers, S. Myers, C. Connolly, D. Neal,
duced by perfusion of the VMH with 100 mM D-glucose can J. Jaspan, and A. Cherrington. 1993. Counterregulation during hypoglycemia is
be attributed to direct osmotic effects, since perfusion of the directed by widespread brain regions. Diabetes. 42:1253–1261.
6. Borg, W., M. During, R. Sherwin, M. Borg, M. Brines, and G. Shulman.
VMH with 100 mM L-glucose had no effect on the counterreg- 1994. Ventromedial hypothalamic lesions in rats suppress counterregulatory re-
ulatory response. Moreover, in previous experiments, perfusion sponses to hypoglycemia. J. Clin. Invest. 93:1677–1682.
of 100 mM D-glucose into the VMH had no effect on counter- 7. Borg, W., R. Sherwin, M. During, M. Borg, and G. Shulman. 1995. Local
ventromedial hypothalamus glucopenia triggers counterregulatory hormone re-
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Ventromedial Hypothalamic Nucleus as a Glucose Sensor 365

Chronic Hypoglycemia and Diabetes Impair
Counterregulation Induced by Localized
2-Deoxy-Glucose Perfusion of the Ventromedial
Hypothalamus in Rats
Monica A. Borg, Walter P. Borg, William V. Tamborlane, Michael L. Brines, Gerald I. Shulman,
and Robert S. Sherwin

Previous studies have demonstrated that the ventro- that normally protect against hypoglycemia are defective. In
medial hypothalamus (VMH) plays a critical role in the early stages of type 1 diabetes, the capacity to release
sensing and responding to systemic hypoglycemia. To glucagon during hypoglycemia is lost in the early stages of the
evaluate the mechanisms of defective counterregulation disease (2) and subsequently because epinephrine responses
caused by iatrogenic hypoglycemia and diabetes per se, may also diminish (3). In addition, intensive insulin therapy for
we delivered 2-deoxy-glucose (2-DG) via microdialysis diabetes causes reversible suppression in sympathoadrenal
into the VMH to produce localized cellular glucopenia
in the absence of systemic hypoglycemia. Three groups responses to hypoglycemia (4,5). This phenomenon is
of awake chronically catheterized rats were studied: thought to be a consequence of iatrogenic hypoglycemia,
1) nondiabetic (with a mean daily glucose [MDG] of 6.9 because even brief periods of mild hypoglycemia in nondia-
mmol/l) BB control rats (n = 5); 2) chronically hypo- betic and diabetic subjects have been shown to reduce coun-
glycemic nondiabetic (3–4 weeks, with an MDG of 2.7 terregulatory hormone responses to subsequent hypogly-
mmol/l) BB rats (n = 5); and 3) moderately hypergly- cemic challenges (6,7). It has been hypothesized that such
cemic insulin-treated diabetic (with an MDG of 12.4 defects might be related to adaptive changes in glucose trans-
mmol/l) BB rats (n = 8). In hypoglycemic rats, both port or glucose metabolism that serve to make the central ner-
glucagon and catecholamine responses to VMH glu- vous system (CNS) less vulnerable to neuroglycopenia (8–12).
copenia were markedly (77–93%) suppressed. In dia- Paradoxically, if the CNS glucose sensors also become resis-
betic rats, VMH 2-DG perfusion was totally ineffective
in stimulating glucagon release. The epinephrine tant to neuroglycopenia, this adaptation might delay and
response, but not the norepinephrine response, was impair activation of protective counterregulatory responses to
also diminished by 38% in the diabetic group. We con- falling plasma glucose levels.
clude that impaired counterregulation after chronic Recent studies from our laboratory indicate that the ven-
hypoglycemia may result from alterations of the VMH or tromedial hypothalamus (VMH) is a dominant center within
its efferent pathways. In diabetes, the capacity of VMH the CNS for sensing of glucopenia (13–15). In rats, focal
glucopenia to activate the sympathoadrenal system is lesioning of the VMH abolishes hormonal response to sys-
only modestly diminished; however, the communication temic hypoglycemia (13), as does selective prevention of glu-
between the VMH and the a-cell is totally interrupted. copenia in the VMH by glucose perfusion via stereotaxically
Diabetes 48:584–587, 1999 placed microdialysis probes (14). Conversely, production of
local neuroglycopenia by perfusion of 2-deoxy-glucose (2-DG)
directly into the VMH of awake rats triggers the release of

S evere hypoglycemia is a major risk of insulin ther-

apy, especially in patients with type 1 diabetes who
are aggressively attempting to achieve near-nor-
moglycemia (1). Patients with diabetes are more
vulnerable to low blood glucose levels, not only because they
are unable to synchronize insulin delivery with meal ingestion
and activity but also because the counterregulatory responses
counterregulatory hormones in the absence of systemic
hypoglycemia (15).
In the present study, we used the microdialysis technique
to perfuse the VMH with 2-DG in awake, unrestrained, and
chronically hypoglycemic nondiabetic rats and spon-
taneously diabetic BB rats. These animal models were cho-
sen because they express similar defects in counterregulatory
hormone secretion (16), as is seen in patients with insulino-
From the Departments of Internal Medicine (M.A.B., W.P.B., M.L.B., R.S.S.) mas (17) and type 1 diabetes (3) during hypoglycemia. The
and Pediatrics (W.V.T.) and the Howard Hughes Medical Institute (G.I.S.), aim was to directly examine whether chronic hypoglycemia
Yale University School of Medicine, New Haven, Connecticut. or diabetes alters the capacity of glucose sensors in the VMH
Address correspondence and reprint requests to Robert S. Sherwin,
MD, Yale University School of Medicine, Department of Internal Medi-
to recognize and respond to local neuroglycopenia.
cine/Endocrinology, Box 208020, FMP 1, New Haven, CT 06520-8020. E-mail: RESEARCH DESIGN AND METHODS
Received for publication 27 July 1998 and accepted in revised form 5 Animals. Male nondiabetic and spontaneously diabetic BB rats were purchased
November 1998. from BB/Wor Laboratories (University of Massachusetts, Worcester, MA). Non-
CNS, central nervous system; 2-DG, 2-deoxy-glucose; MDG, mean daily diabetic BB rats were members of the diabetes- resistant strain of BB rats, of which
glucose; PZI, protamine zinc insulin; VMH, ventromedial hypothalamus. <1% develop diabetes. Animals were housed in an environmentally controlled room

584 DIABETES, VOL. 48, MARCH 1999


with a 12-h light/dark cycle, and they were maintained on a standard ad libitum RESULTS
rat diet (Agway Prolab 3000, Waverly, NY) comprised of 22% protein, 5% fat, and Effect of chronic hypoglycemia. At the onset of the study,
51% carbohydrate (the remaining 22% consists of ash, crude fiber, and moisture).
Three groups of rats (4–6 months old, weighing 330–370 g) were studied. For each as well as during the baseline period of 5 mmol/l VMH glucose
group, the mean plasma glucose was determined in the fed state by measuring perfusion, mean plasma glucose levels for the chronically
plasma glucose from the tail vein at least three times between 0900 and 2400, and hypoglycemic nondiabetic BB rats and their untreated control
often more frequently, every 2–3 days to adequately determine the overall
glycemic response to each insulin treatment, including the times of the greatest
rats were not significantly different (6.9 ± 0.4 and 7.0 ± 0.5
probability of hypoglycemia, normoglycemia, or hyperglycemia. mmol/l, respectively). However, during perfusion of 2-DG into
Group 1 consisted of untreated nondiabetic rats, aged 4–5 months, with a mean the VMH, the rise in plasma glucose concentration in the
plasma glucose level of 6.9 ± 0.5 mmol/l (mean ± SE), that served as the control chronically hypoglycemic rats was markedly suppressed com-
group (n = 5).
Group 2 consisted of nondiabetic rats that at 3–4 months of age were made
pared with the normal control group (P < 0.05, Fig. 1). Similarly,
chronically hypoglycemic using gradually increased doses of protamine zinc the concentrations of catecholamines and glucagon were sta-
insulin (PZI) (Lilly, Indianapolis, IN) over 1 week, followed by 3–4 weeks of treat- ble and not significantly different in the chronically hypogly-
ment with twice-daily injections of 9–10 U/kg PZI insulin, resulting in mean cemic rats and the control rats during the basal and 5 mmol/l
plasma glucose values of 2.7 ± 0.6 mmol/l (n = 5). glucose VMH perfusion periods. In the 2-DG phase of the VMH
Group 3 consisted of diabetic rats, aged 5–6 months, that were in moderate
glycemic control (mean plasma glucose of 12.4 ± 1.0 mmol/l) achieved by daily perfusion, the rise in plasma counterregulatory hormones in the
injections of PZI insulin in doses of ~11–12 U/kg. The doses of insulin were chronically hypoglycemic rats were markedly suppressed. The
adjusted during treatment to avoid hypoglycemia by frequent monitoring magnitude of the suppression of epinephrine and norepi-
throughout the 24-h diurnal cycle (n = 8). nephrine release was 91 and 93%, respectively. Glucagon
Surgical procedures. Rats were anesthetized as previously described (14) and
placed on a stereotaxic frame. The skull was exposed, and holes were drilled bilat-
responses were also reduced by 77% (P < 0.05), particularly the
erally in chosen coordinates (14,15) through which the guide cannulas were low- peak increment at 60 min, which was diminished by 94%.
ered slowly into the brain and then secured with stainless steel screws and den- Effect of diabetes. Plasma glucose levels in the insulin-
tal acrylic. Immediately after stereotaxic surgery, animals underwent an additional treated diabetic animals in the basal state and during 5
aseptic surgical procedure for placement of internal jugular vein and carotid mmol/l glucose perfusion of the VMH were similar to those
artery catheters. At the end of the procedure, both catheters were filled with
heparin (42 U/ml) and polyvinylpyrrolidone (1.7 g/ml) solution, plugged, tun- seen in control animals (6.9 ± 0.3 mmol/l). As shown in Fig.
neled subcutaneously around the side of the neck, and externalized behind the 1, the rise in the peripheral plasma glucose levels in response
head through a skin incision. Animals were then allowed to recover for 5–7 days. to VMH glucopenia was slightly, but not significantly, reduced
Only those animals that appeared healthy and were able to maintain their weight compared with that in control rats. Figure 2 presents the
were used. The evening before the experiment, animals received their last dose
of PZI insulin, and the microdialysis probes (10.5 mm in length) of side-by-side
hormonal changes during the 100 mmol/l 2-DG VMH perfu-
design were inserted into the guide cannulas, as previously described (18). The sion. There was a 38% reduction (P < 0.05) in the overall
exposed microdialysis membrane was 1.0–1.5 mm (approximately the size of the plasma epinephrine response in the diabetic rats that was
VMH), so that we could selectively perfuse this brain region. At 1 h before the significant at the 60-min time point. However, the reduction
experiment, the VMH perfusion medium was loaded into 1-ml syringes and deliv- in the norepinephrine response in the diabetic group was
ered at a flow rate of 2.5 µl/min using a Harvard perfusion pump (model 22; Har-
vard Apparatus, Holliston, MA). not significantly different from that in the control group. In
At the end of each experiment, the accuracy of probe placement was confirmed contrast, the increase in plasma glucagon that normally fol-
histologically by cresyl violet staining. Only those animals that showed bilateral lowed VMH glucopenia in the nondiabetic animals was
probe placement into the desired brain region were included. totally abolished in the type 1 diabetic rats.
Experimental protocol. Each animal was food deprived for ~4 h before the
study. On the morning of the experiment, the vascular catheters were flushed and
maintained patent by a slow infusion of saline (20 µl/min) that contained a small
amount of heparin (1–2 U/ml), and the VMH microdialysis perfusion was initiated.
Rats were conscious and allowed to roam freely in their cages during the experi-
ment. To achieve comparable plasma glucose levels in each group before study, for
~2 h before the experiment, the chronically hypoglycemic animals (group 2) as well
as the diabetic animals (group 3) were brought to normoglycemic levels (see
RESULTS) by intravenous variable infusion of exogenous 20% glucose or a solution
of insulin (20 mU · kg–1 · min–1), respectively. These infusions were discontinued
20–30 min before initiating the experiment. Thereafter, blood samples were with-
drawn for measurement of baseline plasma concentrations of glucose, glucagon,
epinephrine, and norepinephrine. Subsequently, for the first 30 min of the experi-
ment, the VMH probes were perfused with a solution that contained 5 mmol/l glu-
cose; this was designated as the control phase. Thereafter, 100 mmol/l 2-DG perfusion
was introduced and maintained for 60 min. Arterial blood samples for measurement
of plasma glucagon and catecholamines were taken at 15-min intervals during the
control phase (15 and 30 min) as well as during VMH glucopenia (45, 60, 75, and 90
min). To avoid dilution by fluid in the dead space of the catheter during blood sam-
pling, 0.5 ml of blood was withdrawn before sample collection. Subsequently, the
contents of the initial syringe were reinfused to minimize blood losses. Blood
recently obtained from littermates was also transfused during the study to quanti-
tatively replace the blood withdrawn during the experiment. The protocol was
reviewed and approved by the Yale Animal Care and Use Committee.
Analytical methods and calculations. Plasma glucose was measured in dupli-
cate using a Beckman Glucose Analyzer II (Fullerton, CA). Plasma catecholamines
were measured with a radioenzymatic method (Amersham, Arlington Heights, IL),
and plasma glucagon was measured using a double antibody radioimmunoassay FIG. 1. Effect of chronic hypoglycemia and type 1 diabetes on periph-
procedure (Linco Research, St. Charles, MO) as previously described (14). eral plasma glucose levels during VMH glucopenia. Experimental
Data are expressed as means ± SE. Comparison between the experimental groups included nondiabetic BB control rats (n = 5) ( s), chronically
groups over time was made by analysis of variance with a repeated measure hypoglycemic nondiabetic rats (n = 5) (m), and moderately hypergly-
design, followed by the Student’s t test to localize effects. cemic insulin-treated diabetic BB rats (n = 8) (d). *P < 0.05 vs. normal.

DIABETES, VOL. 48, MARCH 1999 585


hormonal responses to severe local VMH glucopenia pro-

duced by 2-DG delivered to the VMH via microdialysis. In pre-
vious studies, we also demonstrated that microdialysis probe
placement, although invasive, did not interrupt the ability of
the VMH to stimulate sympathetic and pancreatic a-cell path-
ways in experiments in which systemic hypoglycemia was
produced (14). The microdialysis probes were perfused with
a high concentration (100 mmol/l) of 2-DG, a nonmetaboliz-
able form of glucose, because only a small fraction (~4%) is
actually delivered across the dialysis membrane to the extra-
cellular fluid space of the VMH (14). In earlier studies in nor-
mal Sprague-Dawley rats, we demonstrated that sufficient 2-
DG is delivered by the microdialysis system to produce local
neuroglycopenia, and that this, in turn, provokes brisk coun-
terregulatory hormone responses and systemic hypergly-
cemia (15). Nearly identical changes were observed in the
nondiabetic diabetes-resistant strain of BB rats (group 1)
used in the current study.
As in humans, we have previously reported that nondiabetic
rats made repetitively hypoglycemic with exogenous insulin
show delayed and impaired release of catecholamines and
glucagon during hypoglycemia, and that these changes are
reversible over time (16). We reasoned that if such alterations
were mediated by an impairment of VMH glucose-sensing
neurons to detect neuroglycopenia, there would be a gener-
alized suppression of the hormonal response to VMH 2-DG per-
fusion, as was observed in the current study. To the extent that
our data can be applied to the clinical situation, it is possible
that the suppression of counterregulatory hormone responses
induced by iatrogenic hypoglycemia in normal subjects (6) or
in intensively treated type 1 diabetic patients (19) may also
involve dysfunction of the VMH or any of its efferent pathways.
This defect appears to be glucose specific. In our previous stud-
ies we showed that glucagon and adrenomedullary responses
in chronically hypoglycemic nondiabetic BB rats are pre-
served to nonspecific stimuli (16). Based on the profound
degree of the suppressive effect, one might speculate that it
FIG. 2. Effect of chronic hypoglycemia and type 1 diabetes on plasma could involve a variety of mechanisms, such as an impair-
counterregulatory hormones during VMH glucopenia. Experimental ment in VMH signaling pathways or neurotransmitter release
groups included nondiabetic BB control rats (n = 5) ( s), chronically
hypoglycemic nondiabetic rats (n = 5) (m), and moderately hypergly-
and/or action as well as an alteration of glucose transport in
cemic insulin-treated diabetic BB rats (n = 8) (d). *P < 0.05 vs. normal. this brain area. The exact nature of this defect cannot be
directly established from the current experiments.
The spontaneous diabetic BB rat used in this study, an
DISCUSSION established animal model of type 1 diabetes (20), displays
The results of the Diabetes Control and Complications Trial counterregulatory defects remarkably similar to that present
demonstrated the long-term benefits of intensive insulin ther- in humans with the disease (21,16). In diabetic BB rats,
apy aimed at near normalization of glucose levels in patients glucagon release during systemic hypoglycemia is totally lost
with type 1 diabetes (1). However, severe and frequent hypo- within a week after disease onset, whereas the epinephrine
glycemia emerged as a serious complication that has limited response is initially preserved but tends to diminish over the
the application of such regimens (1). Defective counterregu- next 6–8 weeks (22). This pattern is similar to that observed
latory responses associated with type 1 diabetes (2,3) as well in human type 1 diabetes; however, the time sequence of the
as suppressed hormonal defense mechanisms induced by changes is much more rapid in the rat model system (3). The
iatrogenic hypoglycemia per se (4) contribute to the observed discrepancy between the appearance of the glucagon and
increased risk of severe hypoglycemia. A better understand- epinephrine counterregulatory defects in type 1 diabetes may
ing of the mechanisms used to sense glucopenia and how imply that different pathophysiological mechanisms may
they are altered by diabetes and iatrogenic hypoglycemia underlie each of these defects. In the diabetic rats, VMH 2-DG
could therefore offer new therapies aimed at reversing these perfusion totally failed to provoke glucagon release. Whereas
counterregulatory defects. this effect was complete, catecholamine release was only
In the current study, we examined how chronic hypogly- modestly diminished. Although undetected hypoglycemia
cemia in nondiabetic rats as well as spontaneous insulin- induced during insulin treatment of the diabetic rats might
deficient diabetes in the rat affects the capacity of the VMH have contributed to the mild epinephrine secretory defect, con-
to act as a glucose sensor. For this purpose, we measured the siderable care was undertaken to avoid hypoglycemia for sev-
586 DIABETES, VOL. 48, MARCH 1999

eral weeks before study, and in no case was hypoglycemia doc- F, Lepore M, Annibale B, Ciofetta M, Bottini P, Porcellati F, Scionti L, San-
umented by glucose monitoring. Thus, it seems likely that in teusanio F, Brunetti P, Bolli GB: Meticulous prevention of hypoglycemia nor-
malizes the glycemic thresholds and magnitude of most neuroendocrine
the diabetic rats, the signal from the VMH was operative, but responses to, symptoms of, and cognitive function during hypoglycemia in
mildly impaired, at some level in the path between the VMH intensively treated patients with short-term IDDM. Diabetes 42:1683–1689, 1993
and the adrenal medulla. In contrast, this VMH signal was 6. Heller S, Cryer P: Reduced neuroendocrine and symptomatic responses to sub-
totally ineffective in activating the a-cells. Whereas the exact sequent hypoglycemia after one episode of hypoglycemia in nondiabetic
nature of this a-cell defect is uncertain, it is intriguing to spec- humans. Diabetes 40:223–226, 1991
7. Dagogo-Jack SE, Cryer PE: Hypoglycemia-associated autonomic failure in
ulate that it may be due to changes in neural innervation of the IDDM: recent antecedent hypoglycemia reduces autonomic responses to
islet, the absence of a b-cell signal, or an intrinsic defect in the symptoms of, and defense against subsequent hypoglycemia. J Clin Invest
a-cell itself. Extrapolation of these observations in the BB rat 91:891–898, 1993
to the clinical setting should, however, be made with caution. 8. McCall AL, Fixman LB, Fleming N, Tornheim K, Chick W, Ruderman NB:
Chronic hypoglycemia increases brain glucose transport. Am J Physiol
For example, in the clinical setting, type 1 diabetic patients 251:E442–E447, 1986
rarely experience persistent hypo- or hyperglycemia. 9. Pelligrino DA, Segil LJ, Albrecht RF: Brain glucose utilization and transport
In summary, our studies demonstrate that chronic hypo- and cortical function in chronic vs. acute hypoglycemia. Am J Physiol
glycemia in nondiabetic rats suppresses the ability of the 259:E729–E735, 1990
VMH to recognize glucopenia or activate hormonal counter- 10. Kumagai AK, Kang YS, Boado RJ, Pardridge WM: Upregulation of blood-
brain barrier GLUT1 glucose transporter expression in diabetes mellitus.
regulation. In diabetic BB rats, the response to VMH glu- Diabetes 44:1399–1404, 1995
copenia is only slightly diminished for catecholamines but 11. Boyle PJ, Nagy RJ, O’Connor AM, Kempers SF, Yeo RA, Qualls C: Adaptation
absent for glucagon. These findings support the hypothesis in brain glucose uptake following recurrent hypoglycemia. Proc Natl Acad Sci
that the impaired counterregulation after chronic hypogly- U S A 91:9352–9356, 1994
cemia may result from alterations of the function of the VMH 12. Boyle PJ, Kempers SF, O’Connor AM, Nagy RJ: Brain glucose uptake and
unawareness of hypoglycemia in patients with insulin-dependent diabetes mel-
or its efferent pathways, and that in diabetes, there is a dis- litus [see comments]. N Engl J Med 333:1726–1731, 1995
tinct independent defect in which the communication 13. Borg WP, During MJ, Sherwin RS, Borg MA, Brines ML, Shulman GI: Ventro-
between the VMH and the a-cell is interrupted. medial hypothalamic lesions in rats suppress counterregulatory responses to
hypoglycemia. J Clin Invest 93:1677–1682, 1994
ACKNOWLEDGMENTS 14. Borg MA, Sherwin RS, Borg WP, Tamborlane WV, Shulman GI: Local ventro-
medial hypothalamus glucose perfusion blocks counterregulation during sys-
This research was supported by grants R01-DK-20495, R01- temic hypoglycemia in awake rats. J Clin Invest 99:361–365, 1997
DK- 40936, and P30-DK-45735 from the Public Health Service. 15. Borg W, Sherwin R, During M, Borg M, Shulman G. Local ventromedial hypo-
M.A.B. is the recipient of a fellowship grant from the Juvenile thalamus glucopenia triggers counterregulatory hormone release. Diabetes
Diabetes Foundation International. G.I.S. is an investigator of 44:180–184, 1995
16. Powell AM, Sherwin RS, Shulman GI: Impaired hormonal responses to hypo-
the Howard Hughes Medical Institute. glycemia in spontaneously diabetic and recurrently hypoglycemic rats:
We appreciate the assistance of Aida Groszmann and reversibility and stimulus specificity of the deficits. J Clin Invest
Andrea Belous in performing the hormone assays. We also are 92:2667–2674, 1993
grateful to Melissa Huang for expert technical assistance. 17. Mitrakou AFC, Veneman T, Perriello G, Calderone S, Platanisiotis D, Rambotti
A, Raptis S, Brunetti P, Cryer P, Gerich J, Bolli G: Reversibility of unawareness
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329:977–986, 1993 19. Amiel SA, Sherwin RS, Simonson DC, Tamborlane WV: Effect of intensive
2. Gerich J, Langlois M, Noacco C, Karam J, Forsham P: Lack of glucagon insulin therapy on glycemic thresholds for counterregulatory hormone
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impaired glucagon and epinephrine secretion. Diabetes 32:134–141, 1983 “BB” Wistar rat: possible relevance to type 1 (insulin-dependent) diabetes in
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DIABETES, VOL. 48, MARCH 1999 587

0021-972X/97/$03.00/0 Vol. 82, No. 6
Journal of Clinical Endocrinology and Metabolism Printed in U.S.A.
Copyright © 1997 by The Endocrine Society

Resistance to Neuroglycopenia: An Adaptative Response

during Intensive Insulin Treatment of Diabetes*
Departments of Internal Medicine, Pediatrics, and Neurosurgery and the General Clinical Research
Center, Yale University School of Medicine, New Haven, Connecticut 06520

ABSTRACT trolled counterparts (P , 0.05), and hormonal responses were sup-

Counterregulation and awareness of hypoglycemia begins at lower pressed compared to those in healthy controls. Similarly directed
plasma glucose levels in insulin-dependent diabetes mellitus (IDDM) changes occurred in the level of circulating glucose required to alter
subjects given intensive insulin treatment. To determine whether cortical evoked potentials during hypoglycemia. A greater reduction
these changes are associated with an alteration in the susceptibility in plasma glucose was required to alter P300 event-related potentials
of the brain to mild hypoglycemia, we compared central nervous in the intensively treated patients (2.2 mmol/L) compared to those in
system responses to hypoglycemia in 8 intensively treated (hemoglo- the conventionally treated and nondiabetic groups (;3.5 and ;3.0
bin A1, 8.3 6 0.2%; normal, ,8%) and 11 conventionally treated IDDM mmol/L, respectively). We conclude that intensively treated IDDM
patients (hemoglobin A1, 14.6 6 1.3%) with those in 10 healthy sub- patients are resistant to changes in cortical evoked potentials induced
jects. Plasma glucose was lowered from ;4.6 mmol/L in 0.5– 0.6 steps by mild hypoglycemia. This may explain why intensively treated
using the clamp technique. Glucose levels triggering hormonal re- IDDM counterregulate and experience hypoglycemic symptoms at a
sponses and perception of hypoglycemic symptoms were significantly lower glucose level than conventionally treated patients. (J Clin En-
lower in intensively treated patients compared to their poorly con- docrinol Metab 82: 1713–1718, 1997)

T HE REPORT of the Diabetes Control and Complication

Trial Research Group (1) has established the long term
benefits of intensive insulin therapy aimed at near normal-
regulation and symptomatic unawareness of hypoglycemia
(7–10). Recent studies suggest that this phenomenon is
caused by iatrogenic hypoglycemia per se. In both healthy
ization of glucose levels in insulin-dependent diabetes mel- subjects and IDDM patients a brief period of moderate hy-
litus (IDDM). As a result, this therapeutic approach has been poglycemia reduces hormonal responses and symptoms
recommended for (2) and is being offered to an increasing during experimentally induced hypoglycemia the following
number of IDDM patients in an effort to prevent or delay day (11, 12). Conversely, it has been reported that in young,
microvascular and neuropathic complications. Unfortu- poorly controlled IDDM patients, hypoglycemic symptoms
nately, the frequency and severity of hypoglycemia are and epinephrine responses can be elicited when glucose is
markedly increased by such regimens despite close medical lowered into the normal range (13).
supervision (1, 3). A key issue is whether the divergent changes in glycemic
The mechanisms contributing to the greater risk of severe thresholds for counterregulatory responses in well con-
hypoglycemia during intensive insulin therapy in IDDM trolled, intensively treated and in poorly controlled, conven-
patients have recently been clarified. Although lowering of tionally treated IDDM patients are mirrored by similarly
target glycemic goals in the face of persisting conventional directed changes in brain function as the availability of glu-
risk factors would be expected to promote iatrogenic hypo- cose via the circulation is reduced. This possibility is sup-
glycemia, impaired counterregulatory defenses against hy-
ported by studies in rats demonstrating that chronic hypo-
poglycemia play an important role as well (4 – 6). In the early
glycemia and hyperglycemia, respectively, increase and
stages of IDDM, the capacity to release glucagon during
decrease the efficiency of glucose extraction by the brain
hypoglycemia is lost (4, 5), and as the duration of the disease
(14 –16). Furthermore, in normal human subjects rendered
increases, epinephrine responses are also diminished in
some patients (6). In addition, intensified insulin treatment mildly hypoglycemic for several days and IDDM patients
aimed at restoring glycemia as close to normal as possible who have been well controlled, brain glucose uptake is more
leads to a further deterioration of hypoglycemic counter- effectively preserved during hypoglycemia (17, 18). It is un-
certain, however, whether these adaptations in brain glucose
uptake during hypoglycemia are accompanied by corre-
Received June 10, 1996. Revision received August 22, 1996. Accepted
February 28, 1997. sponding alterations in brain function. Studies using tests of
Address all correspondence and requests for reprints to: Dr. Robert neuropsychological performance to evaluate the effect of
S. Sherwin, Department of Internal Medicine, Yale University School of diabetes treatment on the central nervous system (CNS) re-
Medicine, 333 Cedar Street, New Haven, Connecticut 06520-8020.
* This work was supported by grants from the USPHS (RR-06022,
sponse to hypoglycemia have generated contradictory re-
RR-00125, DK-20495, and DK-45735) and fellowship grants from the sults (19 –22). Studies were, therefore, undertaken to assess
Juvenile Diabetes Foundation International (to W.P.B. and M.A.B.). this question by combining the hypoglycemic clamp with


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1714 JONES ET AL. JCE & M • 1997
Vol 82 • No 6

measurements of cortical evoked potentials, an end point difficulty thinking, sweating, slowed thinking, pounding heart, and
shown to be sensitive to even modest decrements in plasma shakiness. The sum of these eight items in each subject constituted the
glucose (23). total symptom score at the observation point. In addition, four “filler”
items (i.e. pain in joints, earache, watery eyes, and ringing in ears) were
included to control for nonspecific symptoms not referable to hypogly-
Subjects and Methods cemia. To obtain cortical evoked potentials, scalp electrodes were placed
Subjects at Cz and Pz positions, with a reference electrode placed in the opposite
ear. A bipolar pair of electrodes was placed above the right eye to
A total of 29 subjects were studied, including 19 IDDM patients and monitor for ocular artifacts. P300-evoked potentials were obtained with
10 nondiabetic controls. Eight of the patients with IDDM were receiving an auditory categorization (“oddball”) task, in which subjects were
intensive insulin therapy (multiple daily injections or continuous sub- required to silently count the number of soft clicks. The loud clicks were
cutaneous insulin infusion) for at least 6 months, and 11 were being presented to the ear ipsilateral to the reference electrode, and the soft
treated with conventional insulin regimens (twice daily injections of clicks were delivered to the ear contralateral to the reference electrode.
regular and intermediate acting insulin). They were eligible for study if The proportion of soft clicks varied between 10 –20% of a total of 200
their disease duration was more than 1 yr, they had no symptoms or
clicks. Two replications were obtained at each plasma glucose plateau.
physical signs of autonomic neuropathy, and they were receiving no
medications other than insulin and had no acute illness. The clinical
characteristics of all subjects are shown in Table 1. Each gave written
informed consent to participate in the study protocol, which was ap-
Determinations and analysis
proved by the Yale human investigation committee. Catecholamines were measured by radioenzymatic assay (Upjohn,
Kalamazoo, MI), and free insulin, GH, cortisol, and glucagon were
Hypoglycemic clamp procedures determined using double antibody RIAs, as described previously (10).
Averaged evoked potential waveforms for P300 were calculated on-line
Studies were performed after a 10- to 12-h overnight fast. Subjects
and then analyzed off-line by two investigators who were blinded to the
with diabetes were admitted to the Yale General Clinical Research Cen-
subjects experimental group and to the glycemic levels at which the
ter on the evening before the study. An iv catheter was inserted, and
evoked potentials were obtained. Peaks and latencies of the P300 po-
basal insulin was administered as a continuous iv infusion that was
adjusted during the night based on plasma glucose measurements ob- tential were measured with a waveform cursor. P300 evoked potentials
tained every 30 – 60 min. The plasma glucose concentration did not fall were calculated as the difference between potentials evoked by soft
below 4.0 mmol/L in any of the patients during the overnight period. clicks and those elicited by loud clicks; this procedure minimized po-
The next morning, a modification of the glucose clamp technique was tentials evoked by clicks per se and isolated those associated with the
used to produce a gradual and standardized reduction in plasma glu- counting task.
cose. The methods used for this procedure (hypoglycemic clamp) have Demographic data are expressed as the mean 6 sd, and all other data
been previously described (10). Briefly, two venous catheters were em- are expressed as the mean 6 se. Comparisons of glucose levels, hormone
ployed, one in an antecubital vein for administration of glucose and responses, symptoms scores, and P300 latency and amplitude measures
insulin, and the other in a dorsal hand vein for blood sampling. The hand between the study groups were made using ANOVA with repeated
was placed in a heated (;65 C) box to “arterialize” venous blood (24). measures design, followed by Student’s t test to localize the effects. P ,
After a 60-min basal period during which baseline measurements were 0.05 was considered statistically significant. Hormonal data are pre-
obtained, a primed continuous infusion of regular human insulin was sented as the average of measurements obtained during the last 40 min
given (Novo Nordisk, Princeton, NJ) in a dose of 80 mU/m2zmin. Plasma of each glycemic step of the clamp studies.
glucose was measured at the bedside in duplicate at 5-min intervals
using a Beckman glucose analyzer (Beckman, Fullerton, CA), and target
glucose levels were achieved by varying the rate of an infusion of 20% Results
glucose. In all three groups, plasma glucose was maintained at eugly-
cemic levels (;4.6 mmol/L) for the initial 60 min of the study and then Glucose and insulin levels
was reduced in 0.5– 0.6 mmol/L steps each hour for 240 min. In inten- As shown in Fig. 1, the hypoglycemic clamp procedure
sively treated patients, the study was extended for an additional 60-min
period to allow for an additional hypoglycemic plateau of 2.2 mmol/L. caused plasma glucose levels to decline in a nearly identical
All subjects were masked to the plasma glucose levels during the study. fashion in each of the three groups during the first 240 min
of the study, i.e. ;0.5 mmol/L each hour to a nadir of ;3
Measurements mmol/L. In the intensively treated IDDM subjects, plasma
Blood samples were taken at 10- to 20-min intervals for measurement glucose was further reduced to 2.2 mmol/L for an additional
of insulin and counterregulatory hormones. In the final 20 min of each 60-min period. Steady state plasma free insulin levels during
hypoglycemic plateau (beginning at 4.0 mmol/L), symptoms and elec- the clamp studies were not significantly different between
trophysiological data were recorded. Symptoms were assessed using a
questionnaire presented on a laptop computer that also recorded subject
the groups (nondiabetic, 847 6 36 pmol/L; conventionally
responses. Subjects rated the following symptoms on a scale of 1 (not at treated IDDM, 744 6 60 pmol/L; intensively treated IDDM,
all) to 7 (extreme): headache, difficulty with concentration, weakness, 828 6 72 pmol/L; P 5 NS).

TABLE 1. Clinical characteristic of the study groups and plasma counterregulatory hormones concentration during baseline

Conventionally treated Intensively treated Nondiabetic

IDDM (n 5 11) IDDM (n 5 8) adults (n 5 10)
Age (yr) 23 6 2 (15–32) 30 6 3 (20 – 41) 24 6 1 (20 –28)
Sex (m/f) 6/5 4/4 5/5
Duration of IDDM (yr) 11 6 1 (5–16) 15 6 2 (4 –27)
HBA1 (%) 14.6 6 1.3 (9.9 –22.0) 8.3 6 0.2 (7.6 – 8.9)
Epinephrine (pmol/L) 350 6 65 308 6 51 231 6 35
Cortisol (nmol/L) 342 6 60 329 6 73 295 6 28
GH (mg/L) 6.1 6 1.3 6.3 6 2.4 1.9 6 0.3
Values are expressed as the mean 6 SEM, with the range in parentheses. HBA1 values in nondiabetic subjects range from 4 – 8%.

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FIG. 1. Plasma glucose levels (mean 6

SEM) during the stepped hypoglycemic
clamp procedure in intensively treated
IDDM (8), conventionally treated
IDDM (11), and control (10) subjects.

Counterregulatory hormones and symptoms glucose had been reduced to 2.2 mmol/L. Moreover, P300
The plasma counterregulatory hormone responses of each latency significantly increased by ;9% in the conventionally
group during the hypoglycemic clamp study are summa- treated IDDM and in the nondiabetic controls at the 3.0
rized in Fig. 2. During the hypoglycemic phase of the study mmol/L step (P , 0.05), whereas a small, but insignificant,
a significant rise in plasma epinephrine above basal values increase (;5%) occurred at the 2.2 mmol/L step in the well
occurred at the 4.0 mmol/L step in the conventionally treated controlled IDDM patients.
IDDM patients, whereas there was a small, but also signif-
icant, increase at the 3.5 mmol/L step in the nondiabetic Discussion
subjects (P , 0.05 for both groups). In the intensively treated In keeping with the earlier reports (7, 8, 10), our intensively
patients, a significant rise in plasma epinephrine did not treated IDDM patients required a greater hypoglycemic
occur until plasma glucose was lowered below 3.0 mmol/L, stimulus to trigger counterregulatory hormone responses
and plasma epinephrine levels were substantially lower than and symptoms than did poorly controlled, conventionally
those in the other groups (P , 0.05). Plasma cortisol rose treated patients with IDDM. A key unresolved issue is
significantly at 3.0 mmol/L glucose in both conventionally whether these changes associated with IDDM therapy are
treated IDDM and nondiabetic subjects (P , 0.05), whereas accompanied by a similar shift in the glucose level at which
a glucose level of even 2.2 mmol/L was not sufficient to elicit brain function becomes impaired. This is of importance clin-
a significant response in the intensively treated patients. ically because the consequences of hypoglycemia depend at
Moreover, significant increments in GH first occurred at a least in part on the therapeutic window between counter-
higher glucose level in conventionally treated IDDM and regulatory responses and/or symptoms and onset of CNS
nondiabetic subjects (3.5 mmol/L) compared to the level in dysfunction. The results of the current study suggest that the
the intensively treated patients (3.0 mmol/L; P , 0.05). shift in counterregulatory responses to hypoglycemia in
As shown in Fig. 3, symptomatic awareness of hypogly- IDDM is, in fact, mirrored by similarly directed changes in
cemia followed a similar pattern. Hypoglycemic symptom neuroglycopenia onset, as assessed by P300 measurements.
scores increased significantly (P , 0.05) above baseline at 4.0 Thus, intensive therapy may have led to an adaptation in
mmol/L in conventionally treated patients, and at 3.0 brain glucose metabolism that resulted in greater preserva-
mmol/L in nondiabetic subjects and the intensively treated tion of cortical evoked potentials in the face of subnormal
group. There were no changes in control symptoms in any of glucose levels, levels that caused subtle neuroglycopenia in
the groups throughout the study (data not shown). conventionally treated IDDM patients. This conclusion is
supported by the experimental data in nondiabetic and di-
Electrophysiological measures
abetic rats (14 –16, 25, 26) and in human subjects (17, 18). In
Figure 4 depicts P300 amplitude in the three groups. At rats, chronic hypoglycemia increases and chronic hypergly-
baseline, P300 amplitude was not significantly different cemia decreases the efficiency of brain glucose extraction and
among the groups. During the hypoglycemic phase, the con- metabolism (14 –16). Furthermore, chronically hyperglyce-
ventionally treated IDDM patients showed a significant (P , mic BB rats are more susceptible (25) and recurrently hypo-
0.05) reduction in P300 amplitude at the 3.5 mmol/L glucose glycemic diabetic BB rats are resistant to the adverse effects
plateau, a higher glucose level than that producing a signif- of hypoglycemia on brain stem function (26). Recent studies
icant P300 change in nondiabetic control subjects (3.0 mmol/ suggest that these changes may be due to changes in blood-
L). In contrast, the well controlled IDDM patients failed to brain barrier glucose transport (27). In humans, several days
show a significant change in P300 amplitude until plasma of sustained mild hypoglycemia or intensive insulin treat-

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1716 JONES ET AL. JCE & M • 1997
Vol 82 • No 6

FIG. 3. Total hypoglycemic symptoms score (mean 6 SEM) of each

subject group during hypoglycemic clamp studies. The total symptom
score represents the sum of eight hypoglycemic symptoms. Asterisks
represents significant difference vs. baseline (P , 0.05).

FIG. 4. P300 amplitude during each hypoglycemic plateau in nondi-

abetic, conventionally treated IDDM, and intensively treated IDDm
subjects. Asterisks indicate significant amplitude reduction compared
to baseline (P , 0.05).

have yielded conflicting results (19 –22, 28, 29). It has been
suggested that intensively treated IDDM patients may be
more, rather than less, vulnerable to neuroglycopenia than
poorly controlled counterparts when conventional electro-
encephalogram recordings are used as an end point (22). In
contrast, studies using neuropsychological tests to assess
cognitive performance during hypoglycemia in IDDM pa-
tients have found either no change (19, 21) or a lowering of
FIG. 2. Plasma counterregulatory hormones responses of each sub- the plasma glucose level required to provoke cognitive dys-
ject group to stepped reduction in plasma glucose concentrations function in patients treated intensively (20, 28, 29). It should
during hypoglycemic clamp studies. be emphasized that these discrepancies may be more appar-
ent than real because the various tests of cognitive function
ment leading to near-normal glycated hemoglobin levels in are likely to involve specific brain regions that may have
IDDM patients has been reported to prevent the decline in different glucose requirements.
whole brain glucose uptake (measured by internal jugular In the current study, brain function was assessed using
venous sampling) produced by experimental hypoglycemia P300, an event-related potential elicited during discrimina-
(;3.0 mmol/L) using the stepped hypoglycemic clamp tech- tion of signals with a low subjective probability. The P300
nique (17, 18). waveform, unlike the electroencephalogram, which mea-
Previous studies examining the effect of glycemic control sures the spontaneous electrical output of the brain, is gen-
of IDDM on CNS function during hypoglycemia, however, erated by the active cognitive processing of stimulus infor-

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mation and is not affected by the physical characteristics of 2. American Diabetes Association. 1993 Position statement: implication of the
Diabetes Control and Complication Trial. Clin Diabetes. 11:91–96.
the stimulus. It requires the active participation of the subject 3. The Diabetes Control and Complication Trial Research Group. 1991 Epide-
and involves higher brain centers, particularly the hip- miology of severe hypoglycemia in the Diabetes Control and Complication
pocampus, and the parahippocampal gyrus (30). Although Trial. Am J Med. 90:450 – 459.
4. Gerich J, Langlois M, Noacco C, Karam J, Forsham P. 1973 Lack of glucagon
the P300 may be influenced by such external factors as the response to hypoglycemia in diabetes: evidence for an intrinsic pancreatic
probability of the stimulus and the relevance and difficulty alpha-cell defect. Science. 182:171–173.
of the task, and may only be relevant for specific cognitive 5. Bolli G, DeFeo P, Compagnucci P, et al. 1983 Abnormal glucose counter-
regulation in insulin-dependent diabetes mellitus: interaction of anti-insulin
functions, it is useful in analyzing effects in which the subject antibodies and impaired glucagon and epinephrine secretion. Diabetes.
acts as his/her own control (31). In this context it has been 32:134 –141.
6. Kleinbaum J, Shamoon H. 1983 Impaired counterregulation of hypoglycemia
shown to be sensitive to small decrements in circulating in insulin-dependent diabetes mellitus. Diabetes. 32:493– 498.
glucose (23, 32). These changes appear to be produced by 7. Simonson DC, Tamborlane WV, DeFronzo RA, Sherwin RS. 1985 Intensive
hypoglycemia per se because the infusions of multiple coun- insulin therapy reduces counterregulatory hormone responses to hypoglyce-
mia in patients with type I diabetes. Ann Intern Med 103:184.
terregulatory hormones have no effect on P300 under con- 8. Amiel SA, Tamborlane WV, Simonson DC, Sherwin RS. 1987 Defective
ditions of euglycemia (33). glucose counterregulation after strict control of insulin-dependent diabetes
Our observation that intensively treated IDDM patients mellitus. N Engl J Med. 316:1376 –1383.
9. Ryder REJ, Owens DR, Hayes TM, Ghatei M, Bloom SR. 1990 Unawareness
require a greater and poorly controlled, conventionally of hypoglycaemia and inadequate glucose counterregulation: no causal rela-
treated IDDM patients require a smaller decrement in tionship with diabetic autonomic neuropathy. Br Med J. 301:783–787.
10. Amiel SA, Sherwin RS, Simonson DC, Tamborlane WV. 1988 Effect of
glucose concentration to alter P300 event-related poten- intensive insulin therapy on glycemic threshold for counterregulatory hor-
tials is consistent with data cited above suggesting the mone release. Diabetes. 37:901–907.
development of a CNS adaptation depending on the in- 11. Heller S, Cryer PE. 1991 Reduced neuroendocrine and symptomatic responses
to subsequent hypoglycemia after one episode of hypoglycemia in non-dia-
tensity of insulin treatment (14 –18, 25, 26). Our data are betic humans. Diabetes. 40:223–226.
consistent with those of an earlier report that also used the 12. Dagogo-Jack SE, Cryer PE. 1993 Hypoglycemia associated autonomic failure
P300 to monitor changes in cognitive function during hy- in insulin-dependent diabetes mellitus: recent antecedent hypoglycemia re-
duces autonomic responses to symptoms of, and defense against subsequent
poglycemia in IDDM patients (34). However, in that study hypoglycemia. J Clin Invest. 91:819 – 828.
the assessment of glycemic thresholds for P300 changes 13. Jones TW, Boulware SD, Kraemer DT, Caprio S, Sherwin RS, Tamborlane
was limited by the fact that hypoglycemic stimulus was WV. 1991 Independent effects of youth and poor diabetes control on responses
to hypoglycemia in children. Diabetes. 40:358 –363.
brief, the magnitude of stimulus was not controlled, and 14. McCall AL, Fixman LB, Fleming N, Tornheim K, Chick W, Ruderman NB.
the measurements of P300 were made under rapidly 1986 Chronic hypoglycemia increases brain glucose transport. Am J Physiol
changing, nonsteady state conditions. Recent studies sug- 15. Pelligrino DA, Segil LJ, Albrecht RF. 1990 Brain glucose utilization and
gest that there is a delay before changes in plasma glucose transport and cortical function in chronic vs. acute hypoglycemia. Am J Physiol
alter measurement of P300 (34). 259:E729 –E735.
16. McCall AL, Millington WR, Wurtman RJ. 1982 Metabolic fuel and amino acid
In summary, both ends of the spectrum of glycemic transport into the brain in experimental diabetes mellitus. Proc Natl Acad Sci
control of IDDM influence in a similar way not only the USA. 79:5406 –5410.
level of glucose required to trigger counterregulatory re- 17. Boyle PJ, Nagy RJ, O’Connor AM, Kempers SF, Yeo RA, Qualls C. 1994
Adaptation in brain glucose uptake following recurrent hypoglycemia. Proc
sponses, but also the responsiveness of at least some brain Natl Acad Sci USA. 91:9352–9356.
functions to mild hypoglycemia. Although the nature of 18. Boyle PJ, Kempers SF, O’Connor AM, Nagy RJ. 1995 Brain glucose uptake and
unawareness of hypoglycemia in patients with IDDM. N Engl J Med.
this association is uncertain, its existence may be more 333:1726 –1731.
than just a coincidence. It is conceivable that the same 19. Widom B, Simonson D. 1990 Glycemic control and neuropsychologic function
molecular mechanisms affected regions of the CNS re- during hypoglycemia in patients with insulin dependent diabetes mellitus.
Ann Intern Med. 112:904 –912.
sponsible for glucose sensing (35, 36) as well as others 20. Herold KC, Polonsky KS, Cohen RM, Levy J, Douglas F. 1985 Variable
involved in cortical event-related potentials (30). It should deterioration in cortical function during insulin induced hypoglycemia. Dia-
also be noted that although P300 was preserved during betes. 34:677– 685.
21. Maran A, Cranston I, Lomas J, Macdonald I, Amiel SA. 1994 Protection by
mild to moderate experimental hypoglycemia in inten- lactate of cerebral function during hypoglycemia. Lancet. 343:16 –20.
sively treated IDDM patients, they are at much higher risk 22. Amiel SA, Pottinger RC, Archibald HR, et al. 1991 Effect of antecedent
of severe hypoglycemia in real life than conventionally glucose control on cerebral function during hypoglycemia. Diabetes Care.
14:109 –18.
treated patients. Therefore, this treatment-induced adap- 23. DeFeo P, Gallai V, Mazzotta G, et al. 1988 Modest decrements in plasma
tation may have a clinically adverse effect if plasma glu- glucose concentration cause early impairment in cognitive function and later
activation of glucose counterregulation in the absence of hypoglycemic symp-
cose falls to values well below those that can be compen- toms in normal man. J Clin Invest. 82:436 – 444.
sated for by changes in brain glucose metabolism. 24. McGuire EAH, Helderman JH, Tobin JD, Andres R, Berman M. 1976 Effect
of arterial vs venous sampling on analysis of glucose kinetics in man. J Appl
Physiol. 41:565–573.
Acknowledgments 25. Jacob RJ, Weber AB, Dziura JD, Morgen J, Sherwin RS. 1995 Brainstem
dysfunction is provoked by a less pronounced hypoglycemic stimulus in
We thank Aida Groszmann and Andrea Belous for their help with the diabetic BB rats. Diabetes. 44:900 –905.
measurements of plasma hormones, and Elizabeth Roessler for her tech- 26. Jacob RJ, Dziura JD, Blumberg M, Morgen JP, Sherwin RS. 1995 The brain
nical help with the measurement of evoked potentials. adapts to iatrogenic hypoglycemia in IDDM [Abstract]. Diabetes. 44(Suppl
27. Kumagai AK, Kang YS, Boado RJ, Pardridge WM. 1995 Upregulation of
References blood-brain barrier GLUT1 glucose transporter protein in experimental
chronic hypoglycemia. Diabetes. 44:1399 –1404.
1. The Diabetes Control and Complication Trial Research Group. 1993 The 28. Clarke WL, Gonder-Frederick LA, Richards FE, Cryer PE. 1991 Multifactorial
effect of intensive treatment of diabetes on the development and progression origin of hypoglycemia unawareness in IDDM: association with defective
of long term-complications in insulin-dependent diabetes mellitus. N Engl glucose counterregulation and better glycemic control. Diabetes. 40:680 – 685.
J Med. 329:977–986. 29. Fanelli CG, Epifano L, Rambotti AM, et al. 1993 Meticulous prevention of

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1718 JONES ET AL. JCE & M • 1997
Vol 82 • No 6

hypoglycemia normalizes the glycemic thresholds and magnitude of most of ence of counterregulatory hormones, independently of hypoglycemia, on
neuroendocrine responses to, symptoms of, and cognitive function during cognitive function, warning symptoms and glucose kinetics. Clin Sci.
hypoglycemia in intensively treated patients with short term IDDM. Diabetes. 85:197–202.
42:1683–1689. 34. Ziegler D, Hubinger A, Muhlen H, Gries FA. 1992 Effect of previous glycemic
30. Wood CC, McCarthy G, Squires NK, Vaughan G, Woods DL, McCallum WC. control on the onset and magnitude of cognitive dysfunction during hypo-
1984 Anatomical and physiological substrates of event-related potentials. Ann glycemia in type 1 (insulin-dependent) diabetic patients. Diabetologia.
NY Acad Sci. 425:681–721. 35:828 – 834.
31. Chiappa KH. 1990 Evoked potentials in clinical medicine, 2nd ed. New York: 35. Borg WP, During MJ, Sherwin RS, Borg MA, Brines ML, Shulman GI. 1994
Raven Press; 31. Ventromedial hypothalamic lesions in rats suppress counterregulatory re-
32. Jones TW, McCarty G, Tamborlane WV, et al. 1990 Mild hypoglycemia and sponses to hypoglycemia. J Clin Invest. 93:1677–1682.
impairment of brain stem and cortical evoked potentials in healthy subjects. 36. Borg WP, Sherwin RS, During MJ, Borg MA, Shulman GI. 1995 Local ven-
Diabetes. 39:1550 –1555. tromedial hypothalamus glucopenia triggers counterregulatory hormone re-
33. Kerr D, Diamond MP, Tamborlane WV, Kerr S, Sherwin RS. 1993 Influ- lease. Diabetes. 44:180 –184.

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Enhoncedodrenomedullory response
ond increosedsusceptibility to
neuroglycopenio : Mechonisms
underlyingthe odverseeffects of sugor
i n g e s t i o ni n h e o l t h yc h i l d r e n
TimothyW. Jones,vo, Wolterp. Borg,vo. SusonD. Boulwore.
GregoryMcCorthy,pno.RobertS.Sherwin,vo, ond
WilliomV. Tomborlone,MD
Fromthe Deportmentsof Pediotrics,
InlernolMedicine,ond Neurosurgery
ond the Generol
centers,yole university
clinicolReseorch schoolof Medicine,New Hoven,connecticut

oblecllve: Eoling simple sugors hos been suggesled os hoving odverse behov-
lorol ond c.ognlllve ellecls in children, buf o physiologic me-chonism hos not
been esloblished. rhis srudy wos performed to oddresJ thls lssue.
Deslgn: Metobollc, hormonol, ond symptomofic responEes lo o slonctqrd orol
glucose lood (1.75 gm/kg; moxlmum, r20 gm) were compqred In 25 heolthy
children ond 23 young odults, ond lhe hypoglycemlc clomp, logether wilh
meosuremenls of p300 oudlfory evoked polenliols, wqs used fo ossesswhether
chlldren ore more vulneroble lhon odulls to neuroglycopenlo.
SettlngzChlldren's Cllnicol ReseorchCenler, Yole UniversltySchool ot Medtcine.
Pesults|Bosellne ond olol glucose-siamuloted plosmo glucose ond insulin lev_
els were simllor in bofh groups, including fhe nodir glucose level 3 to s hours of-
ler orql odmlnlslrollon ol glucose (3.4 -r 0.1 mmol/t (6,1r l.g mg/dl) in children
ond 3.5 r- 0.t mmol/t (63 -* l.E mg/dtt in odulfs). rhe lole glucose decleose
sllmuloted o lise ln plosmo epln6phrlne levets thol wos fwololJ hlgher In chlldren
lhon fn odulls (2260 t 269 vs t03t + 147 pmo'/L l4o7 -r 52 vs tg6 + 26 pg/mll, p
<0.0{) ond o slgniflconl increose in hypoglycemic symplom scores in chlldren
(p <0.0f )' bul nol ln qdulls. During conlrol experimenls, In which stx ot the heolthy
chlldren Ingesfed q sugor-tree drlnk, lhere were no slgnlficont chonges In
ploimo glucose levels, hormone concenlrollon!, or hypoglycemlc sympfom
scores. Durlng lhe hypoglycemic clomp, p300 polenilols dld not chonge In ony
ot eighl odull sublecls unlll lhe plosmo glucose concenlrollon wos lowered to
3.0 mmol/L (54 mg/dl), whereos similor chonges ln p300 polenilqls were ob-
served ln slx of seven chlldren of glucose levels 3.6 lo 4.2 mmol/l (65 fo 75
concluslon: Enhonced odrenomedullory responses lo modest reducllons in
plosmo glucose concenlrolion ond Increosed suscepilblllty fo neuroglycope-
nlo moy be impoilonl conltlbuting locfors lo qdverse behqvlorol ond Jognlfive
eftecls ofler sugor ingesllon in heolthy children. (J peoren ig95;126:ll1-71

Supportedby grants RR06022, RR00125, DK20495, and Reprint requests:William V. Tamborlane,MD, Departmentof
HD3067l from the NationalInstitutesof Healthand by a grant Pediatrics,Yale University Schoolof Medicine, 333 Cedar St..
fromthe JuvenileDiabetesFoundationInternational. New Haven,CT 06510-8064.
Copyrighto 1995by Mosby-Year Book, Inc.
for publicationSept.6, 1994;acceptedOcr,.26,1994. 0022-3476lesl$3.00
t + 0 9/2o/6rs76
172 Joneset al. The Journal of Pediatrics
Februarv 1995

The question of whether refined sugar adverselyaffects the healthy, taking no medications, and have no history of di-
behavior or cognitive functions of healthy children is a con- abetesor carbohydrate intolerance. The children included
tinuing source of controversy. Whereas parents and clini- eight boys and 17 girls between 8 and 16 years of age; the
cians have frequently reported that even healthy children adults included 9 men and 14 women between 20 and 30
are prone to the development of behavioral changes and years of age. Subjects with attentional, behavioral, or psy-
cognitive defectsafter eating simple sugars,the outcomesof chiatric problems or with a history of unusual sensitivity to
research studies have been complex and discrepant.l-aIn sugar were excluded from the study. All subjectswere con-
addition, possible physiologic mechanisms for enhanced suming weight-maintaining diets containing at least 115
sensitivity to dietary sugar during childhood have not been gm/mz body surface area of carbohydratesper day before
clear. The presumed negative reactionsto sugar have been the study. Ofthe 25 children, eight were prepubertal (Tan-
attributed to an early rise or late fall in the plasma glucose ner stage I), and the other l7 had Tanner stage II to IV de-
concentration, and to an immunologic responseto sucrose.5 velopment.Bodymassindex(mean + SD)averaged18.2 +
Unfortunately, given the lack of consistentobjectivedata to 2.2kglm2 in the children and22.6 -r 2.8 in the adults. The
substantiatetheseassumptions,all thesehypothesesremain study was approvedby the Yale University School of Med-
open to questron. icine Human Investigation Committee, and informed writ-
The results of our previous studies of counterregulatory ten consentwas obtained from all subjectsand their parents
respons€sto hypoglycemia in children6'7 prompted us to (for the children).
propose a new hypothesis to explain the mechanisms Procedures.All subjectswere admitted to the Yale Gen-
underlying adverseeffectsof sugar ingestionin children. We eral Clinical ResearchCenter, where a history was obtained
hypothesizedthat, although the changesin plasma glucose and a physical examination performed. The studies were
levels after sucrose ingestion do not differ between adults started between8 and 8:30 epr, after the subjectshad been
and children, the children would show enhanced adreno- fasting for l0 to 12 hours overnight. With all procedures,
medullary and symptomatic responsesto these changesin an intravenouscatheter was inserted in a vein of a hand or
comparison with those of adults. Several lines of evidence distal portion of a forearm for blood sampling, and that
hand was kept in a heated box (60o to 65" C) to
ize" the blood.
See related article, p. l7E.
Oral glucose loading. All subjects took part in the oral
glucose loading study. They were allowed to rest for at least
support this hypothesis.Although sugar ingestion does not 30 minutes after insertion of the blood sampling catheter
normally causeseverereductionsin plasma glucoselevelsin
before baselinemeasurementswere obtained. Glucosewas
the late postprandialperiod in children,s'eour previous
ingestedin a dosageof 1.75 gm/kg body weight to a max-
study showed that plasma epinephrineresponsesto identi- imum of 120 gm (actual amount, I l0 + 5 gm in adults and
cal, modest insulin-induced reductions in plasma glucose 86 + 6 gm in children [mean + SD]) with a decaffeinated
are twofold greater in healthy children than in healthy cola preparation (General Medical Corp., Richmond, Va.).
adults.6 In addition, the plasma glucoselevel that triggers Blood sampleswere obtained every l0 minutes from -30 to
epinephrinereleaseand symptoms is substantiallyhigher in 300 minutes for measurementof the plasma glucose con-
children than in adults,T reaching values (plasma glucose centration, and every 30 minutes for measurement of
levels4.4 to 3.4 mmol/L [80 to 6l rng/dl]) that are com- plasma levels of insulin, catecholamines,and other coun-
monly observed 3 to 5 hours after oral glucose ingestion. terregulatory hormones (growth hormone, cortisol, and
To test our hypothesis,we compared the metabolic, hor- glucagon).
monal, and symptomatic responsesof healthy children and Each of the subjectsrated a list of symptoms before and
healthy young adults with a standardized, large glucose every 60 minutes after glucose ingestion, as previously de-
load. In addition, the hypoglycemic clamp technique was scribed. Symptoms (recorded on a lap-top computer) were
combined with serial measurementsof cortical auditory rated on a scale of I (not at all) to 7 (extreme). The eight
evokedpotentials to assesswhether young subjectsare more hypoglycemic symptoms included pounding heart, feeling
susceptibleto the adverseeffectsof reduced glucoseavail- shaky, feeling weak, having difficulty concentrating, head-
ability on cognitive function. aches,feeling anxious, slowed thinking, and feeling sweaty.
The sum of these symptoms at each observation point con-
METHODS stituted the total hypoglycemic symptom score at that time.
Subjects. Forty-eight nonobesesubjects(25 children' 23 In addition, four filler items (earache, pain in joints, watery
adults) were studied. To be eligible, the subjectshad to be eyes,and ringing in ears) were included to account for non-
The Journal of pediatrics
Volume126,Number 2 Joneset al. 173

specificeffects not referable to glucoseand antiinsulin hor-

electrode.The proportion ofsoft clicks varied between l0Zo
aad 20Vaof 200 clicks, and subjectsreported their count of
Control experiments were also performed in three boys
soft clicks at the end of each run. Two replicationswere ob-
and three girls, aged 10 + 3 years (mean r- SD), who had tained at each baseline(before insulin infusion) and at each
participated in the oral glucoseloading studies.These con_ plasma glucose plateau.
trol experiments were performed as describedabove; how_ f)eterminations.Plasma glucose was measuredin dupli_
ever, the oral glucoseload was replacedby an equal volume cate at the bedsidewith a glucose analyzer (Beckman In_
of sugar-free decaffeinatedcola drink (Coca-Cola Co., At_ struments Inc., Fullerton, Calif.). Epinephrine and norepi_
lanta. Ga.). nephrine were measured with a radioenzymatic assay
Hypoglycemic clamping. Eight of the children, aged (Amersham Corp., Arlington Heights, Ill.), glucagon (ICN
ll + 2 years (mean + SD), and eight of the adults,aged Biomedicals,Inc., Costa Mesa, Calif.), cortisol (Diagnostic
24 -t 2 years, who had taken part in oral glucose loading Products Corp., Los Angeles, Calif.), and growth hormone
experiments participated in the clamp protocol. However, (Kallestad Diagnostic Inc., Chaska, Minn.) were deter_
the cortical evoked potentials of one of children could not mined by radioimmunoassay.The intraassaycoefficientsof
be analyzed becauseof technical problems and was not rn- variation for these assayswere as follows: for epinephrine,
cluded in the analysis of electrophysiologicdata. 20.2Voat 175 pmol/L (31.5 pg/ml) and 6.j%oat l168
The methods used for the hypoglycemicclamp have been pmol/L (210 pg/ml); for growth hormone, 6.2Voat 3.5
describedin detail elsewhere.l0For this procedure,a second pg/L (3.5 nglml); for glucagon,9Vaat t22 ng/L (t22 pgl
cathcter was inserted in an antecubital vein in the arm op- ml); and for cortisol, l2go.Plasma insulin was measuredby
posite to the blood-sampling hand for administration of ex- double-antibody radioimmunoassay (Binax Inc., South
ogenous glucose and insulin. Portland, Maine).
After a 30-minute rest period, three blood sampleswere Statistical methods and analyses. Glucose and hormone
obtained during a 40-minute interval for measurementof responsesto orally administered glucoseand during clamp
baselinc glucose and hormone levels.A primed continuous studies were compared betweon groups by analysis of var!
infusion of rapid-acting human insulin (Novolin; Novo ance with repeated measures design. Other comparisons
Nordisk, Princeton, N.J.) was then begun at a continuous were made by analysisof variance,unpaired Student , test,
rate of 480 pmol . m-2 . min-I. and chi-square analysis where appropriate. The p values
After the insulin infusion was begun, the desired plasma (0.05 are reported as significant.
glucose level was achieved by varying the rate of 20Vogltt- Group differencesin symptom scores after glucose in-
cose infusion on the basis of measurementsof plasma glu- gestion were examined with time and at the plasma glu-
cose obtained at S-minute intervals. Blood samples for cosenadir. For the latter comparison,the symptom assess-
measurementof insulin and epinephrinewere taken at 20- ment that occurred at or immediately after the nadir
minute intervals throughout the study. In all subjects, plasma glucoselevel in each subject was used. During the
plasma glucosewas initially maintained at fasting euglyce- hypoglycemicclamp procedure,the plasma glucosethresh-
mic levelsfor 60 minutes and then reducedin hourly -Q.J6 old for epinephrine releasein each individual subject was
mmol/L (10 mg/dl) steps to a nadir of 3.0 mmol/L (54 defined as the glucoselevel at which a sustainedelevation
mg/dl). The subjectswere masked to plasma glucoselevels of >410 pmol/L Qa pg/ml\ above basal values was ob-
during the study. served, as previously described.TThe glycemic threshold
Electrophysiologic recordings were obtained during the for changesin P300 potentials in individual subjectsduring
last l0 to 15 minutes of each glycemicplateau.Scalp elec- the clamp procedure was defined as the plateau plasma
trodes were placed at the C, and P, positions,with a refer- glucoselevel at which a 30Voor greater reduction in p300
ence electrode placed on one ear and the ground electrode amplitude was observed.Demographic data are presented
placed on the opposite ear. A bipolar pair of electrodeswas as mean + SD, whereas other data are presented as
placed above and below the right eye to monitor for ocular mean + sEM.
artifacts. The P300 evokedpotentialswere obtained with an
auditory categorization ("oddball") task in which subjects RESULTS
were required to count silently the number of soft clicks de- Glucose ingestion. Basal plasma glucose levels and plas-
livered in a train consistingof a random mix of frequent loud ma insulin levels (55 + 5 pmol/L [8.8 t 0.8 pUlm|
and infrequent soft clicks. The loud clicks were presentedto in children;60 {- 5 pmol/L [9.6 + 0.E pUlm[ in adults)
the ear ipsilateral to the reference electrode, and the soft were similar in both groups, and the glucose and insu-
clicks wcre deliveredto the ear contralateral to the reference lin profiles were similar in children and adults after glucose

11 4 Jones et al The Journal of Pediatrics
Februarv 1995

Ioble l. Basal and nadir plasma glucoseand basal and Toble ll. Total hypoglycemic and filler (control)
peak hormone concentrations (mean -r SEM) after symptom scoresat baselineand at the postprandial
glucose load glucose nadir

Chlldren Adults Children Adulfs

P l a s m a / g l u c o s e( m m o l / L ) Glucose Glucose
Basal 4.6 + 0.1 4.8+ 0.1 Boseline nodir Soieline nodll
Peak ?.5 + 0.3 8.1 + 0.4
Nadir 3.4 + 0.1 3.5+ 0.1 Total hypoglycemic scores
P l a s m a e p i n e p h r i n e( p m o l / L ) Mean 12.8 22.O' l0.o l 3.o
Basal 229 + 27 I9l + 27 sE 1.0 3.0 0.5 1.7
Peak 2260 + 289* l03l + 147 Filler (control) scores
Plasma growth hormone (pg/L) Mean 5.0 5.5 5.0 5.2
Basal 1.7+ 0.3 l.l + 0.2 sE 0.5 0.6 0.4 0.5
Peak 1 9 . 5+ 2 . 5 1 8 . 3+ 2 . 8 tp <0.01 versus baseline.

Plasma glucagon (ng/L)

Basal 94+12 1 2 0+ l 8
Peak 2 0 1+ 3 l 180 + 25 0.5), or weak (1.9 + 0.3 to 3.8 + 0.5), or having a
To convert to metric units: multiply glucosevalue by 18. epinephrinevalue pounding heart I ."7+ 0.2 to 2.5 + 0.5) (p (0.05 vs base-
by 0.I8. growth hormone value by 1.0, and glucagon value by L0. line).
tp <0.01 versus adults.
ln contrast to the response to glucose ingestion, no
changesin plasma epinephrine levels were observedafter
ingestion (data not shown). Peak and nadir plasma glu- the sugar-free cola drink. As a result, plasma epinephrine
cose levels observed in the postprandial period in individ- levelswere significantly higher after glucoseingestion ver-
ual subjects determined by frequent sampling (every 10 suscontrol valuesat 240 minutes and 270 minutes (p <0.05)
minutes) were virtually identical in children and adults in these subjects (Fig. l). Total hypoglycemic symptom
(Table I). scoresalso did not change from baseline values (ll + 2)
Baseline plasma epinephrine levels were similar, and during the control study, in contrast to the increasein hy-
mean epinephrine concentrationsrose in both groups in re- poglycemicsymptomsin thesesubjects( I I -r 2 to 25 + 4)
sponseto the late postprandial fall in plasma glucose (Ta- after glucoseingestion.
ble I). However, the rise in the plasma concentration of Hypoglycemic clamp studies. As shown in Fig. 2, the
epinephrinewas significantly greater in the children, reach- plasma glucose concentration was gradually reduced in
ing peak values that were nearly twofold higher in the chil- nearly identical 0.56 mmol/L (10 mg/dl) stepsfrom 4.8 to
dren than in the adults (p <0.01) (Table I). On the oth-er 3.0 mmol/L (86 mg/dl to 54 mgldl) in both children and
hand, peak postprandial plasma growth hormone and glu- adults during the 240-minute hypoglycemic clamp proce-
cagon levelsdid not differ betweenthe groups.Basal plasma dure. As expected,Tthis controlled reduction in the plasma
concentrationsof cortisol and norepinephrinewere similar glucoseconcentrationstimulated an exaggeratedrise in the
in healthy children and adults, and levelsof thesehormones plasma epinephrineconcentration in children, in compari-
did not change significantly in either group after oral glu- son with that in adults (e.9.,4830 -t 334 vs 2365 t 3ll
cose (data not shown). Results of subgroup analysesfailed pmol/L [870 -r 60 vs 426 -r- 56 pg/ml] at 240 minutes; p
to demonstrate a significant effect of gender or pubertal <0.011. The plasma glucoselevel that stimulated a rise in
stageon the rise in epinephrinelevelsafter glucoseingestion epinephrinewas also significantly higher in children than in
by the 25 healthy children. adults (3.6 + 0.1 vs 3.0 -r 0.1 mmol/L [65 + 1.8 vs 54 +
Total hypoglycemic and filler (control) symptom scores 1.8 mg/dll; p <0.01), even though steady-stateplasma in-
were similar in both groups at baseline(Table II), and the sulin levelsachievedduring the insulin infusion were sim-
symptom scores remained unchanged in the adults after ilar in both groups (903 + 48 pmol/L [144.5 t 7.6 pU/
glucoseingestion. In contrast, total hypoglycemicsymptom mll in childrenvs 857 * 42pmollL [137 + 6.7 pUlml] in
scoresrose sharply in the children in associationwith the adults).
late fall in plasma glucoseconcentration (p <0.01 vs base- At baseline(before the start of the insulin infusion) the
line). As in the adults, filler (control) symptom scoresdid P300 amplitude was not significantly greater in the children
not changein the children throughout the study. The change than in the adults (l 1.7 t 1.5 vs 9.7 + 1.4, respcctively),
in hypoglycemic symptom scoresin the children was mainly and no change in either group was observed at thc cnd of
due to significantly increased reporting of feeling shaky the initial euglycemic period (plasma glucose level, 4.E
(from 1.7 + 0.2 to 3.2 + 0.6), sweaty(1.5 + 0.2 to 2.8 + mmol/L [86 mgi dl]) (Fig. 3). Most important, no change
The Journal of Pediatrics
volume 126,Number Z Jones et al. I 75

2 10 0

18 0 0

15 0 0
12 0 0
Ep r n e p n n n e
(mmo/L) eoo


0 60 120 180 240 300

TIME (minutes)
Flg' {. Plasma epinephrinelevels(mean + SEM) in six of the healthy children
before and after ingestionof oral glucose
(a) and a sugar-free cola drink (L). Asterisk indicates
significant difference in values between oral glucoseand control
e x p e r i m e n t sI < 0 . 0 5 ) . T o c o n v e r t t o m e t r i c u n i t s , m u l t i p l y e p i n e p h r i n e
value by 0.1g.



o 50 240 3oo
,,i? ,-,",,:::
Fig. 2. Plasma glucoseand epinephrinelevels (mean + SEM) during hypoglycemic
clamp studies in children (tr) and
adults (I). Asrerisk indicates significant difference (p <0.01) versuscorrespondingvalue
in adults. To convert to metric
units, multiply glucosevalue by 18, and e p i n e p h r i n ev a l u e b y 0 . 1 8 .

in P300 potentials was observedin any of the adults until glucoseconcentrationwas lowered to 4.2 mmol/L (75 mg/
the plasma glucose concentration was lowered to the last dl) (p <0.05 vs baseline)and the p300 amplitude remained
step (3.0 mmol/L [5a mg/dl]). In contrast,in the children significantly reduced throughout the remainder of the
the P300 amplitude was significantly reduced when the study. All but one of the children had alterations in cortical

I 76 Jones et al
The Journal of Pediatrics
February 1995

- l

A of P3O0
Amplitude -3

@v) -4




6A5AL 4.E 4.2 3.0 3.0

cLyCEutCLEVEL (mmal/L)

Fig.3. Deltaof P300amplitudeduringeachglycemicplateauinchildren(O)andadults(I). Asterisksindicatesignif-

icant differences versus euglycemia in the children (rp <0.05). Dagger (l\ indicates significant difference versus eugly-
cemia in adults (p <0.01).

evoked potentials at glycemic levels greater than 3.0 propriate term to describethesephenomena.To excludethe
mmol/L (5a mg/dl) (p <0.002 vs adult values). possibility that the findings reported above might have been
caused by nonspecific effects not connected with the oral
glucose load, we performed control experiments with a sug-
Our findings indicate that the late postprandialrise in the ar-free drink on a subgroup of the healthy childrcn wbose
plasma epinephrineconcentration was markedly greater in responses to glucose ingestion were typical of the cntire
children than in adults, whereasthe responsesof other an- group of children studied, and no changes in plasma
tiinsulin, counterregulatory hormones were not different. hormone concentrations or hypoglycemic symptom scores
Epinephrine r€sponseswere exaggeratedin children, even were noted.
though a glucoseload larger than the standard 75 to I 00 gm The P300 evoked potential is a neuroelectrophysiologic
was given to adults and, more important, the peak and na- measureof cognitive function produced when a subject at-
dir plasma glucose levels were nearly identical in the two tends to and discriminates a given stimulus.l3 The cortical
groups. auditory evoked potential has been shown to be adversely
Enhanced adrenomedullary responsesin the children affected by modest reductions in plasma glucose levelsl4 and
were associatedwith an increasein symptoms,such as feel- may therefore provide a sensitiveindex of neuroglycopenia.
ing shaky and sweaty, that are commonly attributed to During controlled reductionsin the plasma glucoseconcen-
stimulation of th€ sympatheticnervoussystem,lI' l2 whereas tration, cortical potentials are normally maintained until a
the scoreson filler items included to control for nonspecific critical threshold glucosevalue is reduced.r5In this study,
fatigue effects did not change. Moreover, adults who had P300 potentials were markedly altered in almost all the
blunted epinephrine responsesremained free of symptoms children when the plasma glucose concentration was low-
throughout the study, even though the lowest plasma glu- ered to values that were equal to or higher than the nadir
cose level in the postprandial period, as determined by fre- plasma glucose level seen after glucose ingestion in this
quent measurements,was nearly identical in the two groups. group. On the other hand, in adults P300 potentials were
These data suggestthat discrepanciesin symptom aware- preserved until the plasma glucose concentration was low-
nessof the late fall in the plasma glucoseconcentrationaf- ered to 3.0 mmol/L (5a mg/dl), values rarely observedin
ter an oral glucoseload are more likely related to differences either group after glucose ingestion. These data suggestthat
in epinephrine responses than to the absolute plasma healthy children are more vulnerable to the effects of hypo-
glucose level achieved. The plasma glucose concentration glycemia on cognitive function than are adults. Becausere-
fell to only 3.4 + 0.1 mmol/L (61 + 1.8 mg/dl) in the lease of epinephrine in responseto a fall in the plasma glu-
children, so "enhanced adrenomedullary responsiveness," cose concentration appears to be mediated through the
rather than "reactive hypoglycemia," might be a more ap- central nervous system,l6' l7 increased susceptibility to neu-

The Journal of Pediatrics
Jones et al. 177
Volume 126, Number 2

roglycopenia may, in turn, account for the higher plasma 6. Amiel SA, Simonson DC, Sherwin RS, Lauritano AA, Tam-
glucose level that triggered release of epinephrine in the borlane WV. Exaggerated epinephrine responsesto hypogly-
children. cemia in normal and insulin dependent diabetic children. J
PEDTATR 1 9 8 7 ; ll 0 : 8 3 2 - 7
The putative adverseeffectsofdietary sugar on behavior
7. JonesTW, Boulware SD, Kraemer DT, Caprio S, Sherwin RS,
and cognitive function in children have been the subject of Tamborlane WV. Independent effects of youth and poor dia_
marked controversy.lE-20 Previous studies in this area that betes control on responsesto hypoglycemia in children. Diabe-
have been focused on children with attention deficit disor- tes l99l;40:358-63.
der have tended to make exaggeratedclaims regarding the 8. RosenbloomAL, Wheeler L, Bianchi R, Chin FT, Tiwary CM,
Gorgic A. Age-adjusted analysis of insulin rcsponses during
global effectsof sugar and other food additives on their be-
normal and abnormal glucosetests in normal children and ad-
havioral problems. Kinsbourne2l noted that the problems olescents.Diabetes 1975t24:820-8.
caused by dietary sugar appear to be much less severeor 9. Grant DB. Serum-insulin levelsin children during glucosetol-
prevalent than some studiessuggest.On the other hand, we erance tests. Acta Paediatr Scand 1968;57:297-9.
10. Amiel SA, Simonson DC, Tamborlane WV, DeFronzo RA,
have shown in this study that consumption of glucosein an
Sherwin RS. Rate of glucose fall does not affect counterreg-
amount roughly equivalent in carbohydrate content to two ulatory hormone responsesto hypoglycemia in normal and di-
l2-ounce cans of Coca-Cola is followed by a fall in the abetic humans. Diabetes 1987;36:518-22.
plasmaglucoseconcentrationsufficient to induce hormonal, I l. Cryer PE, Binder C, Bolli GB, et al. Hypoglycemia in IDDM.
symptomatic, and neurophysiologic changes in healthy D i a b e t e s1 9 8 9 ; 3 8I: 1 9 3 - 9 .
12. Heller SR, Herbert M, MacDonald IA, Tattersall RB. Influ-
children. However, the rise in the plasma epinephrinecon-
enceof sympathetic nervoussystemon hypoglycemic warntng
centration and in associated adrenergic symptoms was s y m p t o m s .L a n c e t I 9 8 7 ; 2 : 3 5 9 - 6 3 .
acute and self-limited, and occurred only in the late post- 13. Sutton S, Tueting P, Zubin J, John ER. Information delivery
prandial period. These data do not indicate that dietary and the sensingevoked potential. Science 1976;155: 1436-9.
sugar is the cause of hyperactivity problems, but they em- 14. DeFeo P, Gallai Y,Mazzotta G, et al. Modest decrementsrn
plasma glucoseconcentrationscauseearly impairment in cog-
phasizethat most children could benefit from eating mixed
1 nitive function and later activation of glucose counterregula-
mealsthat include protein, fat, complex carbohydrate,and tion in the absenceof hypoglycemic symptoms in normal man.
fiber to limit postprandial reductions in plasma glucose lev- J Clin Invest 1988;82:436-44.
cls and increasesin plasma epinephrine levels. [n keeping 15. JonesTW, McCarthy G, Tamborlane WV, et al. Mild hypo-
I with this conclusion,Wolraich et al.l were unableto dem- glycemia and impairment of brain stem and cortical evoked
I onstrate any effects on behavior and cognitive function in
potentials in healthy subjects.Diabetes I 990;39:I 550-5.
I preschooland school-agechildren by adding or subtracting
16. Borg WP, During MJ, Sherwin RS, Borg MA, Brines ML,
Ii sucrosefrom a balanced diet.
Shulman GI. Ventromedial hypothalamic lesionsin rats sup-
press counterregulatory responsesto hypoglycemia. J Clin In-
I vest 1994;93:1677-82.
i 17. Biggers DW, Myers SR, Neal D, et al. Role of brain in coun-
terregulation of insulin-induced hypoglycemia. Diabetes
L WolraichML, LindgrenSD, StumboPJ,SteginkLD, Appel- I 9 8 9 ; 3 8 : 7I -6 .
baum MI, Kiritsy MC. Effect of diets high in sucroseor 18. Rosen LA, Booth SR, Bender ME, McGrath ML, Sorrel S,
on the behaviorand cognitiveperformanceof chil- Drabman RS. Effects of sugar (sucrose)on children's behav-
dren.N Engl J Med 1994;330:301-7. i o r . J C o n s u l t C l i n P s y c h o l1 9 8 8 ; 5 7 : 5 8 3 - 9 .
:. Milich R, Wolraich M, Lindgren SD. Sugar and hyperactiv- 19. Goldman JA, Lerman RH, Contois JM, Udall JN. Behavioral
rty: a critical review of empirical findings.Clinical Psychology
I effects of sucrose on preschool children. J Abnorm Child psy-
R e v i e w1 9 8 6 ; 6 : 4 9 3 - 531. chol 1986:14:565-77.
t ,$
L Crook WG. Food allergy: the great masquerader.Pediatr Clin 20. Wender EH, Solanto MV. Effects of sugar on aggressiveand
! N o r t h A m 1 9 5 1. 2 2 : 2 2 1 - 3 8 .
I. Rapp DJ. Does diet affect hyperactivity? Journal of Learning
inattentive behavior in children with attention deficit disorder $
with hyperactivity and normal children. pediatrics l99l:
D i s a b i l i t i e s1 9 7 8 ; lI : 3 8 3 - 9 . 88:960-6.
i Wcnder E. Review of researchof the relationship of nutritive 21. Kinsbourne M. Sugar and the hyperactivechild. N Engl J Med
swcetenersand b€havior. In: Diet and behavior. Washington, I 994;330:355-6.
D . C . :N a t i o n a l C e n t e r f o r N u t r i t i o n a n d D i e t e t i c s .l 9 9 l : 6 5 -
Yolume126 FEBRITARY1995 Number 2

PEDIffiRICS Medlcol progress 261 Sweat chlorides in Mauriac syndrome polack er al.

163 Allergic colitis in inltnts Odze et al.

263 Transformation of neutropenia into leukemia in child treated
with G-CSF Weinblatt et al.
Orlglnot orilctes
l7l Adrenomedullery responseand susceptibility to neuroglvcopenia 266 Keratoderma and photophobia in tyrosinemia type II
efter suglr ingestion Jones et al. Rabinowitz et al.

178 Effects of hyperglycemig on mental efficiency in adolescentswith 269 Loss of antibody to hepatitis B in immunized patients with
diebetes Gschwend et al. hemophilia Maris, Butler, and Cohen

lE5 Subsequentinsect stings in Hymenoptera hypersensitivity-

Felol oncl neonolql medlclne
Hauk et al.

l9l Relotion between infent feeding and infections 212 EfIect of maternal glucocorticoids on intraventricular
Eeaudry, Dufour, and Marcoux hemorrhage in preterr4 infa.nts Garland, Buck, and ltviton

l9E Effect of neonetel immunization with DT toxoids on responsesto 2E0 Reducing blood donor exposures by use of older red blood cells
Haemophilus conjugete vlccines Lieberntan et al. Lee et al.

20,6 Antibody responses after different sequencesof Haemophilus 2E7 Effects of L-carnitine on fat metabolism in premrture neonates
conjugate vaccinesGreenberget al. Bonner et al.

212 Risk fectors and outcomes in patients hospitalizedwith RSV

infection l|/ang et al. 293 Congenital CMV infection causedby nonprimary diseasein
mother with AIDS Sclueb&e el a/.

220 Hmpitelizrtion rNtes for lower respirrtory tract illness in infants

McConnochre, Roghmann, and Liptak Phormqcology ond lheropeullct

230 Binding oI Pseudomonosto respiratory cells in cystic fibrosis

297 Growth and growth holmone axis in children treated for asthma
Zar et al.
Crowley et al.

23.f Spectrum of herpes simplex encephalitisSthlesinger et al.

304 Corticosteroid therapy for ventricular tachycardia in lymphocytic
myocarditis Ino et al.
242 Lipid rbnormelities in Turner syndrome Ross et al.

246 Lysinuric protein intolennce with bone marrow abnormalities 309 Granisetron vs chlorpromazine plus dexametha$ne to prevent
Parenti et al. ifosfamide-inducedemcsis Hiihlen et al.

252 Reduction in bone mess after severe bttns Klein et al. 313 Amoxicillin-clavulanatcduring URI for preventionof otitis media
Heikkinen et al.
Edllor'r column
Currenl lllelqlule ond cllnlcol lrsues
257 Venom immunotherapy Eierman

317 Antenatal corticosteroid therapy to prevent RDS Ryan and Finer


*"5l,gPlls,*k*"o."9M 327 Acknowledgmenl ol reylewe]3

n t sl i s t e do n p a g e s5 A . 6 A , 7 A . 8 A , 9 A , l 0 A , l l A

lvl vrosuy
llt30 WestlineIndustrialDrive ".i'
St. Ianis! Missoui 531.163318 :..rr-.: t,,ti,Jij,.,
rSSN002di&6: .l:r:,
Ventromedial Hypothalamic Lesions in Rats Suppress
Counterregulatory Responses to Hypoglycemia
Walter P. Borg,* Matthew J. During,t Robert S. Sherwin,* Monica A. Borg,* Michael L. Brines,* and Gerald 1. Shulman*
Yale University School ofMedicine, Departments of * Internal Medicine and tSurgery, New Haven, Connecticut 06510

Abstract center") (7, 8), may also contain glucosensitive tissues that
could mediate the responses to hypoglycemia (9, 10). This
The central nervous system has been implicated in the activa- hypothesis was primarily based on the observations that injec-
tion of counterregulatory hormone release during hypoglyce- tions of 2-deoxy-glucose into the third ventricle causes hyper-
mia. However, the precise loci involved are not established. To glycemia (8). Initially, the VMH was considered a sympathetic
determine the role of the ventromedial hypothalamic nuclei center, controlling mainly catecholamine secretion in response
(VMH) in the hormonal response to hypoglycemia, we per- to hypoglycemia (4, 11 ); glucagon responses were thought to
formed hypoglycemic clamp studies in conscious Sprague- be triggered by intraislet rather than CNS mechanisms ( 12,
Dawley rats with bilateral VMH lesions produced by local ibo- 13). However, Frohman and Bernardis (14) demonstrated
tenic acid injection 2 wk earlier. Rats with lesions in the lateral that electric stimulation of the VMH caused an increase in
hypothalamic area, frontal lobe, sham operated (stereotaxic plasma glucagon. This work has been supported by recent stud-
needle placement into hypothalamus without injection), and ies performed by Biggers et al. ( 1 ) and Havel et al. ( 15). While
naive animals served as control groups. The clamp study had these studies do not address the role of the VMH during hypo-
two phases. For the first hour plasma glucose was fixed by a glycemia directly, they do suggest that the VMH could poten-
variable glucose infusion at euglycemia ( 5.9 mM). Thereaf-
tially serve as a key center for the activation of hypoglycemic
ter, for an additional 90 min, glucose was either allowed to fall counterregulation. However, since that report, there has been a
to (a) mild hypoglycemia ( 3.0 mM) or (b) more severe hypo-

paucity of data examining this specific issue. Indeed, the rela-

glycemia ( - 2.5 mM). Glucagon and catecholamine responses tive role of the CNS or extracerebral glucose sensors in initiat-
of lateral hypothalamic area-, frontal lobe-lesioned, sham ing counterregulatory responses has remained the subject of
operated, and naive animals were virtually identical at each controversy.
hypoglycemic plateau. In contrast, glucagon, epinephrine, and To determine the role of the VMH nuclei in the hormonal
norepinephrine responses in the VMH-lesioned rats were mark- response to hypoglycemia, we combined the hypoglycemic in-
edly inhibited; hormones were diminished by 50-60% during sulin clamp technique with bilateral VMH lesions produced by
mild and by 75-80% during severe hypoglycemia as compared stereotaxic ibotenic acid injection in Sprague-Dawley rats. Le-
with the other groups. We conclude that the VMH plays a sions in the lateral hypothalamic area, frontal lobe, sham oper-
crucial role in triggering the release of glucagon and catechol- ated, and nonlesioned (naive) animals served as controls. This
amines during hypoglycemia. (J. Clin. Invest. 1994. 93:1677- approach allowed us to introduce highly specific lesions within
1682.) Key words: ventromedial hypothalamus * hypoglycemia the chosen brain region and to test their effect under standard-
* glucagon epinephrine * counterregulation
ized hypoglycemic conditions.
The role of the central nervous system (CNS) in the regulation Methods
of counterregulatory responses to hypoglycemia remains con-
troversial. Although several lines ofevidence strongly implicate Animals
the CNS in hypoglycemia detection and counterregulation ( 1- Male Sprague-Dawley rats were purchased from Charles River Breed-
3), the precise brain regions involved are not known. While ing Laboratories (Wilmington, MA). Animals were housed in an envi-
ronmentally controlled room with a 12-h light/dark cycle and were
various nuclei have been implicated, current data suggest that maintained on standard ad lib. rat chow (Prolab 3000; AGWAY, Wa-
counterregulatory responses during hypoglycemia are acti- verley, NY) comprising of 22% protein, 5% fat, and 5 1% carbohydrate
vated, at least in part, via the hypothalamus (4-6). It has (the remaining 22% consists of ash, crude fiber, and moisture).
been frequently suggested that ventromedial hypothalamus
(VMH),' known as a regulator of food intake ("satiety Lesions
Rats (mean body wt 290 g, range 270-310 g) were anesthetized by

Address correspondence to Gerald I. Shulman, M.D., Ph.D., Yale Uni- intraperitoneal injection (1 ml/kg) of a mixture of xylazine (20 mg/
versity School of Medicine, Department of Internal Medicine/Endocri- ml) (AnaSed; Lloyd Laboratories, Shenandoah, IA) and ketamine
nology, 333 Cedar Street, FMP 104, New Haven, CT 06510. (100 mg/ml) (Ketaset; Aveco Co., Fort Dodge, IA) in a ratio of 1:2
Receivedfor publication 5 October 1993 and in revisedform 6 De- (vol:vol). Thereafter, they received bilateral stereotaxic infusions of
cember 1993. 0.12 M ibotenic acid (Research Biochemicals Inc., Natick, MA) dis-
solved in a sterile artificial extracellular fluid solution (NaCl 135 mM,
1. Abbreviations used in this paper: FL, frontal lobe; LHA, lateral hypo- KCI 3 mM, MgC12 1 mM, CaCl2 1.2 mM, ascorbate 200 ,M, and a
thalamic area; VMH, ventromedial hypothalamus. sodium phosphate buffer of 2 mM to pH 7.4) (16). The solution was
infused at a rate of 0.25 1 /min. The needle was placed with the bevel-
J. Clin. Invest. ing directed anteriorly, and 0.5 ytL of ibotenic acid was infused, then
© The American Society for Clinical Investigation, Inc. beveling of the needle was reversed, and 0.5 Al was infused into poste-
0021-9738/94/04/1677/06 $2.00 rior directions. The needle was left inside the tissue for a 3-min period,
Volume 93, April 1994, 1677-1682 to allow diffusion and to prevent backflow of ibotenic acid through the

Role of Ventromedial Hypothalamus in Counterregulation 1677

needle track. All stereotaxic coordinates were determined from the the second set of experiments) and maintained there for 90 min. Exper-
atlas of Paxinos and Watson (17). Five different groups of animals iments were terminated if the plasma glucose (a) fell below 4.5 mM
were prepared as follows. during the last 45 min ofthe euglycemic phase, (b) rose above 3.9 mM
Group 1. VMH-lesioned rats were injected at points located 2.5 mm (secondary to glucose infusion), or (c) inadvertently fell below 2.0 mM
posterior, 0.6 mm lateral in relation to bregma, and 9.6 mm below the during the hypoglycemic phase. Only the studies during which the
horizontal plane passing through bregma and lambda. mean blood glucose level achieved during hypoglycemia was in the
Group 2. Frontal lobe (FL)-lesioned rats were injected using the range of 2.8-3.1 mM (for the first set) or 2.2-2.5 mM (for the second
coordinates: 2.0 mm anterior, 2.0 mm lateral in relation to bregma, set) were analyzed. In the mild hypoglycemia group 18% of FL prop-
and 2.0 mm below the horizontal plane passing through bregma and erly lesioned (as verified histologically) and 40% of naive animals did
lambda. not meet these criteria. In the severe hypoglycemia group 16% of con-
Group 3. Lateral hypothalamic area (LHA)-lesioned rats were in- trol studies (naive rats) were excluded. Histological results and the
jected using the coordinates: 2.0 mm posterior, 2.0 mm lateral in rela- accuracy ofthe clamps were the only criteria for excluding or including
tion to bregma, and 8.4 mm below the horizontal plane passing the clamp studies for the data analysis; exclusions were never made
through bregma and lambda. based on the knowledge of the hormonal results.
Group 4. Sham operated animals were prepared by lowering the Blood samples for measurements of glucagon, epinephrine, and
injection needle in the proximity of VMH using the coordinates 2.5 insulin were taken during the euglycemic (30- and 60-min) and hypo-
mm posterior, 0.6 mm lateral in relation to bregma, and 8.6 mm below glycemic (90-, 120-, 135-, and 150-min) phases of the experiments.
the horizontal plane passing through bregma and lambda, but no toxin During blood sampling, sample dilution by fluid in the dead space of
was infused. the catheter was avoided by withdrawal of 0.5 ml of blood before sam-
Group 5. Control (naive) animals did not receive any surgery. Le- ple collection. This volume exceeded the dead space volume by about
sioned animals were studied 12-16 d after the stereotaxic procedure. At fivefold. A new syringe was then used for sample collection. Subse-
the end of the experiments, lesion placement was verified histologically quently, the initial syringe was reattached, and the contents reinfused.
by cresyl violet staining. Lesions were identified as areas of neuronal Blood obtained from littermates at least 30 min before the study was
loss. Only the animals that showed bilateral destruction of the desired transfused during the clamp to quantitatively replace blood withdrawn
brain regions were included. From all animals studied, in the mild during the experiment. The protocol was reviewed and approved by the
hypoglycemia group 11% of VMH and 29% of LHA did not meet the Yale Animal Care and Use Committee.
histological criteria. In the severe hypoglycemia group 16% of VMH
were excluded. All other animals studied met histological criteria. Analytical methods and calculations
Plasma glucose was measured in duplicate using a Glucose Analyzer II
Surgical procedures (Beckman Instruments Inc., Fullerton, CA). Plasma insulin (Binax,
At 6-10 d before the study and 7-9 d after stereotaxic surgery, animals South Portland, MA), and glucagon (ICN Biomedicals, Inc., Carson,
underwent an additional aseptic surgical procedure for placement of CA) using porcine standard were determined by a double antibody
internal jugular vein and carotid artery catheters under intraperitoneal radioimmunoassay. Insulin measurements during the basal and insulin
pentobarbital anesthesia (Nembutal 35 mg/kg body wt; Abbott Labo- infusion periods were made using rat and porcine standards, respec-
ratories, North Chicago, IL). The polyethylene carotid artery catheter tively. Plasma catecholamines were measured with a radioenzymatic
was extended to the level of the aortic arch, and the silicone internal method (Amersham Corp., Arlington Heights, IL).
jugular vein catheter was advanced to the level of the right atrium. At Data are expressed as mean±SE. Comparison between the study
the end of the procedure both catheters were flushed and filled with groups was made by ANOVA with a repeated measure design, followed
heparin (42 U/ml) and polyvinylpyrrolidone (1.7 g/ml) solution, by the Student's t test to localize effects. Glucose infusion rates during
plugged, tunneled subcutaneously around the side of the neck, and the hypoglycemic steps represent the average values during the desig-
externalized behind the head through a skin incision. Catheters re- nated time interval.
mained sealed until the day of the study. Only those animals that had
recovered completely and showed no signs of infection within 36 h Results
after surgery were used.
Set 1: mild hypoglycemia. As summarized in Table I, basal
Euglycemic/hypoglycemic clamp glucose, insulin, glucagon, epinephrine, and norepinephrine
The hyperinsulinemic glucose clamp technique, as adapted for the rat levels were not significantly different between any of the study
( 18), was used to provide a fixed hypoglycemic stimulus. Two sets of groups. The average weight of the animals on the day of the
experiments were performed: In the first, all five groups of animals study was 290±10 g (VMH), 250±5 g (LHA ), 290±15 g (FL),
were studied, and the plasma glucose concentration was lowered to 290±5 (sham operated), and 290±10 g (naive). The weight of
3.0 mM. In the second, only VMH-lesioned and naive (control) the LHA-lesioned animals was significantly lower as compared
animals were studied, and a more severe hypoglycemic stimulus with other groups (P < 0.05). During the insulin infusion,
( 2.5 mM) was produced.
The rats were fasted for 24 h before the study. On the morning of
- plasma insulin rose to nearly identical levels, and plasma glu-
the experiment, the catheters were flushed with saline and maintained cose was indistinguishable during each phase ofthe study in all
patent by a slow infusion of saline (20 Mll/min) that contained a small groups (Table II). During the hypoglycemic phase ofthe study,
amount of heparin ( 1-2 U/ml). Animals were fully awake and freely the desired level of glycemia (i.e., 3.0 mM) was achieved in the
moving about in their cages, untethered. After a 60-min rest period, first 30 min. When euglycemia (5.9 mM) was maintained, the
blood samples were withdrawn for measurement of baseline glucose, concentrations of glucagon and norepinephrine were not dif-
insulin, glucagon, epinephrine, and norepinephrine concentration. ferent between the studied groups, whereas during the euglyce-
Thereafter, a primed (2,160 pmol over 1.5 min) continuous infusion mic phase, plasma epinephrine was significantly decreased in
( 120 pmol/kg-'* min' ) ofporcine insulin (Eli Lilly & Co., Indianapo- the VMH-lesioned animals as compared with other groups
lis, IN) was initiated and maintained for 150 min. A variable infusion
of exogenous glucose was adjusted based on plasma glucose measure- (0.3±0.01 nM [VMH] vs 0.9±0.1 [LHA], 0.7±0.1 [FL],
ments obtained at 5-min intervals to achieve the desired glucose level. 0.9±0.1 [sham operated], 0.8±0.2 nM [naive]; P < 0.05).
During the first 60 min, the rats were maintained at euglycemia (mean The effects of VMH lesions on hormonal counterregulatory
plasma glucose - 5.9 mM). Thereafter, plasma glucose was allowed to responses to mild hypoglycemia (3.0 mM) are depicted in Fig.
fall to hypoglycemic levels ( - 3.0 mM for the first set or 2.5 mM for
- 1. Despite a comparable hypoglycemic stimulus, epinephrine,

1678 Borg, During, Sherwin, Borg, Brines, and Shulman

Table I. Basal Characteristics of the Animals Studied
VMH FL Naive LHA Sham operated
(n = 8) (n = 9) (n = 6) (n = 5) (n = 5)
(nM) 0.1±0.04 0.1±0.02 0.1±0.02 0.1±0.02 0.1±0.05
(ng/liter) 136±24 122±16 112±10 201±51 191±8
(nM) 0.6±0.1 0.8±0.2 0.8±0.3 0.4±0.2 0.7±0.2
(nM) 0.9±0.2 1.4±0.2 1.3±0.2 1.2±0.2 1.1±0.2

norepinephrine, and glucagon responses in the VMH-lesioned step (3.0±0.7 mg/kg-1*min-' [VMH] vs 1.1±0.2 mg/
rats were significantly blunted (by 50-60%) compared with
kg- . min-' in naive animals, P < 0.05).
the other groups (P < 0.05). LHA-lesioned, FL-lesioned, sham
operated, and naive animals showed indistinguishable hor- Discussion
monal responses to mild hypoglycemia (Fig. 1).
The diminished hormonal response in the VMH-lesioned The current data demonstrate that bilateral selective lesions of
rats was associated with a higher glucose infusion rate required the VMH produce striking reductions in the magnitude of the
to maintain the 3.1 mM hypoglycemic plateau during the final glucagon, epinephrine, and norepinephrine responses to both
30 min of the study (13.8±1.3 mg/kg-'. min-' [VMH] vs mild and moderately severe hypoglycemia. Neither sham sur-
5.2±0.7 [LHA], 6.9±0.7 [FL], 5.8±1.0 [sham-operated], gery nor selective lesions of the LHA or the FL had such an
7.0±1.4 mg/kg'- min-' [naive]; P < 0.05). effect.
Set 2: severe hypoglycemia. To determine ifthe diminished Whereas the role of individual counterregulatory hormones
hormonal responses during hypoglycemia seen in VMH-le- in hypoglycemia correction has been studied extensively ( 19-
sioned animals persisted with a stronger hypoglycemic stimu- 22), the mechanisms that link glucopenia with activation of
lus, plasma glucose was lowered to 2.5 mM. The average weight the counterregulatory system are poorly understood. There is
of the VMH and naive animals on the day of study was 295±10 much controversy concerning the tissues that sense glucopenia
and 270±5 g, respectively. As in set 1, during the insulin infu- and coordinate the counterregulatory responses. In fact, numer-
sion plasma insulin rose to nearly identical levels, and plasma ous neural centers have been proposed to control counterregu-
glucose was indistinguishable during each phase of the study in latory hormone release (2, 23). The hypothalamus has long
the two groups (Table III). During the hypoglycemic phase of been considered a potential center for the integration of the
the study, the desired level of glycemia (i.e., 2.5 mM) was hypoglycemia-induced adrenergic response (4, 24-26). Stud-
achieved in the first 30 min. As shown in Fig. 2, VMH lesions ies have implicated the ventromedial portion of this brain re-
caused a marked reduction in peak elevations of circulating gion, in particular. However, the role of the VMH in modulat-
glucagon (241±45 ng/liter [VMH] vs 796±112 ng/liter in ing counterregulatory hormone release has never been directly
naive animals, P < 0.01), epinephrine ( 12.8±0.9 nM [VMH] tested under hypoglycemic conditions. Rather, it has been
vs 69.3±4.1 nM in naive animals, P < 0.01 ), and norepineph- shown that electrical or mechanical stimulation of the VMH
rine (3.3±0.4 nM [ VMH ] vs 45.1±4.5 nM in naive animals, P caused hyperglycemia (25, 27, 28). Moreover, injection of 2-
< 0.01) in response to the 2.5 mM hypoglycemic plateau. deoxy-glucose into the third ventricle also resulted in hypergly-
In keeping with this finding, the VMH-lesioned animals cemia (8, 29). However, this does not localize the locus for the
required a higher glucose infusion rate to maintain plasma glu- counterregulatory response to hypoglycemia specifically to the
cose at 2.5 mM during the final 30 min of the hypoglycemic VMH. Interestingly, in contrast to traditional opinion that

Table II. Mean Plasma Glucose and Insulin Concentrations during Mild Hypoglycemia
VMH FL Naive LHA Sham operated
(n = 8) (n = 9) (n = 6) (n = 5) (n = 5)
Glucose (mM) 5.8±0.2 5.9±0.1 5.9±0.1 5.9±0.2 5.8±0.2
Insulin (nM) 4.9±0.6 4.7±0.7 4.7±0.5 5.0±0.6 4.8±0.6
Glucose (mM) 2.9±0.1 3.0±0.1 3.0±0.1 2.9±0.2 2.9±0.2
Insulin (nM) 5.0±0.6 4.8±0.4 4.9±0.4 5.0±0.5 4.9±0.4

Glucose values were obtained by averaging all glucose measurements during the euglycemic or hypoglycemic phases of the study. Insulin data for
the euglycemic phase represent the average insulin level of the samples obtained at the end of this phase. Insulin values during hypoglycemia
represent the average of measurements obtained at 90 and 150 min.

Role of Ventromedial Hypothalamus in Counterregulation 1679


(nM) (nM)

(nM) (nM)

* *
* *
(ng/L) 600-
300' (ng/L)
A 200
0 30 60 90 120 135 150
0 30 60 90 120 135 150
Time (nOn)
Tom (mim)
Figure 1. Effect of VMH lesioning on counterregulatory hormone re-
sponse to mild (3.0 mM) hypoglycemia. The asterisk denotes signifi- Figure 2. Effect of VMH lesioning on counterregulatory hormone re-
cant statistical difference as compared with all control groups (P sponse to severe (2.5 mM) hypoglycemia. The asterisk denotes sig-
< 0.05). nificant statistical difference as compared with all control groups (P
only catecholamine release is critically dependent upon the
CNS (2), electrical stimulation of the VMH in rats has been region of the hypothalamus impinge on the endocrine pan-
found to cause an increase in plasma glucagon concentration creas (30).
( 14), consistent with evidence that efferents from the ventral It should be noted, however, that some data do not support
the concept that the neurons controlling the response to gluco-
penia reside in the VMH. In fact, it has been suggested that they
Table III. Mean Plasma Glucose and Insulin Concentrations are not located in the forebrain (which comprises cerebral
during Severe Hypoglycemia hemispheres, thalamus, subthalamus, hypothalamus, and
VMH Naive epithalamus). Cantu et al. (31 ) reported finding glucorecep-
(n= 5) (n= 5) tors located within the spinal cord that are capable of augment-
ing catecholamine secretion during hypoglycemia. Goldfien et
Euglycemia al. (32) also concluded that there are similar centers in the
Glucose (mM) 5.7±0.3 5.8±0.2 cervical portion of the spinal cord. In keeping with this conclu-
Insulin (nM) 4.6±0.5 4.7±0.6 sion DiRocco and Grill (33) have demonstrated that the sym-
Hypoglycemia pathetic response to systemic 2-deoxy-D-glucose was not pre-
Glucose (mM) 2.5±0.1 2.6±0.2 vented by complete decerebration of the rat. Ritter et al. (34)
Insulin (nM) 4.7±0.5 4.8±0.8 showed that the obstruction of the cerebral aqueduct (that con-
nects the fourth ventricle located in the hindbrain with third
Glucose and insulin data were obtained as indicated in Table II. and lateral ventricles situated in the forebrain) failed to sup-

1680 Borg, During, Sherwin, Borg, Brines, and Shulman

press the hyperglycemic response to 5-thioglucose injected into counterregulatory responses. It is noteworthy that chemical le-
the fourth ventricle. Furthermore, under these conditions the sions located in hypothalamus (LHA), but outside VMH,
hyperglycemic response to 5-thioglucose injection into third failed to change the counterregulatory response. Finally, sham
ventricle was abolished. From these data authors concluded operations, which included all manipulation involved in the
that glucoreceptors are located in the hindbrain (which consists chemical lesioning of VMH except ibotenic acid infusions, had
of pons and medulla oblongata) and not in the hypothalamus. no effect on counterregulatory response.
This view is consistent with data generated by Cane et al. (35), In summary, the current studies demonstrate a profound
who reported that counterregulatory responses were not inhib- decrease in the magnitude of hormonal counterregulation after
ited when glucose was infused in the carotid arteries to abolish selective lesioning of the VMH. These data suggest that the
forebrain hypoglycemia during systemic hypoglycemia. How- neurons located in VMH are essential for the integrated hor-
ever, these results may not exclude an important role for hypo- monal response to glucose deprivation. However, it should be
thalamic glucoreceptors. Studies performed by Biggers et al. emphasized that our studies cannot answer the question if glu-
( 1) and more recently by Frizzell et al. (36) demonstrated that cosensors are located in the neurons of VMH. The specific role
bilateral infusion of glucose into the carotid as well as vertebral of these neurons in detection of hypoglycemia and/or coordi-
arteries suppressed counterregulatory hormone responses to pe- nating counterregulatory response remains to be established.
ripheral hypoglycemia. Thus, it is still plausible that the hypo-
thalamus plays a role in hypoglycemia detection or in integra- Acknowledaments
tion of information generated from glucoreceptors localized
elsewhere (37-39). We appreciate the assistance of Aida Groszmann and Andrea Belous
Controversy surrounding the loci responsible for triggering for their help in the measurements of plasma hormones. We also grate-
hypoglycemia-induced counterregulation may at least in part fully acknowledge Jeffrey Tamborlane and Nicole Hamlet for their
be methodology related. Firstly, in many studies, nonphysiolo- expert technical assistance.
gic stimuli were used to generate cellular glucopenia, such as This research was supported by grants from the U.S. Public Health
the use of systemically administered metabolic inhibitors. The Service (DK-20495, DK-40936, DK-45735, and NS-28227) and a
degree of glucopenia with 2-deoxy-D-glucose is difficult to con- fellowship grant from The Juvenile Diabetes Foundations Interna-
tional (W. P. Borg).
trol and therefore difficult to relate to physiologic hypoglyce-
mia. Secondly, it is very difficult to interpret data generated by
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1682 Borg, During, Sherwin, Borg, Brines, and Shulman

Diabetologia (1997) 40: S 75–S 82

 Springer-Verlag 1997

Microdialysis techniques in the study of brain and skeletal muscle

D.G. Maggs, W. P. Borg, R. S. Sherwin
School of Medicine, Yale University, New Haven, USA

Summary Traditionally, plasma measurements have milieu seen by individual tissues (and hormone re-
been used to monitor metabolic events and the ac- ceptors?) may, at least in some instances, be strikingly
tions of hormones that are actually taking place with- different from that in plasma, and as a result, plasma
in tissue beds that are anatomically separated from measurements by themselves may not appropriately
the vascular compartment. It is generally assumed define the contributions of specific tissues to meta-
that the extracellular fluid (ECF) within metaboli- bolic events, and overlook the importance of meta-
cally active tissues is composed of substrates and hor- bolic processes which are largely restricted to individ-
mones in concentrations that closely approximate ual tissue beds. Through the use of microdialysis as a
those in plasma. Indeed, this view is implicit in non- means of directly sampling ECF from metabolically
steady-state tracer calculations. However, through important body tissues and with the evolution of its
the use of microdialysis techniques in the study of tis- use in animal and human research, this technique
sue metabolism this view is being challenged. Our will continue to offer exciting new insights into tissue
data suggest that there may be substantial concentra- metabolism and to investigate fundamental issues
tion gradients for a variety of fuels between plasma that cannot be addressed by other methods. [Dia-
and ECF, i. e. fuels (e. g. glucose) removed from the betologia (1997) 40: S 75–S 82]
circulation being lower and fuels (e. g. glycerol, lac-
tate, some amino acids) produced by tissues being Keywords Brain, skeletal muscle, interstitial fluid.
higher than plasma levels. In short, the metabolic

Microdialysis offers a powerful investigative tool been applied to the study of the limb as a means of
which enables in vivo sampling of extracellular fluid ‘isolating’ skeletal muscle, but these studies are con-
(ECF) from body tissues [1, 2]. Alternative methods, founded by the contributions of other tissues to net
such as positron emission tomography and magnetic metabolic activity [3]. Moreover, arteriovenous dif-
resonance spectroscopy, enable insights into tissue ferences across a tissue bed assess ‘net’ changes but
metabolism, but are indirect and observations are de- may underestimate the turnover of a substrate in the
rived. The Fick principle, through measurement of tissue in question. For example, in the limb, high rates
tissue blood flow and arteriovenous differences, is a of glycerol uptake and release occur, despite a small
more direct approach, however, it has anatomical arteriovenous difference [4, 5].
limitations. In humans, this technique has mostly To date, microdialysis has been used mainly in the
study of animal brain, where the focus has been pre-
dominantly in the area of neurotransmission [6–8].
Corresponding author: Dr. D. G. Maggs, Section of Endocrinol- The application of microdialysis in humans has most-
ogy, Yale School of Medicine, P. O. Box 20 80 20, New Haven,
CT 06520-8020, USA
ly been directed at fuel metabolism in adipose tissue
Abbreviations: ECF, Extracellular fluid; VMH, ventromedial although, more recently, it has also been applied to
hypothalamus; IDDM, insulin-dependent diabetes mellitus; 2- the study of skeletal muscle. From a local standpoint,
DG, 2-deoxyglucose; GABA, g-aminobutyric acid. we have applied microdialysis techniques to the in
S 76 D.G. Maggs et al.: Brain and skeletal muscle microdialysis

vivo study of both animal and human brain as well as 20 mm

the investigation of peripheral tissue metabolism in
both skeletal muscle and adipose tissue. In animal Inlet tube
brain, we have directed our attention to the role of
the ventromedial hypothalamus (VMH) in glucose Outlet tube 250 µm
sensing and the effect of glucose availability on the
release of neurotransmitters. Whereas, in the periph-
ery, we have focused on human skeletal muscle, a tis-
sue that plays a key role in fuel homeostasis. Through Membrane
the unique access to specific tissue beds that microdi- Fig. 1. Schematic representation of the microdialysis probe we
alysis offers, we have applied this method in two main utilize in the study of adipose and skeletal muscle tissues. Fluid
directions: (i) perturbing the local extracellular envi- is perfused (perfusate) via the inlet tube into the cylindrical
chamber comprizing the dialysis membrane and subsequently
ronment of a specific target tissue by the perfusion exits via the outlet tube (dialysate)
of substrates or pharmacologic agents via the micro-
dialysis catheter, (ii) sampling local tissue ECF from
the microdialysis catheter during systemic metabolic
perturbations. concentration and this reflects the ‘recovery’ of the
microdialysis system. Recovery is often expressed as
a percent (i. e. [dialysate]/[ECF] × 100) and is easily
Methodologic considerations calculated in vitro by placing a microdialysis catheter
in an aqueous solution containing a known amount of
In view of our application of this technique in brain substrate. The catheter is perfused at a constant rate
and peripheral tissues, it is important to address and substrate concentration is measured in dialysate
some general methodologic issues that concern the enabling the calculation of in vitro recovery (i. e. [di-
microdialysis process and some further, more spe- alysate]/[solute] × 100). In vivo recovery is generally
cific, issues that we have encountered. Microdialysis less than that seen in vitro, and this is thought to re-
is based on the principle of diffusion of substances flect the reduced capacity for substrates and other
across a permeable membrane between two compart- molecules to diffuse in the ECF space surrounding
ments. Its typical use in vivo involves the perfusion of the membrane when compared with diffusing capac-
an isotonic artificial ECF solution (perfusate) into a ity in an aqueous solution in vitro. However, it is pos-
tissue bed where perfusate equilibrates with the local sible to calculate absolute ECF concentrations, and
tissue ECF across a dialysis membrane; this fluid (di- therefore in vivo recovery, by a number of methods,
alysate) is then collected for measurement of tissue two of which we have utilized in our studies: the ‘no
substrates ex vivo. Although, through the nature of net flux’ [1] and ‘zero flow rate’ [9] procedures. Both
the microdialysis process, it also feasible to deliver of these methods have been validated in the past and
substrates, pharmacologic agents or even hormones we have utilized both procedures in vitro and in
to the local tissue bed via the microdialysis catheter. vivo. The no net flux method is based on the principle
The performance or efficiency of a microdialysis of perfusing a microdialysis system with different
system is dependent on a number of factors including concentrations and, by measuring dialysate concen-
the structural design of the microdialysis catheter. A trations of substrate, it is possible to calculate a stea-
schematic representation of the microdialysis system dy-state where perfusate concentration equals ECF
that we have used in peripheral tissues is shown in Fig- concentration, in other words, a steady-state of ‘no
ure 1, however, other similar prototypes exist, some net flux’. This estimate represents absolute ECF con-
with structural differences. Efficiency is also depen- centrations of the substrate in question. Figure 2
dent on the nature of the dialysis membrane and a shows representative examples of ‘no net flux’ cali-
range of cellulose ester, polysulfate and polycarbonate bration procedures carried out in healthy human sub-
membranes are in use, each with specific physical char- jects for the measurement of glucose in the ECF
acteristics. It is theorized that the nature of the perfu- space of adipose tissue and skeletal muscle.
sate may also influence the dialysis process and that The ‘zero flow rate’ method is based on the princi-
to minimize artifact caused by the perfusate itself it is ple that the relative recovery of substrate across dial-
recommended that catheters should be perfused with ysis membrane is inversely related to perfusate flow
a solution that closely resembles tissue ECF. For this through the microdialysis system, and when flow rate
reason, we use an artificial ECF solution (135 mmol/l is zero the perfusate and ECF are in a state of com-
NaCl, 3 mmol/l KCl, 1 mmol/l MgCl2, CaCl2 plete equilibrium. Therefore, by measuring dialysate
1.2 mmol/l and sodium phosphate to buffer the perfu- concentrations of substrate at different perfusate
sate to pH 7.4) which closely approximates tissue ECF. flow rates, it is possible to plot the inverse relation-
In vivo, the concentration of substances in ship and to extrapolate to zero flow rate by non-linear
dialysate is invariably a fraction of the actual ECF regression, thus estimating absolute concentration of
D.G. Maggs et al.: Brain and skeletal muscle microdialysis S 77

relative delivery of isotopic substrate from perfusate

into local tissue ECF equals the relative recovery of
cold substrate from tissue ECF into dialysate. This
method seems to compare favourably with the no
net flux calibration procedure; however, data pro-
duced by this technique raise some methodologic
concerns [11]. Apparent overestimates of ECF glu-
cose levels in rat adipose tissue measured by this
technique can be explained by an accumulation of
isotope in the local tissue surrounding the microdialy-
sis catheter thereby resulting in an underestimate of
the delivery of glucose isotope to the tissue and artifi-
Fig. 2. Data from ‘no net flux’ calibration procedures carried cially overestimating calculations for cold levels of
out in a non-obese human subject. Data indicates glucose con- glucose in ECF. Finally, with the question of measur-
centrations (mmol/l) expressed as the difference between di- ing absolute ECF levels of susbstrates in mind, Arner
alysate and perfusate concentrations on the y-axis, plotted
and colleagues are utilizing a microdialysis system
k U
against perfusate concentration on the x-axis. Data is plotted
from adipose ( ) and skeletal muscle ( ) dialysate at four which they propose has an approximate 100 % rela-
different perfusate concentrations of glucose (mmol/l). As in- tive recovery [12]. In other words, dialysate concen-
dicated, a regression line is extrapolated through the four trations of substrate are equal to those in ECF. This
data points for each tissue bed and the estimate of absolute high efficiency is allowed because the microdialysis
ECF concentration is the point where the line crosses the point catheters have a large surface area of dialysis mem-
of [dialysate]-[perfusate] = 0 on the y-axis, the point of no net brane (30 mm length) and are perfused at very low
rates (0.3 ml/min).
A further concern with microdialysis studies con-
ducted over many hours is the ‘stability’ of the micro-
dialysis system. In other words, could changes in sub-
strate concentrations observed during study interven-
tions be a manifestation of an artifact created by the
probe itself resulting in a shifting baseline? This is an
area of some contention as it has been suggested by
some that microdialysis may cause a depletion of sub-
strate from the local tissue bed causing a time-depen-
dent shift in dialysate substrate concentration [1].
However, others contest that this phenomenon is not
Fig. 3. Data from a ‘zero flow rate’ calibration procedure car- a significant concern [13]. Furthermore, this potential
ried out in a non-obese human subject. Data indicates (a) glu- phenomenon has only been described for glucose and
cose and (b) lactate concentrations (mmol/l) in adipose ( ) not for other substrates. To attempt to address this is-
and skeletal muscle ( ) dialysate at different dialysis flow
rates (ml/min)
sue, we carried out a series of studies in healthy sub-
jects under basal conditions and found no significant
time-dependent change in skeletal muscle and adi-
pose tissue dialysate concentrations of substrates un-
substrate in ECF. Figure 3 shows representative ex- der basal conditions over a 6 h period.
amples of ‘zero flow rate’ calibration procedures car- An additional concern with the microdialysis pro-
ried out in healthy humans to measure glucose and cess is the possible artifact that locally altered blood
lactate concentrations in adipose tissue and skeletal flow may have on the recovery of ECF substrates. A
muscle ECF. In addition, as further validation of recent study, involving the coupling of laser Doppler
these techniques, we have shown that estimates of pe- flowmetry as a measure of local blood flow with mi-
ripheral tissue ECF concentrations of glucose, lactate crodialysis, showed that insertion of the microdialysis
and some key amino acids by these two methods are catheter into skin caused a short lived hyperaemic re-
virtually identical. sponse which quickly ( < 15 min) resolved [14]. Thus,
A significant drawback with these calibration tech- in the bounds of the short-term metabolic studies we
niques is that they are inherently time consuming and and others conduct, the catheter itself does not seem
the more recent description of an alternate calibra- to significantly alter local blood flow. However, in
tion technique [10, 11] involving isotopic perfusion is the context of metabolic studies involving hyperinsu-
of interest as this method allows an almost ‘immedi- linaemia, especially with concurrent hypoglycaemia,
ate’ estimate of microdialysis recovery. The tech- where one would expect physiologic alterations in
nique is based on the principle that through the addi- blood flow it is important to consider the potential in-
tion of substrate in isotope form to the perfusate, the fluence that blood flow may have on the recovery of
S 78 D.G. Maggs et al.: Brain and skeletal muscle microdialysis

substrates by microdialysis. This issue was first raised diabetes mellitus (IDDM) and, as a result, this ther-
when ethanol clearance from microdialysis perfusate apy has been recommended for, and undoubtedly
was developed and validated [15] as a method of at- will be offered to, an increasing number of IDDM pa-
taining a qualitative measure of local tissue blood tients in an effort to prevent or delay microvascular
flow at the site of microdialysis catheter insertion. and neuropathic complications. Unfortunately, the
Studies demonstrated that, in animals, gross changes frequency and severity of hypoglycaemia is markedly
in tissue blood flow alter the recovery of glucose by increased by such regimens [21]. In light of this, the
microdialysis [16] (i. e. an increase in local blood neurohormonal response to hypoglycaemia in health
flow increases dialysate recovery of glucose) and sim- and diabetes has been extensively investigated in re-
ilar findings have since been reported in human adi- cent years, resulting in a better understanding of the
pose and muscle tissues but with a less predictable ef- defective counterregulatory mechanisms that exist in
fect of altered blood flow on the recovery of ECF lac- IDDM. However, it is still not clear what mechanisms
tate [17]. are involved in glucose sensing and the initiation of
To address this issue, we studied the potential ef- the neurohormonal response to hypoglycaemia.
fects that insulin-induced hypoglycaemia may have A role for both cerebral and extra-cerebral glucose
on the recovery of ECF glucose [18]. We studied heal- sensors has been proposed [22, 23]. Although there is
thy subjects during hypoglycaemic clamps (plasma some evidence that the liver may play a role in the ac-
glucose 2.8 mmol/l) and measured ECF glucose levels tivation of sympathoadrenal activity [24], most data
in peripheral tissues by two different methods. On point to the central nervous system as the dominant
one occasion, we indirectly estimated tissue ECF glu- centre for the sensing and integration of hypoglycae-
cose during hypoglycaemia by calculating absolute mic signals. This view is perhaps best supported by
levels of ECF glucose at baseline and therefore the studies showing that bilateral infusion of glucose
in vivo recovery of our microdialysis system. We into the carotid and vertebral arteries (maintaining
then applied the baseline recovery to indirectly calcu- intracerebral euglycaemia in the face of systemic hy-
late ECF glucose during a hypoglycaemic clamp ([hy- poglycaemia) nearly abolishes hormone release dur-
poglycaemia dialysate glucose] × baseline recov- ing hypoglycaemia [22, 25]. These studies, however,
ery = [hypoglycaemic ECF glucose]). On the second do not define the precise brain regions involved.
occasion, we made direct estimations of absolute While the hypothalamus has long been viewed as the
ECF glucose concentrations during stable hypogly- most likely site [26], others have suggested that the
caemia using the no net flux calibration procedure. glucose sensor is located in the hindbrain or spinal
If the microdialysis recovery of glucose had been al- cord [27, 28]. Recent data from our laboratory pro-
tered by hypoglycaemia itself, then the indirect and vide strong evidence that the VMH is essential for hy-
direct estimates of tissue ECF glucose during hypo- poglycaemic counterregulation [29]. We observed
glycaemia would differ. However, we found no differ- that conscious VMH-lesioned rats (bilateral lesions
ence between the indirect and direct estimations of produced by local ibotenic acid injection 2 weeks ear-
ECF glucose levels under these conditions, therefore lier) had blunted glucagon, epinephrine and norepi-
suggesting that the relative recovery of glucose from nephrine responses to a hypoglycaemic stimulus
the tissue bed by microdialysis was not altered in this (3 mmol/l or 2.5 mmol/l) when compared with re-
particular model of insulin-induced hypoglycaemia. sponses noted in controls. We therefore reasoned
In contrast to these findings, the blood flow and ECF that, if glucosensors are in the VMH, glucopenia lim-
glucose changes that occur during insulin-induced hy- ited to this centre should cause counterregulatory re-
poglycaemia have been described in some detail in sponses despite peripheral normoglycaemia. At this
rat muscle and adipose tissue with evidence that hy- point, we applied microdialysis techniques to study
poglycaemia-induced blood flow changes may affect the role of the VMH in triggering counterregulatory
microdialysis recovery of glucose [19], however, these responses to hypoglycaemia [30]. In the VMH or
studies were done under non-steady-state conditions. frontal lobes (controls) of awake rats (n = 20), 2-de-
oxyglucose (2-DG) or glucose (to serve as a control)
was perfused via microdialysis catheters to produce
Brain localized cellular glucopenia. Perfusion of 2-DG
(but not glucose) into the VMH caused a striking in-
Although the vast majority of work in the field of mi- crease in plasma glucagon (by 200 %), epinephrine
crodialysis has been in animal brain, little attention (by 600 %), norepinephrine (by 400 %) in association
has been directed to the study of brain fuel homeo- with a prompt elevation of circulating blood glucose.
stasis and, more specific to our area of interest, the It should also be noted that 2-DG had no effect
role of the brain as a glucose sensor under conditions when delivered to the frontal lobes. These data there-
of hypoglycaemia. The recent report of the DCCT fore suggest that the neurons sensing glucopenia are
Research Group [20] demonstrated the long-term in the VMH. To follow the findings from this study,
benefits of intensified treatment in insulin-dependent we have used an opposite experimental paradigm
D.G. Maggs et al.: Brain and skeletal muscle microdialysis S 79

again to determine the role of VMH in glucose sens- similarity to that of pancreatic beta cells. Thus, glu-
ing. Under conditions of systemic hypoglycaemia, cose could act as a signalling molecule for at least
we used microdialysis to perfuse the VMH bilaterally some neurons in the central nervous system. Taken
with glucose preventing VMH hypoglycaemia [31]. together, these observations suggest that the VMH
Data indicate that hypoglycaemic rats with ‘euglycae- might function much like the beta cell, a scenario
mic’ VMH have markedly diminished hormonal re- that might account for functional changes in hormone
sponses to systemic hypoglycaemia, again suggesting release seen with chronic hyper- or recurrent hypo-
that the VMH is the dominant glucose sensor for acti- glycaemia.
vation of the counterregulatory response to hypogly- It is well established that glucose passes across the
caemia. blood brain barrier by carrier mediated transport
A concern with these experiments relates to whe- (via GLUT 1 transporters) and unsaturable (diffu-
ther the anatomical positioning of the catheters was sion) mechanisms [34]. Once in the ECF, the concen-
appropriate and whether the local perfusion with 2- tration of glucose seen by neurones and glia is, how-
DG confined its effect to the VMH. The hypothala- ever, probably much lower than that of plasma. Esti-
mus is a small, albeit highly complex, area of the brain mates of brain ECF glucose levels in animals using
and, despite positioning the catheters with stereotactic microdialysis or microelectrodes range from 0.5–
surgery, one must be careful in attributing these find- 2.8 mmol/l [35, 36], suggesting that a steep glucose
ings to an area as small as the VMH. However, the ap- concentration gradient exists between blood and
propriate anatomical localization of the catheters was brain ECF. We have recently had the unique opportu-
confirmed histologically. In addition, in a small num- nity to utilize microdialysis to measure glucose levels
ber of rats (n = 4), further studies were catheters per- in brain ECF of conscious humans undergoing intra-
formed where [3H]2-DG was perfused via the cathe- cerebral depth electrode monitoring for intractable
ters and local radioactivity was measured in VMH epilepsy [37]. Preliminary data suggest that ECF glu-
and surrounding structures. This procedure confirmed cose levels in human brain are very different from
that radioactivity was largely restricted to the VMH; plasma levels; i. e. approximately 30 % of plasma lev-
and the radioactivity in the surrounding tissues was els. These findings imply that neurons are bathed in
only slightly above background levels. In other words, a metabolic milieu different to that seen in the circu-
the local perfusion of glucose via the microdialysis lation.
catheters was indeed confined to the VMH.
The cellular mechanisms used by the VMH to
sense and transduce the glucose signal are unknown. Skeletal muscle
It is intriguing to speculate that the VMH glucose
sensor shares common features with the only glucose Application of microdialysis techniques to the study
sensing cell that has been well characterized, the pan- of peripheral tissues has increased in recent years
creatic beta cell. Recent studies support this view. Jet- and this technique is particularly well suited to these
ton et al. [32] have reported that cells within the me- tissues since larger catheters can be used (than those
dial hypothalamus express glucokinase as well as used in the study of animal brain), substantially in-
GLUT2. Furthermore, VMH neurons contain ATP- creasing the efficiency of the microdialysis process
sensitive potassium channels, a key signal transduc- and therefore the ability to detect metabolic change
tion protein in beta cells [33]. In keeping with this hy- in ECF. Most attention has been directed to adipose
pothesis, we have recently reported, again through tissue, mainly because of easy access, and studies
the utilization of microdialysis catheters in rat sub- have reported the measurement of ECF levels of glu-
stantia nigra, a brain region rich in KATP channels as cose, lactate and glycerol [1, 38–42] and characterized
determined by sulfonylurea binding [33], that this the opposing effects of insulin and sympathetic stimu-
area responds to changes in local glucose availability lation on adipose tissue fat and glucose metabolism
via effects on KATP channels. Perfusing the dialysis [39, 43–45]. The application of microdialysis to the
catheter with 10 mmol/l glucose increased g-ami- study of diabetic or insulin resistant states has been
nobutyric acid (GABA) release two-fold. Local per- limited but work in this area includes study of the ef-
fusion with the sulfonylurea glipizide had a nearly fects of insulin withdrawal [45] and hyperglycaemia
identical effect; whereas perfusion with the specific [46] on levels of substrates in adipose ECF in
KATP channel activator, lemakalim, or 2-DG with oli- IDDM; and abnormalities of glucose and glycerol
gomycin inhibited GABA release by 45–50 %. The metabolism in adipose tissue have also been reported
latter effect was blocked by glipizide. When systemic in obesity [39, 47], patients with liver cirrhosis [48]
hypoglycaemia was produced in awake rats, nigral di- and spinal cord injured patients with mild insulin re-
alysate GABA concentrations decreased by approxi- sistance [49].
mately 50 %, as well. These data suggest that glucose Microdialysis has been applied to a lesser extent in
may modulate nigral GABA release via an effect on skeletal muscle; a tissue that plays a major role in pro-
KATP channels, a pattern which bears a striking tein metabolism and insulin-mediated peripheral
S 80 D.G. Maggs et al.: Brain and skeletal muscle microdialysis

glucose uptake, and that also serves as the major 100 % recovery, Enoksson et al. [12] reported that ba-
source of gluconeogenic precursors under catabolic sal ECF levels of glycerol are indeed higher than plas-
conditions. In rats, a number of studies have shown ma in skeletal muscle as well as adipose tissue but ab-
that microdialysis is applicable to this tissue bed [15, solute levels were not nearly as high as those ob-
19, 50] and we have used microdialysis in both skele- served by us. We have since tested our original micro-
tal muscle and adipose tissue ECF in humans. Our dialysis system side by side with a commercially avail-
studies were firstly notable for the concentration gra- able system in fasted humans during a continuous in-
dients that exist for key metabolic substrates between fusion of stable isotope of glycerol to attain a steady-
the ECF compartment of both tissue beds and the cir- state isotope enrichment of glycerol in both plasma
culation. High glycerol levels were noted in skeletal and adipose tissue ECF. Our preliminary data show
muscle dialysate, suggesting that there is significant that with both microdialysis systems the isotope en-
lipolysis occurring in skeletal muscle and that the im- richment in the adipose dialysate was approximately
portance of the glucose-fatty acid cycle may be under- 10–20 % of plasma enrichments indicating local glyc-
estimated in this tissue bed [51]. In addition, we also erol production. However, the relative enrichment
demonstrated that ECF levels of lactate were higher of isotope in dialysate was at least twofold higher
than plasma levels indicating that there is an efflux (and therefore nearer to plasma enrichments) in the
of lactate from both tissues under fasting conditions. commercially available catheter when compared
Whereas, levels of glucose in muscle and adipose with our original catheter. Accepting these observa-
ECF were approximately 30 % lower than arterial- tions are confined to adipose tissue and that the com-
ized plasma levels indicating a concentration gradient mercially available microdialysis catheter generated
in the opposite direction in keeping with glucose up- data that supports observations made by others, this
take. would suggest our original microdialysis system may
Peripheral tissue ECF glycerol levels were higher have overestimated glycerol levels in skeletal muscle
than levels reported by others [41, 42] raising the pos- and adipose tissues.
sibility that the high glycerol levels were, in part, arti- A further interesting development in the use of
factual. However, in vitro, we confirmed that the microdialysis in the study of peripheral tissues is the
glycerol content of the cuprophan membrane we application of stable or radioactive isotopes in the
used was seemingly negligible. This fact was further study of local tissue metabolism. As mentioned ear-
underlined when we used the same catheters in hu- lier, isotopes added to the perfusate have been used
man brain and found that glycerol levels were immea- as a measure of microdialysis recovery [10, 11] but a
surably low in brain dialysate, a tissue bed where you more recent development has been the addition of
would expect negligible lipolytic activity. In addition, substrate isotope to perfusate and the measurement
the question that elevated glycerol levels in dialysate of the substrate’s metabolite in its isotope form in di-
may be a trauma artifact has been largely refuted by alysate. Henry et al. [53] recently reported the perfu-
past work showing that the initial trauma reaction sion of U-13C glucose via a microdialysis catheter
quickly subsides shortly after catheter implantation into human adipose tissue and the measurement of
[13] and, thereafter, dialysate sampling reflects a sta- C lactate in tissue dialysate as an indicator of local
ble ECF environment for hours. Finally, specific to anaerobic glycolysis [53]. This particular application
skeletal muscle, the disparity between muscle ECF of microdialysis techniques may offer a unique in-
and plasma glycerol raises the question of whether sight into local tissue metabolic processes.
the muscle probes were positioned appropriately as
the presence of high ECF glycerol could imply that
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Local Ventromedial Hypothalamus Glucopenia Triggers
l Counterregulatory Hormone Release
\Valter P. Borg, Robert S. Shen,in, Matthew J. During, i\Ionica A. Borg, and Gerald 1. Shulman

To test the hj1wthesis that nuclei of the ventromedial t'eC1s.)ned thm. if glucosensors arC' 10,';\[,,([ in [hi' \-\IH. this
hypothaJamus (\~nI) playa key role in the detection of manipulation should calise a counrerregU1alory response
(l-Qnnterreg1l1atory responses to h.J.poglycemia. 'we delh'­ clesptte systemic t1ol111ogIycemia. The micmclialysi::; rech­
ered the gIucopen.ic agent 2-deoxyglucose ,ia bilaterally nique allowed LIS to clelh-er :2-deoxyglucose dtrectly iuro a
placed microdialysis probes into the '-"ill of conscious. localized brain region of awake, unrestr:.lined aninJi1ls. elim.
chrollicallv catheterized rats. The goal wa.·,:; to produce inaring such confounding factors as anesrhesia and hypogly­
cellular glucopenia localized to the ''3ill. TIle volume of cerma 1I1 other areas of the body,
brain tissue exposed to 2-deox.,rglncose was determ.ined
by adding CHjZ.deoxygLllcose to the dialysttte: its distri­
bution in cerebral tissue witS almost exclusively limit~d
to the \'-"ill. Rats \\;th microdialysis probes placed into RESEARCH DESIG:\ AND :\IETHODS
the frontal lobes served as a control group. Locttl perfu­ :'lak· Sprague-Dawley rats WE're purch:1.'"d from Ch:lrlt's Ri\'i'!" Labor".
sion of 2-deoxyglucose (bllt lIOt glucose) into the VlVrn {ori.e~. :~nitnab \\'ere housed in "Ul el1\iro{;.menraHy cO[lrroHed l"I.HJ!lt \\i[i:
:1 1:::-!. ilght'(\ark cycle. and werE: maim::unt'd ')l\ st;:tlHiard ad lthirurll fm
caused a prompt twofold increa.';e in plasma glucose in
diet i Pmlab :]0<)0..'\G\\·.-\ Y. \l:a'·erly. :-:~'. l")!llpris"{j ut :'::':"'" pmkin. $'.
a.-;sociation with a striking elevation of plasma glucagon fat. awi '" ["'J carbohyrtralt' (the remai:~tn2 :'::':"" cOllsbts of ,,,,,II. cmd"
(:3.5-fold), epinephrine OO-fold). and norepinephrine fii It" " "nd nlOisture). Rats (bod,' \\1: :2~I)-:.l!O :; J Wer,· ane~rhdiZt·Li b':
(:3.5-fold). No effect was seen when 2-deo~lg1ucose WitS imrap>'l1wneal injeclion (l tlIlikg) of a miC-;lure of c-;'.Iazin,~ ':':'1 Ill"- mi'
delh'ered into the fro mal lobes. We conclude that gluco­ and :':eramlae [ [00 n1&ml) in a ratio o( l:~ l\'oh'ol) 'a:ld placed .'); rh~
penia locallzed to the \~IH triggers the release of COUll­ stt'reOl;lC-;l(:: fnUllE'. Th<' skull was exposed. and holes wen:> c!tiHc-ri
tenegulatory hormones that defend against h}pogly­ bilari'l":l!ly in chosten coordinates. through which the guide c-annulos
cem.ia. Thus, the neurons that sense gluc~ may be were lowered slowly into the brain. Al! :>tereow_xic ('o~rdinates Wt:,.p
situated in the V1\UI. Diabetes 44:180--184e ([t'ter.:liI1ed from the atlas of Pa.xlnos \l:arsol1 18 ,:Two ~rolJPs ~~.
amn,c"s Wf're prepared as follows. r-or group 1. \,}IH cannulas were
pbct'(! by using the coordinares 2.f\ Illnl postetioi:~lnd :1:5 mmlat"t~ll it:
rela:wlI [0 bregma. and at rile angle 0;' ~lr in relation to the IlOn1.0ma:
plan,.. passing through bregma and 1a.1;;b(\a. Fllt group :'!. froI1wl lobe
I FL, ('annl!I,1.~ were placed by using Ihe connEllat!':; :2.'' ' nun antenur ill\(:

hile the role of the- incli,idual cOllnterregula­
::.0 mal lateral in relation [0 bregma at the all!?[e or ~ilJ' in re!atiOll to th~
tory llOl1110neS in h)1)oglycemia correction has \'erric-al bregma-lambda plane, The Ca..·1IllililS Wert' then secured to th~
been srudit,d extfnsil:ely (I), the mechanisms skull with stainless sleel serews ami di?ntal act.....lic. c\pjmab were the~
that link glucopenia with actil:ation of the alio\\'eel to recoq'l' from the stereota.'C(" ;Jrocedtl~·<, for :~-tS ,),1\'S hefor~
counterregulator:r system nre poorly understoocl. Specifi­ st:I(V One (by before each experime~t. standard tnicrodialYsls probe~
or sldt'-h~:,slde destgn (10) wert' i!l.'c1"t<!'d into gUide cnnnulas. Th"
cally. there has been contnwersy conceming the tissues that
leng::1s ot the \,}IH probes and FL probes were 105 amI ::!.<) mm,
sense gLucopenia :md coordinate the counterregulawry re­ l'esp·:("tiwly (ilS measured from bregtrta-brnbda plane I. On Ih,' morniag

1 sponse. Both the cent ral l\l't .....ous system (C:\S) and extra­
cerebral glucose Sfnsors ha,'e been implic:lted in the
01' tIo", ec-;perimem. perfusion mecli\UTI was loalled ilwJ l-ml synnges an'~i
delt'.~",rE'd at a flow rate of 2.5 ILL'min b:: using a H~u,:ard Pt'l1'usiotl !JlIfl1P
I mor.:d :!:.;, Har-'arrl Bioscience). llm'e kinds ot" perfusates Were used, :;
acti,'ation of counrerTeglllalOr.'i' hormonE' release dming hy­
nunol1 glllC!)Se. 100 nlmolll glucose. and 100 mmol!l 2-<:leoc-;,·g1ucQse. :\.
poglycemia (2-, Recent studies from our labomtoty. based sl.:n:<:,. pyrogen-free. arrificial. extracellular. t1nld solmion t·I:].3 mm,)ll
f on chemical lesioning of ,'aJiollS brain regions. have sug­
gested that the neurons located in the \'entromedial hypo­
\aC, :3 mmolt1 KCL 1 mmol!1 .\!gO:- 1.2 mmolil (·aCL. :2l)f) mmoi 1
asc(),·oate. and 2 mmolt1 sodium phospnare bulfel'{O pH 7.":n "er:eu as :l
thalamus (Vt'lIH) are essential for the integrated homlOnal soh·-::nt for these solutions. At the end of each ~xperimt'nt. prob"
response to glucose deprinltion (8). To further clatify the placement was verified histologically by <:re:;yl \'i0\e( staining. Onl';
anitnills thar showed bilateral prob<; placement inro rhe dl'sirf.'d brai~,
role of the v';\IH in hypoglycemia detection and counterregu­ [<>glons Wf.'re included. Of rats with \'}Ill. 5 of IS (2S·".,) and nune of rhe
lation. we delivered 2-deoxyglueose. a metabolic inhibitor rats ·.Ii(h fL probes failed the his(l)10?ical criteria. Ihsrolnt;ical ]'('sl1lt;
commonly used to generate cellular glucopenia. \ia microcli­ were (he only criteria for excluding ')r including the stmiil's f(Jr data
alysis probes directly into the V-:'IH of awake rats. [t was
At G--10 days before study (i.e .. 6-~ clays after stereotnsit: surger,'j
,mimals underwent an additional aseptic sllrgical procedure for plac".
ment of internal jugular vein nnd c;;t[orid arl('l..... c:uhetE'rs under intra.
From rhe DepartmenL~ of \nternal Medicine anti ~lIr~.:ry I~U.D.l. Yale rniwcsity peritoneal pentobarbit:a\ anesthesia (:-:embuta!. ':35 mykg b"dy wt). The
SdlOul of :'ledicine. Ne", H:l'·('n. C'mmc'etit'llt.
",llin'$S corre:;pondenc(' and reprim rettllests to Dr. l~t'ralll l. Shulman. Yale polyethylene L-:U'otid arrery carhett'r was extemkd (0 tht' ·It',·d. or th!'
L"niwrsity School or Mellicine. Departmem ur IlItpmaJ )"'<lieine/Endocrinolol:lY. aomc arch. and Ihe silicone imernal Jugular vein catheter \Va,; nUVall<ll~!
:J:3J C,>dar St.. Fitkin I. :-:t'w Hawn. C1' 1~)~:2th'iir2U. to the le"el of the right atrium. At the eud 1)1' the prol'P(/Url'. hoth
Rect'iwo fOr publication ~S :;"prt'mber Hl!c4 ami alT.'ptetl in ..."ised form ti c[lrheters were /lushe<t and filled with heparin ,·t! Uillli) alUl pIJIYIl'
nylpYITolidone (1.7 glm!) solution. plugged. tttnneled sl!lwU[anI!O\l~h
1 October 19'.4.
eNS. cent!"'.!l nervous system: "MH. ,'(>lttrom",liaJ hyputhalamus: FL. frontal
lobe; UL-\. latem! h}1Jocl1iuamitc area.
around the side uf the neLk, and externalized behind the ht'~ld thr(lI1.•i.
1:1 sl,.in incision. Catheters remained sealed until th.., day Ill' the stUll\'
I nBLE 1

pf'rfomwd. In tlw first group (11


,1. \ ".'.fH proiJe:" were pt'rfU'i-t'I!

sequentially with a soluli!)n that conrained :) mmoL 1 g!uco~" [,)[ rite fir;;;
Basal char;JC[etistics of rht" animals
:30 min. followpd hy II)!) mm!)!:1 gillms!' for 6r) mill. ,') m!ll011 ~!H"l):'"
J- Perfusion rhrough

afpin for :30 min. and filnlly I');) mmoll ::·deoxyg!,lCOS>' [.x rh,.· 1:1.-', ,~;,
min of the srudy. In thl' St'c'ond I',ruup (II 6·i. perfusatc'S wen~ (h'li\"~r.:,<l
in the sanle order a~ in tht~ firSt group. bur rht' FLs wen:, Pt'!1'us~t! in;-:tc';'1':
DIH rL (ren~rsedorder) of the \")IH, In the third :;:;r;up I 1/ .)). l·il} mmoll ::·tlt'11xygluCl)sl' \\;r;:.

introduct'd after tIlt' inin;!1 ;) lHln.)ll giu,·,)se Pt'11·I.tSltJn, .lJlerwl hio.),j

1 6 6

Insulin (pmoL1"l :!-!ti :::; 1)0 =
:3;3-1 61) ::6-l = -l~
samples w.:re withdrawn ,ltuing rnt' p'-'l-tusron to nlt'asl:rt' glue,),;" e ....,,[-.
10 min ami gIUCa~0n. c,rtecho!;:rmines. and insulin t'\~t'[-; :3') nun. [)unn~
Glucagon (ng1) 1:311 :::; S I."):."! - II l·tS - 23

Epinephrine (" mHon) 0.'3 = I).:~

:10,:::; 0;;)
O.S 0:'>
L:! [)
0,1 0.2

1.1 _ 0.3

blood wirhdt<m·a!. ;:;ampl" dilwi<,n hy thud in rill' ,1c:J!! space of rf.-:

catheter wa.s aH)icit'd tn' \'\lfhdraW;lf c'f -·0.,' tn! ,'It Illon.l 1.<"'"1'''
:\orepillepilril1t' (mllov11
eollectioll of "aell blt)"d sample I -0. I mil ,,"Hh a ~t'·,·mld ~'.n:;~,.'
Data are m!?~HlS SE. Sllhsetluenrl,-. tilt' l"l>l1rt'nrs or" [ht' mirial syring·: w,-:',' rt'H\f>:~,-.l t,.'
minimi.,' biOI)cI i<)Ss. [n adl!itl<)!l. i)!<l<ld "braini'd irom !fr;",nn;\tt.'~ ,,",L­
Only rhDse :1nim:ll.s thar had cnmp[I'lely rE.'co"t'rt"j and show"dno signs transfused dUling till' swdy "ja rht' juguJar ,'ein catileter t,l 'lllalHH:1fl·,,·i',
of infection \\.-irhin :]1;' h aftl)f surgt:'ry \\-ere tts~d. reptac" blood \\irhdra\\ll during till' \'xperinl€'IlL
• The rats WNe food·,Jepri'"f'd fol' -2 h bdore rhe start of each The \'olume of br:lin t'xpo~e(l to ::·d"OX\',2Iueo::;t:' C';:L"; ,,$,im'Ht'd h'.
e:'1klllllenr. The catht'lt'rs wert' Hushed wirh saline and maintained including tlw I 'H}2·r!eoxyglu,·ose in tilt' di,{lrS;lr\! ;UHl mea:'il!r!uiZ ir~
p;ltenr by a slo'" infusion or' saline (20 p.Lmin) that comain"d a smull distribution in the cerebral tissue' in fOllr raL~. In tht':><' t"")e[lltl~m,-,
amOlllH of hepaIin (1-:; Ulllll, Animals were fully awake and freely V;\IH prob.:,; ,,"pre pf'r-tnsl'CI wirh a lOr) 11l1110Vl :l-d('ox':;;:luco;e Si)lllti,',r:
mo\ ing about in their cagps..-lit"r a 50-min rest period. arterial blood containing 0.06 f.lCi. mlll( [:Hl:l-dt't)xyglucose for Ill) ;l~tl, At rtl" t'nc! '"
samples ,,-ere \\irhdrawt\ [rom carotid artt;>ry carheter for assay of tbe expelimt?nr. the r:lrs \\'ere ill\t'stherized 'with p.;-nrobariJllal ':\emj),;.
baseline plasma glucose. insulin. glucagon, epin.:;phrint'. and norepi­ tal. :}:) mg;:kg body W!l and decapi!aretl. and the brains wert' [e-t1\""":
nephline concenrrari0ns..-\t the sallle tim". perfusions through tht' and put inco a rodem br:lin slicer. Frozen I·mm coronal sections or ['ral:'
microdialysis probes wert' staned. Three groups of e:---periments were conrainin~ probes werlO' mad.: :md place([ on an k,,·cl)[d fj:u -,un';We

l00mM lOOmM 100 mM

100mM GlUCose
2~O&Oxy·GtUCos, 2-Deoxy-Giucose


::1GLUCOSE (mM) 8oo~
600 I
8 _ _ A "­

1 400

6 FL

: O.
o 30 60 90 120 150 180 0 30 60 90 120 150 180
.~' : TIME (min) TIME (min)

Gluc.".. looml.! 100m'"
GlueOM 2·0e0xy·Gluco$<l
80 30



-0- FL

-0-- FL
10 --+-- VMH


- a--~

120 150 180 0 30 60 90 120 150 180
0 30 60 90
TIME (min) TIME (min) .
FiG. 1. Plasma glucose Ilnd counterregulatory hormone concentrations durlllg microdialysls perrusion!> of the VMU and FLs. -Significant statistical
'<difference as compared with FL perfusion (I' <: O.OS).


~----------~~~ ~~~~

,. 12
(mM) ,/ BOO~ (ngll)
10 •

, , . ::/"
.wO j


o :lO 60 00 :JO 60 00
TIME (min) TIME (min)


, (nM) (nM)
.w~ 150
* ..

:JO 50 00 o :JO 60 00
TIME (min) TIME (min)
FIG. ~. Effect of \")1H perfusion C"ith :!.deoxyglucose introduced at the
beginning of the study. i.e .. after the initial;) ulInoVl glucose perfusion)
on plasma glucose and counterregulatory hormone concentra.tions.
'Significant statistical difference between samples taken during and
after 2·deoxyglucose perfusion. as compared \\ith the baseline and the
i,litial,) mIDol/l glucose perfusion period.

ThE'rt'after. fissue samplt's were collected using :m IS-gauge biopsy

llC'ecllC' from the \~IH and surrounding c£'rebral tissue. including the
inff'110r part of the arcuate nllclE'us. dorsoI1l£'ciial nucleus. perifomical
nucleus. I\l£'dial amygdaloid l\ucleus. ICHNCtl hYPQ[halamic area (LHA).
m\(l third "ertidE': Tissue samples w!:'n: plact'd in scintillation liquid
(Utima-Gold. Packard). sonicated. and counted in a liquid scimi!lation VERTICAL
coumer {Tri·carb 1900; Packard). TIlE' protocol was re\iewed and : ZSO . ..
appro\'etl cby the Yale Animal Care and Cse Committee.
Plasma glucose was measured in duplicatE' by using a Beckman
glucose analyzer II (Beckman). Plasma glucagon (lC';) and plasma
insulin (Bina;,,) concentrations wer!:' dewmlined by a clouble·antibodr
radioimmunoassay, \"ith a porCine srandard anc! a rat standard. respec·
[j\·ely. Plasma concentrations of epinepluine and norepinephrine were
measmed bv a radioenzymatic method I.\mersham).
Dma are 'expressed as means SE. Comparison between the study
groups was made by analysis of variance with a repeated measure
design. followed by Student's I test to localize efl"('Cts.

As summarized in Table 1, basal plasma glucose. insulin,
glucagon. epinephrine. and norepinephrine concentrations
were not significantly differenr between any of the study RIGHT
groups. The average weights of the animals on the day of the
stucly were also not significantly different between the FIG. 3. The results of the measurements of distributions of the
groups (340 :!: 20 g [VM.H perfuSion\. 320 ::':: 30 g [FL radjolabeled 2-~eoxyglucose. perfused through \-;\lH probe in four rats..4.
perfusion],340 30 g [YMH perfusion \\ith reversed order of shows the locanon of the samples: V,l/H; Arr. inferior part of arc-unte
nucleus: D,VD, do,:omedia.l nude us; PeF. perifornical nucleus; LH.
the perfusatesJ). Iate~al hypothalamiC area; JfePD, medial amygdaloid nudei; :IV. third
The effects of YMH and FL perfusions on plasma glucose verticll!_ Band C show the radioacthity (expressed as dpm) uf the
samples from cerebral tissue. 'Signillcam statisti<tal difference betweell
and counterregulatory hormone concentrations are depicted VlIrIH and other samples (P <.: 0.001).
in Fig. L Both VMH and FL perfusions with 5 and 100 mmolJl

(1I.illETES. \"(l!. 14. FEBRt"ARY t:u
J . .

hac! no effect on plasma glucose concen(r:1tion.


Among: rhl' S[l1Ictun's lo':att:'d near the brain n'nuiciL"5. tilt'

Huwcn:!r. pt'rfusion of tOO 011110\;1 :?-deoxyglucose into tht> nucleus of \-:'IIH. h.ilown a'i a of food lnrak,· f)t" tIlt'
\·:'fH L'~\H"c'd a signific::1I1t increase in pbsma gluctJst.' concen· cenft'l" (I:::.l-!i. Ita:; bt'l'!1 pr\.'po'Sl?d tn Cont,H!l I!l!.l­
tratii)n as c()lllpared \\ith that dUling 100 nlIlloL 1 gluL'ose per· COSt'llsi,iw Ih'lIrOnS tlut ("ould tnelii:He rite COI.l;:[ern':!~lb.
rusion (p < 0.0·::; \'5. 100 mmol/l glucose perfusion 1. \Iore· tmy reSpt)!lSl'S to h}pD::dycemia 1 [S. I I)). This pn5;:,il111ir\'
on'!". 2·(I.·'oxyglucose had no suchetrect when deli\'erecl rt) mainly de!1\'es from rhe 0bSt'IYation that mpc'hanlcal 0'1'
[he FLs ,-\.:'i presented in Fig. l. 2-deoxyglucost' pt'rfllsion pjcctlical stimulation ot' tl\\" \'}lH pt'oduces hYlWrgl'.cc·mii\ ll\
inw \ -:\IH caused a marked increase in Circulating glucagon. anl'sthetizeci rats [l·U -;-j Huwt>n:r, se\'eral scudi;:s' b'."(> 1\1)[
t'pint:phl111e. and norepinepl:tri.nt' concentrarions as com­ sllpponed this cUllcepr. Di RI)e,:o and Grill i 1Sj rp[.)()[T,-<I thar
\\·"h FL p~'rr"usion (P. 0.0·')). \0 sllch dc\';:nion Wa" the sym~\,l[hetic respOt:5P to sy'ste:l1lc inlusion or :2-, !.,")X\"­
n/l;-;,'r'.tc'd .lUting- rhe em'li~l~ ljhase of r1w study when:) or 101} glUCOSe' \';<1"-; !lot aboli5h",d c!t"l'E'rebCHli);; f)( tt~c'
mmni 1 glllcose~ was delin?red w the \ -:\IH, [n addition. the rat. Cane' ef aL (HI'j nD""r'>t'd th,l, COLlnre!TP!."u!a:()r~,: rr'~
C<ltlt,'t'l1trarions of the L'OltntetTegulator:r hOl11v)l1t';;o during FL spouses \\ere not it1hihit~;cl when \\'as illt'ust'd i;,tl;e
pt~rfLlsion wirh :::-c1eo);::'>'glucose \\'ere \1nually identical [t) cnrorid ::mt>lit's to aboli",h forebrain hypog[~"Ct>mia during
thar obst'n:ed dtHing 100 mllloIJ1 glucose p~'lfusioIl. hyyoglycellli,L Tn additioll, Ritter et aL I :21)) ,'ih,)'\'t"!~!
\\'hel1 :2~dC'oC\:yglllcose \\'as introduced at rhe' heginning ot that obstnll'rion of rhL' eert'bral aqueduct 1 \\'hit:h l'Of1nt'cts
tht' swriy, perfusion ot" 2·deo",""yglu\.'ose through \ -:\IH alsl) the founh \t'lHlicie ltKart'd III the hindbrain \\-j,h rh,· third
had a l1:-verglyct'mic etfeet :21. FUI111t:'nllOre. unde!' and brerru H'ntril'ies siElwterl in the forebrain) stiliun~ssc'd
tht:'sC' conditions. ele\·ation of the countelTcgulmory hoI" the h~1Jt'rglycel1\il' rt'sp,)I1:-;t! to the metabolic i!1hibit~;· ,C)-tili,.!.
mone has also been noted tFig. glucose injected into the lateral \·t'ntricle. wiwreas rht' 11-:.
DUling rhe FL perfusions. plasma insulin concentrations perglycelllic response to 5-rhioglul'ose injectt'd into tt~t­
\q;>I"P not significamiy different during both Jt)O mOlol1 fottrth wnnicle was attenuatecl. but not abolished. TIH>Sc­
:::-dt'oxyglucose and 100 mmol/l glucose perfusion (.30 15!.; claw suggested that gtucoreceprors might he locar<~[ i in thtc
\·s. :27{;= ;)4 pmo!Jl: .\"S). Also, 2·cleo:,."-:;glucose perfusion into hindbrain (which consists of the pons and medulla obi.):,.
\"11H prodllced no significant change in cin:ulating insulin gata) and not in the h~Vt)rhalal1\llS, HOI\'e\·er. this \1t".'; is nix
COtH:cnrration as compared with that seen during the 100 supported by clata of Frizzell et aL (-±). who shn\\'ed (ha,
tllmut'1 glucose perfusion (11-! - 31) \-s. 150 = IS ptnol!l 0:S1. bilateral glUcose infusion into the \'enebral circulm;'Jtl
Figun: :3 sllows the distribution of radiobllPlecl 2-cleoxy­ nor attt'l1Ltate the C'uUIHt'lTegl1laror::: response ro
glucose in rats perfused through the niH probe, The radio· mia Tht'}" did. ho\\·e\'t~r. :olio\\" that the release of COUlHp!·.
acti\1(Y of samples obtained from the \-:\IH \\'<1S markedly regulatory hOI"11lOlleS in response to hypogl;':C"etnw cottld b"
higher than that of the sUlTounding tissues (i.e,. infetior pan inhibited when glucose was infused into thl'- c<1l'o[id and
of arcuate nucleus: clorsomeclial. perifornical. and mediai \'ertebr~u arteries simultaneol\sly. suggesting that the
amygdaloid nuclei: LHA: and third wrticlt': P < 0.001). sensor for hypoglyct'mia was indeed located \\'ithin til,,> C\'".
Funhennore. the radioacthity fOlmd in each ()f the AlthOllgh it has long been recognizeel that efreren~s t"ru(;,
of tissuE' outside of the \"1(H was not significantly different the \"('nrral region of the hypothalamus inminge on t!;..
from the backgrollnd le\'el ([.;'S). enc\OCt1nt' pancrE'<lS the \ -:\IH \\"as initialh' ~ol~~i(!;::red ,L-,
n sympathetic center. ('omrolIing mainly catecholamine fe.
DlscrsslO:\ sptmses (:'::::-2:)). Thus. when ir \\'as 5ho\'\'1\ that ei<?l'trical
This stud\' clemonstl"ntes that localized glucopenia produced stimulation of the \ -:\IH in rats promoted glucagon ::'c'<":rerii)n
by cleli\'~ry of 2-cleoxyglucose directly into the niH (17), this \\'<lS thought to reflect acti\·ation of the autrmonUi~
a\\'ake rats triggers the release of connterregulmory hor· nelYOllS system, a potent stimulator of glncngon r",le:l~"
mones in the absence of systemic hypoglycemia. These (11.:26), Howe\·er. mort' recent smdies lU\\'e thcu
findings prodde strong e\ictence for me existence of h:,Vo, the'\'?'1H may playa role in the detection and l'oC)r(lin~.
thalamic glucoreceptors. tion of the cOllnterregulatory responses ro h:.vog1ycemia, b
The role of the C;\S in the regulation of cOU[l[t'lTegllJaror:: these e"'l)erimems. bilateml \"1IH It'sions were produced ir~
responses to hypoglycemia is controversial. AJrh.ough several Sprague-Dawley rats local ibotenic acid injection i.s J. Ra.~
lines of e\idence strongly implicated the C.'iS in h.ypoglyce­ with lesions in the LH..--\ and FL. sham·operated (srerf.:'Ora.,\I~·
min detection nnd counterregwation (2.4.8,11), the precise needle placement iIltO hypothalamus \\ithour injenion) Hili.
brain regions lln-olved were not esrablished. Preiolts studi!:"s mals, ,md naive animals selTecl as control groups. (~ll\cag'm.
lli1\'e demonstrated thnt injections of 2-deoxygIlIcose into the epinephline, amI norepinephrine responses to il1sulin~jn­
third ventricle of anesthetized animals causes h:;,'perglycemia clucecl hypoglycemia were markedly inhibited ill the nil!·
(12.13), suggesting that glucosensith'e tissue may be situated lesioned rats as compared with the other control groups Fh
in close proximity to the third \·entricle. Howe\'er, the This srudy '"';"as intended to further clarify the contribllti'J[!
specific nuclei in\oln?d are difficult to determine by using of the \ -:\IH to hypoglycemic counten-egulml'ln. We l"€':lsow.t!
this approach because many important nuclei (arcuate. that. if glucosensors are located in the \ -:'IIH. glllcopellJ~
pelivenrricular. perifomical, dorsomedial, VMH nuclei, etc.) limited to this neural center should catlse a cOtll\terreguia­
are situated near th~ third ventricle. Furthermore. the wide­ tDry response despite pelipheral nonnoglycemia. The min!}­
spread circulation of cerebrospinal fluid throughout the dialysis techniqllE' (10) prm·idecl a means of lieliverin:?
cerebral ventricles makes any conclusion regarding the :?-deoll..·yglucose direC'tly into a specifically defined region .;(
location of the potential glucoreceptors even more uncer­ awake. unrestrained animals. Deli\'ery of 2·deoxyglllc"sl'
tain. For these reasons, such experiments do not prm;de into the VMH caused .1 rapid and marked increa..'>e in cire'U'
precise infomlation about the location of the brain glucosen­ Iating counterregulatOlY hormones accompanied by h:'l)t:r·
sors. glycemia. These responses were not caused by a nonspedlic

hyperosmolar effect of the 100 mmolll 2-deoxyglucose solu­ .) .\iijima A: Glucose sensi(i\-e- atfen~nt llt'n:e fibt~rs in tht.· tI~, {'-r and
rE"gnlarit)f1 o(bl,)od glucose. Brniu R,"s Buil.') ~5qpp[ ~ ·t':":)-t:-:~~ l~)~n
tion. because the same concenrrmion of gillcose perfused as
.) Lauu \\\\': Ht'I);l{le nt.~n. r'::i: a Cf"\leW of rht':r furwri'~n:- tJ.r'.d t'~f';~l'~5 (~"" J
a contre! had no slich effecL Potential nonspecific effer:-ts of Ph!1sil '/ p!trrnll(j("{,f ·),';:;.!O·}-l~:;. U)Sr)
the microdialysis technique per se were excluded b.... apply­ RtLS~t~k ~l ra!11ciparir~n or ltt:p~rk glucort''''::'~lfOt:-; 1:1 ::1t' \'n;:~:-I1; 'J!' {,)oil
int:tk~ .. \-d'IlP' l~)-;-::-fL)IJ<
ing the identical microciialysis procedure to a localized
:' g'>rg WP. DUring 2\1.J. >Iu.'rwin RS. B"r~ ~L\. Bn:l<"s ~[L :'ht;i:::;m f.;1
\'olume of FLs. Finally. the possibility that these changes \·t"n[t'on:t.~dial hn)orhabnltc lesions tn raj;:: ~uppre~~ t'l)H:Hf!-:'t':.:qi~H()r-.
were simply due to a time-dependent phenomenon was rE'5pl)n~l?S to h:'VO,:!!Yft""nlia. .f ("lin {lfP:>\;: :'I:~' l'~:-7~l':6'2 ~~{q" •
excluded by showing the same elfect when 2-deo:-.:yglucose ~'l. PaxlIlo:, (;. \\-~t!son C: 00:' Rill Bnlin if! S/,····01rl.;-/'· ("'J'd'hrul/.'.;:': :':nd ('d
~t"W Yt)f;':..\cadelllil' Press. IDt1L p. ~r.}2 .
was introduced m the begiIUling of rhe study. i.e.. after the P) Dtuin~ ~[J: III \;\0 r~t:urodll)rllisr)'y of ;;:e COn:5.::dl::::- h:u1\;::: hrain:
initial '5 nunolll perfu3ion. :\ote that the degree of !!ltrah!~,~·t\c1tnpal mil.:rodiaiY51s in evtlt~~':-::~-- fa JI~/'!"'!'r::~J"':":: ;" [hi'
glucopenia induced with is usually difficult .\-rfit-#)";'-"·f~rr....-_ Rnhin:-.)n TE, .JusliCt~ .iB .!~'. Ed5, .4"m"'=">~-i!~Ui; c:;"'\1~!".
1;'~.lL p ~~-H-l~
to comrol and. therefore. difficult to relate to physiological II KH,'! ,'1. \"'rlh RC. Dunnrn'.( BE T,!h,,!,~:,-- GE: R.. :'.· f.• ,' ,' ..'. -:hH:lk
h~voglycel1lia . .\'en,>n:heless. urilizmioll of radiolabelecl 2-de· [wr-.. Ot:5 =-,:'-:Sft"Hl (n intTt.)aSe pancft...>.ui,,' St'lTt"~:P!\ d·,:r:::..:. ::iarkt'~
o~-ygillcose perfused rtmmgh a microdialj/sis probe clearly lnsuiir!. mdtH.'ed h:'l)og!yeelnia in d,)gs.. ~1i ~ 1.<'"-: : l..f: ~" ~
!:.:. Onmm:l r Kimura f.:. I)oyama H. ~la<"o T. [~1 :'1. t,:',UU:;'I,:a \ R" .:t'fI.H:al
showt'd that the local glucopenia produced in our e:-..-peli­ aCIi\'lth..~,:, o( n'-"nrroIHt'liial and I~Ht'ral h}V.}d~;")!~HUic ar£<L':' >I( c~tt;,. ,; ,~"tft'"
;lems W[L<; limited to the \ "1[H. 1·j:j·4';4-lS.-,. l~"i.j

[n summary. our s£udies demonstrated thm glucopeni<l I·; '.[.,IU1.' rEo Ellaye!, f.:. HOU!~Uli H. (Jkamc::.t II:. \'anl:,o:. LB. V;,::.d"''; P.
.-'\humr:ld :\\: Honnon::ll anti mt~rabolit~ t'"tc~~,:~S uf nt··ttr,:..,Ut\·Op,:~~::~:.. B,uitt
localized to the \"1IH the re!'case of glucagon and R··..'- I)i . t:!~r-LI)S. 19~"J:3 . ,
catecholamines, These support the hypothesis that 1-1.. f{t"fllt t1:1;;roH ...\.\\', Ran....;;t)n S\\w: The spt)nt~~::t·t)U:S acn~.~:~. ,u~d ,.,.: auak...

the \ "1IH contains l1E'urons that sense glucopenia and that ur rar~ '.\ nh h..\1>Olh,)ia:1tic 1t"5ion:$. A.ft) ..J F';~(j .... ;./I : . ) . ~:~:. :.. '-'~i. ~ .~:;

l . ) Bc.'nzu \. -.-\, ThE" h~l)1)rhal~unus anti hlood ~ll1cn:'>t~ r~'.!'!t,Hz"a .';{'f

this brain region is important for mediating a counteITegula· :j.~:::<;!I~i_~-5 i .3. 19;;;3 ­
tory response to hypoglycemia. tt; Sz:..tbo I).. Szabo ~.u= ~rlldies on tht-. nittt!!~ and m(ltft..• '!l"f:, , . ',f tht<
insuli!l-::'t'flsitlYe gllleor~Ceptor in {he C"':".::":\1 n,,-'"r..<OH:f ~::::;rc:;~ f::.'Jrwfr.,:,
::~"~~::~-"~:~I;. 197.).
ACK;\OWLEDG?IlE::\TS t7 fmh!~l:l:! L\. BemanEs LL: Etrec[ of hy!>.. :~.t!;tnl\c ~lit;n':1f)"(\ '~:. :.iasma
This research \va., supported by L·.S. Public Health Seniee ~!ut.·o"'t· . m~ulin. and g~llC'agotllt~q:IS_ .-tm ,," PI1ll,-,",J/ :!~: . :::,:Il:_:' ,,,:,,;, l~I:-!
Grams DK-20-195. DK--W9:313. DK-!'57:35. and NS-:2S:227 and by l~. OtRO"("(1 R.I. (;till HJ; Tilt'" forebrain IS n!); t'.:--:-iF;>oual (,)f ::~.1np~:r:~.}:t' irenai
h..\:p~'r~! .\l·t~mil" re.::,pnr~5t' (0 gtucuplinlrttl;', -;n'/'ur:,> :'::11'; <ll"':-:::~" 1ft';':'.
a fdlowship grant from the .]lI\'enile Diaheres Foundations l~t, i- "~Hh~ P ...\~1al R.. I3~~n!!l,an R:-':: Pu(ari\"t" ity:\-,)rhai~l!niC' i<'~n;"t.'\'t.'l :.;'~ pl~l";
International (W.P,R, :'LA,R). thj t:·:o.::-t·:Hl~\l rol~ in [!',r! reSpf)f't.'5t" (() nH.. l':~>~'~Ht' !; .. l)u".!:-... ..;t.~mia' J' ti)v(r',
:J..)::2t}~-~:-7. [9:3.') . .
\v-e are indebted to Drs, \Yilliam Tamborlane <md :'lichael 2U.. Ritr r;;'t Rl....... Slusser PC. Slone $: GIlH~nr(·l·t:'~")rt)r..., and
:'kCaleb for comments ancl suggestions, We appreciate thE' blond ~luco$e; locanorr in th~ hindbrain, ,,:;,"'h-/lrt?
assistance of Ajda Groszmann and :illdrea Belolls for their ~ l. '.riller RE: Pancre~riL neuroenciocrinr)Ic,~';:
nisms i:l rhe regulation oftb~ islets orLan~~;'·hat\5.
help in measuring plasma honnones. We also gratefully 1951 .
acknowledge Karen Dmis for e~-pen technical assistance. Hil..n:::WI)rr~ RL H~1>I){h~llamic cOfuroll)t" "h:rt"naiirft-:'~:::ii'~I_':-~rtL)i\ ;::, ::---iPOll:'iV
trbHiinl'lH glucos<,-. .f Ph./jsil/{ ~Of),-t II.....; 17. t' '7r)

~:l .-\nand Sf.:. Chhina (;5. Shanna h:...'\. DU:1 ::.. Sin!!!! B: .','u<ir,· .: .-in~I"
REFERE~CES r.t'llron~ in [he h:'VGrnaltliHic ft"t"liing l..'t'!:·t:'l'S: t,"[rt"v: .;;' '2tlll'I1::o-,* .1I11".J
l. ('1'\,<'1' PE: Gltleo~t' ('tJulIit'ITl'l.(u!a!ion in man. Dj(J/}I'(I'S :)O:~!jI-:;f3..l. W:'I Plu!,i,,; ~07: 11-t,;""1 F~. 191H
.} Biggers mI'. ~IYl'IS SR. ",e'll D. :';rinson R. Conp>?r :;lB. .filSP"" .IB. I ~~::'t"l ::(1-. [n~lt·s DL Gtyeenlic re:5Vl)n~fS indul.. . c"d :',\' h:.r. aj~\mi(~
Williams PE. Chenim;tol1 AD. Frizz",! RT: Rot€' of brain in l'oumeIT€'l:!ll' cHUl!ubaon, '\'~lil'f)~'fr!f)('n"Ilf)I{)yy ~S:~ t~-:':l,j. 1~17~1
Inlion or ii1sulin-indue...d hypn.~lycl?lllia in dogs. DI(lI>I'(I',< :17:7-16. UJS~l ~:; . Frohm,;,ul L..\.. ~agai 't\: Central ner\."i)U5 :;.::...... tf'tll-medt~l::·, t :::.£tltH::.... ~irH\ (d
:3. Woods Sf. Punt: D .k \','ur..ll cotHrolui Ih" encloerill,· P'Ulcn-'as. Ph!!si'"
~lllt:;l~lln~t"Crcrt!Jn ir. [he dog rollowin~ :':·~:t.·I)X~·~.;[IJ\~t . ,,,;, .l/.'ton ~.""," £~r'l;
R"f' ';..\:501}-IH!1. W7-t
:':5"l-l";"-1-l·}2. It)~6
-t Frizzell RT. .Jon<'5 E::\L Da\is S:". Biggt'rs DW. 2\(yers SR. ('unnoHy CC
BLoum SR. Edwartis A \'. \·aughan \.) TIle r"I,· ()i !lit' .l;.·',fl'lIlU'·
\»;:\1 DW, .Jaspl\n JB. Owningron .ill: ['lllntE'ITt'gul:ltjol\ durin,! h}1)ogiy­
innt'"n·;t~lun in rhe control or glucagon !t':t'a."if>' d:.Hin~ :1·.1)tI~h.l\.. ,.:..:nia in
\'I"l\\la is ,lirectE'd hy \\ldt>:;pread hrain regions. Dil/opll!s .jt:lt::;:.l--1~61. It~t)
lh~ c~:r .f Pltysin' ~:)j:i) 11-6:2:3. W7.. . . .






Abraham Roth Professor and Chairman

Department of Obstetrics, Gynecology, and

Reproductive Sciences


Department of Internal Medicine

Temple University School of Medicine

Director, Division of Maternal-Fetal Medicine

Department of Obstetrics, Gynecology, and

Reproductive Sciences

Temple Universiry Hospital

Philadelphia, Pennsylvania


Professor and Chairman

Department of Obstetrics and Gynecology

Brown University School of Medicine

Obstetrician and Gynecologist-in-Chief

Department of Obstetrics and Gynecology

Women and Infants' Hospital

Providence, Rhode Island

Churchill Livingstone
New York, Edinburgh, London, Melbourne, Tok-yo

Roberta A. Bhllard, M.D; Nam H. Cho, Ph.D.

Professor, Department of Pediatrics, University of Associate Professor, Department of Preventive iv!edi­

Pennsylvania School of Medicine; Director, Divisipn cine, Ajou University School of Medicine, Suwon,

of Neonatology, Children's Hospital of Philadelphia Korea

and Hospital of the University of Pennsylvania,

Philadelphia, Pennsylvania C. Andrew Combs, M.D., Ph.D.

Associate Director, Department of Maternal-Fetal

Peter H. Bennett, M.B., F.R.C.P., F.F.C.M. Medicine, Good Samaritan Health System, San Jose,

Chief, Phoenix Epidemiology and Clinical Research


Branch, National Insrin.He of Diabetes and Digestive

and Kidney Diseases, Phoeni"<, Arizona

Joshua Copel, M.D.

Professor, Department of Obstetrics and Gynecology,

Walter P. Borg, M.D.

Yale University School of Medicine; Director, Depart­

Postdoctoral Fellow and Research Affiliate,

ment of Obstetrics, Yale-New Haven Hospital, New

Department of Internal Medicine, Yale University

Haven, Connecticut

School of Medicine, New Haven, Connecticut;

Resident, Department of Internal Medicine, John

I.arry Cousins, M.D.

Dempsey Hospital. University of Connecticut Health

Director, .Maternal-Fetal Medicine, DivisT6n of Peri­

Center, Farmington, Connecticut

natology, Mary Birch Hospital for Women at Sharp

Memorial and Sharp PeriI1atal Cemer, San Diego,

Florence M. Brown, M.D.


Instructor in Medicine, Harvard Medical School; Assoc­

iate Staff Physician, Joslin Diabetes Center, Boston.

Donald R. Coustan, M.D.


Professor and Chairman, Department of Obstetrics

Thomas A. Buchanan, M.D.

and Gynecology, Brown Universiry School of

Medicine; Obstetrician and Gynecologist-in-Chief,

Associate Professor, Departments of Medicine and

Department of Obstetrics and Gynecology, \Vomen

Obsterrics-Gynecology, University of Southern Cali­

and Infants' Hospital, Providence, Rhode Island

fornia School of Medicine; Attending Physician,

Departments of Medicine and Obstetrics and Gyne­

cology, lAC-USC Medical Center, Los Angeles.

Ulf]. Eriksson, M.D.

Associate Professor, Department of Medical Cell Biol­

ogy, University of Uppsala, Uppsala, S""'eden

Marshall W. Carpenter, M.D.

Associate Professor, Department of Obstetrics and

Ann .M. Ferris, Ph.D., R.D.

Gynecology, Brown University School of Medicine;

Professor and Head, Department of Nutritional

Director, Maternal-Fetal Medicine, Women and Infants'

Sciences, Uniyersity of Connecticut College of

Hospital, PrOVidence, Rhode Island

Agriculture and Natural Resources. Storrs, Connecticut


Carbohydrate, Lipid, and

Amino Acid Metabolism In the
Nonpregnant Patient

METABOLISM AND DIABETES which this residual insulin secretion is maintained ap­
MELLITUS pears to be an important factor in determining the
stability of long-term metabolic comroL! Ultimatek,
Di:tbetes mellitus may be defined as a syndrome in endogenous insulin secretion becomes undetect:1bie
which a complex interaction of hereditary and envi­ as residual functioning f3-cells are comoleteh' de­
ronmental factors leads to inadequate action of insulin, stroyed. Abundant evidence now exisrs that ·autOi~mu.
due to decreased insulin secretion or resistance of tar­ nfty plays a critical role in the pathogenesis, of type I
get tissues to irs action. Regardless of the pathogenic
(insulin-dependent) diabetes mellitus:;
factors causing the disease, the e\'enrual metabolic con­ In the patienrs with type II (non-insulin-dependent)

sequences of the diabetic syndrome primarily reflect diabetes. the secretory failure is less severe. Basal insu­

the degree to which there is this absolute or relative lin le\-els are generallv normal or mildl\' increased.

deficiency of insulin. Insulin's critical role in the patho­ whereas glucose-Stimuiaced insulin secretion is dimin~

physiology of diabetes derives from its central role in ished. The magnitude of the insulin secretory defect

regulating the stOrage and release of metabolic fuels, usually correlates with the severity of fasting hyperglr­

namely. glucose, fat, and amino acids. cemia in these patientS. In itS mildest forms (plasma

glucose approximatel;: 140 mg/dl), the l3-cell defecr

involves only the initial secretory phase; the more-de­

Insulin Secretion in Diabetes layed insulin response remains intact. In such individu­

als, the loss of responsiveness to glucose is specific,

The defiCiency in insulin secretory capacity in diabetes

may vary from complete failure to a panial defect that is (Le., normal responsiveness is maintained to Other se­
apparent only in circumstances of increased demands cretagogues such as f3-adrenergic stimulation or amino
such as physical stress, obesity, pregnancy, or aging. acids).4 Consequendy, insulin defiCiency is much less
In the typical insulin-dependent (or type 1) diabetiC pronounced during ingestion of mixed·meals as com­
patient, insulin secretion is either totally defective or pared with glucose. s In patientS with more severe fast­
severely impaired; insulin production varies with the ing hyperglycemia (>200 mg/dt), the capaciry to reo
duration of the disease. For up to 3 to 5 years, these spond to increases in circulating glucose is \-irtually
patientS continue to secrete some insulin, despite their lost. These observations suggest that a specific abnor­
requirement for exogenous insulin.' The extent to mality in recognition of glucose by the l3·cell occurs



in the earliest stages of I:)pe II diabetes and that this stantially among the various insulin-sensitive fuels ar

I defeCt advances as the disease progresses.

The initial reportS by Yalow and Berson6 regarding
target tissues.
In type II diabetic patients, insulin is markedly Ie
plasma insulin levels in [)pe II diabetes emphasized effective. 12 The insulin dose-response curve for au
the presence of h~perinsulinemia, This seeming para­ meming glucose uptake in peripheral tissues is shift(
I dox was subsequently shown to be more apparent than
real when tota:lb.odr weight and ambient blood glu­
to the right (decreased sensitivi[)') and the maxim
response is commonly reduced, particularly in patier.
cose levels ag~considered. Approximately 80 to 85 per­ with more severe h}perglycemia. 13 Other insulin-stir.
cent of eypeI! diiibetics are obese. Obesity per se is ubted processes, such as inhibition of hepatic gluco:
generallr accompanied by hiperinsuIinemia and is as­ production, also sho'!;'" reduced sensitivirj' to insuli
sociated with resistance on the pan of targ~ tissues However, by contrast, hepatic insensiti\"irj: is relative
(muscle. liver, adipose tissue) to insulin? \'I;11en com­ mild and m3.Y be readiii' overcome br larger amour.
parison is made bet:\veen obese [)pe II diabetic pa­ of insulin. 13 It is generally believed that insulin res!
tiems and appropriate weight-matched nondiabetic tance in type II diabetes results from both a reductic
control patients, it is apparent that insulin levels in in insulin receptors and postreceptor defect.H Tr
1 obese diabetic patients are below those observed in causes of insulin resistance in nonobese type II dt
obese subjects with normal glucose tolerance.s An ad­ betic patients do nOt appear to be different from the
ditional factor contributing to the seeming hyperinsuli­ obese counterpartS. Howe\·er, the severirj' of the res:
J nemia is the presence of hyperglycemia itself. In other
words, when glucose levels are raised in nondiabetic
tant state tends to be accenruated by the co-existenc
ofobesity and diabetes. Overall, the available data inc
individuals to stimulate the glucose tolerance curve cate that insulin resistance contributes importantly [

t of the diabetiC patient, the deficiencr of the insulin

response in the diabetes becomes more clearly ev­
the development of I:)pe II diabetes. It remains unce
tain, however, whether insulin resistance or the de:
cieney in insulin secretion is the primary e\'ent leadi:-.
to diabetes in these patients. Regarr;lless, bam disn.:
bances undoubtedly imeraa, thereb;1"magnif;.ring r.r
Insulin Resistance in Diabetes severity of the metabolic derangement Interesting;
the ability of insulin to stimulat~ glucose uptake :
The biologic actiqn of insulin is not only related to its peripheral tissues is also impaired in patientS with [)1'
concentration but is also a function of its ability to I diabetes 15 and in animals rendered panially insuli:
activate cellular events. The first step in the cellular deficient by subtotal pancreatectomy. This· insul'
action of insulin is the binding of the hormone to a resistance is thought to be medi,ned by a poser,
specific receptor on the cell surface, This interaction ceptor defect and is responsh'e to intensi\"e insul:
triggers a cascade of poorly characterized cellular therapy.16 Thus, even in I1pe I diabetes, insulin r,
changes, termedpostreceptor ecenfS, leading to a meta­ sistance may contribute to and impair therapeu,
I bolic response. The concentr:uions of insulin required
to activate metabolic processes vary both with respect
measures directed at improving glucose COntrol. Tr.
defecrs in insulin action in diabetes are summarize
to the target tissues and the substrates involved. For in Table 4-1.
I example, a doubling of systemic insulin levels is suffi­
cient to achieve near-maximal suppression of lipolysis
but has a negligible effecc on glucose uptake by periph­

I eral tissues (muscle and f::u).9 The insulin concentra­

tions needed to promote protein anabolism or sup­
press hepatiC glucose production fall beC"Ween these
Table 4-1. Defects in Insulin Secretion and Actior.
in Diabetes
Ti-pe I but again are much lower than those that Diabetes Type II Dl:lbetes

1 ma;ximaily increase peripheral glucose uptake,lO.n As

might be expected from this siruation, the conse­
Insulin secretion
insulin aaion
Impaired (pa:;;icularl:; to glueQ:;·
Severely imp:lired (receptor :1...:.
postreceptor defec-..s)
quences of incomplete insulin defiCiency differ sub­

1 ..


Table 4-2. Metabolic Actions of Insulin

[Jver Adipose TISSue Muscle

~ciGl(lbolic effeCtS Decre:lSed Decfe:lSe:d Decre::l.Sed

Glycogenolysis Lipolysis Protein bre:t.!.;down
."~'1;lbolic effeCtS Inere:lSed Increased InCTe:lSed
GI;:cogen synthesis Fat synthesis Glycogen and prOtein synthesis
Fat synthesis G~'cerol synthesis Glucose uptake .

Metabolic Effects of Insulin cose use continues at a rate of 200 to 250 gld.l7. The
main site of glucose uptake is the brain, >':hich is criti­
Insulin is the primary hormonal factor that controls cally dependent on an ongoing supply of glucose for
the storage and metabolism of ingested metabolic oxidative metabolism. Despite the lack of exogenous
fuels. After a meal, augmented secretion of insulin facil­ fuel and the ongoing glucose reqUirement, blood glu­
itates the uptake and storage of glucose, fat, and amino cose remains relath-ely stable as the liver releases glu­
acids. Conversel~', a deficiency of insulin leads to mobi­ c.ose at rates sufficient to match those of consuming
lization and endogenous fuels and reduced use of in­ [tssues.
gested nutrients. The action of insulin involves each The hepatic processes involved in the release of glu­
of the three metabolic fuels and is manifested in three cose into the bloodstream consist of glycogenolysis
principal tissues: liver. muscle, and adipose tissue, and gluconeogenesis. It has been estimated that ap­
where insulin exerts both anticatabolic and anabolic proximately 50 to 75 percent of hepatic glucose pro·
effects, ""hich reinforce each other (Table 4-2). duction in the POStabsorptive state is derived from gly·
cogenolysis, with gluconeogenesis contributing the
remalnder. 1 $ The resynthesis of glucose from the gly­
BODY FUEL METABOLISM IN colytiC intermediate, lactate, amounts for"3t least one·
NORMAL SUBJECTS half the gluconeogenic component, and the conver­
Postabsorptive State sion of glycogenic amino acids comp.t;ises most of the
remainder. Alanine, whose release from muscle and
The period after an overnight fast and preceding the
uptake by liver predominates over that of other amino
ingestion of the morning meal is referred to as the
acids, is the main amino acid contributing to glucose
postabsorptive state. At this time, the concentrations of
hormones (insulin and glucagon) and substrates (glu­ S}nthesis. Conversion of fat·derived glycerol and recy­
cose, amino acids, and fat) thar were altered due to cled pyruvate contribute less than 2 percent and 1 per­
meal ingestion during the preceding day h:l\"e reo cem, respectively, to total glucose production. ls Fuel
turned to baseline, and the rate of fuel consumption is homeostasis in the postabsorptive state is summarized
closely matched by endogenous fuel production. The in Figure 4-1.
postabsorptive state thus serves as a useful reference Regarding the hormonal factors regulating glucose
point because it represents the period of transition metabolism in postabsorpuve humans, bOth glucagon
1 from the fed to the fasted condition.
After an overnight fast, the decline in circulating in·
and insulin moderate glucose release by the liver. Per­
haps the most compelling evidence that basal glucagon
sulin leads to a marked reduction of glucose uptake secretion is important in maintaining glucose produc­
I by peripheral insulin-sensitive tissues (e.g., muscle and
fat) and a shift toward the mobilization and use of fatty
tion in the postabsorptive state derives from sLUdies
in which somatostatin is infused to suppress plasmJ
acids as energy·yielding fuels. Glucose consumption, glucagon and circulating insulin is prevented from fall­
nevertheless, continues in the non-insulin-sensitive tiS­ ing by exogenous insulin infusion. In this circum·
sues (e.g., the brain, renal medulla, formed elements stance, a sustained 70 to 75 percent reduction in he­
of blood) and in the splanchnic area, so that total glu­ patic glucose production occurs, indicating the







Fig. 4-1. Glucose homeostasis in the pO$[Jbsorpth'e sate in normal human subjects, FFA. free- fatty acid: A-\.
amino acid(s),

importance of basal concentrations of glucagon in op­ net flux of amino acids thus existS bet";een musci::
posing the inhibitOry actions on this process,19 The tissue and norunuscular tissues where nitrogenou:
restraining influence of basal concentrations of insulin 'waste products (urea and ammonia) are generated,
on postabsorptive glucose production is also e\'ident Ah:hough virrually ali amino adds are rele3Sed b:,
when plasma insulin is suppressed by somarostatinand muscle, alanine and glutamine predominate, accour,:
circulating glucagon is maimained at the basalb'el by ing for more than 50 percent of the lmal amino ac:.
exogenous glucagon infusion. Under these condi[ions. release. 22 The alanine serves as a substrate for hepa::.
hepatic glucose production promptly incre3Ses,~o He­ gluconeogenesis, and glutamine as a precursor f.:::
patic glucose production appears to be regul:ued by a renal ammonia synthesis and as an energy-yielding rue
"push-pull" system, with the opposing actions of insu­ for the gUt. Because alanine and glutamine account fo
lin and glucagon balancing each mher. In the pOSt­ only 10 to 13 percent of the amino acid residues i;­
absorptive state, the hormonal regulation of glucose muscle prmein, their release at higher concentracior::
homeostasis is primarily directed at endogenous has been explained on the basis of de nO\'o symhesl'
production of glucose rather than its uptake by tissues, in muscle_
After overnight fasting. muscle tissue, the main res­ The carbon skeleton of al:mine is largely derh-e.
en'oir of body protein, is in negath'e nitrogen babnce from pyru\'are; it is belie\'ed that most of the pyru\'a:~
as evidenced by a net release of amino acid5,~t This used in alanine syntheSis in muscle is formed via gl;'
net proteolysis in muscle is facilitated by the decline colysis. 23 The branched chJin amino acids (leucino:
in circulating insulin to baseline concentrations, Inas­ isoleUcine, and \~Jline) appear to be the predomina:-:
much as plasma amino acid le\'els remain relatively sources of the nitrogen for alanine formation 2 -l (Fif'
constant; amino acid release from muscle must be JC­ 4·2). In contrast to mher amino acids. these amir::
companied by amino acid uptake by nonmuscubr tis­ acids are metabolized to a greater extem in muscle
sues (liver and, to a lesser e).,em. kidney and gut). A than liver, Furthermore, their oxidation in muscle :!f'


GLUCOSE the dissolution of muscle prOtein and branched chain
1 amino acid oxidation limit the release of glycogenic
amino acids by muscle. therebr complementing its reo

ALANINE «:-(---
stl.lining effea on hepatiC gluconeogenesis.
Finally. the hll in circulating insulin after an over­
+ night fast allows for the release offree fatty acids (FEAs)
Nilregen Greui' from adipose tissue depots (Fig. 4-3), Insulin is ex·
tremely effective in inhibiting hormone·sensitive li­
pase within the fat cell. which cltaivzes the hvdrolvsis
of stored triglycerides and r...l-te libe;ation of ff_-\5. This
BCAA anti[ipolyric action of insulin occurs at concentrations
Fig. 4-2. Alanine synthesis in muscle. The Clfbon skeletcn of insulin well below those neceSSJry to affeCt glu~ose
is m:lin!\' derived from glucose, ""here:lS br:lnched ch:1in trJ,nspon.9 The levels of insulin normally present in
amino a~ids (BC\..l.,.) pby an [mpor..ant role in donating the the posrabsorptive stJ.te lre suffiCiently low, however.
nitrogen group. to permit the flux of Ff.\s from Storlge sites to eX"1race­
rebral tissues, such as muscle. he:lft, renal cortex, and
liver (Fig. 4-3). In these tissues, bay acids are the prin­
pears to be sufficient to provide nearly all the nilrogen cipal energy-yielding fueL The consumption of FFAs
required for alanine formation. z5 The apparent cou· by muscle tissue is an impomm f:lctor in diminishing
pIing bet,,;een branched chain amino acid cat:lbolism muscle glucose upr.a..":e and oxid:uion in the posrab­
and alanine s}l1thesis suggests that the [ate of oxidation sorptive state. FFAs act in this ,;vay by reducing glyco·
of these amino acids may be an imponant factor, mod­ lytiC flux as well as emry of glucose·derived pyruvate
ulating the availability of alanine as a substrJ,te for he­ imo the Kreb's cycle (through the pyruvate dehydro­
patiC gluconeogenesis. Leucine oxidation is inversely genase step ).30 The r:lte of lipolysis and the magnitude
correlated with insulin concentration?6 In contrast to of the insulin decline in porrJ! blood after an overnight
alanine, the carbon skeleton as well as the nitrogen fase is not, however, of sufficient magn!tude to stimu­
source for glutamine is most likely derived from in late appreCiably the rate of hepltic convehion of FFAs
27 to ketones.
situ muscle catabolism of amino acids.
The partern of amino acid uptake by the splanchnic
area (liver and gut) complements that of muscle re­ Metabolic Adaptation During Short~

lease (I.e., alanine and glutamine predominate).23

Term Starvation

However, when hepatic and extrahepatic tissues are

considered indiVidually, mOSt of the alanine is reo Because liver glycogen is Iimired to about 70 g after
moved bv the . liver, whereas glutamine is taken up an overnight fasc,3 1 whereas glucose consumption oc·
by the g~L29 Nevertheless. glutamine contributes (0 curs at a rate of approximately 200 to 250 gld, hepatiC
glucose and urea production by the liver, because a glycogen scores are rapidly dissipated very early in the
ponion of the glutamine taken up by the gut is con· course of fasting. Thus, the ini[ial phase ofstarvation is
vened to alanine, released directly imo the ponal vein, characterized by an acceleration of hepatiC gluconeo­
and ultimately convened to glucose and urea by the genesis to meet ongoing tissue demands for glucose
liver (Fig. 4-1). Alanine is thus nOt only a main vehicle by the brain. The augmem:l[ion of gluconeogenesis
for nitrogen release from muscle but is quantitatively and the maimenance of glucose homeostasis are me·
the most impon:ant protein.derived precursor for he­ diated by both hepatic :Jnd extrahepatic events. The
patic gluconeogenesis. The rate at which afanine and release of alanine and Ocher glycogenic amino acids
other amino acids are convened imo glucose is in large from muscle increases,3 2 and the rate of hepatic con­
pan determined by the balance between glucagon version of alanine to glucose accelerares.2 1 The en­
(stimuhtory) and insulin (inhibitory) in the porral vein hancement of glucose symhesis from amino acids is
as well as the availability of glucose precursors gener­ nOt, however, sofely a function of increased a\-ailabilir. .'
ated by muscle. The restraining effeCts of insulin on of these precursors, since plasm3.levels of alanine and






\~ FFA~--
\1'"01 lJ~o"
CHYlOM1CRON- rG> l'?o~ro'e,,' FFA J
! lIPOPROTEIN- TG lipase \ -;


\ ; ' /- _ _ GLUCOSE

Fig. 4-3. F3.t syntheSiS, storage, and rele3.Se in normJ! humans. Normal fJt homeostasis is dependent on the action
of insulin. \'\'ithin the liver, insulin stimulates the synthesiS of free fatty acids (FFAs) from glucose and their
esterification EO form triglycerides (TG). Both exogenously deri,'ed (dietary) triglycerides (chylomicron.TG) and
endogenous!:' synthesized triglycerides (lipoprotein-TG) are sources of fany acid delivery to adipose tissue. Insulin
acceler::ues the uptake of FFA by adipose tissue by its stimulator:' effect on lipoprotein lipase, Fat storage within
adipose tissue is also enhtlnced by insulin's glycerogenic effects. The amilipolytic actions of insulin (inhibition of
tissue lipase) enhance fat storage as '\vel!, while redUCing the a\-ai/ability of circulating farr:' Jcids. FFAs a;e'released
from adipose tissue when insulin ie':e!s fall and are taken up by muscle, hem, kidney, and liver.

other glycogenic amino acids actually fall despite their of glucose are synthesized under the influence of barh
increased release from muscle. These observations the elevation of glucagon and the depression of porta!
suggest that intrahepatiC gIuconeogenic mechanisms insulin (Fig. 4-4). Hypoinsulinemia further contributes
are stimulated during short-term fasting. An addition::11 to glucose homeostasis by reducing extracerebra! glu­
factor contributing to glucose homeostasis at this time cose consumption and by increasing the a'v'ailabiliry of
is the increased release ofFFAs from adipose tissue. FFAs for oxidative metabolism bv muscle and liver.
Oxidation of fam' acids by muscle spares glucose for The progressive rise in blood ketOnes during Starva·
use by the brain' whereas their oxidation by the liver tion is also regulated by insulin and glucagon36 (Fig. 4­
activates key gluconeogenic enzymes and furnishes [he 4). The development of hyperketonemia im'olves three
energy and reducing power necessary for glucose syn­ distincc mecabolic events: (1) delivery of FFA from adi­
thesis. 33 pose tissue, (2) hepatic oxidation of FFA5 leading to
These metabolic adaptations (namely, increased glu- ketone formation ("ketogenic capacity"), and (3) a re­
COfleogenesis, amino acid mobilization, andlipolysis) duction in ketone uptake by peripheral tissues. Hypo­
are facilitated by a decline in insulin secretion below insulinemia activates each of these steps, whereas
posrabsorptlve levels as well as a modest increase in hyperglucagonemia contributes by enhancing keto­
circulating gluclgon !eve!s.34.35 The former appears to geniC capacity.36 Growth hormone may also contribute
be triggered by a fall in arterial glucose concentration, by promoting JipolySis.3 7
which inhibits insulin secretion, whereas the rise in
glucagon is due to diminution in the race of glucagon
metabolism.35 Hypoinsulinemia enhances protein As in starvation, during exercise there is a' need to
breakdown and thus the delivery of glycogenic amino generate glucose and FFAs from endogenous sources
adds from muscle to liver, where increased quantities to meet tissue demands. Because muscle glycogen


t Insulin

I~~i~\ &
t Amino Acid

t Gluccneogenic Enzymes
t Ketof;enic Capacity


t Ketosis ---.J
-_._. .

t Glucagon

Fig. 4-4. Incer:J.ction of insulin suppression and giucJ.gon stimulation in promoting gluconeogenesis and ketosis
during stlf'l"ltion. ITA. free fatty acid.

stores are rapidly depleted, energy' for working muscle insulin secretion diminishes in associatiQn with the ac­
must be supplied from blood-borne fuels (Fig. 4-5). tivation of sympathetic nervous system ~cti\'i(y :\1so,
Glucose is supplied by the liver, which ma~: increase increments in plasma glucagon, epinephrine, gro\'.th
itS production of glucose three- to fivefold. 38 The rate hormone, and cortisol commonly Otcur, particularly
of hepatic glucose production is precisely regulated to as the intensity of exercise is incre3Sed."o These hor­
supply enough glucose to keep blood levels from fall­ monal changes, acting in concert, promote the mobili·
ing despite the increased glucose extraction by work­ zation of glucose and FFAs from liver and adipose tis·
ing muscle. FFAs are mobilized from adipose tissue at sue. respectivel~'_
dramatically increased rates to minimize the need for
glucose from the lh-er's limited glycogen stores. As ex­
Glucose Ingestion
ercise continues for prolonged periods, the consump­
tion of FFAS assumes an increasingly important role GLucose ingestion involves a variety of homeostatic
in meeting muscle energy requirementS 39 (Fig. 4-5). mechanisms that act to minimize the rise in pb5m~
This spares the liver from further demands for glucose glucose and restore normoglycemia. They include (1)
production, which, by this time, is occurring co a suppression of endogenous glucose production by the:
greater extent from gluconeogenic precursors. includ­ liver, (2) stimulation of glucose uptake by spl:mchnk'
ing protein-derived amino acids. 39 tissues, especially the liver, and (3) stimui::ttion of pe·
Current evidence suggestS that the increased con­ ripheralglucose uptake, particularly in muscle. These
sumption of glucose and FFAs by exercising muscle is homeostatic processes are activated by a rise in circl!'
mediated largely \-[a local non hormonal mechanisms. lating insulin or a rise in blood glucose itSelf. SpecifI·
A coordinated hormonal and neural response is, how­ cally. inhibition of hepatic glucose production is exqu:·
ever, of critical importance for the produaion of ap­ sirely sensitive to small elevations of portal insuli"
propriate quantities of SUbSlTales. During exerCise, concentration lOAI but may also occur in response tC;



75 Musde Glycogen FFA Uptake

/ ----­



/ -
to Total
02 Uptake



o 2 3 4 Hours
'---.--' ,
Glycogen Glucose + FFA Uptake t FFA, .} Glucose Uptake

Fig. 4-5. Time-dependent ch;lOges in the contribution of muscle glycogen and blood-borne fuels (glucose and
free fatty acids [ to [he energy requiremen£s of leg tissues during bicycle exercise.

hyperglycemia per se, provided thar b:15al insulin lev­ ited in muscle tissue. Thus. the muscle and liver bo,'­
els are mainrained.-I2 Glucose upt.:lke by splmchnic tis­ playa crucial role in the homeostatic response to i:J.rg'

sues is stimulated by a rising glucose concentration glucose meals (Fig. 4·6).

and to some extent by hyperinsulinemia.~3 The pres­ A large (75-g) oral glucose load is, however, r.C

ence of basal amoun£s of insulin are required for glu­ representativ·e of either the magnitude of carbohydr:l:'

cose to exert this effecr.-I-l By contrast, peripheral glu­ intake or the amplitude of blood glucose excursio:.

cose uptake is promOted by hyperinsulinemia arid, to observed in healthy individuals ingesting ordinr

a more limited extent, by the mass effect caused br meals. In circumstances of mixed meal intake, hexos

hyperglycemia i£self. 43 Considerably l:1rger amountS of intake is conSiderably less and the blood glucose ge::

insulin are, however, needed to incre:15e peripheral erally varies by no more than 30 mgldl over 24 hou~.

glucose uptake than are required to suppress hepatic This "fine tuning" of blood glucose regulation is dele

glucose production. lO mined to a greater extent by the exquisite sensirjl.:i .

. The elevation in plasma glucose caused by ingestion of the liver to small changes in insulin secretion. \Vhe
of large glucose loads brings into play e:lch of the small amountS of glucose are consumed, peripher.
abm'e metabolic adjustmentS. Hepatic glucose produc· insulin b'els rise modesd)f (less thanrwofold). Ho,
tion is markedly suppressed for several hours until ever, because insulin is released directly into the pore
plasma glucose has returned to baseline. This serves
vein, ponal insulin concentrations rise considera::o
to limit glucose entry into the systemic circulation at
higher. Consequently, hepatic glucose production
a time when the system is overloaded by exogenous
suppressed, whereas peripheral glucose uptake, whi"
glucose. With respect to (he uptake of the exogenous
requires more insulin to be activated 10 is onlv me
glucose load, recent studies indicate that in quantita­
tive terms most of the exogenous glucose load (about estly incre:1Sed. 46 If the quantity of gl~cose co~umc
[v"o-thirdS) is deposited in muscle."; The remainder
is suffiCiently small so that it is compensated for by tr
(abour one-third) is taken up by splanchnic tissues reduction in endogenous glucose production, gluco

(e.g., liver and gut). When one considers that the liver
homeostasis is maintained simply by retaining hep:.;·
also reduces itS endogenous production of glucose by
glycogen stores. Thus, as compared wirh me liver, ITa

more than 50 percent, the net effect is a substantial

de and adipose tissue are involved to "more limit

retention of glucose in the splanchnic are!, which ap­ extent in the metabolic adjustment to very small g:

proaches in magnirude,~e amount of glucose depos- cose loads. This phenomenon is a direct consequer:

. .:.-.





(20g) (20g)


Fig. 4-6. Cumulative rates of hepatic glucose production and glucose disposal by non-insulin-dependent tissues
(brain), the splanchnic bed, and peripheral insulin-sensitive tissues during a 4-hour period either in the pos.absorp·
tive state (fast) or after ingestion of a 75-g glucose load in normal subjects. After glucose ingestion, hepatic glucose
produaion is reduced (by 50 percent) and glucose uptake by splanchnic (Z.5·fold) and peripheral (S·fold) tissues
is increased.

of the disparity of the dose-response CUl>'es of hepatic

and peripheral tissues to insulin. lO
-::: Vaitoe I~1

::!, 300
Protein Ingestion a
f: .-- --1 1
. - 1/
Because muscle is in negative nitrogen balance in the < LeUCH"te ,/
fasting state, repletion of muscle nitrogen depends on

z \ / I holtudne
_- -----1T
a net upr.a.l::.e of amino acids in response to prOtein 200
a '/ -
feeding. The transfer of amino acids from the gut to u
0 / 1----
muscle after prOtein ingestion is facilitated by the ac­ u V
tion of insulin.
= /1 /

In normal healthy subjeCtS, ingestion of a pure pro­ :::;: 100 1 / ,/

tein meal is followed by a large output of amino acids "" / :t
from the splanchnic bed, involving predominantly the
'"<...J /
branched chain amino acids. 47 Valine, isoleucine, and c:. ;;"
leucine together account for more than 60 percent of <l
the total amino acids entering the systemic circulation.
even though they contribute only 20 percent of the
:. :.. TIME [hr,)
total amino acids in the protein meal (Fig_ 4·7). The
glycogenic amino acids, however, are for the most pan: Fig.4-7. Changes in plasma :UTl:no acids after ingestiOn of a
deposited within the splanchnic area. 47 Simultaneous pure protein meal. IncrementS in the branched chain a!Tlino
with the release of amino acids from the splanchnic acids (leucine, isoleucine. and valine) exceed those of Other
bed, peripheral muscle exchange of most amino acids amino :lCids. (Based on unpublished data.)


revens from the net outpm observed in the basal state tions. The net effect of the ancllipolytic, bt symhetic,
toa net upt:lk.e. As in the case of sphnchnic exchange, an.d glycerogenic aaions of meal-stimubted insulin
the net up~1..,",e of amino acids across peripheral muscle elevations is to increase total f:lt stOrage and reduce
tissue is most marked for the branched chJin amino circubting FF.-\.5 and ketone
acids. The laaer account for more tha..'1 hJlf the total
peripherJI amino acid uptake in the first hour and for Role of Gender in Glucose

nearly 90 percent at 2 to 3 hours.~7 Leucine, isoleucine, Homeostasis

and valine thus constirute [he mJin substrate for the

immediate repletion of muscle nitrogen after prOtein Assessment of metabolism h15 gene rail;' been per·
intake. Furthermore, because the branched chain formed wimoU[ regard to the sex steroid hormonal
amino acids comprise only 20 percem of the amino milieu. Recentl:', however, se:era! lines of evidence
acid residues in muscle prorein,25 it is likely tha( these indicate me existence of gende;-rel::ued differences in
amino acids Jre not solely used for protein S}-l1thesis fuel metabolism. In response to exogenous insulin in­
but are c3tJbolized within muscle. Thus, as in the fast­ fusions, the rates of total whole·body gl:Jcose upta.l;:e
ing state mey provide a source of energy for muscle. or glucose upt:lke per unit of ir.sulin have been shown
Interestingly, the high intracellular le;'els of branched to be greater in males than Furthermore
chain amino acids in muscle induced by protein feed­ the response to extended fasting is notably iruluenced
ing may have importance beyond delivery of nitrogen. by the sex of the individuaL \iihereas the f:lll in circulat·
There is evidence that me branched chain amino acids ing glucose in normal men is gradual and glucose b',
have a regulatory role in stimulating protein syn­ els rarely fall below 50 mgfdl. in nonobese ,,'omen
thesis. 48 the process is accelerated and plasma glucose often
decreases to the 40 to 50-mg'dl range after 48 to T2
hours. 53 Although the explanation for these differences
Fat Metabolism has nOt been established, it h1.5 been suggested that
The rise in plasma insulin that normally accompanies the smaller muscle mass of women than men leads [Q
the consumption of mLxed meals accelerates the reo the reduction in insulin-stimubted glucose up;:ake, as
moval of ingested triglyceride by adipose tissue and well as the changes seen during fasting in females. With
serves to promote triglyceride s;.l1thesis in li\'er and respect LO the latter, it has bee;; demonstrated that the
adipose tissue (Fig. 4·3). In the liver, a significant pro­ generation of amino acids f:om muscle is diminished
portion of the dietary carbohydrate taken up is used in fasted fem3les. In keeping with Ll-jis. the fas,ing-in·
for trigl:'ceride formation. In humans, the liver is quan­ duced decline in circularing abnine is more pro·
titativel:" a far more important site of conversion of nounced in women than in mer<.53 The greater decline
dieta[\· carbohydrate into fat than is adipose tissue. in glucose concentrations leads to greater suppression
Howe~er, bOth' exogenously derived (or dietarr) tri­ of insulin secretion and, in ru~n. to a more pronounced
glycerides (chylomicron-TG) and endogenously syn­ rise in blood ketones in fasted wome:1. Despite the
thesized triglycerides (lipoprotein·TG) provide a evidence that women achieve a lower glucose le\'e!
source of FFA for triglyceride synthesis in adipose tis· during fasting than men, the literature contains con­
sue. Insulin accelerates the uptake of FFA by adipose flicting data regarding the e:fect of the gender on
tissue by virtue of itS stimulatory effea on lipoprotein coumerregulatory response [Q hypoglycemi:l. Se\'er:tl
lipase, an enzyme that breaks dO' Circulating triglyc­ reports have demonstrated that epinephrine, norepi·
erides.-i9 Triglyceride storage within adipose tissue is nephrine, and grow1h hormone responses to hypogly­
also enh::mced by insulin's glycerogenic effeCtS. A large cemia and other stimuli were Significantly higher in
proportiOn of insulin-mediated glucose uptake in the males. 54 .5S By COntrast, Other i:westigators have failed
fat cell is used for the formation of a-glycerophos. to demonstrate such differerl':es%
phate, which is necessary for the esterification of fatty Another situation in which u'1ere is variation in the
acids to form ([iglycerides. The amilipolY1ic actions of sex hormones milieu is the follicular and lute:11 phases
insulin (inhibition of hormone·sensitive lipase) also of me menstrual cycle in 'women. Assessment of carbo·
enhance fat storage while redUCing FFA concentra­ hydrate metabolism under hyperglycemic conditions

have demonstrated greater glucose uptake in the pre· lin deficiency is apparent (br definition) only after
o'fulatorr period. s7 - s9 However, no detectlble differ· meal ingestion, when augmented glucose uptake by
ences in the basal rate of glucose rurnO\'er, insulin· peripheral insulin-sensitive tissue is required to com­
stimul:ued glucose uptake, or the insulin·induced pensate for increosed glucose emry into the circub·
suppression of hepatic glucose production has been tion. The postabsorptive glucose level is norm:ll be·
obse[\'ed be(ween 1:\....0 phases of the menstrual cause basal insulin secretion is adequate to pre....ent
cvc!e. 60 - 62 Also, the cOI,mterregulatory hormone reo glucose overproduCtion by the li\'er. a process that is
SPonses [0 hypoglycemia. are simibr in the follicular \'ery sensitive to minor incremencs of insulin.
and luteal phase of'the cyde.
63 When absolute or re!Jri\'e insulin deficieoC\' OCcurs
in the basal state, an elevmion in fas;ing blood'glucose
ensues, In patients with r;.pe II diJ.betes. norm::ll .or
even increased basal le\'e[s of insulin ma\' be mlin·
tained but only at the expense of fasting h~perglyce.
mia, In such patients, hepatic glucose production (as
Metabolic Dysfunction determined by radioacc[\'e tracers) ma:: be normal but
The metabolic alterations observed in diabetes primar­ is generally increased in propor-ion to the magnitude
ilv reflect the degree £0 which there is an absolute or of the blood glucose elevation6~ Because only mild
r~lati\'e deficiency of insulin. Viewed in the context of h:perglycemia in normal individuals is sufficien't to in­
the role of insulin as the main storage hormone, a hibit hep:ltic glucose production,46 the ~pe II diabetic
minimal deficiency results in a diminished abilicy to patient with fasting h~pergly'cemia is always in a state
increase effectively the storage reservoir of body fuels, of relative or absolute glucose overproduction. In pa·
because of inadequate disposal of ingested foodstuffs tients with type 1 diabetes, portal insulin dencienC\' is
(e.g., postprandial hyperglycemia, hyperaminoacide· invariably present, and thus hep3cic glucose produc.
mia, and ele\'ated trigly'cerides). In its most severe tion is more consistently elevated. In this situation, the
form (diabetic ketoacidosis), there is overproduction insulin deficiency leads to hypersecretion of glucagon
of glucose and marked acceleration of catabolic pro­ and gro\'.'h hormone, which furll~er accentuate glu·
cesses (lipolysis and proteolysis). cose overproduction. 6s .66 Because glucose UDG1..l(e (in
contrast to glucose production) in the posrabsorptive
scate largely occurs in non-insulin-sensilive tissues. it
Postabsorptive State is nOt unexpected that total body glucose uptake tends
After an overnight fast, substrate homeostasis in [he to be increased under fasting conditions as a result of
patient with diabetes resembles that obser....ed during the mass action of hyperglycemi::!. This underscores
starvation in nondiabetic individuals, Thus, diabetes the crUCial role that the liver pla\'s in determining the
might be thought of as a st:1te of "accelerated starva­ fasting glucose level in diabetes (Fig, 4~8),
tion," a situ::uion not unlike that seen in normal preg­ The increased hepatiC glucose production :lccompa·
lianC\', This should nOt be surprising considering that nying diabetes is characterized by an alteration in the
the hormonal milieu of diabetes and stan'alion share relative comribution of glycogenolysis and gluconeo·
mam' common features, namely, insulin deficiency in genesis [0 rotal hepatic glucose production. In type I
asso~iation with relative or absolute glucJgon and diabetes. the presence of glucagon and the absence of
grO\'.'h hormone excess. The principal difference from a restraining effect of insulin increase the hepatic up·
the metabolic standpoint is that blood glucose in the take ofglycogenic substrates and facilit::ue theircom'er·
diabetic patient is elevated rather th:ln reduced as it is sion ro glucose in the liver. As a result. gluconeogene­
in the case during starvation. Such discrepancies may sis accounts for a substantially larger proportion of
be due to the persistence of liver glycogen Stores in hepatiC glucose production in such pJtiems as com·
the diabetic that aHow glycogenolysis to be maintained pared with [hat in normal subjects; the rebti',e contri·
in conjuncrionwith increased rates of gluconeo, bution of gluconeogenesis is increased [\'.·ofo!d,6- Br
genesis, contrast, the magnitude and pattern of amino acid reo
In the patient with glucose intolerance, relative insu· le:lse from muscle in h:perglvcemic r;.pe [ diabetic

'f. 6
!­ 4
(f) 2
1= 0
Fig. 4-8. Influence of diabetes and diabetic ketOacidosis (DKA) on hepatic glucose produaion.

subjeCtS is similar to healthy controls,67 implying thar in diabetic keroacidosis or non1::etmic hyperosmolar
changes in intrahepatic process rather in muscle are Coma,
primarily responsible for augmented gluconeogenesis In the postabsorptive state, patientS wim insulin·de·
in diabetes. Ke\'ertheless, it is possible that increased pendent diabetes exhibit hyperaminQacidemia. Exami­
recycling of glycolytic intermediates (e.g., laerate) may nation of individual amino acid levels reveals that the
occur as well. Because FFAs are oxidized at an acceler­ increment in Circulating amino acids in these patienCE
ated race in the muscle of diabetic patienES (as a result is due almost entirely to a rise in .the branched char:­
of their high cirCulating levels), glucose-derived pyru­ amino acids (leucine, isoleucine, and valine)6:- (Fig. q.
vate cannot readily emer the Kreb's cycle. Similar 9). By contrast, plasma alanine levels may be reducec
changes have also been reported in type II diabetes. 68 particularly when insulin defiCiency is se\'ere arid he
The presence of incre35ed gluconeogenesis in type II patic removal of alanine is accelerated 70 The speci~
diabetes is consistent with the finding that greater tissue site accounting for tllis rise in plasma branche,
amountS of insulin are necessary to inhibit gluconeo­ chain amino acids has nm been fully'clarified. For ex
genesis as compared with glycogenolysis in Iiver. 69... ample, there is no demonstrable incre35e in the ne
In the extreme situation of total insulin lack, an e\'er­ release of branched chain amino acids from eithe
increasing fasting blood glucose level fails to elicit a 1eg67 or splanchnic tissues in these subjectS 70 Ne'.'e~
secretory response, The absence of insulin together theless, recent studies using radioacti,'e tracers der:
with the excessive rele35e ofa variety of coumerregula­ onstrate that the delivery of branched chain amin
to["\' hormones (glucagon, catecholamines, gro"Lh .acids into the Circulation is augmented in poorly co;
ho~mone, and cortisol) that ensues causes hepatic glu· trolled type I diabetic individuals. 71 Considering th
cose production to increase threefold or more above the branched chains are essential amino aCids. the:
normal, largely as a consequence of accelerated gluco­ studies imply that [mal body protein breakdown is i
neogenesis. Because of the hypoinsulinemia and creased in such patientS even though they show or;
the insulin resistance produced by the elevated levels moderate insulin deficiency. Such changes are nor us
of insulin-antagonistic hormones, compensatory in­ ally discerned in the clinical serung. perhaps becau
creases in glucose disposal (other than renal) are vir· compensarory increases in prmein synthesis may mi:
tually paralyzed. The clinical correlate of this sequence mize the loss of body prmein. This is nm the C:!.S::'
of eventS is profound hyperglycemia, as is observed insulin deficiency becomes more se\'ere, as evidenc


600 II p<O.025




'CHAIN 400
-' .


250 "*

RISE 150
(p.M) 100


Fig. 4-9. Fasting and postprandial incrementS (after mixed me-J! ingestion) of total branched ch:1in ami~o acids
(SCM) in eype I diabetic patientS and healthy controls. The dilbetic patientS were studied before (during poor
contro!) and after 7 and 14 ooys of intensive insulin therapy using a portable pump. (Based on the dara of
Tamborbne et af.1° 2)

by the stunted groMh of young diabetic patienrs in the The abnormaUties of branched chain amino acid
preinsulin era and the marked protein wa5cing of the metabolism described for insulin·dependent patients
diabetic individual in ketoacidosis. may not be evident inpatients with t:-~e II diabetes.
The elevated circulating levels of branched chain This may be because' the metabolism of branched
amino acids also lead to an acceleration of leucine, chain amino acids is more sensitive to the action of
isoleucine, and valine oxidation in the diabetic state.'l insulin than is peripheral glucose metaboHsm. l l
In diabetic animals, oxidation of branched chain amino In addition to hyperglycemia and hyperaminoacide­
acids is increased by 50 percent. 26 The increased in mia, the levels ofFFAs are frequently elevated in post­
situ catabolism of these amino acids provides muscle absorptive diabetic patientS.72 This phenomenon ap­
tissue with the nitrogen groups necessary for alanine pears to be a consequence of accelerated mobilization
symhesis,24 thereby increasing substrate availabiliry for of body fat stores and can primarily be attributed to
gluconeogenesis. 'In' this way, the accelerated break­ deficiency of insulin. In type II diabetic subjects. FfA
down of amino acids in muscle contributes indirectly elevations occur in the presence of normal or in­
to hyperglycemia in diabetes. creased le,'els of insulin,n suggesting resiStaIlCe to in­


sulin's inhibitory effect on lipol:.:sis. The increased (v1.DL) triglyceride, -,;\'hich m:1y be seen in milder
availability of FFAs leads to their oxidation b\' muscle forms of ty-pe II diabetes as well as in insulin-deficient
tissues :l~d in turn causes a concomit:mt di~inution patients, It would appear th:lt the mech:lr'lism responsi­
in the rate of glucose oxidation,30 Although FFAs on­ ble for h:-penriglyceridemiJ vJries in these groups, In
not be direcdv convened ro glucose, they promote h~-. the former situa<ion, elevated portal insulin le\-els rna\'
perglycemia in'diabetes by providing the liver ",vith promote \ l.DL triglyceride symhesis, and in the I::me~
energy-yielding fuel and the necessary cofacrors ro Circumstance, lipoprotein lipase, an insulin-sensitive
support gluconeogenesis and b~' interfering with glu· enzyme. is deficient. leading to decreased triglyceride
cose consumption by muscle,73 ' removal from the circul:uion"s--7
In r;.-pe II diabetes. the presence of endogenous in­
sulin secretion allows for sufficient le\'e!s of insulin in Glucose Ingestion
the ponal vein to suppress ketogenic processes in the
liver. In the p:uiem with r:-pe [ diabetes, however, mo­ The ingesrion of glucose triggers a varier::' of homeo­
bilized FFAs are very readily convened to kerone bod· st::ttic responses in nondiabetic subjeCts (see above)
ies, Lack of insulin in the porral circulation and the that are directed to-,;\'ard minimizing the rise in blood
presence of gluc:lgon suppresses fat synthesis in the glucose concenrrations_ Ther include suppression of
lh'er and thus intrahepatic Ie\'els of malonyl co-enzyme endogenous glucose production, as well as the uptake
A The latter together with increased availabilir:' of car­ of the exogenous glucose load. by splanchnic and pe­
nitine stimul::ues the activity of hepatic acylcarnitine ripheral tissues (nninlr muscle). Because these re­
transferase, This enzyme facilitates the transfer of long sponses are brgely dependent on insulin. diabetes,
chain bttv acids inro mitochondri::l, where they are bro­ e\'en in its mildest forms, is invariably accompanied
ken dO"l\:n \·iJ f3-oxidation and converted to ketone by postprandial h}-perglY'cemia,
bodies. 36 Bv ,'irtue of itS inhibitory effect on ketone Patients with impaired glucose tolerance a.ppear to
turnover, h;-poinsulinemia in the r;.-pe I diabetic indi­ have relari\-e1y intact insulin secretory responses [0
vidual enhances the magnitude of the ketosis for an~' glucose but demonStrate reduced sensith'it\- to insu­
given level of increased ketone production.''; As are· lin,13 Postprandial hy-pergl}-cemia. in thes~ patients
suit. blood kerones are generally ele":ared in r:-pe I mainly deri\-es from a reduction in glucose uptake by
diabetic patientS (Fig, 4·10). although usually not ro peripheral tissues, .-\similar partern is obser,ed in r:pe
the extent that acid-base balance is affected, II di::tberic subjecrs with fasting h:-perglycemia.. al·
Finally', patientS with diabetes commonly exhibit ele­ though the magnitude of the pancreatic defect is more
vated fasting concentrations of lipoproteins, Most strik­ pronounced.-a This is because (l) insulin secre[o~­
ing is the elevation in \-err low-demir:' lipoprotein response is markedly reduced in these patients and

MUSCL '/f'.
~0ii /',j


Fig_ 4-10, The development of hyperketonemi:l in diabetes is a consequence of three distinct merabolic ev<.!ms;
(1) :lCcelerated delivery of free fat~ acids (FFAs) from adipose tissue, (2) augmented ~·o:\idation of FFAs to ketones
as a result of elevated cminine levels and reduced concentrations of mJlonyl co-enzyme A. and (3) :l reduction
in ketone use in muscle. Each of these processes is reversed by the action of insulin.


:;.: ..

(2) the magnitude of the defea in insulin aaion on and the movement of glucose-derived pyru'(ace into
peripheral lissues is greater (these patienlS can no the Kreb's cycle,73 In type It di3betic subjectS, despite
longer overcome the defea with larger amounlS of the J','aibbiliry of some insulin. there is much less
insulin and t.'1us presumably have an impairment in suppression of lipolysis during glucose ingestion be·
postrecepcor processing of the insulin signal),}} How­ cause of insulin resistance?:! Simil3r changes are ob·
c:\'er, patienlS with type II diabetes have sufficienr levels served in l:}'Pe I diabetic p:1tiems, brgely beclUse of
of insulin in their portal 'circulation to allow the rising h~poinsulinemia (although resist:mce to insulin m,1\'
glucose le\'el t9 suppress glucose production and also contribute). The failure of diabetiC patients to sup.
promote glucose upt:1ke by the liver. This tends co re­ press bt oxid1cion during consumption of glucose
duce the postprandial glucose excursion somewhat, at contributes to the peripheral insutin resist:lr.ce of dia·
ie:l5t as compared with insulin-deficient I:}pe I diabetic betes and actS to block the oxidation of glucose that
patients, The type I di3betlc individuJ.l characteristr: enters muscle tissue.
calk shows the mos, marked and prolonged elevations
in blood glucose concemrJ.tion after ingestion of car­ Protein Ingestion
bohvdrate, These individuals, because ther fail to se­
cret~ insulin e\-en in the postabsorpth'e state, have con­ In addition to the increased use of amino J.cids for
Siderably lower portal insulin levels than patienlS with gluconeogenesis and release of branched chJin amino
type II diabetes. This loss of the porol-peripheral insu­ acids in the posw.bsorptive state, repletion of muscle
lin gradient is not readily re,'ersed by conventional nitrogen is impaired in the cype I diabetic patient After
subcutaneous insulin therapy, Consequently, the insu­ ingestion of a protein meal, the net splanchniC reie:lse
lin-deprived liver bils to reduce its glucose production of indi\'idu:l1 amino acids in insu[in-dependem di:t­
or take up glucose in response to a rising circulating hetic subjects is simiklr to that observed in he:tlthv
glucose level.6~ In addition, glucose uptake by periph­ comrols,":- Although the systemic delIvery is nm ai.
eral tissues is grossl~' impaired beclUse of the lack of (ered, postprandi:ll elevations in arterial amino acids
an insulin secretOry response and the de\'elopmem of are exaggeratedY This protein-induced hyperamino­
insulin resistance at the postreceptor level after acidemia is solely accoumed for bY the branched chain
chronic insulin depriv:1tion,ls The net result of chis ::Imino acids (Fig, 4-9), In comr(lS'( to the ongoing ner
mulrifaceted disturb:mce is a gross defect in metabolic upta.l.(e of branched chain amino acids bv muscle tis·
glucose disposal that is only partially compensated for sues seen in normal subjeclS, in di:lbeticlndi\'idu::t!s a
by increased renal gl:'cosuria (Table 4-3). net uptake is only transiemlr obsef\'ed."7 As a conse­
In normal subjects, the rise in pl:l5ma insulin caused quence, the total uptake of these amino acids bv mus­
by glucose ingestion also inhibitS Hpot:-sis, which in cle is decreased, resulting in their accumul:.!c'ion in
t:.:rn decreases the a\'aiIabilir:- of FFAs for oxidation in plasma, T}pe 1 diabetes thus may be \-iewed as a disor­
muscle. This facilitateS glucose upt;Li.::e and oxidation der of prOtein tolerance as well as glucose tolerance,
because the oxid:ltion of FFAs interferes with glycolysis This \'iew is in keeping with the kno""n c:1p:lcicy of
insulin to inhibit: the net rele:l5e of branched chain
amino acids from muscle tissue,~9 Because the capacity
Table 4-3. Homeosca(ic Response to Glucose
to rele:l~e insulin in response to systemic h:.perami­
Ingescion in Diabetes
noacidemia is commonlr inmct in patients witb type
It diabetes,5 it is unlikely that comparable defects in
protein disposJ.1 would occur in such pJ.tiems, This,
however, has not been full:' in\'estigated.
Suppression of gluco»e Protein feeding also produces abnormalities in glu­
cose regulation in the insulin·deficient di;tbetic sub­
Scimubtion of $pbnchni<.:
glucose uptake
jects, In norm:ll subjects, protein ingestion induces a
Stimublion of peripherJl ! ! ! ! modest rise in insulin secretion, which offse[s the stim­
-.?!ucose upoke ubtory effects of glucagon (and the amino acid IO:ld
Abbre\'[:1liOn5: :-'1., norr;ul. ,,'I.. !, norml! or belo"" normal. itself) on hepatic glucose production_~-;' As a result,






c 0- 0 t:i- a
-<5 '0
I 90
30 60 120 150 18Q"

Fig,4-11. Hyperglycemic effec( of protein feeding in patients with type I diabetes, (Based on unpublished d:Ha.)

blood glucose levels remain at basal values, B~- con­ quem!y, postprandial elevations of plasma triglycer­
trast, in diabetic subjects protein ingestion produces ides may be increased or prolonged in insulin­
a large (albeit transient) incr~e in 'hepatic glucose deficient diabetic patients because of deficient triglyc­
production due to the rise in glucagon in a selling in eride removal. This situation is most evident during
which insulin is deficiemf7 Consequemly, there is a ingestion of carbohydrate-comaining mb:ed meals,
substantial increase in blood glucose (Fig, 4-11). This when the discrepancies becween insulin levels in non·
exaggerated glucose response in this setting also partly diabetic and diabetic individuals are most pronounced.
reflects the failure to metabolize the glucose released
by the liver due to the failure of insulin secretion.
The rapid fall in blood glucose levels commonly ob­
Fat Ingestion
served in diabetic patients after vigorous exercise has
Consumption of fat-containing foods leJds to the for­ traditionally been the basis for recommending exer­
mation of chylomicrons. which provide a means of cise to diabetic patients. This acute glucose-lo,,-eriog
transferring triglycerides from the gut to adipose tis­ action is much more pronounced in insulin-treated
sue. The ultimate disposal of exogenous triglycerides diabetic subjeCts and occurs only if insulin. merapr is
(like endogenous triglycerides) is regulated by the ac­ sufficient to prevent marked hyperglycemia (Le., >300
tivity of insulin-sensitive lipoprotein lipase. Conse­ mgldl) or ketosis. so The importance of insulin avail­


, 1

abili"l in mediating the exercise-induced £:Ill in glu­ NOR.'AAL

cose levels is underscored by two observations in insu­
lin-treated patients. First, it is well recognized that the
magnirude of the decline in blood glucose is increased
if coincides with the time of the peak action
of the insulin preparation used. Second, exercise may
accelerate insulin absorption from its injection site,
and when this occurs, the fa!ling blood glucose is more
pronounced. 81 .' .
Recently-. swdies using regional catheters and radio­
labeled glucose ha\'e helped elucidate the mechanism
of the glucose-lowering effect of acute exercise in dia- ­
betes, Normally. physical exercise causes a marked in­
crease in glucose uptake by muscle. 38 Blood glucose
levels nevertheless remain suble, because hepatic glu­
cose produGion incre3.5es to match exactl;: the rate of
glucose consumption. This process is mediated by a
decline in circulating insulin and by an acth'ation of
the sympathetic nerY'ous system 3.5 well as counterreg­
uhHory hormone release:iO In the diabetic individual
receiving insulin exogenously, the circulating level of Fig.4-12. Mechanism of exercise-induced hypoglycemiJ in
the insulin may, at times, be inappropriately high and insu!in·tre:lted di3bet[c patients. The f3Hure of insulin levels
fail to decline during exercise, Consequently, this 10 fall during exercise and the potential for insulin to rise
"finely tuned" homeostatic mechanism is disrurbed_ because of accelerated absorption pre\'ents the no~mal com­
Exogenous hypednsulinemia has been shown to po­ pensator;' increase in hep3tic glucose production and mav
tentiate the stimublOry effect of exercise on glucose potemilce glucose upt:l..l:e bv muscle, The net ef­
uptake,8~ Even more import:3.nt, the compensatory in­ fect is a reduction in blood'glucose le"e[s.
crease in glucose release from the liver is blocked
under these conditions, resulting in a fall in glucose
concentration (Fig, 4-12).
In poorly regulated ketotic diabetic individuals, ex­
The usefulness of the acme glucose-lowering effect
ercise accentuates hyperglycemia and mav increase ke­
of intermirtent exercise in rrpe I diabetes is limited.
tonemia as "'ell, particularly if exercise is·prolonged. s3
because the effect is reJarivelr short-lived (generally
This may be explained by the low levels of insulin in
se\'eral hours) after exercise is terminated. Unless ex­
these patients and because that poor control causes an
erCise is regular and of appropriate intensity and dura­
tion, there is Hrtle long-term "carry-over" effect that exaggerated release of counterregul:l.tory hormones in
can be expected to aid in diabetic care. Furthermore, response to exercise. s,; These hormonal changes (cou­
the magnitude of the fall is not easily titrated, so hypo­ pled with the diabetic's hyperresponsiveness to coun­
glycemia is a frequent complication of vigorous exer­ terregulatory hormones66 ) potentiate the exercise-in­
cise. The rapid rise in counrerregulatory hormones duced increase in hepatiC glucose production as well
that ensues, coupled with the increased responsive­ as lipolysis and ketone production by the li\'er (Fig, 4­
ness of the diabetic individual to these hormones 66 and 13). Because the stimulation of glucose upta.l::e in mus·
a tendency to overeat when hypoglycemic symptoms cIe occurs independent of insulin (namely, local [3C­
occur, often leads to a rebound increase in blood glu­ cors, as described above), glucose consumption by ex­
cose to hyperglycemic levels, Thus, if the diabetic sub­ ercising muscle is relatively un:lifecred during poor
ject is unable to adjust her diet and insulin dose, acute control and thus does not contribute to a rise in blood
Intermirtent exerCise mav cause fluctuations in glucose le,"els.85
blood glucose levels rat'her than impro,'e glycemic The differences in the rate of gluconeogenesis be­
ControL tween nondiabetic and rype I diabetic are fur-


I NSUUN ) l' Glucose Production

DEFICIENCY l' Hepatic Ketogenesis

l' Counrerregulatory /
Hormones /

\: t Hepatic Sensitivity
to Counterregulatory

Fig. 4-13_ ~lecb:mism of exercise-induced h~:rerglrcemiJ md ketosis in poorl)' comrolled type I diabetic parien""

ther increased during exercise. During short-term ex­ hormonal changes combine to promme glucose pro­
erCise, the compensatory elevation in hepatic glucose duction and return the plasma glucose level w>'."::lrd
production in nondiabetics is mediated by an accelera· normal. 88
tion of glycogenolysis; gluconeogenesis remains un­ In type I diabetes. insulin levels are incapable of
changed. 38 By contrast, the diabetic individual demon­ responding to phrsiologic signals and the glucagon
strates a rapid increase in gluconeogenesis that is onl}' response to hypoglycemia is lost.89 Although some pa­
seen in normal subjects when exercise is extended for tients retain the ability to release glucagon du:-ing hy"
prolonged periods (2 to 4 hours).8o Thus, the effect poglycemia early in the COurse of their disease,90 the
of exercise in insulin-deficient diabetic patients is to ultimate failure of glucagon secretion to'this panicular
exaggerate [he excessive rate of gluconeogenesis that stimulus is a consistent finding in patients who have
characterizes di::lbetes. had rype I diabetes for several years. 89•91 .A..:s a result,
such patients :.Ire largely dependen'[ on epinephrine
for acute glucose counterregulation (Table 4-..f) Con­
Glucose Counterregulation sequently. they may show impaired glucose recovery
The main risk of insulin therapy in diabetes is hn)og!y­ when f3-adrenergic blocking agents are adminisfered v2
cemia. It is now appreciated that patients with diabetes or when there is coexisting autonomic neurop:lth~·.93
are more vulnerable to hypoglycemia, not only be­ Some long-standing diabetic patients show defective
cause they are unable to normally- synchronize insulin catecholamine secretion in response to hypogl:,.·cemia
delivery with meal ingestion and activity but also be­ without overt clinical evidence of diabetic neuropa.
cause the hormonal responses that normally protect thy.94 and more important, intensive insulin therapy
against hypoglrcemia are defective. In normal subjects, aimed at normalizing blood glucose levels mJ.:; itseti
insulin administration produces a rapid decline in diminish epinephrine responses to hypogl~·cemia.95
plasma glucose concentration. The glucose fall is due The latter may be especially important during preg­
to a suppression of hepatic glucose production and a nancy. when such treatment regimens have become
rise in peripheral glucose uptake.s6 The former pro­ standard clinical practice.
cess is more sensitive to insulin (see above). and this
is mainly responsible for hypoglycemia when insulin
Table 4-4_ Hormonal Defense Mechanisms Against

elevations are relatively smal1.87 Glucose recovery re­

Acute Hypoglycemia

quires the re\'ersal of these responses-most impor­

Hormone Secrerion I"ormal
tam. a stimulation of hepatic glucose production. Once
circulating glucose levels fall below 60 (0 70 mgtdl.
Epinephrine i
secretion ofglucagon and epinephrine is triggered and
endogenous insulin secretion is suppressed. These

Effect of Intensive Insulin Therapy With respect to other insulin-se:15iti\'e fuels, the ele­
on Body Fuel Metabolism vated basal concentrations of branched chain amino
acids are reduced to nomul in conjunction with im­
described above, poorly controlled diJbetes is ch:lr­
pro\'ed glucose regulation (Fig, 4·9), Tracer studies
acterized by a variety of metabolic and hormonal ab­
hJ\'e demonS([Ilted th::u the accelerated leucine deliv­
normJ.lities that ma:' contribute [0 the long·term com·
ery into plasma observed during cOl1\'emiomI treat·
plicJ.tions of the dise'ase,. The introduction of improved
mem is decreased,-:-l accounting for the observed
methods for quantifying'blood glucose control and de·
li\'ering insulin in' more physiologic mJoner with
ch::mges in circubting amino acids, Furthermore, in­
rensi,'e treatment of r:-pe I diabetes reverses the exces­
multiple injections or pomble infusion pumps has reo
si\'e postprandial\e~'el:) of branched chain amino acids
cently allowed im'estig3tors to examine the extent to
after ingestion of mixed meals (Fig. 4·9) :lnd lh~ -de­
which these abnormalities mJ!, be reversed by sys­
cre:.lSed clearance of intravenoush' Jdministered leu·
temic insulin delivery, cine seen in convemionally tre::ued p3rients.100JO~ Sim­
~IJny studies h:t\'e shown th:1t imensi\-e insulin tre:.!,­
ilarly, increased levels of FF..-\s and triglycerides are
mem restores blood glucose ie\'e\s to near-normJl val,
reduced tow:lrd normal. 100.101
ues,96-98 Because these regimens pro\'ide a relati\'ely
E\e\'ations in growth hormone and glucagon con­
constam bJ5ai \e\'el of insulin throughout the night in
centrations observ'ed during 24-hour monitoring in p::t­
amountS sufficient to restrain hepatiC glucose produc­
tiems with poorly controlled diJbetes are diminished
tion, they normalize posrabsorpcive glucose concemrJ­
afrer instirution of StriCt gl:'Cemic COntro1. 65,103 In addi,
tions. postprandial glucose eb'ations are also dramati,
tion, excessive incrementS in CJtecholamines and
cally reduced but not conSistently normalized, In
gro\\l:h hormone during mild exercise are re\'ersed 9()
nondiabetiC patientS, fluctuations in blood glucose are
(Fig. 4-14). It follows, therefore, thar some of the mera­
minimized by the concomitant suppression of endoge,
bolic benefitS of intensive insulin regimens mav be
nous glucose production from the !i\·er. 46 This fails to
derived from their coumerregubror.· hormone'lo~ver­
occur in convemionally treated ry-pe I diabetic subjecLS
ing effectS. This may be particubrh: true with regard
because, in this siruarion, the liver is less sensitive to
to grov.m hormone. When g[O,\th' horm'one w~ in­
small increments in circulating insulin99 and unable
fused as hourly intravenous pulses to a grooD of dia­
to respond to the inhibitory effects of hyperglycemia
betic subjecrs who were intensi\'el~' treat~d uSi~g a por­
per se on glucose production 67 After intensive insulin
table insulin pump, serum grmnh hormone was raised
tf}erapy, these abnormalities are corrected;e.99 and
to le\'els similar to those observed in poorly comrotled
therefore the endogenous contribution to circulating
diabetes. Under these conditions, glycemic control·
glucose levels can be effecti\'ely suppressed, In addi­
m:1rkedly decerioratcd while circubtor.· b[tY acids, ke­
tion. the premeal bolus doses of insulin allow for ade·
tOnes, and branched amino acids al~o i~creJ5ed.65
quare portal insulin levels to promore glucose uptake
Thus. gro\\th hormone elevations can themselves re­
by splanchnic tissues. As noted above, this process reo
quires some insulin but is mJinly driven by the magni­ produce the entire spectrum of poor diabetic control
tude of hy-perglycemia:H The anticipated result is the despite previously optimized insulin treatment, This
restoration of the liver's capaciry to handle adminis­ serves to emphasize the multifaaorial nJture of the
tered glucose, Howe\'er, the rise in circubting insulin met:1bolic disturbances of diabetes as well as the pri­
after subcutaneous hormone injection is debyed, and mary role of insulin deficiency ill initi:l.ting them.
peak levels are lower as compared with normal sub,
jects.lOO Furthermore, peripheral resistance to the ac, Role of Insulin~like Growth Factor 1

tion of insulin is only p:.mi:l.lly reversed by imensi\'e Deficiency

insulin therapy,lOl It is, therefore, unlikely that periph,

eral glucose uptake in response to glucose ingestion The classical studies of Froesch and colleagues demon­
is restored to normal. Because this is the m:1in site of strated thar somatomedin C. the put:uj;-e mediatOr of
exogenous glucose disposaV 5 it is not une:-:pected th::u gro\\th hormone's somatOtroDhic acrio,b, exerted
postprandial glucose levels commonly exceed normal metJbolic effectS in vitro that closeh' resembled those
values. of insulin, Hence, itS redesignilti;n as insuHn-fike
* p<O.OI
1 20



10 *
"T" _








Fig. 4-14. Resting levels and exercise·induced increments of grov,w hormone. epinephrine, and'norepinephrine
in diabetic and healthy control subjects. The diabetic patients were studied before (during poor control) and a.'i:e,
7 and 14 days of intensive insulin treatment using a portable pump. (Based on the daca of Tamborlane et al. 8 ")

I nsulin Deficiency


Fig.4-15. Hypothetical role of insulin-like gro....w factor-I (lGF-I) deficiency in the pathology of insulin-depen­
dent diabetes mellitus. GH, hormone.



ro"Lh faCtor 1 (rGF-1). Later studies ofZapf and Guier lions of insulin on forearm met::lbolism: persis:ence of
gtearlv established IGF-l's unique position as the only its aCtion on potassium and free fatty acids without its
~:J.(ur~l!;: occurring hypoglycemic hormone besi~es in­ effeCt on glucose.) Clin Invest 43:950, 1964
sulin. The buLLe of data indicates that convenuonally 10. Rizz:! R, Mand:uino L, Gerich): Dose·response charac-.
treated insulin·dependent diabetes mellitus (lOOM) teristics for effects of insulin on produaion and utiliza.
tion of glucose in man. Am) Physiol 240:E630, 1981
p:ui.entS have subnormal levels of IGF·1,104.105 ~pe·
11. Fukagawa NK, Min::t.I.;er KL, Rowe)E et al: Insulin-me,
clalh- during puberty, It has been suggested that raised
diated reducrion of whole body protein brea..~down:
gro~Lh hormone. levels in IDOM ar~, .in pan:, a com· dose· response effects on Ieucir.e metabolism in postab­
pensatoryacrempt to overcome a blOCK In IGF-l synth~. sorptive man.) Clin Invest 76:1306. 1985
sis caused by insulin deficiency and poor me(:lbotlc 12. DeFronzo R-\., Ferrannini E. Koivisto V: New concepts
controL 6 5 Although it is uncertain whether IGF-l d~fi. in the pathogenesis and rreatfnenc of non·
ciencv has direct adverse metabolic effectS in IDO.\t, pendent diabetes meHirus. Am J Med 74:52, 1983
to th~ extent that it contributes to gro';'.th hormone 13. Olefsk.-y TM, Ciaraldi TP, Kolterman OG: Mech:l.nism of
hypersecretion, it undoubtedly conrribuce: ~o diabe:ic insulin resistance in non·insulin·dependent ('0 pe 1I) di­
hyperglycemia (see above). Furthermore, It IS COnCel\" abetes. Am J /lIed 79:12, 1985
able that IGF·l deficiency plays a role in the hyperglu· 1.£. Kolcerman OG, Gray RS, Griffin) et al: Receptor and
POSt receptor defects contribute to the insu lin res istance
cagonemia of poorly controlled IDO:'L Thus, [( is in­
in non-insulin-dependent diJbetes meltitus. J Clin In­
triguing to speculate that insulin resistance associated \'esc 68:957, 1981
with poorly controlled IDOM may, in part, be due to
15. DeFronzo R.-\., Hendler R, Simonson D: Insulin resis­
IGF·1 defiCiency, which in turn promOtes gro';'.th hor· t:lOce is a prominent fearure or insulin-dependent dia­
mone and glucagon hypersecretion (Fig. 4·15). betes_ Diabetes 31:795, 1982
16_ Sca.rlett JA, Gray RS, Griffin) et al: Insulin tre:ltmem

reverses the insulin resistanCe of ~pe II diabetes melli.

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type I diabetes. Ann Intern Med 103:18-i. 1985 Endocrinol Met:lb 63:651, 1986

Ii Serono Symposia USA

Norwell, lVlassachusetts

Marc R. Blackman S. Mitchell Harman

Jesse Roth Jay R. Shapiro


Basic and Clinical Advances
With 54 Figures

I Springer-Verlag
New York Berlin Heidelberg London Paris
Tokyo Hong Kong Barcelona Budapest



\VILLlA~1 H. ADLER. Clinical Immunology Section. :\ation:;11 I;btilute '0:1

Aging. National Institutes of Health. Baltimore. Maryland. USA.

JOSEPH A. ALOI. Di\'ision of Endocrinology and i\.kraDolism. Dep:mment

of l\[edicine, University of Virginia Health Sciences Center.
Charlottesville. Virginia. CSA.

MICHELE F. BELl.... :-.:TO:-.:1. Department of Medicine. Johns Hopkins

University School of Medicine. Baltimore. i.\!arylo.nd. USA.

BARRY B. BERClJ. Section of Pediatric Endocrinology. Department of

Pediatrics and Department of Pharmacology and Therapeutics. Unh'ersity
of South Florida. College of i-.fedicine. Tampa. and All Children's
Hospital. SL Petersburg. Florida. USA.

MARC R. BLACK~I.-\:-;. Division of Endocrinology and ~letaboli:>m. Francis

Scott Key 1>.!edical Center. Department of Medicine. Johns ""Hopkins
University School of r.!edicine. Baltimore. Maryland. USA. '

WALTER P. BORG. Department of Internal Medicine. Section of Endo­

crinology, Yale University School of Medicine. New Haven. Connecticut.

SUSAN D. BOULWARE. Department of Pediatrics. Section of Pediatric

Endocrinology. Yale University School of rvledicine. New Haven. Con­
necticut. USA.

J.... N BUSBy-WHlTEHE.... D. Di\'i5ion of Geriatric Medicine. University of

'North Carolina School of Medicine. Chapel Hill. Korth Carolin'l. USA.

GArl BUTIERFIELD. Geriatrics Research. Education and Clinical Cemer.

VA Medical Center. Palo Alto. California. USA.

GrAN PAOLO CEO..\" University of Parma. G. Sruard Hospital. Parmn.




t I 18
Metabolic Effects of IGF-I:
Implications for the Therapy of
Diabetes Mellitus

When the radioimmunoassay for insulin was developed. i[ Oe:::m:.c

apparent that serum contained far more insulin-like biologic :!ccj\'[,:
than could be accounted for simply by its content of immunore:;':cl\e
insulin (1). This insulin-like activity was initially termed by Froes:h e:
al. IlOllStippressible insutin-like activity (NSILA) (2). Later. the ~e;m
somaromedill was imroduced to denote the uncharacterized bet.)r lh~H
displayed insulin-lik<; activity and mediated the action of G H i:1 tee
stimulation of somatic growth (3). Further studies of Froesch a:1::! C('11­
leagues demonstrated that somatomedin C exerted metabolic e(fe:ts :n
vitro that closely resembled those of insulin (4-9); hence. its redesig,:,,:}[t\.)n
as insulin-like groll'th factor I (IGF-I).
More recently. the availability of recomoinanr hllman IG F-l (rhIGF- Ii
synthesized by recombinant DNA techniques has pro\'ided the im?ecus
for a systematic assessment of rhIGF-rs metabolic actions in \'j''-o. Zao:
et al. (10) were the first to show that rhIGF-I had hypoglycemic e;:ec[s if:
normal rats that were not inhibited by anti-insulin antibodies. Guk~ et at.
(11) subsequently demonstrated a similar acute hypoglycemic e(rect in
healthy human volunteers. These studies clearly established IGF-I's unique
position as the only other naturally" occurring hypoglycemic hormone
besides insulin. It is noteworthy that rhIGF-Ihas been used successful!\'
to improv'e protein balance in hypercatabolic:states associated \\'ith iosufi;
resistance (12). suggesting that rhIGF-I may be useful in impro\'ir:g body
fuel and metabolism in other conditions associated with defecti\'e insulin
action. such as diabetes mellitus (13).


196 R.S. Sherwin et al.

Potential Role of IGF-I in the Pathology of

Type I Diabetes Mellitus
The bulk of data indicate that conventionally treated ins(I/in.depelldem
diabetes mellillis (IDD~1) patients have subnormal levels of lGF-l (l-l.,
15). especially during puberty. This may not be the case in pariems with
advanced retinopathy (16). although this finding has been disputed (17)
This issue will need to be resolved if rhIGF·I is to be used as a thei;:;peutic
agent in diabetes. Moreover. low-molecular weight IG F-/ binding protein
(IGFBPs) (Le., IGFBp·l) h,l\'e been reported to be increased in exper­
imental diabetes in rodents (1S) and in patients with IDDr.[ after insuliil
has been withdrawn (19). Since low-molecular weight BPs h:l\e acceS5 to
the extracellular fluid bathing lGF-I-responsive tissues. this might rurther
limit the availability of biologically active IGF-I in patients with 1DD:--1.
We have previously suggested that raised GH levels in IDD\l Qre. in
part. a compensatory attempt to overcome a biock in IGF-I synrhesis
caused by insulin deficiency and poor metabolic control (10). Although i~
is uncertain whether IGF-I deficiency has direct adverse metJboiic effects
in IDDM. to the e:\L\~nt that it contributes to GH hypersecre;lon. i,
undoubtedly contributes to diabetic hyperglycemia. E\'en til;: modest GH
elevations seen in poorly controlled diabetes have been shown to p~'Jdu;;e
marked hyperglycemia and metabolic decompens:ltion in IDD:-..r patient;
in the face of intensiye treatment with the insulin pump C21). Further·
more. IGF-I deficiency may play ;.\ role in the hyperglucagone:nia 0:
poorly controlkd IDD~f as well. Therefore. insulin resistance associ:Hd
with poorly controlled IDDM may. in pan. be due to fGF-I ddc:e:lc:.
and its stimulatory effect on GH and gluc;.\gon secretion (Fig, 1S.'),
IGf-1 Deiiciency


t Glucagon & ~ GH

Insulin Deficiency

Insulin Resistance

Hyperg Iycemia

FIGC"RE IS.1. Hypothetical role of IGF·I deficiency in the IMr.Ophy,:,~ingy '):

IDD:VL Imulin resiswnce associated with poorly cOrl;rol!ed IDD\I may r:: In po'::

due to IGF-I deficiency and its stimu:<lrory cikct on GH and gil.l'::lgL)1l :::;:>:n:lio::,

18. Metabolic Eff:::cts of IGF-I 197

Despite IGF-l's potential for improving metabolic control in IDDM.

little is known about its effects in IDDM. In the BB rat, a model of
spontaneous rDD\L we have reported that acute administration of
rhIGF-I exerted a potent stimulatory effect on glucose uptake that was
indistinguishable from that seen in nondiabetic controls (22). This Occurred
in the face of insulin resistance; insulin-stimulated glucose uptake in the
diabetic animals was reduced -30%. These findings imply that rGF-I
might be effective in human diabetes. For example. administration of low
...., . doses of rhIGF-I sufficient to restore basal IGF-I k\els to normal in
IDDM might lower glucagon and GH levels. as well as improve cellulJr
metabolism in muscle. As a result. insulin might become more effective.
thereby ameliorating insulin resistance in IDD;"!. It is noteworthy that
administration of insulin may in a similar fashion the metabolic
effect of IGF-I. Insulin rapidly lowers IGFBP-I and. therefore. may
increase tissue access to free IGF-I (23). Such complementary effects
of IGF-I and insulin might facilitate restoration of normal substrate
me tabolism in ID D:-'L

Ivletabolic Effects of IGF-I Under Different

Glycemic Conditions
The potenEial role of IGF-I on the pathophysiology of lDDM !ed us ro
initiate studies designed to define more precisely [he mechanisms for
IGF-l"s effects. In our preliminary studies we administered rhIGf-l to
awake. chronically catheterized. 2-l-h-fasted rats using the eugiycemic
damp technique (2-+). Effects of rhIGF-I were compared to rhose of
insulin using doses of each hormone that produced compilrable stimulation·
of glucose uptake (i.e .. that were biologically equi\·alent).; Although
rhIGF-I and insulin increased glucose uptake to a similar extent (by
design), in the rat their effects on hepatic glucose production (as measured
by 3-3 H-glucose) were strikingly different. In contrast to insulin. rhIGF·I
failed to suppress hepatic glucose production Or lipolysis. In further
studies. to avoid the potential limitation of species differences in the
response to the hormone. we examined the metabolic effects of rhIGF-I
in normal humans under various glycemic condirions.

To examine rhIGF-I's actions withom the confounding changes pro­
duced by hypoglycemic counterregulation. \ve studied 9 healthy human
volunteers (aged 19-34) during a primed-continuous IV infusion of
rhIGF-I for 3h (prime. 20llg/kg; continuous rate. O.-l~tg/kg/min) (25).
Plasma glucose was damped at euglycemic levels (90 mg/dL) using a
variable infusion of exogenous glucose. During the IGF-I infusion total
198 R.S. Sher."'in et al.

IGF-I increased 3-fold. and free IGF-I levels rose from undetectable to
-70 ng/mL. There was marked suppression of islet hormones; plasma
insulin fell by 32%, C-peptide by 58%, and glucagon by 40% (P < 0.01).
Glucose uptake (measured by 3- 3H-glucose) increased 2- to 3-fold over
baseline (P < 0.001), glucose oxidation (measured by indirect calorimetry)
increased by 50% despite the decline in insulin secretion, and amino acid
concentrations feU by 40%-60% (P < 0.05). Unexpectedly. IGF-I
also suppressed hepatic glucose production (P < 0.01) (Fig, 18.2) and
circutating FFA levels by 70% (P < 0.001). Indirect calorimetry showed a
shift in fuel oxidation from lipids to carbohydrate,
The overall pattern of response to IGF-I seen in these studies is
remarkably similar to that seen with insulin. The similarity of the pattern
of mewbolic response to IGF-I and insulin suggests that either hormone
may act via binding to the insulin receptor (or hybrid insulinflGF-I
receptors) and/or that both hormones activate a similar cascade of post­
receptor events after e:lCh binds to its own specinc receptor.



(mg·kg- 1 min- 1 )

o Basal
o IGr-l Infusion

(mg·kg- 1 min-' )


FIGURE 18.2. Effect of rhIGF·I on glucose uptake and h:!patic glucose production,
During this study plasma glucose was damped at eugfycemic (e\'els (90 mgiJLl
using a \"ariable infusion of exogenous glucose. The asterisks denote a significant
change as compared to basal values: • P < 0.001 and" P < 0.01.

18. Metabolic Eff~cts of IGF-r 199

j To determine whether IGF-rs inhibitory effect on insulin secretion (see
above) persists during glucose-stimulated insulin secretion. healthy.
nonobese subjects received either rhIGF-I (OA ~lg:kgimin) or saline for
3 h (26). After an initial euglycemic phase. plasma glucose was raised to
two different levels of hyperglycemia (clamp technique) to evaluate insulin
., responses to a standard stimulus. In one group. glucose was raised
: +50 mg!dL (n = 6). and in the other. + 125 mg!dL (II 8) above baseline.
At the +- 50-mgdL step. C-peptide levels we re suppressed by -W%
during rhIGF·I infusion. while insulin levels were suppressed by 30o~
(P < 0.05). Despite the reduction in insulin secretion. the rate of glucose
metabolism was ]·fold higher in the IGF·r·jnfused group (P < 0.00l) . .-\5
depicted in Figure IS.3. at the higher glucose stimulus (+125mgidl).
rates of glucose metabolism were 30% -35% higher during IGF·I infusion
(P < 0.01): suppression of insulin secretion by IGF·I was also noted. bu~
it was less remarkable than with the +- 50·mg/dl step.

j ao 0



20 "'.­

(mg'kg- 1 min- 1 )

F,GURE l8.3. Effect of rhlGF·[ on glucose-stimulated insulin release and the rate
of glucose me[aboli~m (M) during + 125·mg/dL hypergl;cemic clamp. Rates of
glucose mewbolism were 30% -35% higher during IGF·L and suppression of
insulin secretion by [GF·I was noted.
~oo R.S. Sherwin et a1.

These data demonstrate that rhlGF-rs inhibitory effect on glucose­

stimulated insulin secretion is subst,:mtially overcome by increasing the
hyperglycemic stimulus. Despite the decrease in insulin secretion. glucose
disposal is actuatly accelerated by IGF-I's ability to stimulate glucose
metabolism. These findings suggest that the insulin-lowering effect of
IGF-I is not likely to limit its usefulness as a glucose-lowering agent in
patients with preexisting capabilities for insulin secretion.

Hypo glycem fa
To determine if the effeCts of rhIGF-I on hormonal counterregulation
were distinguishable: from those of insulin. we compared infusions of
rhIGF-I (O.7mg!kglmin) and insulin (O.SmUfkgfmin) for 120min in 10
healthy volunteers (plasma glucose allowed to fall freely) (27). Despite
similar plasma glucose nadirs (2.6 = 0.1 vs. 2.7 ± 1mr-.r). glucagon and
GH responses to hypoglycemia were clearly lower with rhIGF-I than with
insulin. while epinephrine rele:lse was unaffected (Fig. 18.4). In conrrast.
the norepinephrine response was enhanced by rhIGF-I (data not shown).
When the IGF-I and insulin iniusions were discontinued. the rebound
increase in plasma glUCOSe was delayed with IGF-I as compared to insulin.
This delay in glucose reco\ery was mainly due to persistent suppression of
hepatic glucose production. as leas! in part because of the failure of
glucagon levels to rise significantly.
The absent glucagon response to hypoglycemia observed during the
free-fall study was also confirmed when we used the glucose damp tech­
nique to produce a sustained. standardized. hypoglycemic stimulus
(50mgidL). Surprisingly. the magnitude of the inhibitory- effect on
glucagon release was substantially greater than that see.f), for GH. Once
again. IGF-I generated a more pronounced rise in circulating norepineph­
rine as compared with toe insulin study. IG F-I also caused a greater
increase in heart rate than insulin. as well as greater awareness of hypo­
glycemia. findings that suggest enhanced stimulation of sympatheric
These data suggest that the clinical use of rhIGF-I may be limited by
its ability to cause hypoglycemia and diminish glucose recovery due to
inhibition of glucagon secretion. Paradoxically. a\I;areness of hypoglycemia
is enhanced with rhIGF-I. presumably due to more pronounced stirr.u­
lation of sympathetic nervous system actidty. It is interesting to speculate
that this pattern of response to rhlGF-I could be advantageous in lOOM
patients with hypoglycemia unawareness syndrome and defecti\e sym­
pathetic responses to hypoglycemia. The inhibitory effect of IG F-I on
glucagon secretion is minimized in such patients since their capacity £0
release glucagon during hypoglycemia is already lost.

18. Metabolic Effects of IGF·I 201




o Insulin
~ rhlGF-l





FIGt:RE 18.-1-. Glucagon. G H. and epinephrine responses to hypoglycemia produced

bv rhIGF·L The asterisk denotes a significant cham,e in hormoneleve! as com­
p~red to basal values (P < 0.05). whereas the d-;gger indicates a significant
difference between the hormonal response to hypoglycemia achieved by insulin or
rhIGF-I infusion (P < 0.05).

Concluding Remarks
Taken together. our studies in normal human subjects support the vie'.v
that IGF-I might be effective in human diabetes. especially when used to
restore to normal the typically low basal IGF-Ilevels seen in IDD~L This
might be particularly true during the pubertal period. when the magnitude
202 R.S. Sherwin et al.

of IGF-I deficiency is most pronounced. In this setting IGF-I replacement

might lower glucagon and GH levels and act directly to improve cellular
fuel metabolism. thereby overcoming the profound insulin resistance of
100M and making it easier to optimize metabolic control.

A ckno wledgmef!(s. This work was supported by grants from the U_So
Public Health Service (DK-20~95 and DK-~5735) and a fellowship grant
from The JU\'enile Diabetes Foundations International (\V.P.B.).

L Sara \'. Hall K. fnsulin-like:: growth factors and the::ir binding prote::ins P~1\siol
Re\' 1990:70:591-61-1.
2. Froe::sch ER. Burgi H. Ramseirer EB. Bally P. Labh:lrl. Antibody supre~sible::
and non-suppressible:: insulin-like activitie::s in hum:lr1 serum and the::ir physio­
logical signincance. J Clin Invest 1963A2:1S16-3-L
3. Daughday WK. Hall K. Raben Salmon \VD Jr. \'an den Br~nde:: JL. Van
Wyk JJ. Somatome::din: propose::d de::sign:Hion for sufphation factor. :'\ature
4. r.kuli c. Froe::sch ER. Insulin and non suppressible:: insulin· like ac,i\'ity
(NSILAs) stimulated the same glucose transport syste::m \'ia two sepJ.rJ.te::
re::ceptors in rat he::art. Biochem Biophys Re::s Commun 1977:75:689-95.
:; Zapf J. Schoenle:: E. Froe::sch ER. fnsulin-like:: growth factors f and fr. some::
biological actions and receptor binding characleris;i:s of two puritie::d con­
stituents of non suppre::ssible:: insulin-like activity ot" human serum. cur J
Biochem 1975:87:265-96.
6. Poggi C. Le:: :--'1archand-Brustel Y. Zapf J. Froesch ER. Freyche; P. Effects of
binding of insulin-like growth bctor I in the isolated soleus rr:~sde or iean
and obese mice: comparison with insuiin. Endocrinology 19.79:10-1:723-30.
7. Zapf J. Froesch ER. Humbel RE. The insulin-like grov.:th factors (Gr) of
human serum: chemical and biological ch:lracteriz:!tion and aspects of their
possible physiological role. Curr Top Cell Regul 19S1:19:257 -309.
8. Rechler \1:'.1. ~issley SP. King GL. e::t ::ll. :vrultiplic:lrion stimulating :lci\'ity
(SA) from BRL3A rat liver line: relaxation to human somatomedins :lnu
insulin. J Supramol Struct Cell Biochem 1981:15:253-$6.
9. Guenther HL. Guenther HE. Froesch ER. Fleisch H. Efkcts of insulin-like
growth factor on collagen and glycosaminog~ycan synthesis by rabbit :1rticuiar
chondrocytes in culture. Experientia 1982:38:979-80.
10. Zapf J. H~uri C. Waldvogel M. Froesch ER. Acute metabolic effe::cts and
half-lives of intr::lvenously administered insulin-like growth factors I anu rr in
normal and hypophosectomized rats. J Clin Invest 1986:77: 1763-75.
II. Guller HP. Zapf J. Froesch ER. Short-term me::t::lboiic effects of recombinant
human insulin-like growth factor I in he::llthy adults. ?-.: Engl J !\led IYS7:3 17:
12. Ross RJM. :V1iell JP. Buchanan CR. Avoiding au[Oc::lnnibalism. Sr .'.fed J
1991 :303: 1147-8.


18. Metabolic Effects of IGF-f 203

13. Froesch ER. Guier HP. Schmid C. Binz K. Zapf 1. Thtrapcucic potential of
insulin like gro\'th factor 1. Trends Endocrinol Mdab 1990:l:25~-60.
14. Amiel SA. She:r\\ln RS. Hintz RL. Ge:rtner JM. Press CM. Tambor!anc \\'V.
Effect of diabetes and its control on insulin-like i!rowth factors in the: \'Qun"
subjects with type I diabetes. Diabetes 198-1:33:1175-9. -"
15. Tan K. Ba:<ler RE. Serum insulin-like growth factor I Ie:vels in adult diabetic
patients: the df~ct of age. J Clin Endocrinol Met:lb 1986:63:651- 5.
16. Merimee Tl. Zapf J. Froesch ER. Insulin-like growth factors: scudies in
diabetics with :!;,\c without rerinopathy. ~ Engl J :'Ied 1983:30-k527-JO.
17. Arner P. Sjo~e~g ~1. GjOlterberg M. Skottner A. Circui;Hing i<lsulin-like
growlh facror I in type I diabetic patients \\ith retinopathy. Oi:!oe:tologia
IS. Unterman TG. Patd K. 1\:umJr Mahathre \'. RJjamuhan G. Oehler OT.
Becker RE. R;:;:ularion L)t 10\\' molecubr weight insltlin-Itke growth factOr
binding prottin:, in ex;:aimentJI diabelts me:l!iw>. Endc)crinotogy ['-)'11):126;
19. Holly J:'IP. BiG,,;i::'::ombe RA. Ounger DB. tt ,,/. Circldian \ariation of GH­
independent IGF-Qli1dii1g protein in diaberes mellitus lnd it::; rd:Hionship to
in5ulin: a new [G[c: (or insu!in'? Clin EndocrinollOxfl [()SS:~9;667 75.
20. Tamborfane: \\Y. Hintz RL Bergman :'1.1. Gene! :'1. Felig P. Sh;:f\\in RS.
Insulin infusion pump trelement or diabetes: inftuem:e or im;:ro\'ed metabuli.:
control on plasmJ. sOnlJ.tomedin Itvels. N Engl J \led 19S1:305:303-7.
21. Press M. Tamboriane \\·Y. Sherwin RS. Importance t)t r:tised grou'lh hormone
lends in mediat:n~ ,he metabolic derangemcms or diabetes. ~ J :'\[ed
")") Jacob RJ. Sherwi:1 RS. Bowen Let aI. :\letabolic eff~-::s of IGF· [ :wd insulin
in spontaneously ,jiaveric BB \\ [illS. Am J Physioi 199t:2bO:E21)2-",
23. Smder OK. Cle:::-,;non OR. Insulin-dependent re!lubtion or in:iuHn-like ,zrowth
f;ctor-binding FO[~in- L 1 Clin Endocrinol Met;b 199U::-l:i6':;2-I)~ ~.-
2-1. Jacob R. Barre:: E. P!ewe G. Fagin 1\:0. Sherwin RS. ACute effects ot
insulin-like gro";n factor I on glucost and amino acid mer::bolism in the
awake fasted rJ[: ,;omp:Hison with insulin. 1 Clin Invest! 9S9:S3:'lil -;- - 2:;.
25. Boulware SO. TJmboriane \\Y . .'.fanhews LS. She:rwiD RS. Di\'e~se eifec:s
of ins.ulin-like :!~~'wrh factor I on glucose_ lipid ilnd ;tmi:lO :lcid m;:[:lQoiism.
Am] PhvsioI19 0 2::::5:EI30-3.
:!6. Rennert ·N). C<!,nio S. Sherwin RS. Insulin-like growth r'acwr t inhibits
glucose stimulated insulin secretion but does not imp~ir glueD,e me:abolism
in normal humans J Clin Endocrinol MetJb 199}:76:S0~-6.
27. Kerr D. Tamborbne \\'\'. Rife F. Sherwin RS. Effect of insuiin-like \l.rowth
faccor I on the response to and recognition of hypoglycemia in hu;ans: :!
comparison wilh insulin. J Clin Invest 1993:91: 141- 7.

Tlte: Diabetes Atlntwll9

S.H i'.\:ll'<;h:lti. P.O. Home. RA. Rizz:l (editors)
© 1995 ElscvierScicnce 8.V. All rights resC[vd 57

3 Metabolic effects of insulin-like

growth factor 1



Insulin-like growth factor I (lGF-l) is a 7649 Da polypeptide hormone chaf<lc­

terized by high structural homology \vith insulin (I). Its existence was first sug­
gested by the discovery that serum cont<lined much more insulin-lik-e,bioiogic<ll
activity than conld be accounted for by the level of immunoreacti\'e insulin
alone (1). It is now appreciated that this "additional" or "non-suppressible" in­
sulin-like activity (2), is largely, if not exclusively, mediated by two members of
the family of growth-promoting hormones, termed somatomedins. Because these
polypeptide hormones exerted both insulin-like and growth-promoting effects.
they are currently designated as IGF- [ (formerly called somatomedin A) and
IGF-2 (formerly called multiplication-stimulating activity or MSA) to emph<l­
size their dual biological actions.
In this review we focus on IGF-I, the growth hormone responsive IGF. The
recent availability of recombinant human IGF-I has paved the way for numerous
I studies that have provided new insights inco both the scope of its metabolic ef­
j fects and how they might potentially be used in the treatment of diabetes melli­

Pharmacokinetics ofIGF-l

In a contrast to insulin. which is synthesized and released into the bloodstream

exclusively from the pancreatic B-cells, IGF-I has many sites of production.
Although the liver is the major source of the circulating IGF- I (3), this peptide is
also locally produced in many other tissues of the body (4-7). Consequently
IGF-\ exerts its physiological actions in two ways: as a classical endocrine hor­
mone and also as an auto-paracrine factor.
IGF-I is further distinguished from insulin by its association with specific
binding proteins (8). There are six majorIGF binding proteins (IGFBPs) recog­
nized, among which IGFBP-3 is dominant within the circulation (9). This pro­

tein plays a pivotal rok by virtue of its ability to maintain a resen'oir of IGF-I in
the vascular space. IGFBP-3 forms a complex bet\veen its acid labile subunit
(ALS) and the IGF-l molecule producing alSO kDa heterotrimer. which is ~
biologically inactive "storage" form of IGF-l (9). Interestingly, while IGFBP-3
is produced mostly by endothelial cells, ALS is synthesized in the liver under the
control of growth hormone (GH), much as IGF-l. For [his reason, a decreJ.sed
level of GH diminishes hepatic production of both IGF-I and ALS. This leads to
a reduction in IGF-I bound to [he 150 kDa complexes and the 50 kDa complexes
(consisting of IGF-! combined with BP-2 and BP-3) which facilitate passage of
IGF-l to the extracellular compartment. The net effect of these changes is a
transient increase in IGF-I availability, followed ultimately by depletion of the
total circulating IGF-I. The biological actions of IGF-l are further complicated
by the fact that IGFBPs are synthesized and released locally by the various tis­
sues. In this setting. IGFBPs may play an important role not only in storing bu.;
also in inhibiting, enhancing ;lnd/or targeting the IGF-l to the specific receptors
Clearly. IGF- I is a hormone characterized by unusually complex pharma­
cokinetics. Because it has the capacity to modify the IGF-lIIGFBP system
through direct and indirect (via GH) effects on the levels of IGFBP, and to exe"
dual endocrine and paracrine effects on responsive tissues, its actions may
change over time. thereby complicating attempts at defining its biological role
and possible pharmacological use.

i Physiological actions of IGF·l in animals

Early in vitro studies demonstrated that IGF-I can mimic the capacity of insulin
to promote glucose uptake and metabolism in isolated muscle and adipose tissue
J (13-16), stimulate glycogen synthesis in rat he~lIT and diaphragm muscle (15.
17), increase amino acid transport in fibroblasts (17), and increase net protein
synthesis in cartilage (18). Initial in vivo studies using partially purified IGF-I
I showed a glucose lmvering action, in hypophysectomized and adrenalectomized
rats (19, 20). More recently, the availability of human IGF-I synthesized by re­
combinant DNA techniques has made a more extensive analysis of IGF-l's ef­
I fects on fuel metabolism possible. Zapf and colleagues (2 J) were the first to
show that recombinant IGF-I had hypoglycaemic effects in normal rats that
were not inhibited by anti-insulin antibodies. In addition, medium-term infusions
of IGF-I were shown to stimulate growth in hypophysectomized rats (22), im­
plying that IGF-l may exert protein anabolic effects as well.
To examine the mechanisms underlying its glucose lowering action, we gave
a primed-continuous infusion of human recombinant IGF-l to consciolls, long­
term catheterized 24-h fasted normal rats (23). IGF-l produced a sustained fall
of plasma glucose (from -6.2 to 3.4 mmoll- I ) which was due to a rise in glucose


uptake; hcp:1tic glucose production changed littk. if at all. To further aSSeSs IGF­
I effects in the absence of hypoglycJ:emic counterregubtion. we comp:lrt~d IGF­
I with insulin using the euglycJemic clamp technique. Doses of IGF-I :1Od insu­
lin were specifically chosen to produce a comparable twofold stimulation of glu­
cOSe uptake. Although IGF-I and in5ulin h3d similar effects on glUCOSe uptake,
IGF-I was less effective suppressing hep:Hic glucose production. IGF-I also had
no detectable effect on non-esterified fany acids (NEFA) levels, a feature thot
further distinguished IGF-l from insulin.
The disparate effects of IGF-I on glucose uptake vis-a-vis other insulin­
sensitive processes, implies that IGF-l may be acting through its own specific
recepwr. However, the in vivo effects of IGF-l on circulating substrates and
hormones may themselves have contributed to the unique pattern of response to
IGF-1. For example, IGF-I produced a marked decline in circulating insu! in
concentrations. even when euglycaemia was maintained, and this may have
contributed to IGF-l's failure to reduce hepatic glucose production or l\EFA
levels in normal rats. These data rJised the possibility that IGF- I met:lbolic
effects might be quite different in people with insulin-treated diabetes.
In contrast to the disparate effects of IGF-I and insulin on hepatic' glucose
production and NEFA concentration, IGF- I '5 effects on plasma. amino acids.
leucine kinetics. and leucine incorporation imo protein closely resembled those
of insulin (23). IGF-I, like insulin. cJused generalized hypoaminoacidaemia and
suppressed leucine flux. whereJs bbelled leucine incorporation into he:1rt,
skeletal muscle, and liver proteins declined. These datJ suggest that the amino
;)cid lowering effect of both IGF-l Jnd insulin results from a diminution in pro­
teiQ breakdown rather than an incre:lse in protein synthesis. It should be empha­
sized that the failure to observe :In effect of IGF-l on protein synthesis may be
due to the concomitant decrease in tissue availability of amino acids, since we
have observed stimulation of protein synthesis by IGF- I in the normal rat when
hypoaminoacidaemia is prevemed by exogenous amino acid infusion
(unpublished data).
Despite IGF-I's potential for improving metabolic control in insulin-treated
patients, little is known about its effects in diJbetes. ScheilwiUer and colleagues
(24) reported that medium-term low dose infusions of IGF-l restored growth,
but had negligible glucose-lowering effects in young streptozocin diabetic rats,
To further examine the influence of diabetes on the metabolic response to IGF-I,
we recently administered "bioequivalem" doses of IGF-I or insulin to awake
chronically catheterized diabetic and non-diabetic BB rats under "clamped"
euglycaemic conditions (25). Again doses of IGF-I and insulin were titrated to
produce equivalent increases in glucose uptake in non-diabetic BB rats. As
shown in Fig.!, insulin resistance was observed in these BB rats with spontane­
ous autoimmune diabe~es, with insulin-stimulated glucose uptake reduced by
-30% (P < 0.05 versus non-diabetic rats). In contrast, IGF-I's capacity to pro­



• IGF·l




1 FIG. 1. IGF-/ and insulin slinllllared glucose uptake in non·diaberic and ciialx[ic BB
rars. *p < 0.05 versus lIon·diabetic rats.

mote glucose uptake was identical in diabetic and non-diabeYic BB rats. Fur­
thermore, in diabetic BB (but not normal) rats IGF-I suppressed hepatic glucose
production as well as ketones by -75% (Fig. 2). These observations indicate thal
the in vivo metabolic response to IGF-l is not diminished, and may even be en­
hanced, in insulin-dependent rats that are insulin resistant. The findings of a
more diverse action of IGF-l in IDDM rats is consistent with the hypothesis that
IGF-l' s metabolic actions in normal rats had .been offset by suppression of insu­
J lin secretion.
The demonstration of potent acute effects on glucose and ketone metabolism
I in IDDM rats prompted us to undertake further studies using medium-term sub­
cutaneous infusion of IGF-I (-I mg/day). In these experiments diabetic BB rats
(n = 8) were given low doses of insulin (-1.5 Ufday) adjusted to maintain

I chronic hyperglycaemia for 1 week, and then subcutaneous IGF-l infusion was
superimposed for 1 week. Mean plasma glucose levels fell from -235 to
-12.0 mmolfl and nitrogen balance improved by -15% following IGF-l (26).

I These findings imply that IGF-I might be effective as adjunctive therapy in

Type 1 (insulin-dependent) diabetes.

Physiological actions ofIGF·l in humans


In a preliminary study Gu ler and colleagues (27) demonstratfd that the acute


IS ;6

12 12
• IGF·1

= Insulin


e..... ~ru:uQn InfusIon a.aal InfuSIOn 8a5oa1 TntuSlon

(mM) ,i..

SaMI :rttus,ora
! i

B.l:w:1 InfuSion

FIG, 2. Comparison of rhe effects of lGF-l and insulin infw;ior! on the hepatic glucose
production and {3.hydroxybutyrate levels in non-diaberic and diabeti,c BB rats.

administration of IGF- [ rapidly lowers blood glucose levels in healthy human
i volunteers. To more precisely compare the effects of recombinant, human IGF- I
(rhIGF-l) on hormonal counter-regulation with those of insulin, we infused
rhIGF-I (0.7 pglkg per min) and insulin (0.8 mU/kg per min) for 120 min inro
10 healthy volunteers (plasma glucose allo';ved to fall freely) (28). Both hor­
mones produced a prompt fall in plasma glucose due to a simultaneous decrease
in hepatic glucose production and increase in glucose uptake. Despite similar
plasma glucose nadirs (2.6 ± 0.1 versus 2.7 ± I mmolll), glucagon and gro\'·(th
hormone (GH) responses to hypoglycaemia were clearly diminished with rhIGF­
I compared with insulin, while epinephrine release waS unaffected. In a contrast.
the norepinephrine response was enhanced by rhIGF-l (Fig. 3), When the IGF-l
and insulin infusions were discontinued, the rebound increase in plasma glucose
was delayed with IGF-l'as compared to insulin. This delay in glucose reco\:ery
was mainly due to persistent suppression of hepatic glucose production. at least
in part because of the failure of glucagon levels to rise significantly .


(;.IgIL) (ngIL) 101.11

200 .' Insulin


FIG. 3. Comparison 0/ the changes ill glucagon. growth hormone wid norepinephrine
levels caused by hypoglycaemia produced by rhIG F-l. or insulin. The as[erisk I ~) indio
cates a sigl!ificant difference between the hormonal response 10 hypog/ycaemia acilit'l'ed
by insulin or rhIGF-l ill/usion (P < D.DJ).

The absent glucagon response to hypoglycaemia was also confirmed when \Ve
used the glucose clamp technique to produce a sustained standardized hypogly­
caemic stimulus (2.8 mmolll). Surprisingly, the magnitude of tbe inhibitory ef­
fect on glucagon release under these conditions was substantially than
that seen for GH. Once again IGF-l generated a more pronounced rise in circu­
lating norepinephrine as compared with the insulin study. IGF-I also caused a
greater increase in heart rate than insulin as well as greater awareness of hypo­
glycaemia. findings suggesting enhanced stimulation of sympathetic activity.
These data suggest that the clinical use of rhIGF-1 may be limited by its ability
to cause hypoglycaemia and diminish glucose recovery due to inhibition of glu­
cagon secretion. Paradoxically, awareness of hypoglycaemia is enh;:mced with
rhIGF-I, presumably due to more pronounced stimulation of sympathetic nen'­
ous system activity. It is interesting to speculate that this pattern of response to
rhIGF-I could be advantageous in insulin-dependent diabetic (lOOM) p:ltients
with hypoglycaemia unawareness and/or defective sympathetic responses to hy­

poglycaemiu. In such patients the inhibitory effect of IGF-l on glucagon secre­
tion is minimized since their capacity to release glucagon during hypoglycaemia
is already lost.

• Euglycaemia

To examine rhIGF-I' s actions without the confounding changes produced by

hypoglycaemic counter-regulation, we studied nine healthy human volunteers
(aged 19-34 years) during a primed-continuous intravenous infusion of rhIGF-l
for 3 h (prime 20 .uglkg, continuous rate 0.4 .uglkg per min) (29). Plasma glucose
was clamped at euglycaemk levels (5 mmoll1) using a variable infusion of ex­
ogenous glucose. During the IGF-infusion total IGF-l increasfid threefold, and



fre.: IGF-l le\'els rose from undetectable to -3,9 mmolll. There was marked
suppression of islet hormones: plasma insulin fell by 329'c, C-peptid.: by 580(.
and glucagon by 409c (P < 0.01). Glucose uptake (measured by [3- 3 HJglucos.:)
increased two- [Q threefold over baseline (P < 0.001), glucose oxidation (meas­
ured by indirect calorimetry) increased by 50% despite the decline m insulin
secretion and amino acid concentrations fell by 40-609c (P < 0.05). Cnexpect­
edl;;, IGF-I also suppressed hepatic glucose production (P<O.OI) as well as
circulating NEFA levels by 70% (P < 0.001). Indirect calorimetry showed a shift
in fuel oxidation frorr, lipids to carbohydrate. This study demonstrated that in
humans as compared to rodents the pattern of IGF-I metabolic actions is much
closer to that of insulin.
The above findings prompted uS to compare the metabolic actions of rhIGF-1
and insulin in 21 healthy young subjects (2..+ ± 1 years) and 14 healthy middle­
ag.:d subjects (48 ± 2 years) during euglycaemic clamp studies Llsing: one of
three doses of rhIGF-l (0.2,0.4, 0.8,llg/kg per min) or insulin (0.2, mUi
kg per min) on separate days (30). These doses of the rhIGF-I and iosulin v"ere
cha;en to induce equivalent increases in gluca;e uptake and to compare '[he meta­
bolic actions of the hormones in the low end of the insulin dose-response curve.
As anticipated, these doses of IGF-I and insulin produced an equivalent in­
crease in glucose uptake. However, these hormones also had remarkably similar
effects on a variety of other metabolic processes. Both hormones (in these doses)
increased non-oxidati\'e and oxidative glucose metabolism identically and also
suppressed glucose production. NEFA, and fat oxidation similarly. In contrast.
rhIGF-I had a more pronounced inhibitory effect on islet function and produced
a greater decline in plasma amino acids than did insulin, despite the presence of
hypoinsulinaemia. implying that IGF-I may have a more potent protein anabolic
effect than insulin. The finding of a similar inhibitory effect on hepatic glucose
production was also unexpected, since human hepatocytes are virtually devoid of
IGF-I receptors. Possible mechanisms for this effect include a spillover effect of
rhIGF-l leading to stimulation of the insulin receptor, rhIGF- I stimulation of
IGF- Iiinsulin hybrid receptors or an indirect effect due to reduced circulating
Remarkably, basal IGF-I concentrations in the middle-aged subjects were
only one-half of those measured in the younger subjects, while insulin and C­
peptide levels were 25% higher in the middle-aged compared to younger sub­
jects. The response to rhIGF-l infusions in the older subjects was no different
from that seen in the younger subjects, glucose uptake and inhibition of glucose
production were similar. However, there was blunting of these responses to in­
sulin. These data suggest that middle age is characterized by basal IGF-l defi­
ciency and hyperinsulinaemia, but normal rhIGF-I responses and blunted insulin
action. IGF-l deficiency, as well as insulin resistance, may thus be responsible
for some of the adverse metabolic changes accompanying the aging process.



f-lipe rglvcaemia

To determine \vhether IGF-l's inhibitory effect on insulin secretion (see :lbove)

persists during glucose-stimuLlted insulin secretion, he:llthy non-obese subjects
recei\'ed either rhIGF-I (OA per min) or saline for 3 h (31). After an initial
euglycaemic phase, pl:lsm:l glucose W<lS raised to two different levels of hyper­
glyc<lemia by the cbmp technique to eV:lluate insulin responses to a Standard
stimulus. In one group, glucose was mised -2.8 mmoi/l (11 == 6) and in the other
-7 mmol/l <lbove baseline.
At the -2.8 mmolll step, C-peptide leve!s were suppressed by 40% during
rhIGF-I infusion, while insulin levels were suppressed by 30% (P < 0.05). De­
spire the reduction in insulin secretion, the rate of glucose metabolism was two­
fold higher in the IGF-l infused group (P < 0.00 I). At the higher glucose
stimll [us (-7 mmolll), rates of glucose metabol ism were 30-35% higher duri ng
IGF-l infusion (P < O.Ol). Suppression of insulin secretion by IGF-I also was
noted, but was less marked than with -2.8 mmolll step. _
These data demonstmte that rhIGF-l's inhibitory effect on glucose-stimulated
insulin secretion could be partially overcome by increasing the hype'rglycaemic
stimulus. Despite the decreJse in insulin secretion, glucose dispoSJ! during hy­
perglycaemia was accelemted by IGF-I, findings in accordance with the obser­
varions of Zenobi and colleagues (32) who demonstrated that while rhIGF-1
suppressed fasting and meal stimu!Jted insulin levels, glucose tolerance did not
change. They suggested that IGF-I may improve glucose disposal by increasing
tissue sensitivity to insulin. however it is also possible that this phenomenon re­
nects the combined stimulatory effects of both IGF-I and insulin rather than a
direct change i'ninsulin action.
Regardless of the mechanisms involved, these findings suggest that [he insu­
lin lowering effect of IGF-l is not likely to limit its usefulness as a glucose low­
ering agent in patients with the pre-existing capacity for insulin secretion.

Role ofIGF-I in the pathology and therapy ofIDDM

It is now recognized that conventionally-rreated patients with Type I diabetes

mellitus (IDDM) are chJracterized by reduced levels of IGF-I (33.34), espe­
cially during puberty. Furthermore. experimental diabetes in rodents (35; nnd
insulin deficient patients with IDDM (36) exhibit increased levels of lov" mo­
lecular weight IGF-I binding proteins such as IGFBP-I which might further
limit the tissue availability of biologically active IGF-l in IDDM.
Indeed, the raised GH levels in IODM appear to be, for the most part, a com­
pensatory attempt to overcome a block in IGF-l synthesis caused by insLllin de­
ficiency and poor metabolic control (37). Thus, while it is uncertain whether

IGF-I deficiency directly contributes to the ad\'ase metabolIC effects in IDDM.

to the extent th:lt it contributes to GH hypersecretion it undoubtedly contributes
to diabetic hyperglycaemia. Even the modest GH elevations seen in poorly con­
trolled diabetes have been shown to produce marked hyperglycaemia and meta­
bolic decompensation in IDDM patients in the face of optimized intensive insu­
lin treatment (38). Furthermore. IGF- I deficiency may play il role in the hyper­
glucagonaemia of poorly controlled IDDr.'L Insulin resistance associated with
poorly controlled IDDM may. therefore be. at least in part, due to IGF-I defi­
ciency. and in turn the loss of its inhibitory effect on GH and glucagon secretion
(Fig, 4). Theoretically. administration of low doses of I sufficient to re­
store basal IGF-I levels to normal in IDDM, might lower glucagon and GH lev­
els as well as improve cellular metabolism in muscle. As a result insulin might
become more effective. thereby ameliorating insulin resistance in IDDM.
It is noteworthy that administration of insulin may in a similar fashion aug­
ment the metabolic effect of IGF-I. Insulin rapidly lowers I. and there­
fore may increase tissue access to free IGF-I (39). Such complementaQ' effects
of IGF-l and insulin might facilitate restoration of normal substrate membolism
in IDDM. The potential therapeutic value of combined insuIin/rhIGF-1 admini­
stration in young patients with Type I diabetes has recently been ie'sted in ado­
lescence. Bach and colleagues (40) have compared the impact of short term IGF­
I administration on GH. IGF- I. and insulin requirements in nine IDDM adoles­
cent patients and six pubertal-stage matched healthy volunteers. Subcutaneous

r -_ _ _ _ .lnsulin Deficiency _ _ _ _ _~

IGF-l Deficiency

Insulin Resistance

t Glucose
t Amino Acids
t FFA, Ketones

FIG. 4. Potential role of the deficiency of fGF-} in the pathogenesis of insulin resistance
in poorly controlled fDDM. '.
infusion of IGF-I (20 u g/kg per h for the lO h) applied for two consC'cuti \'c da','s
resulted in a 60% dC'('rease in the insulin dose necessary to m::rimJin baseli~e
blood glucose level in diabetic patients. In addition a marked suppression of
spontaneous overnight and ::rrginine stimulated GH secretion \\(;15 noted. Simi­
larly, Cheetham and colleagues (41) using a double-blind placebo controlled
design. showed that a single subcutaneous injection of relatively small doses of
rhIGF-l (40pg/kg body \veight) in the evening, reduced nocturnJl GH levels
and insulin requirements throughout the night in pubertal diabetic p:l.tiems.
Although these results are encouraging, their clinical implications are limited
by the fact that IGF-I was administered by relatively brief periods. Whether
more long-term administration of IGF-l directed toward the replacement of the
depleted IGF-l in Type I diabetes might reduce the metabolic bbility com­
monly seen in Type I diabetic patients, especially during puberty reminds still to
be determined. Furthermore. [he potential risks of IGF- I. perhaps in accelerating
retinopathy, need to be examined before its long-term use can be recommended
for the management of diabetes mellitus.

IGF-l and the therapy of NIDDM

The potential value of IGF-I as therapeutic agent to control hypcrgl;cac:mia and

dyslipidaemia associated with Type 2 diabetes is suggested by short term studies
in NIDDM patients (32). Ie has been demonstrated that short term IGF- I treat­
ment in Type 2 diabetes results in decreased hyperglycaemia following mixed
meals, as well as decreased circulating levels of insulin, C peptide, total triglyc­
erides, VLDL-triglyceride. LDL-cholesterol and lipoproteina (32, 42). Consider­
ing that NIDDM patients are at particular risk for the development of the acce!­
er;ted atherosclerosis. the reduction in these atherosclerotic risk factors is par-"
ticularly noteworthy. Unfortunately, a subsequent study examining the thera­
peutic efficacy of I in obese NIDDM patients who had required insulin to
prevent severe hyperglycaemia had to be terminated because of the development
of adverse side effects of this therapy (43). In that study, which lasted from 4 to
52 days, two large daily doses of IGF-I (120 or 160,uglkg) were administered to
seven obese Type 2 diabetic patients who had been treated previously with high
doses of insulin. While the glucose control achieved with high doses of IGF-I
was similar to that accomplished during previous insulin treatment. side effects
of IGF-l administration appeared including facial and upper extremities oedema,
mild weight gain. dyspnoea, bilateral jaw tenderness, arthralgias, myalgias, fa­
tigue. tachycardia, flushing, orthostatic hypotension, and local burning in the
injection site. .
These studies indicate that obese, insulin resistant patients requiring insulin
for metabolic control also require large pharmacological doses of IGF-l if IGF-I

is to be used as a substitute for insulin. and that [his produces unacceptable side
effects. On the other h;:md, the use of smaller replacement doses mie:ht still be
potentially beneficial in 0.'IDDf.,L By virtue of its insulin and glucagon lowering
effects, its ability to reduce dyslipidaemia and increase leJn body mass and in­
sulin action, IGF-l could potentially serve a useful function in less obese. or
milder forms of NIDD~L


Current studies suggest that IGF-I might be an effective therapeutic agent in

various pathological conditions, especially when used [Q restore to normal ab­
normally low levels of IGF-l caused by relative growth hormone or insulin de­
ficiency, such as in diabetes, malnutrition, and catabolic states. In those settings
IGF-I replacement might lower glucagon and GH levels as well as act directly to
improve cellular fuel metabolism, thereby overcoming insulin resistl:Ulce. De­
spite the recent research on the actions of IGF-l, clinical experience with the
IGF-I is still quite limited. As a result, the therapeutic benefits and risks of IGF­
1 require further investigation before they can be appropriately defi'ri'ed.


This work was supported by grants from the U.S. Public Health Service (DK­
20495 and DK-45735) and a fellowship grant from The Juvenile Diabetes Foun­
dations International (W.P.B.).


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j .~ '- .


The AmericanAssociationof Clinical Endocrinologistsis pleasedto presentthe frfth edition ofthe AACE Endocrine
CodingManual.This manualwas desiped and compiledby clinical endocrinologistsfor clinical endocrinologists,
d tlus creatinga resourcethat shouldbe higbly relevantto tle needsof our real-world practices.
tt Specificfeaturesof interestinclude comprehensivesectionsincorporatingall primary codesfor 15 categoriesof
endocrine-specificdisorders;formatting by diseasestates;a detailedindex; and a "coding quick referenceguide" for
t easyuse.

t In designingthe manual,the AACE Socioeconomicand MemberAdvocacy Committeereviewedthe entire

compendiumof CPT@,ICD-9-CM, andHCPCScodesfor their clinical relevanceto endocrinology.The Committee
quickly realizedthat all codesofpotential interestcould simply not be includeddue to spacelimitations, and
selectivelychosecodeswitl the greatestanticipatedclinical utility.

t: Diagnosticandtherapeuticadvancesoccur daily; therefore,periodic updatesof tlis resourcewill be madeavailable

regularly.Watch AACf, plilings or AACE Online ( information on updates.
This outstandingmanualcould not havebeencreatedandproducedwithout the hard work and dedicationof the
ill AACE Socioeconomicand MemberAdvocacy CommitteeandAACE staff. Particulargratitudeis extendedto R.
Mack Harrell, MD, FACP, FACE, Chair andA. Jay Cohen,MD, FACE, Vice Chair for their exemplaryleadershipof

n the Socioeconomicard MemberAdvocacy Committeein making this importantand valuabledocumentavailablefor

practicingclinical endocrinologists.
AACE welcomesfeedbackand suggestionsto makesubsequenteditionsofthe manualevenmore useful and
E beneficialto the practiceof endocrinology.

il Sincerelv.

StevenM. Petak,MD, JD, FACE, FCLM, President

AACE 2006-2007Socioeconomicand Member Advocacv Committee

E R. Mack Harrell, MD, FACP. FACE" Chair
A. Jay Cohen, MD, FACE, Vice Chair Arthur N. Lurvey, MD, FACE
Walter P. Borg, MD Tilak Kumar Mallik, MD, FACE
William Conway, MD Eric A. Orzeck,MD, FACP, FACE
= Marc J. Laufgraben,MD, FACE S. SethuK. Reddy, MD, MBA, FACE
Bill Law Jr., MD, FACP, FACE SharonE. Selinger,MD, FACE
: Anne-Marie Lee, MD, FACE W. PatrickZeller, MD, FACE
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til AACE Staff

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PAGE  The First Messenger • March/April 2008

FDA Takes a Stand on Bioidentical Hormones

Walter P. Borg, MD • Chair, Reproductive Medicine Committee
Continued from page 1 no place in endeavors addressing public • Performance of reliable clinical tri- approach. The First Messenger.
safety issues. Moreover, as it was already als and basic research in the area of American Association of Clinical
The FDA expressed explicit concerns about
pointed out by some legislators, issue of BHRT; and Endocrinologists. 2007;16(2):10.
those claims, since the above statements are
public safety should always be balanced 4. AACE Reproductive Medicine
scientifically unsupported. The FDA is con-
with the individual freedom to make in- • Formulation of the enforceable legis- Committee Position Statement on
cerned because those false statements can
formed personal choices (9). Obviously, lation to address the issue of BHRT. Bioidentical Hormones. The First
mislead women and health care profession-
such informed, personal decisions should Such legislation and its enforcement Messenger. American Association
als. In fact, the FDA regards the use of the
be based upon objective information that may become a model solution for of Clinical Endocrinologists.
term “bioidentical” as nothing more than a
addressing similar therapeutical con- 2007;16(5):14.
clever marketing ploy that lacks scientific is not tainted by any commercial or doc-
troversies that are likely to follow. 5. American Medical Association. FDA
substantiation and is used only to boost the trinal bias. Therefore, those programs
oversight of bioidentical hormone
sales of the product. Therefore, the FDA is should include efforts on the part of med-
Last but not least, in view of the recent (BH) preparations. Resolution 706
encouraging patients to use FDA-approved ical community, legislature, enforcement
development - it would be most prudent (I-06). AMA House of Delegates
drugs for menopause- related complaints agencies (especially FDA), and public Meeting. 2006: Las Vegas, NV.
for any patient on bioidentical hormone
whenever possible, instead of relying on advocates, and consist of: 6. The Endocrine Society. Bioidentical
replacement therapy prescribed by a phy-
compounded drugs that are not reviewed Hormones Position Statement.
• Performance of surveys for purity and sician to contact his/her doctor about the
by the agency for safety and effectiveness.
dosage accuracy of all compounded safety and efficacy of this treatment. October 2006.
It has to be noted that several major 7. American College of Obstetricians
medical societies, including AACE, have “bioidentical hormone” formulations; and Gynecologists Committee.
recently published statements reflecting Opinion #322: Compounded
their concerns regarding BHRT. The • Requirements for mandatory report- 1. Borg W. “Bioidentical” hormones- bioidentical hormones. Obstet
statements are consistent with those ing by all drug manufacturers, includ- the need for an objective Gynecol. 2005;106(5 Pt 1):1139-40.
expressed by the FDA (4,5,6,7,8). ing compounding pharmacies, of adverse approach. The First Messenger. 8. MacLennan AH, Sturdee DW. The
However, in its recent statement, the FDA events related to the use of BHRT; American Association of Clinical ‘bioidentical/bioequivalent’ hormone
went further than expressing just a mere Endocrinologists, 2006;15(5):1,7. scam. Climacteric. 2006; 9(1):1-3.
criticism of the BHRT concept. Warning • Creation of a registry of such adverse 2. Borg W. AMA calls for reality check 9. Senate Hearings: bioidentical
letters issued by this agency clearly stated events; on bioidentical hormones. The First hormones: sound science or bad
that the specific pharmacy operations Messenger. American Association medicine? United States Senate
• Requirements for inclusion of uni- of Clinical Endocrinologists. Special Committee on Aging. April
violate federal law by making false and
form patient information (warnings 2007;16(1):3. 19, 2007. FM
misleading claims about BHRT.
and precautions), in packaging of 3. Borg W. Comments to the author
compounded bioidentical hormones; re the need for an objective
There is a clear change in the attitude
to the issue of BHRT by the scientific
medical community and governmental
Cardiometabolic Risk:
Seventeenth Annual
oversight agency. For many years after Meeting and

New Insights and Perspectives

Clinical Congress
its emergence, the concept of biodentical Satellite Symposium

hormones has been proliferating steadily

in the consumer market without being in- on the Management of Dyslipidemia, Obesity, and Type 2 Diabetes
dependently reviewed or debated. Even- A 1.5 AMA PRA Category 1 Credit™ activity in conjunction with the AACE Seventeenth Annual Meeting and Clinical Congress
tually, it became so pervasive that it could
not be ignored any longer. Yet, despite all Wednesday Evening, May 14, 2008 Goal
Walt Disney World Dolphin Resort To provide clinical endocrinologists who treat patients with
the described initiatives, we are still far diabetes and its related conditions with an update on the
1500 Epcot Resort Boulevard Buffet dinner: 6:30 PM – 7:15 PM
away from the so much needed objective Lake Buena Vista, Florida Symposium: 7:15 PM – 8:45 PM advancement of preventive and treatment strategies for
approach to this important matter. patients with multiple cardiometabolic risk factors.
Agenda Objectives
• Targeting Cardiometabolic Risk: A Pathophysiologic Overview and Update on Participants will be provided with clinically relevant,
There are serious dangers of BHRT use Lipid Treatment Strategies evidence-based information. Upon completion of this activity,
that have not been sufficiently evaluated. • Adipose Tissue and Metabolism: Pathophysiology and Clinical Implications participants should be able to:
The primary concern about bioidentical • Dysglycemia and Type 2 Diabetes: Targeted Interventions to Optimize Outcomes
• Identify metabolic risk factors such as obesity, diabetes, and
hormone use is patient safety. These sub- dyslipidemia, and describe the role of each in increasing
cardiovascular risk
stances have not been shown to be clini- Faculty
• Initiate interventions to prevent the development/progression of cardiovascular
cally effective. In addition, utilization Sergio Fazio, Osama B. Hamdy, Paul S. Jellinger,
disease, particularly targeting dysglycemia, dyslipidemia, and obesity
of these formulations may be associated Professor of Medicine and Medical Director PROGRAM CHAIR • Target recommended lipid levels for optimal cardiometabolic risk reduction,
including the full spectrum of lipid components, to optimally reduce residual risk
with various risks inherent to the com- Pathology Obesity Clinical Program Professor of Medicine,
of end-organ impact
Co-Director, Atherosclerosis Joslin Diabetes Center Voluntary Faculty
pounding process, including variability Research Unit Instructor in Medicine University of Miami • Identify individuals needing weight-reduction interventions and initiate
in potency, high potential for contamina- Director, Lipid Laboratory Harvard Medical School Miami, FL interventions targeting abdominal adiposity that blunt its cardiometabolic impact
Vanderbilt University
tion, and impurity of the preparations. Medical Center
Boston, MA Past President, American • Assess specific mechanisms leading to the dysglycemia of type 2 diabetes and
College of Endocrinology formulate management programs targeting those etiologic mechanisms to blunt
Nashville, TN
cardiometabolic risk.
The issue of BHRT is no longer ignored.
Accreditation and Designation of Credit You must be registered for the AACE Annual Meeting to attend this symposium.
However, additional initiatives are neces- The American Association of Clinical Endocrinologists is accredited by the Accreditation Council for Please select Satellite Symposium #8 on the official registration form and mail or fax your form
sary to help protect the health and safety Continuing Medical Education (ACCME) to provide continuing medical education for physicians. directly to AACE. If you need a meeting registration form, please visit the AACE website at
The American Association of Clinical Endocrinologists designates this educational activity for a or call AACE at 904-353-7878.
of the public. This should be approached maximum of 1.5 AMA PRA Category 1 Credit™. Physicians should only claim credit This activity is sponsored by the American Association of Clinical Endocrinologists. Program
with utmost diligence and objectivity. commensurate with the extent of their participation in the activity. management services provided by Joslin Diabetes Center. This activity is supported by an
educational grant from Merck & Co., Inc.
Both positive and negative biases have
Vol. 17, No. 2 • PAGE 


As we head down the road of accelerat-

ing Medicare cuts, private payors are
becoming very creative in decreasing
physicians’ reimbursements as well. Phy-
sicians’ cognitive services are severely
underpaid. Just maintaining compensa-
tion in accordance with their contracts
has become an ongoing challenge for
most doctors. In this setting, the need to

Managing Medical Vendors optimize the efficiency of business opera-

tions is critical.

Our professional viability ultimately re-

quires us to operate at a financial profit.
Walter P. Borg, MD • Socioeconomics & Member Adovocacy Committee Member A profit is the difference between real re-
imbursement and the costs of running the

xcessive prices of indispensable tical references on vendor management. ly to patients’ needs. Consequently, phy- practice. Although very little can be done
medical supplies contribute great- The purpose of this article is to introduce sicians develop the mindset that con- to significantly increase reimbursement,
ly to exponentially rising costs of the reader to the most basic aspects of frontation should be avoided, otherwise, effective Loss Prevention Policy is abso-
medical services. The rise in the cost of controlling costs through vendor man- they may be labeled as “difficult” and lutely critical. This concept is so obvious,
doing medical business has occurred in a agement. From the practical standpoint, yet it is usually overlooked by physicians
setting of declining reimbursement. Med- this includes two critical elements: and office managers alike.
icine is the only industry in which a pay- “Many practices allow
ment of 30 cents per one dollar charged is • Understanding the duality of the vendors to mark up lab Physicians are supposed to supervise
considered acceptable. At the same time, medical practice their accounts payable. In reality, how-
however, medical office managers tend to and office supply pricing ever, they are overwhelmed by direct pa-
pay exorbitant prices for medical supplies • Effective loss prevention policy more than 20% without tient care, compliance issues, and endless
without ever questioning the validity of reimbursement appeals. Most doctors
the bills. Many practices allow vendors
any protest.”
DUaLITY OF still assume that they should generate ad-
to mark up lab and office supply pricing MEDICaL PRaCTICE ditional revenue by seeing more patients
more than 20% without any protest. This their medical reputation and career may rather than “sweating the small stuff.”
is well known as “price gouging” in the Most clinical endocrinologists in the U.S. be jeopardized. Medical training also Monthly charges under $100 are often in-
rest of the business world. In medicine, it have both professional and business re- nurtures a natural human tendency to be