Untitled

Regulation
of
Carbohydrate
Metabolism
Volume II
Editor
Rivka Beitner, Ph.D.

Associate Professor of Biochemistry
Department of Life Sciences
Bar-Han University, Ramat-Gan
Israel
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THE EDITOR
Rivka Beitner, Ph.D., is Associate Professor of Biochemistry in the Department of Life

Sciences at Bar-Ilan University at Ramat-Gan, Israel.
Dr. Beitner obtained her training at Bar-Ilan University, Ramat-Gan, Israel, receiving the
B.Sc. degree in 1961 and the M.Sc. degree in 1963. She received the Ph.D. degree in 1970
from McGill University, Montreal, Canada. She served as a Research Fellow at the Lady
Davis Institute for Medical Research of the Jewish General Hospital and McGill University,
Montreal, Canada, from 1967 to 1970 and as a Lecturer, Assistant Professor and Associate
Professor of Biochemistry at Bar-Ilan University from 1970 to the present. It was in 1979
that she assumed her present position.
Dr. Beitner is a member of the Israeli Society for Biochemistry, the Israeli Society for
Endocrinology, and the Israeli Society for Diabetes. She has been the recipient of many
research grants from the Muscular Dystrophy Association of America; the U.S.-Israel
Binational Science Foundation; the Medical Research Council of Canada; the Ministry of
Commerce and Industry, Israel, and the Research Authority, Bar-Ilan University.
Dr. Beitner is the author of more than 70 papers and abstracts. Her current major research
interests relate to the regulation of carbohydrate metabolism.
CONTRIBUTORS
Ramon Bartrons, Ph.D. Hans Werner Hofer, Prof. Dr.

Department of Biochemistry University of Konstanz
Faculty of Medicine Faculty of Biology
University of Barcelona Konstanz, West Germany
Barcelona, Spain
Nava Bashan, Ph.D. Louis Hue, M.D., Ph.D.

Pediatric Research Laboratory Hormone and Metabolic Research Unit
Soroka Medical Center International Institute of Molecular and
Faculty of Health Sciences Cellular Pathology
Ben Gurion University of the Negev University of Louvain Medical School
Beer Sheva, Israel Brussels, Belgium
Rivka Beitner, Ph.D. Norman Kalant, Ph.D.

Associate Professor of Biochemistry Lady Davis Institute for Medical
Department of Life Sciences Research
Bar-Ilan University Sir Mortimer B. Davis-Jewish General
Ramat-Gan, Israel Hospital and
Frank Clarke, Ph.D. Department of Medicine,
Lecturer McGill University
Griffith University Montreal, Quebec, Canada
School of Science
Nathan, Australia Don Morton, Ph.D.
Principal Research Scientist
Valentine A. Duruibe, M.S. Meat Research Laboratories
Graduate Research Associate CSIRO Division of Food Research
Department of Pharmacology Cannon Hill, Australia
College of Medicine
The Ohio State University
Shimon W. Moses, M.D., Ph.D.
Columbus, Ohio
Professor of Pediatrics
Erich Eigenbrodt, Prof. Dr. Pediatric Research Laboratory
Institut fur Biochemie und Endokrinologie Soroka Medical Center
Fachbereich Veterinarmedizin und Faculty of Health Sciences
Tierzucht Ben Gurion University of the Negev
Justus-Liebig-Universitat Giessen Beer Sheva, Israel
Giessen, West Germany
M. Reinacher, Prof. Dr.
P. Fister, Ph.D.
Institut fur Veterinar-Pathologie
Medizinisch Wissenschaftliche Abteilung
Fachbereich Veterinarmedizin und
Biotest Pharma
Tierzucht
Frankfurt, West Germany
Justus-Liebig-Universtat Giessen
Alisa Gutman, M.D. Giessen, West Germany
Professor of Biochemistry
Department of Biochemistry G. Rijksen, Ph.D.
Hadassah University Hospital and Department of Hematology
Hebrew University-Hadassah Medical Division of Medical Enzymology
School Academic Hospital
Jerusalem, Israel Utrecht, The Netherlands
Eleazar Shafrir, Ph.D. Petra Stephan, M.S.
Professor and Head Postgraduate Student
Department of Clinical Biochemistry Griffith University
Hadassah University Hospital and School of Science
Hebrew University-Hadassah Medical Nathan, Australia
School
Jerusalem, Israel Gopi A. Tejwani, Ph.D.
Assistant Professor
Alfred E. Slonim, M.D. Department of Pharmacology
Pediatric Department The Ohio State University
North Shore University Hospital College of Medicine
Manhasset, New York Columbus, Ohio
G. E. J. Staal, Ph.D. John Weidemann, B.Sc.

Professor in Medical Enzymology Scientist
Department of Hematology Meat Research Laboratory
Academic Hospital CSIRO Division of Food Research
Utrecht, The Netherlands Cannon Hill, Australia
John E. Wilson, Ph.D.

Professor
Department of Biochemistry and
The Neuroscience Program
Michigan State University
East Lansing, Michigan
TABLE OF CONTENTS
Volume I
Chapter 1
Glucose-1,6-Bisphosphate—The Regulator of Carbohydrate Metabolism 1
Rivka Beitner
Chapter 2
Role of Fructose-2,6-Bisphosphate in the Control of Glycolysis in Liver,
Muscle, and Adipose Tissue 29
Louis Hue and Ramon Bartrons
Chapter 3
Regulation of Mammalian Hexokinase Activity 45
John E. Wilson
Chapter 4
Hexokinase in Health and Disease 87
G. Rijksen and G. E. J. Staal
Chapter 5
Phosphorylation of Phosphofructokinase—The Possible Role of Covalent
Modification in the Regulation of Glycolysis 105
Hans Werner Hofer
Chapter 6
Regulation of Pyruvate Kinase in Normal and Pathological Conditions 143
G. E. J. Staal and G. Rijksen
Index 161
Volume II
Chapter 1
Glycolytic Enzyme Organization Via the Cytoskeleton and Its Role in Metabolic
Regulation 1
Frank Clarke, Petra Stephan, Don Morton, and John Weidemann
Chapter 2
Regulation of Glycogen Metabolism 3
Alisa Gutman
Chapter 3
Effects of the Abnormal Carbohydrate Metabolism Present in Glycogen Storage
Disease on Intermediary Amino Acid and Lipid Metabolism 53
Shimon W. Moses, Nava Bashan, and Alfred E. Slonim
Chapter 4
Effect of Ethanol on Carbohydrate Metabolism 67
Gopi A. Tejwani and Valentine A. Duruibe
Chapter 5
Effect of Sucrose and Fructose on Carbohydrate and Lipid Metabolism and
the Resulting Consequences 95
Eleazar Shafrir
Chapter 6
New Perspectives on Carbohydrate Metabolism in Tumor Cells 141
E. Eigenbrodt, P. Fister, and M. Reinacher
Chapter 7
Insulin Binding and Metabolism by Hepatocytes in Primary Culture 181
N. Kalant
Index 201
Taylor & Francis
http://taylorandfrancis.com
Volume II 1
Chapter I
GLYCOLYTIC ENZYME ORGANIZATION VIA THE CYTOSKELETON

AND ITS ROLE IN METABOLIC REGULATION
Frank Clarke, Petra Stephan, Don Morton, and John Weidemann
TABLE OF CONTENTS
Historical Perspective 2
II. The Nature of the Adsorbent 3

A. Muscle: The Role of Actin and the I-Band 3
B. The Role of Tropomyosin (TM) and Troponin (TN) 4
C. The Specificity of Enzyme Binding Sites 5
D. Piggy-Back or Indirect Binding and Glycolytic Enzyme Organization.... 10
E. Nonmuscle Cells and Cytoskeletal Actin II
III. Dynamics of Enzyme Organization 15

A. Metabolic Dependence of Enzyme Binding 15
B. Effectors of Binding 18
C. Genetic Determinants of Binding 20
IV. Functional Significance of Binding 20

A. Influence on Catalytic Expression 20
B. Structural Considerations 24
References 26
2 Regulation of Carbohydrate Metabolism
I. HISTORICAL PERSPECTIVE
While the classification of the glycolytic enzymes as soluble is an operational definition

which reflects the relative ease with which these proteins are extracted from cells and tissues,
the term also has the conceptual connotation that these enzymes exist free in solution within
the cell. Unfortunately this implied intracellular distribution is still widely held despite the
wealth of accumulated evidence that these enzymes are represented in both the particulate
and soluble phases of the cell.
Many of the early reports' - ' 4 of glycolytic enzyme associations with cell particulate frac-
tions occurred at a time when investigations of organelles as multienzyme complexes were
at their height.' The observation of glycolytic enzymes bound to subcellular structures
naturally led speculation in the direction of the existence of a discrete glycolytic complex.
This viewpoint was encouraged by the demonstration by Green et al.'6 of the isolation of
membranous components from both red cell and yeast which were capable of catalyzing the
complete glycolytic sequence. Whatever expectations there were for the existence of a
complete glycolytic complex, the notion was grievously injured by de Duve in 1972 who
compared the sedimentation of some glycolytic enzymes in a "cell sap" (in fact a conven-
tional supernatant fraction) of liver with that of the purified enzymes and found that they
sedimented identically and so concluded that complex formation between these enzymes in
the cytoplasm did not occur." When this was coupled with a recognition'" that most of the
in vitro studies of glycolytic enzyme binding to subcellular structures have been performed
only under very low ionic strength conditions," then it has been convenient to dismiss not
only the notion of glycolytic enzyme complexes but also enzyme binding to structural
components as nonspecific and irrelevant.
The soluble fraction (cell sap) of classical subcellular fractionation procedures'' cannot
be equated with the cytoplasm of the cell even with due attention to the ionic strength. It
is now acknowledged that the cytoplasm of eukaryotic cells is a highly concentrated, thix-
otropic protein solution with a high degree of structure imposed by the all-pervasive cyto-
skeletal network formed by the actin, microtubule, and intermediate filament systems.'`' 2'
Indeed it has been the extensive studies of this cytoskeletal apparatus over the last
decade24--" that have finally brought about general acceptance of this view of the cytoplasm
— a view it should be remembered which has been held by a number of workers long before
the modern concepts of the cytoskeleton.' ' There is no more telling testimony to the nature
and structure of the cytoplasmic environment than to view the recent electron micrographs
provided by Porter,'"' Heuser and Kirschner," and others.4"2 Keeping pace with these
developments is the clear demonstration that interactions of glycolytic enzymes with cyto-
skeletal components can and do occur within this cellular environment.23-43-49
Despite these advances, however, we still lack a clear perspective as to the purposes
served by enzyme binding. In skeletal muscle, for example, of the ten enzymes which
catalyze the conversion of glucose-6-phosphate to lactate, at least four, aldolase (ALD),
phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GPDH), and py-
ruvate kinase (PK) are capable of binding very strongly to actin-containing filaments, while
the other six bind to a lesser but no less appreciable degree.22-23 This binding varies between
different types of muscle and even within the same muscle in response to different phys-
iological stimuli (Table 1). This level of complexity in just one type of tissue serves to
obscure both the relevance and role of enzyme binding in the glycolytic economy.
In this article we seek to establish the principles involved in glycolytic enzyme binding
and then to consider the metabolic and structural purposes served by the interaction of these
enzymes with the cytoskeletal apparatus of the cell.
Volume II 3
Table 1
GLYCOLYTIC ENZYME BINDING LEVELS IN
DIFFERENT MUSCLES'
Percent enzyme bound
Muscle PFK ALD GPDH PK LDH
Rabbit skeletal (white)

Resting 27 44 41
Stimulated 89 55 49
Rabbit skeletal (red)
Resting' 35 40 54
Stimulated 45 51 70
Sheep skeletal
Resting' 13 35 15 14 12
Stimulated 26 46 39 15 15
Sheep smooth (uterine)`' 38 77 86 37 35
Rat cardiac
Normal' 13 29 19 17 15
Ischemic 25 76 18 19 13
Enzyme binding as determined by Clarke, F. M., Morton, D. J., and

Shaw, F., Biochem. J., 186, 104, 1980 (Reference 97).
• Enzyme binding as determined by Walsh. T. P., Clarke, F. M and
Morton, D. J., unpublished data (Reference 144).
Enzyme binding as determined by Walsh, T. P., Masters, C. J., Mor-
ton, D. J., and Clarke, F. M., Biochem. Biophys. Acta, 675, 29, 1981
(Reference 98).
• Enzyme binding as determined by Clarke, F. M.. Morton, D. J.,
Hamilton, D., and Huxham, G., unpublished data (Reference 145).
• Enzyme binding as determined by Clarke, F. M., Stephan, P., Hux-
ham, G., Hamilton, D., and Morton, D. J., Eur. J. Biochem., 138,
643, 1984 (Reference 142).
II. THE NATURE OF THE ADSORBENT
A. Muscle: The Role of Actin and the I-Band

In skeletal muscle, a proportion of the enzymes of glycolysis are discretely localized on
the structural framework provided by the actin-containing filaments of the contractile ap-
paratus. The physiological reality of this binding was established principally by Pette and
co-workers. Following on the original observation of Bucher" that a significant portion of
the activity of many glycolytic enzymes was not readily extractable from muscle, Pette and
his colleagues applied a series of histochemica1, 43.5' immunofluorescent," and biochemical
techniques52-54 to show that most of the glycolytic enzymes are localized within the I-band
of muscle fibers.
The histochemical and immunofluorescent studies revealed that phosphorylase, phos-
phoglucomutase, glucose phosphate isomerase, phosphofructokinase (PFK), triosephosphate
isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GPDH), pyruvate kinase (PK),
and lactate dehydrogenase (LDH) were all discretely localized within the I-band, whereas
hexokinase (HK) showed a distribution in keeping with its known association with mito-
chondria.55 While these cytological studies by themselves cannot unambiguously distinguish
between a localization in the interfilamentary space or the overlying sarcoplasmic reticulum,
Pette and others have adduced a number of arguments to support their conclusion that the
glycogenolytic and glycolytic enzymes are localized in the region of, or directly associated
with the thin filaments of the contractile apparatus. Arnold and Pette found that many of
these enzymes interact quite strongly with F-actin, the major structural protein of the I-band
thin filaments.52 '4 This was confirmed by Clarke and Masters who established that the
interactions can occur under physiological conditions of pH and ionic strength.' Although
there have been reports of enzyme associations with sarcoplasmic reticulum membranes,
these interactions appear to be weak and nonspecific insofar as they have been reported only
under conditions of low ionic strength.56-57 Sigel and Pette" point out that there are great
variations in the relative amount of sarcoplasmic reticulum in different muscles, and indeed
there is an inverse relationship between the amount of reticulum and the level of glycolytic
enzymes in different insect muscles.38 On the other hand, a constant ratio between sarcomere
length and the content of glycolytic enzymes has been found in these muscles. Such ob-
servations together with the cytological and binding data all support a major localization of
the glycolytic enzymes at the site of the thin filaments.
Not all glycolytic enzymes interact directly with actin despite the fact that all, except
hexokinase, are localized within the I-band. Some like adolase (ALD), phosphofructokinase
(PFK), and pyruvate kinase (PK) bind to pure actin very strongly, glyceraldehyde-3-phos-
phate dehydrogenase (GPDH), phosphoglycerate kinase (PGK), and lactate dehydrogenase
(LDH) more weakly, and triosephosphate isomerase (TPI) hardly at all. 54 Furthermore, the
binding of these individual enzymes is modulated by a number of environmental factors
including pH and the concentration of metabolites." The influence of metabolites is usually
quite specific for a given enzyme and is discussed in detail later.
B. The Role of Tropomyosin (TM) and Troponin (TN)

Since the original studies of Pette it has become apparent that actin is not the only I-band
protein capable of interacting with glycolytic enzymes. The thin filaments of the I-band are
a highly ordered complex of F-actin and two accessory proteins, tropomyosin (TM) and
troponin (TN) which serve to regulate the interaction of myosin with the actin in response
to Ca" . The extended tropomyosin dimer (41 nm) lies in the groove of the F-actin double
helix, while troponin is regularly situated along the filament at 38 nm intervals.59 61 A role
for these accessory proteins in binding aldolase can be readily inferred from the lattice
structure shown in Figure 1. Filaments reconstituted from actin, tropomyosin, and troponin
were mixed with aldolase, then negatively stained and examined by electron microscopy.
The actin filaments are assembled into a lattice with aldolase cross-links at 38 nm intervals
corresponding to the distribution of the troponin along the filament.65
The involvement of tropomyosin and troponin in enzyme binding was first demonstrated
by Clarke and Masters, when it was shown that F-actin-tropomyosin-troponin filaments were
better adsorbents for glycolytic enzymes than F-actin.47 Since then a direct interaction
between enzyme and tropomyosin and troponin has been observed. For example, physical
techniques have established that aldolase interacts with both tropomyosin and troponin when
these proteins are free or when they are associated with F-actin.62." Quantitative studies
show that aldolase binds to pure F-actin with a stoichiometry of one aldolase per 14 actin
monomers, i.e., one aldolase per repeat of the actin helix. When tropomyosin is present on
the filament, the ratio doubles to 2:14 so that tropomyosin is contributing an additional
binding site. With F-actin-tropomyosin-troponin, the stoichiometry increases to 3:14 or 4:14
in the presence or absence of Ca" , respectively, which suggests an aldolase-troponin
interaction on these filaments. Electron microscopy has proven particularly useful in dem-
onstrating the troponin and tropomyosin binding sites for aldolase.64-66 When aldolase in-
teracts with isolated actin-containing filaments, it cross-links the filaments into ordered
paracrystalline filament bundles which have proved to be amenable to detailed structural
analysis. As shown in Figure 2A, the filament bundles formed with aldolase and F-actin-
tropomyosin have a single light transverse band every 38 nm. By contrast, the bundles
Volume 11 5
FIGURE I . Electron micrograph of aldolase-F-actin-tropomyosin-troponin mixture. The aldolase mol-

ecules cross-link the filament at regular 38 nm intervals to form a two-dimensional lattice structure.
formed between aldolase and F-actin-tropomyosin-troponin filaments are quite different in

appearance (Figure 2B). In this case there are two transverse bands every 38 nm: a major
band interpreted as aldolase binding to troponin and a minor band due to cross-linking at
the actin-tropomyosin site. As shown in Figures 2C and D, troponin has a similar influence
on the placement of both pyruvate kinase and phosphofructokinase on actin filaments; this
is particularly so with pyruvate kinase where the paracrystalline bundles are similar in
appearance to those formed with aldolase. Glyceraldehyde-3-phosphate dehydrogenase and
lactate dehydrogenase interact with F-actin-tropomyosin-troponin filaments in such a way
that only limited cross-linking occurs (Figures 2E and F).
Troponin and tropomyosin are, thus, clearly established as binding sites for individual
enzymes and the examples shown in Figures 2A to D indicate how they influence the
placement of enzymes along the filament. Both tropomyosin and troponin are long asym-
metric molecules,6'.68 and so could possess spatially distinct binding sites for individual
enzymes. This would then provide for a spatial organization of enzymes along the filament.
C. The Specificity of Enzyme Binding Sites

Studies on binding of individual enzymes lack the dimension of considering whether there
are separate sites for each enzyme or whether competition exists for a common site. In one
of the few studies of competition, Yeltman and Harris69 observed direct competition between
aldolase and glyceraldehyde-3-phosphate dehydrogenase for binding to pure F-actin which
indicates that there is a common binding site for both enzymes on this structural protein.
We have approached the question of specificity and competition using myofibrils as the
adsorbent. Myofibrils are a suitable analog of the cellular adsorbent, since the actin-
tropomyosin-troponin filaments are held in the same three-dimensional array as found in
intact muscle. The results of these studies are shown in Figures 3 and 4. When aldolase
FIGURE 2. (A) Three-dimensional filament bundle formed by aldolase cross-linked F-actin-tropo-

myosin filaments. Note parallel arrangement of filaments and the regular transverse bands at approxi-
mately 38 nm due to the cross-linking enzyme. (B) Filament bundles formed by aldolase cross-linking
F-actin-tropomyosin-troponin filaments. Note prominent cross-striation at 38 nm intervals interpreted
as aldolase binding to troponin; and the fainter cross-striations intermediate between the major ones
interpreted as the aldolase cross-linking at an actin-tropomyosin site. (C) Filament bundles formed by
pyruvate kinase cross-linking F-actin-tropomyosin-troponin filaments. Comparison with B reveals sim-
ilarity to structures formed between aldolase and these filaments. (D) Complexes formed between
phosphofructokinase and F-actin-tropomyosin-troponin filaments revealing the cross-linking of the fil-
aments and the regular placement of the enzyme molecules along the filament. (E) Electron micrograph
of glyceraldehyde-3-phosphate dehydrogenase/F-actin-tropomyosin-troponin mixture. Note the limited
formation of bundles and less-ordered structure of those which do form when compared with those
shown in A to D. (F) Lactate dehydrogenase/F-actin-tropomyosin-troponin mixture. Note very limited
cross-linking of filaments by this enzyme.
alone binds to isolated myofibrils it does so with an ultimate stoichiometry corresponding

to two aldolase per repeat of the actin filament (Figure 3). Glyceraldehyde-3-phosphate
dehydrogenase alone also binds to the thin filaments but with lower affinity and a stoichi-
ometry of only one enzyme molecule per repeat of the actin (Figure 4). When aldolase
binding is examined in the presence of saturating concentrations of glyceraldehyde-3-phos-
phate dehydrogenase, the stoichiometry of aldolase binding is decreased (Figure 3). In fact,
one aldolase binding site per repeat is lost, suggesting that one of the two binding sites
Volume 11 7
FIGURE 2C
FIGURE 2D
which aldolase may occupy is preferentially a binding site for glyceraldehyde-3-phosphate

dehydrogenase. The reverse experiment in which glyceraldehyde-3-phosphate dehydrogenase
binding takes place in the presence of saturating aldolase is shown in Figure 4 and confirms
this interpretation. Although aldolase competition for the glyceraldehyde-3-phosphate de-
hydrogenase site occurs at low glyceraldehyde-3-phosphate dehydrogenase concentrations,
it is readily overcome at higher concentrations. Thus it would appear that the actin-containing
filaments of the myofibrils possess at least two binding• sites per repeat, one which is an
aldolase binding site and a second which displays a greater specificity for glyceraldehyde-
3-phosphate dehydrogenase. The situation is summarized diagrammatically in Figure 5.
Given the lack of specificity in the binding of aldolase and glyceraldehyde-3-phosphate
dehydrogenase to F-actin observed by Yeltman and Harris,69 the specificity found with
isolated myofibrils undoubtedly is due to the involvement of tropomyosin and troponin in
the binding process.
The involvement of these accessory proteins may also be important in determining the
enzyme binding patterns in different muscle fiber types. Tropomyosin and troponin display
ri
FIGURE 2E
FIGURE 2F
considerable polymorphism which could contribute to significant variations of adsorbent in

different muscles and tissues." There are two different forms of the tropomyosin subunit a
and 13 which differ considerably in primary sequence." The proportion of a and 13 forms
is a characteristic of muscle fiber type with a-tropomyosin present in fast, type II fibers and
cardiac muscle, while 13-tropomyosin is associated with slow, type I fibers. Despite extensive
studies, no significant functional differences have been found in the ability of tropomyosin
isoforms to mediate the calcium regulation of the actin-myosin interaction.72 Recently, we
have shown that isolated tropomyosin has distinct sites for aldolase and glyceraldehyde-3-
phosphate dehydrogenase binding. 143 Moreover these enzymes bind with different affinities
to the different multiple forms of tropomyosin (a2, e(3, 132), suggesting a key role for this
accessory protein in determining the enzyme binding patterns of different tissues. Such a
role for tropomyosin as a determinant of enzyme binding may explain the persistence of
this structural protein in those muscles and other cell types which do not have a thin-filament
based system for the Ca' regulation of the actin-myosin interaction.
Volume II 9
300-
— 0 ALD
-2:14
ALDOLA SE BOUND pmoles
200-
0
ALD in presence
• of 42[LPA GDPH
100-
1k) 20 30 40
µM
FREE ALDOLASE
FIGURE 3. Aldolase binding to myofibrils. Rabbit psoas myofibrils samples, 400 itg each, were incubated with
increasing concentrations of aldolase in a final volume of 50 iite of 10 mM imidazole. 40 mM KC1, I mM MgCl,,
0.5 mM DTT, pH 6.8 at 37°C. Following centrifugation the amount of enzyme bound was determined as described
by Clarke et al.'" Each point represents mean of triplicate determinations. SDs shown are typical. As fluorescent-
labeled enzyme revealed binding was occurring to the thin filaments, the stoichiometry of binding is also expressed
(right hand axis) as the ratio of enzyme molecules bound per 14 actin monomers, i.e., per repeat of the filament
structure. (0) Aldolase binding alone; (0) aldolase binding in presence of 42 p.M GPDH when there is one GPDH
bound per repeat.
300
2:14
GPDH BOUND pmoles
200
GPDH in
presence of
17.6 LOA ALD
--GPO
100
10 20 30 40 50
FREE [GPDH]
FIGURE 4. Glyceraldehyde-3-phosphate dehydrogenase binding to myofibrils. Enzyme binding was determined

as described in legend to Figure 3. (V) GPDH binding alone; (0) GPDH binding in presence of 17.6 p.M aldolase
when there is one aldolase bound per repeat.
TN TM
(a)
(b)
(c)
(d)
FIGURE 5. Diagrammatic representation of the binding of aldolase, glyceraldehyde-3-

phosphate dehydrogenase, and triosephosphate isomerase to the thin filaments of my-
ofibrils as deduced from experimental results of Figures 3, 4, and 6. (A) The thin filament
has two binding sites per repeat of its structure — one specific for aldolase, the other
for GPDH. (B) When aldolase binds alone (e.g., Figure 3) it may occupy both its own
and the GPDH site. (C) When aldolase binds in presence of saturating GPDH, aldolase
(e.g., Figure 3) is restricted to its own specific site. (D) TPI may only bind indirectly,
by piggy-backing on an aldolase or GPDH molecule which is directly bound to the filament
(e.g., Figure 6).
While much remains to be explored in this area, it is clear that it is the accessory structural
proteins which dictate the extent and specificity of enzyme binding to actin filaments.
D. Piggy-Back or Indirect Binding and Glycolytic Enzyme Organization

As noted before, not all glycolytic enzymes when studied in isolation are capable of
interacting directly with actin-containing filaments. However, the histochemical and im-
munofluorescent studies already reviewed show that all are localized on the I-band of the
muscle fiber."'" So if they do not interact directly with the filaments, what is the basis of
their rather precise localization at the site of these filaments? A recent observation of
Volume II 11
Bronstein and Knull provides us with an important insight into the localization and organ-
ization of this group of glycolytic enzymes." It was noted that when examined individually
the purified enzymes triosephosphate isomerase (TPI), glucose-6-phosphate isomerase (PGI),
and phosphoglucomutase did not bind to F-actin-tropomyosin. When examined in a complex
mixture consisting of all other glycolytic enzymes, significant binding of triosephosphate
isomerase (TPI), glucose phosphate isomerase, and phosphoglycerate mutase (PGM) was
observed. This suggests that these enzymes were interacting indirectly by binding ("piggy-
backing") to other enzyme molecules already directly bound to the filament. A demonstration
of such indirect binding of phosphoglycerate mutase via lactate dehydrogenase was provided.
We have recently demonstrated a similar phenomenon involving triosephosphate isomer-
ase. As shown in Figure 6, the binding of pure triosephosphate isomerase to isolated
myofibrils is minimal. However, if either aldolase (ALD) or glyceraldehyde-3-phosphate
dehydrogenase (GPDH) is present, then significant triosephosphate isomerase binding is
observed and in both instances the binding is approximately equimolar with the aldolase
and glyceraldehyde-3-phosphate dehydrogenase. The simplest interpretation of these results
is that triosephosphate isomerase becomes associated with the filament indirectly by piggy-
backing to the already bound aldolase or glyceraldehyde-3-phosphate dehydrogenase. There
have been previous reports suggesting a weak complex formation between these enzymes
in solution.' 7' The results shown in Figure 6 indicate that the affinity of triosephosphate
isomerase for aldolase and glyceraldehyde-3-phosphate dehydrogenase is greatly enhanced
when these latter enzymes are bound to the actin-containing filament. Thus the binding sites
for the firmly or directly bound enzymes (aldolase, glyceraldehyde-3-phosphate dehydro-
genase) can be seen as a blueprint for the building of a more complex organization of
glycolytic enzymes utilizing piggy-back interactions as a key element in the construction
strategy. A diagrammatic representation of these principles is provided in Figure 7.
E. Nonmuscle Cells and Cytoskeletal Actin

Structural proteins (actin, tropomyosin, etc.) similar to those already established as binding
sites for some of the glycolytic enzymes in muscle, also occur in nonmuscle cells where
they play a central role in the structure of the cytoskeletal apparatus and the expression of
motile functions. 24-3' The potential for enzyme binding and consequent organization also
exists in nonmuscle cells and there have been numerous reports of associations between
glycolytic enzymes and the various particulate fractions, but rarely has the actual adsorbent
been identified.77-" The exceptions here are the well-established association of hexokinase
with mitochondria8•ss.s3 and the possible delineation of the binding sites for some enzymes
on the red cell membrane'" which is discussed below. In all other cases only indirect
indications of the adsorbent have been obtained; for example, for the binding of aldolase to
particulate fractions of rat brain homogenates, a correlation was noted between aldolase
binding and actin distribution.78 Knull has also commented on actin-containing structures
as the likely adsorbent of these enzymes following extensive studies on the association of
glycolytic enzymes with the particulate material of nerve ending particles.82
A similar association for enolase (ENOL), creatine kinase, aldolase (ALD), and pyruvate
kinase (PK) in nerve axons has been deduced by Brady and Lasek by the use of a noninvasive
approach to demonstrate that these interactions occur within the intact axon. 23 They found
that these enzymes are transported with slow component b of axonal transport in guinea pig
optic nerves. The enzymes move down the axons as a discrete front at 2 mm/day, a behavior
not to be expected of a soluble enzyme free to move by diffusion. Instead Brady and Lasek
suggest that these enzymes are associated with actin, clathrin, and related proteins forming
a structure which they define as the axoplasmic matrix — a structural matrix visualized as
being analogous to the actin-containing microtrabecular system described by Porter and
300
2:14
TPI B OU ND pmoles
200
TPI in
presence
of 22.2
M ALD
100
TPI
10 20 30 40
FREE [TPI]
300
2:14
200
TPI BOUND Moles
ENZYME:ACTIN
RATIO
1:14
TPI in
presence
100 of 39.8 pMGPIDli
TPI
_ ‘-- --- -6-
10 20 NM 30 40
FREE [TPI]
FIGURE 6. Binding of triosephosphate isomerase to myofibrils. Binding determined as described in legend to

Figure 3. (A) A, TPI binding alone: •, TPI binding in presence of 22.2 p,M aldolase when there is approximately
one aldolase bound per repeat. (B) A, TPI binding alone: •, TPI binding in presence of 39.8 p,M GPDH when
there is approximately one GPDH bound per repeat.
Volume II 13
TN TM
TM,TN AND ACCESSORY PROTEINS

PROVIDE SPECIFIC BINDING
SITES FOR KEY ENZYMES.
THIS DETERMINES THEIR

SPATIAL ORGANISATION ALONG
THE FILAMENT.
AND PROVIDES THE BLUEPRINT

FOR THE BUILDING OF HIGHER
ORDER COMPLEXES OF GLYCOLYTIC
ENZYMES THROUGH 'PIGGY-BACK'
BINDING.
FIGURE 7. Diagrammatic representation of the principles involved in the construction of clusters of glycolytic
enzymes along actin-containing filaments.
colleagues:38.39 As important and provocative as these studies are, the evidence for the nature
of the enzyme adsorbent remains indirect.
In a recent study, we have established a direct correlation between the distribution of a
number of glycolytic enzymes and that of actin in fetal calf brain homogenates and extracts
under physiologically relevant conditions of pH, ionic strength, and temperature." Direct
evidence was obtained that cytoskeletal actin and/or actin-associated proteins are the major
adsorbent in this tissue. A role for the actin cytoskeleton in binding glycolytic enzymes is
supported by double immunofluorescent studies of actin and enzyme distribution in fibro-
blasts (Figure 8). These studies show that there is an almost identical distribution for actin
and pyruvate kinase in these cultured cells. The distribution of pyruvate kinase along the
actin stress fibers is evident as is the association of enzyme with the more diffuse areas of
actin staining. Glyceraldehyde-3-phosphate dehydrogenase also shows the same pattern of
distribution," while similar but more limited associations of triosephosphate isomerase and
lactate dehydrogenase with the cytoskeleton have been revealed by this technique.
It is interesting that exposure of the cells to 2-deoxyglucose, an inhibitor of glycolysis,
induces the disruption of the actin cytoskeleton. At the same time the enzyme distribution
is realigned to that of the dispersed actin. Gibbins has provided direct evidence that 2-
deoxyglucose arrests cell motility functions, particularly membrane ruffling.87 Taken together
with the ability of certain glycolytic enzymes to organize actin-containing filaments into
A B
FIGURE 8. Rabbit muscle fibroblast double labeled for immunotluorescence by (A; left) antipyruvate
kinase (fluorescein-indirect method) and (B; right) by tetramethyl rhodamine labeled anti-actin.
ordered supramolecular structures (Figures 1 and 2) these results lead to the suggestion that
enzyme binding may have a second and structural function in modulating the actin cytoskeleton.
The ubiquitous distribution of actin in eukaryotic cells implies that glycolytic enzyme
binding to actin and its associated structural proteins is a general phenomenon. The failure
to observe such associations in the past has probably been due to choice of experimental
conditions. Processing at low temperatures, together with absence of precautions to control
the free Ca" concentration favors extensive depolymerization of actin," " while the use
of relatively high dilution of cellular constituents leads to disruption of labile associations
(see Figures 3 and 4). The demonstration of actin (and in all probability actin-associated
structural proteins) as a major adsorbent in nonmuscle tissue suggests that the basic principles
so far defined as governing enzyme organization in muscle apply to other cells and tissues
as well.
There may be one major exception to this general conclusion and this concerns the
association of glycolytic enzymes with the red cell membrane, a phenomenon of considerable
attention ever since Green et al.' suggested that the entire glycolytic sequence was mem-
brane-bound in these cells. Many glycolytic enzymes are found associated with isolated red
blood cell ghosts, notably glyceraldehyde-3-phosphate dehydrogenase,9"-9' aldolase,' and
phosphofructokinase." Indeed glyceraldehyde-3-phosphate dehydrogenase is one of the ma-
jor membrane-associated proteins (band 6) of erythrocyte ghosts prepared by conventional
procedures." Steck and colleagues have studied the binding of several enzymes to isolated
ghosts and have concluded that the cytoplasmic domain of the major transmembrane protein
band 3 is the exclusive site for the binding of aldolase, glyceraldehyde-3-phosphate dehy-
drogenase, and phosphofructokinase."-"-90.9' The association of these enzymes with band
3 proteins has so far been reported using very low ionic strength conditions. Steck et al.
Volume II 15
have commented on the extreme electrostatic character of the interaction between the very
acidic N-terminal domain of band 3 and these essentially basic enzyme proteins." While
band 3 may well prove to be an important binding site for enzymes on these membranes,
the question of the site (or sites) of enzyme binding should be held open at least until enzyme-
band 3 interactions have been demonstrated using physiologically relevant conditions of
ionic strength and enzyme concentration. Such caution is warranted given the artifactual
associations between glycolytic enzymes and very acidic macromolecules which occur at
low ionic strength." It is important to note that actin is also a major protein associated with
erythrocyte ghosts and in association with spectrin and other polypeptides forms the mem-
brane associated cytoskeleton of the red cell." Given the well-established interactions of
glycolytic enzymes with actin and actin-associated proteins, it is reasonable to suspect that
the actin-containing red cell cytoskeleton may well provide sites for enzyme binding. The
work of Yeltman and Harris' on the association of aldolase and glyceraldehyde-3-phosphate
dehydrogenase to red cell actin highlights this possibility and suggests that further investi-
gations are needed to unambiguously identify the enzyme binding sites in red cells.
The possible involvement of the other major cytoskeletal elements, microtubules, and
intermediate filaments in enzyme binding remains largely unexplored. Certainly in fetal calf
brain, the evidence does not favor any association between microtubules and glycolytic
enzymes."
III. DYNAMICS OF ENZYME ORGANIZATION
A. Metabolic Dependence of Enzyme Binding

Having established that each cell has a given quantity and pattern of potential binding
sites, defined mainly by the nature of the cytoskeletal proteins, it is important from a
functional point of view to assess to what extent that potential is expressed and what influence
that expression may have on metabolic control. An examination of the data in Table 1,
which documents the extent of binding in a variety of tissues, indicates that the expression
of binding varies both from tissue-to-tissue and from enzyme-to-enzyme. Part of this variation
must be due to different adsorbents, but it is now evident that the enzyme binding pattern
is also dependent on the metabolic state of the tissue. As well as showing that binding is a
dynamic process, it indicates that the expression of enzyme binding capacity may be mod-
ulated by environmental and metabolic factors.
The dependence of enzyme binding on metabolic state was first demonstrated in 1967 by
Starlinger who found that electrical stimulation of rat skeletal muscle in the live anesthetized
animal led to a large increase in aldolase binding, which quickly returned to resting levels
on cessation of stimulation." Much later, this finding was substantiated in a study of enzyme
binding as a result of electrical stimulation of post-mortem bovine psoas muscle.' In addition
to increases in aldolase binding, marked increases in phosphofructokinase and glyceralde-
hyde-3-phosphate dehydrogenase binding were observed in association with an accelerated
glycolytic flux. In the post-mortem situation the binding was not reversed upon cessation
of stimulation.
Subsequently we have examined in detail the reversible binding of glycolytic enzymes in
various skeletal muscles of the live anesthetized sheep under a variety of metabolic condi-
tions.' Figure 9 shows the effect of tetanic electrical stimulation on the binding of these
enzymes in sheep semitendinosus muscle. During the 4-min stimulation period there was a
marked decrease in muscle glycogen and a concomitant increase in tissue lactate indicative
of a considerable activation of glycogenolysis and glycolysis. Concurrently there was a
significant increase in the binding of three enzymes, aldolase, phosphofructokinase, and
glyceraldehyde-3-phosphate dehydrogenase. None of the other enzymes displayed detectable
changes in binding. The increases in aldolase, phosphofructokinase, and glyceraldehyde-3-
Sheep M. Semitendinosus
00 q Control
1. Stimulated
40
* p <0 005
BOUND
30
20
HK GPI PFK A LD TPI GPDH PGK PGM ENOL PK LDH
FIGURE 9. Binding of glycolytic enzymes in control and tetanically stimulated semitendinosus muscles from
the live, anesthetized sheep. (Taken from the data of Walsh, T. P., Masters, C. J., Morton, D. J., and Clarke,
F. M., Biochem. Biophvs. Acta, 675, 29, 1981. With permission.)
phosphate dehydrogenase binding were rapidly reversible and binding levels quickly returned
to control values, if the muscles were allowed to recover in the live animal following the
stimulation.
The increase in enzyme binding levels on stimulation was not restricted to M. semiten-
dinosus, but was observed with other muscles as well. Figure 10 compares the enzyme
binding patterns in four sheep hind limb muscles either at rest, after tetanic stimulation, or
following tetanic stimulation under ischemic conditions. After tetanic stimulation, charac-
teristic response patterns are observed; M. semitendinosus, M. biceps femoris, and M.
semimembranosus all respond with an increase in the binding of phosphofructokinase, al-
dolase, and glyceraldehyde-3-phosphate dehydrogenase. In each muscle, considerable de-
pletion of glycogen and accumulation of lactate occurs. Under the same conditions, M.
gracilis, a muscle anatomically opposed to the others fails to show any evidence of glyco-
genolysis or anaerobic glycolysis and is characterized by no observable increase in enzyme
binding levels. The metabolic response of a given muscle to stimulation depends upon a
number of physiological parameters, not the least of which is the provision of adequate
blood supply. Marked reductions in regional blood supply to muscles occur during sustained
contractions which leads to decreased oxygen tension and consequently a greater dependence
on glycogenolysis and anaerobic glycolysis to meet the energy demands of the working
muscle. This appears to be the situation in three of the four muscles examined in the tetanic
stimulation experiments summarized in Figure 10, and in each case an increase in enzyme
binding occurs. The different anatomical location of M. gracilis opposed to the other three
muscles presumably results in a less restricted blood flow during tetanic stimulation which
may explain the absence of both glycogenolytic and enzyme binding responses.
A corollary to these considerations is that muscle working under enforced ischemic con-
ditions should exhibit even greater changes in enzyme binding and this appears to be the
case (Figure 10). When stimulated under ischemic conditions all muscles including M.
gracilis show highly significant glycogen loss and lactate accumulation and all respond to
the situation by displaying a maximum increase in the level of enzyme binding. Particularly
noteworthy are the very high levels of phosphofructokinase binding at 80 to 90%. The link
between glycogenolysis, anaerobic glycolysis, and the enzyme binding response is further
Volume 11 17
100 ST
80
8
03
60
Lu
40
N
20
ALDOLASE GPDH GLYCOGEN

30
25
20
CIS
PFK ALDOLASE GPDH LACTATE GLYCOGEN

100 SM 30 30
25 25
20 20
ts,
15
10 10
D
0
ALDOLASE GPDH
100
80
03
60
12
.
20
0
PFK ALOOLASE GPOH LACTATE GLYCOGEN
FIGURE 10. Comparison of glycolytic enzyme binding and metabolite levels in four hind limb muscles of the
anesthetized sheep following (0) no stimulation; tetanic stimulation; and (U) tetanic stimulation under ischemic
conditions. ST, M. semitendinosus; B.F, M. biceps femoris; SM, M. semimembranosus; and G, M. gracilis. (Data
taken from Walsh et al., Biochem. Biophys. Acra, 675, 29, 1981 and Walsh et al., unpublished data. With
permission.)
strengthened by experiments involving prior glycogen depletion or by the use of trained or

preconditioned animals. The prior removal of muscle glycogen stored by acute adrenalin
treatment prevents the usual changes in enzyme binding induced by a subsequent tetanic
stimulation." In trained muscles, where there is a shift to greater aerobic capacity and
decreased demand for glycogenolysis upon stimulation, there is also a diminished enzyme
binding response.
The dynamics of enzyme binding can be studied most satisfactorily in the ischemic heart
of anesthetized sheep. The heart may be exposed and sequential biopsy samples can be
rapidly obtained from the same region of the heart, following induction of ischemia. Ho-
mogenization and separation of the soluble and particulate phases may begin within 10 sec
of obtaining samples. Results obtained with three different sheep hearts are shown in Figure
I 1 and these illustrate the dynamic nature of enzyme binding. The time course of enzyme
binding shows that there are rapid fluctuations in the binding levels of aldolase, phospho-
fructokinase, and glyceraldehyde-3-phosphate dehydrogenase following the onset of is-
chemia. These oscillations in enzyme binding levels are reminiscent of and follow a com-
parable time course to the fluctuations in glycolytic intermediates and glycolytic flux known
to occur under such conditions.99 '°' While there are some not unexpected differences of
detail in the responses of individual hearts, the overall similarity of the enzyme binding
response patterns is striking and collectively they serve to establish that rapid oscillations
in glycolytic enzyme organization occur in response to changing metabolic demands.
B. Effectors of Binding
As to the mechanisms by which enzyme adsorption may be modified, it has been found
that the binding in vitro is dependent on pH and the level of specific metabolites. Aldolase
(ALD) binding to actin-containing filaments is sensitive to low, but physiological, concen-
trations of its substrate fructose bisphosphate which with increasing concentration causes
elution of bound enzyme."'" On the other hand, fructose bisphosphate has little or no effect
on the binding of other enzymes which are influenced in turn by the level of other specific
metabolites or effectors. For example, glyceraldehyde-3-phosphate dehydrogenase (GPDH)
binding is sensitive to adenosine-5'-triphosphate (ATP) and NADH levels, more so than
any other enzyme.' The protocol developed by Scopes for the purification of glycolytic
enzymes using sequential elution by metabolites of the enzymes bound to a cellulose phos-
phate column is probably a useful guide to which effectors are significant for any particular
enzyme.'
It has been proposed"." that the distribution of enzymes between soluble and particulate
phases in vivo is controlled by the level of specific function-dependent metabolites. Direct
demonstrations of the operation of this mechanism in vivo are scarce. One of the few
satisfying demonstrations was provided by Knull et al. '°3 who observed that the binding of
hexokinase (HK) in brain was dependent on the energy status of the tissue. The partitioning
of the enzyme between free and bound forms was well correlated with the intracellular levels
of ATP and glucose-6-phosphate and the known influence of these metabolites on hexokinase
binding to mitochondria in vitro. On the other hand, in recent studies of variations in
glycolytic enzyme binding in stimulated sheep skeletal muscle, no simple correlations were
found." In the case of aldolase, binding increased in both tetanized ischemic and tetanized
normal muscles (Figure 9), but in the first instance fructose bisphosphate levels rose while
in the second they fell. The failure to observe the expected correlation between binding and
metabolite levels warrants particular consideration. First, enzyme binding in vivo may be
influenced by the simultaneous operation of two or more competing effects. Aldolase, for
example, is susceptible to elution by fructose bisphosphate, but at the same time it is sensitive
to pH so that as the pH falls aldolase binding increases. The net effect is that as the pH
falls sensitivity to fructose bisphosphate elution declines. Thus the metabolite fructose bis-
Volume II 19
100 100
,GPDH
90 ,/ ALD
90
r PFK
/PFK
80 80
ALD
70 70 _
%ENZYMEBOUND
60 60 PDH
50 50
40 40
//
30 30 /
20 20 _
10 10_
11111
100 150 200 250 50 100 150 200 250
TIME ( seconds) TIME (seconds)
100
90
80
70
%ENZYME BOUND
60
50
40
30
20 PFK
10
1 1 I I 1 1 I 1 I 1 1
50 100 150 200 250
TIME (seconds)
FIGURE I I. Comparison of glycolytic enzyme binding in three sheep hearts following onset of ischemia. Enzyme
binding levels in serial biopsy were determined as described previously. (Data. taken from Walsh et al., Biochem.
Biophys. Acta, 675, 29, 1981. With permission.)
phosphate is not the sole determinant of aldolase binding and its influence may be modulated
by other effectors.
The second consideration is whether metabolites influence binding in the complex mixtures
found intracellularly as they do in simpler in vitro systems. There is evidence that while
bound aldolase is susceptible to elution by fructose bisphosphate in a simple two component
system, this is not the case in more complex mixtures. Aldolase binding to actin filaments
carried out in the presence of a concentrated muscle extract (myogen) is increased, in fact,
by the addition of fructose bisphosphate." Furthermore, recent studies of aldolase binding
to myofibrils reveal that sensitivity to fructose bisphosphate elution is markedly decreased
in the presence of glyceraldehyde-3-phosphate dehydrogenase when both enzymes are bound
at the same time. We must conclude that while the potential exists for specific metabolites
and effectors to modulate enzyme binding, the conditions under which these effects are
expressed remain to be determined.
Enzyme binding may be modified not only by specific effectors of enzyme structure but
also by effectors of the adsorbent structure. For example, we have already noted that aldolase
binding to actin-tropomyosin-troponin filaments is sensitive to micromolar concentrations
of Ca' ."." It is entirely possible that the mechanical signal of the interaction of the myosin
head with the actin filament could induce changes in the nature and affinity of enzyme
binding sites.
Still other mechanisms can be proposed to account for the regulation of the metabolic
dependence of binding. An attractive candidate is the phosphorylation of either the enzyme
or the adsorbent. The phosphorylation of both tropomyosin and troponin have been de-
scribed,'" although no clearcut evidence has been forthcoming as to the consequences
of the phosphorylation as far as Ca" regulation and muscle function are concerned. Sim-
ilarly the phosphorylation of a number of glycolytic enzymes is known to occur, notably
phosphofructokinase as well as enolase and lactate dehydrogenase." 10 As yet there is no
available information as to the influence of this covalent modification on enzyme binding.
C. Genetic Determinants of Binding

The enzyme binding pattern of different cells and tissues is influenced by polymorphism
of both the enzyme and the adsorbent protein. The influence of the various isoforms of
tropomyosin and troponin has already been dealt with in Section II.C. Glycolytic enzymes
also display considerable polymorphism as there are tissue specific isoenzyme forms of
aldolase, lactate dehydrogenase, phosphofructokinase, pyruvate kinase, and others."' 14
There is certainly evidence of differential binding properties within a number of these
isoenzyme systems. Aldolase A binds much better than aldolase C,77-7 while the muscle
form of lactate dehydrogenase LDH-5 is adsorbed more strongly than any of the other lactate
dehydrogenase isoenzymes. "5 The permutations of isoenzyme variation and adsorbent poly-
morphism provide an extensive repertoire of patterns of enzyme binding available for each
cell.
IV. FUNCTIONAL SIGNIFICANCE OF BINDING
A. Influence on Catalytic Expression

It has been amply demonstrated that the kinetic and control parameters of enzymes such
as phosphofructokinase, "6'117 glyceraldehyde-3-phosphate dehydrogenase,'' and aldolase' '9
are substantially altered when bound to structural proteins. On this basis, it has been suggested
that partitioning of enzymes between free and bound forms may constitute an important
mechanism for enzyme regulation.5' " For example, aldolase when bound to F-actin-tro-
pomyosin-troponin filaments has markedly altered kinetic parameters. The Vr„a, increases
approximately fourfold and the K0, increases from 1 to 100 µM in the absence of calcium
Volume II 21
1.0 2.8
2.6
.9
2.4
.8 2.2
.7 2.0
RELATIVE ACTIVITY
1.8
FRACTIONBOUND
.6
1.6
.5 1.4
1. 2
.4
1.0
.3 .8
20
.2 .6
.4 10
.1
.2
I I I
.2 .4 .6 .8 1.0 1.2 1.4 1.6 1.8 2.0 .2 .4 .6 .8 1.0 1.2 1.4 I.6 1.8 2.0
X X
A B
FIGURE 12. Potential influence of binding on the expression of aldolase activity. Part B shows the expression
of aldolase activity at different substrate concentrations as a function of a hypothetical binding effector x on the
assumption that aldolase binding depends solely on x as shown in part A, i.e., according to the function:
x"
Fraction aldolase bound = where in the example shown n = 4
I 4- x"
Consequently the rate of the aldolase reaction (v) at any value of .v is defined by
V, [FBP] V, [FBP]
v = [Fraction soluble] • + [fraction bound]
K, + [FBPJ K2 + [FBP]
x" V, [FBP] .v" + V, [FBP]
- I +
I + xn K, + [FBP] I + .v" K, + [FBP]
where V, = V„„„ of soluble enzyme; K, = K,,, of soluble enzyme; V, = V,,„ of bound enzyme; and K, = K,,,
of bound enzyme.
In the numerical examples shown in part B the following parameters have been set, V, = I. V2 = 4, K, = 1
p.M, and K, = 100 p.A4 (see Walsh et al. "9), and the aldolase activity (relative to that of the soluble enzyme) has
been calculated as a function of x at the substrate concentrations indicated.
ions. As a consequence, the bound enzyme may be more or less active than the soluble
enzyme depending on the prevailing fructose bisphosphate concentration. As a result, par-
titioning of aldolase between free and bound forms could provide a versatile mechanism for
modulating catalytic activity. The potential of this mechanism is illustrated by the numerical
example in Figure 12 which shows the influence of binding on aldolase activity over a range
of substrate concentrations using the kinetic parameters for free and bound enzyme previously
obtained by Walsh et al.' It can be seen that binding can lead either to inhibition at low
fructose bisphosphate concentrations or activation at high concentrations.
A further observation is that when bound to F-actin-tropomyosin-troponin filaments, the
kinetic parameters of aldolase are altered not only compared to the free enzyme, but they
are rendered calcium sensitive. In the presence of low (100 pLM) concentrations of Ca' ,
the K,, of the bound enzyme changes from 100 to 20 RM with only minimal effects on the
V„,a„, a change which results in higher catalytic activity at lower fructose bisphosphate
concentrations. Calcium ions have no effect on the kinetic properties of the soluble enzyme.
The influence of Ca" on bound aldolase must be indirect and arises as a result of Ca"
binding to troponin. Presumably some form of conformational change in filament structure
is transmitted to the bound enzyme leading to a kinetically modified form of the aldolase.
The end result of binding may not only be simple modifications of kinetic properties, but
also an indirect allosteric regulation by effectors which have no access or influence upon
the soluble form of the enzyme.
While these concepts are of general application they may be particularly relevant to the
role of aldolase in the regulation of glycolysis. In the past a possible regulatory role for
aldolase has sometimes received comment,' on the basis that it is frequently the enzyme
with the lowest catalytic activity in the pathway. Other studies have shown that the intra-
cellular reaction catalyzed by aldolase is not at equilibrium.121-1" In the absence of a known
allosteric mechanism for the regulation of aldolase, it has not been recognized as a point of
control. The active partitioning of aldolase between free and bound forms on the other hand,
may just provide the means for such a role. The case for the involvement of aldolase in the
control of glycolysis has received support from the detailed computer simulation studies of
Garfinkel and Achs.""'•1 O1 A major conclusion from their analyses of ischemic glycolysis in
the heart is that aldolase is an important control element and this control might be exerted
through interaction of aldolase with structural proteins. The dynamic character of aldolase
binding in ischemic cardiac muscle (Figure 11) is consistent with the interpretation by
Garfinkel and Achs, and oblige a reassessment of aldolase in terms of control function.
Phosphofructokinase, on the other hand, has long been held as the key regulatory enzyme
of glycolysis due to the role of its substrates and other metabolites as allosteric effectors of
its catalytic capacity.'" As phosphofructokinase is known to bind to actin-containing fila-
ments with high affinity," and further, as there are large fluctuations in phosphofructokinase
binding as a function of metabolic state,"." it is relevant to examine how reversible binding
may effect the regulatory features of this enzyme. The accumulating evidence suggests that
the effects indeed may be profound. Karadesh and Uyeda' '6 examined phosphofructokinase
bound to red cell membranes and Liou and Anderson"' examined the enzyme bound to
actin-containing filaments; both groups report the desensitization of the bound enzyme to
allosteric inhibition. Binding to structural proteins appears to partially reverse inhibition by
high ATP, decrease the apparent affinity for fructose-6-phosphate (F6P), augment activation
by adenosine-5'-phosphate (AMP), and decrease inhibition by citrate. The sum total of these
effects would suggest that the bound enzyme may be a more efficient catalyst under intra-
cellular conditions. It has been calculated on the basis of the kinetic properties of the isolated
enzyme that the activity of a soluble phosphofructokinase would be almost negligible in the
presence of physiological concentrations of substrates and negative effectors. 125 Relief from
this allosteric inhibition with binding would provide an effective means for the rapid activation
of phosphofructokinase. Two metabolites, glucose-l,6-bisphosphate1L6•' -7 and the more re-
cently discovered fructose-2,6-bisphosphate,'25• 128.'" fulfill a similar function as potent ac-
tivators of phosphofructokinase activity by desensitizing the enzyme to allosteric inhibition.
Indeed, fructose-2,6-bisphosphate is now considered to be the most potent and significant
of the regulators of phosphofructokinase activity. While there is no denying the importance
of this metabolite in regulating phosphofructokinase and glycolysis in many circumstances,
Volume II 23
e.g., in liver metabolism, there are other situations where activation of phosphofructokinase
and glycolysis cannot be explained either by the action of fructose-2,6-bisphosphate or by
the more traditional allosteric regulators.109-125 In cardiac muscle during the induction of
anoxia or in skeletal muscle during electrical stimulation, there is a rapid activation of
phosphofructokinase and glycolysis, yet at the same time the level of fructose-2,6-bisphos-
phate falls.'" As Hue points out, when the provision of energy by glycolysis becomes an
issue, fructose-2,6-bisphosphate is probably not the major regulator of phosphofructokinase
activity. 125 It is precisely in these circumstances that increased phosphofructokinase binding
may exert its most important effect as it has the potential to bring about the rapid activation
of this enzyme. On the other hand, Hue has indicated that given the relatively low catalytic
capability of phosphofructokinase-2/fructose-2,6-bisphosphatase, changes in fructose-2 ,6-
bisphosphate concentration are relatively slow and require several minutes for completion.
Such an effector system is not well suited when rapid changes in flux are required. Activation
of phosphofructokinase by binding and activation by fructose-2,6-bisphosphate may have
complementary roles in regulation depending on the metabolic circumstances. It remains to
be determined whether fructose-2,6-bisphosphate can effect either the binding or kinetics of
bound phosphofructokinase.
Up to this point we have dealt only with the functional properties of single enzymes
oscillating between free and bound forms. We can now examine the characteristics of those
enzymes which form functional groups or clusters. In Section II.D it was shown that when
aldolase and glyceraldehyde-3-phosphate dehydrogenase bind, triosephosphate isomerase
may also complex by piggy-backing. This means that not only is there the juxtapositioning
of two enzymes carrying out sequential reactions in glycolysis, but also the integration of
triosephosphate isomerase as the enzyme responsible for a third catalytic function at this
branch point of glycolysis. Figure 13 illustrates this filament-based enzyme cluster. The
incorporation of phosphoglycerate kinase into this mini-complex as preliminary evidence
suggests, results in enzyme clusters which would have interesting functional attributes. This
form of enzyme complex allows for a phenomenon which Welch has termed 'the spatial
translocation of intermediate metabolites' otherwise known as metabolic channeling in which
the product of one enzyme is transferred directly to the active site of the next enzyme in
the pathway, without the release of the product to the medium.'9 Patthy and Vas have already
provided evidence of the channeling of glyceraldehyde-3-phosphate between aldolase and
glyceraldehyde-3-phosphate dehydrogenase when these enzymes form a complex in solu-
tion. '31 It is not unreasonable to suppose that channeling would be facilitated by the formation
of an adsorbent-linked complex as suggested in Figure 13. Among the relevant features of
metabolic channeling are that it allows a reduction of transit times, the maintenance of low
intracellular concentrations of intermediates, and the segregation of potentially competing
pathways. In the example shown in Figure 13, the metabolic channeling produced by a
filament-based aldolase-triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogen-
ase-phosphoglycerate kinase system could be envisaged as providing ATP directly at the
site of utilization which is the actomyosin ATPase. Such a process of directional or vectorial
translocation of metabolites has been termed vectorial catalysis by Welch.' As early as
1957, Kamiya and Hatano established that the immediate energy source for protoplasmic
streaming in Physarum was 'ATP synthesized by the process of fermentation' .'"-I" Gibbins
found that the inhibitor 2-deoxyglucose caused an abrupt cessation of membrane ruffling in
epithelial cells." In a recent study Bricknell et al. found that in perfused cardiac muscle,
ATP produced by glycolysis was better able to delay or prevent the onset of ischemic
contracture than was ATP generated by oxidative phosphorylation.134 This may well imply
that the ATP supplied by glycolysis is provided directly to the contractile apparatus. A
filament-based organization of enzymes would provide for the efficient delivery of the ATP.
In theory, actin-adsorbed enzyme clusters could exhibit unique regulatory features not
FIGURE 13. Glycolytic enzyme organization via the cytoskeleton — metabolic channeling. An illustration of a
possible actin-filament based glycolytic enzyme cluster involving aldolase, GPDH, TPI, and PGK indicating its
potential for metabolic channeling and direct delivery of ATP to the actin-myosin interface. Note that PFK stands
alone and is not integrated into the proposed cluster. See text for full discussion.
shared by the component enzymes. We have already shown that the association of aldolase
with F-actin-tropomyosin-troponin renders the kinetic properties of the enzyme sensitive to
Ca — . The organization and, hence, the function of enzyme clusters could also be controlled
by Ca" in the same way. Since the binding of a myosin head to an actin-containing filament
is known to cause considerable changes in the filament structure,59 °' this mechanical signal
might well result in a change in the ordering of enzyme binding sites and so induce the
formation of multienzyme clusters. If this was so, then there would be a mechanism for
directly integrating the metabolic and motile behavior of cells. The relevance of such a
mechanism is that it would serve to account for the very close coupling observed between
the initiation of muscle contraction and the activation of glycolysis. Using NMR techniques
on muscle, Wilkie et al.'" have shown that changes in ATP, ADP, and AMP cannot account
for the rapid activation of glycolysis induced by muscle contraction. Consequently, they
concluded that glycolytic activation is directly linked to the contractile cycle.
It should be noted that phosphofructokinase has not been integrated into the proposed
mini-complex shown in Figure 13. The reasoning for this is based upon a consideration of
the molar quantities of phosphofructokinase present in cells which is at least an order of
magnitude less than any other glycolytic enzyme." Quite simply, there would not be a
sufficient number of molecules of phosphofructokinase available for integration into the
enzyme clusters we have postulated. The role of phosphofructokinase would be in the
provision of substrate for these clusters and achieve control by way of its well-established
regulatory features as discussed previously.
B. Structural Considerations
Glycolytic enzymes comprise close to 20% of total muscle protein' so changes in enzyme
binding not only imply translocation of enzyme activity, but also the sequestration of sub-
Volume II 25
stantial amounts of protein on the actin filament. This is particularly so in the case of aldolase
and glyceraldehyde-3-phosphate dehydrogenase which together represent 40% of the total
glycolytic enzyme molecules present in muscle. By contrast phosphofructokinase constitutes
less than 0.3%. If we consider the binding of aldolase and glyceraldehyde-3-phosphate
dehydrogenase in bovine muscle there are approximately 20 nmoles of each enzyme bound
per gram of muscle and this increases to about 30 nmoles bound following electrical stim-
ulation." It can be calculated that this would represent one of each of these enzyme molecules
bound per repeat of the actin helix, i.e., one aldolase and one glyceraldehyde-3-phosphate
dehydrogenase for each 14 actin monomers. Simple modeling studies predict that this increase
in mass of the actin filament should be readily detectable by X-ray diffraction techniques.
This was substantiated by a study of the equatorial X-ray diffraction pattern of glycerinated
muscle in the absence and presence of aldolase and glyceraldehyde-3-phosphate dehydro-
genase when changes in diffraction intensities of the magnitude predicted were obtained. '1'
Preliminary studies show that enzymes are bound in such a way that there is no interference
with the essential actin-myosin interaction.
Examination of electron microscope images of longitudinally sectioned muscle sometimes
show periodic densities along the I-band filaments, which are generally attributed to the
presence of troponin on the filament at 38 nm intervals. L" This density is certainly not
invariably present on the filament while we must presume that troponin invariably is. On
this account, we suggest that the origin of the structure seen is a cluster of enzyme molecules
as proposed in the earlier sections. Whether these enzymes are capable of cross-linking the
I-band filaments to stabilize the three-dimensional array of actin filaments, remains to be
determined.
Nonmuscle cells have an elaborate cytoskeletal network, consisting of four principal
elements. These are the microtubules,32 the intermediate filaments,33 and two actin-based
systems, microfilaments29.31." and the microtrabecular lattice recently described by Por-
ter.'0.21 •38-39 Cytoskeletal actin may thus be organized into a variety of forms ranging from
large stress fibers through thick filamentous networks to the fine microtrabecular lattice.
The stress fibers are relatively large three-dimensional bundles of actin filaments held together
by cross-linking proteins. In appearance and dimensions, they are not dissimilar to the
structures formed when aldolase or pyruvate kinase cross-link actin-containing filaments
(Figure 2). A number of actin-binding proteins have been described which could possibly
be the cross-linking agents for the formation of stress fibers.' " We are attracted to the
possibility that glycolytic enzymes, which were the first actin-binding proteins to be de-
scribed,52 participate in cross-linking. The localization of glyceraldehyde-3-phosphate de-
hydrogenase and pyruvate kinase on the stress fibers seen in Figure 8 is encouraging evidence
of such an involvement in stress fiber structure.
Actin is also to be found in the cell cortex in association with the cell membrane. In this
situation the actin network is involved in membrane ruffling'-`'-"' and membrane receptor
mobility. "9-'40 Glycolytic enzymes are shown to be localized in these same regions of the
cell cortex by immunofluorescence.' The abrupt arrest of epithelial cell membrane ruffling
by the glycolytic inhibitor, 2-deoxyglucose observed by Gibbins," suggests the involvement
of glycolytic enzymes may go beyond the provision of energy for the process. In recent
publications Porter and colleagues2O.21 have alluded to the possible involvement of glycolytic
enzymes and other "soluble" proteins as structural elements of the metastable microtra-
becular lattice.
While it will prove to be difficult to dissect the energy yielding aspects of glycolysis from
the structural properties of glycolytic enzymes, we can observe the suitability of enzymes
as cross-linking agents. The glycolytic enzymes which are most firmly bound are all multi-
subunit proteins with multiple sites for binding to structural proteins." As multivalent ligands
they could promote the formation either of large filament bundles or gel-like networks formed
with smaller oligomeric or monomeric forms of structural proteins.' Then modification of

cross-linking by enzyme effectors would lead to considerable effects on the organization of
the cytoskeleton. In this context, metabolites, Ca", and other effectors of binding take on
an added significance as potential modifiers of cytoskeletal structure as well as metabolic
activity. In this way, modifiers of glycolytic behavior and this would include a wide range
of hormones and cell regulators, could influence cell functions dependent upon actin. Gly-
colysis with the functional duality of its component enzymes could provide the integrating
element in many cell structure-function relationships. It is perhaps no coincidence that cells
with extensive motile properties such as macrophages and tumor cells also have a high
glycolytic potential.
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thin filaments. Biochemistry, 19, 2684, 1980.
118. Dagher, S. M. and Hultin, H. 0., Association of glyceraldehyde-3-phosphate dehydrogenase with the
particulate fraction of chicken skeletal muscle, Eur. J. Biochem., 55, 185, 1975.
119. Walsh, T. P., Clarke, F. M., and Masters, C. J., Modification of the kinetic parameters of aldolase on
binding to actin-containing filaments of skeletal muscle, Biochem. J.. 165, 165. 1977.
120. Scrutton, M. C. and Utter, M. F., The regulation of glycolysis and gluconeogenesis in animal tissues.
Ann. Rev. Biochem., 37, 249. 1968.
121. Newsholme, E. A. and Start, C., Regulation in Metabolism, John Wiley & Sons. London, 1976.
122. Ottaway, J. H. and Mowbray, J., The role of compartmentation in the control of glycolysis. Curr. Top.
Cell. Regul., 12, 107, 1977.
123. Williamson, J. R., Glycolytic control mechanisms. II. Kinetics of intermediate changes during the aerobic-
anoxic transition in perfused rat heart, J. Biol. Chem., 241. 5026, 1966.
124. Uyeda, K., Phosphofructokinase, Adv. Enzymol., 48, 193, 1979.
125. Hue, L., Role of fructose-2,6-bisphosphate in the regulation of glycolysis, Biochem. Soc. Trans., II. 246,
1983.
126. Beitner, R., Haberman, S., and Livni, L., Complementarity in the regulation of phosphoglucomutase,
phosphofructokinase, and hexokinase: the role of glucose- I ,6-bisphosphate, Biochem. Biophys. Acta. 397,
355, 1975.
127. Beitner, R., The role of glucose-1,6-diphosphate in the regulation of carbohydrate metabolism in muscle,
Trends Biochem. Sci., 4, 228, 1979.
128. van Schaffingen, E., Jett, M. F., Hue, L., and Hers, H. G., Control of liver 6-phosphofructokinase by
fructose-2,6-bisphosphate and other effectors, Proc. Natl. Acad. Sci., 78, 3483. 1981.
129. Uyeda, K., Furuya, E., and Luby, L. J., The effect of natural and synthetic D-fructose-2.6-bisphosphate
on the regulatory kinetic properties of liver and muscle phosphofructokinase, J. Biol. Chem., 256. 8394.
1981.
130. Hue, L., Blackmore, P. F., Shikama, H., Robinson-Steiner, A., and Exton, J. H., Regulation of
fructose-2,6-bisphosphate content in rat hepatocytes. perfused hearts, and perfused hind limbs, J. Biol.
Chem., 257, 4308, 1982.
131. Patthy, L. and Vas, M., Aldolase-catalysed inactivation of glyceraldehyde-3-phosphate dehydrogenase.
Nature (London), 276, 94, 1978.
132. Kamiya, N., Nakajima, H., and Abe, S., Physiology of the motive force of protoplasmic streaming.
Protoplasma, 48, 94. 1957.
133. Hatano, S. and Takeuchi, I., ATP content in myxomycete plasmodium and its levels in relation to some
external conditions, Protoplasma, 52. 169, 1959.
134. Bricknell, 0. L., Daries, P. S., and Opie, L. H., A relationship between adenosine triphosphate, glycolysis
and ischemic contracture in the isolated rat heart, J. Mol. Cell. Cardiol., 13. 941, 1981.
135. Wilkie, D. R., The control of glycolysis in living muscle studied by nuclear magnetic resonance and other
techniques, Biochem. Soc. Trans., II, 244. 1983.
136. Stewart, M., Morton, D. J., and Clarke, F. M., Changes associated with glycolytic enzyme binding in
the equatorial X-ray diffraction pattern of glycerinated rabbit psoas muscle, Biochem. J., 183. 663. 1979.
137. Franzini-Armstrong, C., Details of the I-band structure as revealed by the localization of ferritin. Tissue
Cell, 2, 327, 1970.
138. Schliwa, M., Proteins associated with cytoplasmic actin, Cell, 25, 587, 1981.
Volume 11 31
139. Nicolson, G., Transmembrane control of the receptors on normal and tumor cells. I. Cytoplasmic influence
over cell surface components, Biochem. Biophvs. Acta, 457, 57, 1976.
140. Mescher, M. F., Jose, M. J. L., and Balk, S. P., Actin-containing matrix associated with the plasma
membrane of murine tumor and lymphoid cells, Nature (London), 289, 139, 1981.
141. Magri, E., Zaccarini, M., and Grazi, E., Versatility of G-actin as the building block of biological
structures, FEBS Lett., 89, 276, 1978.
142. Clarke, F. M., Stephan, P., Huxham, G., Hamilton, D., and Morton, D. J., Metabolic dependance
of the glycolytic enzyme binding in rat and sheep heart, Eur. J. Biochem., 138, 643, 1984.
143. Clarke, F. M., Stephan, P., and Morton, D. J., unpublished observations.
144. Walsh, T. P., Clarke, F. M., and Morton, D. J., unpublished data.
145. Clarke, F. M., Morton, D. J., Hamilton, D., and Huxham, G., unpublished data.
Taylor & Francis
Volume II 33
Chapter 2
REGULATION OF GLYCOGEN METABOLISM
Alisa Gutman
TABLE OF CONTENTS
I. Introduction 34
A. Functions of Glycogen in Specific Tissues 34
1. Liver 34
2. Muscle 34
3. Other Tissues 35
B. Enzymes of Glycogen Metabolism — Overview of Recent
Developments 35
1. Glycogen Synthase 36
II. Phosphorylase Kinase 36
A. Muscle Phosphorylase Kinase 36
1. Structure 36
2. Activity in Resting Muscle 36
a. Phosphorylase Kinase a 36
b. Phosphorylase Kinase b 37
3. Activity in Contracting Muscle 37
a. Effects of Calmodulin 37
b. Effects of Troponin 38
c. Phosphorylase Kinase Deficiency 38
B. Liver Phosphorylase Kinase 38
III. Glycogen Synthase Kinase 39
A. Classification 39
B. Muscle Glycogen Synthase Kinases 39
I. Hormonal Effects 39
a. Epinephrine 39
b. Insulin 40
C. Liver Glycogen Synthase Kinases 41
D. Other Tissues 41
IV. Phosphoprotein Phosphatase 42
A. Development of Concepts 42
B. Classification of Protein Phosphatases 42
1. Type-I Protein Phosphatase 43
2. Type-2 Protein Phosphatases 43
a. Protein Phosphatase 2A 43
b. Protein Phosphatase 2B 43
c. Protein Phosphatase 2C 44
C. Tissue Distribution of Phosphatases 44
D. Heat-Stable Inhibitors 44
1. Inhibitor-1 44
a. Phosphorylation 44
V. Summary and Conclusions 46
References 47
I. INTRODUCTION
Studies on the regulation of glycogen metabolism during the last 25 years have provided
important insights into the basic aspects of metabolic regulation. Regulation of enzyme
activity by phosphorylation-dephosphorylation was first described by Krebs and Fischer' for
the reversible activation of phosphorylase and has since been shown to be a widespread
mechanism for metabolic control.' Cyclic AMP, which is now known to be involved in the
regulation of most known metabolic pathways, was first isolated by Sutherland' in connection
with its role as an activator of phosphorylase and the mechanism of its action as a "second
messenger" of hormone action was established when it became evident that the hypothetical
"phosphorylase kinase kinase" was in reality a nonspecific protein kinase that was activated
by cyclic AMP.'
Synthesis of glycogen depends on the availability of substrate, usually glucose, and on
the existence of hormonal or metabolic signals that will channel these substrates to glycogen
by activating glycogen synthase, the rate limiting enzyme on the pathway between glucose-
6-phosphate and glycogen. Conversely, glycogen breakdown depends on the availability of
glycogen stores and on hormonal or other signals which will cause activation of the rate
limiting enzyme — phosphorylase.
The state of activity of these opposing pathways at any given moment depends on the
hormonal and metabolic make-up of the different tissues which, in turn, is intimately related
to the function of glycogen in the specific tissue. In a very broad sense it is possible to
divide tissues of higher organisms into three broad categories.
A. Functions of Glycogen in Specific Tissues

I. Liver
The liver serves as a buffer to maintain a constant level of blood glucose by uptake of
glucose and its conversion to glycogen when glucose supply is abundant and through release
of glucose by breakdown to glycogen between meals. Both glycogen synthesis and glycogen
breakdown respond to changes in the metabolic (nutritional) or hormonal environment. The
importance of liver glycogen synthesis and breakdown for overall metabolism can be seen
by looking at subjects in whom liver glycogen breakdown does not take place because of a
congenital enzyme deficiency. These patients cannot maintain blood glucose levels during
fasting and are absolutely dependent on a constant supply of glucose or glucose precursors
from external sources.5
2. Muscle
Muscle glycogen serves as an important source of energy for muscle contraction, although
a major portion of the energy supply of muscle comes from breakdown of fatty acids. In
the absence of muscle phosphorylase (McArdle's disease) patients are unable to carry out
strenuous exercise which would depend on anaerobic breakdown of glycogen.5 Although
the obligatory participation of glycogen in muscle contraction is limited, glycogen breakdown
does normally occur during muscle contraction and is immediately followed by resynthesis.'
Glycogen breakdown is coordinated with muscle contraction by the action of calcium ions
on activation of phosphorylase kinase.' The second mechanism activating this system is
activation of phosphorylase kinase by cyclic AMP-dependent protein kinase.' In this case,
cyclic AMP levels are increased as a result of a "fight and flight" response of the sympathetic
nervous system acting through catecholamine induced activation of adenylate cyclase.9 Re-
synthesis of glycogen following muscular contraction is facilitated by inactivation of phos-
phorylase and, to some extent, accumulation of glucose-6-phosphate.6 On the other hand,
muscle takes part in the Cori cycle and plays a role in the postprandial maintenance of blood
Vohone // 35
glucose concentration by an insulin induced coordinated increase in glucose uptake' and

activity of glycogen synthase."
3. Other Tissues
In most other tissues glycogen seems to be of lesser importance for the specific functions
of the tissue, and little is known about metabolic or hormonal factors affecting it. In adipose
tissue glycogen synthase is activated by insulin and by glucose'2 ' 4 and large quantities of
glycogen accumulate during refeeding after fasting, prior to renewed synthesis of fatty
acids.I 5 Endometrial glycogen almost disappears during the first half of the menstrual cycle
and increases markedly during the luteal phase and this hormone induced accumulation of
glycogen may be a prerequisite for successful implantation. '6 Glycogen metabolism of uterine
muscle also seems to be under control of sex hormones and is markedly increased during
the last stages of gestation." In polymorphonuclear leukocytes phagocytosis may be the
physiological stimulus for activation of phosphorylase and subsequent breakdown of gly-
cogen, although it is also possible to demonstrate effects of catecholamines. '8
B. Enzymes of Glycogen Metabolism — Overview of Recent Developments

Recent progress in our understanding of the mechanism by which hormonal or other
factors affect enzyme activities has been mainly in the field of glycogen synthase activation
and inactivation. In the area of glycogen breakdown the most important recent work has
been on the interaction of cyclic AMP and calcium as activators of phosphorylase kinase
and this topic will be further discussed in Section II.
I. Glycogen Synthase
In spite of the above-mentioned differences in the hormonal and metabolic regulation of
glycogen metabolism in different tissues, the changes occurring at the molecular level in
the various tissues studied are remarkably similar. Glycogen synthase was first described in
1957 by Leloir and Cardini.19 It soon became apparent that the enzyme existed in two forms
which could be distinguished by their dependence on glucose-6-phosphate for activity. Villar-
Palasi and Lamer" demonstrated that incubation of diaphragm with insulin increased the
activity measured in the absence of the activator glucose-6-phosphate, without changing
total activity measured at high glucose-6-phosphate concentration. The concept which de-
veloped out of these studies in the early 1960s was that glycogen synthase existed in a
phosphorylated form that depended on glucose-6-phosphate for activity (D) and a nonphos-
phorylated glucose-6-phosphate independent (I) form." It also became apparent, mainly
through the work of Hers and his group at the University of Louvain,'-' that in the liver the
D (called b by this group) form was inhibited by physiological concentrations of phosphate
ions and that its activity was not regulated by the intracellular glucose-6-phosphate concen-
tration. Piras et al.22 found that the activity of the D form of the muscle enzyme was inhibited
(competitively with glucose-6-phosphate) by adenine nucleotides, especially by ATP. Glu-
cose-6-phosphate could activate the D form only when its concentration was very high, a
situation which occurred only for very short periods immediately following muscular
contraction.6
The general concept emerging from these studies was that in vivo only the I (or a) form
of glycogen synthase was active and that glycogen synthesis was regulated by an on/off
mechanism, coordinated through cyclic AMP-dependent protein kinase, that activated phos-
phorylase kinase and inactivated glycogen synthase." It was also understood that reactivation
of glycogen synthase and inactivation of phosphorylase were the result of the activities of
two separate phosphatases, synthase phosphatase and phosphorylase phosphatase. There
were many data that did not exactly fit into this scheme, but real progress in our understanding
of the complexities of activation and inactivation of glycogen synthase was made only when
purification of the phosphatases and kinases revealed that instead of one cyclic AMP-
dependent kinase phosphorylating glycogen synthase at one site, several kinases, including
phosphorylase kinase," were involved in phosphorylation of glycogen synthase at multiple
sites.'S-31 Activation of glycogen synthase by dephosphorylation was found by most authors
to result from the activity of one or more phosphoprotein phosphatases which acted on
phosphorylase a and on phosphorylase kinase as well as on glycogen synthase.32-34 The
situation was further complicated by the discovery that phosphatase activity can be modified
by specific inhibitors of phosphoprotein phosphatase activity' and that the activity of one
of these inhibitors is under hormonal control.'"
II. PHOSPHORYLASE KINASE
Phosphorylase has been known for many years to exist in a less active (nonphosphorylated)
b form and in an (active) phosphorylated, a form.' The mechanism of activation of muscle
phosphorylase by phosphorylase kinase and the interrelations between activation of this
enzyme and the electrical stimulus initiating muscle contraction have been clarified in recent
years and will be discussed in this section. The inactivation of phosphorylase and of phos-
phorylase kinase will be discussed together in Section IV.
A. Muscle Phosphorylase Kinase

I. Structure
Muscle phosphorylase kinase is composed of four subunits, designated alpha, beta, gamma,
and delta having subunit molecular weights of 145,000, 130,000, 45,000,'y4° and 17,000,
respectively.4 ' Cyclic AMP-dependent protein kinase phosphorylates one serum residue on
the beta and alpha subunits, respectively, but only the phosphorylation of the beta subunit
is important for activity."'" " The 45,000 dalton gamma subunit is the catalytic unit. The
delta subunit, which is tightly bound to the gamma subunit, has recently been shown to be
identical with calmodulin and is responsible for the Ca" dependence of the enzyme."
2. Activity in Resting Muscle

a. Phosphorylase Kinase a
The concentration of Ca" ions in muscle sarcoplasma is 0.1 µ/14 or less in resting muscle
and increases to the micromolar range after electrical stimulation." According to Cohen et
al] the A 0.5 of phosphorylase kinase b for Ca" is 20 µM; activity of the enzyme, even
after electrical stimulation, depends on conversion to the a form, which has an A 0.5 for
Ca' of 1.6 µM,7 or on the presence of factors which increase the affinity of the b form
for Ca". A phosphorylation induced decrease in the requirement of phosphorylase kinase
for Ca' was also found by Brostrom et al." but the A 0.5 for Ca' of phosphorylase kinase
found by these authors was lower by an order of magnitude. A relatively low A 0.5 of
phosphorylase kinase b for Ca" may have been caused by trace limited proteolysis,' and
further work will be necessary to resolve these discrepancies.
Conversion of phosphorylase kinase b to a (or a') can be achieved by several mechanisms
which modify the alpha and beta subunits:
1. Phosphorylation by cyclic AMP-dependent protein kinase.35.47.4"

2. Autophosphorylation in the presence of ATP-Mg and Ca' ."
3. Limited proteolysis by an endogenous Ca" dependent proteinase" or by trypsin (a'
form).39 The a' enzyme species is partially active even in the absence of Ca' .39
The physiologically important mechanism for conversion of phosphorylase b to a is

phosphorylation by cyclic AMP-dependent protein kinase. In muscle, catecholamine binding
Volume II 37
to beta adrenergic receptors is the only physiologically significant mechanism for increasing
the concentration of cyclic AMP.5' 54 At physiological pH and Ca" concentrations prevailing
in electrically stimulated muscle, the activity of phosphorylase kinase a is about a hundred
times higher than that of phosphorylase kinase b.7 On the other hand, at the Ca" concen-
tration of resting muscle (less than 0.1 p,M), activity of phosphorylase kinase a would be
only 5% of maximal activity.' Since phosphorylase b is converted to phosphorylase a
following administration of epinephrine to resting muscle,5 ' 55 it has to be assumed that
under these conditions additional factors are operative. Such factors could be 56
1. An epinephrine induced increase in sarcoplasmic Ca" concentration to levels which

are below the threshold necessary to induce muscle contraction — In liver, epinephrine
induces an increase in cytoplasmic Ca" levels which is mediated by binding of the
hormone to alpha adrenergic receptors.57 In muscle, binding of epinephrine has been
shown to be to beta receptors only," and no hormonal effects on Ca' levels have
been demonstrated.
2. Inhibition of phosphatase activity — Activity of phosphoprotein phosphatase-1, which
dephosphorylates the beta subunit of phosphorylase kinase and phosphorylase a' is
inhibited by inhibitor-1.58 Activity of this inhibitor depends on phosphorylation by
cyclic AMP-dependent protein kinase which has been shown to be increased after
administration of epinephrine to ratS. 4638'58 A full discussion of protein phosphatase
inhibitors will be given in Section IV.
b. Phosphorylase Kinase b
In resting muscle virtually all the enzyme is in the b form which, under standard conditions
of assay, is almost inactive at physiological pH (6.8), but is active at pH 8.2.52.53 The activity
ratio at pH 6.8/8.2 is, therefore, used to distinguish between the b form which has an activity
ratio of 0.01 and 0.02 and the a form which has an activity ratio of 0.36.39 Phosphorylase
kinase b has been shown to contain 2.5 to 3 mol of Pi/monomer (alpha, beta, and gamma),
but the origin and localization of these Pi found in phosphorylase kinase b has not been
clarified.59 Cyclic GMP-dependent protein kinase has recently been found to phosphorylate
phosphorylase kinase in vitro, but it is doubtful whether this activity is of physiological
importance."
3. Activity in Contracting Muscle

a. Effects of Calmodulin
Electrical stimulation of muscle results in muscle contraction and activation of phospho-
rylase kinase without conversion to the a form.52.53 The activity of phosphorylase kinase
under these conditions results from the increase in Ca' concentration from less than 0.1
pM to 1.5 pM.45 In the absence of additional factors this concentration of Ca" would not
be sufficient to account for the activity of phosphorylase kinase observed in vivo. Recent
work of Cohen et al. in vitro has demonstrated that in the presence of Cat ', addition of
calmodulin to the assay increased the activity of phosphorylase kinase.7.6'.62 The added
calmodulin binds to the alpha and beta subunits, and binding can be prevented by the addition
of trifluoperazine or of the calmodulin binding protein calcineurin.63 The activity of the
native enzyme that contains calmodulin (the delta subunit) bound to the gamma subunit is
not affected by trifluoperazine or by calcineurin. 62.63 As expected, binding of calmodulin is
not found after removal of the alpha and beta subunits by trypsin.7 64 The observation that
calmodulin inhibits phosphorylation of the beta subunit of phosphorylase kinase by cyclic
AMP-dependent protein kinase may be taken as additional proof that the binding of cal-
modulin is to the beta subunit of phosphorylase kinase.'
b. Effects of Troponin
Activation of phosphorylase kinase b can also be observed after addition of troponin,
which is 50% homologous to calmodulin, to the assay.'''." The concentration of troponin
in muscle is about 100 1.i./144' and the concentration required for activation of phosphorylase
kinase is 1 p.M.24 The suggestion by Cohen et al.7.61 -" that troponin, rather than calmodulin,
is the physiological activator of phosphorylase kinase b is based on several arguments:
1. The A 0.5 for Ca' of the calmodulin activated enzyme is 20 p.M and little activation
of phosphorylase kinase b can, therefore, be expected with physiological concentrations
of Ca".
2. Calmodulin bound to phosphorylase kinase as the delta subunit makes up about 35 to
40% of the total amount of calmodulin in fast twitch muscles." Binding of additional
calmodulin to phosphorylase kinase would leave little calmodulin for other functions
in which it is known to be involved.
3. The A 0.5 for Ca' in the presence of troponin is 4 which is close to the
concentration prevailing in muscle, following electrical stimulation. At 1 p.M Ca'
addition of troponin to phosphorylase kinase b caused a 15-fold stimulation, whereas
conversion to the a form increased activity 150-fold. At 3µM Ca' the troponin or
phosphorylation induced increases in activity of phosphorylase kinase were 25- and
100-fold, respectively. Both calmodulin and troponin had little effect on the activity
of phosphorylase kinase a.7.61
Stimulation of phosphorylase b activity by troponin can provide the link between electrical
stimulation of muscle leading to increased levels of Ca" in the sarcoplasm and activation
of glycogenolysis. If, in addition to the electrical stimulus, sympathetic activity is increased,
conversion of phosphorylase kinase b to a would lead to a further fourfold increase in
phosphorylase kinase activity (at 3 p.M Ca" ).'
c. Phosphorylase Kinase Deficiency

In ICR/An mice, which lack muscle phosphorylase kinase, electrical stimulation of muscle
does not result in conversion of phosphorylase b to a.67 •6" Nevertheless, after a lag period
of several seconds, glycogenolysis takes place."' The lag period seems to be necessary for
the accumulation of metabolites that enable phosphorylase b to be active. The most likely
metabolite responsible for the stimulation of phosphorylase b activity in these animals is
5'IMP which increases markedly during muscular contraction."
B. Liver Phosphorylase Kinase

Liver phosphorylase kinase is present at a much lower concentration than the muscle
enzyme, and has not been as extensively studied. In general, the liver enzyme seems to
resemble muscle phosphorylase kinase." It is interesting that the Ca' requirement of the
liver enzyme is in the range occurring in liver cytosol, which is lower than in muscle
sarcoplasma.69 Conversion to the a form by cyclic AMP-dependent protein kinase has been
demonstrated." In vivo glucagon is the most important activator of liver adenylate cyclase.''
The effects of catecholamines are species-dependent: in rat liver epinephrine, angiotensin
II and vasopressin stimulate glycogenolysis by mobilization of Ca' from intracellular
stores.57.72 In rats deficient in liver phosphorylase kinase (gsd/gsd), cytoplasmic Ca" levels
increase in hepatocytes incubated with the alpha adrenergic agonist phenylephrine, but
activation of phosphorylase does not take place.' In contrast to the active glycogenolysis
occurring in the muscle of phosphorylase kinase deficient mice," there is virtually no
breakdown of glycogen in the liver of phosphorylase kinase deficient rats.'
Volume II 39
III. GLYCOGEN SYNTHASE KINASE
A. Classification
The in vivo activity of glycogen synthase depends on the affinity of the enzyme for the
substrate UDP-glucose, the activator glucose-6-phosphate, and other activators and inhibitors
(mainly ions and adenine nucleotides) normally present in the cell. Dephosphorylation of
glycogen synthase causes an increase in affinity of the enzyme for glucose-6-phosphate and
UDP-glucose and changes in the affinity for other ligands,21 •22 resulting in higher activity
under in vivo conditions in which concentrations of effectors are nonsaturating.6 The most
widely used working definition of the activity state of glycogen synthase is based on the
activity ratio ( — ) glucose-6-phosphate/( +) glucose-6-phosphate which is as low as 0.04 in
a highly phosphorylated enzyme" and may be as high as 0.85 to 0.90 in a maximally
dephosphorylated form." Following the discovery of multiple phosphorylation of glycogen
synthase, it became necessary to correlate phosphorylation of specific sites with changes in
the activity ratio or with changes in the K for glucose-6-phosphate or the K,„ for UDP-
glucose.
Glycogen synthase is phosphorylated by at least three different classes of kinases:
1. Cyclic AMP-dependent protein kinase27.29."-"

2. Cyclic AMP-independent protein kinases25 3°.76
3. Phosphorylase kinase24."
B. Muscle Glycogen Synthase Kinases

The most detailed analysis of the various kinases involved in phosphorylation of muscle
glycogen synthase, and of the sites on the 90,000 dalton subunit of the enzyme that are
phosphorylated by the different kinases, has been presented by Cohen et al.29 According to
these authors six different kinases are involved in the inactivation of glycogen synthase. In
addition to cyclic AMP-dependent protein kinase and phosphorylase kinase, they have sep-
arated from rabbit skeletal muscle 3 cyclic AMP-, Ca' -, and calmodulin-independent
kinases termed glycogen synthase kinase (GSK) 3, 4, and 576 as well as a Ca' -dependent
kinase, which is not identical with phosphorylase kinase.'
In accordance with other investigators,"2•"6 Cohen et al.29 also found that phosphorylation
of the 90,000 dalton subunit of glycogen synthase took place on an amino acid near the
amino terminal end of the peptide chain, designated CB-1 or trypsin insensitive domain,
and on a larger (approximately 140 residues) fragment near the COOH terminal of the chain
termed CB-2 or trypsin sensitive domain. The cyanobromide fragment CB-2 is not always
equivalent to the fragment obtained by treatment with trypsin, since the exact site of cleavage
by trypsin has been shown to depend on the concentration of trypsin used.' Further treatment
of the CNBr fragments with trypsin or chymotrypsin, and two dimensional electrophoresis
of the fragments obtained, enabled the exact localization of 7 phosphorylation sites29 (Table
1).
I. Hormonal Effects
a. Epinephrine
Most of the work on the characterization of the various kinases has been carried out on
skeletal or cardiac muscle, in which glycogen synthesis is activated by insulin and inactivated
by epinephrine.75•85-88 The physiological importance of the different kinases was studied by
following the phosphorylation pattern and kinetic properties of the enzyme obtained after
treatment of rabbits with epinephrine or insulin. In nonstimulated muscle, phosphorylation
of glycogen synthase by epinephrine was found to be 2.4 and 3 mol Pi/90,000 subunit by
Sheorain et al." and Cohen et al.," respectively. The administration of epinephrine resulted
Table 1
PHOSPHORYLATION SITES ON GLYCOGEN SYNTHASE
Distance from N
terminus Distance from
Site (residues) COOH terminus Kinase(s)
la 53 c'AMP dependent
lb 40 Protein kinase
2 7 Phosphorylase kinase, cal-
modulin dependent phos-
phorylase GSK4
3a 110
3b 106 GSK3
3c 102
5 94 GSKS
Data from Ingebritsen, T. S., Stewart, A. A.. and Cohen, P.,

Eur. J. Biochem., 132, 297, 1983. With permission.
in an increase in phosphorylation of 1.5 to 2.0 mol Pi/subunit in rat54 and rabbit muscle.75
Sheorain et al." found that 76% of the additional Pi was in the trypsin sensitive domain
(assumed to be in sites la + b which is phosphorylated by cyclic AMP-dependent protein
kinase).
On the other hand, Parker et al.75 analyzing the phosphate content of all 7 sites found
similar increases in phosphate content of sites 1 a + b (phosphorylated by cyclic AMP—
dependent protein kinase), site 2 (phosphorylated by cyclic AMP-dependent protein kinase,
phosphorylase kinase, glycogen synthase kinase 4, and calmodulin-dependent kinase), and
of sites 3a, b, and c (phosphorylated by the cyclic AMP-independent GSK3). Since the
localization of site 3 in the trypsin sensitive or insensitive region depended on the conditions
under which the treatment with trypsin was carried out,75 these results of Parker et al. were
not necessarily at variance with those of Sheorain et al.," but they raised the question how
an increase in the concentration of cyclic AMP, induced by treatment with epinephrine,
could increase phosphorylation of a site phosphorylated by a cyclic AMP-independent kinase.
Parker et al.75 suggested that the increase in Pi content of site 3 was due to a decreased
activity of phosphatase-1, the major phosphoprotein phosphatase acting on glycogen syn-
thase, phosphorylase, and phosphorylase kinase." Activity of phosphatase-1 is inhibited by
inhibitor-1 which is activated by cyclic AMP-dependent protein kinase; increased phosphor-
ylation of inhibitor-1 in response to epinephrine administration in vivo has been
demonstrated.3"
b. Insulin
The effect of insulin on glycogen metabolism is, in general, opposite to that of epinephrine.
Although an effect of insulin on the level of cyclic AMP or on the activity of cyclic AMP-
dependent protein kinase has been postulated,9° insulin can activate glycogen synthase with-
out any involvement of cyclic AMP, as has been shown in the perfused heart." Analysis
of fragments obtained by treatment of purified muscle glycogen synthase from insulin treated
rabbits demonstrated that insulin reduced the phosphorylation in the trypsin insensitive
domain of the enzyme, in which only cyclic AMP-independent kinases are located."'
Similar results have been obtained by Parker et al.,87 who found that the administration of
insulin to rabbits results in a specific dephosphorylation of sites 3a, b, and c and no change
in the phosphorylation state of any other site. This effect could not be ascribed to activation
of phosphoprotein phosphatase-1 or -2 since these phosphatases would have, at least in vitro,
dephosphorylated site 2 as well as, or even better than, site 3.32
Volume II 41
The hypothesis put forward by Parker et al." was that since GSK3 specifically phosphor-
ylated sites 3a, b, and c, the effect of insulin on the phosphorylation of these sites was due
to inhibition of GSK3 by insulin. This effect of insulin is in contrast to the effect of
epinephrine, that was assumed to promote phosphorylation of sites 3a, b, and c by a cyclic
AMP-mediated activation of phosphatase- l inhibitor." An alternative explanation may be
that insulin, or the recently discovered "second messenger" produced by interaction of
insulin with the cell membrane,"" "2 modifies the site on glycogen synthase that is phosphor-
ylated by glycogen synthase kinase, making it thereby less accessible to the kinase. The
involvement of GSK3 in the activation of phosphoprotein phosphatase- l will be further
discussed in Section IV. D.2.
C. Liver Glycogen Synthase Kinases

Glycogen synthase kinases acting on the liver enzyme have not been studied in as much
detail as the muscle system, but the existence of cyclic AMP-dependent and -independent
kinases has been demonstrated.94.95 In addition, a Ca2 ±-calmodulin dependent glycogen
synthase has also been found in liver.96 Recent work by Strickland and his colleagues has
stressed the role of phosphorylase kinase as an indirect modulator of glycogen synthase
activity in liver."' Using both normal and phosphorylase kinase deficient rats, these authors
have shown that the addition of ATP-Mg to a Sephadex® filtrate from normal liver slowed
the rate of glycogen synthase activation and that this effect was Ca2 + -dependent since it
could be blocked by the addition of EGTA. Furthermore, ATP-Mg had no effect in filtrates
from phosphorylase kinase deficient rats or in preparations from which phosphorylase had
been removed. These results were considered as confirming the hypothesis originally pro-
posed by Hers and his colleagues98-99 that liver glycogen synthase activity was regulated by
an inhibitory effect of phosphorylase a on glycogen synthase phosphatase. According to
Hers"' the activity of phosphorylase a could be modified by cyclic AMP-induced activation
of phosphorylase kinase and by glucose induced changes in the conformation of phospho-
rylase a, which increased its rate of dephosphorylation by phosphorylase phosphatase.1 O1 'O4
Activation of liver glycogen synthase by glucose has been demonstrated by Hers and his
group both in vivo99.w4." and in vitro,9831°" and this observation has been confirmed in other
laboratories. 106 109 " I 39 Such a mechanism can be considered as being extremely efficient for
"pulling" glucose (or glucose-6-phosphate) in the direction of glycogen synthesis when
glucose concentration in the portal vein exceeds a threshold value of 7 mM.1 °""0S 0 Although
an effect of glucose on activation of glycogen synthase can also be demonstrated in other
tissues, 14,108.110 it is doubtful whether glucose concentration in the systemic circulation of
the normal organism would ever reach the required threshold. The data presented by Strick-
land et al.' suggest a direct effect of phosphorylase a on the activity state of glycogen
synthase and provide a mechanism to explain Ca"-dependent effects on the activity of liver
glycogen synthase. It should, however, be mentioned that the notion that inactivation of
phosphorylase is a prerequisite for activation of glycogen synthase has been challenged by
work from several laboratories,106.109,111 113 and that the data presented by Strickland et al."
that showed activation of glycogen synthase in the liver of phosphorylase kinase deficient
rats to be inhibited by the residual phosphorylase a activity in these preparations have been
differently interpreted by other investigators. 13
D. Other Tissues
It is not known at present whether the same phosphorylation sites are present on glycogen
synthase from other tissues. Cardiac glycogen synthase kinases have been found to be similar
to the skeletal muscle kinases."4 Glycogen synthase from polymorphonuclear leukocytes
can be phosphorylated by cyclic AMP-dependent and -independent kinases which also seem
to be similar to the muscle enzyme.31 •84 Inactivation of glycogen synthase by cyclic AMP-
dependent protein kinase seems to be a general phenomenon, but no detailed information

on cyclic AMP-independent glycogen synthase kinases in other tissues is available.
IV. PHOSPHOPROTEIN PHOSPHATASE
A. Development of Concepts
Inactivation of phosphorylase by a "prosthetic group removing enzyme" (PR enzyme)
was described by Cori and Green"' as early as 1943, and this enzyme was shown by
Sutherland and Wosilait' '" to be a phosphatase that catalyzed the removal of Pi from phos-
phorylase a. Glycogen synthase phosphatase activity was first demonstrated by Friedman
and Larner" in 1963, and phosphorylase kinase activity was described by Riley et al.' ''
in 1968. Although it was originally assumed that dephosphorylation of the three enzymes
was catalyzed by different enzymes, early attempts at purification of glycogen synthase
phosphatase revealed that the partially purified enzyme had a broad substrate specificity and
was able to act on phosphorylase a and phosphorylase kinase as well as on histone and
casein. "8 '" These observations raised the possibility that dephosphorylation of the three
enzymes was controlled by a single-broad specificity phosphoprotein phosphatase, but further
work showed the presence of multiple phosphoprotein phosphatases of widely varying mo-
lecular weights, which could be separated from each other by gel filtration or ion exchange
chromatography. 121-123
Nevertheless, the concept of a single phosphoprotein phosphatase gained support when
Lee et al. '24-' 28 showed that a high molecular weight phosphoprotein phosphatase-H (Mr
260,000) could be broken down by treatment with 80% ethanol to Mr 30 to 40,000 fragments,
which were later designated phosphoprotein phosphatase-C.126 The same group isolated from
tissue extracts a heat stable and trypsin sensitive inhibitor of phosphorylase phosphatase and
proposed the concept that the multiple forms of phosphoprotein phosphatase were complexes
of the catalytic unit (phosphoprotein phosphatase-C) with the heat stable inhibitor. ' 27 Further
work by Huang and Glinsmann35 that was later confirmed by other laboratories 129.130.112.167
revealed that skeletal muscle contained 2 heat stable inhibitors, called inhibitors 1 and 2,
respectively. Inhibitor I was active only after phosphorylation by cyclic AMP-dependent
protein kinase.35.129,131.132.167 A detailed discussion of these inhibitors will be given at the
end of this Section (IV .D).
In recent years the simple concept of a single phosphoprotein phosphatase of broad
specificity acting on glycogen synthase, phosphorylase a, and phosphorylase kinase had to
be modified following results obtained by different groups working in this field (for detailed
reviews see References 33, 34, 59, and 135). One of the main difficulties encountered in
attempts to purify phosphorylase (or glycogen synthase) phosphatase is the extreme variability
in molecular weight of the enzyme(s) obtained, which makes it difficult to compare prep-
arations obtained by different laboratories. Other criteria to characterize the various enzyme
preparations such as cation dependence, sensitivity to inhibition by the heat-stable inhibitors,
and activity on well-defined substrates have not been used widely enough to draw clear
conclusions on the identity of phosphatases purified by different groups of investigators. An
additional factor responsible for differences between proteins isolated by different groups is
related to the methods employed for purification. The presence of Mg" or Mn' ions has
a stabilizing effect, whereas ethanol may selectively inactivate some enzyme activities.'34
Another factor adding to the prevailing confusion is the presence or absence of cations such
as Mn' , Mg" , or Ca" in the assay mixture, and the use of phosphorylase kinase phos-
phorylated in the alpha or beta subunit by only one laboratory.133
B. Classification of Protein Phosphatases

The most comprehensive classification of phosphoprotein phosphatases of skeletal and
Volume II 43
heart muscle, liver, brain, and adipose tissue has been proposed by Cohen and his col-
leagues.32 ' 35 The basis for classification in this system is the specificity of the protein
phosphatases for the alpha or beta subunits of phosphorylase kinase and the sensitivity to
the heat-stable inhibitors 1 and 2. Type-1 protein phosphatase is defined as acting on the
beta subunit of phosphorylase kinase and being subject to inhibition by inhibitors 1 and 2.
Type-2 protein phosphatases (2A, 2B, and 2C) act on the alpha subunit of phosphorylase
kinase and are not inhibited by the heat-stable inhibitors.12.89.'35
I. Type-1 Protein Phosphatase

In skeletal muscle, phosphoprotein phosphatase-1 accounts for 80 to 90% of phosphatase
activity against phosphorylase a, 70 to 80% of the activity against glycogen synthase, and
more than 90% of phosphatase activity acting on the beta subunit of phosphorylase kinase.
About 50% of the enzyme is bound to the glycogen pellet. "5 The Mr of the catalytic subunit
is 33,000,'' and its activity is not dependent on metal ions." Regulation of the activity of
protein phosphatase-1 by the heat-stable inhibitors will be discussed in Section IV.D.
2. Type-2 Protein Phosphatases

a. Protein Phosphatase-2A
The three type-2 protein phosphatases, act on the alpha subunit of phosphorylase kinase
and are not affected by the heat-stable inhibitors. Protein phosphatase-2A has the same
substrate specificity as protein phosphatase-1, and like protein phosphatase-1 it is not de-
pendent on metal ions for activity. During purification of protein phosphatase-2A, three
high molecular weight fractions 2A0, 2A1, and 2A2, all of which yielded the same Mr
34,000 catalytic subunit, were obtained." The high molecular weight protein phosphatase-
2A1 is apparently identical with protein phosphatase IB isolated by Tsuiki and his colleagues
from rat liver.137• 13" This enzyme is composed of three polypeptide chains of Mr 69,000,
58,000, and 35,000, respectively. Protein phosphatase 2A2, which is similar to the II form
of Tamura et al.,'" seems to have been generated from protein phosphatase-2A1 during
purification by dissociation of the Mr 58,000 subunit.'35 Similar enzymes have also been
isolated from rabbit liver and canine heart by Lee33 and Li 34 and their co-workers, respec-
tively. Protein phosphatase-C of Lee and his co-workers'-0 has been resolved into two (Mr
32,000 and 34,000) components corresponding to protein phosphatases-1 and 2A,
respectively.''
b. Protein Phosphatase-2B
Protein phosphatase-2B is a Ca"- and calmodulin-specific enzyme that has no significant
activity on any protein with the exception of the alpha subunit of phosphorylase kinase,
heat-stable inhibitor-1, and myosin light chains.'33 " This enzyme has been shown to be
similar to the calcium binding protein isolated from the brain called calcineurin.63 The enzyme
is activated by micromolar concentrations of Ca' (found in contracting muscle but not in
muscle at rest"), and is also active in the presence of (nonphysiological) concentrations of
manganese ions. Calmodulin increases the activity of this enzyme against inhibitor-1 but
has no effect on the activity against phosphorylase kinase, which contains calmodulin as an
intrinsic part of the enzyme (the delta subunit). Protein phosphatase-2B has been resolved
by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate into two
major subfractions of Mr 61,000 and 15,000 designated A and B, respectively.'6' On the
basis of work by Klee et al.63 on calcineurin, Cohen and his colleagues' speculated that
component A was the catalytic subunit and component B was a calcium ion binding protein.
In the presence of Ca", calmodulin could bind to an additional site on the catalytic unit,
thereby increasing activity tenfold.
c. Protein Phosphatase-2C
Protein phosphatase-2C is a Mr 46,000 magnesium ion-dependent enzyme which de-
phosphorylates the alpha subunit of phosphorylase kinase and glycogen synthase, but has
almost no activity against phosphorylase a."' This phosphatase seems to be identical with
protein phosphatase IA of Hiraga et al. 134 and with a similar Mr 43,000 protein phosphatase
isolated by McKenzie et al.'6
C. Tissue Distribution of Phosphatases

Analysis of the tissue distribution of the protein phosphatases (Table 2) revealed that
skeletal muscle was the only tissue in which protein phosphatase (PP)-1 was the predominant
enzyme, and that had virtually no PP-2C activity. In all other tissues examined, PP2A was
the major protein phosphatase and there was also a significant amount of PP-2C which may
be regarded as a "glycogen synthase phosphatase". '3' The isolation and characterization of
PP-2C will perhaps resolve the dispute between most investigators who had found only
broad spectrum protein phosphatases (PP-1 and PP-2A), and others who had evidence for
the existence of a separate glycogen synthase phosphatase in liver. 134,143,164 Cohen and his
colleagues tried to assess the relative contribution of the different phosphatases to dephos-
phorylation of the enzymes involved in glycogen metabolism.'' In resting muscle only PP-
1 and PP-2A had any significant activity. PP-2A contributed only 6 and 12% to dephos-
phorylation of the beta subunit of phosphorylase kinase and of phosphorylase a, respectively,
whereas its relative contribution to dephosphorylation of sites 1 a, 2, and 3a + b + c was 35,
24, and 24%, respectively. At Ca" concentrations prevailing during muscular contraction,
PP-2B was the major phosphatase acting on the alpha subunit of phosphorylase kinase and
on inhibitor-1. In the liver PP-2A was the predominant phosphatase but, as in muscle, its
contribution to dephosphorylation of the beta subunit of phosphorylase kinase was relatively
small (21%), and its activity towards site la on glycogen synthase was markedly higher
than on sites 2 or 3a+ b + c. PP-2C was responsible for 25% of the dephosphorylation
of site 2 and for less than 10% of the activity towards sites 1 a or 3a + b + c.''
In the liver more than 50% of PP-1 is associated with the glycogen pellet, and this enzyme
contributes only little to dephosphorylation of phosphorylated enzymes not directly involved
in glycogen metabolism. PP-2A is the major phosphatase involved in dephosphorylation of
pyruvate kinase, acetyl CoA carboxylase, and ATP-citrate lyase; PP-2C is the only phos-
phatase acting on HMG CoA reductase and HMG CoA reductase kinase.'"
D. Heat-Stable Inhibitors
I. Inhibitor-1
a. Phosphorylation
The two heat-stable inhibitors originally described by Lee and his colleagues' 27 and by
Huang and Glinsmann35 are small proteins that have been purified to homogeneity.'"'"
Inhibitor-1 is a specific inhibitor of protein phosphatase-1 that is active only after the
phosphorylation by cyclic AMP-dependent protein kinase".'" of a threonine'67 located 35
residues from the N terminal end of the molecule. Available evidence seems to indicate that
the activity of inhibitor-1 is under hormonal control.' 3"
Phosphorylation of inhibitor-1 is increased by the addition of epinephrine to muscle
perfusates.3" Foulkes et al.37 have shown that the effect of epinephrine could be antagonized
by insulin,'" but Khatra et al.' have not found an effect of insulin on the state of phos-
phorylation of inhibitor-1 in perfused rat muscle. In vivo, Shenolikar et al.'" have used
specific antibodies which recognize only the phosphorylated form of inhibitor-1 to dem-
onstrate an effect of insulin and glucagon on the phosphorylation state of inhibitor-1 in liver
and similar effects of estrogen in uterus. The existence of inhibitor- I under physiological
Volume II 45
Table 2
DISTRIBUTION OF PROTEIN
PHOSPHATASES IN RABBIT TISSUES
PP-1 PP-2A PP-2B PP-2C

(%) (%) (%) (%)
Skeletal muscle 61 19 19 1
Heart 23 67 5 5
Brain 18 61 18 3
Adipose tissue 15 60 22 3
Liver 28 59 9 4
Data from Ingebritsen, T. S., Stewart, A. A., and Cohen, P.,

Eur. J. Biochem., 132, 297, 1983.
conditions has been questioned by Laloux and Hers's' who could not detect inhibitor effects
in freshly prepared homogenates. This discrepancy may be related to the observation of
Ingebritsen et al.135 that the sensitivity of protein phosphatase-1 to inhibitor-1 in a concen-
trated tissue extract was lower than in dilute preparations.
b. Dephosphorylation of Inhibitor-1
In vitro inhibitor-1 can be dephosphorylated by protein phosphatase-1 as well as by protein
phosphatases-2A and by the Ca"-dependent phosphatase-2B.""" In contrast to the metal
ion independence of PP-1 activity against all other substrates, activity of PP-1 against
inhibitor-1 is dependent on Mn' .'3' This dependence on Mn" , which is not present in
high concentrations in cells, makes it seem unlikely that PP-1 acts on inhibitor-1 in vivo.
A "deinhibitor" isolated by Merlevede and his colleagues from dog liver's' can abolish the
Mn" dependence of PP-1, but it is not known whether this substance is present in tissues
other than dog liver. "5 In the presence of micromolar concentrations of Ca' PP-2B is the
most active phosphatase acting on inhibitor-1.32 Stewart et al.'" suggested that Ca" initiated
dephosphorylation of inhibitor-1 by PP-2B could be a mechanism for activation of PP-1
leading to replenishment of glycogen stores during muscular contraction. In the liver, PP-
2A seems to be the major phosphatase acting on inhibitor-1.
2. Inhibitor-2
Inhibitor-2 is a competitive inhibitor of the dephosphorylation of phosphorylase a by PP-
1 152 physiological role and mode of regulation of this inhibitor is unknown. Recent
studies by Cohen and his colleagues indicate that inhibitor-2 may affect the activity of PP-
1 by an additional mechanism which can be detected only at very low concentrations of the
inhibitor.16•'4° Several years ago, Merlevede and his colleagues isolated a phosphorylase
phosphatase (Fc) which was active only after preincubation with ATP-Mg and with a Mr
50,000 activating factor (Fa),I53 Further work of Merlevede with Cohen and his colleagues
demonstrated that Fc had the same substrate specificity as PP-1,I 54 and that Fa was identical
with glycogen synthase kinase 3.155 The relationship of this ATP-Mg-dependent phosphatase
to PP-1 was not clear until the demonstration by Hemmings et al. 16 that incubation of PP-
1 with low concentrations of inhibitor-2 led to the slow formation of a complex which was
identical with the ATP-Mg-dependent phosphatase. The inhibition of phosphatase-1 activity
by inhibitor-2 was abolished by phosphorylation of the inhibitor by ATP-Mg and glycogen
synthase kinase 3 (Fa).'"
A model accounting for the interrelationship between the catalytic subunit (Fe), protein
phosphatase-1, inhibitor-2, and GSK-3 (Fa) has been proposed by Merlevede and his
colleagues'" and by Ballou and Fischer. 157 According to this model, phosphorylation of the
modulator (inhibitor-2) by GSK-3 causes a conformational change in Fc which results in
activation. The activated phosphatase is able to dephosphorylate the modulator, thereby
reversing the activation of Fc. This model accounts for the observed continuous hydrolysis
of ATP during the activation process and suggests the existence of a continuous phospho-
rylation-dephosphorylation cycle. The phosphatase-modulator complex can also be activated
by proteolytic degradation of the modulator by trypsin followed by activation of the catalytic
subunit by Mn-+ .157-158
V. SUMMARY AND CONCLUSIONS
Developments during the last few years have provided many interesting data on the
regulation of glycogen metabolism by phosphorylation and dephosphorylation, but have not
contributed much to an understanding of the molecular basis and site of action of the effects
of insulin.
The "state-of-the-art" at the present time can be summarized as follows:
1. Hormones which activate adenylate cyclase induce phosphorylation of sites 1 a, 1 b,

and 2 on glycogen synthase. Protein phosphatase-2A, which is not known to be under
hormonal control, seems to have a relative preference for dephosphorylation of site
I a.
2. Phosphorylation of sites 3a+ b+ c (phosphorylated by GSK-3) is increased by epi-
nephrine and decreased by insulin treatment. Hormone mediated changes in the in-
hibition of protein phosphatase-1 by inhibitor-1 may be involved in these effects of
hormones. The relatively greater contribution of protein phosphatase-1 to dephos-
phorylation of sites 3a+ b+ c, compared to site la, would be in line with this
possibility.
3. The physiological importance of the inhibitor-2 induced transformation of protein
phosphatase-1 into the Mg-ATP-dependent phosphatase and the role of GSK-3 in this
process will have to be clarified in the future.
4. The predominant protein phosphatase of skeletal muscle is protein phosphatase-1.
Inhibition of this enzyme by inhibitor-1 may be the mechanism providing for control
of phosphatase activity by hormones.
5. In liver, the heart, and other tissues, protein phosphatase-1 (and the heat-stable in-
hibitors) are of lesser importance. Dephosphorylation of phosphorylase and glycogen
synthase in these tissues is carried out by the broad specificity protein phosphatase-
2A and by protein phosphatase-2C, which is essentially a glycogen synthase phosphatase.
6. The involvement of troponin in Ca"-induced activation of phosphorylase kinase b
and the phosphorylation of site 2 on glycogen synthase by phosphorylase kinase provide
a mechanism for activation of glycogen breakdown and inhibition of glycogen syn-
thesis, during the initial stages of muscular contraction which does not involve effects
of hormones.
Volume Ii 47
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Volume 11 49
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liver by administration of glucose, Eur. J. Biochem., 2, 50. 1967.
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108. Nuttall, F. Q., Gallon, M. C., and Earner, J., Oral glucose effect on glycogen synthetase and phos-
phorylase in heart, muscle and liver, Physiol. Chem. Phys., 4 , 497, 1972.
109. Goldstein, D. E. and Curnow, R. D., Effect of starvation on hepatic glycogen metabolism and glucose
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phosphatase activities in rat adipose tissue, Biochim. Biophys. Acta, 481, 86, 1977.
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rat, Life Sci., 23, 2429, 1978.
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a prerequisite for the activation of liver glycogen.synthase, FEBS Leo., 99, 321. 1979.
113. Watts, C., Redshaw, J. R., and Gain, K. R., The activation of glycogen synthase in hepatocytes from
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phosphorylation. Catalysis of inactivation by cAMP-dependent and cAMP-independent protein kinases and
comparison with the phosphorylation of skeletal muscle enzyme, Arch. Biochem. Biophys., 214. 311, 1982.
115. Cori, G. T. and Green, A. A., Crystalline muscle phosphorylase II. Prosthetic group, J. Biol. Chem.,
151, 31, 1943.
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(London), 175, 169, 1955.
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activation, J. Biol. Chem., 243, 2209, 1968.
118. Nakai, C. and Thomas, J. A., Substrate specificity of glycogen synthase phosphatase from bovine heart:
action on phosphorylase a and histone, Biochem. Biophys. Res. Commun., 52, 530, 1973.
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kinase by the same phosphoprotein phosphatase, Biochem. Biophys. Res. Commun., 50. 872, 1973.
120. Kato, K. and Bishop, J• S., Glycogen synthetase-D phosphatase. I. Some new properties of the partially
purified enzyme from rabbit skeletal muscle, J. Biol. Chem., 247, 7420, 1972.
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93, 1977.
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on glycogen synthase, phosphorylase and histone, J. Biol. Chem., 249, 6459, 1974.
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Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver, Biochim. Biophys. Acta, 377.
343, 1975.
124. Brandt, H., Killilea, S. D., and Lee, E. Y. C., Activation of phosphorylase phosphatase by a novel
procedure: evidence for a regulatory mechanism involving the release of a catalytic, subunit from enzyme
inhibitor complex(es) of higher molecular weight, Biochem. Biophys. Res. Commun., 61, 598, 1974.
125. Brandt, H., Capulong, Z. L., and Lee, E. Y. C., Purification and properties of rabbit liver phosphorylase
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126. Lee, E. Y. C., Mellgren, R. L., Killilea, S. D., and Aylward, J• H., Properties and regulation of liver
protein phosphatases, in Regulatory Mechanisms of Carbohydrate Metabolism. Vol. 42. Esmann, V., Ed..
Pergamon Press, New York, 1978, 327.
127. Brandt, H., Lee, E. Y. C., and Killilea, S. D., A protein inhibitor of rabbit liver phosphorylase phos-
phatase, Biochem. Biophvs. Res. Commun., 63, 950, 1975.
128. Khandelwal, R. L. and Zinman, S. M., Purification and properties of a heat-stable protein inhibitor of
phosphoprotein phosphatase from rabbit liver, J. Biol. Chem., 253, 560, 1978.
129. Cohen, P., Rylatt, D. B., and Nimmo, G. A., The hormonal control of glycogen metabolism: the amino
acid sequence at the phosphorylation site of protein phosphatase inhibitor-I, FEBS Lett., 76. 182. 1977.
130. Nimmo, G. A. and Cohen, P., The regulation of glycogen metabolism. Purification and characterization
of protein phosphatase inhibitor-I from rabbit skeletal muscle, Eur. J. Biochem., 87, 341, 1978.
131. Toth, G., Gergely, P., Parsadanian, H. K., and Bot, G., Regulation of phosphorylase phosphatase from
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phorylase phosphatase dependent on and independent of protein kinase, Eur. J. Biochem., 91, 457, 1978.
133. Cohen, P., The role of protein phosphorylation in neural and hormonal control of cellular activity, Nature
(London), 296, 613, 1982.
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glycogen synthase phosphatase (phosphoprotein phosphatase IA) from rat liver, Eur. J. Biochem., 119,
503, 1981.
135. Ingebritsen, T. S., Stewart, A. A., and Cohen, P., The protein phosphatases involved in cellular
regulation. VI. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues;
an assessment of their physiological roles, Eur. J. Biochem., 132, 297, 1983.
136. Hemmings, B. A., Resink, T., and Cohen, P., Reconstruction of a MG-ATP dependent protein phos-
phatase and its activation through a phosphorylation mechanism. FEBS Len., 150, 319, 1982.
137. Tamura, S., Kikuchi, H., Kikuchi, K., Hiraga, A., and Tsuiki, S., Purification and subunit structure
of a high molecular weight protein phosphatase (phosphatase II) from rat liver, Eur. J. Biochem., 104,
347, 1980.
138. Tamura, S. and Tsuiki, S., Purification and subunit structure of rat liver phosphoprotein phosphatase
whose molecular weight is 260000 by gel filtration phosphatase IB, Eur. J. Biochem., III, 217, 1980.
139. Ingebritsen, T. S., Foulkes, J. G., and Cohen, P., The broad specificity protein phosphatase from
mammalian liver. Separation of the Mr 35000 catalytic subunit into two distinct enzymes, FEBS Lett., 119.
9. 1980.
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regulation. V. Purification and properties of a /calmodulin dependent protein phosphatase (2B) from
rabbit skeletal muscle, Eur. J. Biochem., 132, 289, 1983.
141. Stewart, A. A., Ingebritsen, T. S., Manalan, A., Klee, C. B., and Cohen, P., Discovery of a Ca-
and calmodulin-dependent protein phosphatase. Probable identity with calcineurin (CaM-BP„(,), FEBS Lett..
137. 80, 1982.
142. Mackenzie, C. W., Bulbulian, G. J., and Bishop, J. S., Use of fluoride to inactivate phosphorylase a
phosphatase from rat liver cytosol. Presence of fluoride insensitive glycogen synthase specific phosphatase,
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The non-identity of native phosphorylase phosphatase and synthase phosphatase. Eur. J. Biochem., 92. 15,
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phosphorylase phosphatase and histone phosphatase activity in the rat liver, Biochim. Biophys. Acta, 522,
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from rabbit skeletal muscle, Eur. J. Biochem., 126, 235, 1982.
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monally directed regulation of protein phosphatase inhibitor-I, C/in. Res., 31, 529A, 1983.
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FEBS Lett., 105. 239, 1979.
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inhibitors on dog liver phosphorylase phosphatase, FEBS Lett., 79, 125, 1977.
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inhibitor-1 and inhibitor-2 on the activity of protein phosphatase-I, Eur. J. Biochem., 132, 309, 1983.
153. Goris, J., Defreyn, G., and Merlevede, W., Resolution of the ATP-Mg-dependent phosphorylase phos-
phatase from liver into a two protein component system, FEBS Lett., 99, 279, 1979.
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protein phosphatase and protein phosphatase-1 have identical substrate specificities, Eur. J. Biochem., 115,
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phosphatase in pig heart, Fed. Proc., 42, 2027, 1983.
Volume II 53
Chapter 3
EFFECTS OF THE ABNORMAL CARBOHYDRATE METABOLISM

PRESENT IN GLYCOGEN STORAGE DISEASE ON INTERMEDIARY
AMINO ACID AND LIPID METABOLISM
Shimon W. Moses, Nava Bashan, and Alfred E. Slonim
Many of Claude Bernard's pioneering observations, made 128 years ago regarding fun-
damental principles of glycogen metabolism in the mammal, are still valid today.' However,
it has since been established that the synthesis and breakdown of glycogen is a highly
complex and tightly controlled process involving substrates, intermediates, cofactors, acti-
vators, inhibitors, enzymic interconversions, and hormonal balances. Glycogen metabolism
occurs in many tissues, however, these various controls are different in each tissue and are
adapted to the particular environment and metabolic requirements of each organ. For ex-
ample, regulation of glycogen synthesis and breakdown in the mammalian liver differs
markedly from its regulation in muscle, each one serving a different function; whereas
glycogenolysis in the liver leads primarily to the liberation of glucose into the blood,
glycogenolysis in the muscle provides energy for vigorous muscle contraction.
The existence of glycogen turnover can also be demonstrated in other tissues such as red
blood cells (RBC), leukocytes and adipocytes; however, the exact controls of glycogen
metabolism in these tissues have not been studied to the same extent as in liver and muscle.
Not only do controls differ among tissues, but structurally different enzymes catalyzing
similar reactions have been shown to exist among tissues. This has been known for several
decades in respect to muscle in contrast to liver phosphorylase and has recently also been
demonstrated in regard to glycogen synthetase.2 Other tissues such as RBC contain an active
glycogen metabolism that does not lead to glycogen accumulation unless an enzyme defect
such as amylo-1,6-glucosidase, or phosphorylase deficiency is present, leading to marked
accumulation of glycogen. This deposition apparently does not compromise RBC function
or survival to a major extent.'
Glucose occupies a unique position in mammalian carbohydrate metabolism by not only
providing a substrate for glycolysis, but by being intimately involved in the regulation of
glycogen synthesis and breakdown in addition to its stimulative effects on the production
and secretion of hormones related to carbohydrate homeostasis.
Glycolysis represents the only pathway for anaerobic adenosine-5-triphosphate (ATP)
production. Some tissues such as RBC are completely dependent on glucose for ATP pro-
duction, whereas other tissues such as the brain, though normally utilizing glucose as its
main substrate, can, after a period of adaptation in the presence of low blood glucose levels,
utilize other metabolites such as lactate and ketone bodies to meet their energy requirements.'
However, in the nonconditioned individual, the brain is very sensitive to sudden decreases
in blood glucose levels. Therefore, it is not surprising that a tightly controlled mechanism
that includes several backup systems exists in order to maintain glucose homeostasis under
varying metabolic conditions. For instance, in the liver, once glycogenolysis has depleted
the easily mobilizable glycogen stores, metabolite and hormonal settings initiate gluconeo-
genesis to provide blood glucose, which, after prolonged fasting is supplemented by renal
gluconeogenesis. These backup systems provide sufficient glucose in the normal adult human
individual to sustain prolonged periods of fasting without becoming hypoglycemic. However,
this is not invariably the case in infants, where the limited availability of gluconeogenic
reserves are more likely to lead to a fasting hypoglycemia than in the adult.5 This limited
capacity to maintain euglucemia in the presence of fasting is even more compromised in
the malnourished infant, who has limited resources to provide for gluconeogenic amino
acids. In addition to the group with a normal enzymic complement but limited gluconeogenic
resources, cases with enzymatic defects either in the glycogenolytic or the gluconeogenetic
pathway are notoriously incapable of maintaining glucose homeostasis during fasting. Among
these two groups of enzymopathies, inborn errors of glycogen metabolism are more prevalent,
have been studied to a greater extent and will be discussed below.
Mutational defects have been described in most enzymes involved in glycogen breakdown
leading to complex clinical syndromes which have been designated as glycogen storage
diseases. Each one of these diseases has been numerically typed according to its specific
enzyme defect. More than a dozen different enzyme defects affecting glycogen metabolism
have been demonstrated so far. The different types can be subdivided into one group affecting
primarily the intermediary blood carbohydrate metabolism with secondary effects on lipid
and protein metabolism such as glucose-6-phosphatase deficiency (GSD type I) and amylo-
1,6-glucosidase deficiency (GSD type III), both of which will be discussed. In contrast,
other enzymatic defects are mainly characterized by myopathic features such as muscle
phosphorylase (GSD type V) and phosphohexose isomerase deficiency (GSD type VIII).
GSD type III can present both muscle and liver involvement'
Each type of glycogen storage disease has been shown to occur, in different cases, with
variably expressivity, which is not always explicable on the basis of the degree of the
enzymic inactivity. Secondary effects of the enzymopathy on intermediary metabolism have
to be considered. In addition, subtle controls of glycogen metabolism have recently been
studied in the normal mammal, such as the different kinases, both Ca" -activated and cyclic
AMP-dependent, the protein phosphatases, and their respective inhibitors. It has not been
established whether and to what degree these regulators play a role in glycogen storage
disease.
GSD type I and GSD type III are the two most common forms of glycogen storage disease.
They have many clinical similarities and are sometimes difficult to distinguish from each
other. In the young, both manifest episodes of hypoglycemia, hepatomegaly, and growth
failure. Usually, but not always, the clinical findings in GSD type I are more severe than
those found in GSD type III. Biochemically, although there are a number of similarities,
there are also clear metabolic differences that distinguish these two conditions. The clinical
manifestations of these conditions are primarily a consequence of the specific hepatic enzyme
deficiency of each condition. In GSD type I, there is a deficiency of the enzyme glucose-
6-phosphatase, which hydrolyzes glucose-6-phosphate to glucose. This enzyme is normally
present in liver, kidney, and intestinal mucosa and is deficient in these tissues in GSD type
The deficiency of hepatic glucose-6-phosphatase does not permit endogenous glucose
production by specific dephosphorylation of glucose-6-phosphate. During fasting episodes,
this leaves three potential alternative sources for the provision of glucose to the blood.
Nonspecific phosphatase can be involved in the hydrolysis of small amounts of glucose-6-
phosphate.' Acid a-glucosidase, present in lysosomes, can liberate glucose from glycogen
without traversing the glucose-6-phosphate step. This lysosomal pathway has recently been
quantitatively assayed.' Furthermore, Sadeghi-Nejad demonstrated an increased incorpora-
tion of lactate into glycogen and then subsequently into glucose, as a result of a dynamic
pool of lactate recycling between glycogen and lactate.' This cycling procedure is thought
to be the mechanism by which ,hepatic glucose production is able to be maintained to some
extent in GSD type I." The release of free glucose from the liver probably occurs mainly
by the action of amylo-1,6-glucosidase at the branch points."
However, at least in the young age groups, the fasting tolerance of glucose-6-phosphatase
deficient patients is very limited and hypoglycemia develops 2 to 6 hours postprandially.
Glucose-6-phosphate which cannot be converted into glucose is consequently directed along
the glycolytic pathway leading to increased pyruvate, lactate, alanine, acetyl CoA, and
Volume II 55
triglyceride formation." In normal subjects, circulating lactate is largely derived from red
cell and muscle glycolysis, and is utilized for resynthesis of glucose via the Cori cycle.'"
However, in GSD type I, the increased lactate formation primarily originates in the liver.'
This lactate has been shown to be utilized by muscle or brain as measured by arteriovenous
differences.4-' 2 This represents a unique situation in which lactate is produced by the liver
to be utilized by peripheral tissues, constituting a reversal of the normal Cori cycle.
In a previously published study, we were able to show high serum alanine levels in fasting
GSD type I patients." Two factors appear to contribute to the increased circulating alanine
levels in GSD type I. The enhanced hepatic glycolysis that occurs in GSD type I not only
leads to increased lactate formation, but also results in increased generation of alanine via
the pyruvate aminotransferase reaction. In addition, uptake and utilization of alanine by the
liver from peripheral tissues is inhibited by lactic acidosis which is universally associated
with hyperalaninemia.'4 The degree that peripheral tissues such as skeletal muscle contribute
to the hyperalaninemia and hyperlacturia of GSD type I is uncertain. However, since these
patients are generally in a state of acidosis and muscle catabolism similar to what exists in
uncontrolled insulin-dependent diabetes,' some increase in release of lactate and alanine
from peripheral tissues would also be expected." It has been shown that the lactic acidemia
found in this disease is a result of an increased liver glycogenolysis which does not result
in glucose formation but rather floods the Embden Meyerhof pathway. The severe lactic
acidemia is characteristically accompanied by a striking hyperuricemia. The hyperuricemia
has been attributed to a decreased renal clearance of uric acid resulting from competitive
inhibition by elevated levels of lactic acid in the renal tubular transport system.'' It was
subsequently demonstrated by isotope studies that GSD type I patients had also an increased
net uric acid production.'6 This has been considered to be due to an increased PRPP pro-
duction as a result of a greater fraction of glucose-6-phosphate being converted to ribose
because one of its alternative pathways is blocked."
Different studies have shown that under certain metabolic conditions, the gluconeogenetic
pathway can be highly active.' " However, both glycogenolysis and glucogenosis are, to
a large extent, futile cycles in terms of preventing fasting hypoglycemia. Persistent hypo-
glycemia is accompanied by low insulin and high glucagon levels which lead to an excessive
lipolysis from adipose tissue releasing large amounts of free fatty acids into the blood.''-"
Hyperlipidemia is a prominent feature in these patients. The operation of the Embden
Meyerhof pathway results in the generation of NADH while the phosphogluconic pathway
generates NADPH. Reduction of pyruvate to lactate requires either one of these cofactors
and fatty acid synthesis requires both. NADPH is apparently the rate-limiting factor in
cholesterol synthesis. Fatty acid and cholesterol synthesis require acetyl CoA which is
produced from pyruvate generated from glycolysis. The presence of acetyl CoA and oxal-
oacetate are required to drive the citric acid cycle. Thus, the substrate, cofactors, and reduced
coenzyme required for lactate, fatty acid, and cholesterol. synthesis are made available by
the operation of glycolysis, oxidative pathways, and the citric acid cycle. Thus, elevated
levels of serum triglycerides, phospholipid, and cholesterol are possible consequences of
glycogenolytic responses to hypoglycemia in a liver containing a block in the glucose-6-
phosphatase reaction. It is of interest to note that the fasting glucose-6-phosphatase deficient
patients do not show increased ketone body formation. This may be due to an increased
glycolysis which leads to high pyruvate levels which accounts for adequate rates of oxal-
oacetate synthesis. Thus, acetyl CoA enters the citric acid cycle and does not accumulate
and is not channeled into the 3-ketobutyryl CoA pathway to form ketone bodies.'
Whereas the development of fasting hypoglycemia in GSD type I is obvious, the mech-
anism of the accumulation of liver glycogen is not so easily explained. The metabolic block
is several steps removed from glycogen breakdown. In the absence of glucose-6-phosphatase,
two alternative pathways, the Embden Meyerhof and the phosphogluconic pathways, are
available for the removal of glycolytic metabolites. Hormonal profiles of low insulin and
high glucagon levels, as demonstrated by Lockwood" and separately by Okada' are not
supportive of glycogen synthesis, but rather of glycogenolysis. Glycogen synthesis is ex-
pected to result from an accelerated gluconeogenesis, shown by Sadeghi-Nejad'" to exist in
this disease in addition to marked postprandial steep and prolonged elevations of blood
glucose which stimulate glycogen synthesis.
In GSD type III, the deficiency of the debrancher enzyme amylo- 1 ,6-glucosidase limits
glycogen breakdown resulting in an increased accumulation of limit dextrin among tissues.
As the normal steady-state between glycogen synthesis and breakdown is upset by the
deficiency of a glycogenolytic enzyme, this accumulation of limit dextrin is not surprising.
Once the outer branches of the glycogen molecule have been broken down, such patients
reach a stage of limited availability of glycogen-derived glucosyl moieties for glucose pro-
duction. This leads to a fasting hypoglycemia unless gluconeogenesis is switched on to
provide blood glucose.
In the young patient, this activation of the gluconeogenic pathway is not effective in
preventing fasting hypoglycemia in most cases. For some poorly understood reasons, both
in GSD type I, but more strikingly in GSD type III, the fasting hypoglycemia improves
with age so that most adult GSD type III patients tolerate fasting both clinically and bio-
chemically well in that blood glucose remains at the lower limit of normal range. However,
augmented lipolysis continues to occur as expressed by an increased ketogenesis which can
be explained in the following way. In amylo-1,6-glucosidase deficient patients, the inter-
mediary metabolism during fasting is markedly different from glucose-6-phosphatase defi-
cient individuals. Once outer branches have been broken down, glycogenolysis is blocked.
These patients, in order to supply glucose to the blood, rely heavily on liver gluconeogenesis
which causes a drain on amino acids and citric acid cycle intermediates such as oxaloacetate.
Fatty acid oxidation is active; however, the lack of oxaloacetate does not permit channeling
of acetyl CoA from fatty acid oxidation into the citric acid cycle, diverting its excess into
ketone bodies. This process is reversed upon feeding.''
Yudkoff et al. were able to show, utilizing stable isotopes, that in patients of both GSD
type I and GSD type III, in the presence of an inadequate glucose supply, an increased
breakdown of amino acids to urea occurred which was more pronounced in GSD type III
and which could be prevented by an adequate glucose supply.'
Amylo-1,6-glucosidase deficiency has been found to manifest with a marked heteroge-
neity, which is reflected in its distribution among tissues. This provides the basis for sub-
dividing cases into one category in which the liver enzyme activity is deficient, but muscle
enzyme activity is retained, as opposed to other cases where both muscle and liver enzymes
are affected. The distribution of these two categories varies among European case material
collected by Van Hoof and Hers," cases collected by Brown and Brown in St. Louis,' and
cases diagnosed in Israel.' In addition to the heterogeneity of enzyme activity in liver and
muscle, cases are on record which displayed a heterogeneity in terms of their enzyme activity
in leukocytes or erythrocytes:27.28 This heterogeneity is also manifested by the clinical
expression of this disease. Whereas in most patients the hepatic involvement dominated the
clinical picture, patients are on record who developed different degrees of myopathy, reflected
by functional deficits and electromyographic changes. Biochemically in such patients, muscle
amylo-1 ,6-glucosidase was deficient, glycogen was increased and of the limit dextrin type.
Among the many cases with biochemically proven muscle amylo- I ,6-glucosidase deficiency,
ten patients with clinically manifest severe myopathy have been reported." These patients
had documented muscle as well as liver debrancher enzyme deficiency. As patients are more
extensively studied, it appears that the enzyme defect is generalized, occurring not only in
liver and muscle, but also in heart, erythrocytes, leukocytes, and cultured fibroblasts.'" It
is not clear why clinical myopathy should occur only in a few patients with debrancher
Volume II 57
deficiency yet not in others who have a similar degree of muscle enzyme deficiency. Sug-
gested mechanisms by which debrancher myopathy may develop include: (1) impaired muscle
glycogenolysis with resultant lack of readily available glucose-6-phosphate for muscle gly-
colysis, and (2) disorganization and replacement of muscle fibers by excessive glycogen
deposition." Although these processes may play a part in the development of myopathy,
they do not adequately explain the functional differences, since glycogen accumulation and
morphological changes are present in muscles of debrancher deficiency patients both with
and without myopathy.'"
It seems possible that an imbalance in muscle protein synthesis and protein degradation
may play a role in the pathogenesis of debrancher myopathy.' " Muscle proteins are in a
dynamic state, and under normal circumstances there is a net balance between the rates of
muscle protein synthesis and protein degradation. This balance is of physiological importance
in overall energy homeostasis, since it determines whether there is net uptake or net release
of amino acids by skeletal muscle, which contains most of the body's protein reserves.'
Muscle protein synthesis and protein degradation are subject to precise hormonal and nu-
tritional regulation. As shown by Yudkoff et al.,' in the absence of adequate glucose
provision, an increased muscle protein catabolism is present in this disease which may play
a role in the pathogenesis of myopathy.
Types I and III glycogenosis respond quite differently to the administration of varying
substrates such as glucose, fructose.35 galactose, and glycerol.'' The difference in response
to a glucose tolerance test is based on the different lactate responses seen in the two
conditions.' The different responses seen in the latter three substrate tolerance tests are
based on the ability of GSD type III patients to utilize these gluconeogenic substrates,
causing a rise in plasma glucose and a modest rise and then fall in plasma lactate. On the
other hand, since GSD type I patients cannot effectively utilize gluconeogenesis for glucose
production, ingestion of any of these gluconeogenic substrates leads to a fall in plasma
glucose and a concomitant rise in plasma lactate.'9.32-35 We have recently extended these
types of studies to examine the plasma amino acid and hormonal responses to differing
substrates in these two conditions.""
Each patient underwent an oral glucose tolerance test (GTT — 1.75 g glucose/kg body
weight) and an oral protein tolerance test (PTT — 4 g broiled lean beef steak/kg body
weight), after a 4 hr fast in the case of GSD type I patients, and after an overnight fast in
GSD type III patients. The results were compared to responses obtained in normal control
subjects. As seen in Table 1, the postabsorptive concentrations of several amino acids in
GSD type III patients were significantly below those of normal controls, including threonine,
alanine, methionine, valine, isoleucine, leucine, tyrosine, phenylalanine, and lysine. Most
of the amino acids in GSD type I patients were similar to those of normal controls except
alanine, which was significantly elevated. The marked difference in alanine levels in these
two conditions is illustrated in Figure 1. The plasma glucose, insulin, lactate, alanine, and
valine responses to an oral glucose load in GSD type I and GSD type III patients and in
control subjects are illustrated in Figures 2 and 3. Both GSD type I and GSD type III patients
demonstrated moderate glucose intolerance, followed by a fall to hypoglycemic levels. The
insulin response in GSD type I was delayed and somewhat attenuated, while in GSD type
III there was an enhanced insulin response. In GSD type I, the basal lactate, and alanine
levels were significantly elevated and fell towards normal on ingestion of glucose. In GSD
type III, the glucagon response was similar to that of control subjects, while the lactate and
alanine levels rose considerably and then returned to baseline. The pattern of fall in plasma
valine (being typical of all three branched chain amino acids) following glucose ingestion
was similar in the three groups of patients, but the levels in GSD type III were consistently
lower than that found in control subjects.
The response to a protein load in the three groups of patients is illustrated in Figures 4
Table 1
POSTABSORPTIVE PLASMA AMINO ACID
CONCENTRATIONS IN GSD-III AND GSD-I
PATIENTS AND IN CONTROL SUBJECTSa
(MEAN ± SE, µm/t)
GSD-III GSD-I Normal controls

Amino acid (n = 11) (n = 8) (n = 27)
Taurine 49 ± 5.8 72 ± 14.5 46 ± 4.2

Aspartic acid 18 ± 3.2 16 ± 2.8 14 ± 1.8
Threonine 57 ± 7.3' 73 ± 15.1' 112 ± 5.8
Serine 90 ± 8.5 81 ± 8.4 102 ± 4.3
Glycine 164 ± 31.5 215 ± 34.3 206 ± 9.3
Alanine 127 ± 15.9' 719 ± 21.5h 311 ± 14.9
Valine 100 ± 13h 241 ± 18 238 ± 8.7
Cystine 37 ± 4.4 58 ± 3.6 47 ± 4
Methionine 14 ± 1.8' 23 ± 4.5' 37 ± 2.3
lsoleucine 34 ± 4.66 78 ± 7.1 68 ± 3.5
Leucine 64 ± 8h 137 ± 13.4 114 ± 10.5
Tyrosine 32 ± 3.5' 55 ± 14.8 54 ± 1.9
Phenylalanine 31 ± 3.1' 52 ± 6.4 56 ± 2.8
Ornithine 52 ± 3.3 39 ± 2.3 49 ± 3.4
Lysine 98 ± 9.9h 180 ± 18.3 197 ± 15
Histidine 68 ± 4.2 70 ± 3.7 83 ± 7.3
Areinine 64 ± 6.5 69 ± 3.4 79 ± 8.5
Total 1,099 2,178 1.813
Since glutamine is partly converted to glutamate these amino acids are

not reported.
= Significance of difference from controls, p < 0.01.
From Slonim, A. E. et al., Metabolism, 32, 70, 1983. With permission.
800
600
PL. ALANINE
J.Jrnol/L
400
200
0
NORMAL GSD- I GSD- III
I. Postabsorptive plasma alanine levels (mean ± SE) in control subjects (n =27), GSD-I patients
(n =8), and GSD-II1 patients (n = I I).
Volume II 59
GSD-I
200 x---x
•---• CONTROL
150
GLUCOSE
(mg/di)
100
50
150
INSULIN 100
(pU/mI)
50
I0
LACTATE
(mM) 5
0
HOURS POST GLUCOSE
FIGURE 2. The plasma glucose, insulin, glucagon, and lactate responses (mean ± SE)
to glucose ingestion ( I .75 g/kg body weight) in control subjects, GSD-I patients, and
GSD-III patients.
and 5. In GSD type I, protein caused a fall in plasma glucose and insulin and a sharp rise
in lactate, alanine, and valine. Conversely, in GSD type III, protein ingestion caused a
distinct rise in plasma glucose with an associated rise in insulin, while the rise in lactate,
alanine, and valine were attenuated compared to that observed in normal controls.
The acute improvement in many of the metabolic disturbances in GSD type I following
glucose administration (Figures 2 and 3) indicates that the majority of the biochemical
disturbances are secondary to hypoglycemia. This observation formed the basis for treatment
of GSD type I with total parenteral nutrition' or continuous high carbohydrate enteral
nutrition" thereby preventing the development of hypoglycemia. Both of these methods of
treatment led to a marked improvement in the metabolic disturbances found in GSD type I.
Unfortunately, neither of these methods of treatment was practical for long-term therapy,
800
o-• -0 GSD -I
GSD-IIT
700 •--• CONTROLS
600
500
PI.
ALAN I NE
(j.Jrnol/1)
400 400
300 300
1\ PI .
T '
e \ -r VALINE
/1_ \)( cumoVI) 200
200 /
/ _1_\\
/ \I
1
II
100 i loo
O 111111 0-111L11
0 I 2 3 4 5 0 I 2 3 4 5
HOURS POST GLUCOSE INGESTION
FIGURE 3. The plasma alanine and valine responses (mean ± SE) to glucose ingestion ( I .75 g/kg body weight)
in control subjects, GSD-I patients, and GSD-III patients.
however, a modification of this type of therapy, in the form of nocturnal intragastric therapy
(NIG), in which the patient receives continuous intragastric high carbohydrate feeding over-
night and frequent high carbohydrate feeds during the day, has proven to be eminently
suitable for GSD type I.' The use of this therapy has proven to be highly effective in
improving metabolic disturbances (Table 2 and Figure 6),36 clinical features and growth of
these patients. The mechanism by which continuous carbohydrate administration corrects
the abnormal intermediary metabolism can be explained as follows: by keeping blood glucose
at 4 to 5 mM concentrations, glucose is provided to peripheral tissue, without invoking either
massive glycogen synthesis or glycogenolysis and subsequent glycolysis in the liver.
Malonyl CoA, an intermediate in fatty acid synthesis from pyruvate, inhibits carnitine-
acetyl-transferase, the regulatory step in fatty acid oxidation. Stanley et al." have suggested
that in untreated GSD type I patients, accelerated glycolytic and tricarboxylic acid cycle
activity resulting in increased acetyl CoA and subsequent malonyl CoA formation, may
inhibit oxidation of the large quantities of fatty acids delivered to the liver from enhanced
adipose tissue lipolysis. This would account for the massive hepatic fat accumulation39 and
impaired ketogenesis":21 that occurs in untreated patients. Nocturnal intragastric therapy
would greatly decrease the glucagon/insulin ratio and inhibit the constant glycogenolysis
and glycolysis that exists in the untreated state. The consequent decrease in accumulation
of glycolytic intermediates and lypolytic products cause a return to a normal, or near-normal
biochemical situation.'
It has been found by Sidbury' and subsequently confirmed by others that the administration
Volume II 61
0---0 GSD-I
x---x GSD-III
150
•--• CONTROL
100
GLUCOSE
(mg/di) ST
IFSIs
50 •
100
INSULIN
50
(NU/m1)
I0
LACTATE
(mM)
5
ce•
0
I I I 1 I I
HOURS POST BEEF
FIGURE 4. The plasma glucose, insulin, glucagon, and lactate responses (mean ± SE) to
beef ingestion (4 g/kg body weight) in control subjects, GSD-I patients, and GSD-III patients.
of unboiled, unacidified cornstarch at a concentration of 1.5 to 2.0 g/kg in H-,0 at the end
of each meal prolonged the fasting tolerance of such patients. The exact mechanism of this
effect has not been established; however, if undigested cornstarch is broken down only
slowly in the digestive tract, this could lead to the prolonged provision of glucose to the
blood with its salutary effects on intermediary metabolism.
Figure 7 shows a typical response of a GSD type III patient to oral administration of
cornstarch. This patient had an average fasting tolerance after a meal or glucose administration
of 2 to 3 hr. With the help of cornflour, the patient can lead a normal daytime feeding
regime with one added cornstarch meal at midnight. Fernandes et al.42 found and we have
subsequently confirmed that the oral administration of medium chain triglycerides to such
patients prolonged their fasting tolerance. As fatty acids cannot be converted to glucose,
the sustaining of blood glucose levels has to be attributed to a peripheral glucose sparing
effect caused by the administration of triglycerides. These beneficial dietary effects affect
both patients with GSD type I and GSD type III.
In GSD type III, the considerable rise in blood glucose following protein ingestion supports
the contention that enhanced gluconeogenesis exists in this condition.32 Furthermore, since
glycogenolysis is impeded, gluconeogenesis is instituted quite early after fasting (4 to 5 hr)
to maintain euglycemia. The low circulating amino acid levels and in particular that of
900
0-0 GSD-I
X--- GSD-III
•—• CONTROLS
80
700
600 600
500 500
Pl. PI.
ALANINE VALINE
(jmo1/1) (prno1/1)
400 400
300 300
- I
200 200
100 100
I I I I I 0
1 2 3 4 5 0 1 2 3 4 5
HOURS POST BEEF INGESTION
FIGURE 5. The plasma alanine and valine responses (mean ± SE) to beef ingestion (4 g/kg
body weight) in control subjects. GSD-I patients. and GSD-III patients.
alanine, the principle gluconeogenic amino acid, suggests that there is a constant drain of
muscle amino acids by the liver, to maintain adequate hepatic glucose production. The rise
in plasma alanine and lactate following glucose ingestion supports this contention. This
unusual alanine response to glucose may be explained by the differing effect of insulin on
hepatic and muscle alanine metabolism. Insulin has been shown to inhibit hepatic alanine
uptake' but have little effect on release of alanine from skeletal muscle." In normal subjects,
where only a small amount of alanine is constantly being released from muscle, glucose
ingestion causes little change in circulating alanine levels. However, in GSD type III where
there is a constant large release of alanine from muscle, glucose ingestion resulted in a
marked rise in alanine. We postulated that if this constant loss of gluconeogenic amino acids
from muscle plays a role in the pathogenesis of myopathy and severe growth failure, then
these complications should be improved by providing adequate amino acid replacement.
Indeed, marked improvement of myopathic and severe growth failure was achieved in four
childhood GSD type III patients by the administration of high protein enteral feedings
overnight and high protein feeds during the day."' This therapy increased muscle strength,
induced a growth spurt, reversed myopathic EMG, reversed abnormal EKG, and restored
most of the circulating amino acids to normal. This apparently came about by providing an
enhanced supply of amino acid substrate for the needs of both hepatic gluconeogenesis and
muscle protein synthesis.
It is evident from this presentation that different types of glycogen storage disease, though
being clinically akin, do present major metabolic differences which affect not only the
carbohydrate but also the amino acid and lipid intermediary metabolism.
Table 2
BIOCHEMICAL VALUES IN GSD-I PATIENTS IN THE UNTREATED STATE AND FOLLOWING NIG THERAPY
S Uric
S Lactate acid S Triglycerides Phospholipids SGOT S Calcium S Phosphate RBC GSH
Patient (mM/e) (mg/de) (mg/de) (mg/de) (1U/e) (mg/de) (mg/de) (p(mol/g Hb)
T.D.
Untreated 9.30 16.0 2300 540 140 11.1 2.9 10.9
NIG 2.20 8.9 450 372 30 10.0 5.5 8.0
J.P.
Untreated 5.80 8.0 551 330 185 10.7 4.5 8.4
NIG 2.60 6.6 140 220 71 10.4 6.6 5.4
S.V.
Untreated 13.30 10.2 3167 1130 120 11.2 2.5
Portacaval shunt 6.14 8.6 482 480 125 10.8 4.8
Obstructed portacaval shunt 6.40 10.0 404 520 80 10.9 4.5 11.6
NIG 2.10 6.7 330 410 65 9.3 5.4 8.6
A.H.
Untreated 13.40 5.4 1080 365 159 11.6 2.1 10.9
NIG 2.10 3.7 270 256 24 10.2 5.3 7.8
C.A.
Untreated 25.00 10.1 2500 357 10.4 1.4 10.2
NIG 3.30 3.0 180 170 45 10.0 5.7 7.2
Mean ± SE
Untreated 13.4 ± 3.2 9.9 ± 1.7 1920 ± 480 591 ± 185 192 ± 42 11.0 ± 0.2 2.7 ± 0.5 10.4 ± 0.6
NIG 2.5 ± 0.2 5.8 ± 1.1 274 ± 55 314 ± 45 47 ± 9 10.0 ± 0.2 5.7 ± 0.2 7.4 ± 0.6
P <0.05 <0.02 <0.05 NS <0.05 <0.05 <0.01 <0.001
Upper limit of normal 2.0 7.0 150 300 40 11.0 2.5' 7.5
Note: Abbreviation - S, serum; NS, not significant.
Lower limit.
From Slonim, A. E. et al., Metabolism, 28, 707, 1979. With permission.

n-5
GSD - I mean ± S.E.
GLUCOSE GLUCAGON INSULIN H GH A LANI NE PROUNE GLUTAMATE

mg/dI pg/ml nOn1 p moV L pmol/ L prnoV L
200
150
100
50 40 40
20 20
0 0
PRIOR TO TREATMENT
9 NIG THERAPY — — — — MEAN OF NORMAL CONTROLS
FIGURE 6. The mean ( ± SE) plasma glucose, glucagon, insulin, HGH, cortisol alanine, proline. and glutamate
levels of samples drawn every 4 hr over a 24-hr period, prior to therapy (shaded bars), and following high
carbohydrate NIG therapy (open bars), in GSD-I patients. Interrupted lines indicate mean levels of normal controls.
mg %
110
-El-
100
BLOODGLUCOSE
90
80
D
70 \
00 \
50
40
30
20
10
To 0.5 1.3 2.0 25 30 3,5 4.0 4.3 3.0 3.3 6.0 6.0
HOURS
FIGURE 7. Cornstarch tolerance curve in a patient with GSD-Ill. Cornstarch 1.8 g/kg body weight in water;
T„= after a 2.5 hr fast: interrupted line: blood glucose concentrations after cornstarch administration; and dotted
line: blood glucose concentration upon fasting.
Volume II 65
In recent years, much progress in the understanding of these diseases has been made
which enabled more rational approaches to handling such patients, which in turn had a major
effect on their survival and well-being. However, not all the biochemical features of these
diseases are as yet fully understood. Recent progress in the evaluation of the delicate hormonal
interplay and the subtle metabolic regulations of normal glycogen metabolism may, once
applied to cases with glycogen storage disease, shed additional light on the understanding
of these inborn errors of metabolism.
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2. Larner, J., Smith, C. H., Rosenkrans, A. M., Miller, T. B., Jr., Huang, L. C., Villar-Palasi, C.,
and Rebhun, L., Studies on glycogen synthase and its control by hormones, in Metabolic Interconversion
of Enzymes, Fischer, E. H., Krebs, E. G., Neurath, H., and Stadtman, E. R., Eds., Springer Verlag,
Berlin, 1974, 63.
3. Moses, S. W., Bashan, N., and Gutman, A., Glycogen metabolism in the normal blood cells, Blood,
90, 836, 1972.
4. Fernandes, J., Personal communication.
5. Chaussain, J. L., Georges, P., Olive, G., and Job, J. C., Glycemic response to 24 hour fast in normal
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10. Sadeghi-Nejad, A., Presente, E., Binkiewicz, A., and Senior, B., Studies in type I glycogenosis of the
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13. Slonim, A. E., Coleman, R. A., Moses, S. W., Bashan, N., Shipp, E., and Mushlin, P., Amino acid
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14. Marliss, E. B., Aoki, T. T., Toews, C. J., Felig, P., Connon, J. J., Kyner, J., Huckabee, W. E.,
and Cahill, G. F., Jr., Amino acid metabolism in lactic acidosis, Am. J. Med., 52, 474, 1972.
IS. Wahren, J., Hagenfeld, L., and Felig, P., Splanchnic and leg exchange of glucose, amino acids, and
free fatty acids during exercise in diabetes mellitus. J. Clin. Invest., 55, 1303, 1975.
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17. Howell, R. R., Hyperuricemia in childhood, Fed. Proc., 27, 1078, 1968.
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neogenesis and glycolysis in glycogenesis of the liver, J. Pediatr., 76, 561, 1970.
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intravenously administratered glycerol, N. Engl. J. Med., 279, 958, 1968.
20. Shafrir, E. and Gutman, A., Prolonged elevation of serum free fatty acids following epinephrine admin-
istration to patients with glycogenosis, Israel J. Med. Sci., I, 84, 1965.
21. Fernandes, J. and Pikaar, N. A., Ketosis in hepatic glycogenosis, Arch. Dis. Child., 41, 47, 1972.
22. Lockwood, D. H., Merimee, T. J., Edgar, P. J., Greene, M. L., Fujimoto, W. Y., Seegmiller, J.
E., and Howell, R. R., Insulin secretion in type I glycogen storage disease, Diabetes, 18, 755, 1969.
23. Okada, S., Seino, Y., Kodama, H., Yutaka, T., Inui, K., Ishida, M., Yabuuchi, H., and Scino, Y.,
Insulin and glucagon secretion in heptic glycogenosis. Acta Pediatr. Scand., 68, 735, 1969.
24. Yudkoff, M., Nissim, I., Stanley, C., Baker, L., and Segal, S., Glycogen storage disease: effects of
glucose infusion on ('5N) glycine kinetics and nitrogen metabolism, J. Pediatr. Gastroenterol. Nutr, 3,
81, 1984.
25. VanHoof, F. and Hers, H. G., The subgroups of type III glycogenosis, Eur. J. Biochem., 2, 275, 1967.
26. Moses, S. W., Bashan, N., Gadoth, N., and Slonim, A. E., Myopathic features in glycogen storage
disease type III (Abstr.), Pediatr. Res., 16, 700, 1982.
27. Gutman, A., Agam, G., and Deckelbaum, R., Type III glycogen storage diseases with normal enzyme
activity in erythrocytes, Israel J. Med. Sci., 7, 1212, 1971.
28. Williams, C. and Field, J. B., Studies in glycogen storage disease III limit dextrinosis, a genetic study.
J. Pediatr., 72, 214, 1968.
29. DiMauro, S., Metabolic myopathies, in Handbook of Clinical Neurology, Vol. 41, Vinken, P. J. and
Bruyen, G. W., Eds., North-Holland, Amsterdam, 1980, 175.
30. DiMauro, S., Hartwig, G. B., Hays, A., Eastwood, A. B., Franco, R., Olarte, M., Chang, M., Roses,
A. D., Fetell, M., Schoenfeldt, R. S., and Stern, L. Z., Debrancher deficiency: neuromuscular disorder
in 5 adults, Ann. Neurol., 5, 422, 1979.
31. Murase, T., Ikeda, H., Muro, T., Nakao, K., and Sugita, H., Myopathy associated with type Ill
glycogenosis, J. Neurol. Sci., 20, 287, 1975.
32. Fernandes, J. and Van-De Kamer, J. N., Hexose and protein tolerance test in children with liver
glycogenosis caused by a deficiency of the debrancher enzyme system, Pediatrics, 41, 935, 1968.
33. Slonim, A. E., Weisberg, C., Benke, P., Evans, 0. B., and Burr, I. M., Reversal of debrancher
deficiency myopathy by the use of high-protein nutrition. Ann. Neurol., I I, 420. 1982.
34. Goldberg, A. L. and Chang, T. W., Regulation and significance of amino acid metabolism in skeletal
muscle, Fed. Proc., 37. 3201, 1978.
35. Fernandes, J., Huijing, F., and Van-De Kamer, J. H., A screening method for liver glycogen diseases.
Arch. Dis. Childh.. 44, 311, 1969.
36. Slonim, A. E., Lacy, W. W., Terry, A., Greene, H. L., and Burr, I. M., Nocturnal intragastric therapy
in Type I glycogen storage disease: effect on hormonal and amino acid metabolism, Metabolism, 28. 707.
1979.
37. Folkman, J., Phillipart, A., Tze, W. J., and Crigler, J., Jr., Portacaval shunt for glycogen storage
disease: value of prolonged intravenous hyperalimentation before surgery, Surgery, 72. 306. 1972.
38. Burr, I. M., O'Neill, J. A., Jr., Karzon, D. R., Howard, L. J., and Greene, H. L., Comparison of
the effects of total parental nutrition, continuous intra-gastric feeding, and portacaval shunt on a patient
with type I glycogen storage disease, J. Pediatr., 85, 792, 1974.
39. Greene, H. L., Slonim, A. E., O'Neill, J. A., Jr., and Burr, 1. M., Continuous nocturnal intraeastric
feeding for management of type I glycogen storage disease. N. Engl. J. Med., 294. 423. 1976.
40. Stanley, C. A., Mills, J. L., and Baker, L., Intragastric feeding in type I glycogen storage disease: factors
affecting the control of lactic acidemia, Pediatr. Res., 15, 1504, 1981.
41. Chen, Y. T., Cornblath, M., and Sidbury, J• B., Corn starch therapy in type I glycogen storage disease
(abstr.), Pediatr. Res., 17, 208A, 1983.
42. Fernandes, J., Berger, R., and Smith, G. P. A., Lactate as a cerebral metabolic fuel for glucose-6-
phosphatase deficient children, Pediatr. Res., 18, 335, 1984.
43. Felig, P. and Wahren, J., Influence of endogenous insulin secretion on splanchnic glucose and amino
acid metabolism, J. Clin. Invest., 50, 1702, 1971.
44. Pozefsky, T., Felig, P., Soeldner, J. G., Cahill, G. F., and Cahill, G. F., Jr., Insulin blockade of
amino acid release by human forearm tissues, Trans. Assoc. Am. Physicians, 81, 258, 1968.
45. Slonim, A. E., Coleman, M. A., and Moses, S. W., unpublished data, 1983.
Volume 11 67
Chapter 4
EFFECT OF ETHANOL ON CARBOHYDRATE METABOLISM
Gopi A. Tejwani and Valentine A. Duruibe
TABLE OF CONTENTS
I. Introduction 68
A. Effect on Carbohydrate Digestion and Absorption 68
B. Effect on Glucose Metabolism 68
II. Ethanol Metabolism 69
III. Effect on the Modulators of Glucose-6-P Pool 73

A. Insulin 73
B. Glucagon 73
C. Catecholamines 74
D. Redox Potential 75
E. Energy Charge 77
IV. Effect on the Levels of Metabolic Intermediates and Substrates of Glucose

Metabolism 77
V. Effect on the Activities of the Rate Limiting Enzymes of Glucose Metabolism 79

A. Chronic Ethanol Intake 80
B. Ethanol Perfusion 81
C. Acute Ethanol Administration In Vivo 84
VI. Summary and Conclusions 87
Acknowledgments 89
References 89
I. INTRODUCTION
The hypoglycemic effect of ethanol was first described by Brown and Harvey in 1941.'
For many years, it was thought that this hypoglycemia was a result of the hepatotoxic effect
of denatured alcohol solvents. In 1963 it was shown that pure ethanol can be used to reproduce
the syndromes observed in clinical alcohol hypoglycemia.' The decrease in blood sugar level
of chronic alcoholics with neurological abnormalities and in deep coma following the inges-
tion of an alcoholic beverage was also known. The effectiveness of intravenous glucose
injection in the restoration of neurological normalcy and consciousness in these patients
suggested that hypoglycemia was the cause of such abnormalities.3.4 Alcohol-induced hy-
poglycemia became a matter of concern in children, as it was also shown that ethanol caused
hypoglycemia in fed children.5 Ethanol is known to induce hypoglycemia when glycogen
stores are low.' This observation is true even for healthy, fasted individuals.`''
On the other hand, the infusion of ethanol can cause an increase in the hepatic glucose
output and a decrease in glucose utilization in fed animals.H.'" In fed rats, ethanol-induced
hyperglycemia is accompanied by an increase in the conversion of lactate to glucose while
in starved rats ethanol increases the formation of lactate from pyruvate.''' The concentrations
of some hormones and modulators of blood glucose are also influenced by ethanol.'" - "'
The purpose of this chapter is to discuss some of the effects of ethanol on carbohydrate
metabolism in mammals. We will also look at some possible mechanisms involved in the
glycemic effects of ethanol.
A. Effect on Carbohydrate Digestion and Absorption

Most carbohydrates injested by mammals are in the form of the plant polysaccharide
starch and small amounts of the disaccharides lactose and sucrose. Digestion of starch begins
during its passage through the stomach. Starch is partially digested by the enzyme salivary
amylase as it passes through the stomach. Further digestion is carried on by pancreatic
amylase released into the small intestine. The two enzymatic reactions yield the disaccharide
maltose and some polyglucose chains. The dissachrides: maltose, sucrose, and lactose, are
broken down to the monosaccharides, glucose and galactose by plasma membrane bound
enzymes. Glucose and galactose are actively transported across the intestinal epithelium into
the blood.
It has been reported that rats receiving ethanol-rich diet for a period of 3 months exhibit
a decrease in pancreatic amylase.' Ethanol also decreases the active transport of glucose
in the small intestine of fasted animals by up to 80%." This inhibition of active transport
is probably due to the nonspecific alteration of intestinal membrane permeability by ethanol.
B. Effect on Glucose Metabolism

Blood glucose concentration is a measure of the equilibrium between glucose entry into
the blood and glucose removal from the blood. Within hours after taking a meal, glucose
entering the blood comes either from the breakdown of glycogen (glycogenolysis) or through
synthesis of new glucose (gluconeogenesis). Some glucose leaving the blood goes through
the Cori cycle resulting in glucose production in the liver. The amount of glycogen in the
liver of an adult has been estimated to be about 50 to 75 g.'8 This is enough to sustain the
body glucose requirement for only a few hours. It has been reported that ethanol can decrease
liver glycogen by more than half in animals fed nutritionally adequate diets.'" If ethanol
caused the breakdown of glycogen to glucose, up to a 2.5-fold increase in glucose output,5-'6
and a decrease in glucose utilization"" (Table 1) one would expect to see a shift towards
increased blood glucose level. It is, however, important to note that in a perfusion experiment
in which ethanol increased glucose output and decreased glucose utilization, there was no
change in the liver glycogen level.' The lack of glycogenolytic effect in the perfused liver
Volume II 69
Table 1
METABOLIC RATES AND REDOX RATIOS IN LIVER
FROM FED RATS PERFUSED WITH 2 mM ETHANOL
Ethanol-
Control treated
(p.mol/min/g) (20 min) % Change
Metabolic rates
Oxygen consumption 2.25 2.45 +9

Glycolysis 1.88 0.58 — 69
Glucose production 1.18 1.99 + 69
Glycogenolysis 2.13 2.30 +8
Ketogenesis 0.13 0.25 + 92
Ethanol uptake 1.73
Redox Ratios
Lactate/pyruvate 4.3 38 + 884

3-Hydroxybutyrate/ 0.32 0.55 + 72
acetoacetate
From Soboll, S., Heldt, H., and Scholz. R., Hoppe-Sevler's Z. Physiol. Chem.,
363. 5247, 1981. With permission.
may be related to the absence of glucagon, as will be described later. In fasting animals
there is evidence that ethanol decreases blood glucose° 7 '2()21 with some exceptions.'' It is
possible that ethanol interferes with at least one step in the glucose metabolizing pathways.
In the mammalian cell, glucose is mainly phosphorylated to glucose-6-phosphate (G6P)
by the enzymes hexokinase (HK) and, glucokinase (GK) in the presence of ATP. Further
metabolism of G6P takes place through any or all of the following four primary pathways
(Figure 1).
1. Hydrolysis of G6P back to glucose by the enzyme glucose-6-phosphatase (G6Pase).
2. Oxidation of G6P in the presence of NADP to yield 6-phosphogluconolactone (6PG);
a reaction catalyzed by the enzyme glucose-6-phosphate dehydrogenase (G6PDH).
This is the first step in the oxidation of glucose through the hexose monophosphate
pathway which leads to the production of pentoses and the NADPH required for
reductive biosynthesis.
3. Conversion of G6P to glucose- 1-phosphate (GIP); an initial step in the ultimate syn-
thesis of various nucleotides, diphosphate esters of sugars and glycogen. The enzyme
phosphoglucomutase (PGM) catalyzes this initial step. The final step in glycogen
synthesis is catalyzed by two interconvertible enzymes, G6P-dependent glycogen syn-
thetase (GS-D) and the G6P-independent form (GS-I).
4. Isomerization of G6P to fructose-6-phosphate (F6P) catalyzed by phosphoglucose
isomerase (PGI); a first step in glucose oxidation through the Embden-Meyerhof path-
way (glycolysis), citric acid cycle, and oxidative phosphorylation. This pathway yields
water and carbon dioxide as the final products of glucose catabolism, while simulta-
neously releasing high energy in the form of ATP.
At this point it is necessary to review the metabolism of ethanol before looking at the
effect of ethanol or its metabolites on glucose metabolism.
II. ETHANOL METABOLISM
Although the body can produce very small amounts of ethanol, the major source of this
GLUCOSE CATEC HO LAM I NES

BLOOD Ar" ti
INSULIN B- RECEPTOR oC-RECEPTOR
CYTOSOL ACTIVATION ACTIVATION

F174—GLUCAGON
AMP, CO2 +
GLUCOSE
ATP
I+1)
I GS -II IGS-DI + G6P
1117433 IG 6 Pose I
1 ,...-. up pG
UDPGPz pi
6-P - GLUCONOLACTONE GLUCOSE 6-P GLUCOSE I - P GLYCOGEN
106 PDHI [PGMI
i
16 PGDHF.— NADPH PHPHOSPH a
FRUCTOSE 6-P
+4-(CAMP, CO 2+ )
ATP,_ -(ATP, CITRATE )
HEXOSEMONO- P [F 2, 6P, A MP, ADP, Pi, NH +4]
SHUNT CONTINUED PHOSPH b AMP
FRUCTOSE - I , 6- P,
4
4
4
P-ENOL PYRUVATE
1-1 NA DH , ATP, CAMP )
PYRUVATE
FIGURE I. Pathways of glucose utilization at the level of glucose 6-P, and the intermediates, modulators. and
enzymes regulating the glucose 6-P pool. The positive ( + ) and negative ( - ) modulators are shown behind the
arrows, which point to the enzymes or reactions being modulated. The enzymes are shown in the boxes, adjacent
to the arrows, which point in the direction of the enzyme-catalyzed reactions. Hexokinase (HK). glucokinase (GK).
glucose 6-P dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), phosphoglucomutase (PGM),
UDP-glucose pyrophosphorylase (UDPGP), UDP-glucose (UDPG), glucose 6-P-independent glycogen synthetase
(GS-I), glucose 6-P-dependent glycogen synthetase (GS-D), phosphorylase (PHOSPH a), phosphorylase b (PHOSPH
b) (note the absence of phosphate (P) on this enzyme as compared to PHOSPH a), phosphofructokinase (PFK),
and pyruvate kinase (PK). Note that glucose 6-P inhibits HK but not GK. Also note that glucagon and p-adrenergic
receptor activators act through cAMP while et-adrenergic receptor activators act through Cat - .
compound is exogenous. Ethanol is rapidly absorbed through the gastrointestinal tract. The
blood ethanol level is dependent on the size of the individual and the quantity of ethanol
consumed. This level is also an approximation of tissue levels of ethanol throughout the
body." Blood ethanol levels in humans have been estimated to range from 0 to 295 mg/
100 mf under various conditions of ethanol intake.21-26 These levels seem to increase in
chronic alcoholics depending on the duration of alcoholism."
The disappearance of ethanol from the blood follows zero-order kinetics.' When ethanol
(8% v/v) was administered in man by constant intravenous infusion and the time course of
blood ethanol concentrations followed, the curve showed a V,,„x of 0.232 mg/mf/hr and a
K,, of 0.0821 mg/mf.' However, in individuals receiving oral doses of ethanol, daily
variations and circadian rhythmicity of the slope of ethanol disappearance curve were reported
to occur.'
Only 2 to 10% of absorbed ethanol is eliminated through the kidneys and lungs; the rest
is oxidized in the body.3' In most animals, including man, ethanol is primarily metabolized
in the liver. Approximately 75% of total ethanol metabolism in man and 90% or more in
other species occur in the liver.3'-" Infusion of ethanol at a concentration of 24 mmol/mf
of blood results in an ethanol utilization rate of about 2.8 limol/min/g of perfused liver from
rat.8 Ethanol may also be metabolized in other body tissues including intestine, lung, kidney,
and possibly brain.'-33 However, ethanol is neither stored nor metabolized in large amounts
in the peripheral tissues, and its oxidation lacks a feedback contro1.31 Hepatocytes oxidize
ethanol at an average rate of 0.85 limol/min/g wet weight of cells. This rate is, however,
Volume Il 71
less than the rate observed in perfused liver8 and it is probably due to the depletion of some
important intermediates during cell preparation." The physiological mechanism of ethanol
metabolism has been reported to be active even at ethanol levels much lower than 10 mM
(46 mg/I00 me blood).'
The metabolism of ethanol in the liver can be carried out by three enzymatic pathways,
these include:
1. The alcohol dehydrogenase reaction in the cytosol

2. The microsomal ethanol oxidizing system (MEOS) in the endoplasmic reticulum (ER)
3. The NAD(P)H oxidase/catalase system also in the ER 36-38
Although the exact pathway of ethanol metabolism has been controversial, it is generally
accepted that ethanol is metabolized mainly by oxidation to acetaldehyde, catalyzed by the
Zn"-linked enzyme alcohol dehydrogenase (ALDH, EC 1.1.1.1) in the presence of NAD+ .
The ability of the other pathways to regulate ethanol metabolism depends on the extent to
which they contribute to the conversion of ethanol to acetaldehyde. The MEOS has a higher
K„, for ethanol (9 to 10 mM) than does alcohol dehydrogenase with a K„, of 0.5 mM. Most
of the alcohol dehydrogenase activity is located in the cytosol of the liver.' The oxidation
of ethanol through this pathway (Figure 2) can be summarized as follows:
Zn"
CH,CH,OH + NAD+ > CH3CHO + NADH + H+ (1)
ALDH
Alcohol dehydrogenase catalyzes the transfer of one hydrogen from ethanol to NADH
to generate acetaldehyde and NADH. Acetaldehyde is oxidized primarily in the mitochon-
dria in the reaction catalyzed by another enzyme, aldehyde dehydrogenase (ADH, EC 1.2.-
1.3).36-40-42 This reaction can be summarized as follows:
CH,CHO + NAD+ + H2O -> CH COOH + NADH + H+ (2)

ADH 3
In this reaction, acetaldehyde is oxidized to acetic acid and NADH produced. Acetaldehyde
oxidation in the cytosol occurs only at very high acetaldehyde concentrations (1 to 10 mM).42
It follows from the above reactions that for each molecule of ethanol which is completely
oxidized, two equivalents of NADH are formed.
The rate of oxidation of ethanol varies with species, route of administration, and dos-
age of ethano1.4" It has also been shown that the alcohol dehydrogenase activity is influ-
enced by the state of nutrition. During 12 to 19 hr of starvation, the total activity of
alcohol dehydrogenase was decreased by 40% and the rate of ethanol metabolism also de-
creased.' 46 Pyrazole and substituted pyrazoles inhibit the alcohol dehydrogenase reaction.
Pyrazole inhibits this enzyme by competing with ethanol. Rat alcohol dehydrogenase has a
K, of about 0.05 mM for pyrazole.47 Acetaldehyde oxidation can be inhibited by mitochondrial
substrates (fatty acids, 13-hydroxybutyrate, etc.) and disulfiram (Antabuse). Oxidation of
acetaldehyde results in the formation of acetyl CoA either directly or through acetate for-
mation. This is followed by the complete combustion of acetyl CoA to CO, and H,0 in the
citric acid cycle.33-48
Ethanol oxidation and elimination rates are enhanced by chronic intake of ethano1,49 5"
phenobarbita1,48.81.82 fructose,82 .88 glucagon,57 and ascorbic acid. 58 Fructose which has been
found to be most effective in increasing ethanol elimination' does not affect acetaldehyde
clearance when ethanol is present in the blood.88.86 It has been reported that a decrease in
the ethanol level in blood does not result in an immediate decrease in the acetaldehyde or
GLUCOSE
BLOOD
CYTOSOL
GLUCOSE
ADP HEXO (GLUCO)KINASE

ATP)1
GLUCpSE 6-P
FRUCTOSE 6-P
ATP
PHOSPHOFRUCTOKINASE
FRUCTOSE I, 6- P2
GLYCERALDEHYDE - P DI HYDROXYACETONE- P
NAD
GAPDH
NADH
I , 3 - BISPHOSPHOGLYCERATE
ADP MDH - GLYCERO-P
ATP "
3 - PHOSPHOGLYCERATE OAA MAL ATE
4
2 - PHOSPHOGLYCERATE
P- ENOLPYRUVATE
ADP PYRUVATE K I NASE
ATP
LACTATE PYRUVATE
DHAP
ETHANOL ACETALDEHYDE
MITOCHONDRION
ACETATE ACETALDEHYDE
NADH
SHUTTLES
NADH
CITRIC
ACID
CYCLE
FIGURE 2. The pathways of ethanol oxidation and glycolysis, showing substrates, intermediates, rate limiting
enzymes, enzymes catalyzing redox reactions, and two NADH shuttle systems. The enzymes numbered I, 2, and
3 catalyze the rate limiting and essentially irreversible reactions in glycolysis. The enzymes in the boxes catalyze
NAD'/NADH-dependent reactions. Glyceraldehyde-P dehydrogenase (GAPDH), a-glycero-P dehydrogenase (a-
GPDH), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), alcohol dehydrogenase (ALDH), aldehyde
dehydrogenase (ADH), and oxaloacetic acid (OAA). Dihydroxyacetone-P (DHAP) and GAP are interconvertible
by the enzyme triosephosphate isomerase. Each molecule of glucose oxidized yields two molecules of each of the
products below GAP in the glycolytic pathway. The oxidation of a-glycero-P to DHAP, resulting in NADH
shuttling, is a membrane process. Proton generated through this, and during ethanol oxidation, can be used in the
citric acid cycle and in the electron transport system.
Volume II 73
acetate levels in blood. Both of these metabolites attend plateau levels which remain in the
case of acetaldehyde until the blood ethanol level is as low as 24 mM,3g and in the case of
acetate, as long as ethanol is present.'
Having reviewed the possible destiny of a casual drink of ethyl alcohol (ethanol) or a
forced parenteral administration of ethanol in experimental animals, let us turn to our initial
purpose of understanding the mechanism of ethanol induced changes in glucose levels.
III. EFFECT ON THE MODULATORS OF GLUCOSE-6-P POOL
Among the modulators of glucose-6-phosphate pool are blood glucose, insulin, intra-
cellular glucose, G6P, glucagon, catecholamines (a- and (3-adrenergic receptor stimulators),
cAMP, Ca' , AMP, ATP, citrate, F2,6-P,, NADPH, NADH, Mg' , and NH4 • (Figure
1).
A. Insulin
Insulin is produced by the (3-cells in the pancreatic islets of Langerhans in response to
increased blood glucose. On the other hand, insulin stimulates glucose uptake"" and gly-
cogenesis in the liver. It has been shown that ethanol (4.5 g/kg body weight or more)
ingestion decreases plasma insulin in a dose related fashion in the fed or fasted anesthetized
rats.'" Plasma glucose was increased only in the fed rats.'" Similar lack of effect of low
doses of ethanol on plasma insulin and blood glucose has been reported by other authors
using ethanol (0.4 g/kg) in 18 hr fasted rats."' However, ethanol at even lower doses than
4.5 g/kg has been shown to increase the response of insulin to the administration of glucose
in the fed"2-" and to decrease the response in the fasted"' and diabetic' animals. Insulin
release from pancreatic islets in response to glucose is also inhibited by ethanol administered
by different routes.65 67 These effects were also found to be dose related" 67 and dependent
on the length of fast.6'68 It is believed that the ethanol effects on insulin release is more
direct than through its metabolites.66.67 These effects can be enhanced by pyrazole67." and
do not require ethanol metabolites.'" In addition, it requires higher than in vivo levels of
acetaldehyde to inhibit insulin release, and acetate does not decrease insulin release even at
high doses."
It has been suggested that ethanol may decrease glucose-induced insulin release by sta-
bilizing the microtubules which provide a pathway for the movement of insulin granules out
of the islet cells."' The contraction of these microtubules is associated with the movement
of insulin to the cell surface and an increase in Ca2 + has been shown to prevent the ethanol
effect on insulin."
B. Glucagon
Acting opposite to insulin is the pancreatic a-cell secreted hormone, glucagon. Glucagon
release is stimulated by decreased blood glucose levels and it, in turn, causes an increase
in blood glucose levels. Fasting also increases plasma glucagon." Ethanol (4.5 g/kg or more
post-oral), which decreases plasma insulin, increases plasma glucagon in both fed and fasted
rats.' 6 In the fasted (56 hr) man, the increase in plasma glucagon due to ethanol can be as
high as three times that due to fasting alone." An increase in glucose resulting from glucagon
stimulation is accompanied by an increase in substrate cycling at the level of F6P and
fructose-1,6-bisphosphate (Fru-P2).72 In addition to stimulating glycogenolysis, glucagon is
also known to increase gluconeogenesis. It exerts its control by enzyme induction or by
phosphorylation via the cAMP-stimulated protein kinase.' 2 Glucagon also stimulates the
ethanol oxidation rate." Pretreatment with pyrazole enhances ethanol stimulated glucagon
release.69 However, acetaldehyde and acetate have also been reported to increase glucagon
release from perfused rat pancreas.' Thus, ethanol can inhibit glycogenesis while simul-
taneously enhancing glycogenolysis in the fasted animal, through its modulation of insulin
and glucagon secretion.
C. Catecholamines
Catecholamines, especially 13-adrenergic agonists like isoproterenol and epinephrine (ad-
renaline), stimulate glucose uptake in adipose tissue." Catecholamines also cause hyper-
glycemia.' As shown in Figure 1, both a- and 13-receptor activation results in the inhibition
of glycogenesis and enhancement of glycogenolysis. The a-receptor effect is mediated
through Ca" while the I3-receptor effect is mediated through the stimulation of adenyl
cyclase. Adenyl cyclase is an enzyme responsible for the synthesis of cAMP from ATP.
Both cAMP and Ca" enhance the phosphorylation of enzymes involved in glycogen me-
tabolism. The unphosphorylated form of glycogen synthetase (GS-I) is the active form while
the phosphorylated form is a less active form and is dependent on G6P stimulation for its
activity. However, in the case of enzymes responsible for glycogenolysis the phosphorylated
form phosphorylase a (Phosph a) is the active form while the unphosphorylated form phos-
phorylase b (Phosph b) is less active unless stimulated by AMP. Therefore, phosphorylation
results in the inhibition of glycogenesis and stimulation of glycogenolysis.
It has been reported that ethanol (1.13 g/kg) given 1 hr prior to the injection of cate-
cholamines inhibits the hyperglycemic response to these compounds in fasted animals."
However, in fed animals in which the hyperglycemic effect of isoproterenol seems to be
inhibited by insulin release, ethanol unmasks the hyperglycemia. Ethanol has been reported
to increase the urinary excretion of catecholamines and their major metabolites.' However,
the reported increase was not proportional to ethanol levels in the blood. It was also found
that 4-methylpyrazole normalized the excretion of catecholamines, suggesting that ethanol
metabolism was involved. In man, however, there was no significant change in the urinary
excretion of catecholamines following a higher dose of ethanol (1.5 g/kg) in man.'
Other investigators have suggested that ethanol may induce adrenergic subsensitivity.
Intragastric ethanol (6g/kg) or dose of ethanol causing a blood ethanol level of up to 68 mg/
dt inhibited an isoproterenol induced increase in cAMP." There are other reports that
intragastric ethanol (5 g/kg) in fasted rats resulted in an increase in hepatic cAMP, while
decreasing liver glycogen and glucose as well as adipose cAMP." Again, the effect on
cAMP concentration did not correlate with the blood ethanol concentration. However, when
blood ethanol in fasted rats was about 82 mg/dC the plasma cAMP level reached a level
approximately three times that observed in the control animals not receiving ethanol or other
drugs.'' Ethanol may also cause a decrease in calcium and magnesium levels." Even the
adrenergic-stimulated uptake of glucose in adipose tissue has been reported to decrease
following treatment with ethanol.' It was suggested that ethanol may modify the coupling
of the adrenergic receptors to glucose transport_
As mentioned earlier, the oxidation of ethanol results in the generation of reducing equiv-
alents in the cytosol. This is accompanied by an increase in NADPH" " which can inhibit
G6P utilization through the hexose monophosphate pathway by inhibiting G6PDH and
6PGDH catalyzed oxidations. Glucose-6-phosphate is an important intermediate in glucose
metabolism. Its function in the regulation of glycogenesis has been mentioned. As shown
in Figure 1, G6P also regulates hexokinase (HK) activity through allosteric inhibition. Ethanol
increases the level of G6P in the liver." It has been reported that ethanol inhibits the activity
of HK in fed rats given chronic ethanol' and of GK in the isolated perfused rat liver.'" As
will be discussed later, we have shown that acute ethanol (1 to 5 g/kg body weight) i.p.
decreases the activity of hexokinase in the fed rats but produces no significant effect on the
already depressed enzyme activity in the fasted rats." The enhancement of G6Pase activity
by ethanol in fed rats has also been reported."' In the same animals ethanol seems to
selectively decrease the activities of G6PDH (42%) and UDPGP (62%) in males only, while
Volume II 75
inhibiting PGM (29%) in the females only.' The ratio of G6Pase/HK(GK) was increased
in both chronic' and acute' ethanol-treated fed rats. However, there was a decrease in
blood glucose in the chronic ethanol-treated male rats. Similarly, an ethanol-induced decrease
in blood glucose has been observed in some well-nourished adult humans.82 This finding
indicates that low carbohydrate is not required for ethanol to induce hypoglycemia especially
in chronic alcoholics. However, it does not dispute the ethanol-induced hypoglycemia in
animals receiving low carbohydrate diets' or fasted animals whose liver glycogen has been
depleted.7'21
Since the activities of G6PDH, PGM, and UDPGP were decreased in the animals, it was
suggested that G6P was mainly being utilized through a pathway other than hexose mon-
ophosphate shunt or glycogenesis. The most likely pathway is glycolysis.'9 An increase in
glycolysis would result in an increase in pyruvate, lactate, and citric acid cycle intermediates.
Ethanol increases lactate formation from pyruvate as follows:
ALDH>
Ethanol + NAD± Acetaldehyde + NADH + (3)
LD H
Pyruvate + NADH Lactate + NADI- (4)
Such an increase in blood lactate is commonly observed in ethanol-treated animals including

man.' 28'4x.83.84 This may eventually result in an increase in ethanol oxidation rate. It has
been shown that when the lactate concentration is 0.5 to 2 mM and lactate/pyruvate ratio is
10, a dose related increase in the rate of ethanol oxidation occurs, reaching up to three times
the basal rate.' However, further increase in lactate results in no apparent change in the
ethanol oxidation rate nor a significant increase in glucose synthesis.' The increase in
gluconeogenesis found especially in fed animals can be accounted for by an increase in the
conversion of lactate and pyruvate to glucose.8'85 Other ways in which ethanol can influence
glucose metabolism at the level of glycolysis and gluconeogenesis include:
1. Changes in NADH/NAD ± concentration ratio

2. Alteration in the concentrations of enzyme modulators involved in glucose metabolism
3. Change in the concentrations of metabolic intermediates and other substrates
4. Direct effect on the activities of rate limiting enzymes
Let us look at each of these possibilities and at some of the evidence in support of such
a mechanism of ethanol action on glucose metabolism. Figure 2 depicts the steps, inter-
mediates, regulatory enzymes, and modulators of glucose oxidation through the glycolytic
pathway. It also shows how ethanol oxidation by ALDH and ADH reactions can interfere
with glucose metabolism through changes in NAD±/NADH ratio. How ethanol oxidation
can influence gluconeogenesis is shown in Figure 3."
D. Redox Potential
As previously mentioned, the complete oxidation of ethanol through the ALDH reaction
results in the generation of two equivalents of NADH from NAD± . The consequence of
this reaction is an increase in the cytosolic and mitochondrial NADH/NAD± ratios.' From
Figure 2 we can see that such a decrease in NAD± concentration will impair the NAD +-
dependent conversion of glyceraldehyde phosphate to 3-phosphoglycerate (3PGA),84 while
GLUCOSE
BLOOD
CYTOSOL
GLUCOSE
Pi
FATTY ACIDS - 11
PHOSPHOLIPIDS GLUCOSE 6-P
• TRIGLYCERIDES
NADH+H+
From Ethanol FRUCTOSE- 6-P
Metabolism
Pi
GLYCER -P 12_1
cNiAD
nA D :11 H 20
ACETYL Co A
FRUCTOSE I, 6-P2
AMINO ACIDS
H FATTY
ACIDS DIHYDROXYACETONE P•—•GLYCERALDEHYDE -3 -P
CITRATE
GTP GDP 1,3- BISPHOSPHOGLYCERATE
OXALOACETATE .PHOSPHOENOLPYRUVATE
NAD+ NADH
MALATE
t LACTATE.' • PYRUVATE
MALATE ADP ATP
KETONES . ,..,;:y___/
OXALOACETATE • PYRUVATE
4
CITRATE • ACETYL CC°o2A'---------
i [
MITOCHONDRION ALAN1NE
FIGURE 3. The pathway of gluconeogenesis, showing substrates, intermediate rate limiting enzymes, redox
reactions and how the products of ethanol oxidation can affect gluconeogenesis. The numbers I to 4 in the boxes,
indicate the rate limiting enzymes, pyruvate carboxylase, phosphoenolpyruvate carboxykinase fructose- I ,6-bis-
phosphatase, and glucose-6-phosphatase, respectively. The redox reactions are catalyzed by the same enzymes
indicated in Figure 2. The NADH and F1' resulting from ethanol metabolism, can be used to generate fatty acids,
triglycerides, and ct-glycero-P. They can also enhance the NADH-dependent reductive reactions. For a more detailed
description of the gluconeogenic pathway, see the section on gluconeogenesis.
simultaneously providing the NADH necessary for the conversion of dihydroxyacetone

phosphate (DHAP) to a-glycerophosphate (GP). It has been reported that ethanol increases
the a-GP/DHAP ratio and the DHAP concentration, while decreasing the concentration of
3PGA."' From these observations one can conclude that the NADH, generated during
ethanol oxidation, was used for the reactions resulting in the conversion of 3PGA to DHAP.
Such an effect would decrease glycolysis and enhance gluconeogenesis.
An increase in NADH can inhibit the formation of pyruvate from phosphoenolpyruvate
(PEP) while simultaneously enhancing the conversion of pyruvate to lactate. As stated
previously, there are reports which suggest the increase in the lactate/pyruvate ratio in both
fed and fasted animals treated with ethanol. ' 28 82.85,86 Further support is lent to this by the
effect of ethanol in patients suffering from type I glycogenosis of the liver (a deficiency in
the G6Pase activity and an abnormally high lactate concentration). In these patients, ethanol
decreases the levels of lactate and pyruvate. However, the inhibitory effect. of ethanol was
greater on pyruvate than on lactate, resulting in an increase in the lactate to pyruvate
The increased conversion of pyruvate to lactate would result in a decrease in the quantity
of pyruvate channeled toward the synthesis of glucose through the gluconeogenic pathway
(Figure 3). However, the inhibition of pyruvate formation would also result in the inhibition
of glycolysis at the step involving the conversion of PEP to pyruvate. It must, however, be
recognized that the removal of NADH from the cytosol by the malate dehydrogenase (MDH)
and a-glycerophosphate dehydrogenase (a-GDPH) shuttles (Figure 2) may ameliorate the
influence of NADH on the cytosolic reactions. It has also been reported that ethanol increases
the concentrations of malate and other intermediates of the citric acid cycle.28
Volume II 77
E. Energy Charge
An interesting point about this NADH-dependent ethanol effect is that it does not seem
to affect the F6P and Fru-P2 interconversion reactions directly. If ethanol acts only through
the increase in NADH, it should cause a recycling of F6P and Fru-P,. This would result in
a wasteful utilization of ATP through the F6P-Fru-P, recycling, a futile cycle. It has been
reported that ethanol (10 mM) decreased the PFK flux during gluconeogenesis from dihy-
droxyacetone, but had no significant effect on the rate of gluconeogenesis.87 Also, feeding
ethanol (equivalent to 20 to 30% of the total dietary caloric intake) resulted in a 50% drop
in hepatic ATP. However, this decrease in ATP has been attributed to a decrease in ATP
synthesis." In the fasted rats, ethanol (1.7 and 3.0 g/kg body weight) was reported to cause
a slight increase in ATP, while decreasing ADP and inorganic phosphate, hence increasing
the lAT131/([ADP][Pi]) ratio."
It is possible that the increase in ATP was a result of an increase in oxidative phosphorylation"
following the movement of NADH into the mitochondria through the shuttle system indicated
in Figure 2. In carrying out this ATP generation, ethanol is also reported to stimulate cell
membrane ATPase activity, thus generating ADP. This cycle may also result in the waste
of energy by ethanol and an increased oxygen consumption."' As mentioned above, ethanol
can decrease ATP synthesis. This decrease in ATP synthesis can be contributed to by a
decrease in glycolysis with a concomitant decrease in the phosphorylation of ADP to ATP.
This unphosphorylated cytosolic ADP is translocated into the mitochondria, where it con-
tributes 23 to 50% of the increased oxygen uptake." Recently, in the fed male rats, ethanol
was observed to increase the concentration of AMP by 71% but did not affect significantly
the concentration of ATP, ADP, or Pi.92
It is known that ATP serves both as a substrate and a modulator of the PFK catalyzed
reaction and as a modulator of PK reaction as will be discussed later. As shown in Figure
1, an increase in ATP will result in inhibition of the PFK and PK catalyzed reactions. Such
inhibition could result in a decrease in the rate of glycolysis. However, while ethanol may
increase ATP in fasted animal, it also increases AMP, citrate, and cAMP.".28-75 An increase
in AMP and F2,6-P2 can override the ATP inhibition of PFK, by increasing the enzyme
affinity for F6P.93-°4 The extent of an ethanol-induced increase in these modulators including
citrate will at least partially determine the effect of ethanol on PFK reaction.95-96 However,
the increases in ATP, cAMP, and NADH would point toward the inhibition of pyruvate
kinase activity. Table 2 summarizes the effect of ethanol on the modulators of glycolysis
in fed and fasted animals.
IV. EFFECT ON THE LEVELS OF METABOLIC INTERMEDIATES AND

SUBSTRATES OF GLUCOSE METABOLISM
Changes in the levels of G6P, F6P, Fru-P2, PEP, pyruvate, and lactate can influence
glycolysis and gluconeogenesis. An increase in G6P as previously mentioned will result in
the inhibition of HK, the first enzyme in glycolysis."" Increased G6P is also likely to result
in increases in G6Pase activity and in F6P level.
Fructose 6-phosphate and Fru-P2 are important substrates and modulators of PFK and Fru-
P2ase. They are more effective modulators in the presence of AMP and F2,6-P2;94 whereas
F6P acts cooperatively with AMP and F2,6-13, to enhance the PFK reaction, Fru-P2 inhibits
the Fru-Pease reaction in the presence of AMP and F2,6-P2.93'99 Therefore, in the presence
of AMP and F2,6-P2, an increase in the levels of F6P and Fru-P2 will favor glycolysis at
the expense of gluconeogenesis. It has been reported that depending on the concentration,
Fru-P2 can also enhance or inhibit PFK reaction." However, at high levels of Fru-P2, the
inhibitory effect predominates. More detailed discussions on this reciprocal regulation of
Table 2
EFFECT OF ETHANOL ON THE
CONCENTRATION OF ENZYME
MODULATORS (nmol/g) AND REDOX
RATIOS IN RAT LIVER'2,28.",9'
Ethanol-
Modulators Control treated
AMP 380 (122)" 432 (209)

ATP 2230 (3250) 2460 (3380)
Citrate 320 540
cAMPb 1.1 1.46
NH4 - 510 530
[NAD - /NADF]` 495 (8.1) 200 (52)
INAD - /NADHr 7.31(0.6) 7.04 (1.2)
INADP/NAPHr 0.0068 0.0049
Values in parenthesis were obtained in fed rats, others

were obtained in fasted rats.
h Values expressed as pmol/mg.
Values in cytosol.
Values in mitochondria.
PFK and Fru-P,ase can be found in some recent publications.'. °°"°1 Fru-P, is an activator
of PK, the enzyme responsible for the conversion of PEP to pyruvate.
Phosphoenolpyruvate is itself an inhibitor of F2,6-132 and F6P synthesis1 °'2•'°' in most
tissues excluding lung.104 It is also the substrate for the PK reaction. Therefore, changes in
the concentration of this intermediate will influence both the reciprocal regulation of PFK
and Fru-P,ase and the production of pyruvate. Pyruvate is at a key position in glycolysis
and gluconeogenesis. Anything that influences the rate of pyruvate formation or utilization
will influence gluconeogenesis.104 The effect of ethanol on the concentrations of the above
intermediates is shown in Table 3. The results presented were found in experiments with
fasted female rats.' It is possible that the results may vary depending on sex and state of
nutrition. For example, it has been reported that ethanol decreases lactate in fed animals
and increases it in the fasteds animals. This is supported by our observation that ethanol
decreases the activity of these enzymes in fasted animals.8 ' The decrease in levels of most
of the above intermediates may be due to an increase in the rate of glycolysis in the fasted
animals. This may also be a result of a decrease in gluconeogenesis.
As mentioned earlier, there has also been reports of an increase in G6P level in animals
treated chronically with ethanol.'" This may also indicate an increase in the HK activity, or
increased glycogenolysis due to ethanol as previously mentioned.
Increases in malate and a-glycerophosphate levels following ethanol administration in
fasted animals' suggest the shunting of substrates away from the gluconeogenic pathway.
The reported ethanol-induced decrease in hepatic oxidation of fatty acids and the decrease
in mobilization of amino acids and glycerol' could result in low concentrations of acetyl
CoA and oxaloacetate (OAA) in fasted animals. Since acetyl CoA is an allosteric activator
of pyruvate carboxylase 1 °6 and OAA is the immediate precursor of PEPCK reaction,w6
lowering of these substances will decrease the activities of the two enzymes. This can also
impair the conversion of pyruvate to PEP and thus decrease gluconeogenesis from pyruvate.
Acetyl CoA is also important for the synthesis of triglyceride (Figure 3).
As shown in Table 4, it has also been reported that ethanol decreases gluconeogenesis
Volume II 79
Table 3
THE EFFECT OF ETHANOL (4 g/kg BODY WT)
ON THE CONCENTRATIONS
(nmol/g WET WT LIVER) OF KEY
INTERMEDIATES OF GLYCOLYSIS AND
GLUCONEOGENESIS IN THE LIVER OF
FASTED (48 hr) RATS
Ethanol-
Control related % Change
Glucose-6-phosphate 126 43.2 —65.7

Fructose-6-phosphate 30.6 14.9 —51.3
Fructose-1 ,6-bisphosphate 18.7 13.3 —28.8
Phosphoenolpyruvate 57.2 42.8 —25.1 (NS)4
Pyruvate 61.7 77.5 +25.6 (NS)
Lactate 1465 1671 +14 (NS)
NS, not statistically significant.
From ZaKim, D Proc. Soc. Exp. Biol. Med., 129, 373, 1968. With
permission.
from other substrates especially in the fasted animal.105 It was found that 10 mM ethanol
exerted the most inhibitory effect on gluconeogenesis and that pyrazole removed such in-
hibition, dose dependently.'
V. EFFECT ON THE ACTIVITIES OF THE RATE LIMITING ENZYMES OF

GLUCOSE METABOLISM
So far, enzyme activities have been inevitably linked with glycemic effects of ethanol.
Ethanol can alter the enzyme activity by any or all of the following ways.
1. Ethanol may alter the levels of

a. Substrates
b. Activators
c. Inhibitors
2. Influence the transition of an active-inactive form of enzyme
3. Change the total content of enzyme by altering:

a. The rate of synthesis
b. The rate of degradation
c. Both rates of synthesis and degradation to different extents; for example, ethanol
may inhibit PFK activity by:
1. Decreasing the concentration of F6P in the liver
2. Converting the active form to the inactive (or less active) form"'
3. Decreasing the total content of the enzyme
By assaying the enzyme activity in the presence and absence of its specific activators,
one can postulate the mode of its inhibition by ethanol. As stated above, a change in the
level of substrates, activators, or inhibitors of an enzyme in vivo may affect the activity of
the enzyme. Such changes in the effector concentration could also transform the enzyme to
Table 4
EFFECT OF ETHANOL (10 mM) ON THE
RATE OF GLUCONEOGENESIS FROM
VARIOUS SUBSTRATES (10 mM) IN THE
PERFUSED RAT LIVER
Rate of gluconeogenesis
(µmol/min/g wet wt of tissue)
Ethanol- Decrease by
Substrate Control treated ethanol (%)
None 0.14 0.05 65

Lactate 1.06 0.42 60
Glycerol 0.48 0.17 65
Dihydroxyacetone 2.07 0.97 53
D-Fructose 2.68 1.90 29
L-Proline 0.55 0.16 71
L-Serine 0.98 0.73 25
L-Alanine 0.66 0.49 26
Galactose 0.36 <0.10 >72
From Krebs, H. A., Freedland, R. A., Hems, R., and Stubbs,

M.. Biochem. J., 112, 117, 1969. With permission.
an altered form, which may be either more or less active. The activity obtained when an
enzyme is assayed in vitro without any added effectors may reflect to a certain extent the
enzyme activity in vivo. However, in order to detect the change in total content of enzyme,
it is appropriate to assay the activity of the enzyme in the presence of its activators which
gives the maximum activity of the enzyme.
For example, Fru-132 within the range of 0.01 to 1.00 mM will activate PK by completely
correcting any defect in the kinetic characteristics without affecting its K, for PEP.''°" If
hepatic PK from ethanol-treated rats is assayed in the presence of Fru-P„ any alteration in
the conformation of the enzyme by ethanol will be corrected by Fru-P,. Thus, the activity
of the enzyme will be dependent on the total content of the enzyme. However, if the enzyme
is assayed in the absence of Fru-P, one would find a significant difference in the activity
of the enzyme compared to the control assuming that ethanol alters the conformation of this
enzyme. This latter difference will be a result of the summed effects of the altered nature
of the individual enzyme molecule and the change in the concentration of the enzyme.
Another example is the activation of fructose-I ,6-bisphosphatase (Fru-P,ase) by EDTA.
Such activation seems to depend on the removal of endogenous Zn2 + by EDTA.'09-"0 Zinc
concentration has been reported to decrease in tissues of ethanol-treated rats, including
liver."' By assaying Fru-P,ase in ethanol-treated rats in the absence of EDTA, one would
find a significant difference in the activity of the enzyme compared to the control due to a
lower endogenous concentration of Zn" in the cytosol. However, by assaying the enzyme
activity in the presence of EDTA, the activity obtained will be independent of the Zn'
concentration in the cytosol. This is because EDTA will chelate Zn' in both the control-
and ethanol-treated samples.
A. Chronic Ethanol Intake

The effect of chronic administration of ethanol on some of the key enzymes of glucose
metabolism has already been mentioned.'`' In animals treated chronically with ethanol, the
activities of HK and GK are decreased while that of G6Pase is increased.'`'"' In addition
to G6Pase, the other key enzymes of gluconeogenesis, PC, PEPCK, and Fru-P,ase may also
Volume 11 81
be influenced by chronic ethanol. It has been reported that chronic ethanol increases the
activities of PC and PEPCK. "2 The reactions of these two enzymes bypass the irreversible
PK reaction. They catalyze the conversion of pyruvate back to PEP via oxaloacetic acid as
shown in Equations 5 and 6, and in Figure 3.
AcetylCoA + Me + Me'
Pyruvate + CO2 + ATP > Oxaloacetate + ADP + Pi
PC
(5)
Mn'
Oxaloacetate + GTP PEPCK > Phosphoenolpyruvate + CO2 + GDP (6)
The increase in the activities of these key gluconeogenic enzymes and the decrease in the
activities of some of the key glycolytic enzymes may partially explain the higher blood
glucose levels in alcoholics as compared with nonalcoholics. It has also been suggested that
hyperglycemia may be caused by increase in glycogenolysis and that hypoglycemia may
follow complete glycogen depletion."
However, there is a reported lack of increase in the activities of the glycogenolytic enzymes
after 40 to 60 days of chronic alcoholism.'` As shown in Table 5, the activity of phosphorylase
a is decreased by chronic ethanol ingestion. The reported increase in the G6Pase/IHK(GK)1
activity ratio suggests that chronic ethanol feeding may favor glucose production over glucose
utilization. The mechanism of hyperglycemia in this situation may be similar to that operating
in the case of diabetes. That is, there may be glucose hyperproduction and underutilization."4
The decrease in activities of enzymes involved in glycogen synthesis (Table 5) supports a
possible increase in the threshold for stopping glucose release into the circulation."' Since
the activity of PGM is very high in these animals, it is likely that the rate limiting effect
will be due to the activity of UDPGP. In males, UDPGP is inhibited by 62% due to chronic
alcohol ingestion.' Such inhibition compared with the inhibition of phosphorylase a (21%)
will slow down glycogenesis more than glycogenolysis in the male animals. The net effect
may be a negative glycogen balance with possible glycogen depletion and a decreased glucose
level in blood. It will be interesting to do a time response study on the levels of glucose
and glycogen during chronic alcoholism to determine the period of onset of hypoglycemia
in these animals.'`'
B. Ethanol Perfusion
Changes in the activities of some enzymes of glucose metabolism following perfusion of
tissues with ethanol has been reported by different authors.
The analysis of ethanol-perfused livers shows decreases in the levels of GK and PFK .8°
HK and PK activities have been reported to be unaffected by such ethanol treatments. The
activities of most of the gluconeogenic enzymes were reported to increase in the ethanol-
perfused liver. The activity of G6Pase was reported to increase slightly in the perfused
liver."'"" Increase in the PC and PEPCK activities in the perfused kidney were reported to
be comparable to those observed in chronic alcoholism."'
A decrease in the activities of some key glycolytic enzymes and an increase in the activities
of key gluconeogenic enzymes, reported above, may result in hyperglycemia. An increase
in glucose concentration following ethanol perfusion of liver from fed rats has been reported.'
As shown in Table 6, the increase in the blood glucose level may be due to an increase in
glucose output.
It was reported that ethanol perfusion does not change the level of glycogen in the liver
Table 5
THE EFFECT OF CHRONIC ETHANOL
INGESTION ON THE ACTIVITIES
(nmol/min/mg PROTEIN) OF SOME ENZYMES OF
GLYCOGEN AND GLUCOSE METABOLISM IN
RAT LIVER
Ethanol-
Enzyme Control treated % Change
UDPG Pyrophosphorylase
Male 17.5 6.6 -62
Female 55.1 52.8 -4
Phosphoglucomutase
Male 476.8 489.9 +3
Female 204.9 146.0 -29
Phosphorylase a
Male 72.8 57.2 -21
Female 44.7 26.3 -41
Phosphorylase a (in presence
of I mM AMP)
Male 81.7 80.8 -1
Female 62.4 57.0 -9
G6P Dehydrogenase
Male 65.7 38.1 -42
Female 145.0 146.1 <1
Glucose-6-phosphatase
Male 181.1 260.1 +44
Female 193.1 296.2 +53
Hexo(gluco)kinase
Male 22.6 13.3 -41
Female 44.5 24.4 -45
Glucose-6-phosphatase/
hexo(gluco)kinase
Male 8.0 19.5 +149
Female 4.3 12.1 + 181
From Winston, G. W. and Reitz, R. C Life Sci., 26, 201, 1980. With
permission.
of fed rats. However, the lactate content of the perfusate was reported to decrease during
the perfusion.' This supports the involvement of gluconeogenic enzymes in the ethanol-
induced hyperglycemia in fed animals. A similar study has shown that oxygen consumption
increases in the ethanol-perfused liver of fed rats (Table 1).9' Such an increase has been
explained in terms of an ethanol-induced decrease in glycolysis and subsequent decrease in
the production of ATP. This supports the findings that some key glycolytic enzymes are
inhibited in ethanol-perfused liver. Therefore, the inhibition of these enzymes may contribute
to hyperglycemia by decreasing glucose utilization.
In the liver from fasted animals, the effect of ethanol on these enzymes may be different
from that in fed animals. The general observations are a decrease in gluconeogenesis,' and
an increase in lactate. However, there is at least one report on the inhibition of PFK reaction
by infusion of 10 mM ethanol in liver from fasted rats." The question remains as to whether
the hypoglycemic state occurring in the fasted animals receiving ethanol is due to decreased
gluconeogenesis alone, or if an increase in glycolysis plays a role. Even when this question
is answered in the isolated liver, another arises. That is whether or not the same results can
be obtained in the in vivo condition following acute ethanol treatment. We tried to answer
Volume II 83
Table 6
EFFECT OF ETHANOL PERFUSION IN LIVERS OF FED
RATS ON THE CONCENTRATION AND OUTPUT OF
GLUCOSE
Glucose
Concentration in perfusate Output

(p.mol/mf of blood) (p.mol/hr/g of liver)
Time of
perfusion Ethanol- Ethanol-
(min) Control treated % Change Control treated
0 8.4 9.1 +8
15 7.2 8.8 + 22 -23.1 + 3.1
30 6.7 9.3 +39 + 0.1 +30.0
45 6.5 10.0 +54 + 3.3 +27.3
60 6.5 10.7 +65 + 9.7 +28.3
From Topping, D. L., Clark, D. G., Stover, G. B., Trimble, R. P., and Illman, R.
J., Biochem. J., 184, 97, 1979. With permission.
Table 7
CONCENTRATION OF ETHANOL
IN BLOOD OF FED RATS, 1 HR
AFTER INTRAGASTRIC OR
INTRAPERITONEAL
ADMINISTRATION OF DIFFERENT
DOSES OF ETHANOL
Blood alcohol
(mmol/t )'
Ethanol
(g/kg) I.G. I.P. LP.'
1 19.4 36.52 18.9

2 25.9 41.30' 42.4
3 60.3 72.39 67.4
From Reference 65.

From Reference 115.
From Reference 45.
this question by measuring the activities of key gluconeogenic and glycolytic enzymes in
livers of rats given ethanol.8'
As shown in Table 7, the administration of ethanol through an intragastric or intraperitoneal
route results in a dose dependent increase in the blood ethanol concentration. Also as shown
in Table 8, the blood concentration of ethanol 1 hr after intraperitoneal injection of this drug
is similar for both fed and fasted rats. This blood concentration has been reported to decline
at later times as also shown in the table. However, as shown in the lower section of Table
8, the liver concentration of ethanol, measured at the same time after injection, in the fed
rats is less than that in the livers of fasted rats. This is probably due to a faster rate of ethanol
metabolism in the fed as compared to the fasted rats. The effect of ethanol on the enzymes
Table 8
CONCENTRATION OF ETHANOL IN
BLOOD AND LIVER OF FED AND
FASTED RAT FOLLOWING
INTRAPERITONEAL INJECTION OF
2 g ETHANOL/kg BODY WEIGHT
Ethanol level in blood (mil/)a
Hours after Starved rats (48

injection Fed rats hr)
I 41.3 41.3
2 33.7 37
3 26.1 (41.7)h 32.6 (46.5)
Ethanol concentration in liver

(m/IT)°
Hours after Starved rats (48

injection Fed rats hr)
I 35 39
2 26 33
From Reference 45.

Intraperitoneal injection of 3 g ethanol/kg body
weight.
From Reference 116.
From Reference 117.
of glucose metabolism may vary in the animals depending on the state of nutrition. This
may be reflected in the extent to which the glucose level is altered when ethanol is given
during different durations of starvation. For example, the dose of ethanol related to increase
glucose level in fed animals, did not conclusively decrease the level of blood glucose in
animals fasted for less than 24 hr.16.48.61.68 However, following longer periods of starvation,
equivalent doses of ethanol cause a decrease in blood glucose.' 7-'8-77
C. Acute Ethanol Administration In Vivo

When 1 or 3 g of ethanol per kilogram body weight is injected intraperitoneally into fed
rats and liver enzymes analyzed in vitro, HK is inhibited more than any other key glycolytic
enzyme (Table 9). However, a high dose of ethanol (5 g/kg) inhibits PFK most, followed
by PK, then HK and GK. Thus ethanol (5 g/kg) decreases the activities of all the key
glycolytic enzymes in vivo in the fed rats.
Ethanol causes a relatively mild inhibition of key gluconeogenic enzymes except PC, and
G6Pase in the presence of its activator, phosphatidylcholine. Of these enzymes, PEPCK
appears most sensitive to the inhibition by ethanol." The activity of PC increases (7 to 28%)
in the liver of fed rats given ethanol. The inhibition of PEPCK and Fru-P,ase range from 7
to 28%. The G6Pase activity seems least sensitive to the effect of ethanol in the liver of
fed rats. This low sensitivity seems to be characteristic of G6Pase from liver of fed rats as
it was also observed in the perfusion experiments mentioned earlier.80 -H 2 In the in vivo
study, the activation of G6Pase (20%) occurs only with a high dose of ethanol in the presence
of phosphatidylcholine.81
Fasting for 48 hr decreases the activities of GK, HK, and PFK while increasing that of
PK as shown in Table 10. The activities of all the gluconeogenic enzymes, except Fru-
P,ase, have also been shown to increase during 48 hr fasting in rats." It appears that ethanol
Volume II 85
Table 9
EFFECT OF ETHANOL ON THE ACTIVITIES OF KEY GLYCOLYTIC AND
GLUCONEOGENIC ENZYMES FROM LIVERS OF FED RATS
Enzymes µmol/min/mg protein

Ethanol
(g/kg body wt) GK HK PFK PK PC PEPCK Fru-Pease G6Pase
0 0.33 0.017 0.006 0.29 0.14 0.071 0.29 0.38

1 0.034 0.013 0.005 0.22 0.15 0.061 0.27 0.30
3 0.032 0.012 0.006 0.022 0.18 0.065 0.25 0.39
5 0.026 0.013 0.003 0.33 0.17 0.053 0.27 0.36
From Duruibe, V. and Tejwani, G. A., Mol. Pharmacol., 20, 621, 1981. With permission.
Table 10
EFFECT OF FASTING (48 hr) AND
ETHANOL (1-5 g/kg BODY WT) ON THE
ACTIVITIES OF RATE LIMITING ENZYMES
OF GLYCOLYSIS AND GLUCONEOGENESIS
Ethanol in fasted
Enzymes Fed Fasted 1 g/kg 3 g/kg 5 g/kg
Glucokinase 0.033 0.022 0.017 0.016 0.012

Hexokinase 0.017 0.0037 0.0028 0.0034 0.0037
Phosphrofructokinase 0.006 0.005 0.004 0.005 0.006
Pyruvate kinase 0.29 0.79 0.78 0.70 0.65
Pyruvate carboxylase 0.14 0.168 0.185 0.163 0.175
Phosphoenolpyruvate- 0.071 0.18 0.18 0.25 0.3
carboxykinase
Fructose bisphosphatase 0.29 0.20 0.22 0.26 0.21
Glucose 6-phosphatase 0.38 1.47 1.36 1.69 1.15
From Duruibe, V. and Tejwani, G. A., Mol. Pharmacol., 20, 621, 1981. With permission.
given to the fasted animals inhibits the activities of hepatic GK and PK without significantly
affecting HK. A high dose of ethanol (5 g/kg) seems to increase the activity of PFK in the
fasted rats. This increase can be as high as 40% in the presence of the activators of PFK,
and is important since PFK is the major rate limiting enzyme in glycolysis.
Of all the key gluconeogenic enzymes in the liver of fasted rats, the activity of PC seems
least sensitive to the effect of ethanol.' High doses of ethanol (3 to 5 g/kg) increase the
activity of PEPCK in fasted rats. The increase in the activity of PEPCK may be due to
metabolic acidosis occurring in the animals. 12 The reaction of Fru-Pease to ethanol treatment
in these rats may depend on the presence or absence of Zn2 ± . In the absence of EDTA
which chelates Zn2 ± , the activity of this enzyme does not significantly vary between ethanol-
treated rats and the control. However, analysis of Fru-P2ase activity in the presence of EDTA
revealed an increase in the activity of this enzyme in the livers of ethanol-treated, fasted
rats. The lack of effect of ethanol on the activity of Fru-P,ase under in vivo conditions may,
at least, partially explain the lack of effect of ethanol on gluconeogenesis from DHA in the
Table 11
EFFECT OF ETHANOL ON KEY
GLUCONEOGENIC AND GLYCOLYTIC
ENZYMES
% change in the specific activity

of the enzyme in the livers of
rats given 5 g/kg ethanol
Fasted
Enzymes Fed (48 hr)
Glycolytic
Hexokinase —24
Glucokinase —20 —46
Phosphofructokinase —50 ( — 29)" —20 ( + 40)
Pyruvate kinase —38 ( — 17) —18 ( —23)
Gluconeogenic
Pyruvate carboxylase +21 +4
Phosphoenolpyruvate- —25 + 66
carboxykinase
Fructose-bisphosphatase —8 ( — 25) 0 ( + 48)
Glucose-6-Phosphatase —5 ( + 20) —22 (0)
Gluconeogenic/glycolytic % activity ratio'

(PC + PEPCK)/PK 1.58 1.64
Fru-P,ase/PFK 1.9 0.83
G6Pase/(HK + GK) 1.22 1.00
Percent change in presence of specific activators is shown in

parenthesis.
All ratios were divided by the ratios obtained in controls. For
example, control (PC + PEPCK)/PK = (100 + 100)/100 =
[(121 + 75)/62]
2; ethanol-treated fed (PC + PEPCK)/PK
2
= 3.16/2 = 1.58.
From Duruibe, V. and Tejwani, G. A., Mot. Phurmacol., 20, 621,

1981. With permission.
fasted rats." Our studies showed that a high dose of ethanol (5 g/kg) inhibits the activity
of G6Pase in the fasted rats." Such inhibition may be reversed by the phospholipid, phos-
phatidylcholine (10 mM).
The percentage changes in the activities of key glycolytic and gluconeogenic enzymes in
livers of fed and fasted (48 hr) rats, 1 hr after the intraperitoneal injection of a high dose
of ethanol (5 g/kg) are listed in Table 11. This table suggests that the flux of substrates
through the three cycles between glycolysis and gluconeogenesis will favor hyperglycemia
in fed and hypoglycemia in fasted animals receiving ethanol. These fluxes are expressed as
the ratios of percentage activities of gluconeogenic vs. glycolytic enzyme(s) at each possible
futile cycle."5 Expression of the total activity ratios for gluconeogenic/glycolytic enzymes
(Table 12) also supports the possibility of hyperglycemia in fed and hypoglycemia in fasted
animals treated with high doses of ethanol. Such glycemic effects of ethanol have been
reported in the fed and fasted animals receiving ethano1.7.9'
Therefore changes in the activities of key glycolytic and gluconeogenic enzymes may
play an important role in the mechanism of ethanol induced changes in blood glucose level.
Volume II 87
Table 12
EFFECT OF ETHANOL (5 g/kg BODY WT) ON THE TOTAL
ACTIVITIES AND ACTIVITY RATIOS OF KEY
GLUCONEOGENIC AND GLYCOLYTIC ENZYMES IN RAT
LIVER
Fed animals Fasted animals
Enzymes Control + Ethanol Control + Ethanol
G6Pase 12.2 13.8 29.3 36.3

HK + GK 0.89 + 0.26 0.71 + 0.13 0.5 + 0.07 0.26 + 0.06
(10.6) (16.4) (51.4) (113.4)
Fru-P2ase 8.2 7.3 3.73 3.76
PFK 0.2 0.06 0.065 0.07
(41) (121.6) (57.4) (53.7)
PC + PEPCK 4.36+1.5 4.5+1.1 4.24 + 3.05 2.7+2.6
PK 6.9 3.4 11.34 13.71
(0.85) (1.6) (0.64) (0.38)
Note Numbers in parenthesis indicate the ratios of the activities of the gluconeogenic
enzymes to the glycolytic enzymes. Data expressed in units/g liver wet wt.
VI. SUMMARY AND CONCLUSIONS
Ethanol-induced hypoglycemia was first described in 1941. It occurs in alcoholics, healthy,

fasted adults, and in children. This hypoglycemia is reversed by intravenous glucose. Hy-
perglycemia may also occur in fed animals receiving alcohol. These glycemic effects result
from the alteration in the rates of glycogen and glucose turnover (utilization and synthesis).
Such alterations may be due to changes in the concentrations of hormones and modulators
of blood glucose.
Ethanol may also decrease the bioavailability of products of carbohydrate digestion. It
inhibits pancreatic amylase and also decreases the active transport of glucose across the
small intestine. The storage form of carbohydrate, glycogen, is depleted by ethanol. Ethanol
stimulates the breakdown of glycogen to glucose-6-phosphate.
Liver, which is the main organ for glucose synthesis, is also responsible for metabolizing
75 to 90% of total ethanol in the body. Other organs like the lungs, kidneys, and brain
metabolize only about 2 to 10% of the ethanol. The metabolic products of ethanol include
acetaldehyde, acetate, and NADH, Most intermediary metabolic effects of ethanol have
been linked to the generation of reducing equivalents NAD(P)H by ethanol metabolism.
Some of the blood glucose modulators which can be affected by ethanol include insulin,
glucagon, catecholamines, cAMP, Ca" , AMP, citrate, F2,6-P2, NADPH, NADH, and
Mg". Such alterations can influence glucose metabolism.
Ethanol decreases plasma insulin in both fed and fasted animals. At least 4.5 g/kg body
weight of ethanol intake is necessary to cause a significant decrease in the concentration of
plasma insulin. However, even lower doses of ethanol can increase insulin response to
glucose administration in fed animals. In fasted animals, ethanol decreases insulin response
to glucose loading. Ethanol decreases pancreatic release of insulin. Ethanol increases plasma
glucagon in both fed and fasted animals. The effect on glucagon may be caused by both
ethanol and its metabolites. By varying the levels of insulin and glucagon, ethanol can affect
changes in blood glucose levels.
Urinary excretion of catecholamines and their metabolites is increased by ethanol. In

fasted animals, ethanol inhibits the hyperglycemic effects of catecholamines but in fed
animals it unmasks the catecholamine-induced hyperglycemia. Ethanol may also induce
adrenergic subsensitivity, decreasing plasma cAMP response to catecholamines. In fasted
rats, the hepatic cAMP level is increased by ethanol and this is accompanied by decreases
in glycogen and glucose levels.
The concentrations of calcium and magnesium may be decreased by ethanol. AMP is
slightly increased by ethanol in fasted rats. This increase in AMP may activate phosphorylase,
thus increasing glycogenolysis.
The generation of NADH during ethanol oxidation is associated with an increase in
NADPH. This can inhibit glucose utilization through the hexose monophosphate pathway
at the levels of G6PDH and 6PGDH.
Ethanol increases the level of G6P and also decreases the activity of HK in fed animals.
G6P is an allosteric inhibitor of HK. Glucokinase activity may also be decreased by ethanol.
The effect of ethanol on UDPGP, G6PDH, and PGM may be dependent on the gender of
the animal. Low carbohydrate does not seem to be absolutely required for ethanol to induce
hypoglycemia, especially in chronic alcoholism. However, ethanol is more likely to induce
hypoglycemia when liver glycogen is already depleted.
Glycolysis seems to be the most likely route of an ethanol-induced increase in glucose
utilization. Pyruvate produced during glycolysis may be rapidly converted to lactate using
NADH generated by ethanol oxidation. Lactate and pyruvate may also be converted to
glucose in fed animals. The increased NADH/NAD+ ratio in these animals will also increase
all reductive reactions at the expense of oxidative reactions. The increase in NADH may
also inhibit the conversion of PEP to pyruvate while also increasing formation of lactate
from pyruvate. This will produce an increase in the lactate/pyruvate ratio resulting in the
inhibition of gluconeogenesis.
The NADH shuttles may remove excess NADH from the cytosol, thus decreasing their
influence on glucose metabolism in the cytosol. Ethanol decreases PFK flux during glu-
coneogenesis from DHA, but does not increase the rate of gluconeogenesis from this sub-
strate. A decrease in glycolysis due to ethanol can cause a decrease in ATP synthesis and
subsequent increase in oxygen uptake. The levels of substrates and intermediates of glucose
metabolism can also be altered by ethanol. Some of these substrates and intermediates are,
themselves, modulators of key enzymes of glucose metabolism. In fasted rats, it has been
shown that ethanol decreases the concentrations of most gluconeogenic intermediates. This
may be due to increased glycolysis, decreased gluconeogenesis, shuttling of intermediates
away from gluconeogenic pathway, or all of the above. In the fasted rats, ethanol also
decreases gluconeogenesis from other substrates. Ethanol-induced changes in the levels of
substrates, activators, or inhibitors of an enzyme may affect its activity. The enzyme may
be transformed to an altered form which may be more or less active.
Chronic alcoholism increases the activities of the gluconeogenic enzymes G6Pase, PEPCK,
and PC while decreasing the activities of the glycolytic enzymes HK and GK. Such changes
may partially explain the high blood glucose levels found in some chronic alcoholics.
Although both the glycogenic and glycogenolytic enzymes are inhibited by ethanol, it appears
that the inhibition of the former enzymes is more than the inhibition of the latter. This will
result in the depletion of glycogen, and lead to hypoglycemia in chronic alcoholics receiving
an inadequate carbohydrate diet. Acute ethanol treatment can also alter blood glucose. Again,
such alteration may depend on the state of nutrition. Ethanol perfusion decreases the activities
of GK and PFK while increasing the activities G6Pase, PEPCK, and PC. In the livers of
fed rats, ethanol perfusion increases glucose output without affecting the glycogen level. In
the livers from fasted rats, ethanol perfusion decreases gluconeogenesis.
In in vivo studies, ethanol effect on enzymes of glucose metabolism has been found to
Volume II 89
vary depending on the state of nutrition and dosage of ethanol. Intraperitoneal injection of
ethanol into fed rats results in the inhibition of key glycolytic enzymes, while increasing
the activities of some key gluconeogenic enzymes such as PC and G6Pase. The activities
of most of the key glycolytic enzymes are decreased while those of key gluconeogenic
enzymes are increased by fasting. High doses of ethanol in fasted animals may increase the
activity of PFK which is the major rate limiting enzyme of glycolysis. The other glycolytic
enzymes are slightly inhibited or unaffected. The key gluconeogenic enzymes, PC and Fru-
P2ase, are not very sensitive to ethanol in fasted rats in situ. However, G6Pase activity is
decreased by ethanol in these rats while PEPCK activity increases.
The ratios of the activities of gluconeogenic:glycolytic enzymes at key points in glucose
metabolism support hyperglycemia in fed and hypoglycemia in starved animals. Such gly-
cemic states have been reported in studies in which the blood glucose level was monitored
in fed and fasted animals given ethanol.
In conclusion, ethanol can influence carbohydrate metabolism at the steps of digestion,
conversion to glucose-6-phosphate, utilization for energy production, and synthesis of other
biological molecules such as other sugars, nucleotides, and citric acid cycle intermediates.
The level of glucose in the blood is altered by ethanol in fed and fasted animals. This
alteration may mean an increase or a decrease in blood glucose level.
Such changes may be due to the effects of ethanol and its metabolic products on modulators,
substrates, intermediates, and enzymes of glucose metabolism. In the fed animals these
changes result mostly in hyperglycemia, while in fasted animals the result is hypoglycemia.
These glycemic states may result from ethanol-induced changes in glucose utilization, pro-
duction, or both.
ACKNOWLEDGMENTS
This work was supported by grants from the Distilled Spirits Council of the United States,
Inc. and the Weight Watchers Foundation, Inc. We thank Ms. Carol Jones for the typing
of this manuscript and Ms. Catherine Rowley for her help in reviewing the manuscript.
Duruibe was partially supported by the Federal Government of Nigeria.
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92. Richards, C. S., Furuya, E., and Uyeda, K., Regulation of fructose-2,6-P, concentration in isolated
hepatocytes, Biochem. Biophys. Res. Commun., 100(4), 1673, 1981.
93. Van Schaftingen, E., Jett, M. F., Hue, L., and Hers, H. G., Control of liver 6-phosphofructokinase
by fructose-2,6-biophosphate and other effectors, Proc. Natl. Acad. Sci. U.S.A.. 78(6), 3483, 1981.
94. Muniyappi, K., Leibach, F. H., and Mendicino, J., Reciprocal regulation of fructose-I ,6-bisphosphatase
and phosphofructokinase by fructose- I ,6-bisphosphate in swine kidney, Life Sci., 32, 271, 1983.
95. Tejwani, G. A., The role of phosphofructokinase in skeletal muscle contraction, Arch. Bio/. Med. Exp.,
12, 617, 1979.
96. Van Schaftingen, E., Hue, L., and Hers, H. G., Study of fructose 6-phosphate/fructose-1,6-phosphate
cycle in the liver in vivo, Biochem. J., 192, 263, 1980.
Volume II 93
97. Lindros, K. 0. and Stowell, A., Effects of ethanol-derived acetaldhyde on the phosphorylation potential
and on the intramitochondrial redox state in intact rat liver, Arch. Biochem. Biophys., 218(2), 429. 1982.
98. Weil-Malhatbe, H. and Bone, A. D., Studies in hexokinase. I. The hexokinase activity of rat brain extract,
Biochem. J., 49, 339, 1951.
99. Van Schaftingen, E. and Hers, H. G., Inhibition of fructose-I ,6-bisphosphatase by fructose-2,6-bis-
phosphate, Proc. Natl. Acad. Sci. U.S.A., 78, 2861, 1981.
100. Hers, H. G. and Van Schaftingen, E., F2,6-bisphosphate 2 years after its discovery, Biochem. J., 206,
I, 1982.
101. Tejwani, G. A., Regulation of fructose-bisphosphatase activity, Adv. Enzymol. Relat. Areas Mol. Biol.,
54, 121, 1983.
102. Van Schaftingen, E., Davies, D. R., and Hers, H. G., Inactivation of phosphofructokinase 2 by cyclic
AMP-dependent protein kinase, Biochem. Biophys. Res. Commun., 103, 362, 1981.
103. Tejwani, G. A. and Ramaiah, A., Properties of phosphofructokinase from the mucosa of rat jejunum and
their relation to the lack of Pasteur effect, Biochem. J., 125, 507, 1971.
104. Tejwani, G. A. and Mousa, S., Allosteric properties of rat lung phosphofructokinase, Enzyme, 26, 306,
1981.
105. KrebS, H. A., Freedland, R. A., Hems, R., and Stubbs, M., Inhibition of hepatic gluconeogenesis by
ethanol, Biochem. J., 112, 117, 1969.
106. Freedman, A. D. and Kohn, L., Pyruvate metabolism and control, factors affecting pyruvate carboxylase
activity, Science, 145, 58, 1964.
107. Kreb, H. A., The regulation of the release of ketone bodies by the liver, Adv. Enzyme Regul., 12, 339,
1966.
108. Ramaiah, A. and Tejwani, G. A., Interconvertible forms of phosphofructokinase of rabbit liver. The role
of effectors on the interconversion, Eur. J. Biochem., 39, 183, 1973.
109. Paglic, D. E. and Valentine, W. M., Additional kinetics distinctions between normal pyruvate kinase and
mutant isozyme from human erythrocytes. Correction of the kinetic annomaly by fructose-1,6-diphosphate,
Blood, 37, 311, 1971.
110. Tejwani, G. A., Pedrosa, F. 0., Pontremoli, S., and Horecher, B. L., Dual role of Zn2 " as inhibitor
and activator of fructose-I ,6-bisphosphatase, Proc. Natl. Acad. Sci. U.S.A., 73, 2692, 1976.
I 1 1. Ahmed, S. B. and Russel, R. M., The effect of ethanol feeding on zinc balance and tissue zinc levels in
rats maintained on zinc deficient diets, J. Lab. Clin. Med., 100, 211, 1982.
112. Chan, A. W. K., Leong, F. W., and Schanley, D. L., Acute and chronic effects of ethanol on hepatic
and renal gluconeogenic enzymes. Subst. and Alcohol Actions/Misuse, I, 43, 1980.
113. Kailas, P. and Sellers, E. M., Blood glucose in intoxicated chronic alcoholics, C.M.A., 112, 590, 1975.
114. Garfield, S. A. and Cardell, R. A., Hepatic glucose-6-phosphate activities and correlated ultrastructural
alterations in hepatocytes of diabetic rats, Diabetes, 28, 664, 1979.
115. Bloom, F., Lad, P., Pittman, Q., and Rogers, J., Blood alcohol levels in rats: non-uniform yields from
intraperitoneal doses based on body weight, Br. J. Pharmacol., 75, 251, 1982.
116. Smith, M. E. and Newman, H. W., The rate of ethanol metabolism in fed and fasting animals, J. Bio/.
Chem., 234, 1544, 1959.
117. Lumeng, L. et al., Advances in Experimental Medicine and Biology, Thurman, R. G., Ed., Plenum, New
York, 1980, 489.
Taylor & Francis
Volume II 95
Chapter 5
EFFECT OF SUCROSE AND FRUCTOSE ON CARBOHYDRATE AND LIPID

METABOLISM AND THE RESULTING CONSEQUENCES
Eleazar Shafrir
TABLE OF CONTENTS
I. Introduction 96
II. Intestinal Metabolism of Sucrose and Absorption of Fructose 96
III. Pathway of Fructose Metabolism 98
IV. Regulation of Fructose Metabolism 101

A. Fructokinase and Related Enzymes 101
B. Metabolic Response to Acute Fructose Excess 103
C. Enzymatic Adaptation to Long-Term Sucrose or Fructose Diets 106
V. Sucrose (Fructose)-Induced Hyperlipidemia 109
VI. Adipose Tissue Response to Sucrose or Fructose Diets 111
VII. Hormonal Alterations on Sucrose Diet Inducing Increased Energy

Consumption 112
VIII. Features of the Specific Effect of Fructose on Lipogenesis and Lipid

Metabolism 115
IX. Deleterious Effects of Sucrose Diets 118

A. Sucrose and Diabetes 118
B. Hyperlipidemia and Atherosclerosis 119
C. Pregnancy, Perinatal and Survival Aspects 120
D. Hepatic Effects 122
X. Concluding Comments with Thoughts on Future Research on Sucrose Nutrition

and Metabolism 123
References 125
I. INTRODUCTION
The disaccharide sucrose is widely distributed in plants, fruits, and vegetables and con-
stitutes an important share of dietary carbohydrate. Its consumption rises steadily with the
affluence and acculturation of the human population in various countries."' It is the pre-
dominant source of the ketohexose, fructose, which is responsible for the specific effects
of sucrose on the changes in enzymes and hormones affecting mainly carbohydrate and lipid
metabolism, distinct from those produced by glucose or starch.
The daily intake of fructose (almost all as sucrose) has been estimated to amount 50 to
100 g,' from 1/6 to 1/3 of the total dietary carbohydrate. Excessive parenteral administration
of fructose is fraught with dangers and long-lasting intake of sucrose has been shown to
induce hyperlipidemia, glucose intolerance, and other detrimental reactions.
On the other hand, fructose may have beneficial effects as a low caloric sweetener (it is
1.7 times sweeter than sucrose) which does not induce dental caries. The purpose of this
article is, therefore, to review the pathways of fructose assimilation and the specific enzymatic
and hormonal adjustments related to, or following sucrose absorption and fructose metab-
olism, which may provide the background for the observed impact of this carbohydrate and
guidance in its use. Apart from the above-mentioned monographs, comprehensive reviews
of fructose metabolism and its regulation have been contributed by Hers,' Herman and
Zakim,5 Nikkila and Huttunen,6 and Van den Berghe.7
II. INTESTINAL METABOLISM OF SUCROSE AND ABSORPTION

OF FRUCTOSE
Upon ingestion, sucrose is hydrolyzed to glucose and fructose by sucrase, secreted by

the intestinal cells along with other disaccharidases. Sucrase is not a sucrose-specific enzyme,
it also splits the 1:6 bonds on a-dextrins and nowadays it is referred to as sucrase-a-dextrinase
(isomaltase).8 Conklin et al.' were able to show that sucrase is a hybrid enzyme (mol wt
280,000), composed of immunologically distinct two glycoprotein chains, one with sucrase,
the other with isomaltase activity (mol wt 125,000 and 115,000, respectively). The probable
advantage of having two enzyme activities on one molecule is that it may facilitate a
cooperative hydrolysis of adjacent 1:4 and 1:6 bonds on a-dextrin.
The activity of sucrase-a-dextrinase is in the range of 40 to 165 µmol/min/g intestinal
protein or 3 to 14 µmol/min/g tissue wet weight. This exceeds the activity of lactase,--2-
fold but is only —1/3 of that of maltase. The K, to sucrose is 20 mM, similar to that of
lactase, but —5-fold that of maltase. The high activity of the enzyme and its broad pH
optimum (5.0 to 7.0) enables it to cope with high loads of ingested sucrose. Little is known
on regulatory influences on sucrase and other oligosaccharidases as well. It appears to be
maximally active already at birth'" and does not decrease with age—as often happens with
lactase. Feeding of sucrose or fructose increases the activity of jejunal sucrase and maltase
after 2 to 5 days,'" which indicates that there is no specific induction of the enzyme protein
but that the sucrase secreting cell is affected (this is the approximate turnover time of the
intestinal digestive absorptive cell, whereas t,,, of sucrase is ---11 hr).' In man, fructose
seems to be the specific component inducing the sucrase activity" and feeding a high sucrose
diet enhances fructose absorption.'2
Glucocorticoid treatment accelerates the appearance of the activity of sucrase and of other
disaccharidases during the early intestinal development but does not induce the activity of
sucrase in the adult rat.I6 The increase in jejunal sucrase activity in rats, prematurely weaned
onto sucrose, is also attributed to steroid hormones, since it was abolished by adrenalec-
tomy."7 Progesterone treatment of 2-month-old rats does not appreciably increase sucrase
activity while promptly augmenting that of lactase and maltase,18 a finding compatible with
similar changes occurring in pregnancy. '9 Streptozotocin-induced diabetes is associated with
Volume II 97
a marked rise in total sucrase activity, together with that of other disaccharidases,'9.2° but
this appears to be related to the hyperphagia-enhanced overall intestinal growth.
Intolerance to sucrose has been hitherto considered to be a rare disease (-100 published
cases up to 1973).2 ' However, more reports continue to appear with better diagnostic means
and awareness to its specific symptoms, which are generally similar to those of lactase
deficiency. In contrast to lactase deficiency, there seems to be no ethnic predisposition with
the exception of Greenland Eskimos,22^-10% of whom are affected. The deficiency is severe
in infants or young children, in which the enzyme activity drops to <5 p.mol/min/g protein,
but the tolerance to sucrose increases with age. Both subunits of the enzyme are missing,
since quantitative radioimmunoassay with ['25I]-labeled sucrase-a-dextrinase and monospe-
cific antiserum did not reveal any cross-reacting material, indicating a total deficiency of
the enzyme protein.' Starch is tolerated in the diet probably because its indigested a-dextrin
portion represents a small part of the molecule with osmotic activity too low to cause intestinal
discomfort.
Sucrase action produces equimolar quantities of glucose and fructose. Glucose is absorbed
from the intestine by an active transport mechanism, whereas fructose absorption proceeds
by diffusion, 24,25 since it is not inhibited by phlorizin, dinitrophenol, fluoride, or cyanide.
Later it was found that it is transported via a stereospecific, saturable, protein carrier-
facilitated diffusion.'
From the entry into the blood the metabolism of sucrose is actually represented by the
metabolism of fructose, which requires a special metabolic pathway located in the liver, but
evidence exists that already at the point of entry some fructose may be converted to glucose.
Enzymes of fructose metabolism, glycolysis as well as G-6-Pase are present in the mammalian
intestinal mucosa27-" but their quantitative share in total fructose metabolism varies with
species and with the size of the load. For example, human fructose absorption is generally
slower than that of glucose but at the rate of supply of 1 g/min/ 30 cm of jejunum it was
estimated to be equivalent to that of glucose.' Of 50 g of oral fructose given to patients
with liver cirrhosis, most was found in the portal blood as fructose, judging from similar
amounts recovered after 50 g of glucose.' In other studies it was shown that upon an oral
30 g fructose load, there is a peak in the portal vein at 30 to 60 min, which precedes the
systemic blood rise in glucose (peak at 60 to 90 min), strongly indicating that the bulk of
fructose is absorbed as such and converted to glucose by subsequent metabolism."
Species differences may be illustrated by comparing guinea pig and rat intestine. In the
former, of 100 mg fructose —80% was absorbed as glucose in the portal vein and 8 to 30%
was recovered in the intestinal fluid as lactate.' In rats —50% was recovered as fructose
in the portal vein, 25 to 40% as lactate, and only —15% converted to glucose." This
difference was attributed to the variation in the intestinal G-6-Pase activity.'
The metabolism of a fraction of the absorbed fructose in mucosal cells during sucrose
feeding was postulated to facilitate its transport through an effect on jejunal permeability."
Fructose, like glucose, enters the liver by insulin-independent diffusion.39 However, fruc-
tose does not enter the hepatic cell freely — a gradient exists between plasma and intracellular
concentrations.' As calculated from liver perfusion experiments, there is a carrier-mediated
transport with a Km considerably higher than that of fructose phosphorylation and may
constitute a limiting factor of its metabolism.'
Pancreatic insulin secretion is not appreciably stimulated by fructose as shown by a follow
up of insulin secretion after a fructose load in the whole organism in various species,4'-44
and by lack of fructose effect on isolated pancreas preparations.'" The small amounts of
insulin which appear in the plasma after in vivo sucrose intake are derived from the pancreas
stimulation by the glucose moiety."
GLYCOGEN
\ I
SUCROSE G -1 -P
glucokinase phosphogluco-
,eocros mutase
.hexokinase
96p dehydrogenase
GLUCOSE 04-P 6
grrirr isomerase
hexokinase
F-S-P
FilucTosE fructokinase
r F-1,542P
aldolase aldolase
GA
DHAP 4 isorf OA-3
glycerol I
)I
triokinase I
ga deh
YUioDen
GLYCEROL ase )-CF/) •
41/1.,
l-3-P -44-03.‘
glyceroogenase
dehydr
GLYCERATE -2 -P
GLYCEROL-3-P
PEP
I
pyruvate
ki nese
Id h
PYRUVATE ----LACTATE
MITOCHONDRIAL I pdh
NADH - FAD
SHUTTLE
ACETYL-CoA
•
•
FATTY ACIDS,
CHOLESTEROL
FIGURE 1. Metabolism of sucrose-derived fructose in the liver. The main pathway after fructose
absorption is fructokinase-catalyzed phosphorylation to F-1-P, which is then cleaved by F-I-P aldolase
to glyceraldehyde (GA) and dihydroxy acetonephosphate (DHAP). While DHAP directly enters the
glycolytic flow — GA is metabolized by several pathways, the most active of which is phosphorylation
to glyceraldehyde phosphate (GA-3-P) by triokinase. Since fructolysis starts below the phosphofruc-
tokinase (pfk)-committed step of glycolysis, the flow towards pyruvate formation and entry into the
mitochondrial citrate cycle is preferred, resulting in lipogenesis enhancement. This is abetted by the
increased supply of glycerol-3-P (for more detailed discussion see Section III).
III. PATHWAY OF FRUCTOSE METABOLISM
As illustrated in Figure 1, it has long been recognized that the first step of fructose
metabolism upon its absorption is the ATP-dependent phosphorylation to F-1-P by a specific
fructokinase (ketohexose-l-phosphotransferase), located almost exclusively in the liver."--"
Although glucokinase and hexokinase (hexose-6-phosphotransferases) can also phosphorylate
fructose5a-56 the Km of these enzymes toward fructose is high (-12 triM57 and —2 mM,5s
respectively, compared to 0.2-0.5 mM of fructokinase), and glucose competitively inhibits
the 6-phosphorylation of fructose. Therefore, at the ambient plasma hexose concentrations
after a sucrose load of 1 g/kg (1 to 3 mM fructose and 5 to 7 mM glucose), phosphorylation
of fructose by enzymes other than fructokinase is negligible and may be disregarded as a
Volume II 99
Table 1
LIVER HEXOSEPHOSPHOTRANSFERASE
ACTIVITIES IN SEVERAL SPECIES
Glucokinase
FK
Species (no.) Fructokinase hexokinase GK + HK
Spiny mice (14) 38.6 ± 2.8 7.6 ± 0.4 5.1

Albino mice' (10) 56.2 ± 2.4 16.1 ± 0.9 3.5
Albino rats" (12) 48.8 ± 2.1 16.8 ± 0.7 2.9
Humans' (6) 16.6 ± 2.8 4.2 ± 0.6 4.0
Note: Activities in the table are means ± SE from several experiments

and from unpublished investigations, expressed as nmol/min
(37°C) per mg protein in liver cytosol (100,000 g supernatant
fraction). Glucokinase and hexokinase activities were deter-
mined by the method of Sharma et al." and that of fructokinase
according to Adelman et al."' All animals were ad libitum fed
on a regular laboratory chow.
Acomys cahirinus from the Jerusalem strain.

Mice and rats from the Hebrew University strain.
Liver samples from surgical biopsies obtained under anesthesia and
12 hr fast taken from patients suspected for unrelated genetic enzyme
deficiency.
meaningful pathway of physiologic utilization. The K„,, of fructokinase for ATP is quite low
(- 1 mm59.60 compared to that of hexokinase of 0.2 mM58) and at the hepatic ATP concen-
tration, ranging from 2 to 3 p,mol/g mM)61 " the intracellular fructokinase activity would
be expected to be close to maximal, at saturating fructose concentrations.
The dominant role of fructokinase in fructose phosphorylation is also due to the fact that
the capacity of this enzyme exceeds two- to fourfold the combined capacities of glucokinase
and hexokinase toward glucose in rodent56•63.' and human65.66 liver (see Table 1). This has
been amply confirmed by substrate channeling studies6'.66.6" and it may be generally estimated
that >80% of circulating fructose is converted to F-1-P in the liver, although this value may
vary according to species.
To join the main glycolytic or glucogenic flow, F- I -P has to be converted to F-1,6-DP.
Direct, repeated phosphorylation of F-1-P is precluded in the mammalian liver because of
the absence of 1-phosphofructokinase activity. This enzyme has been isolated from bacteria69.7"
and utilized for in vitro F-I-P and fructokinase assay."-" Some activity of 1-phosphofruc-
tokinase was found in muscle extracts, perhaps as a side activity of F-6-P-phosphofructo-
kinase,74 but its usefulness is doubtful because of the absence of F-1-P in this tissue. Thus,
the conversion of F-1-P to F-1,6-DP or to other glycolytic intermediates requires indirect
pathways which have been clarified mainly by Hers, Leuthardt, Heinz, Lamprecht, and their
collaborators.4-5 ' '52'65'75 79
F- I -P is promptly cleaved in the liver by aldolase to D-glyceraldehyde (GA) and dihy-
droxyacetone phosphate (DHAP). Liver aldolase activity has been extensively investiga-
ted.80-88 It cleaves F-I-P and F-1,6-DP at the ratio of 1:1 and differs from muscle aldolase
in several aspects, particularly in its high activity and affinity towards F-1-P. Both aldolases
are composed of four subunits which can be dissociated and recombined to form hybrid
variants with varying activities towards F-I-P and F-1,6-DP."' Deficiency of certain subunits
in liver aldolase is indicated by the selective deficiency of the F-1-P cleaving activity in the
genetic fructose intolerances' and by the different ratios of F-1-P to F-1,6-DP aldolase
activities in muscle and liver.
The DHAP produced by aldolytic cleavage of F-1-P can condense with GA-3-P to form
F-1,6-P, catalyzed by the reverse action of aldolase, and proceed to glucose and glycogen,
or can be converted to GA-3-P by the action of triosephosphate isomerase and follow the
glycolytic flow (see Figure 1). DHAP can be also reduced to glycerol-3-P by a NAD-
dependent, cytosolic glycerol-P dehydrogenase." Glycerol-3-P is important for two meta-
bolic pathways: (1) transport of cytosolic reducing equivalents into mitochondria ("glycerol-
3-P-DHAP shuttle") and (2) esterification of fatty acids to glycerides and phospholipids
(see below).
The GA may be metabolized by three enzymes. The chief route appears to be direct
phosphorylation to GA-3-P by the cytosolic triokinase,m-65.75.9' 9' which then may join the
glycolytic or gluconeogenic metabolite flow. Triokinase matches fructokinase with respect
to V ax and K„, and interestingly, may utilize to a certain extent ITP and GTP as the phosphate
donor.' It also exhibits an adaptive behavior similar to that of fructokinase.'4°
The second possibility for GA metabolism is conversion to 0-glycerate by a mitochondrial
GA dehydrogenase,64 66'94'95 followed by phosphorylation to glycerate-2-P by a glycerate
kinase widely distributed among the tissues."'" The activity ratio glycerate kinase/GA
dehydrogenase in rat liver is 2:164 and would favor such reaction, whereas in the human
liver, glycerate kinase activity was reported to be low65 and glycerate phosphorylation may
be rate limiting. In fact, this is an important argument in favor of participation of this
pathway since glycerate accumulates in the liver after a fructose load."
GA can also be oxidized to glycerol by a NAD-dependent dehydrogenase similar in action
to the dehydrogenase converting acetaldehyde to ethanol"' or by a NADP-dependent de-
hydrogenase.'"'h" Glycerol then enters the hepatic metabolism via glycerol kinase.m5 h°6
The quantitative significance of the GA conversion to glycerol-3-P is not known and appears
to be small in comparison to the other GA pathways. It may be added that there also exists
a NADP-dependent glycerol dehydrogenase in muscle' 17 with an equilibrium toward glycerol
rather than GA but its role in fructose metabolism in this tissue is doubtful.
Studies with [ 1-'4Q-fructose77 and 16-'4Q-fructose"." show that a large portion of
glucose recovered from glycogen has an interconverted label in the 1,6 positions. This
sustains the conclusions reached above, that fructose must be cleaved to trioses and reas-
sembled as F-1,6-DP in order to obtain this randomization and corroborates the F-1-P-
aldolase-triokinase pathway. Little labeling in the 3,4 positions on the glucose residues in
glycogen argues against the importance of the glycerol phosphate pathway, because of the
stereospecific action of glycerol kinase. Additional evidence on the predominance of the
triokinase-catalyzed GA phosphorylation was obtained from mouse liver glycogen labeling
patterns after [4-3H, 6-'4C1 fructose administration.'
Fructose metabolism in extrahepatic tissues proceeds mainly by phosphorylation to F-6-
P by hexokinase. This reaction is minimal at low fructose concentration, particularly in the
presence of glucose, since the affinity of this enzyme to fructose is 1/20 that to glucose."'
It may have some importance, however, at high fructose concentrations. This is typical of
adipose tissue,"2 h" or muscle,'" in the presence of insulin,' or brain,"' which are all
devoid of fructokinase activity. An exception is the kidney'' "6." and the proximal
intestine-9°3h' which contain low but appreciable activities of fructokinase and other
enzymes specific to fructolysis, e.g., F-1-P aldolase and triokinase.
Another pathway by which fructo-glucose interconversion may occur is mediated by the
ubiquitous enzymes aldose reductase and sorbitol dehydrogenase.'"4-' '8 It has been suggested
that this pathway is of importance in the seminal tissue,19 lens,'2() nerves,' 21 and placenta,' 22
but it is mainly directed to fructose formation from glucose rather than fructose metabolism,
except when excess sorbitol or glucose are available. A special case may be the dystrophic
muscle, in which an increased utilization of glucose and fructose via the polyol pathway
was observed. 123
Volume II 101
IV. REGULATION OF FRUCTOSE METABOLISM
A. Fructokinase and Related Enzymes

Liver fructokinase was first investigated by Leuthardt, Testa, and Kuyper (see Hers') who
found that besides fructose it does phosphorylate also several ketohexoses (L-sorbose, D-
tagatose), as well as certain pentuloses and heptuloses but it does not phosphorylate aldoses.
It is an enzyme sensitive to activation or inhibition by SH-group-affecting agents.
Fructokinase is activated by Mg" and K ions i24-1" and inhibited by ADP.59 This finding
explains the earlier observations that fructose phosphorylation was more effective under
aerobic than anerobic conditions, under which the oxidative rephosphorylation of ADP was
retarded. Because of the strong ADP inhibition, any procedure for the assay of this enzyme
has to include an ATP regeneration system (CP and creatine phosphokinase or PEP and
pyruvate kinase) to prevent ADP accumulation. Mg" concentration must be equimolar or
exceed that of ATP to attain maximal fructokinase activity. ATP concentration > Mg" is
strongly inhibitory" and at a ATP/Mg + molar ratio of 5:1 the activity disappears. 59•'24 The
sensitivity of fructokinase to ATP/ADP ratio, and to the modulating effects of Mg" , K+
and
and Na+ on this dependency, particularly in the proximity of the enzyme-substrate lc
region, make it most probable that the intracellular variation of these factors under various
physiological situations exerts a regulatory effect on fructokinase activity.
Fructokinase is not inhibited by its product F- I -P,59' 129 as suggested,' in contrast to the
inhibition of low K„, hexokinase by G-6-P."3
Fructokinase activity appears to be also modified by extracellular hormonal or nutritional
conditions. Activity of the enzyme was reported to be depressed by androgens in male rats
and by progesterone in female rats. '3 ' Fitch and Chaikoff132 •'33 found that high fructose diets,
given for 7 days, enhance rat liver fructokinase activity and this finding was shared by
Chevalier et in 8 day fructose-fed rats. However, Adelman et al.'' reported that 48
to 72 hr of fasting, feeding a 70% fructose diet, alloxan diabetes, adrenalectomy, and cortisol
administration failed to influence the specific activity of fructokinase and all changes in total
liver enzyme activity could be related to changes in liver size. In contrast, these conditions,
especially insulin deficiency, markedly influence the high K„, glucokinase activity. 68,136 138
Despite these "nonadaptive" properties, it should be noted that refeeding of fasted rats with
a 70% fructose diet effected a complete restoration of fructokinase activity by 24 hr.'"
Refeeding with a high glucose diet resulted only in partial recovery, and with a high protein
diet no fructokinase recovery was evident. Other fructose metabolism enzymes, F-1-P al-
dolase and triokinase, showed a similar behavior. This specific effect of fructose was also
evident on long-term maintenance, which resulted in a much higher hepatic level of fruc-
tokinase and of the associated enzymes than on maintenance on fat or protein-rich diets. '35
It is also pertinent that the activity of fructokinase in adrenalectomized or hypophysectomized
rats was at the fasting level of intact rats and was not affected by fasting or fructose feeding,
whereas that of aldolase and triokinase remained at the intact rat level, decreased on fasting
and did not recover on fructose feeding.'" These manipulations indicate a direct substrate
effect on fructokinase and possibly an indirect, metabolite-mediated effect on aldolase and
triokinase, discernible only in certain situations.
Sillero et al.139 found a 20% increase in fructokinase activity in rats maintained for 3 days
on a 60% fructose diet, while other enzymes concerned with fructose metabolism remained
without change. These investigators were also hesitant to regard fructokinase as an adaptive
enzyme and ascribed the small rise in its activity to a protective effect of substrate. They
concluded that the fructose metabolism pathway is of a constitutive character. Zakim et al.63
also failed to obtain a significant rise in fructokinase activity upon 48 hr feeding of fructose
to rats, but in other experiments from the same laboratory a significant increase was noted
Table 2
EFFECT OF LONG-TERM SUCROSE FEEDING, DIABETES AND
FASTING ON HEPATIC FRUCTOKINASE ACTIVITY IN RELATION
TO OTHER GLYCOLYTIC ENZYMES
Species and treatment (no.) Fructokinase Glucokinase Hexokinase Pyruvate kinase
Spiny mice'
Regular chow (12) 43 ± 4 3.9 ± 0.3 4.8 ± 0.4 63 ± 9
Sucrose (50%) (9) 86 ± 6* 6.1 ± 0.6* 5.4 ± 0.7 792 ± 38*
Albino mice'
Regular chow (12) 53 ± 3 16.4 ± 0.8 6.7 ± 0.7 407 ± 38
Sucrose (50%) (9) 101 ± 8* 19.8 ± 1.0* 9.0 ± 1.1 1338 ± 110*
Albino rats'
Regular chow (20) 48 ±2 18.8 ± 0.9 7.2 ± 0.5 619 ± 48
Sucrose (50%) (14) 97 ± 8* 22.6 ± 1.3* 8.9 ± 0.9 1781 ± 85*
Fructose (25%) (6) 83 ± 6* 20.3 ± 1.7 8.5 ± 1.0 2085 ± 104*
Glucose (25%) (6) 56 ±4 22.0 ± 1.2* 7.9 ± 0.8 1145 ± 90*
48 hr fasting (6) 43 ±2 13.3 ± 1.7* 8.1 ± 0.9 345 ± 50*
Diabetes (8) 26 ± 2* 5.1 ± 0.8* 7.6 ± 0.9 241 ± 28*
Diabetes + insulin (6) 53 ±4 18.0 ± 1.9 9.1 ± 1.2 552 ± 74
Note: Activities in the table are means ± SE, expressed as nmol/min/mg of cytosolic protein (100,000
g liver supernatant fraction), for the number of animals given in parentheses for each group.
Asterisk (*) denotes a significant difference from control animals maintained on the regular chow
(p < 0.05 at least) Values taken from Shafrir and Orevi+4" Sucrose feeding (American Institute
of Nutrition 50% sucrose pellets) was for 3 months. The 25% fructose or glucose diets were
supplemented with corn starch. Diabetes was induced by i.p. injection of 65 mg/kg streptozotocin
and the rats used 14 days later (blood glucose 410 ± 28 mg/M). Other explanations see Table
1.
Acomys cahirinus from the Jerusalem colony.

Mice and rats from the Hebrew University strain.
both on fructose and sucrose diets, given for 3 days.' It should be emphasized that almost
all the results mentioned above were based on relatively short-term fructose feedings. We
have recently measured fructokinase activity in rodents maintained on a 50% sucrose vs.
regular diet for a period of 3 months.'" As demonstrated in Table 2, a twofold rise in hepatic
fructokinase activity was seen in spiny mice, albino mice, and rats. A 50% glucose diet or
48 hr of fasting did not elicit significant changes in the enzyme activity, whereas a long-
term 25% fructose diet produced changes similar to those of 50% sucrose. After 14 days of
streptozotocin-induced diabetes in rats, a 46% decrease in fructokinase activity was noted,
with reversal to normal range after 1 week of insulin treatment.
Parallel, but smaller changes were seen in glucokinase activity on the sucrose diet. There
were no changes in the low hepatic hexokinase activity, whereas that of pyruvate kinase
became markedly enhanced (-11-fold in spiny mice and -2-fold in albino mice and rats).
The effect of sucrose or fructose diet on pyruvate kinase, compared to that of the glucose
diet, was more pronounced. In other experiments'" (not shown in Table 2) the sucrose effect
on pyruvate kinase activity was evident already after 2 to 3 days, while the increase in
fructokinase activity was insignificant.
It may, therefore, be concluded that the hepatic fructokinase adaptively responds to its
substrate. The effect requires a week or more of fructose feeding and is less extensive than
that of other glycolytic or lipogenic enzymes (see later), but it is specific and independent
since each enzyme responded with different activity increments.
Volume II 103
In connection with the adaptive nature of liver fructokinase, the inductive behavior of the
intestinal enzyme should be mentioned in rat"." and man.14 ' A marked increase in the
activity of fructokinase was recorded in the proximal segment of rat intestinal mucosa and
in human jejunum on a high fructose or sucrose diet, compared with regular, glucose, or
casein diets.38 Similarly, increases were encountered in the activity of aldolase (with a
significant increase in the F-1-P/F-1,6-DP aldolase ratio, triokinase, glucose-6-phosphatase,
and pyruvate kinase), but no changes were observed in that of hexokinase, phosphofruc-
tokinase, triose-phosphate isomerase, F-1,6-DPase, or G-6-P dehydrogenase.36-38 The in-
duction of mucosal fructokinase by a 3-day substrate feeding may be related to the high
protein turnover rate characteristic of this tissue, which is instrumental in the rapid response
of the whole fructose metabolism pathway to nutritional adaptation. In addition a twofold
rise in rat jejunal and liver fructokinase activity was seen upon sucrose feeding, which could
be prevented by actinomycin, indicating a transcriptional regulation of the synthesis of this
enzyme in both organs.'"
B. Metabolic Response to Acute Fructose Excess

An acute intravenous fructose load to human subjects or rats, or fructose perfusion of
isolated rat liver, were shown to cause a hepatic accumulation of F-1-P, depression in adenine
nucleotides, especially ATP, drop in inorganic phosphate (P,) levels, and a tendency to
hypoglycemia. 61,62,143-145 Incorporation of leucine into liver proteins''''' and of orotic acid
into RNA' was reduced, attesting to the limitation imposed on the energy-dependent
synthetic reactions. An increased production of uric acid and/or allantoin was observed'43.'46
indicating accelerated degradation of preformed nucleotide purines. Similar results were
obtained with L-sorbose and D-sorbitol'45-1" but not at all with aldohexoses. The activity of
sorbitol dehydrogenase, which catalyzes the formation of fructose from sorbitol, is higher
than that of fructokinase' which explains the similarity of sorbitol and fructose effects on
liver metabolites.
It is of interest, that a combined intravenous injection of equimolar amounts of glucose
and fructose resulted in a similar depression of ATP and total nucleotides as that produced
by fructose alone.'46 Although this would indicate that coincident presence of glucose does
not moderate the acute metabolic upheaval created by fructose, oral administration of sucrose
does not produce drastic metabolite changes, due to the slower delivery of the load and
mitigating effect of glucose. Hypoglycemia has not been observed after sucrose loads.
The cellular mechanism of the changes following sudden fructose availability is outlined
in Figure 2. The key to these changes is the rapid phosphorylation to F-1-P and its retention
within the ce11.6''' The accumulation of F-1-P after fructose loading has been known even
before the fructose pathway was clarified,'49 however, the mechanism of F-1-P retention
remained unknown. It was suggested that F-1-P aldolase activity might be rate limiting,'5°
but later it was shown that the maximal capacity and K,, of this enzyme in rat liver (1.6 to
3,4 µmol/min/g and 0.35 mM) is similar to that of fructokinase (2.2 to 3.2 umol/min/g and
0.2 to 0.5 mM).-59-6I 65
The striking effects in rat liver 10 min after perfusion with 10 mM fructose are6'
1. A rise in F-1-P from 0.23 to 8.7 µmol/g (lasting at 7.7 Flinol/g at 40 min)
2. A protracted loss in total adenine nucleotides from 3.3 to 1.4 µmol/g, coupled with
a decrease in P, from 4.3 to 1.7 fiLmolig
3. An increase in IMP from 0.17 to 1.14 µmol/g and in glycerol-3-P from 0.13 to 1.1
µmol/g
Intracellular UTP, UDP-glucose,62 and GT1315' concentrations were also found decreased
after fructose injection.
GLYCOGEN
phosphorylaseii)lie Pi j
G-1-P
0-6-P
I g6pase
GLUCOSE
SUCROSE I
F-6-P
fructokinase
(
FRUCTOSE —irc F-1-P F-1, 6-DP
aldolase
ATP ADP
' P, 4
GA PYRUVATE
Regulationof Carbohydrate Metabolism
1
DHA P — GA-3P — - -
IMP NADH NADH
1
t
URIC NAD NAD
ACID
GLYCEROL- 3-P t LACTATE
FIGURE 2. Metabolic events in hepatic cells on a sudden fructose load. Fructose phosphorylation consumes
most of the available ATP and inorganic phosphate (P,), due to F-I-P accumulation. Rephosphorylation of ADP
is retarded as a result and ADP leads to partial fructokinase inhibition. The decrease in ATP and P, deinhibits
nucleotide breakdown mechanisms causing IMP accumulation which, in turn, inhibits F-I-P aldolase and thus
contributes to F-I-P accumulation. In addition, loss of P, and excess of F-I-P inhibit glycogen phosphorylase
resulting in a tendency to hypoglycemia. The redox state is markedly altered with increasing NAD/NADH ratio
(for detailed discussion see Section IV.B).
Volume II 105
The cause for F-1-P accumulation is attributed to an effective inhibition of F-1-P aldolase
by IMP,'' accumulating as a result of the coincident depletion of ATP and P. Both intra-
and extracellular P, levels fall down, and Mg is released into the circulation.15'
These decreases have a powerful effect on nucleotide metabolism. ATP is an inhibitor of
5-nucleotidase1 " and P, of AMP deaminase.' 54 -'55 The deinhibition of these enzymes at low
ATP and P1 concentrations results in tissue IMP accumulation and in irreversible degradation
of AMP and IMP to inosine, xanthine, uric acid, and allantoin, which appear in plasma and
urine. Although P, is liberated during this nucleotide catabolism, the trapping in F-1-P and
in other esters outweighs the release due to dephosphorylation. For a detailed review on the
mechanism of the effect of fructose on the catabolism of purine nucleotides, the enzymatic
regulatory aspects, and hyperuricemia see Reference 7.
The failure to observe ATP or total adenylate fall in liver perfused with fructose by some
investigators,'"' may be ascribed to the fact that they used a high phosphate medium which
has offset the P, trapping effect and prevented IMP increase. Injection of equimolar amounts
of fructose and P, partially prevented the loss in total liver adenosine nucleotides.'" The
decrease in ATP was nearly the same as with fructose alone but AMP level even increased,
as did that of F-1-P (see Woods in Reference 6), thus supporting the role of P, in the fructose-
induced metabolic derangement. The role of P, depletion is crucial, since mitochondrial
ADP rephosphorylation cannot proceed in its absence.
In addition to the strong inhibition by IMP an inhibitory effect on F-1-P aldolase of other
metabolites, accumulating under fructose load, is suggested by the observation that F-1-P
buildup precedes the rise in IMP on intravenous fructose administration to mice. '5' It is also
possible that both fructokinase and F-1-P aldolase act more rapidly than the subsequent
disposal of triose phosphates. 157
The ATP depletion and P1 fall after a fructose load are moderate in the kidney and absent
in the heart,' concordant to the fact that kidney has a low fructokinase activity and muscle
is practically devoid of it.
The effect of fructose on glycogen metabolism is a complex one. The salient feature after
a fructose load is decreased glycogen breakdown reflected by hypoglycemia. The drop in
P, impairs the activity of glycogen phosphorylase'" which has indeed been shown to be the
underlying cause of hypoglycemia in hereditary fructose intolerance (F-I-P aldolase defi-
ciency). The K„, of phosphorylase a and the cellular P, concentrations are of a similar
magnitude and loss of P1 would have, therefore, a far-reaching effect.I 59 In addition, F-1-P
has been demonstrated to be a competitive inhibitor of liver and muscle phosphorylase a
activity, especially potent at low P, concentrations.'" Of other metabolites which accumulate
after a fructose load, glycerol-3-P was also shown to inhibit glycogen phosphorylase a at
low P, concentrations, though to a smaller extent than F-1-P.'"
The combination of low P, and high F- 1-P levels, together with other inhibitory metabolites,
effectively abolishes glycogenolysis after a sudden fructose burst or in F-1-P aldolase
deficiency.
Notwithstanding the block in glycogenolysis in the face of fructose excess, evidence is
also available that fructose promotes a rapid activation of phosphorylase kinase (conversion
of phosphorylase b to a16' - '64) and a rapid reduction of glycogen synthase phosphatase activity,
curtailing the capacity for glycogen synthesis.I64 These effects have been observed at a lower
than optimal ATP/Mg" ratio on incubation of fructose with isolated hepatocytes, in perfused
liver, upon intravenous fructose administration,' or in liver cytosol.'65 Adenylate cyclase
activation and cyclic AMP involvement have been proposed,'' but are unlikely at the
prevailing low ATP level; any cyclic AMP rises are inconsistent and seem to occur only at
very high (30 mM) fructose concentrations."- '"
A possible explanation for these findings, conflicting with the overall effect of fructose
which does result in liver glycogen increment rather than loss, is that the phosphorylase
stimulating and glycogen synthase depressing effects are short and transient. They probably
occur prior to the cellular P,, ATP, and Mg" depletion. In support of this contention, it
may be added that the stimulation of hepatic glycogenolysis by glucagon is inhibited by
fructose (but phosphorylase b to a conversion is not'62), an observation characteristic of
hereditary fructose intolerance. The phosphorylase inhibitor is most probably the increased
cellular F-1-P,188 as demonstrated in in vitro experiments.' 56
In addition, activation of glycogen synthase simultaneous with glycogen phosphorylase
was found in hepatocytes incubated with fructose alone's' and glycogen synthase activation
seems to prevail.161.168 Glycogen deposition was recorded despite the elevated activity of
phosphorylase a and lack of correlation between glycogen synthesis and phosphorylase
activity was noted. With glucose present together with fructose (or other ketohexoses),
activation of glycogen synthase was higher than with either hexose alone'64 and the relative
activity of these enzymes was related to the proportion of hexoses in the medium. This
finding is of importance bearing in mind that, physiologically, a rise in fructose in the
circulation is usually accompanied by a rise in sucrose-derived glucose.
Of interest is the effect of fructose load on the post-triose phosphate metabolites in
comparison to the upward flow. Increases were recorded in glycerate-3-P, lactate, and
pyruvate after intraperitoneal fructose gavage compared with small or no increments in
F-1,6-DP, F-6-P, and G-6-P.6 L621' Glucose administration did not result in an increase in
post-triose metabolites, but augmented the concentration of hexose phosphates62 as would
be expected of a substrate with a point of entry above the triose phosphate level. However,
the production of lactate exceeded by far that of glucose both in vivo and in vitro.169-172
The possibility that the increase in post-triose metabolites is influenced by inhibition of
gluconeogenesis rather than due to the forced downstream flow is unlikely, since it was also
observed in fructose perfused liver,' where no external source of lactate or pyruvate was
available.
The result of the increased pyruvate flow leads to increased acetyl-CoA levels in livers
perfused with fructose'' as wel as increased channeling of fatty acid precursors up to the
secretion of lipoproteins.'" This may be due either to the direct pyruvate influence on the
activation of pyruvate dehydrogenase175 or to the effect of decreased ATP/ADP ratio, known
to influence the interconversion of active and nonactive forms of this enzyme.' 76
It should be mentioned that increases in glycerol-3-P were not associated with changes
in DHAP, but the ratio between them rose from 18:1 to 1000:1.62 This was accompanied
by minor changes in lactate/pyruvate ratio, although both were increased in concentration,
indicating that coupling to another NAD-NADH dependent enzyme — metabolite system
was apparent. In this condition most of glycerol-3-P was probably directed to fatty acid
esterification or to mitochondrial oxidation by the FAD-dependent glycerol-3-P oxidase.62
Fructose increases the rate of ethanol oxidation and ethanol metabolism is well known to
require a large NAD supply. Thus, the interaction of several coupled redox systems would
be affected including sorbitol/fructose, glycerol-3-P/DHAP, pyruvate/lactate, glycerol/GA,
and even malate/OAA. These influences and their impact on metabolism are extensively
discussed elsewhere.7J" '8°
Availability of fructose to hepatocytes from fed rats activated the futile cycles glucose/
G-6-P and F-6-P/F-1,6-DP as observed by the follow-up of detritiation of 3 H-labeled glu-
cose.181 This acute effect was associated with heat production unaccounted by 0, uptake
and is of interest since it occurs prior to any adaptive increases in the activity of G-6-Pace
or F-1,6-DPase (see Section IV.C). These changes were not seen in perfused liver of fasted
rats'" and their mechanism remains to be elucidated.
C. Enzymatic Adaptation to Long-Term Sucrose or Fructose Diets

Prolonged fructose or sucrose feeding does not induce the changes in the hepatic steady-
Volume II 107
Table 3
SUCROSE-DIET INDUCTION OF ENZYMES RELATED TO
LIPOGENESIS IN LIVER AND ADIPOSE TISSUE OF SPINY
MICE AND RATS
NADP-malate ATP-citrate Acetyl-CoA Fatty acid

Species and diets dehydrogenase lyase carboxylase synthetase
Liver
Spiny mice
Regular chow 9.3 ± 1.3 1.2 ± 0.2 2.6 ± 0.2 4.1 ± 0.4
Sucrose 197 ± 17* 15.8 ± 1.9* 14.3 ± 0.9* 33 ± 4*
Starch 108 ± II* 4.8 ± 0.3* 6.9 ± 0.6* 16 ± 2*
Rats
Regular chow 42 ± 4 14 ± I 6.8 ± 1.0 6.2 ± 0.4
Sucrose 165 ± 15* 62 ± 4* 26.1 ± 4.8 22.5 ± 1.5*
Starch 118 ± 13* 40 ± 2* 18.5 ± 1.2* 12.5 ± 0.8*
Adipose Tissue
Spiny mice
Regular chow 36 ± 4 1.9 ± 0.1 2.8 ± 0.6 5.6 ± 0.7
Sucrose 83 ± 6* 6.0 ± 1.1* 3.4 ± 0.5 6.8 ± 0.9
Starch 61 ± 4* 5.2 ± 1.0* 3.6 ± 0.4 7.8 ± 1.0
Rats
Regular chow 18 ± 2 56 ± 3 11.2 ± 0.8 13 ± 2
Sucrose 48 ± 5* 78 ± 5* 16.5 ± 2.2 16 ± 1
Starch 36 ± 4* 75 ± 2* 31.5 ± 1.9* 28 ± 4*
Note: Values are means ± SE expressed as nmol/min/mg cytosolic protein. Based on previous
results of Shafrir et al.'97 Shafrir,'" and unpublished data. Asterisk (*) denotes a
significant difference (p < 0.05 at least). Spiny mice were maintained on 50% sucrose
or starch diets; rats on 70% sucrose or regular diets.
state, like ATP depletion, encountered after a single fructose load.'" Fructose injection to
diet-adapted animals induces only marginal metabolic changes.184 Animals can tolerate diets
containing 50 to 70% of sucrose or up to 60% fructose without suffering diarrhea.
The main features on diets rich in fructose is an increase in lipogenesis and in the activity
of enzymes regulating glycolysis and lipogenesis, resulting in elevation of circulating lipids.
Early studies on hepatic enzyme activities in the rat by Fitch, Chaikoff, and their
collaborators115,116,185,186 revealed after 3 days of feeding fructose, compared to glucose, a
pronounced increase in the activity of G-6-P dehydrogenase, 6-P-G dehydrogenase, and
NADP-malate dehydrogenase.
These studies were later extended to show that the rise in the activity of G-6-P and
6-P-G dehydrogenases represents a transcription level induction of synthesis of enzyme
protein' and that the activity of a host of other glycolytic and lipogenic rate-limiting enzymes
is enhanced on long-term sucrose, compared with glucose or starch diets. Representative
studies documenting these effects in various strains of rats and laboratory animals concern
pyruvate kinase, 188-191 ATP-citrate lyase,I90 192 fatty acid synthase,'83.'84 and acetyl-CoA
carboxylase.I94
Of interest is the remarkable extent of induction of these enzymes in rodents domesticated
from the desert and offered ad libitum the affluent diet as exemplified by the spiny mice -
in comparison with laboratory-bred albino mice or rats'95- '98 (Table 3). The table illustrates
that in spiny mice the activity of 4 hepatic enzymes related to lipogenesis became induced
6- to 20-fold after 3 months on a 50% sucrose diet as compared with 3- to 11-fold increases
on an isocaloric starch diet. The largest increases were seen in the activity of NADP-malate
dehydrogenase and ATP-citrate lyase. In rats the activity increases were lower, 4-fold on
sucrose and 2- to 3-fold on starch. It should be noted that the initial enzyme activities were
lower in the spiny mice than in the rat, but the maximal activities reached were generally
higher in the rats.
The effect of the sucrose diet on adipose tissue enzymes was less striking. Both in spiny
mice and rats, only NADP-malate dehydrogenase and ATP-citrate lyase were induced two-
to threefold and the extent of induction did not differ appreciably between sucrose and starch.
Adipose tissue acetyl-CoA carboxylase and fatty acid synthetase activities were not signif-
icantly affected in spiny mice, but were induced two- to threefold on the starch diet in rats
(Table 3). The small extent of dietary induction in adipose tissue on the sucrose diet may
be linked to the limited peripheral uptake of this sugar, as well as to the small increment
of insulin, to which adipose tissue is highly sensitive. Indeed insulin treatment produced
activity rises considerably greater than sucrose feeding, particularly in the spiny mice.192
The marked increase in hepatic pyruvate kinase activity on the sucrose diet, is not only
due to increase in synthesis of enzyme protein, but in vivo it is further enhanced through
the feed-forward activation of this enzyme by fructose-derived cellular effectors, especially
F-1,6-DPI99 and F- I-P.2w Thus, the glycolytic flow supplying the citrate cycle precursors
is markedly potentiated during fructose metabolism.
This is well documented by the measurement of hepatic metabolites in rats refed with
fructose' or in spiny mice maintained on high sucrose diets,192 showing significant increases
in pyruvate, lactate, malate and citrate, and acetyl-CoA compared with regular starch or
glucose diets. The remarkable rise in hepatic NADPH generation and citrate cleavage capacity
emphasizes the greatly increased lipogenesis occurring in the liver. Increased channeling of
various lipid precursors to fatty acids and cholesterol in rats and other species has been
amply demonstrated in multiple studies. 67.201-207
The fortified function of the cellular pathways leading to lipogenesis is made remarkable
by the observation that it is not affected by superimposing dietary fat on sucrose.2"8 It is
well known that on a regular or glucose diet, an increase in the proportion of fat effectively
suppresses lipogenesis from carbohydrate.
It is also important for stressing the specific action of fructose, to point out that in contrast
to glucose, fructolysis and induction of hepatic enzymes2°9•2 '" proceed in diabetic animals
in virtual absence of insulin, restoring lipogenesis from acetate2 I (see also Table 2). Feeding
glycerol to diabetic animals produces results similar to fructose with respect to the adaptive
increases in enzymes, including acetyl-CoA carboxylase.2 '2
The response of hepatic enzymes of the gluconeogenesis pathway is a divergent one. PEP-
carboxylase activity decreases,194 whereas that of G-6-Pase"51 213 and F-1,6-DPase'92.214 in-
creases. The extent of G-6-Pase elevation may be dependent on rat strain and appears to be
negatively correlated with the extent of metabolite flow to oxidation and lipogenesis.215 This
divergent behavior of G-6-Pase and PEP-carboxylase is a result of the two-pronged metabolite
flow following the entry of fructose at the triose-phosphate level — part of the flow pro-
ceeding to glucose and probably involved in activation of G-6-Pase, another part proceeding
downstream and exerting a suppressing effect of PEP-carboxylase and even alanine ami-
notransferase. This situation is illustrated, both in spiny mice and rats, in Table 4 and
represents a marked contrast to the condition of increased gluconeogenesis in diabetes or
glucocorticoid excess, where the activity of all four enzymes characteristic of gluconeoge-
nesis is enhanced. The selective segmental induction and repression of the gluconeogenic
enzymes was also achieved by fructose and glycerol feeding of diabetic animals.2 I6 This
proves that these enzymes are not controlled by a single genome and entails the possibility
of independent regulation at different levels. Thus, similarly to the glycolysis segments, the
gluconeogenic pathway does not constitute an inflexible functional grid regulated only by
Volume II 109
Table 4
DIVERGENT EFFECT OF SUCROSE DIET ON HEPATIC
ENZYMES RELATED TO GLUCONEOGENESIS
Glucose-6 Phosphoenol-pyruvate Alanine

phosphatase° carboxylaseb aminotransferaseb
Spiny mice
Regular chow 96 ± 8 319 ± 17 440 ± 41
Sucrose 142 ± 11* 204 ± 16* 205 ± 19*
Rats
Regular chow 122 ± 9 176 ± 14* 367 ± 22
Sucrose 165 ± 12* 130 ± 11* 188 ± 12*
Note: Values are means ± SE for groups of 12 animals maintained on sucrose diet for 3 months.
Asterisk (*) denotes a significant difference (p <0.02 at least).
a Expressed as nmol/min/mg protein in whole liver homogenate.

Expressed as nmol/min/mg cytosolic protein.
central influences, which could be physiologically disadvantageous and wasteful for coping
with the natural diversity of available nutrients.
Time-course enzyme assays during refeeding of fasted BHE rats with labeled sucrose217
indicated, that the initial flow of the carbons is directed to glycogen; then a sharp decrease
occurs coupled with the rise in G-6-Pase activity and glucose release. Only later the NADPH-
generating enzymes and the flow into lipids become predominant. This sequence of events
illustrates and stresses the importance of segmental regulation of the glycolytic and glucogenic
pathways.
V. SUCROSE (FRUCTOSE)-INDUCED HYPERLIPIDEMIA
As early as 1916 Higgins received the first hint that fructose has a greater tendency to
serve as a lipid precursor than glucose, from simple measurements of the respiratory quotient
in intact animals.218 Since then, the remarkable hepatic lipogenic capacity, acquired on
sucrose diet, was recognized to be connected with hyperlipidemia2'9-227 and was extensively
investigated in various species228-238 with regard to its kinetics, duration, sex, diurnal vari-
ations, and age of animals,239-246 as well as to characterization of triglyceride (TG), choles-
terol, and lipoprotein elevations.247-249
An important observation was that fructose feeding of guinea pigs and pigs, which effi-
ciently convert fructose to glucose in the intestine,24•30 did not evoke hypertriglyceridemia227
but rather promoted gain in adipose tissue, in contrast to other animals, which metabolize
fructose in the liver. This observation strengthens the conclusion that it is the intensity of
fructose-stimulated hepatic lipogenesis, which determines the level and duration of
hyperlipidemia.25°
Again the spiny mice, originating from the desert environment, were found susceptible
to highest rises in serum TG and cholesterol on sucrose diets,236-237 linked with a retention
of these lipids in the liver (Table 5). Especially high liver lipid retention on sucrose was
also seen in another related desert species, the golden spiny mice, Acomys russatus. 238
It is of interest that the lipogenic effect of fructose extends beyond the fatty acid synthesis
pathway. A promoting effect on fatty acid esterification is exerted by the availability of
glycerol-3-P (see Section VI), as well as by the esterifying system located in the endoplasmic
reticulum: fructose enhances the activity of glycerol-3-P acetyltransferase and of phospha-
Table 5
HYPERLIPIDEMIA AND LIVER LIPID DEPOSITION ON
SUCROSE DIET
Serum Serum Liver Liver

triglycerides cholesterol triglycerides cholesterol
(mg(dt ) (melt) (mWg) (mWg)
Spiny mice
Regular chow 136 ± 9 110 ± 8 9±I 3.1 ± 0.3
Sucrose 486 ± 35* 295 ± 15* 39 ± 3* 6.9 ± 0.5*
Rats
Regular chow 105 ± 7 70 ± 4 11 ± I 2.6 ± 0.2
Sucrose 189 ± 13* 98 ± 6* 18 ± 1* 3.4 ± 0.2*
Note: Values are means ± SE for groups of 12 animals maintained on sucrose

diet for 3 months. Asterisk (*) denotes significant difference (p < 0.02 at
least).
tidate phosphohydrolase. 251.252 Corticosterone was implicated as a mediator of fructose and

sorbitol-induced rise in the activity of the latter enzyme on rats.253
One of the questions arising with regard to the fructose-induced hyperlipidemia is the
contributory effect of delayed peripheral removal of TG from the circulation as a factor,
additional to the basic determinant of increased synthesis and release of TG from the liver. 254
On the basis of short-term experiments, which were too early for the full expression of
hepatic lipogenesis, fructose diet was not found to depress TG removal, compared to glu-
cose,224 even if the levels of insulin, the hormone inducing the activity of adipose tissue
lipoprotein lipase (LPL), an important regulatory enzyme of tissue TG uptake, were different.
However, this does not appear to hold on long-term diets on which LPL induction on glucose
exceeded that on fructose three- to fivefold.227
Niklcild254 estimated that the lipids elaborated from fructose account for only a small
fraction of its overall metabolism and postulated that other factors, related to fructose
consumption, contribute to the hyperlipidemia. One of these factors may be adipose tissue
lipolysis, which is not restrained to the same extent on fructose as on glucose or starch,
with FFA mobilization supplying the hepatic TG precursors. In this regard two observations
are valid: the hepatic synthesis of VLDL on a fructose-rich diet was found in a large measure
to be derived from recirculated FFA incorporated into the VLDL-TG rather than from de
novo synthesized hepatic fatty acids.255 Secondly, the level of circulating FFA in sucrose-
fed animals and men tends to be higher than on regular diets, indicating an increased turnover
and mobilization of adipose tissue FFA (see Section VII).
In most human trials the substitution of sucrose or fructose for starch resulted in serum
TG and/or cholesterol increases in normal subjects. There is a large variety of experimental
design, regarding the amount of carbohydrate substituted, age and sex of subjects, duration
of surveillance, permanence of the effect, field or metabolic ward study, whether the lipid
changes were measured as a result of sucrose supplement, exchange or withdrawal, in fasted
or in postprandial condition. The quoted References(219. 220. 225. and 256 to 268) are not exhaustive
and just give a representation of the widespread work done. Some may be open to criticism,
as serum lipid changes were associated with body weight loss, but there are studies pointing
out that weight reduction and carbohydrate restriction are independent determinants of TG
decrease' and small weight loss need not interfere with sucrose-induced TG rise.270 How-
ever, there are negative studies showing that sucrose substitution of 7, of carbohydrate intake
for up to 14 days, in healthy volunteers, did not induce hyperlipidemia.27°'
Volume II 111
One of the impressive studies on the positive side of the argument involved the admin-
istration of r 4C1-labeled sucrose to human subjects, first after 1 week on a starch diet, then
after 1 week on an isocaloric sucrose diet."' Peak incorporation into plasma TG after the
starch period was at 3 to 6 hr. It was higher in magnitude and more protracted on sucrose.
The r 4C1 increment was mainly in the fatty acid moiety of the TG, indicating the derivation
from hepatic lipogenesis. If a retarded removal of plasma TG was responsible for the increase
in TG label, then a symmetric involvement of the fatty acid and glycerol moieties would
be expected. "CO, elimination was also higher on sucrose. This positive temporal correlation
between oxidative and lipogenic processes suggested that it was related to coincidentally
increased flow of sucrose metabolites through oxidative as well as lipogenic pathways, rather
than to shunting from one to the other. It was suggested that the increased sucrose uptake
and metabolism might have been related to adaptively increased intestinal sucrase activity
and/or stimulation of enteric insulin secretagogues and not necessarily to an impact on the
liver.
The net plasma TG increase was significant but not as high as the label increase. Hy-
pertriglyceridemia patients showed a smaller response but tended to convert more sucrose
to lipids already on the starch diet. Thus, '4C-sucrose uptake test in these patients was
valuable for the detection of an abnormal lipogenic predisposition.
Similar studies with [3H]-glycerol-labeled VLDL in patients on high sucrose diet in the
postabsorptive state272 revealed a significantly decreased fractional turnover rate, a moderate
increase in TG pool size but unchanged net turnover, Vmax and Km of the removal system.
These results confirm that the removal mechanism was not retarded by sucrose feeding, at
least in the postabsorptive state, and did not contribute to the expansion of the TG pool.
In studies with hypertriglyceridemic subjects, generally a rise in plasma lipids was found
on sucrose feeding 7 222.269.273 275 however, some groups were maintained on unphysiologically
high fructose or sucrose diets, and differing effects were reported in studies with diets
varying in sucrose content. 279.28° The hypertriglyceridemic effect of sucrose vs. fructose was
well illustrated in a metabolic ward study.' Therefore, the negative results encountered
both in normal and hypertriglyceridemic subjects282-285 require reinterpretation in terms of
quantity of sugar consumed as sucrose or fructose within the total dietary intake.
It may be concluded that the majority of investigations points to a specific effect of sucrose
on TG elevation in humans but the wide variety of experimental conditions does not permit
a clear-cut decision as to the effect of "normal" fructose consumption. Additional, well-
planned experiments are needed also with respect to the quantitative contributions of increased
hepatic lipogenesis, decreased peripheral TG uptake, and of increased FFA mobilization to
lipid retention in the circulation (or the liver), particularly in cases of exacerbation of
preexisting hyperlipidemia.
VI. ADIPOSE TISSUE RESPONSE TO SUCROSE AND FRUCTOSE DIETS
As demonstrated in Table 3, in contrast to the remarkable stimulation of hepatic glycolysis

and lipogenesis pathways on the sucrose diet in spiny mice, adipose tissue enzymes did not
respond extensively. This is also apparent in rats in correlation with the fact that there is a
decrease in the size of adipose tissue and a lower total body weight gain on sucrose compared
to starch in these species. 184.194.198.286 Other investigators also recorded reduced adipose tissue
glycolytic and lipogenic enzyme activities, on fructose compared to glucose, in contrast to
their marked induction in the liver. 17'287-289 These results may be interpreted as a shift of
lipogenesis from adipose tissue to the liver in association with low fructose uptake ability
of adipose tissue. ''''29°.291 Lipogenesis from acetate was found not to be diminished in adipose
tissue in one study and reduced in another,292 but it seems more plausible that the rate-
limiting step in lipogenesis was the uptake and conversion of fructose (or glucose) to acetyl
CoA rather than located in the fatty acid synthesis pathway.
Incorporation studies with labeled glucose and fructose also pointed out that 1 week of
sucrose feeding suffices to reduce the uptake and conversion of fructose or glucose to lipids
by rat adipose tissue. This difference became accentuated rather than abolished in the presence
of insulin."' Moreover, rats on fructose-rich diets released more FFA from adipose tissue,
probably due to disturbed reesterification with glycerol-3-P, in connection with reduced
substrate uptake. 294 Although glycerol release was not increased, sensitivity of adipose tissue
to insulin was reduced as judged from glucose incorporation pattern.'-95 In spiny mice fat
uptake capacity, as inferred from LPL activity, was not increased on a prolonged sucrose
diet'98 and fructose in contrast to glucose feeding also failed to enhance LPL activity in
rats."' Contrariwise, lipolysis, measured by in vitro glycerol release, was increased in
adipose tissue of spiny mice on sucrose diet, which was also reflected by increased serum
FFA levels. '98 In addition, the metabolite ratios controlling fatty acid esterification in adipose
tissue of rats were conducive to FFA release on sucrose diet,297 and F-1,6-DP was found to
have a lipolysis stimulating effect in in vitro systems.' All these findings indicate that TG
storage is decreased and FFA mobilization increased on fructose-rich diets, implying a faster
TG turnover, most probably in relation to the reduced amount and/or effectiveness of cir-
culating insulin.
Two other observations fit well with the above findings. Livers from rats fed 10% fructose
for 14 weeks secreted twice as much VLDL-TG, derived from the uptake of external FFA,
compared to regularly fed animals; fructose feeding did not result in altered uptake of plasma
FFA or preferential secretion of TG from endogenously synthetized fatty acids:255 In human
subjects, transferred for 2 weeks to sucrose, the basal and noradrenaline-stimulated lipolysis
became increased and the antilipolytic effect of insulin reduced.'-99
Different observations have been made in strains of rats generally predisposed to obesity:
Osborne-Mendel rats responded to sucrose supplementation in drinking water with fat ac-
cumulation and weight gain, whereas in control S 5B/Pl rats sucrose induced weight loss.299'
Sucrose feeding was also more effective than starch in the fattening of a strain SHR/N
corpulent rats. 299b In Sprague-Dawley rats, the sucrose or "cafeteria diet"-induced hyper-
phagia resulted in overall weight gain which was less than expected from the caloric intake,
but there was a particular increase in brown adipose tissue SiZe,299c. 299d. 379-3s' indicating
increased thermogenesis.
VII. HORMONAL ALTERATIONS ON SUCROSE DIET INDUCING

INCREASED ENERGY CONSUMPTION
The intriguing finding of slow body weight gain and adipose tissue loss on the sucrose
diet, often associated with increased food consumption, prompted a search for the endocrine-
metabolic background of this phenomenon. As demonstrated in Table 6, fructose-rich diets
elicited a significant increase in serum triiodothyronine (T,) together with a decrease in
thyroxine (T4) level, most probably mediated by the negative thyrotropin feedback. These
changes suggested an increased extrathyroidal monodeiodination of T4, possibly induced by
fructose or one of its metabolites, since changes in T,/T., ratio were much less striking on
other affluent diets (starch or fat). '".238
The metabolic expression of the increased T3 levels was apparent in the liver by the
impressive induction of FAD-dependent mitochondrial glycerol-3-P oxidase. This enzyme
is tightly regulated by T33' 3' and even slight changes in circulating T, result in large
alterations of glycerol-3-P oxidase activity, thus being a very sensitive indicator of thyroid
action.'
The increased T, levels on the sucrose diet, may provide the basis for a compensatory
Volume II 113
Table 6
EFFECT OF SUCROSE DIET ON SERUM AND LIVER THYROID
HORMONE INDICES
Liver mitochondria!
FAD-glycerol-3-phosphate
Serum T, Serum T4 oxidase
(ng/mf) (p.g/c1f) (p,g formazan/min/mg protein)
Spiny mice
Regular chow 1.04 ± 0.08 3.76 ± 0.18 1.81 ± 0.10
50% Sucrose diet 1.84 ± 0.12* 2.72 ± 0.16* 4.75 ± 0.37*
25% Fructose diet 2.14 ± 0.14* 2.13 ± 0.12* 6.85 ± 0.42*
Rats
Regular chow 0.68 ± 0.05 3.74 ± 0.20 1.48 ± 0.13
50% Sucrose diet 1.16 ± 0.07* 3.10 ± 0.18* 3.47 ± 0.28*
25% Fructose diet 1.64 ± 0.08* 2.83 ± 0.16* 4.15 ± 0.31*
Note: Values are means ± SE for groups of 14 to 20 animals maintained on diets for 3 months.
Asterisk (*) denotes a significant difference (p < 0.05 at least). Data based on experiments
of Shafrir,'" Shafrir and Adler,' and unpublished observations.
mechanism against weight gain on sucrose-rich diets, similar to what has been observed in
other experiments related to weight gain or loss and thyroid hormone economy."' '7 The
increased activity of mitochondria! FAD-glycerol-3-P oxidase contributes to the reoxidation
of cytosolic NADH by channeling the reducing equivalents into the mitochondria through
increased turnover of the glycerol-3-P shuttle and helps to relieve the accumulation on the
fructose-rich diets. But at the same time this results in a lower yield of ATP:" since the
terminal oxidative phosphorylation chain starts, in part, from FADH instead of NADPH
(Figure 3).
The induction of FAD-glycerol-3-P oxidase exemplifies only one of the numerous energy-
consuming metabolic cycles activated by T3 . Other sites of energy dissipation are ATPase
activation' 3" and cycling through opposing metabolic pathways, such as coincident en-
hancement of cytosolic fatty acid synthesis together with their mitochondrial oxidation,
acting in fact as an "ATPase".312 NADH-NADPH transhydrogenation is also excessively
promoted by repetitive malate-pyruvate shuttling through the T3-induced, coincident rise in
the activities of pyruvate carboxylase3133'4 and NADP-malate dehydrogenase." 2.3'5 Since
G-6-Pase activity was found also increased on sucrose as well as during T3-treatment,316 the
hexokinase-G-6-Pase couple represents an additional ATP consuming "futile" cycle.
The previously discussed lipolysis on fructose-rich diets (Section VI) may be also, at least
in part, attributed to the T3-activation of the intracellular ("hormone sensitive") adipose
tissue lipase.317•3 ' This constitutes another energy wasting cycle and protective mechanism
against obesity during abundant carbohydrate intake, since the FFA release compromises
the simultaneously T3-activated fatty acid synthesis in adipose tissue.'
As seen in Table 6, the rises in serum T3 level and hepatic FAD-glycerol-3-P oxidase
during fructose-rich diets were most pronounced in the spiny mice and were smaller, though
significant, in rats.
Of interest is the observation that rats consuming sucrose supplements showed improved
resistance to cold.' This was attributed to increased brown adipose tissue cellularity, but
may be linked as well to the elevated T3 in the circulation.
As seen in Table 6, the rises in serum T, level and FAD-glycerol-3-P oxidase were most
pronounced in the spiny mice on fructose-rich diets and smaller, though significant, in rats.
In animals with genetic obesity, like the C57BL/6J oblob mice, serum T3 values and
hepatic FAD-glycerol-3-P oxidase activity were found to be already increased on a regular
NADH
GP DHAP
FIGURE 3. Illustration of the effect of increased glycerol-3-P shuttling on the mitochondrial ATP formation.
Sucrose, or T3-stimulated activity of FAD-dependent mitochondrial glycerol-3-P oxidase (Fp,) induces an increased
entry of cytosolic glycerol-3-P into the mitochondria, which is converted to DHAP. The electron transport chain
oxidation starts from FADH instead of NADH resulting in two rather than three molecules of ATP formed per O.
thus operating at less than full efficiency (for additional discussion see Section VIII).
diet 3 '9.3" and when placed on a sucrose diet the increments were only marginal..'-° Adipose
tissue lipolysis was not increased on sucrose,'" indicating a strong TG retention capacity.
The calorigenic response to direct thyroid hormone administration was normal, except FFA
mobilization, but the endogenous T3 response to cold stress was defective."'
The response of the oblob mice to sucrose diet was also unique in the sense that their
serum TG levels fell rather than increased, as expected on sucrose diet.3201" It is difficult
to explain this absence of hypertriglyceridemia on sucrose in view of ample hepatic lipo-
genesis."20•3" An increased TG removal is unlikely but a decreased FFA inflow to the liver
may be responsible in part, as well as the remarkable hepatic TG accumulation in oblob
mice on sucrose,3" perhaps, due to their inability to release TG commensurately to endog-
enous synthesis from fructose and other precursors or to their exogenous availability, as
demonstrated in a perfusion system.324 Fatty liver on sucrose feeding was also seen in
genetically obese Zucker (fa/fa) rats,' with accumulation of VLDL-like particles in the
hepatocytes326 and a lowering of their hypertriglyceridemia. 326a These rats were likewise
found to have no T3 response to high carbohydrate-low protein diets,327 although they did
not lose weight or hepatic lipogenic capacity on sucrose diet, even if their adipose tissue
response was lacking.'
Concordant with these observations, a significant rise in the (Na'/K±)-ATPase-mediated
potassium uptake was found both in liver and muscle upon sucrose-induced hyperphagia in
non-obese mice, whereas in the oblob mice, the sucrose supplement did not elicit an increase
in (Na+/K±)-ATPase activity. 329 The common denominator of all these results appears to
be the extent of insulinemia. The sucrose-induced and T3-mediated metabolic cycling and
thermogenesis are inherent and readily elicited in low or normoinsulinemic spiny mice and
rats. They seem to be overtaxed or close to the peak of their response capacity in the
hyperinsulinemic ob/ob mice or fa/fa rats, due to the insulin-induced hyperphagia, already
on the regular diet.
Volume 11 115
Although this effect may also be due in part to species difference, it is more likely that
hyperinsulinemia, the essential factor of obesity from the early developmental stage, pro-
motes fat accretion not only by the enormous rise in glycolysis and lipogenesis potential,
but also compromises the protective energy dissipation reactions in the liver and restrains
TG lipolysis in the adipose tissue.
The fact that T, production increases on sucrose diet is of interest not only in connection
with activation of energy dissipation processes but also the effects of T, excess and of high
carbohydrate diets may overlap, or even exhibit synergism in their pattern of induction of
hepatic enzymes of glycolysis and lipogenesis.'° T3 treatment induces NADP-malate de-
hydrogenase, G-6-P dehydrogenase, ATP-citrate lyase, acetyl-CoA carboxylase, and fatty
acid synthetase312315 as well as G-6-Pase,316 all of which also rise on the sucrose diet.
However, T3 and sucrose diverge in their effects on gluconeogenesis, sucrose diet causing
a suppression of PEP-carboxylase in rats and spiny mice (Table 4), while T3 enhances the
activity of this enzyme."' Moreover, fructose effects were not prevented by thyroidectomy
and were additive to T, treatment.33' Dissimilarities also occur in effects on glycogen, as it
is well known that T, promotes glycogen breakdown, while sucrose preserves or increases
glycogen synthesis.
Glucocorticoids, too, are known to produce some of the hepatic enzyme activity increases
characteristic of fructose-rich diets, notably in G-6-Pase and F-1,6-DPase332 and in NADPH-
generating and lipogenic enzymes.333 However, it is highly unlikely that the fructose effects
are mediated by glucocorticoid hormones, which also induce pronounced increases in all
gluconeogenic enzymes. Cortisol-induced rises in G-6-Pase and F-1,6-DPase were sup-
pressible by insulin, whereas those elicited on fructose-diets were not.'32 Changes in many
other activities induced on fructose-rich feeding could be obtained in adrenalectomized
animals as Wel1. 334335
VIII. FEATURES OF THE SPECIFIC EFFECT OF FRUCTOSE ON

LIPOGENESIS AND LIPID METABOLISM
From the physiological point of view the meaningful difference in the conversion to fatty
acids of various carbohydrates ingested as starch, glucose, sucrose, and fructose, depends
on the amount of the hexose available to the lipogenic tissue(s) upon its entry into the
circulation and the rate of the conversion which can be attained. Some aspects of this problem
have been already previously discussed.336 The following points based in part on new
information, should be considered, in particular, when comparing the metabolism of fructose
vs. glucose and their effects on lipoprotein metabolism.
1. Distribution of hexose uptake between the liver and peripheral tissues and the relative
contribution to lipogenesis of these compartments.
2. Relative rates of phosphorylation upon the entry into liver.
3. Possibility to by-pass the slow phosphofructokinase step and the relative rates of triose
metabolism, leading to the entry into citrate cycle.
4. Formation and carboxylation of acetyl-CoA by acetyl-CoA carboxylase as the rate
limiting step of lipogenesis.
5. Esterification of long-chain fatty acids.
6. Influences of fructose vs. glucose at the stage of VLDL removal and metabolism.
1. The glucose moiety of sucrose, upon its absorption, is available to most body tissues
— some using it as an obligatory substrate, some metabolizing glucose along with other
fuels, notably the FFA. Estimates of the distribution of a glucose load between the liver
and periphery, in man and laboratory animals, vary from 40 to 60% for the liver. In contrast,
the metabolism of the fructose moiety is virtually confined to the liver and, thus, increases
the hepatic share in the total carbohydrate uptake and compels an adaptive synthesis of
fructolytic, glycolytic, and lipogenic enzymes, together with a considerable increase in liver
size. Since adipose tissue mass remains constant or decreases on fructose-rich diets, the total
lipogenic capacity of the liver markedly increases in relation to the periphery. All this implies
that, while most of the glucose is metabolized elsewhere to support the obligatory energy
needs of a wide spectrum of tissues, the bulk of fructose is converted in the liver to lipogenic
precursors rather than channeled to gluconeogenesis or oxidation.
In addition, the lipogenic potential of the liver should be considered according to species.
In most rodents it is estimated that the total adipose tissue mass contributes 30 to 50% of
body TG by in situ lipogenesis from glucose — the balance being acquired by the transport
of liver-elaborated TG, through very low density lipoproteins (VLDL). That the fructose
ingestion increases the hepatic contribution to body fat synthesis is of particular importance
in humans, in whom only —10% of body TG is derived from lipogenesis within adipose
tissue, due to its low fatty acid synthesis capacity. Since the TG accretion occurs by deposition
of preformed VLDL-TG, the increase in liver lipogenic capacity and liver mass on fructose-
rich diets would tend to accentuate the transport of TG in the human circulation. However,
any comparison of the extent of rodent vs. human hyperlipidemia should take into the account
that the fructose-rich diets in the latter do not approach the —50% level used in animal
experiments.
2. As discussed in Section III, the rate of phosphorylation of glucose is lower than that
of fructose. This is based on comparison of the respective enzyme capacities and on substrate
flow studies." The activity of hepatic fructokinase on regular glucose or sucrose diets or
even in fasting or diabetes always exceeds that of glucokinase + hexokinase, from four to
ten times according to different sources.63 65.139.140 This difference in maximal activities would
be further widened in vivo by the rise in G-6-Pase activity on fructose-rich diets, which
renders phosphorylation by hexokinases even less efficient as a result of increased glucose
— G-6-P cycling and thus accentuates the advantage that fructose has in its entry into the
glycolytic pathway.
3. As demonstrated on short- and long-term fructose administration, there is a preferred
formation of triose phosphates with fructose, since its entry is below the rate-limiting step
of phosphofructokinase and the F-1-P aldolysis together with GA phosphorylation are rapid
reactions. The preferential downstream flow of triose metabolites is facilitated both by the
pronounced induction of pyruvate kinase and its feed-forward activation by fructose phos-
phate esters, as well as by the decreased ATP/ADP ratio. This situation is well supported
by the findings of a crossover point between the cellular lactate + pyruvate and PEP
levels.63-'" and the consequently increased ratio of active/inactive forms of pyruvate
dehydrogenase'" '" facilitating the entry into the mitochondria and formation of acetyl-
CoA.
4. Most studies reviewed in Sections IV and V point out that fructose is a better precursor
of fatty acids and also promotes the incorporation of acetate and 3H,0 to fatty acids.33'- 3 '
The amount of the fatty acid pathway enzymes is markedly increased on carbohydrate-rich
diets, notably acetyl-CoA carboxylase,"9•34° together with the auxiliary enzymes ATP-citrate
lyase, providing the extramitochondrial acetyl-CoA, as well as G-6-P dehydrogenase and
NADP-malate dehydrogenase, generating the mandatory reducing equivalents for fatty acid
synthesis.
That the concentration of hepatic citrate cycle metabolites is preferentially increased on
fructose-rich diets (including several dicarboxylic acids and citrate) signifies not only the
enhanced flux of extramitochondrial acetyl-CoA precursors, but also an activating influence,
sustaining the polymeric (active) form of acetyl-CoA carboxylase.341
The rate of flux of labeled precursors clearly illustrates the increased channeling of fructose-
Volume II 117
derived carbons. In rats refed for 48 hr with fructose vs. glucose, the ratio of hepatic fructose/
glucose label conversion was, 5.2 vs. 2.9 into CO2 and 6.7 vs. 2.1 into fatty acids, re-
spectively.' In experiments with fructose vs. glucose incubated liver slices of chow-fed rats
these ratios were —3 for conversion into lactate, pyruvate, CO„ and fatty acids and as high
as 19 into TG-glycero1.67
Upon intravenous injection of labeled hexoses to rats, treated with Triton WR 1339 to
prevent the peripheral lipid removal by LPL inhibition, a two- to threefold difference in
label incorporation into plasma and liver lipids was observed with fructose compared with
glucose."'
In human liver slices the fructose/glucose carbon ratio, after incubation with the respective
substrates, ranged from 3 to 7 in CO, and from 7 to 29 in fatty acids.' In another series,
in which the patients received 1 day preoperatively 400 g fructose, there was a 4-fold increase
of label incorporation into TG-fatty acids and a 3-fold increase in TG-glycerol.342
These results appear to be the most convincing with regard to the differential effect of
fructose. The rate of flux is the important indicator, since rises in enzyme activities, even
if most impressive, reflect response to metabolite availability but do not cause it. Increase
in metabolite levels are a valuable indicator but decisive only when a clear crossover pattern
is obtained. Their uneven distribution within the cellular compartments does not allow the
assessment of their actual concentration in relation to the K,, of the pertinent enzyme. Here
it should be said that the fructose effect may not be qualitatively different from that of other
carbohydrate if metabolized at a rate providing a quantity of precursors and rapidity of flow
towards acetyl-CoA formation similar to fructose. The unique effect of fructose is based,
thus, on its preferential phosphorylation and entry at the triose phosphate site, which produces
the special situation of hepatic metabolite excess, not attainable under physiologic conditions
by aldohexoses.
5. Esterification of newly synthesized, or inflowing, preformed FFA takes place on the
hepatic endoplasmic reticulum and fructose was shown in various systems to en-
hance this process, together with the increase in hepatic glycerol-3-P content.61.62.67.174.141 350
This metabolite has been demonstrated to be an important determinant of the esterification
rate.35' 353 However, the elevation in glycerol-3-P was not always persistent; in some long-
term sucrose or fructose feeding studies an elevation was noted,'" but in other feeding
studies no increase or even a decrease in glycerol-3-P was seen.66-'97
There is no doubt that fructose increases hepatic FFA esterification, however, this effect
may be not necessarily directly correlated to glycerol-3-P concentrations. The level of this
metabolite is labile as a result of changing esterification requirements, resulting from
differences in the rates of fatty acid and glycerol-3-P synthesis, and other multiple factors
such as phosphorylation rate of external glycerol by glycerol kinase, redox state (con-
version to DHAP),62 and transport of cytosolic NADH into the mitochondria through the
glycerol-3-P shuttle!'
Additional effect of fructose on FFA esterification is located at the enzymatic steps at the
endoplasmic reticulum involving the coupling of glycerol-3-P with long-chain fatty acyl-
CoA esters and formation of TG and phospholipids, as mentioned above.251-253
The increased esterification capacity may have several implications — increased transfer
of newly synthesized fatty acids to VLDL formation on proximal sites in the same cellular
compartment; trapping of long-chain fatty acyl-CoA and preventing their routing to mito-
chondrial oxidation and ketone body formation; or relieving acetyl-CoA carboxylase from
possible feedback inhibition by long-chain fatty acyl-CoA.354 Fatty acid oxidation and ketone
body formation is indeed reduced by fructose,175.345-35° but a direct link to glycerol-3-P was
not as yet decisively demonstrated. Although a drop in cellular long-chain fatty acyl-CoA
on fructose feeding has been noted,355 their removal causing a deinhibition of acetyl-CoA-
carboxylase remains also an assumption, since there is no certainty that this enzyme is
specifically inhibited by long-chain fatty acyl-CoA under the adequate physiological pro-
tection of the high cytosolic protein content.356.357
The inverse relationship between fatty acid esterification and oxidation is, thus, fairly
well established but an interesting finding has to be considered with respect to the role of
fructose.358 Fatty acid oxidation in isolated mitochondria could be slowed down by the
addition of cytosol from liver of fructose-fed rats, in absence of the microsomal esterifying
system. This suggests fructose metabolite(s) as inhibitor(s) of oxidation or a competitive
utilization of fructose-derived acetyl-CoA.
6. Because the fraction of fructose converted to the circulating lipids was suggested to
be small,' the hyperlipidemic influence of fructose is thought to extend beyond its intra-
hepatic lipogenic and oxidation-sparing action.
Retention of lipids in the circulation has been attributed to weak suppression by fructose
of the FFA outflow from adipose tissue, which contributes to plasma TG elevation through
hepatic recirculation as VLDL (see Section V). This has been related to lower insulin secretion
and/or less efficient restraint by insulin of adipose tissue lipolysis, on one hand, and smaller
TG storage through the lack of induction of LPL activity, on the other hand. This aspect
may be of particular importance, if fructose is superimposed on preexisting alimentary or
familial hypertriglyceridemia, when the peripheral VLDL-TG removal system is saturated.
The differential effect of fructose in causing these handicaps is underscored by the obser-
vations that isocaloric glucose diets tend to reduce the circulating TG levels despite increasing
the hepatic lipogenesis, by eliciting stronger lipolysis inhibition and enhancing TG uptake.
IX. DELETERIOUS EFFECTS OF SUCROSE DIETS
A. Sucrose and Diabetes

The first observation which suggested an insulin-independent fructose utilization in dia-
betes came in 1893 from Minkowski 359 who noted that fructose may be utilized for glycogen
synthesis in the depancreatectomized dog. Joslin in his classic book36° believed that fructose
may be beneficial in the treatment of diabetics as a substitute for glucose and to counteract
ketosis. This was extensively discussed,36' '65 the rationale being that fructose metabolism
is insulin-independent, insulin sparing and antiketogenic by slowing down (3-oxidation even
in the absence of insulin.
However, adverse effects of increased lacticacidemia, low phosphate levels, hyperuricemia
and deepening ketoacidosis on fructose infusion have been reported.366 369 All these previously
described effects of acute fructose load (Section IV.B) may become critical if superimposed
on the ion deficient, poor metabolic state of insulin deficiency. Therefore, although the
clinical antiketogenic action of fructose has been confirmed,' its parenteral use in diabetic
ketoacidosis has now been virtually abandoned, also on the ground that it is not more
efficient that the earlier applied glucose + insulin infusion.'
Since fructose is almost twice sweeter than sucrose, its use as a calorie-saving large-scale
sweetener for diabetics was also extensively debated but in a recent editorial not advised.172
Another aspect of fructose or sucrose nutrition is whether it produces glucose intolerance
and eventually diabetes. Long-term administration of fructose-rich diets to rats results in
varying degree of deterioration in glucose tolerance which is usually compensated by hy-
perinsulinemia.373-37" The reduced glucose tolerance in rats maintained on the so-called
"cafeteria diet" was also ascribed to its large sucrose content.'".'' Glucose intolerance is
also accentuated when sucrose, even in low percentage, is given together with a fat-rich
diet3" or is fed to animals with genetic obesity.'
On the other hand, 42 g fructose or sucrose, given as part (^25 cal%) of a single test
meal, did not significatly affect the extent of individual hyperglycemia or hyperinsulinemia
Volume 11 119
curves in healthy or diabetic persons in comparison with equicaloric meals containing other
carbohydrates.""
Since only the tolerance of an oral glucose load was impaired in the hands of some
investigators, while i.v. glucose disappearance was not,384.385 the involvement of intestinal
hormones was suggested.
As discussed earlier'94 the reduced glucose tolerance on a high sucrose diet may ensue
from the bifurcated metabolite flow after fructolysis. The upward flow of hexose phosphates
in the direction of gluconeogenesis3" may saturate the initial steps of the hepatic glycolytic
pathway. The low glucokinase and elevated G-6-Pase and F-1,6-DPase activities, causing
an increased "futile" cycling (see Figure 1), may also retard the hepatic glucose uptake.
A relation between glucose tolerance and glucokinase activity was in fact demonstrated,
although glucose tolerance was not disturbed on short-term fructose refeeding.387
Spiny mice also show glucose intolerance and reduced insulin sensitivity on long-term
sucrose feeding but do not progress to overt diabetes on this regimen. '97'198'236 It is of interest
that in this low insulin responder sucrose diet stimulated pancreatic insulin synthesis, while
insulin secretion remained low.'" Sucrose stimulation of insulinogenesis, with increased
insulin release, was also seen in albino mice'" and rats.'"
By combination of sucrose-induced glucose intolerance and selective inbreeding, Cohen
and collaborators3" were able to isolate a strain of rats with overt diabetes demonstrating
the interaction between affluent nutrition and genetic factors in expressing the diabetic
condition. They could also establish that the responsible component was fructose by ge-
netically selecting a similar strain through repetitive mating of fructose-fed pairs with low
glucose tolerance.389 Many microangiopathic complications resembling those of human di-
abetes were demonstrated in the sucrose-diabetic rats: nephropathy,390•391 retinopathy,392 and
testicular degeneration.393 Perhaps underlying these effects are changes in the properties of
membranal collagen, detected in the fructose-fed rat. 394
The sucrose-based selective breeding of Cohen's rats388 needs, however, a clarification
since the synthetic sucrose-rich diet used for this purpose has been found to be also copper
deficient.395 Absence of copper reduces plasma insulin response to an oral glucose load,
glucose incorporation into diaphragm glycogen, and adipose tissue lipids both on starch and
sucrose diet.395 The exact role of copper during the sucrose-induced diabetes in these rats
remains to be determined. The typical sucrose treatment effects of nondiabetic rats have not
been appreciably affected by the deficiency of another trace element, the chromium.396.3"
The development of microaneurysms of the capillary retinal bed was also demonstrated
in sucrose-fed non-inbred rats, glucose intolerant but not overtly diabetic, which were similar
to the lesions appearing in streptozotocin-diabetic rats;398 the fructose moiety of sucrose was
convincingly implicated as the retinopathic agent.399 In rats fed a 55% sucrose diet for 8
months, irregular thickening of the glomerular basement membrane was also observed, again
similar in appearance to that in diabetic rats.400.40' Increased posttranslational collagen gly-
cosylation was suggested, on the basis of increased content of hydroxylysine, with a larger
percentage occupied by hexose units, and higher activity of glucosyl transferase. Interest-
ingly, in diabetic rats lipogenesis was decreased in the liver and increased in the kidney —
on sucrose it was increased in both tissues with resultant higher renal lipid content. Since
these glomerular changes occurred at normal blood glucose levels, in sucrose but not in
starch-fed control rats, intriguing fructose-initiated intracellular protein-hexose interactions
may be the speculative mechanisms of nonhyperglycemic microangiopathy.
B. Hyperlipidemia and Atherosclerosis

Among the challenges leveled against the trend of changes in modern nutrition is the
hypothesis promulgated by Yudkin and others, mainly in the 1960s ,402-410 that sucrose is a
major factor in the contemporary rise in the incidence of coronary heart disease due to its
hyperlipidemic, diabetogenic, and thrombotic effects.
The claim is based not only on epidemiologic correlations of sucrose intake, morbidity,
and mortality in different population groups ;404,407,408 but on comparisons of sucrose use
among survivors from myocardial infarction and control subjects,409 or an effect of habitual
sucrose intake on platelet adhesiveness in association with hypertriglyceridemia, hyperin-
sulinemia, and peripheral vascular disease in susceptible individuals.4" 412
A pertinent but not well-understood observation is that patients with signs of atherosclerosis
given a sucrose meal show higher plasma fructose but not glucose level than in control
subjects,4" suggesting an abnormality, possibly related to hyperlipidemia.414
Since atherosclerosis is a slowly developing syndrome of diverse origin, comprising many
predisposing factors, such as hyperglycemia, hyperlipidemia, lack of exercise, smoking,
etc., a more objective view would be, that sucrose-induced changes should be regarded
as an adjunct to these multiple risk factors. For example, rises in lipogenesis and circulating
lipids due to sucrose could be shown in some experiments only when the diet included an
increased amount of fat,4 '5 so as to overtax the lipid removal mechanism but the hyperli-
pidemia was blunted in animal trials with polyunsaturated fat supplementation or estrogen
treatment' which accelerate TG removal. Likewise, an elevation of plasma cholesterol was
observed, particularly in rats fed sucrose together with a cholesterol-rich diet.'01
In other surveys no direct relationship was found between sugar consumption and coronary
heart disease, but men and women who smoked used more sugar than nonsmokers and those
with anginal symptoms were found to consume more sugar than those without.417 4"
The effect of fructose or sucrose feeding on potentiation of hyperlipidemia is apparently
dose and duration dependent and its long-lasting effect on vascular deterioration is yet to
be documented. Animals with experimental diabetes maintained on fructose-rich diets promptly
show an exacerbation of hyperlipidemie9-42' but addition of small amounts of fructose to
the regular diet requires a 6-week period for significant plasma TG increases to be noted.28 '
In untreated diabetic type II patients, substitution of 80 to 100 g starch with fructose produced
variable, individual potentiation of hypertriglyceridemia and hyperglycemia, whereas in
insulin-treated type II diabetics, uniform but slight increases in plasma TG, cholesterol, and
FFA were noted.28' While, on the basis of these results, it might be construed that moderate
fructose consumption may be relatively safe in diabetes, it should also be remembered that
diabetic patients spill over more fructose-derived glucose in urine and insulin treatment did
not enhance fructose metabolism neither in diabetic nor nondiabetic rats.422
The relation of fructose-induced hyperlipidemia to atherosclerosis entails an interesting
concept. It is well known that heart LPL activity is inversely related to that of adipose tissue.
On fasting, heart LPL activity rises, especially its heparin-releasable fraction, which cleaves
VLDL-TG at the luminal surface of the capillary endothelial cells; on carbohydrate-rich
nutrition it returns to normal. In fasted rats, intubated with glucose solution, the heart
releasable LPL was found to decrease by 85% already within 90 min, whereas it remained
high in rats intubated with fructose.423 Even without ascertaining whether the retention of
the LPL activity is attributable to continuing insulinopenia, this observation may be relevant
to fructose involvement in atherosclerosis causation. Since, as a result of high LPL activity,
more VLDL-TG are diverted for heart uptake, it was suggested424,425 that the local concen-
tration of partially cleaved VLDL-TG particles increases and that these "remnants", known
to be avidly deposited in the arterial smooth muscle cells, initiate the formation of ather-
osclerotic plaques. If heart LPL activity remains persistently elevated on fructose-rich diets,
this mechanism presents a plausible possibility.
C. Pregnancy, Perinatal, and Survival Aspects

It has been reported that the activity of fructokinase is absent or very low in fetal rat
Volume II 121
liver"' and glycogen formation from fructose is also very low,427 F-1-P aldolase activity
was, however, found to be present.428 Conversion of fructose to glucose proceeds in fetal
liver on day 22 of gestation via fructokinase, as in adult liver, judging from substantial
relocation of 1-'4C-fructose radioactivity to the 6-'4C position in glycogen.' In the 20 day
fetus, there is no label relocation and any fructose taken up is metabolized by hexokinase.
Any pathway of conversion of fructose to glucose via sorbitol may be excluded in fetal liver
since sorbitol dehydrogenase and aldose reductase activities are negligible.'" Fructokinase
and triokinase were shown to have a parallel time-course of development in fetal rat liver
and in the postnatal period up to 35 days.428 On the other hand, F-1-P aldolase activity at
birth was similar to adult rats, which may reflect the general rather than fructolysis-specific
role of this liver enzyme. On the basis of these findings it has been postulated that fructokinase
and triokinase have a coordinated genic expression.' This postulate is supported by the
similar adaptive responses of these two enzymes.'
Investigation of the effect of sucrose feeding during pregnancy on the maternal and fetal
metabolism yielded somewhat contradictory results. In one study43° sucrose feeding did not
augment the maternal hyperlipidemia in the rat: VLDL-hypertriglyceridemia of endogenous
origin and of similar extent was present both on starch and sucrose, although liver weight
and its TG content were higher on sucrose. The important finding was lower fetal weight
on sucrose, probably because of inability of the fetus to metabolize fructose. In another
study,"' dietary sucrose and fructose produced significantly higher plasma TG and FFA
concentrations in nonpregnant, pregnant, and lactating rats, than did starch or glucose. This
was accompanied by enhanced hepatic lipogenesis and depression of adipose tissue lipo-
genesis, particularly by fructose as assessed by the activity of several lipogenic enzymes.
Studies on the maternal dietary influence on the offspring and early dietary experience of
weanlings were also conducted. The 142-day-old BHE rats, which were fed a 65% sucrose
diet from weaning to 50 days had higher serum insulin and TG levels and hepatic glycerol-
3-P dehydrogenase than those fed starch. The animals initially fed sucrose and switched to
starch had higher liver TG and cholesterol levels than those fed starch or sucrose continuously.
These results indicate that the kind of carbohydrate fed during the postweaning period may
have long-lasting effects on the metabolic pattern, even if the particular carbohydrate is
discontinued. 432
In a similar study the influence of materal sucrose feeding of the BHE rat was tested on
the progeny at 142 days.433 Maternal sucrose intake resulted in significantly lowered serum
TG and insulin levels in the progeny fed starch after the weaning, but in higher TG levels
in those fed sucrose during the postweaning period. Hepatic NADP-linked enzymes were
not affected by the maternal history but differed according to the diet given to the weanlings.
These results show that the gestational and lactating period diet does leave an imprint on
the progeny even if they are later fed a different diet until 142 days of age. This imprint is
in accord with another group's results on the lasting effects of maternal sucrose diet on the
growth and metabolism of the newborn mice.434 The progeny of sucrose-fed mothers had
significantly lower body weight gains as found by others."'
It may also be mentioned that substitution of starch with sucrose in a low protein diet of
weanling rats led, in addition to the growth retardation as in the control animals, also to
lower plasma albumin levels' along with increased liver size, which appears to be related
to the reduction in the capacity of hepatic protein synthesis. A contrary effect of maternal
sucrose feeding on newborn weight was found in caffeine consuming maternal rats. In this
case, caffeine ingestion reduced newborn weight but sucrose supplementation of the diet
prevented this reduction,' probably by the sole increase in energy intake.
Sucrose feeding may have a deleterious effect on the fetal development, fertility, and
survival of parents and weanlings in several species. Female normal and sucrose-inbred
diabetic rats,388 mated with nondiabetic males and maintained on 72% sucrose or regular
diets during gestation, showed a high fetus resorption rate and a significant number of
malformations (exencephaly, caudal regression, ventricular septal defect) irrespective whether
the rats were diabetic or normoglycemic.''
Homozygous diabetic C57 BL/KsJ oblob mice were maintained after weaning on a 60%
sucrose vs. 60% casein diet.'38 Those on sucrose exhibited pancreatic necrosis and a 57%
mortality between the 4th and 5th month of life, whereas those on casein remained healthy
up to the 6th month of life. While no control diet with carbohydrate other than sucrose was
used, the results do implicate sucrose as the critical factor rather than the caloric intake,
since restricting the diet by one half failed to prevent the pancreatic necrosis.
In pairs of spiny mice maintained for 18 months on isocaloric 50% sucrose or starch diets,
the total number of pups born was lower by 43% on sucrose.439 In each productive pair the
litter size was not diminished but —96% of the pups survived until weaning on starch and
only —80% on sucrose. Parent mortality on sucrose was also higher: 22 vs. 6%. The effect
of sucrose was underscored by repeating the experiment and adding mice pairs on a natural
fat-rich seed diet. The number of pups born on sucrose was 36% of those born on the fat
diet, and 44% on the regular diet. Pup survival until weaning was 67% on sucrose, 86%
on fat, and 96% on the regular diet.
These effects of sucrose on fertility, survival of pups, and longevity of parents may be
carried to the extreme side when occurring in a desert species transferred to an affluent diet,
but they do present a model for pinpointing the exact cause(s) of the detrimental lesions,
which may be any one or a combination of those mentioned in Section IX.A — or sucrose
feeding may have induced maternal metabolic deficiencies reducing the capacity to support
the fetus and the newborn. The animals were not hyperglycemic except on the fat diet, on
which no untoward effects were recorded. Of the possible causes affecting the survival of
spiny mice a few reports on sucrose-induced rat mortality are worth mentioning. Substitution
of even 15% dietary carbohydrate with sucrose in a strain of nephropathy-susceptible Wistar
rats shortened the life span from 81 to 69 weeks in males and from 87 to 83 weeks in
females.44° BHE rats, derived from the Osborne-Mendel strain, which show at maturity
hyperlipidemia, glucose intolerance and kidney disease"' survive on sucrose diet for 68 vs.
88 weeks."2
D. Hepatic Effects
The long-term maintenance on sucrose diet is associated with a marked liver enlargement
_ 197 ' 198 In spiny mice after a 3-month 50% sucrose diet it may amount
in rats, 184.194,289 or mice
to —60% (from 3.3 ± 0.1 to 5.1 ± 0.2 g/100 g body weight). This large increase is not
due to glycogen or lipid deposition which may contribute only a small fraction of the
increment. It entails an increase in almost all liver constituents and appears to represent a
functional adaptation to the role of the liver as virtually the only tissue capable of fructose
metabolism in the majority of mammalian species. This involves both an increased content
of cytoplasmic enzymes required for fructolysis, gluconeogenesis and lipogenesis, and of
the endoplastic reticulum components, to maintain their increased synthesis. Large liver size
increases are well known in other situations characterized by extensive hepatic enzyme
adaptations and lipoprotein secretion as under glucocorticoid excess or in association with
increased synthesis of extracellular proteins in nephrotic syndrome!'" Little is known on
human liver size on sucrose feeding; it may not be increased as extensively, since the dietary
sucrose content in human nutrition does not reach the percentage used in animal experiments.
Phillips et al.' examined the ultrastructural changes in rat hepatocytes after intraportal
infusion of 3 to 5 mf of 20% fructose solution for 1 to 2 hr and noted a marked hypertrophy
of the smooth endoplasmic reticulum, hyaline rarefaction, loss of ribosomes, formation of
cytolysosomes, and concentric membranal arrays. These changes were similar to those
Volume .11 123
induced by oral fructose feeding to a patient with F-1-P aldolase deficiency,445 and were
not obtained in the same experimental setup upon glucose infusion.
Some of these observations may be considered to manifest acute toxicity resulting from
the accumulation of fructose and its metabolites after a large fructose load. Ribosome loss
may be related to inhibition of protein and RNA synthesis due to ATP depletion,446 hyalo-
plasm hydration, and cytolysosome appearance connected with F-1-P accumulation. Whereas,
these changes could be transient and deleterious, the reticulum proliferation may represent
an adjustment to increased fructose availability. Apart from the stimulation of synthesis of
the numerous cytosolic enzymes involved in fructose metabolism, the endoplasmic reticulum
is required for the increased fatty acid activation, esterification, and assembly of lipoproteins
as well as for the increased synthesis of G-6-Pase, located in this organelle.
Support for the adaptive nature of the endoplasmic reticulum and cytolysosome multi-
plication is obtained from the changes in mice after 3 days of fructose in contrast to glucose
feeding: the marked increase in smooth endoplasmic reticulum in the absorbing cells of
jejunal villi was correlated with increased in G-6-Pase activity.''
X. CONCLUDING COMMENTS WITH THOUGHTS ON FUTURE

RESEARCH ON SUCROSE NUTRITION AND METABOLISM
Fructose as a component of human and animal nutrition poses problems of entwined

beneficial and deleterious effects unlike other oligomeric or polymeric aldohexoses. An
intelligent assessment of the effect of fructose utilization is possible nowadays, thanks to
the great advances achieved during the last three decades in the understanding of the reg-
ulation of metabolism of this ketohexose and of the dietary influences affecting its disposal.
On the "positive" side is the fact that fructose dissimilation (virtually limited to one organ
— the liver) occurs without insulin facilitation and is not associated with stimulation of
pancreatic insulin secretion, thus sparing insulin for other actions. The "negative" impact
is based on the impressive capacity of fructose phosphorylation and fructolysis, which by
far exceeds that of glycolysis, with a clear preference for the downward metabolite flow
and entry into the mitochondria. This results in a plethora of lipogenic precursors, conducive
for channeling into TG and cholesterol synthesis with consequent hyperlipidemia and hepatic
steatosis.
One could ask, from the historical — teleological point of view, is this inborn, preferential
routing of fructose to lipogenesis a phylogenetic rudiment of prehuman metabolism indicating
that fructose was the main (even if quantitatively scarce) nutrient source compared with
starch? Did the intermittent, gorging-type ingestion provide a benefit of rapid energy storage
as fat for extended supply? Such survival advantage is not meaningful today when fructose
is consumed (in excess) with other calorie-rich nutrients and helps to express the detrimental
effects of glucose intolerance leading to overt diabetes, hyperlipidemia associated with
atherosclerosis and microangiopathy, and hyperuricemia aggravating the predisposition to
gout. As stated in the previous sections of this review, these effects become conspicuous
when fructose is ingested in quantity on top of other high carbohydrate or fat diets in
genera1.281
It must be repeatedly stressed though, that in many of these deleterious effects, such as
the coronary heart disease, the cause-and-effect relationship of high sucrose consumption
and consequent specific fructose metabolism is suggestive, and still remains to be unequi-
vocally proven. Most of the demonstrative animal experiments, although quite convincing,
were performed with rations of sucrose exceedingly higher than the average human con-
sumption, under conditions which may tend to exaggerate these effects.
The gout-susceptible persons deserve a special caution since the activities of nucleotidase
and deaminase may become persistently increased after excessive fructose intake due to the
lowered cellular levels of their inhibitors (ATP, GTP, and P,), enhancing purine degradation.
When superimposed on a latent defect of increased purine synthesis, this may induce not
only increased purine breakdown but a "compensatory" enhancement of de novo purine
synthesis and uric acid formation."'
Because of potential genetic proneness to the detrimental effects, sucrose should be
included as an ingredient in the environmental cocktail of risk factors for atherogenic diathesis
but not dealt with as a lone component.
Certain advantages of fructose consumption should be further explored. One is the pos-
sibility to include fructose in the diet of diabetic and/or overweight persons who have to
watch their calorie intake and who cannot renounce their need for sweetening. Fructose,
being 1.7 times sweeter than sucrose, when consumed in an amount of —100 g/day, could
save —300 cal/day and this might be important, especially when used as a beverage sweet-
ener. Fructose may be useful in the preparation of palatable meals of obese diabetics, where
restriction of total calories is more important than reduction in carbohydrate intake."' In
any use of pure fructose or fructose-rich regimens, however, special care must be exercised
to prevent sudden heavy loads, to determine the upper limit of intake, and to contraindicate
its use in susceptible individuals, such as overt diabetics with vascular complications, persons
with familial hyperlipidemia, gout, predisposition to early atherosclerosis, especially with
tendency to ischemia, renal failure requiring hemodialysis,45° and possibly liver disease and
alcoholism. The role of fructose metabolism in pregnancy and lactation and in the devel-
opment of the newborn also requires special attention.
Precautions in the use of fructose or fructose-containing nutrients in diseases with con-
genital deficiency of fructolysis enzymes are discussed in detail elsewhere."'
Another potentially beneficial use of pure fructose as a sweetener might be in prevention
of dental caries. 452 "3 Bacteria are mainly responsible for the breakdown of sugars to calcium-
leaching organic acids in the oral cavity, and various hexose residues are components and
potential initiators of dental plaque formation. Since these organisms appear to be devoid
of fructokinase activity, the replacement of glucose or sucrose-containing sweets and con-
diments with fructose could possibly reduce oral glycolysis and caries incidence. However,
any such widespread use must be thoroughly explored and controlled to prevent the untoward
systemic effects of permissive fructose intake.
With regard to the use of sucrose as an animal nutrient, a careful species by species
evaluation is required, since a large variation exists in main sites of fructose metabolism.
The pig and guinea pig seem to reside at one end of the spectrum with large intestinal
capacity of fructose to glucose conversion, relatively small liver involvement in fructose
metabolism, and marked adipose tissue gain. Most rodents, primates, and particularly human
species require major hepatic involvement in fructose metabolism with the corrollaries of
excessive fructose utilization described here.
Nutritional habits and food supply sources are difficult and slow to change and sucrose
consumption has not peaked even in the developed, industrialized countries, while sharply
rising in the developing ones. Future research should perhaps concentrate, therefore, on the
definition of judicious limits of intake of pure fructose and fructose-rich diets and their long-
term effects at low to moderate intake levels.248-285378
Volume II 125
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Volume II 141
Chapter 6
NEW PERSPECTIVES ON CARBOHYDRATE METABOLISM IN TUMOR

CELLS
E. Eigenbrodt, P. Fister, and M. Reinacher
TABLE OF CONTENTS
I. Principal Concepts of Aerobic Glycolysis in Proliferating Cells 142

A. Aerobic Glycolysis in Its Historical Retrospect 142
B. A New Concept: The Role of Carbohydrate Metabolism in Cell
Proliferation and Tumor Formation 142
II. Glycolysis 143

A. Altered Control of Glycolysis in Tumor Cells as a Consequence of
Induction of Isoenzymes with Special Features 143
B. Influence of Different Carbohydrate Substrates on Aerobic Glycolysis 149
III. Glutaminolysis 150

A. Lactate Production from Glutamine by Special Isoenzymes in
Tumor Cells 150
B. Interaction of Glutaminolysis and Aerobic Glycolysis 150
C. Possible Function of Glutaminolysis and Aerobic Glycolysis in
Tumor Cell Formation 152
IV. Pyruvate Oxidation 153

A. Interaction of Pyruvate Oxidation and Glutaminolysis 153
B. The Selective Advantage of Low Pyruvate Oxidation Capacity for
Tumor Cells 154
I. Resistance Against High Oxygen Levels in Cell Culture 154
2. Optimal Supply of Acetyl-CoA for Lipid Synthesis 155
V. Metabolic Strategy of Tumor Cells for Survival Under Conditions of

Extreme Variability in the Cellular Environment and Nutrient Supply 155
VI. Cell Proliferation 156

A. Role of Glycolysis and Glutaminolysis in Cell Proliferation 156
B. Intermediates Prerequisite for Proliferation 156
I. Aspartate 156
2. Serine 157
3. Glycerol 3-Phosphate 159
4. Fructose- I ,6-B isphosphate 159
5. P-Ribose-PP 160
C. Enzymes of Carbohydrate Metabolism Potentially Involved in Control
of Normal Cell Proliferation 161
D. Changes in the Function of Metabolic Enzymes may Trigger Cell
Proliferation 162
VII. Cell Transformation 163

A. Modified Control of Carbohydrate Metabolism Induced by pp60•"--
Kinase 163
B. Chemical Carcinogenesis: Carbohydrate Metabolism in Hepatocytes,
Preneoplastic Hepatocytes, and Hepatocarcinoma 164
C. Onc-Genes, Multistep Transformation, and Malignancy from the
Viewpoint of Carbohydrate Metabolism 165
VIII. Perspectives 168
Acknowledgments 169
References 169
I. PRINCIPAL CONCEPTS OF AEROBIC GLYCOLYSIS IN

PROLIFERATING CELLS
A. Aerobic Glycolysis in Its Historical Retrospect

Since the original observation that malignant cells exhibit a high rate of glycolysis,
considerable effort has been devoted to elucidation of the role of glycolysis in normal and
tumor cell proliferation.' It could be demonstrated that well differentiated, slowly growing
morris hepatoma cells show a low rate of glycolysis, whereas the fast growing, poorly
differentiated cells have a high glycolysis. Besides their increased capacity for lactate for-
mation in anoxic conditions, a characteristic property of tumor cells is their high level of
lactate production in the presence of oxygen, the so called "aerobic" glycolysis.'-3
The inhibition of glycolysis by oxygen has been named the Pasteur effect. This is a
mechanism for adjusting the consumption of glucose in the presence of oxygen to the energy
needs of the cells. One mole glucose yields under anaerobic conditions 2 mol of ATP
compared to 38 mol of ATP in the presence of oxygen. Hence, respiration is the most
economical mechanism for the synthesis of ATP. The concept of aerobic glycolysis means
that the pyruvate production from glucose exceeds the capacity of the tricarboxylic acid
cycle and respiratory chain, but it also implies that the tumor cell uses only 5% of the energy
available from glucose. From the standpoint of the energy requirement, this is a senseless
waste of the resources by tumor cells. An elevated aerobic glycolysis is, however, not unique
to tumor cells, since intestinal mucosa, renal medulla, retinal cells, and cells stimulated by
growth hormones to proliferate show the same phenomenon of high aerobic glycolysis as
wel1.4- ' 5 Originally, Warburg postulated an altered respiratory capability as a specific property
of tumor cells to explain elevated lactate production, which he thought to be caused only
by glycolysis. It has become clear, however, that gross defects in respiration are neither a
general property of tumor cells and proliferating normal cells, nor the cause of a high rate
of glycolysis. '"
Subsequently, Racker put forward the explanation that the high aerobic glycolysis in tumor
cells is sustained by an aberrant Na -ATPase supplying ADP and P, as the driving
force. This is an interesting explanation, but, at the moment, it remains largely speculative,
since no convincing results for establishing this theory have been presented.'6 19 '255
B. A New Concept: The Role of Carbohydrate Metabolism in Cell Proliferation and

Tumor Formation
Because of the findings and assumptions discussed in this paper, we propose a new concept
for the function of carbohydrate metabolism in cell proliferation and tumor formation.
Volume 11 143
1. Although high aerobic glycolysis takes places not only in tumor cells, but also in
normal proliferating cells and in the quiescent cells of retina, renal medulla, and
erythrocytes, the reason for this common behavior is not the same for each cell type
(Figures IA, B and C).'- ' 5
2. In all proliferating cells, lactate is generated not only by glycolysis, but also in large
measure from glutamine (Figures IC, 3, and 4). 77,78,88108
3. The enhanced formation of lactate in normal proliferating cells is caused by an increased
glycolysis and/or glutaminolysis; the oxidation of pyruvate is not decreased. In tumor
cells, however, additionally, pyruvate oxidation is decreased, not owing to a gross
defect of mitochondria, but as a selective alteration (Figures 1, 3, and 4).4 15 '88
108,110.115,116,124-128
4. In all proliferating cells, the levels and/or the availability of the glycolytic and other
intermediates are enhanced, thus channeling substrates to the synthesis of DNA, pro-
teins, and other macromolecules (Figures IC, 3, 4, and 6C, and Table 2).8.9.54,105-108
5. The enhanced levels of intermediates are caused by changed activities and affinities
of glycolytic and glutaminolytic enzymes for their substrates. ' -12,49-55.88-96
6. The change of the activities and affinities in normal proliferating cells is reversible
and a consequence of, e.g., enzyme phosphorylation.192-1" In tumor cells, this change
is additionally caused by a modified isoenzyme pattern.-31 -38,88-96,197.256 These differing
isoenzymes also exhibit a priori different kinetic features. Therefore, this change is
irreversible, at least, it is constant for a longer time (Table 1).
7. Tumor cells are different from normal proliferating cells mainly in terms of the fol-
lowing: enhanced aerobic glycolysis'- ' 5 and glucose uptake,78 enhanced glutamino-
lysis,76-7888 108 enhanced nucleic acid synthetic capacity 24-27 and enhanced lipid
synthesis ,67,130-132,217 reduced pyruvate and acetyl CoA oxidation rate, ' 21-126 lower sen-
sitivity to oxygen, 111-114,136,213-215 and a lower growth hormone requirement.218'22 '
8. Tumor formation is a multistep process, with the single exception of virus-induced
transformation, e.g., by a tyrosine-specific protein kinase oncogene. 208-212 These steps
allow tissues with low or no proliferation rate like liver, brain, and muscle to regain
proliferation potency. 224,225 In hepatocytes, this elevated proliferation potency is char-
acterized by a higher capacity of enzymes channeling carbohydrates to nucleic acids
and phospholipids. 224-241 Immortalization is presumably linked to a reduced pyruvate
and acetyl-CoA oxidation,'2 ' 126 lower sensitivity to oxygen,111-114,136,213-215,222 and in-
creased glycolysis rate. 28,29,220 Transformation is mainly linked to reduced growth
hormone requirement ,218,221,250,251 and the degree of malignancy correlates with the
reduced growth hormone requirement, enhanced aerobic glycolysis,23-77 glutamino-
lysis,88-91 -96 and enhanced nucleic acid24-27 and phospholipid synthetic capacity.257
This concept is supported by the findings which will be described and interpreted below.
II. GLYCOLYSIS
A. Altered Control of Glycolysis in Tumor Cells as a Consequence of Induction of

Isoenzymes with Special Features
High aerobic glycolysis can be attributed mainly to changes in the isoenzyme patterns of
two enzymes: hexokinase at the starting point of the glycolytic sequence and pyruvate kinase
at the end (Figure IA).
Isoenzymes catalyze the same reaction, but they differ structurally, and in most cases,
they differ in their response to metabolites. There is a general agreement that metabolic
modulation of hexokinase, phosphofructokinase, and pyruvate kinase activities must be
necessary for the inhibition of glycolysis by oxygen (Pasteur effect). Thus, a simple expla-
NORMAL CELL
GLUCOSE
07
ek Glucose -6-P
Fructose -6-P
r hospho-
Fructose 1-6 dphosphatase 0TO f ructoki nose
Fructose 1,6 - P
Glyceraldehyde 3-P
+ Dihydroxyacetone-P
g
Phosphoenol Pyruvate
0110
Pyruvate
A
FIGURE I. (A) Regulation of glycolysis by oxygen in normal cells. With sufficient oxygen, normal cells adjust
pyruvate production to their level of acetyl-CoA consumption and energy needs. The regulation occurs mainly as
a result of the ATP inhibition of phosphofructokinase, which leads to a decrease in the conversion rate of fructose
6-phosphate to fructose-I ,6-bisphosphate. Steady-state levels of fructose-I ,6-bisphosphate are low, since this
metabolite is rapidly converted to pyruvate (high glycolytic capacity from fructose-I ,6-bisphosphate to pyruvate)
or to fructose 6-phosphate (by fructose-I ,6-bisphosphatase). Low levels of fructose-I ,6-bisphosphate are not suf-
ficient to overcome the ATP inhibition of phosphofructokinase. Glucose 6-phosphate, which accumulates as a
result of the inhibition of phosphofructokinase, in turn blocks its own synthesis by hexokinase. Therefore, the
mitochondrial ATP production governs the glycolytic sequence mainly through phosphofructokinase inhibition
(Pasteur effect). Under anaerobic conditions, mitochondrial ATP production by oxidation of acetyl-CoA is blocked.
Thus, ATP levels decrease, phosphofructokinase is deinhibited, and fructose-1,6-bisphosphatase is blocked by the
increased AMP levels (not shown). Fructose-I ,6-bisphosphate accumulates and further stimulates the phospho-
fructokinase and pyruvate kinase activities. The resulting fall in the glucose 6-phosphate levels enhances the
hexokinase capacity. The concerted effect of all these mechanisms allows the cell to utilize the total glycolytic
capacity for ATP production under anoxic conditions. Lactate is formed under these conditions from pyruvate in
order to reoxidize the NADH formed via the glyceraldehyde 3-phosphate dehydrogenase reaction. (B) Regulation
of glycolysis by oxygen in tumor cells at high glucose concentration. In tumor cells, enhanced activities of hexokinase
(the mitochondrially bound form), phosphofructokinase, and pyruvate kinase ensure a high glycolytic capacity.
Pyruvate kinase of the isoenzyme type M, (tumor type) is inhibited by alanine, phenylalanine, and ATP. The
fructose- I ,6-bisphosphate formed is only slowly converted to pyruvate until Fni- I ,6-P2 levels exceed a concentration
necessary to overcome the ATP inhibition of PK-M2. The accumulated fructose-1,6-bisphosphate, thereby, over-
throws the mitochondrial control of glycolysis. The fully activated hexokinase (not inhibited by glucose 6-P),
together with deinhibited phosphofructokinase and pyruvate kinase, leads to a drastic increase of the glycolytic
intermediates from glucose 6-P to glyceraldehyde 3-phosphate, which results in a high aerobic glycolytic rate.
Since the fructose- I ,6-bisphosphatase activity is sharply reduced in tumor cells, the fructose-1,6-bisphosphate levels
and aerobic glycolysis remain permanently elevated. Tumor cells, in contrast to normal cells, constantly use almost
the total glycolytic capacity regardless of the oxygen tension. (C) Regulation of glycolysis by oxygen in tumor
cells at low glucose concentrations or with alternative substrates. At low glucose concentrations (50 pM) or with
other substrates, levels of fructose-1,6-bisphosphate and P-ribose-PP are extremely low compared to high glucose
conditions (5000 p,M). Therefore, pyruvate kinase is functionally inactive. No phosphoenolpyruvate is converted
to pyruvate, no ATP is synthesized in the glycolytic pathway, and no pyruvate is available for ATP production
by pyruvate oxidation. When ATP is used by enzymes, it is decomposed to ADP and P. The resulting P, activates
the phosphate dependent glutaminase, and glutaminolysis begins, yielding 5 ATP + I GTP per mole glutamine
under aerobic conditions. As a result, the ATP levels are restored, and the small amounts of glucose are available
for serine, aspartate, and P-ribose-PP synthesis. At this point, proliferation can start.
Volume II 145
TUMOR CELL
Cytoplasmic
/ ..a.'
Hexokinase
=GLUCOSE
000
00
.--.)1
O
Glucose-
11
Fructose -6-P
Phosphofructcknase ~
®
FRUCTOSE 1,6 - P
11
Glyceraldehyde 3- P
+ Dihydroxyacetone - P
e 11
Phosphoenol Pyruvate
Pyruvate Kinase
„tumortype
type M2
Pyruvate LACTATE
FIGURE I B
TUMOR CELL
Glutamine
CO2. H2O
Pi- dependent
Hexokinase Glutaminase
OCO
Glucose OCO
Glutamate
Glucose-6-P Succi nate
Ma late
Fructose-6-P
Phosphofructo - NAD- dependent
kii.ase Purine Matic enzyme
Pyrimidine
Fructose 1,6-P
P-ribose-PP
Pyruvate
Glyceraldehyde 3-P
f Dihydroxyacetone- P
'••••••10. Serine Aspar tate

i --.
Phosphoenol Pyruvate ---"P ?
Pyruvate j
kinase
Lactate
FIGURE IC
nation for the phenomenon of high aerobic glycolysis would be that the usual regulatory
mechanisms described above are selectively altered in these cells following induction of
isoenzymes which respond differently to metabolites. Indeed, there are several observations
supporting this hypothesis (Figures 1B and C):
1. Hexokinase — All cell types with high aerobic glycolysis such as retinal, renal
medulla, and tumor cells have a special type of mitochondrial hexokinase (type II)
that is not inhibited by concentrations of glucose 6-phosphate below 0.6 mM and that
is regulated by glucose-1,6-bisphosphate levels (differently from other hexokinase
isoenzymes).31-38 All tumor cells tested (H-91 hepatoma cells, chicken embryo fibro-
blasts transformed with the Schmidt-Ruppin strain of Rous sarcoma virus, Ehrlich
ascites tumor cells, leukemic lymphocytes) contain a higher amount of this special
form of hexokinase than the normal cells from which they are derived.33
2. Phosphofructokinase — Although phosphofructokinase plays a central role in the
regulation of glycolysis by oxygen, no characteristic isoenzyme is expressed in cells
with high aerobic glycolysis. Because the three isoenzymes of phosphofructokinase
(type L, F, and M) have similar kinetic characteristics (ATP-inhibition, Fru-1,6-P,-
activation, AMP-activation), all of them would allow the same rate of aerobic gly-
colysis .39-46
3. Pyruvate kinase — All cells with high aerobic glycolysis contain a type of pyruvate
kinase (type M,) that has a lower affinity to phosphoenolpyruvate and a stronger
inhibition by alanine than other pyruvate kinase isoenzymes." 5 ' This isoenzyme can
be phosphorylated and inactivated by a cAMP-independent protein kinase in vivo and
in vitro.51 .52 Under certain culture conditions (under 100 )1/1/ glucose or with fructose),
the M, pyruvate kinase isoenzyme should be functionally inactive in proliferating and
tumor cells.
At first glance, it may appear paradoxical that tumor cells with a high flux from glucose
to lactate possess a pyruvate kinase isoenzyme with such features. A possible explanation
for this phenomenon would be that inactive pyruvate kinase dams up phosphoenolpyruvate.
There is indirect evidence from experimental investigations and theoretical considerations
that inhibition of pyruvate kinase, enolase, or phosphoglyceromutase leads to an accumu-
lation of fructose-1,6-bisphosphate and glycerol 3-phosphate, thus channeling increased
amounts of glycolytic metabolites into three synthetic pathways: serine synthesis from gly-
cerate 3-phosphate, phospholipid-, triglyceride-, and sphingomyelin-synthesis from glycerol
3-phosphate, and ribose 5-phosphate for phosphoribosyl pyrophosphate synthesis from gly-
ceraldehyde 3-phosphate (Figures 1A, B, C; 3, 4, and 5). 58-73 In this context, it is important
to note that functionally inactive type M, pyruvate kinase in tumor cells can be totally
reactivated by micromolar concentrations of fructose-1,6-bisphosphate or millimolar con-
centrations of serine. Also the phosphorylation and inactivation of pyruvate kinase by protein
kinase in tumor cells is inhibited by millimolar concentrations of fructose-1,6-bisphosphate
or phosphoribosyl pyrophosphate (Figures I A, B, and C).49 51 There is compelling evidence
that the enhanced aerobic glycolysis in tumor cells can be explained in the following way.
The inhibition of glycolysis by oxygen (Pasteur effect) in normal cells is largely a result of
allosteric inhibition of phosphofructokinase by ATP, whose generation depends on the
presence of oxygen. This inhibitory action of ATP can be reversed by increased levels of
fructose-1,6-bisphosphate.34-74 The activity of phosphofructokinase affects the other two key
glycolytic enzymes, i.e., hexokinase and pyruvate kinase. First, utilization of fructose 6-
phosphate leads to a decrease in the cellular levels of glucose 6-phosphate, a potent inhibitor
of hexokinase, resulting in hexokinase activation.343" Secondly, the activation of phos-
Table 1
CHANGES OF PATHWAYS AND ENZYME ACTIVITIES IN NORMAL PROLIFERATING AND TUMOR CELLS
COMPARED TO NORMAL QUIESCENT CELLS MEASURED UNDER HIGH GLUCOSE CONDITIONS
Normal proliferating cells
Activity Tumor cells
Early G, Late G, Isoenzyme Isoenzyme

phase phase pattern Activity pattern Ref.
Glycolysis
Lactate formation 0 T TT 5-7, 42, 46, 111-114
Pasteur effect 0 1 11 8, 9. 111—I14
Enzyme activities
Hexokinase T Slightly changed II Modified I I. 28, 33, 36, 37
Phosphofructokinase 0 T Unchanged T Unchanged 5, 6, II, IS, 39-42
Enolase T Unchanged T Modified II. 15, 197
Pyruvate kinase I T Slightly changed T T Modified 11, 15, 28, 47, 48, 256
Glutaminolysis T TT 88-96, 110, 234-236

Enzyme activities
Glutaminase T Slightly changed Modified 78, 88-93, 186, 236
Malic enzyme T Slightly changed Modified 78, 94, 96, 236
Pyruvate oxidation T 1 97, 98, 115, 116, 123-126
Acetate oxidation T 1
Note: The differences to normal quiescent cells are given and indicated by symbols: I , two- to threefold increase , three- to tenfold increase; 1 , decrease;
and zero (0) unchanged.
Table 2
CHANGES OF THE LEVELS AND THE AVAILABILITY OF METABOLITES FOR PROLIFERATION IN NORMAL
PROLIFERATING AND TUMOR CELLS COMPARED TO NORMAL QUIESCENT CELLS
Levels (01) Availability for synthetic processes
Non proliferating
and quiescent Proliferating Proliferating Tumor
Metabolites cells cells Tumor cells Synthesis of cells cells Ref.
P-ribose-PP 3-40' 140-820' 1000-3000" Nucleic acids TT TTT 54, 72. 169-177,
625' 15000' 45000' 262, 263
2' 4 6' 5-30°
Fru, I,6-P, 5-28' 267-940' 150-2800" (Activator of protein 5-10, 21, 54, 56,
25' --18' ,---18' synthesis: 164, 165) 76, 77, 166, 237
Glycerol 3-P 28-700 ND 1090-2600 Lipids 0 TTT 56, 64 68. 217, 257
Serine ND 400-860 1900 Purine T TTT 60-63, 119, 144-146,
Phospholipids T T 238, 239, 241, 242
Cysteine, GSH T T
Regulationof Carbohyd rate Metabolism
Aspartate 150-2500 8-35 150-650 Pyrimidines T T 99-101, 177, 178,

201-207, 260
NADPH, 0.04 6 8-24 6-18 (Activator of protein 77, 83, 237, 260
synthesis: 165, 200)
NAD' 230-760 369-1580 170-600 ADP ribosylation T T 54, 81-84, 139-
142, 260
NADH, 190-140 240-1740 1 I 00-2000 (Inhibitor of protein
degradation: 261)
ANA 0.001 I I (Activator of DNA- 201-203
polymerase: 202)
Note: Measurements were made under a variety of preparation protocols and used several different assay procedures. The approximate metabolite concentrations in
µA/ were calculated from: I µM = I nmol/g wet weight = 5 pmol/mg homogenate protein = 20 pmol/mg soluble protein after centrifugation = I nmol/
10' cells. Differences in the availability of precursors for synthetic processes compared to nonproliferating and quiescent cells are indicated by the following
symbols: zero, unchanged; T , two- to threefold increase; T T , three- to tenfold increase; and T T T , tenfold increase. ND, not determined.
Levels in mammalian cultured cells with 5000-10000 µM glucose in the medium.

Levels in chicken cells; in general, they seem to be 100-fold higher than in rat cells, since purine biosynthesis de novo in avians serves as the major catabolic
pathway for nitrogen.
Levels in mammalian cultured cells or tissue slices with 50-100 µA4 glucose, 10000 µM fructose, or 10000 µAi galactose in the medium.
Volume II 149
phofructokinase elevates the levels of fructose-1 ,6-bisphosphate, which even in micromolar

concentrations activates pyruvate kinase (Figures IA and B).49-54
In extracts of tumor cells, strongly enhanced activities of hexokinase (mitochondrial form),
phosphofructokinase, and type M2 pyruvate kinase are measured under optimal assay con-
ditions. Although this isoenzyme content endows the tumor cell with a high glycolytic
capacity, it may not be used for the cells energy demands immediately. Inhibition of hex-
okinase by glucose 6-phosphate and inhibition of phosphofructokinase by ATP may actually
cause a low glycolytic rate. Furthermore, because of its special features (low affinity for
PEP, inhibition by alanine, phosphorylation), pyruvate kinase type M2 under cellular con-
ditions is virtually inactive. Hence, fructose-1 ,6-bisphosphate, once formed in the cell, is
only slowly converted to pyruvate, and therefore, like the other glycolytic intermediates,
dammed up. When the level of fructose-1 ,6-bisphosphate has risen high enough, the inhi-
bition of pyruvate kinase type M, by alanine, phosphorylation, etc., is presumably overcome.
The accumulated fructose-1 ,6-bisphosphate also reverses the ATP inhibition of phospho-
fructokinase and, thereby, the mitochondrial control of glycolysis. As a further consequence,
hexokinase is also fully activated (no longer inhibited by glucose 6-phosphate). The cellular
activation of these enzymes leads to high glycolytic rates. These rates are only possible
because of a drastic expansion of all glycolytic intermediates from glucose 6-phosphate to
glyceraldehyde 3-phosphate. Since fructose- 1 ,6-bisphosphatase activity is strongly reduced
in tumor cells, the fructose-1 ,6-bisphosphate levels and aerobic glycolysis stay ele-
vated.13.24.27,54 In contrast to normal cells, tumor cells use nearly the total available glycolytic
capacity even when oxygen is present and possess high levels of glycolytic intermediates.
The levels of fructose-1 ,6-bisphosphate and P-ribose-PP are high enough to inhibit efficiently
the inactivation and phosphorylation of pyruvate kinase. 49.5° Any increased activity of the
cAMP-independent protein kinase that phosphorylates pyruvate kinase type M 2 would result
via inactivation of pyruvate kinase activity in the elevation of all intermediates between
phosphoenolpyruvate and glucose 6-P, P-ribose-PP, and serine. 49.5°
B. Influence of Different Carbohydrate Substrates on Aerobic Glycolysis

The presence of other carbohydrate sources in cell cultures — such as galactose, ribose,
fructose, uridine, inosine — leads to a drastic decrease in the overall lactate formation. The
small amount of residual lactate found under these conditions is derived from glutamine by
glutaminolysis."-" The carbon of 14C-ribose and '4C-galactose, however, was not detected
in pyruvate and lactate, but in serine (Figure I C)."-79 Because serine is derived from glycerate
3-phosphate, there must be a block under these conditions that prevents the degradation of
glycerate 3-phosphate to pyruvate (Figures 1 and 4).54-58 Three enzymes are involved in the
conversion of glycerate 3-phosphate to pyruvate: phosphoglyceromutase, enolase, and py-
ruvate kinase. Under the conditions of limited supply of glucose, one of these enzymes must
be functionally inactive in normal proliferating and tumor cells.
With ATP/ADP and NADH/NAD quotients normally found in proliferating cells,
phosphoglycerate kinase and glyceraldehyde 3-phosphate dehydrogenase strongly favor the
reversal of the glycolytic direction. 4,23,80-84 Therefore, estimation of the levels of phosphoen-
olpyruvate, glycerate 2-phosphate, and glycerate 3-phosphate would give no exact answer
to the question which of those enzymes contributes most to the decrease of the flux from
glycerate 3-phosphate to pyruvate. This question is in any case only interesting from a
theoretical standpoint. Certain metabolic experiments on the kinetic behavior of the type M2
isoenzyme of pyruvate kinase in normally proliferating and tumor cells point to pyruvate
kinase as the important site of control in the flux of glycerate 3-phosphate to pyruvate under
limiting glucose conditions, when the formation of fructose-1 ,6-bisphosphate is not sufficient
to overcome the inhibition of type M2 pyruvate kinase. When proliferating cells are selected
for growth on oxaloacetate or lactate as the only carbon source, they express a highly active
mitochondrial phosphoenolpyruvate carboxykinase." This enzyme is absolutely necessary
for the synthesis of phosphoenolpyruvate from carbon sources like oxaloacetate (Figure 2)
or lactate. Under these growth conditions, phosphoenolpyruvate is the unique precursor for
the synthesis of all macromolecules derived from glycolytic intermediates (DNA, RNA,
phospholipids, complex carbohydrates).
Active mitochondrial PEP-carboxykinase seems not only to be a special feature of cell
mutants growing on oxaloacetate or lactate, but is also found in several other normal pro-
liferating cells."." In human fibroblasts, the specific activity of this enzyme increased during
the log phase and remained constant during the stationary phase." This active PEP-car-
boxykinase could explain why in addition to serine, aspartate is also highly labeled when
'C-galactose is offered (Figure IC) as carbon source:" the reaction of PEP-carboxykinase
is reversible under physiological conditions, and oxaloacetate can be directly transaminated
to aspartate." If pyruvate kinase alone was functionally inactive under these conditions, no
radioactivity should be found in pyruvate and its transamination product alanine, but a high
amount in oxaloacetate and aspartate. Therefore, this protocol could be used as an indirect
assay for the estimation of pyruvate kinase activity in normally proliferating and tumor cells
(Figures IC and 2).
III. GLUTAMINOLYSIS
A. Lactate Production from Glutamine by Special Isoenzymes in Tumor Cells

Under conditions of carbohydrate limitation, all lactate produced is derived from glutamine
(Figure IC). The conversion of glutamine to lactate is catalyzed by seven enzymes, five of
which are part of the tricarboxylic acid cycle. Three of these catalyze irreversible reactions
under physiological conditions: the first enzyme of glutaminolysis, glutaminase, the 2-
oxoglutarate dehydrogenase, and the last enzyme of glutaminolysis, malic enzyme." As
with the glycolytic pathway, the glutaminolytic pathway in normally proliferating and tumor
cells is characterized by special characteristic isoenzymes.
1. The glutaminase in normal proliferating and tumor cells is strongly activated by phos-
phate, in contrast to the isoenzyme found in resting cells of tissues with the exception
of the kidney. "" 91.216
2. The malic enzyme in normal proliferating and tumor cells is located intramitochon-
drially, using either NAD+ or NADP+ to catalyze the irreversible decarboxylation of
malate to pyruvate. This ability to use NAD+ is in contrast to other mammalian
isoenzymes of malic enzyme. In addition, NAD±-linked malic enzyme exhibits prop-
erties of a regulatory enzyme, as would be expected of a "pace maker" enzyme at
the end of a pathway. The enzyme is activated by metabolites like succinate, fumarate,
and isocitrate; ATP and ADP are competitive inhibitors for malate (Figures IC and
4). 78
B. Interaction of Glutaminolysis and Aerobic Glycolysis

At high glucose concentrations (5000 p,M), most tumor cell lines metabolize more moles
of glucose than of any other organic molecule in the medium. About 80% of the glucose is
converted into lactate, while only 10% is oxidized in the tricarboxylic cycle.76 In spite of
this high glycolytic rate, under high glucose conditions (5000 µM), only 50% of the cells
energy requirements can be met by glycolysis; 40% are provided by glutaminolysis and 10%
by the oxidation of pyruvate and fatty acids. Next to glucose, glutamine is the main energy
source in proliferating and tumor cells.'" Reduction of glutamine concentrations at sat-
urating glucose concentrations further enhances the aerobic glycolytic rate. It is known from
Uridine
Uf
Hypoearilhine
Ribose
Ribose RK Ribose I - P
Ribose 5-P e--• Xylulcisip 5-P Siedoheptulose 7-P
I Erythose 4-P
6 -Phomitiogluconat•
HK PFX
Maltose ---• Glucose —• Glucose 6-P Fructose 6-P Fructose 1.6 P 4—\Cityciraitclehyde 3P-• letyperaTh°
FDPase
Sleirch Marines* 6-P
II G3 PhosPh091 Ywal• Swine
Golactos• Mannino Fructose —a Fructose I-P -*Chhydrowyoorlone P Pcm Glyclne
t TOK 2 Phosphoglyceratip
Dihydroxyacel one
Galactose EN 1
GDP CO2
Manna,. GTP
131-1 2 PhRIRRRR"R
ysyruvate
Fructose
) • Summate Furnaratelealatee-e0Noloacelote PDC
Dihydioxy- utomat• ftsmKetoglutarate
mAk PK
ocetOne • olio
CO2 TRICJLITEICIVLIC ACID CYCLE I t PC
Pyruvatie
•
Isocitrot• *---• Citrate Acetyl CoA
Asportale
Pruvat•
•
• Lactate
Glutamine Praline lactate AspayagIn•
FIGURE 2. Diagrammatic representation of the differential utilization of alternative carbohydrate sources by proliferating cells. Apart
from glucose, the following substrates can usually support proliferation: ribose, uridine, inosine, starch, maltose, galactose, fructose, and
mannose. Dihydroxyacetone supports only the growth of cell mutants with high thiokinase activities, TOK (EC 2.7.1.28), and fructose-1,6-
bisphosphatase activities, Fru-P2ase (EC 3.1.3.11). Cells growing on pyruvate, lactate, asparagine, and glutamine are additionally equipped
with high pyruvate carboxylase, PC (EC 6.4.1.1), and phosphoenolpyruvate carboxykinase, PEPCK (EC 4.1.1.32). Pyruvate, lactate,
asparagine, and glutamine are converted to oxaloacetate and, thereafter, by the PEP-carboxykinase to phosphoenolpyruvate. The active
fructose-1,6-bisphosphatase and inactive pyruvate kinase allow the reverse flow of carbon atoms in the glycolytic sequence to glucose 6-
phosphate, thereby, ensuring the supply of glycolytic intermediates which are essential as precursors for biosynthetic processes (nucleotides,
lipids, complex carbohydrates).
studies of mutant cells with distinct lesions in the respiratory chain, which drastically reduce
the glutaminolytic capacity, that the cells' total energy requirements could be met just by
doubling the glycolytic rate.1 °' '" Such mutants exhibit a high dependency on external
glucose, on CO2/HCO3 - , and on aspartate. Therefore, in proliferating cells, glutaminolysis
is dispensible for purposes of energy production, but not for anabolic processes like aspartate
production.102-1"
Thus it is apparent that glycolysis alone can provide tumor cells with sufficient energy
and metabolites for survival and growth. Reduction of glucose concentrations (5000 i.M to
50 p,M) at constant high glutamine concentrations (5000 11,M) resulted in a total block in
the formation of pyruvate and lactate from glucose. All lactate formed under these conditions
was derived from glutamine, and 90% of the energy requirement was met by glutaminolysis.
Under such conditions, carbohydrate carbons were only detectable in nucleotides, amino
acids, and in macromolecules like RNA, DNA, phospholipids, proteins, and complex car-
bohydrates.1 °' The concentration of glucose that induces half-maximal inhibition of glutamine
oxidation is in the same range as the concentration of glucose needed for half-maximal
aerobic lactate production. This observation suggests that glutamine oxidation is mainly
regulated by the energy production.98 This reciprocal regulation of glycolysis and glutam-
inolysis has been found in several in vitro animal cell systems, and no exception has been
observed regardless of time in culture, state of proliferation, or of transformation. Thus,
glutaminolysis as a feature of proliferating cells gives no further insights in the function of
carbohydrate metabolism in normal proliferating cells as compared to tumor cells.
C. Possible Function of Glutaminolysis and Aerobic Glycolysis in Tumor Cell Formation

Limitations on external glucose or its substitution with other hexoses resulted in a marked
reduction in lactate formation without a proportional decline in rate of proliferation." These
studies and others together argue against the hypothesis that for cell proliferation a high rate
of carbon flux from glucose to lactate is an essential requirement.'"- "" Although lactate
formation from glucose is strongly enhanced after serum stimulation of resting cells, and
although most tumor cell lines show a good correlation between aerobic glycolysis and
malignancy, the high flux through glycolysis seems not to be absolutely a prerequisite for
cell proliferation.
The same doubts exist as to the essential nature of glutaminolysis for cell proliferation.
It has been reported that serum stimulation enhances the lactate production from glutamine
and that glutaminase and NAD-dependent malic enzyme activities correlate with growth
rates and degrees of malignancy of several rat tumors.88-9" A correlation between glutamine-
and glutamate-dependent respiration in isolated tumor mitochondria, the activity of mito-
chondria! glutaminases, and the malignancy of hepatomas suggest that glutamine may be
an important respiratory fuel for tumor cells. It has been possible, however, to isolate cell
mutants with drastically reduced glutaminolytic activities which, nonetheless, grow at rates
comparable to the normal cells from which they are derived. These cells could be transformed
chemically and were oncogenic in nude mice.'02-1"8 Thus, in the last analysis, a high glu-
taminolytic activity is not essential for cell growth or tumor formation.
We have pointed out that a common feature of normal proliferating and tumor cells is
that they can rely on the energy produced either by glycolysis, if glutamine is limited, or
by glutaminolysis, if carbohydrates are limited. There are, however, remarkable differences
between normal proliferating and tumor cells. Normal proliferating cells show enhanced
rates of glycolysis and glutaminolysis only after serum stimulation to growth. "° Tumor cells
have a constitutively high rate of glycolysis and glutaminolysis. Furthermore, in normal
proliferating cells, the enhanced glycolytic enzyme activities and the glycolytic rate are
strongly depressed by high oxygen levels, whereas tumor cells are insensitive to the effect
of high concentrations of oxygen on glycolysis. "1-'14
Volume II 153
Pro line Glucose

•
Lipid
NADP
Glyceratdehyde 3-P litt Glycerol 3-P
NADPH2 Fatty acid Fatty acid
degradation synthesis
PEP Lactate
- pyrtoline - Scartaoxylate /1IF I /
Pyruvate CoA Acetate
Aspartate *eel Aspartate Oxoloacetate
Lactate Malate Citrate

OZ
Cortisol
succinate Isocitrate
tg"
1111114le.
GlutOnline 4.111. Glutamate Oxoglutarate
t
Aminooxyacetate
FIGURE 3. Diagrammatic representation of the influence of pyruvate oxidation on glutaminolysis, on lipid

metabolism, and NADPH regeneration. Some cultured cells and tumor cells use glutamine in preference to pyruvate
for energy production since impairment of pyruvate oxidation in normal proliferating cells is induced by superoxide
formed from oxygen. Superoxide preferentially inactivates pyruvate dehydrogenase and isocitrate dehydrogenase
but does not affect succinate dehydrogenase, resulting in a reduction of pyruvate oxidation by an enhanced glutamine
oxidation. Hence, glutamine is either converted to lactate and aspartate or to proline but not to citrate or isocitrate.
Proline is excreted to get rid of the surplus hydrogen from NADPH produced in the ribose 5-phosphate synthesis.
This mechanism allows an optimal ribose 5-phosphate synthesis. Under these conditions, no ATP is synthesized
by glycolysis or pyruvate oxidation, and addition of aminoxyacetate blocks the last possibility for energy production
and thus kills the proliferating cells (Figure IC). When the intracellular pyruvate concentrations are high, pyruvate
is oxidized to acetate and excreted under these conditions. The carbon of glutamine is not converted to aspartate
but to citrate. Therefore, the aspartate levels fall while the citrate and isocitrate pools are increasing. The low
pyruvate oxidation rate protects tumor cells from oxygen toxicity (Chapter 4) and guarantees an optimal acetyl-
CoA supply for lipid synthesis under variable nutrient supply.
Aminooxyacetate, a specific inhibitor of the aspartate aminotransferase reaction and,

therefore, of glutaminolysis, is lethal only for normal cells grown at high oxygen levels in
the presence of cortisol (Figure 3).15•"6 In normal proliferating cells, glycolytic enzyme
activities and the glycolytic rate are strongly reduced by high oxygen levels."' " When
such cells suffer an additional block in pyruvate oxidation after cortisol treatment, they have
to rely on glutaminolysis for energy production.15'"6 Addition of aminooxyacetate to normal
proliferating cells cultivated with high oxygen levels blocks the last possibility of these cells
for energy production and is thus lethal. Aminooxyacetate does not, however, effect cell
viability when normal proliferating cells are cultivated at low oxygen levels. Under these
conditions, there is a sufficiently high glycolytic rate in order to meet all energy requirements
for survival . "5 In contrast, if sufficient glucose is available, tumor cells are insensitive to
glutamine limitation since the high glycolytic rate is not depressed by high oxygen
levels.' 2.113.115
IV. PYRUVATE OXIDATION
A. Interaction of Pyruvate Oxidation and Glutaminolysis

As discussed above, many tumor cell types and certain cultured cell lines derive much
of their energy for proliferation from glutamine rather than pyruvate, even though pyruvate
may be present at high concentrations. We have seen that the rate of glutaminolysis is
inversely proportional to the sum of energy production from glycolysis and pyruvate oxi-
dation. Some cell lines and tumor cells actually use glutamine in preference to pyruvate, a
phenomenon which has led to the following hypotheses.' '5 .1 ' In the first, it was postulated
that a general defect in the respiratory chain in tumor cells might block pyruvate oxidation.
Intensive investigation, however, could not find any characteristic defect in mitochondria]
function of tumor cells.'" As a second hypothesis, it was proposed that the shuttle system
transferring reducing equivalents and carbohydrate carbons from the cytosol to the mito-
chondria might be impaired. Indeed, it could be demonstrated that the glycerol phosphate
shuttle was absent in several tumor cell lines and normal proliferating cell types, but the
capacities of the malate-aspartate and the malate-citrate shuttle should be sufficiently large
to allow a high pyruvate oxidation rate. 117 118120 As will be discussed below, there is some
evidence that during normal proliferation the aspartate level might be too small to allow an
adequate shuttle." Ku In such a case, the cytosolic hydrogen formed in the glycolytic gly-
ceraldehyde 3-phosphate dehydrogenase reaction must be transferred to pyruvate, in order
to excrete the surplus hydrogen in the form of lactate. Consistent with the idea of a shuttle
defect in tumor cells at limiting aspartate concentrations, experiments with the retro-virus-
induced tumor cell system indicated that manipulations leading to an enhanced supply of
precursors for aspartate like oxaloacetate, citrate, isocitrate, fumarate, and malate (see below)
reduced the excretion of lactate drastically and enhanced the pyruvate oxidation rate.'2 '.'24
Unfortunately, no data are available on the actual aspartate levels under these experimental
conditions.
A third hypothesis proposed that the low pyruvate oxidation rate might be due to the fact
that a special isoenzymic form of pyruvate dehydrogenase exists in tumor cells which is
activated by AMP.'''-'22 Recent metabolic experiments, however, indicate that in addition
to the pyruvate oxidation the conversion of acetyl-CoA to oxoglutarate is also reduced in
tumor cells. This means that fatty acids or acetoacetate, which are converted to acetyl-CoA
without the pyruvate dehydrogenase reaction, can be only slowly oxidized in normal pro-
liferating cells and not at all in tumor cells (Figure 3).97' Furthermore, pyruvate dehydro-
genase activity cannot be rate limiting since in several tumor cell lines with low thiokinase
activity acetate is excreted.'26 '" In contrast to this interpretation, systematic investigations
with adrenocortical cells showed that the decrease in pyruvate oxidation and the simultaneous
increase in glutamine respiration is caused by an inhibition of pyruvate dehydrogenase and
isocitrate dehydrogenase.' '" The same investigation, however, casts doubts on the tumor
specifity of the low pyruvate oxidation rate since the rate of decrease in the oxidation of
[14g-pyruvate relative to the increase of ["C]-glutamine correlates best with the in culture
time of the cells (Figure 3).'15.1 '" It seems that the gradual loss of pyruvate oxidation capacity
in culture correlates with continued exposure to the high levels of oxygen found in monolayer
cultures.
B. The Selective Advantage of Low Pyruvate Oxidation Capacity for Tumor Cells
I. Resistance Against High Oxygen Levels in Cell Culture
As discussed above, pyruvate oxidation is highly sensitive to oxygen. There is generally
agreement that this effect of oxygen is caused by the formation of superoxide radicals 0,-
during mitochondrial respiration.''' The highly toxic superoxide is converted by the super-
SOD
oxide dismutase (SOD) + +2H H2O, + 02) to hydrogen peroxide
(H2O,), which is further detoxified by the catalase reaction (2 H2O, —> 2 H2O + 02) or by
the glutathione peroxidase reaction (H2O, + 2 GSH —p2 H,0 + GSSG). The mitochondria]
glutathione peroxidase needs reduced glutathione and selenium as a cofactor. The oxidized
Volume II 155
glutathione is regenerated by NADPH + H+ and glutathione reductase. High concentrations

of superoxide radicals (O,) cause the peroxidation of membrane lipids leading to alterations
in the normal membrane function which can be lethal for the cell. The addition of substances
like tocopherols, selenous acid, ubiquinones, and antioxidants reduces the effects of super-
oxides and enhances the life time of cells in culture. '27- ' 29.222 Lower concentrations of
superoxide, which are not toxic for proliferating cells, reduce the activity of mitochondrial
NAD-dehydrogenase but do not effect the FAD-dependent succinate dehydrogenase, with
the consequence that pyruvate respiration decreases and glutamine respiration increases
(Figure 3)."5-' 16 Therefore, addition of substances reducing the effect of superoxide, such
as tocopherols, ubiquinones, selenium, and antioxidants, prevents the decrease in pyruvate
oxidation rate during cell culture. Such cells are highly dependent on the supply of tocopherol,
selenium, or antioxidants for pyruvate oxidation and cell growth. Permanent cell lines or
tumor cells with sufficiently high aerobic glycolytic or glutaminolytic capacities have a
selective advantage for survival and cell division in being independent of pyruvate oxidation
and mitochondrial respiration and, thereby, independent of tocopherol and selenium. Since
glycolysis produces no superoxide radicals, these permanent cell lines and tumor cells are
less sensitive to high levels of oxygen. Furthermore, the superoxide dismutase activities are
directly proportional to the superoxide production rate. '27 Hence, tumor cells should have
lower superoxide dismutase activities. Indeed, there are several reports that tumor cells have
low superoxide dismutase and catalase levels.128 Interestingly, the enhancement of superoxide
dismutase activity in tumor cells to the levels of normal cells causes the transformation
specific morphology to return to normal, thus indicating that superoxides modulate not only
pyruvate oxidation rates but also influence the cell morphology. 128
2. Optimal Supply of Acetyl-CoA for Lipid Synthesis

An advantage of the reduced acetyl-CoA oxidation appears to be the better supply of
acetyl-CoA for lipid synthesis. Thus, it has been reported that Morris hepatoma cells show
an impaired CO, formation from labeled acetate, but acetate incorporation into cholesterol
and fatty acids was normal or even enhanced as compared to normal liver. 1 " The de novo
lipid synthetic capacity of Rous sarcoma virus-transformed fibroblasts is high enough to
ensure a sufficient supply for cell growth.131 One would expect that in tumor cells an enhanced
de novo fatty acid synthesis might also affect the degradation of fatty acids. Indeed, tumor
cells incorporated 97% of labeled fatty acids into lipids, whereas normally proliferating cells
incorporated only 45% into lipids and metabolized the residual 55%.97 This is a remarkable
difference between the metabolism of tumor cells and normal proliferating cells, although
both cell types showed an enhanced synthetic capacity for fatty acid, cholesterol, and
phospholipids.132 One may conclude from these data that pyruvate oxidation and fatty acid
synthesis are regulated differently in tumor and normally proliferating cells.
In summary, it seems that a low pyruvate oxidation rate has nothing to do with cell
proliferation but is a metabolic strategy of permanent and tumor cell lines for survival under
extreme oxygen conditions and for conserving acetyl-CoA for lipid and cell membrane
synthesis. The acetyl-CoA conserving effect is especially important if tumor cells grow under
limited glucose supply where all pyruvate is derived from glutamine. Some tumor cells
oxidize acetyl-CoA so sparingly that with the increased pyruvate or fatty acid supply the
surplus acetyl moiety is excreted as acetate (Figure 3). 125,126
V. METABOLIC STRATEGY OF TUMOR CELLS FOR SURVIVAL UNDER

CONDITIONS OF EXTREME VARIABILITY IN THE CELLULAR
ENVIRONMENT AND NUTRIENT SUPPLY
From observations with cell mutants on one hand and glucose, glutamine, and oxygen
limitation experiments on the other, it has been recognized that tumor cell populations,
unlike normally proliferating cells, can survive extreme variabilities in their environ-
ment.26.105.1 I 1-114,134 137,153 They can survive either anaerobic conditions, if sufficient glucose
is available, or hypoxic or aerobic conditions, if either glutamine or glucose is supplied in
adequate amounts.234.235 In contrast to tumor cells, normal proliferating cells cannot survive
in the presence of oxygen with high glucose and low glutamine levels. Furthermore, the
growth rate, in contrast to normally proliferating cells, is less dependent on tocopherols,
selenous acid, preformed lipids, and serum factors.
It is generally accepted that highly malignant tumor cells grow under conditions of poor
vascularization.'34-13" According to the distance from the blood vessels, tumor cells are
starved either for oxygen, or glucose, or other nutrients. They are, however, always suf-
ficiently supplied with glutamine."' Therefore, the high glycolytic rate, unresponsive to
oxygen, and the simultaneous high glutaminolytic rate, both independent of serum growth
factors, are selective advantages enabling tumor cells to survive and to grow. This feature
is an essential metabolic prerequisite of tumor cells necessary in order to form metastases.
Such behavior is not necessary for normally proliferating cells growing in an organized
tissue with good vascularization.
VI. CELL PROLIFERATION
A. Role of Glycolysis and Glutaminolysis in Cell Proliferation

The carbohydrate limitation experiments have been a helpful tool for obtaining insight
into the role of glycolysis and glutaminolysis in the physiology of cell proliferation.
1. There is compelling evidence that flux through neither pathway is really "essential"
for cell proliferation. Rather, both present opportunities for a metabolic strategy fa-
vorable for survival and growth under extremes in oxygen or nutrient supply. 26 ' 105.106' " 1
114.134-137,153
2. Carbohydrate limitation experiments point to intermediates which are required for

proliferation. It is likely that such essential intermediates are synthesized preferentially
from carbohydrates or glutamine. It is to be expected that proliferating cells try to
ensure an optimal supply of those intermediates using modulations in the enzyme
activities responsible for their synthesis or degradation. The collection of possible
enzymes involved can be narrowed down by successive shifts from limiting carbo-
hydrate to excess carbohydrate supply.
3. Comparisons of metabolite levels which are strongly enhanced during excess carbo-
hydrate supply in nonproliferating-, proliferating-, and tumor cells indicate which
alterations in supply of metabolites or precursors are absolutely necessary for cell
division. Such intermediates would be good candidates for internal signals for preparing
the shift from the Go- into the G,-phase and further into the S-phase of cell cycle, and
enzymes involved in their synthesis or degradation are candidates as targets for growth
hormones and for oncogenic transformation. In the following section, such interme-
diates will be discussed (Table 2).
B. Intermediates Prerequisite for Proliferation

1. Aspartate
Investigations with cell mutants defective in the glutaminolytic pathway have demonstrate
that aspartate formation is the only essential function of glutaminolysis in cell proliferation.
Under conditions of limited carbohydrate supply, synthesis of aspartate is assured both from
glutamine and from the residual carbohydrates. At low glucose concentrations, glutamine
Volume II 157
is preferentially channeled into lactate and aspartate but not into citrate (Figure 3). 99-101
Aspartate is formed from oxaloacetate by a transamination step. Despite the fact that aspartate
is an essential precursor for the synthesis of purines and pyrimidines, its level remains
extremely low during proliferation. Aspartate is only increased when the cells in culture
become confluent.'" The suggestion that a decrease in the utilization of aspartate for
purine and pyrimidine synthesis leads to an accumulation of aspartate after confluence could
not be confirmed since the addition of preformed purines and pyrimidines has no measurable
effect on the aspartate levels. '°' Comparisons of flux measurements at high and low glucose
concentrations point to the acetyl-CoA supply from pyruvate as the main regulator of ox-
aloacetate availability for aspartate synthesis. If elevated acetyl-CoA levels result from
enhanced pyruvate oxidation, a higher consumption of oxaloacetate for citrate synthesis,
thereby, leads to decreased availability of oxaloacetate for aspartate production."'°' Besides
its function as a precursor, aspartate is also involved in the transfer of reducing equivalents
from cytosol to mitochondria, the so called malate-aspartate shuttle. Very low levels of
aspartate would disrupt this shuttle leading to an enhanced cytosolic NADH/NAD quotient.
The NADH/NAD ratio is high in proliferating cells with extremely low aspartate levels,
while the NADH/NAD ratio falls when cells stop proliferation and aspartate accumu-
iates.81-84.139.140 There is convincing evidence that a rising NADH/NAD ratio is one metabolic
84,139-142
signal inducing the progression from the Go- to the G,-phase of the cell cycle.81
Interestingly, transformation of embryonal cells with Rous sarcoma virus or Simian virus
40 leads to abnormalities in their malate aspartate shuttle. 23J' In summary, all enzymes
involved in the malate, oxaloacetate, and pyruvate metabolism are good candidates as targets
of regulation by growth hormones and by oncogenic viruses leading to a reduced dependency
on growth hormones.
2. Serine
Serine is formed from the glycolytic intermediate glycerate 3-phosphate and is an essential
precursor for glycine and for active methyl groups used in synthesis of purines, pyrimidines,
and the methylation of adenosyl homocysteine for methionine synthesis. Homocysteine
released from adenosyl homocysteine is removed by condensation with serine, thereby
forming cystathionine. Cystathionine is hydrolyzed to cysteine and homoserine. A high
serine synthetic capacity guarantees a high adenosyl methionine to adenosyl homocysteine
rate, a requirement for cell proliferation.58-63.'43-159 Serine is, furthermore, an essential pre-
cursor for ethanolamine, choline, sphingomyelin, i.e., phospholipid synthesis."'" Because
serine supplies all carbons necessary for hypoxanthine synthesis, it is possible to synthesize
hypoxanthine solely from glucose and nitrogen being supplied from glutamine (Figure 4). 55
The synthetic capacity from glycerate 3-phosphate is high enough in all proliferating cells
to meet the entire serine requirement.88-63'147-235 The only exceptions are lymphocytes and
other leukocytes which enjoy an assured constant serine supply from their environment in
the blood.59'143
At glucose concentrations under about 40 RM, human diploid fibroblasts stop cell division.
Addition of metabolites derived from serine (hypoxanthine, glycine, and thymidine) restores
cell proliferation to nearly normal rate.'" At 50 RA/ glucose, no hypoxanthine, glycine, or
thymidine are needed to ensure cell proliferation rates comparable to that with 5000 RM
glucose."' Interestingly, the life span of the cells is drastically reduced if they grow in 50
RM glucose or in 25 RM glucose supplemented with hypoxanthine, glycine, and thymidine. '°8
These data fit well with the thesis that a high glycolytic capacity is part of a metabolic
strategy of cells for survival of environmental stress produced by cell culture conditions or
poor vascularization in solid tumors.
Addition of preformed nucleotides to proliferating cells incubated with 50 RM radiolabeled
glucose reduced by 50% the incorporation of label into macromolecules, especially DNA
Glucose
ribose-PP Ribose 5-P 4 P-ribose-PP
Serine
Purine Pyrimidine
synthesis synthesis
PEP
lirPK
P-ribosylamine
A
/0...Malate Pyruvate
Succinate
MDH n ()rotate
Oxa loacetate
I
d.- Oxoglutarat Lactate
Glutamine ,..sti Glutamate

...,< P.Aspatate • Carbamyl
T •
NH 4 CO2
Lactate Aspat ate
FIGURE 4. Diagrammatic representation of the metabolic fate of glutamine and glucose as precursors for purine
and pyrimidine ribonucleotides. Glucose and glutamine are essential precursors for purine and pyrimidine nucleotide
synthesis. Glucose is primarily converted to P-ribose-PP and serine, which supply together all carbon and nitrogen
atoms necessary for purine synthesis. Glutamine is the donor of all carbon and nitrogen groups for pyrimidines
with the exception of the methyl groups of thymidine, which can be supplied by serine. The carbon atoms of
glutamine are converted to aspartate. Aspartate, carbamylphosphate, and P-ribose-PP are used for synthesis of
UMP. Therefore, under conditions of limited carbohydrate supply, most glucose carbon atoms are found in
nucleotides. RNA, and DNA, and most glutamine carbon is found as aspartate, lactate, pyrimidine nucleotides,
and lipids (Figure 3).
and RNA.'" Without preformed nucleotides, the incorporation of labeled serine into DNA
and RNA becomes strongly enhanced after serum stimulation of cell proliferation (Figures
IC, 3, and 4).60 One important function of the glycolytic pathway in proliferating cells is
the adjustment of serine production to the different demands made during the cell cycle and
to extremes of carbohydrate supply. The serine activation of pyruvate kinase points to
pyruvate kinase as the main control site in the regulation of serine synthesis from carbo-
hydrates."-" At low glucose concentrations (50 pLM) and, hence, low fructose-1,6-bis-
phosphate and P-ribose-PP levels, pyruvate kinase is functionally inactive. This is in accordance
with the data that no pyruvate and lactate is derived from glucose under these conditions
but only from glutamine (Figure 1C).76'" Instead, glucose is converted to serine and P-
ribose-PP for nucleotide synthesis (Figure 1C). Because no phosphoenolpyruvate is converted
to pyruvate, no ATP is synthesized in the glycolytic pathway, and no pyruvate is available
for pyruvate oxidation. Thus, the cellular ATP levels are low, and consequently the con-
centration of inorganic phosphate is elevated. This activates the phosphate-dependent glu-
taminase, and glutaminolysis rises yielding 5 ATP + 1 GTP per mole under aerobic or 1
GTP per mole under unaerobic conditions between glutamine and lactate. As a result, ATP
levels are restored, and proliferation can start. A rise in glucose concentration above 50 pLM
should increase not only the concentration of fructose 1,6-bisphosphate but also serine
Volume II 159
synthesis and levels. Pyruvate kinase becomes activated by serine, thus, the conversion of
glycerate 3-phosphate to pyruvate rises, the glycerate 3-phosphate concentration falls, and
serine synthesis slowed.
Proliferating cells prevent serine over-production in order to avoid the "methyl trap":
serine is split by the serine hydroxymethyltransferase reaction to glycine and N5.Ni"-meth-
ylene-tetrahydrofolate; N5,N10-methylene-FH4 is reversibly converted to N'0-formyl-FH4 for
purine and pyrimidine synthesis or irreversibly to N5-methyl-FF14. (45_152.15i 157 From this last
step, tetrahydrofolate can only be released following methionine synthesis from homocy-
steine. If the supply of active methyl groups from the serine pool is too high, the tetra-
hydrofolate is irreversibly channeled to N5-methyl-tetrahydrofolate, the methyl trap, and is
not further available for nucleic acid synthesis.I45 152 '155 157 It is conceivable, but not proven,
that the enhanced folic acid requirement and S-adenosyl methionine decarboxylase activity
in tumor cells are a consequence to the methyl trap of excessive serine synthetic capacity
(Figure 5). '47.'54,'58-'60,18'
3. Glycerol 3-Phosphate
Glycerol 3-phosphate levels regulate the phospholipid and triglyceride synthesis in
cells. 64-66 Unfortunately, no data for their levels and synthetic rates in nonproliferating,
normal proliferating, and tumor cells are available. However, one remarkable difference has
been recognized between normally proliferating and tumor cells with respect to glycerol 3-
phosphate availability for the triglyceride formation when cells are incubated with preformed
fatty acids. In normally proliferating cells, the glycerol 3-phosphate supply is equally small
whether cells are incubated at high or low glucose concentrations. In several tumor cell
lines, however, the supply with glycerol 3-phosphate is low only at glucose limitation but
unlimited at glucose excess." A likely explanation is that the high hexokinase and phos-
phofructokinase activities found in tumor cells ensure a high flux from glucose to glycerol
3-phosphate (Figures 1 and 3).23.31-'8
4. Fructose-1,6-Bisphosphate
Fructose-1,6-bisphosphate is not a precursor for any synthetic pathways, but it is a regulator
of several enzymes and metabolic pathways. In particular, it activates in millimolar con-
centrations its own synthesis by activation of phosphofructokinase and its own degradation
by activation of pyruvate kinase in proliferating and tumor cells.34- 38-53.74 In quiescent cells,
the levels of fructose-1,6-bisphosphate are too low to cause such effects. Fructose-1,6-
bisphosphate inhibits cytosolic malic dehydrogenase and, thereby, influences the malate-
aspartate shuttle.' It inhibits adenylo-succinate synthetase and, thereby, the formation of
adenosine monophosphate from inosine monophosphate. 162,163 Fructose-1,6-bisphosphate in
millimolar concentrations stimulates protein synthesis and inhibits the phosphorylation of
pyruvate kinase type M2 by a cyclic AMP-independent protein kinase or the pp60'rc kin-
ase.49-52.164 Moreover, fructose-1,6-bisphosphate accumulates in normally proliferating cells
and tumor cells at concentrations of 200 to 1000% compared to nonproliferating cells (Table
2)." At these concentrations, fructose-1,6-bisphosphate is a competitive inhibitor of 6-
phosphogluconate dehydrogenase, explaining the inverse relationship between the fructose-
1,6 bisphosphate levels and the flux through the oxidative pentose phosphate shunt.76-77.217
It is for this reason that under limited glucose supply, when fructose-1,6-bisphosphate levels
are drastically reduced, all the ribose 5-phosphate is generated via the oxidative pentose
phosphate shunt.76-77 Serum addition to cells in the resting phase (G„) of the cell cycle leads
to a five- to tenfold increase of the fructose-1,6-bisphosphate levels. 5.6.8-10,46.166.167,179,I80
Fructose-1,6-bisphosphate in millimolar concentrations or a high flux through the pentose
phosphate shunt favor (by a yet unknown mechanism) the protein synthesis. 133•164-'65 There-
fore, an enhanced fructose-1,6-bisphosphate level or a high flux through the oxidative pentose
TetrahydrofoliC acid (FH4)
Serine Glycine
NH3_ COOH N,H3

Serinhydroxy -
HO - CH2 C methyltransf erase HC — COOH
H H
N 5 NI° -Methylen FH4 NADPH • H

5 NI° Met h ylen -
4,
NADP ~ N
5 5 510 - Methylen FH4
NADP reductase
dehydrogenaseNADP1
1
Thyrndsne 411=MM N 5 N10 - Methenyt FH 4 N 5 Methylen FH4

Storage form of
FH4 and one-carbon
Cyclohydrolase units
Homocy s ne
inosine -Forrnyl FH4
411."Mil 510 Formyt FH4 Synthetase
FH 4 • For miat• • ATP 5- Adenosyl- ¤ te Methionlne FH4

methionine
Reversible Purone-Pyrrnidine -direction
of Tetrabydrofot.c metabolism Irreversible N5 510 Alethylen-FH 4
and Methionine-direction of
Tetrahydrofolic metabolism
FIGURE 5. Diagrammatic representation of the influence of serine biosynthetic rate on tetrahydrofolic acid
metabolism. Serine is synthesized from glycerate 3-phosphate and metabolized by the serine hydroxymethyltrans-
ferase reaction (EC 2.1.2.1) to glycine and is/5,M 0-methylene-tetrahydrofolate. N',N10-Methylene-tetrahydrofolic
acid is reversibly converted by the is/5,N10-methylene FH, dehydrogenase reaction to N'"-formyl FH, for purine and
pyrimidine synthesis. The majority of N5,N10-methylene FH, is channeled to N5-methyl FH, because the 1‘15 ,N11)-
methylene FH, reductase reaction is irreversible under cellular conditions. Particularly, when the supply of active
methyl groups by serine synthesis is strongly enhanced, tetrahydrofolic acid is irreversibly channeled to N5-methyl
FH,, the methyl trap, and is lost for further nucleic acid synthesis and cell proliferation. Free FH, can only be
released from N5-methyl FH, by the methionine synthetase reaction (EC 2.1.1.13). The methyl trapping of FH, is
prevented by two mechanisms: first, s-adenosylmethionine inhibits the N5,N10-methylene reductase and activates
the methionine synthetase; secondly, serine overproduction is prevented by the activation of pyruvate kinase type
M,.
phosphate shunt are conditions which may provide internal signals triggering the activation
of protein synthesis and, thus, the transit from the G„- to the G,- and S-phase of the cell
cycle.
5. P-Ribose-PP
P-ribose-PP is formed from ribose 5-P and ATP by the P-ribose-PP synthetase. The levels
and the availability of P-ribose-PP are determined by two factors: (1) the activity of P-ribose-
PP synthetase and the levels of ribose 5-P are directly related; and (2) the P-ribose-PP
synthetase is inhibited by various nucleotides and activated by phosphate; a decrease in the
levels of nucleotides and an increase in phosphate concentrations elevate P-ribose-PP syn-
thesis. 10-1'
' Ribose 5-phosphate is formed either from glucose 6-phosphate via the oxidative
pentose phosphate shunt, thereby, generating 2 mol NADPH per mol, or from fruc-
tose 6-phosphate and glyceraldehyde 3-phosphate via the non-oxidative pentose phosphate
shunt.' The decision which of the pathways is actually used in proliferating cells depends
on the quantity of carbohydrates available. At high glucose levels, 80% of the total ribose
is formed by the non-oxidative pentose phosphate shunt." Fibroblasts with a defect in the
enzyme glucose 6-phosphate dehydrogenase synthesize all ribose via the non-oxidative pen-
Volume II 161
tose phosphate shunt. At limiting glucose concentrations, all ribose 5-P is formed via the
oxidative pentose phosphate shunt, thereby, generating a maximum of NADPH." To ensure
a sufficiently high ribose 5-P production under these conditions, NADP is generated from
NADPH by the pyrroline-5-carboxylate reductase reaction. 1R2 This enzyme converts I-
pyrroline-5-carboxylate, formed from glutamate, to proline, which can be excreted to elim-
inate surplus hydrogen from NADPH (Figure 3). 99.'" An increased availability of P-ribose-
PP and a subsequently enhanced rate of the de novo synthesis of purine nucleotides has been
observed within the first hours after serum addition to lymphocytes or resting fibroblasts."-73
At high glucose concentrations, the levels of P-ribose-PP are 10-fold higher 1 hr after serum
stimulation. 14-1" High levels of P-ribose-PP, therefore, represent an early event induced by
mitogenic hormones. In tumor cells, frequently even higher levels (10- to 100-fold higher
than those of normal cells at high glucose levels) of P-ribose-PP are maintained continu-
ously. 169-172
P-ribose-PP is not only a precursor of purine and pyrimidine biosynthesis de novo but
also a precursor in the salvage pathway of purine nucleotide synthesis and in NAD-synthesis.
Hence, the fact of enhanced P-ribose-PP levels explains that shortly after addition of growth
hormones, the levels of purines, pyrimidines, and the sum of NADH plus NAD increase
(Table 2).81-84.1 " As discussed previously, there is evidence that the rise in the P-ribose-PP
levels and presumably of the pools of purines, pyrimidines, and the sum of NADH + NAD
is caused by a block in the flux from glycerate 3-phosphate to pyruvate. 49.50.54-55
C. Enzymes of Carbohydrate Metabolism Potentially Involved in Control of Normal

Cell Proliferation
In normally proliferating and tumor cells, the supply of the three metabolites aspartate,
serine, and P-ribose-PP is generally assured even under conditions of limiting carbohydrate
supply. Because all three metabolites are precursors for nucleic acid synthesis, we have
previously termed such cells "nucleigenic".54 The glycerol 3-phosphate supply, however,
is only guaranteed in tumor cells but not in normally proliferating cells.67 Hence, one may
conclude that glycerol 3-phosphate is not involved in the regulation of normal cell prolif-
eration. The levels of fructose-1,6-bisphosphate and NADPH are inversely regulated, and
both compounds stimulate protein synthesis. During onset of proliferation, the levels of
fructose-1,6-bisphosphate and P-ribose-PP are strongly enhanced; the changes in the levels
of glycerol 3-phosphate and serine are not known.5-'°•'74-'77•'79 As discussed above, we can
postulate that during onset of proliferation the conversion of glycerate 3-phosphate to pyruvate
is reduced but that the reduction is overcome by high fructose-1,6-bisphosphate and P-ribose-
PP levels.49.50,54 Because three enzymes are involved in the conversion of glycerate 3-
phosphate to pyruvate, one may postulate that pyruvate kinase, enolase, or phosphoglycer-
omutase are inactivated and that this inactivation may be reversed by fructose-1,6-bisphos-
phate and P-ribose-PP. Thus, glycolysis is only possible at high levels of all glycolytic
intermediates.'
Furthermore, one has to postulate that while the levels of aspartate are lowered during
the onset of proliferation, the supply of aspartate for synthetic processes is optimal. The
complexity of aspartate metabolism makes it difficult to define exactly which enzymes might
be altered during the onset of proliferation. The enzyme forming aspartate is the aspartate
aminotransferase. This enzyme transfers the nitrogen from glutamate to oxaloacetate, thereby,
generating oxoglutarate and aspartate. Flux measurements have given indications that the
main regulator of aspartate synthesis is the oxaloacetate availability. 1°°,101,115,116 In addition
to its precursor function for aspartate, oxaloacetate can be converted to citrate by condensation
with acetyl-CoA or to malate by reduction (Figures 3 and 4). Comparison of flux measure-
ments at high or low glucose concentrations points to the acetyl-CoA supply from pyruvate
as the main regulator of oxaloacetate availability for aspartate synthesis. High levels of
acetyl-CoA, from an enhanced pyruvate oxidation, lead to a higher consumption of oxal-

oacetate for citrate synthesis and, thereby, to a decreased availability of oxaloacetate for
aspartate production. In agreement with the postulated low aspartate levels are an enhanced
pyruvate oxidation rate and increased citrate levels found during onset of cell proliferation;
hence, one may assume that pyruvate dehydrogenase is activated by growth hor-
mones. ''''.1"'-'7918`) Most tumor cells, on the other hand, exhibit reduced pyruvate oxidation
rates; pyruvate is converted just to acetate.97.115.116,124 126 It is, therefore, conceivable that
other enzymes involved in pyruvate, oxaloacetate, or malate metabolism may be altered in
tumor cells as compared to normally proliferating cells.78.10 '"3 This possibility requires
further investigation.
D. Changes in the Function of Metabolic Enzymes may Trigger Cell Proliferation

Many investigators working on the mechanism of growth induction by growth hormones
are convinced that proliferation is induced by alterations in the function of (Na ,K+)-
ATPase.17 " This speculation appears very attractive because Na+ and K concentrations
influence many enzymes and proteins, and it would be easy to explain how so many enzymes
and pathways are altered during the onset of cell proliferation. To date, no clear experimental
data prove or disprove this concept. Two findings, however, make this speculation unlikely:
careful investigations have shown, that there are no differences in the equilibrium intracellular
concentrations of Na+ and K+ between normal and tumor cells;'".1 '5•255 in addition, it was
shown that protein synthesis is not increased by experimentally elevated intracellular Na+/
K' ratios either in normal nor in tumor cells.'`'
Normal proliferating cells have a sensible mechanism for budgeting energy and nu-
trient supply during the onset of proliferation. Cells starting cell division have several inter-
nal check and decision mechanisms to allow a stop of the cell cycle, thereby, pre-
venting a nutrient limitation which might be lethal for actively proliferating
1 "- ' 91 .2 '' Recent data show that growth hormones (EGF, PDGF,
insulin) bind to cell membrane receptors, thereby, activating protein kinases. 192 1" Such
protein kinases are unusual in phosphorylating proteins in the amino acid tyrosine.
Since phosphorylation in tyrosine is a relatively rare protein modification, its appearance in
a protein may be a direct indication for involvement in cellular growth control. More than
ten proteins are modified in phosphotyrosine following the addition of growth hormones.'"
There is good evidence that two of these proteins are pyruvate kinase type M, and enolase,
both of which are phosphorylated and inactivated during transformation by Rous sarcoma
virus.4".50.197 -'" This phosphorylation and inactivation is prevented by millimolar concen-
trations of P-ribose-PP or fructose-1 ,6-bisphosphate.".5"
Control over the flux from glycerate 3-phosphate to pyruvate makes possible a mechanism
that checks whether there is a sufficient supply of glucose and/or glutamine for the energy
requirements of the cell during proliferation. The signals for sufficient supply are high levels
of fructose-1 ,6-bisphosphate or NADPH, both of which in millimolar concentrations stim-
ulate protein synthesis and induce plasminogen activator secretion.133,164,165.199 At high glu-
cose levels, with both low and high glutamine concentrations, the inactivation of the flux
from glycerate 3-phosphate to pyruvate dams up fructose-1 ,6-bisphosphate to the millimolar
range, a signal for protein synthesis.'3"64-1 " Under conditions of low glucose with high
glutamine supply, the fructose-1,6-bisphosphate levels are low and the glycolytic flux is
decreased. Because of the low fructose-1 ,6-bisphosphate and high glutamine levels, however,
all glucose is channeled through the oxidative pentose phosphate shunt, thus, enhancing the
NADPH levels:6'77.99-10' This also constitutes a signal for protein synthesis.'13 " 5•2°' How-
ever, when both glucose and glutamine supply are low, neither fructose-1 ,6-bisphosphate
nor NADPH levels will rise, and no signal for elevated protein synthesis can re-
suit.133.168,189.190.204 Protein synthesis is believed to induce DNA synthesis because of Ap4A,
Volume II 163
which is produced as a by-product of aminoacyl tRNA deacylation and is believed to induce

DNA synthesis."1-203 This control represents the first internal check of whether the energy
supply justifies the activation of protein synthesis.
The second reaction of the cell upon stimulation is to make available sufficient precursors
for RNA and especially DNA synthesis in sufficient quantities. If either glucose or glutamine,
or both, are supplied in adequate amounts, an expansion of cellular nucleotide pools in par-
allel with the above-mentioned protein synthesis follows (Figure 4). 8" 83.133 153 '17" l2."" 2171-2"7
This expansion is also probably a result of the block in the flux from glycerate 3-phosphate
to pyruvate, thus, channeling carbohydrates to P-ribose-PP, serine, and aspartate.5"5
Furthermore, DNA-synthesis and nuclear protein ADP-ribosylation, necessary for mitosis,
can only proceed if sufficient NAD is available at low NAD levels.'" "2.191 Those conditions
were obtained when NADH/NAD ratios rose while the total NADH pools are being expanded.
As discussed above, such metabolic conditions develop when the malate-aspartate shuttle
is limited by low aspartate levels and, at same time, a high P-ribose-PP supply elevates
NAD-synthesis." •''' The supply of sufficient nicotinamide and NAD is, therefore, presum-
ably the last check and decision point before a commitment to cell division. This proliferation
mechanism with its built-in metabolic checks prevents a lethal effect of growth hormones
during oxygen or nutrient starvation.
VII. CELL TRANSFORMATION
A. Modified Control of Carbohydrate Metabolism Induced by pp60r"-Kinase

At first glance, it may seem curious that further developments in the study of carbohydrate
metabolism should benefit from the help of the virologist. Nonetheless, it is so. This is
because of the problem of finding tumor material together with truly equivalent normal
tissues for use as controls. The study of tumor-specific metabolic events requires a side-by-
side comparison of tumor and normal cells and tissues. We have seen above (Section III)
that in culture normal cells change, as can be especially well documented with respect to
glutaminolysis. Most insights elaborated by virologists come from experiments with avian
retroviruses, especially Rous sarcoma virus. This virus not only causes sarcomas in chickens
and several other species but also transforms embryonic chicken fibroblasts in cell culture.
Virologists have isolated temperature sensitive mutants able to infect cells, but unable to
transform them. Comparison of these mutants with the wild type virus revealed that only
one gene, the src-gene, is responsible for transformation.2°8 This gene codes for a phos-
phoprotein with a molecular weight of 60 kDa, which exhibits an unusual protein kinase
activity with specificity for tyrosine, the (v)iral pp601°'-s"-kinase. 2°9.2'2 A very slightly dif-
ferent protein kinase is also found endogenously in normal cells in much smaller amounts
and is called (c)ellular pp60'°'-s"-kinase.210•21 It is believed that Rous sarcoma virus picked
up a slightly modified cellular src-gene, leading to a tenfold higher level of gene expression
in the infected cell.'96.210 The existence of temperature sensitive mutants of Rous sarcoma
virus furnishes not only an easy supply of directly comparable low passage RSV-infected
transformed cells and normal, uninfected sister cultures, but also allows a kinetic approach
to study the metabolic events following onset of transformation, using temperature shift to
achieve a synchronous transformation.
The pp60m-s"- and pp60(c)-sr`-kinases phosphorylate the amino acid tyrosine in proteins,
a relatively rare protein modification. 50,195,196.212 The enhanced phosphotyrosine content of
total cell proteins after a temperature shift gives an unequivocal indication of which proteins
might be directly involved in transformation. The number of these phosphotyrosine-con-
taining proteins does not exceed 8-10.195, '" Also, the addition of growth hormones like
platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and multiplication-
stimulating activity (MSA) to quiescent cells enhances the phosphotyrosine content of some,
but not all of the proteins modified by Rous sarcoma virus-transformation.195-19°
Transformation with Rous sarcoma virus not only generates all internal signals triggering
cell division, thereby making the transformed cells independent from growth hormones (like
EGF and PDGF), but it also induces a variety of metabolic alterations in addition to those
prerequisite for cell proliferation; these may be helpful, however, for the formation of
metastases (see Chapter 5 in Volume I). These metabolic alterations are not specific for
Rous sarcoma virus transformed cells but represent a common characteristic of most tumor
cell lines derived from a variety of species, including man. These metabolic alterations are
enhanced aerobic glycolysis," glutaminolysis," '8'88-1' lipid synthetic capacity,67130'132
glucose uptake,' lowered pyruvate and acetyl-CoA oxidation rates,''-'- '26 and, finally, re-
sistance against high oxygen tension. 111-114.136.211 215 From the many enzymes involved in the
regulation of these metabolic pathways, none has been defined so far as essential for cell
proliferation and tumor formation. The Rous sarcoma virus system and other similar oncogene
containing retrovirus host systems seem to be necessary for deciding exactly which of the
proteins and enzyme modifications are absolutely necessary to inducing cell proliferation or
tumor formation. We have discussed above evidence that an unlimited supply with P-ribose-
PP and serine from glucose for purine, pyrimidine, and NAD synthesis is an internal signal
for triggering cell proliferation. The inactivation of pyruvate kinase seems to be involved
therein, because this enzyme appears to regulate the supply of these precursors. Using
temperature shift experiments with Rous sarcoma virus mutant-infected cells, we found an
inactivation of pyruvate kinase and enolase together with an enhanced phosphotyrosine
content at a time before DNA-synthesis occurs.'" Future experiments will have to determine
in flux measurements if, indeed, the enhanced phosphorylation and partial inactivation of
these enzymes really does channel carbohydrates to nucleic acids and to NAD. Furthermore,
the inactivation of enolase and pyruvate kinase type M, is counteracted by the enhanced
synthesis of these isoenzymes.°".'" Apparently, the inactivation of these isoenzymes leads
to an elevated synthesis as a host response, leading to increased activity, thus, establishing
an aerobic glycolysis.
We have presented evidence above for the possibility that the malate-aspartate metabolism
might furnish a further signal which triggers cell division. The aspartate metabolism is
directly linked to glutaminolysis, and, indeed, glutaminolysis is induced by transformation
of cells with Rous sarcoma virus or by treatment with EGF.10 '"6 Cell mutants which have
lost the growth response to EGF have also lost their glutaminolytic activity.' Because a
38 (36196) kDa protein is found to be more strongly phosphorylated in phosphotyrosine either
after EGF treatment or after transformation by Rous sarcoma virus infection, it is conceivable
that the phosphorylation of this 38 kDa protein is involved in the stimulation of glutami-
nolysis.'95.'" This speculation is consistent with the fact that this 38 kDa phosphoprotein
copurifies over several purification steps with another 38 kDa protein, namely the cytosolic
malic dehydrogenase.I83 The active c-MDH could be separated only by chromatography on
Blue Sepharose from the phosphorylated 38 kDa protein. Both proteins, active c-MDH and
phosphorylated 38 kDa protein, exhibit the same isoelectric point, pH 7.5, but they differ
partially in their peptide structure as revealed by proteolytic cleavage. As yet, the function
of the 38 kDa protein is not clear, but it is still a possible candidate for the regulation of
pathways linked to glutaminolysis such as pyruvate oxidation and the malate-aspartate
metabolism.
B. Chemical Carcinogenesis: Carbohydrate Metabolism in Hepatocytes, Preneoplastic

Hepatocytes, and Hepatocarcinoma
The best investigated tumor model from the viewpoint of carbohydrate metabolism has
been liver carcinogenesis. In contrast to Rous sarcoma virus induced transformation, where
Volume II 165
only one step seems necessary, liver tumor formation needs two steps for transformation.
The first step, the so called initiation step, leads to characteristic alterations in the enzyme
equipment of hepatocytes. 224-226 Such hepatocytes are termed putative preneoplastic hepa-
tocytes. There is some evidence that these preneoplastic hepatocytes are converted in a
second step to tumor cells.225 The preneoplastic cells are characterized by a selective change
in their carbohydrate enzyme equipment compared to normal liver cells. Glucose 6-phos-
phatase, pyruvate kinase type L, and serine dehydratase are lost, while the cytosolic PEP-
carboxykinase is reduced. 226.240 The activities of glucose 6-phosphate dehydrogenase, cy-
tochrome P-450 reductase, and glucuronyl transferase are strongly enhanced. 224 226 In contrast
to normal hepatocytes (Figure 6A), preneoplastic hepatocytes channel glucose to glycogen,
to glucuronate, and to ribose 5-phosphate, producing, thereby, excess NADPH.222-243 Because
pyruvate kinase type L is lost and the PEP-carboxykinase activity is reduced, phosphoen-
olpyruvate is dammed up to glycerate 3-phosphate and converted to serine (Figures 6A and
B). In the normal hepatocyte, serine is degraded to pyruvate by the serine dehydratase
reaction, but this enzyme is lost in preneoplastic cells.2" Therefore, serine can only be used
for glutathione synthesis or nucleic acid synthesis (Figures 6A and C).241.242 A high precursor
supply for glucuronate, for NADPH, and for glutathione synthesis, together with a high
cytochrome P-450 activity and -y-glutamyl-transferase activity are essential for optimal de-
toxification of several chemical substances, including carcinogens.' 233 It seems conceivable
that the cells in the preneoplastic islet are selected liver cells with an optimized metabolism
for detoxification of carcinogens and other chemicals. This metabolism, however, also
resembles that of proliferating cells and, in consequence, explains why preneoplastic islets
have a higher proliferating potency than normal liver cells. 224.225 There are, however, two
important differences between the metabolic patterns of preneoplastic cells and normal
embryonal cells which are stimulated to growth by hormones: the ribose 5-P once formed
is not quantitatively converted to P-ribose-PP or serine since fructose-1,6-bisphosphatase is
still active (Figures 6C and B). Therefore, ribose 5-phosphate is recycled in the pentose
phosphate shunt to CO2 and NADPH or by a still active cytosolic PEP-carboxykinase from
PEP to oxaloacetate and aspartate (Figure 6C). If both the fructose-I ,6-bisphosphatase and
the cytosolic PEP-carboxykinase are lost, all carbohydrates must be channeled to P-ribose-
PP, serine, and glycerol 3-phosphate, and a metabolic pattern like that in transformed
fibroblasts is established (Figures I A, C, and 3).54 This could explain why the loss of
fructose-1,6-bisphosphatase and cytosolic PEP-carboxykinase are markers for the selection
of stable hepatoma cell lines in culture.243 In contrast to transformed fibroblasts, where the
distinct metabolic pattern observed in proliferating cells is caused by an inactivation of
pyruvate kinase type M2 and enolase, in hepatomas, the phenotype results from a total loss
of pyruvate kinase type L. Therefore, the particular metabolic situation seems to be more
relevant for the triggering of cell proliferation and tumor formation than the way in which
it comes about.
C. Onc-Genes, Multistep Transformation, and Malignancy from the Viewpoint of

Carbohydrate Metabolism
Transformation with Rous sarcoma virus is induced by a single onc-gene product, the
pp60`"-kinase. This process is, therefore, called one step carcinogenesis. After the detection
of the pp6Os`c-kinase, several other onc-gene products (abl, yes, fps, fes) were found to have
a phosphotyrosine specific protein kinase activity. 2"-212 Other onc-genes, however, without
detectable protein kinase activity (myc, large T, Ad1A, ras, B lym-1) have also been
isolated. 244.249 Onc-genes coding for a tyrosine kinase activity (src, yes, abl, fps, fes) are
transforming in one step, while onc-genes coding for proteins without protein kinase activity
(myc, large T, Ad1A, ras, B lym-1) often seem to require at least the expression of two
separate onc-genes in one cell for transformation. 246-249 One onc-gene or similar elements
Hepatocytes
Glycogen Glucose
6- Phosphatase
Glucose * Glucose - 6 - P Glucose

Gtuco
kinase 11.111.1.111110 Fatty acids
Triglycerid -
NADP
synthesis
_Lat.Fructose - 6 - P 1_,NADPH2
PFK
Fructose-1-6 -P FDPase
Serine
4 4
PEP
PEPCK
Nr ia
r
Malate
NADP*
NADPH2
Oxaloacetate
SDH I Pyruvate
4
Lactate Pyruvate Acetyl - CoA
Amino acids
FIGURE 6. Diagrammatic representation of alterations in the carbohydrate enzyme activities of hepatocytes during
chemical hepatocarcinogenesis. (A) Normal hepatocytes. High pyruvate carboxylase, phosphoenolpyruvate car-
boxykinase, fructose-1,6-bisphosphatase, and glucose 6-phosphatase activities together with low type L-pyruvate
kinase, phosphofructokinase, and glucokinase activities ensure a high gluconeogenetic capacity. A high NADP-
dependent malic enzyme activity and a high fatty acid synthesis capacity allow a high fatty acid synthetic rate from
amino acids and lactate. At high blood glucose levels, glucose is either converted to glycogen, fatty acids, or
amino acids. At low blood glucose levels, glucose is released from glycogen or is synthesized from lactate or
amino acids by gluconeogenesis. The main function of carbohydrate metabolism in liver cells is the homeostasis
of glucose and fatty acid output. (B) Preneopla.stic hepatocytes. Pyruvate kinase type L. serine dehydratase (EC
4.2.1.13). and glucose 6-phosphatase (EC 3.1.3.10) activities are sharply reduced. The activities of glucose 6-
phosphate dehydrogenase (EC 1.1.1.49), cytochrome P,„ and glutamyltransferase (EC 2.3.2.2) are strongly en-
hanced. Carbohydrates are either trapped into glycogen or are used for serine and cysteine synthesis as precursors
for cystathionine or for NADPH production by recycling through the oxidative pentose phosphate shunt. Because
glucose 6-phosphate dehydrogenase is associated with cytochrome P,„ NADPH can be used immediately for the
hydroxylation of chemical carcinogens. In preneoplastic hepatocytes, carbohydrates are channeled to precursors
essential for detoxification of chemical substances. It is conceivable that preneoplastic hepatocytes are selected cell
mutants with the optimal capabilities for detoxification of chemical substances. (C) Hepatomas. Hexokinase and
phosphofructokinase type L activities are strongly enhanced, while the amount of pyruvate kinase type L can be
either low or greatly elevated. The total loss of fructose-I ,6-bisphosphatase and cytosolic phosphoenolpyruvate
carboxykinase is a selective marker for stable hepatoma cell lines. NADP-dependent malic enzyme is reduced,
while the NAD-dependent enzyme is enhanced together with the capacity to convert glutamine to lactate (glutam-
inolysis). The stored glycogen and the ,i-glutamyl transferase activities are in most instances lost. Pyruvate is
converted to phospholipids or excreted as acetate. All carbohydrates are channeled to nucleic acid and phospholipid
synthesis, and a carbohydrate metabolic pattern identical to that in RSV-transformed fibroblasts is constructed.
Volume 11 167
Preneoplastic Neon tocyte
Glycogen GLucuronic acid R - Glucuronic

acid
R - OH
Glucose ummonelli. Glucose-6-P
NADP
Cytochrom
P450
110.NADPH2
I
Fructose --6.-P R-H Cancerogenes
E
Ribose b -P
Fructose 6p P-ribose-PP
Cysteine
R - S -G
Serine Glycine GSH R -Cysteine
Gluta mine
PEP 4- — — — R- CH3
Oxotoocetate
Lactate try Pyruvat imL Acetyl - CoA R CH2 COOH

Amino acids
FIGURE 6B
Hepatom Hepatocarcinom ?
Hyaluronic ac id
Glucose mwtmmiltt Glucose - 6 - P
00,P- Ribose- PP T Purine

synthesis
Fructose-6-P
4
Fructose -1,6 -P
Fatty acids tiel
Phospho -
Lipids
Glycine
Serine
Methyl- FH4
Thymidine
PEP Methylation
Acetate Acetyl-CoA DNA

Aspartate
RNA
Protein
Lactate 4 Pyruvate OxaLoacetate 4 GLutamine
FIGURE 6C
(myc, large T, Ad I A protein, B lym-1) seem to be responsible for the establishment of

immortalized or permanent cells, and a second one-gene (ras, sis, middle T) is required for
the full expression of the one-gene phenotype.' 25' In agreement with these data are ex-
periments with chemical carcinogens which induce immortalization of diploid fibroblasts.
These cells can be ultimately transformed following transfection with the normal human c-
Ha-ras 1 - gene.252 From metabolic data, it is conceivable that enhanced glycolysis, reduced
pyruvate oxidation, and resistance against high oxygen tension are linked to the immortal-
ization of tumor cells since these features have been reported for permanent cell lines and
tumor cell lines after extensive cultivation of primary cells in vitro.'.115.116,229 Furthermore,
the main characteristics of transformation are reduced serum or growth hormone require-
ment.21"-250.25 ' This could be due to the production of growth hormones by the transformed
cells (transforming growth factor, TGF; sarcoma growth factor, SGF; or p28`'` (related to
PDGF)) or by expression of a higher tyrosine kinase activity, which is presumably involved
in the transmission of the growth signals from the cell membrane into the cells. 192 195.221.250.251
The essential metabolic changes induced by growth hormones are a channeling of carbo-
hydrates to nucleic acids and phospholipids, together with a high NADH/NAD ratio (Table
2, Chapter 6). Therefore, from the metabolic viewpoint, transformation should be charac-
terized by a constitutive channeling of carbohydrates to nucleic acids and a permanent high
NADH/NAD ratio.54.8'
The in vivo malignancy of a tumor correlates mainly with the degree of aerobic glycol-
ysis,2-2' the rate of glutaminolysis," 9I-" and phospholipid synthesis"' as well as the capacity
for synthesizing nucleotides' and the reduced growth factor requirement.'s0-25 ' It is generally
accepted that highly malignant tumor cells grow under conditions of poor vasculariza-
tion."4."7 According to the distance from the blood vessels, tumor cells are starved either
for oxygen, glucose, or other nutrients, but they are well supplied with glutamine. '3' There-
fore, the high glycolytic rate, unresponsive to oxygen tension, and simultaneous high glu-
taminolytic rate are selective advantages enabling tumor cells to survive and to grow (Chapter
5). Therefore, although cell mutants lacking glutaminolysis can form tumors, they show an
extremely low proliferation rate in the host."' Likewise, mutant cells defective in glycolytic
enzymes can be tumorigenic but exhibit also a lowered proliferation potency in the host.
Hence, the amounts of the marker enzymes for aerobic glycolysis, such as pyruvate kinase
type M, and enolase, correlate with the malignancy of tumors.47-48-'97.256
VIII. PERSPECTIVES
To establish the new concept given in this article, there are some postulates that still have
to be proven. Furthermore, it has to be shown that all the changes in metabolism reported
from many different experiments under many different conditions are relevant for prolif-
eration when reduced to a single system.
To measure the true activities of enzymes in the cell and their changes during proliferation,
the commonly applied measurements of enzyme assays are unsuitable; instead, the carbon
fluxes through the corresponding enzymes or series of them represent the true enzyme
activities in metabolism. The most important fluxes that have to be measured can be listed
as follows:
1. The carbon flux from dihydroxyacetone to pyruvate, lactate, and alanine define the
activity of the lower part of glycolysis, especially pyruvate kinase.
2. The flux from dihydroxyacetone to aspartate — when pyruvate kinase activity is low
or the mitochondrial pyruvate transport is inhibited — shows the flux through phos-
phoenolpyruvate carboxykinase in the inverse direction.
3. The simultaneously measured flux from dihydroxyacetone to serine, glycerol, P-ribose-
Volume II 169
PP nucleic acids, etc. should be inversely proportional to the flux through the lower
part of glycolysis.
4. To produce high fructose-I ,6-bisphosphate levels, the incubations should be performed
with high glucose levels; for low fructose-1,6-bisphosphate concentrations, fructose
should replace glucose in the incubation medium.
As important as these flux measurements will be the search for the signals and mechanisms
that cause a change in these fluxes during proliferation. In addition to the flux measurements,
concentration shifts which are an essential part of the new concept must be determined.
Determinations of these pools should include the intermediates of glycolysis, pentose phos-
phate metabolism, and lipid metabolism, amino acids, nucleotides, and nucleic acids. All
these measurements must be done in normal quiescent cells in comparison to cells in early
GI and late G, phase as well as in cells infected by temperature sensitive mutant tumor
viruses before and after temperature shift and in cells transfected with different onc-genes.
The most important question, however, is that of the mechanism by which the isoenzyme
patterns in transformed cells are modified. This makes necessary measurements on the
expression of the corresponding mRNAs and on the signals regulating the levels of these
mRNAs and their translation products. 258 .259
ACKNOWLEDGMENTS
We would like to thank Dr. R. R. Friis for many helpful discussions and for critically
reading the manuscript. We are grateful to Dr. W. Schoner for his interest and support.
Financial support by the Bundesministerium fiir Forschung and Technologie (CMT 32),
by DFG (Scho 139/15-5), and by the Justus Liebig-Universitat Giessen (Preis der Justus
Liebig-Universitat 1981, Dr. E. Eigenbrodt) is gratefully acknowledged.
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Volume II 179
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Pharmacol. , 31, 1989, 1982.
Taylor & Francis
Volume II 181
Chapter 7
INSULIN BINDING AND METABOLISM BY HEPATOCYTES IN PRIMARY

CULTURE
N. Kalant
TABLE OF CONTENTS
I. Introduction 182
II. The Nature of Binding of Insulin to Cultured Hepatocytes 182

A. Binding 182
B. Dissociation 183
C. Relation of Binding to Stimulation of Glycogenesis 185
III. Degradation of Insulin 185

A. Degradation of Insulin by Lysosomal Enzymes 187
B. Fate of Intralysosomal Insulin 187
C. Susceptibility of Receptor-Bound Insulin to Cytosolic Insulin Degrading
Enzymes 188
D. Effect of Chloroquine on Insulin Degradation 188
IV. Effects of Exposure to Insulin 191

A. Down-Regulation of Binding 192
B. Effect on Insulin Degradation 193
C. Effect on Insulin-Stimulated Glycogenesis 195
V. Discussion and Conclusions 197
Acknowledgments 198
References 198
I. INTRODUCTION
In the past decade there has been an increasing use of primary cell cultures to study
biochemical and biological characteristics of hepatocytes. This cell preparation offers a
number of advantages over freshly isolated hepatocytes and long-term cultures: freshly
isolated hepatocytes suffer variable damage to their plasma membrane during the process
of isolation, leading to a decrease in hormone binding' and to leakage of enzymes into the
incubation medium;' culture for a short period permits repair of this damage. Furthermore
because these cells survive for a number of days in culture, it is possible to carry out certain
types of study which are not feasible in short-term incubations of freshly isolated hepatocytes.
On the other hand, cells in primary culture retain normal hepatocyte morphology and major
liver-specific functions such as albumin synthesis, gluconeogenesis, urea cycle activity, and
hormone-specific responses, whereas many of these characteristics are lost in established,
replicating cell lines, derived usually from hepatomas.
Liver cell preparations have been extensively used to study the mechanism of action of
insulin. It is generally accepted that the first step in this action is the binding of insulin to
specific receptors on the plasma membrane of the cells; this is followed by internalization
of the insulin complexed to its receptor. These concepts have led to three areas of research:
(1) the nature and metabolism of the receptor itself, (2) the metabolic fate of the insulin
molecule, and (3) the steps by which the binding process leads to the biological effects of
insulin. Though these questions are clearly interrelated, it is the last which is most directly
concerned with the mechanism of action of insulin. The specific sequence of events following
binding to the membrane receptors is in doubt: one hypothesis is that such binding causes
the release of a "second messenger" which brings about the biological effects of insulin,
another is that following binding and internalization either intact insulin or a major fragment
derived from it is directly responsible for the biological effects.3.4
We have used hepatocytes in primary culture to examine some aspects of these questions;
the results support the view that internalized insulin is responsible for at least one major
effect of insulin, stimulation of glycogen synthesis.
II. THE NATURE OF BINDING OF INSULIN TO CULTURED HEPATOCYTES"
A. Binding
The binding or association of insulin to a variety of intact cells, including hepatocytes,
adipocytes, monocytes, and erythrocytes has been thoroughly investigated; for the most part
the results have been interpreted in the context of binding to specific cell surface receptors.*
The binding process gives rise to a curvilinear Scatchard plot which has been interpreted as
evidence of negative cooperativity of bindings or as evidence of two types of receptors.6
Although there is considerable evidence favoring the latter view, extraction and purification
of receptors has provided only one class of molecule. The problem is compounded by the
fact that after interacting with the cell surface receptor, insulin is internalized:7 9 since there
are insulin binding sites on intracellular organelles such as the Golgi, endoplasmic reticulum,
and nucleus (for review see Reference 10) any measurement of insulin binding to whole
cells must include the insulin which has been bound intracellularly. In an attempt to clarify
* The term "receptor" has a specific connotation — that of a molecular species whose function is to recognize
the ligand and react with it as a first step in a sequence of events which leads to the biological effect of the
ligand — and therefore, should be restricted to cell surface sites which first "see" the hormone. The general
term "binding sites" is more appropriate to include both the cell surface receptors and those intracellular sites
which also complex with the ligand.
Volume 11 183
CELLS PLASMA MEMBRANE

0
0 0.3
Z .°
0 0
0 ti 0
0. 0.2
Z CD
E
D `•••. A
cei 0.1
Z c •
0
30 60 90 120 30 60 90 120
• A MINUTES
0 24 48 72 0 24 48 72
o HOURS
FIGURE 1. Effect of temperature on specific binding of Plhodoinsulin to cultured hepatocytes and hepatocyte
plasma membrane. Cells (1.5 x 106) were incubated with 1.7 x 10 - ' 6 M insulin in I me medium; purified plasma
membrane (26 to 33 pig) was incubated with 8.3 x 10 - " M insulin in 1.0 me medium. 0, 4°; A, 22°; and •.
37°.
these findings we have compared the binding of insulin to whole cells and to the isolated
plasma membrane."
The time course of insulin binding to cultured hepatocytes and to hepatocyte plasma
membrane is quite similar (Figure 1). As the binding temperature is increased from 4 to 37°
the amount bound decreases, by over 90% in the case of cells and over 80% for the membrane.
Equilibrium binding to whole cells gives a curvilinear Scatchard plot which can be resolved
into two components differing in their binding capacity and in their apparent affinity for
insulin. However, membrane binding gives a rectilinear Scatchard plot with an apparent
affinity very similar to that of the "high affinity" sites of whole cells (Figure 2).
B. Dissociation
Dissociation of insulin bound to cultured cells is biphasic with slow and fast components;
the rapidly dissociating component accounts for about 30% of the bound insulin. Insulin
bound to isolated plasma membrane dissociates with a single-rate constant which is very
similar to that of the rapid component of whole cells (Figure 3). Well over 90% of the
insulin which dissociates from the plasma membrane or from cells is precipitable by 10%
trichloroacetic acid and can bind to fresh membrane.
These results suggest that cultured hepatocytes, like other cell types, contain two classes
of binding sites, one of high apparent affinity and low capacity and the other of low apparent
affinity and high capacity. It would be anticipated that dissociation would then show a large
rapidly dissociating component and a smaller slowly dissociating component. However, as
pointed out by Kahn et al. ,6 this is inconsistent with the fact that the rapidly dissociating
component is in fact the smaller of the two. This, together with the observations that by
both association and dissociation plasma membrane contains only one class of binding sites,
its apparent affinity is similar to that of the "high affinity" cell sites, and the dissociation
rate is very similar to that of the rapidly dissociating cell sites, suggests that the "high
0.8
CULTURED HEPATOCYTE PLASMA
1.5
HEPATOCYTES MEMBRANE
\.
0.6
—4,
•,41
1.0
K a = 6.7 x 108 M-1
•
•
\
Ka = 6.7 x 108 M-1
•
0.5 •
0.2 •
K o = 8.4 x tog b4-1
•
0 \ t
0.2 0.4 0.6 0.8 1 5 10 15
B B
FIGURE 2. Scatchard plot of insulin binding to cultured hepatocytes and hepatocyte plasma membrane at 22°.
Cells (1.5 x 10°) were incubated with 1.7 x 10- "' Mr5 thodoinsulin and unlabeled insulin up to 1.3 x 10 "
M; membrane (16 ii.g) was incubated at an insulin concentration of 8.3 x 10- " M FIllodoinsulin, together with
unlabeled insulin. Maximum binding capacities (ng/mg protein) were 0.42 and 1.28 for high and low affinity
binding to cells and 14.4 for membrane.
affinity" sites of the intact cells are identical to the membrane sites and that the "low
affinity" sites are not on the plasma membrane.
This possibility has been examined in two ways: ( I ) cell cultures were incubated with
insulin at 4°, then plasma membrane and cytosolic fractions were prepared. The plasmalemma
contained 30 to 32% of the total cell-bound insulin, an amount very similar to the component
found to dissociate rapidly from the cells. (2) The effect of chloroquine on binding char-
acteristics was determined. This agent, which blocks lysosomal enzyme action by elevating
the intralysosomal pH, leads to intracellular accumulation of insulin. '2 When cells were
incubated with insulin in the presence of chloroquine (Table 1), the apparent affinities of
insulin binding were not altered but the amount of insulin bound to the "low affinity" sites
was substantially increased. Thus intracellular insulin appears to be bound to the "low
affinity" sites.
The binding and dissociation characteristics of whole cells and plasmalemma, the similarity
of the amounts of insulin bound to "high affinity" sites and to the plasmalemma fraction,
and the effects of chloroquine on "low affinity" binding all lead to the conclusion that
insulin that appears to bind to sites with a low apparent affinity is in fact intracellular,
presumably bound to cell organelles such as the Golgi membrane and the nucleus.
Estimates of affinity based on Scatchard and other related plots are based on the assumption
of free access of the ligand to the macromolecular binding site. If the "low affinity" sites
are intracellular this assumption is not valid, since extracellular insulin would have to be
internalized and released from its complex with the membrane receptor to be available for
interaction with intracellular sites. Under these circumstances, assessment of affinity would
require knowledge of the concentration of free intracellular insulin. Similarly the apparently
Volume Ii 185
100
80
60
I NS ULINBOUND (%)
40
20
60 120 60 120
MINUTES
FIGURE 3. Dissociation at 22° of VIliodoinsulin bound to cultured cells. (A) Cells were incubated at 4° for
various periods, as shown, with an insulin concentration of 1.7 x 10 -10 M. For comparison, dissociation from
isolated membrane incubated under identical conditions is shown (7). (B) Same as A, but cells were incubated
at 22°.
slow phase of dissociation may be due to the necessity for insulin released from intracellular
sites to be externalized to the medium. Posner et al. have shown that the binding affinity
of isolated Golgi particles is approximately the same as that of plasma membrane." If this
is characteristic of all intracellular binding, the concept of "low affinity" binding sites in
hepatocytes may be incorrect.
C. Relation of Binding to Stimulation of Glycogenesis

Insulin stimulates glycogenesis in cultured hepatocytes in a dose-dependent manner. The
dose-response curve coincides with the concentration-dependent curve of occupancy of the
"low affinity" binding sites, with the maximum glycogenic response attained when about
50% of such sites are occupied (Figure 4).14 If, as suggested above, the "low affinity" sites
are intracellular then the stimulation of glycogenesis would appear to be a function of
intracellular insulin.
III. DEGRADATION OF INSULIN15
There are three known enzymatic mechanisms of insulin degradation: lysosomal enzymes,
glutathione-insulin transhydrogenase (GIT), and cytosolic insulin protease. '6.17 Preparations
of liver plasma membrane have some insulin-degrading capacity which has been described
as similar to GIT, '8 but its K„, and susceptibility to N-ethylmaleimide are similar to those
of insulin protease. '9 In addition, there has been disagreement as to whether the degrading
activity and the binding function of plasma membrane are separate2O•21 or linked.22 Regardless
Table 1
EFFECT OF CHLOROQUINE ON
EQUILIBRIUM BINDING OF INSULIN TO
CULTURED HEPATOCYTES
K.
(M-') (ng/mg protein)
Without chloroquine 7.4 x 10° 0.39

7.0 x 107 1.30
With chloroquine 2.5 x 10° 0.27
6.4 x 107 2.90
Note: Culture dishes were divided into two groups, and bind-
ing studies were carried out at 22° for 90 min with
1.7 x 10 '°M rthodoinsulin and total insulin con-
centrations ranging from 1.7 x 10 10 to 3.3 x 10 -°
M. Chloroquine was added simultaneously with the
insulin to provide a concentration of 0.2 mM. Shown
are the mean values from two experiments, with tri-
plicate plates at each of eight insulin concentrations.
From Ozaki, S. and Kalant, N., Endocrinology, 112, 381,

1983. With permission.
100
o—
RECE PTOROCCU PANCY•
z
75 -
Z
▪ E
0
0 .E
50 g
100 V-
E
0
▪ o
0
z
25 50
)7-
0 0
10-b° 10-9 10-8
INSULIN CONCENTRATION
(M)
FIGURE 4. Relation of insulin concentration to occupancy of high and low affinity binding sites and to stimulation
of [14C] glucose incorporation into glycogen, by cultured rat hepatocytes. Occupancy was calculated from maximum
binding values estimated from Scatchard plots.
of its exact nature the amount of degrading activity on plasma membrane is small, it varies
with the method of preparing the membrane fraction, and it can readily be explained by
contamination of the membrane with other cell components.23 It is therefore likely that the
plasma membrane is not the physiological site of insulin degradation.16
Volume II 187
It has already been noted that freshly isolated hepatocytes frequently leak insulin-degrading
enzymes, so that with suspensions of such cells insulin may be degraded extracellularly.
Cultured cells do not show this enzyme loss to the medium:"•24 when cells in primary culture
were incubated for 2 hr in fresh medium and the medium then transferred to clean tubes
and incubated with insulin for 2 hr, 96 to 100% of the insulin remained precipitable by
trichloroacetic acid and could bind to preparations of placental membrane. Thus degradation
in these preparations is an intracellular process.
There is currently much interest in and controversy about the role of lysosomes in insulin
metabolism. On the one hand there are several lines of evidence implicating lysosomes as
the site of degradation: (1) serum LDL is bound to surface receptors, endocytosed, and then
degraded in lysosomes;25-27 since insulin also undergoes binding, internalization and asso-
ciation with lysosome-like bodies's it is reasonable to assume that degradation occurs within
the lysosomes, and (2) lysosomotropic agents such as chloroquine decrease the rate of insulin
degradation." On the other hand there are observations which argue against the involvement
of lysosomes. (1) There is morphological evidence that insulin administered in vivo rather
than to isolated hepatocytes becomes associated after internalization with the Golgi vesicles,
rather than with lysosomes;3° insulin incubated with isolated organelles is bound both to
Golgi vesicles and to a class of vesicles which are similar to but distinct from lysosomes.3'
(2) There are several enzymatic pathways of insulin degradation as noted above;'6' '7 since
inhibitors of the nonlysosomal pathways are more effective inhibitors of insulin degradation
than are lysosomal inhibitors,32 the lysosomal pathway does not appear to be of major
importance:6 Because of these conflicting results the role of lysosomes requires further
examination.
A. Degradation of Insulin by Lysosomal Enzymes

If receptor-bound insulin is internalized by endocytosis and the endocytic vesicles fuse
with lysosomes, the insulin-receptor complex would be introduced into a milieu whose pH
is 5.5 to 6.0; the complex would be expected to undergo rapid dissociation at this pH. To
determine if the complex is a suitable substrate for lysosomal hydrolases, free insulin and
insulin bound to solubilized receptor were incubated with broken lysosome preparations at
pH 7.6. The receptor-bound insulin was not degraded as rapidly as free insulin and ^-,75%
of the degradation observed could be accounted for by dissociation of the complex and
degradation of the resulting free insulin (Figure 5). When the pH was lowered to 5.5, the
rate of degradation of free insulin increased about fourfold; however, at this pH the receptor-
complex dissociated immediately to an equilibrium level at which 75% of the insulin was
in the free form. (Figure 6).
B. Fate of Intralysosomal Insulin

These results show that any insulin-receptor complex within the lysosomes at their normal
pH would be rapidly dissociated and the free insulin would be subject to degradation. To
determine the extent of such degradation, lysosomes were loaded with insulin at pH 7.6 and
the pH was then adjusted to 5.5. Loading was accomplished by incubation of intact lysosomes
with [125I1 insulin at 37° for 30 min; specific uptake of insulin was 70 ± 20 pg/mg protein.
When the loaded lysosomes were disrupted and the lysosomal membrane pelleted, the insulin
was recovered (82 to 86%) in the supernatant, largely in an intact form (-80%). Similarly,
under conditions for dissociation, recovery of insulin was >99% after 60 min (Figure 7)
and >85% of the released insulin was intact. Under the same conditions lysosomal membrane
showed no specific binding and during dissociation released only 11% of the insulin that
had been bound nonspecifically. These findings indicated that insulin taken up by intact
lysosomes is not bound to the membrane and presumably is, therefore, intralysosomal. When
the pH of loaded lysosomes were abruptly changed to 5.5 the associated insulin was released
p rotein )
0.3
INSU LINDEGRADATION(ng /mg
0.2
0.1
30 60 90 120
MINUTES
FIGURE 5. Degradation of insulin and insulin-receptor complex by broken lysosomes.

[ 251]Insulin (0) or [1251]insulin-receptor complex (A) (3.4 x 10 - " M) was incubated at
pH 7.6 with 300 lig of lysosomes lysed by sonication. Degradation was assessed by solubility
in cold 10% trichloroacetic acid. Degradation of insulin-receptor complex by lysed lysosomes
was estimated (El) on the basis of the observed rate of degradation of unbound insulin and
the rate of dissociation of the complex under the same incubation conditions.
very rapidly, with recovery >99% within 5 min; >85% of this was intact. Thus free insulin,
within lysosomes at pH 5.5 would be released from the lysosomes much more rapidly than
it could be degraded.
C. Susceptibility of Receptor-Bound Insulin to Cytosolic Insulin Degrading Enzymes

The evidence that internalized insulin is not degraded in the lysosomes implies that
degradation occurs through the action of the cytosolic enzymes. Since insulin is internalized
bound to the plasma membrane receptor, it is pertinent to know if insulin in that form is a
substrate for the enzymes. Free and receptor-bound [125] insulin were incubated with
cytosolic preparations; the degradation of the bound insulin was ^-,60% slower than that of
free insulin and the rate could be accounted for by assuming that the complex dissociated
and the free insulin was then degraded (Figure 8). Further, when excess unlabeled insulin
was added to the incubations, degradation of free labeled insulin was decreased, as anticipated
from competitive inhibition, while degradation of bound labeled insulin was increased, due
to competitive inhibition of reassociation of label with the receptor.
D. Effect of Chloroquine on Insulin Degradation

As described above, chloroquine increases the amount of insulin bound to "low affinity"
sites in the cell. It also decreases the rate of degradation, though its effectiveness in this
regard is variable (inhibition of degradation is 60 to 75% in cultures of chick29 and fetal
rat32 hepatocytes and of hepatoma cells" but it is minimal in cultures of adult rat cells24).
It can be inferred from these two effects that insulin bound to the "low affinity" sites is
Volume II 189
100
• 100 •
• • • •
0
50 50
pH 7.6 pH 7.0
0 0
0 5 10 15 0 5 10 15
U 100
•
~• 100 •
•
•
•
0
•
0 50 50
0 0 0
INSULINBOUND
C 0
•
pH 6.5 pH 6.0
o.
0 0
0 5 10 15 0 5 10 15
100 •
•
\•
•
50
pH 5.5
0
0 0
0
0 5 10 15
MINUTES
FIGURE 6. Effect of pH on degradation of [1251]insulin by lysed lysosomes and on dis-

sociation of insulin-receptor complex. For degradation studies [125I]insulin (4.7 x 10 - " M)
was incubated with broken lysosomes in Tris-HC1 buffer at pH shown; the amount of un-
degraded material remaining was measured as label insoluble in cold 10% trichloroacetic
acid. For dissociation studies [125I]insulin-receptor complex was prepared in 4 me of buffer,
pH 7.6 at 0°; the pH was then adjusted by rapid (30 sec) addition of 1 N HC1, and incubation
was carried out at 37°.
the substrate for degradation. The relationship of binding to degradation and the effect of
chloroquine on this were, therefore, examined to test this possibility.
When cells are incubated with increasing concentrations of insulin there is a curvilinear
relationship between the amount of insulin bound at equilibrium and the amount of insulin
100
50
25
•
\•
io \•
•
D
0 5
co
pH 5.5
• I
0 10 20 30 40 50 60
MINUTES
FIGURE 7. Insulin dissociation from lysosomes. Lysosomes were labeled by incubation

for 15 min with [ 1251]insulin at 37° in buffer, then washed three times at 4° to remove
unbound insulin and resuspended in fresh buffer. Loss of insulin was studied at 37°, pH
7.6 and pH 5.5.
degraded, analogous to the curvilinear Scatchard plot: at low levels of binding, when binding
is predominantly to "high affinity" sites, degradation increases modestly with increasing
binding; over the range in which binding is predominantly to "low affinity" sites degradation
increased markedly with increased binding (Figure 9). Chloroquine decreased degradation
in relation to the amount of insulin bound to the "low affinity" sites. These results strongly
suggest that insulin bound to such sites is indeed the preferred substrate for the intracellular
process of degradation. A similar conclusion was arrived at by Caro and Amatruda35 on the
basis of the similarity between the Kd value of "low affinity" binding sites and the K„, for
insulin degradation, in freshly isolated hepatocytes. Failure of others to observe a significant
effect of chloroquine on degradation2436 may have been due to the use of low concentrations
of insulin when the binding would be primarily to the "high affinity" sites.
Taken together these results indicate that degradation is inhibited by a lysosomotropic
agent, but free insulin within the lysosomes would be rapidly released without undergoing
significant degradation. This paradox is resolved by the observation that if the insulin-
Volume II 191
70
(p g /mg cytosolic p rote in/ 30 min )

1-INSULINDEGRADATION 60 CONTROL
50 • INSULIN ADDED
40
30
20
125
10
INSULIN INSULIN- INSULIN-

RECEPTOR RECEPTOR
CALCULATED
FIGURE 8. Cytosolic degradation of ['-5I]iodoinsulin and insulin-receptor complex, alone and

in the presence of unlabeled insulin. Labeled insulin or insulin-receptor complex (4.5 x 10 "
M) was incubated at 37° in buffer containing 2 mg cytosolic protein with or without 40 lig of
unlabeled insulin. Insulin degradation was measured by solubility in 10% cold trichloroacetic
acid. Estimates were made of the degradation of insulin-receptor complex in the presence and
absence of unlabeled insulin, on the basis of the measured rate of dissociation of insulin-receptor
complex and the rate of degradation of free insulin.
receptor complex is internalized and transferred to lysosomes, the complex would be im-
mediately dissociated, with release of insulin into the extralysosomal milieu; here the free
hormone might bind to intracellular sites such as the Golgi or nucleus, or be degraded by
cytosolic enzymes for which it is the preferred substrate. This interpretation is consonant
with the fact that N-ethylmaleimide and bacitracin, which do not inhibit lysosomal proteases,
are nevertheless potent inhibitors of insulin degradation.32 Lysosomes would thus be an
essential intermediary in the degradation process, by virtue of their low pH rather than of
their proteolytic enzymes. This is in agreement with and suggests a mechanism for the
observations of Varandani et al.,37 that intralysosomal "processing" of insulin is essential
for its activity but that degradation of insulin is an extralysosomal function. Chloroquine by
raising the lysosomal pH inhibits complex dissociation; this causes an intracellular accu-
mulation of complexed insulin, which would appear to be bound to "low affinity" sites.
At the same time this decreases the availability of free insulin for degradation and for
biological effects so that stimulation of glycogenesis is inhibited,33.34 and it diminishes the
rate of recycling of receptor to the plasma membrane.38
IV. EFFECTS OF EXPOSURE TO INSULIN"
There is an inverse relationship between the plasma insulin concentration and the insulin-
binding capacity of cells, a phenomenon referred to as down-regulation of binding. This has
been observed in patients with noninsulin-dependent diabetes or with obesity who have
10
INSULINDEGRADE D (ng /mg protein / 90 m in )

8
0
0.5 1.0 1.5
INSULIN BOUND (ng/mg protein)
FIGURE 9. Relationship between insulin degradation and specific insulin binding to

cells in the presence (0) and absence (11) of chloroquine (0.2 mM). Cultured hepatocytes
were incubated for 90 min at 22° with [125I]iodoinsulin at concentrations from 1.7 x
10-10 to 3.3 x 10 -s M. Degraded insulin in the medium was measured by solubility in
10% trichloroacetic acid.
hyperinsulinemia, and it has been suggested that the decreased binding may be responsible
for the decreased insulin responsiveness which is characteristic of such patients. Down-
regulation has been demonstrated in vitro with cultured rat hepatocytes"' and rat hepatoma
cells.' In the latter it is accompanied by a decrease in insulin-stimulated amino acid transport,
attributed to postreceptor changes rather than to the decreased binding per se.43 However,
the effect of insulin exposure on other biological actions of insulin and on the metabolism
of insulin itself, have not been clarified. Since there are species differences in insulin binding
and processing by hepatocytes" a comparison of human and rat hepatocytes may throw some
light on the significance of down-regulation in noninsulin-dependent diabetes.
A. Down-Regulation of Binding
The characteristics of equilibrium binding of insulin to cultured human hepatocytes are
very similar to those of rat cells giving a curvilinear Scatchard plot which can be resolved
into two apparent affinity classes. The rates of dissociation of bound insulin are also similar.
Volume II 193
100
•
INSULIN BOU ND ( % of contro l)
75
50
25
tI 1
0 1 1
io-9 10-8
INSULIN CONCENTRATION (M)
FIGURE 10. Down-regulation of insulin binding by prior exposure to insulin for 24 hr. Cultured
human (A) and rat (0) hepatocytes were incubated with increasing concentrations of insulin as
shown, then binding was measured with 1.7 x 10-10 M [12511 insulin for 90 min at 22°. Values
are means of triplicate dishes. Open symbols show mean ± SE of five experiments each in
triplicate for human (A) and rat (0) hepatocytes; difference between means is significant at p
< 0.005.
On exposure to insulin overnight followed by assay of binding at a low insulin concen-

tration both species of hepatocyte responded by a decrease in binding. The response increased
as a function of the insulin concentration to which the cells were exposed. The maximum
response of the human and rat cells was of the same magnitude but the human cells reached
the maximum at a lower concentration of insulin (Figure 10). This increased sensitivity of
the human cells was also evident from equilibrium binding studies over a range of insulin
concentrations: the maximum binding capacity of rat cells fell by ,-50% while that of human
cells fell '80%. This difference was explained by the Scatchard analysis: rat cells showed
a decrease in the capacity of both classes of binding site while the human cells showed no
decrease in the "high affinity" sites but complete loss of "low affinity" sites (Figure 11).
In neither case were the apparent affinities altered.
B. Effect on Insulin Degradation

The rate of insulin degradation by control and insulin-exposed cells was assessed by two
criteria: loss of ability to bind to placental membrane and solubility in trichloroacetic acid.
The former is the more sensitive criterion, being evident earlier and increasing more rapidly
than the latter. By both criteria insulin-exposed cells degraded insulin more rapidly than
control cells (Figure 12). Furthermore, the 2600 g supernatant of cell homogenates prepared
from insulin-exposed cells also degraded insulin more rapidly than that of control cells
(Figure 13).
As described earlier, in intact control cells there was a curvilinear relationship between
B B
FIGURE 11.. Effect of prior exposure to insulin on Scatchard plot of specific binding to cultured human and rat
hepatocytes. Cells were exposed to 2 x 10-9 M insulin for 24 hr, washed free of medium then incubated for 90
min with PIliodoinsulin and increasing concentrations of unlabeled insulin. Control cells (0); insulin-exposed
cells (0).
0.4
BINDING ASSAY
INSULINDEGRADATION
0.3
(pmoles/mg protein)
0.2
0.1
I
1 II
5 10 15 30 60 30 60 120
MINUTES
FIGURE 12. Effect of insulin exposure on degradation of insulin by rat hepatocytes. Cells were cultured for 24
hr with 2 x 10-9 M insulin, washed, then incubated at 37° with iodoinsulin. Degraded insulin in the medium was
measured by solubility in 10% cold trichloroacetic acid and by loss of ability to bind to placental membrane.
Control cells (111); insulin-exposed cells (0). Difference from control cells significant at p < 0.01 (5*) and at p
< 0.001 (***).
Volume 11 195
0.4
(p mo le s /mg p rotein )
IN SU LINDEGRADED
0.3
0.2 O
•
0.1
0
I I I
30 60 90 120
MINUTES
FIGURE 13. Degradation of insulin by 2600 g supernatant of control (0) and insulin-exposed (0)
hepatocytes. The supernatant was incubated with ['"I]iodoinsulin (1.7 x 10 - '°M); degradation was assessed
by solubility in cold 10% trichloroacetic acid. The difference between the regression lines is significant at
p < 0.005.
the rate of degradation and the extent of insulin binding. Down-regulated human cells had
only that component of the curve corresponding to binding to the "high affinity" sites, and
for this component there was increased degradation in relation to the amount bound (Figure
14). Rat cells also showed increased degradation in relation to binding, particularly in the
range where "low affinity" binding predominated. However, in both cases since total binding
was diminished, the amount of insulin degraded was decreased in relation to insulin
concentration.
C. Effect on Insulin-Stimulated Glycogenesis

Human cells are somewhat more responsive than rat cells to the glycogenic action of
insulin, reaching their maximum response at 250 µU/rn€ while rat cells required 750 to
1500 RU/mt (Figure 15). Prior exposure to insulin completely abolished the response of
human cells to insulin and reduced the maximum response of rat cells by about 50%. These
effects coincide with the abolition in human cells and the reduction in rat cells of "low
affinity" binding following insulin exposure.
These results demonstrate that prolonged exposure to insulin leads to a decrease in binding
capacity, to an increase in the cytosolic insulin degrading activity and to an inhibition of
the glycogenic action of insulin. If, as suggested above, the "low affinity" binding sites
are intracellular, then the major response of human hepatocytes is an inhibition of inter-
nalization of the insulin-receptor complex, leading to loss of intracellular ("low affinity")
binding and of the glycogenic response. Rat hepatocytes show a reduction in internalization
with the consequences noted. In both species there is an increase in insulin degradation in
relation to the amount bound, but this is balanced to some extent by the decrease in the rate
of internalization and thus of availability of insulin for degradation, so that the absolute rates
of degradation are not greatly altered.
(ng/mg protein/ 90 m in)

INSULINDEGRADED
0.5 1
INSULIN BOUND (ng/mg protein)
FIGURE 14. Relation between specific insulin binding and insulin degradation by control and insulin-exposed
(2 x 10' M insulin for 24 hr) cultured hepatocytes. Cells were washed free of medium, then incubated for 90
min at 22° with labeled insulin and increasing concentrations of unlabeled insulin. Degraded insulin was measured
by solubility in 10% cold trichloroacetic acid. Control (0), insulin-exposed (111).
HUMAN RAT
200
GLYCOGENSYNTHESIS
I
( % of control)
100
.\
0
0 250 750 0 250 750
INSULIN (pU/m1)
FIGURE 15. Insulin stimulation of rClglucose incorporation into glycogen in control and insulin-exposed (2
x 10-9 M insulin for 24 hr) hepatocytes. Control cells, q ; insulin-exposed cells, M.
Volume II 197
R DEGRADATION
INSULIN PRODUCTS
INSULIN C)
li
1 t
R —.- DEGRADATION PROTEASE
INSULIN , INSULIN ?
'1?
80
4
GOLGI
FIGURE 16.. Intracellular fate of insulin. R = receptor; IR = insulin-receptor complex; and ? = interaction
with unknown sites, producing biological effects. Effects of exposure to insulin are indicated numerically: I =
decrease in membrane receptor; 2 = inhibition of internalization, and 3 = increase in insulin protease activity.
Chloroquine inhibits the intralysosomal dissociation of IR, leading to an increase in the concentration of intracellular
IR and decreased degradation in relation to this concentration.
V. DISCUSSION AND CONCLUSIONS
Binding to recognition sites or receptors on the cell surface is the first step in a sequence
of events that leads to the biological actions of insulin. Much is now known about the
receptors but very little is known with certainty about the events subsequent to receptor-
binding; electron microscopic autoradiography has provided evidence that insulin is inter-
nalized but the location of intracellular insulin is in question"." and its fate cannot be
determined by such techniques. At the present time, other, less direct techniques must be
used to study these questions, and the interpretation of the results must be largely inferrential.
Within this limitation the observations described above led to several conclusions: (1) that
the binding sites of apparently low affinity are intracellular, (2) that the glycogenic action
of insulin is related to the occupancy of these sites by insulin, (3) that the role of lysosomes
in the intracellular disposal of insulin is to split the insulin-receptor complex after its inter-
nalization, while the actual degradation is carried out by cytosolic insulin protease, and (4)
prolonged exposure to insulin leads to a decrease in cell surface binding, to inhibition of
internalization and to an increase in the rate of degradation of the insulin which does become
internalized.
These concepts may be integrated into the schema shown in Figure 16, which emphasizes
the importance of the intracellular events in determining the response to insulin. The discovery
of the cell surface receptors initially led to the view that the magnitude of the biological
response to insulin was determined by the availability of such receptors. When it became
apparent that "postreceptor events" might also influence these responses, it was postulated
that the interaction of the hormone with its receptor generated a "second messenger" which
brought about the metabolic effects of insulin. This has been termed the receptor-transducer
model of insulin action.3 In support of this hypothesis it has been found that a peptide able
to mimic some of the major insulin effects is released from plasma membrane by insulin.45
Furthermore, insulin receptor antibodies, which compete with insulin for binding sites on
the receptor, mimic many of the effects of insulin; this suggests that receptor occupancy by
either antibody or insulin triggers the biological actions characteristic of insulin and thus
supports the receptor-transducer hypothesis. However, the effect of antibody is only about
50% of the maximum effect produced by insulin' so this mechanism cannot completely
explain insulin action.
The evidence, both direct"-'O and indirect,"2 that insulin is internalized and becomes
associated with various intracellular organelles suggested the possibility that insulin itself
or a product derived from it is directly responsible for at least some of the biological actions
of insulin; this may be termed the receptor-internalization model.' The present results are
best explained by this model, offering indirect evidence of the role of internalization in
explaining the apparent heterogeneity of binding affinity, the effects of exposure to insulin,
and glycogen stimulation in hepatocytes. This view is in agreement with the results of
experiments with hepatoma cells' and with a line of cultured kidney cells,' both lacking
plasma membrane receptors for insulin; insulin bound to the B chain of ricin was internalized
via the ricin receptor and the intracellular insulin hybrid was found to stimulate amino acid
transport" and glycogenesis.' The concept that internalized insulin is responsible for one
or more of the biological actions of insulin is not necessarily incompatible with the finding
that insulin releases a peptidic second messenger from the plasma membrane," since this
action of insulin could occur at the inner surface of the membrane, after internalization.
While this discussion has been concerned with events in hepatocytes, a similar situation
has been described for adipocytes. Thus insulin is internalized and becomes associated with
intracellular vesicles, including lysosomes; some of the lysosomal-associated insulin is re-
leased in an intact form, some is degraded by lysosomal action and some undergoes extra-
lysosomal degradation." Adipocytes and hepatocytes may differ with regard to the relative
contributions of these pathways to insulin metabolism, just as there are differences in their
receptors, as manifested in affinities and specificities for labeled insulins.49 However, in
both cases, and presumably in other target cells as well, the complexity of intracellular
processing of insulin and its importance to the biological actions of insulin are becoming
clearer.
ACKNOWLEDGMENTS
The work described here was carried out with Dr. Shiro Ozaki; we are grateful for the
collaboration of Dr. N. Fukushima, Dr. H. Maekubo, and Dr. B. Mitmaker, and for the
technical assistance of M. Cohen-Khallas. Supported by a grant from the Medical Research
Council of Canada.
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43. Heaton, J. H. and Gelehrter, T. D., J. Biol. Chem., 256, 12257, 1981.
44. Bonnvie-Nielsen, V., Polansky, K. S., Jaspan, J. J., Rubenstein, A. N., Schwartz, T. W., and Tager,
H. S., Proc. Natl. Acad. Sci. U.S.A., 79, 2167, 1982.
45. Larner, J., J. Cyclic Nucleotide Res., 8, 289, 1982.
46. Van Obberghen, E., Spooner, P. M., Kahn, C. R., Chernick, S. S., Garrison, M. M., Karlsson, F.
A., and Grunfeld, C., Nature (London), 280, 500, 1979.
47. Roth, R. A., Maddux, B. A., Wong, K. Y., Iwamoto, Y., and Goldfine, I. D., J. Biol. Chem., 256,
5350, 1981.
48. Kono, T., Recent Prog. Horm. Res., 39, 519, 1983.
49. Podlecki, D. A., Frank, B. H., Kao, M., Horikoshi, H., Freidenberg, G., Marshall, S., Ciarajdi,
T., and Olefsky, J. M., Diabetes, 32, 697, 1983.
Taylor & Francis
Volume II 201
INDEX
A Aldolase-triosephosphate isomerase-glyceraldehyde-
3-phosphate dehydrogenase-phosphoglycerate
kinase system, 23
Abiquinones, 155
Alpha adrenergic receptors, 66
Absorption
Alpha subunits
carbohydrates, 68
of calmodulin, 37
fructose, 96-97
of phosphorylase kinase, 36
Acetaldehyde, 71
Amino acids, see also specific amino acids
Acetate, 73
in GSD, 57
Acetyl-CoA, 78, 106, 115, 161
in substrate, 62
oxidation of, 155, 164
Aminooxyacetate, 153
supply of, 157
Ammonium and ethanol, 78
Acetyl-CoA carboxylase, 44, 116
AMP, see Adenosine-5'-phosphate
Acomys russatus (golden spiny mice), 109
Amylase, 68
Actin, 2-4, 10, 22, 23
Amylo-1,6-glucosidase deficiency, 56
cytoskeletal, 11-15, 25
Anaerobic glycolysis, 16
depolymerization of, 14
Angiotensin 11, 38
double immunofluorescent studies of, 13
Antioxidants, 155
myosin interaction with, 8, 25
Aspartate, 156-157, 163, 168
proteins associated with, 15
for synthetic processes, 161
Actin-tropomyosin-troponin, 20
Atherosclerosis, 119-120, 123
Activity ratio, 37
ATP, see Adenosine-5'-triphosphate
Actomyosin ATPase, 23
ATPase, see Adenosine-5'-triphosphatase
Acute ethanol administration, 84-86
Axonal transport, 11
Adenosine diphosphate (ADP), 24, 77, 150
Axoplasmic matrix, 11
Adenosine diphosphate (ADP)-ribosylation, 163
Adenosine-5'-phosphate (AMP), 22
ethanol and, 78 B
Adenosine-5'-triphosphatase (ATPase), 113
Adenosine-5'-triphosphate (ATP), 18, 23, 24, 53
Backup metabolic systems, 53
ethanol and, 74, 77, 78, 82
Beta adrenergic receptors, 37
glutaminolysis, 150
Beta subunits
glycolysis, 146
of calmodulin, 37
sucrose and, 124
of phosphorylase kinase, 36
Adenosine-5'-triphosphate (ATP)-citrate lyase, 44
Biceps femoris, 16
Adenosine-5'-triphosphate (ATP)-magnesium, 41
Binding
Adenosine-5'-triphosphate (ATP)-magnesium-de-
down-regulation of, 191
pendent phosphatase, 45
insulin, 195, 197
Adenosyl homocysteine, 157
insulin to cultured hepatocytes, 182-185
Adenosyl methionine, 157
Blood ethanol, 83
Adenylate cyclase, 34, 38
circadian rhythms and, 70
Adenylo-succinate synthetase, 159
Bound forms of enzymes, 23
Adipose tissue, 34, 108
Bovine psoas muscle, 15
fructose and, 111-112
Brain
sucrose and, 111-112
fetal calf, 13, 15
ADP, see Adenosine diphosphate hexokinase in, 18
Adrenalin, 18, 74
rat, 11
Aerobic glycolysis, 142-143, 150-152, 164, 168
cell transformation and, 164-168
Affinity of triosephosphate isomerase, 11 C
Alanine, 149, 168
ethanol and, 80 Cafeteria diet, 112, 118
Alcohol, see Ethanol Calcineurin, 37, 43
Alcohol dehydrogenase, 71 Calcium, 4, 14, 20, 22, 24, 26
Alcoholism, see also Ethanol, 80-81, 88 binding and, 8
ALD, see Aldolase glucose-6-P and, 74
Aldolase (ALD) regulation of metabolism, 36, 37, 39, 43
fructose and, 103 Calmodulin, 36-38, 43
isoenzyme forms of, 20 Calmodulin-binding protein calcineurin, 37
in liver, 99 Calmodulin-dependent kinase, 40
Calmodulin-independent kinase, 39 D
Carbohydrate limitation, 156
Carcinogenesis, 168
chemical, 164-165 Debrancher myopathy development mechanisms, 57
one step, 165 Deficiencies
Cardiac glycogen synthase kinase, 41 amylo-1,6-glucosidase, 56
Cardiac muscle, 23 glucose-6-phosphatase, 54
Catalase, 154 phosphorylase kinase, 38
Catalytic expression, 20-24 Delta subunits
Catecholamine, 34, 35, 38 of calmodulin, 37, 38
ethanol and, 74-75 of phosphorylase kinase, 36
Cell culture oxygen, 154-155 Dental caries, 124
Cells, see also specific types 2-Deoxyglucose, 13, 23, 25
cycle of, 157, 159 Dephosphorylation of phosphoprotein phosphatase,
epithelial, 23 45
eukaryotic, 2, 14 Depolymerization of actin, 14
nonmuscle, 11-15, 25 Detoxification, 165
proliferation of, 142-143, 156-163 DHAP, see Dihydroxyacetone phosphate
red, see Red blood cells Diabetes
transformation of, 163-168 ethanol and, 73, 81
tumor, see Tumor cells fructose and, 108
Channeling, 23 sucrose and, 118-119, 123
Chemical carcinogenesis, 164-165, 158 type II, 120
Chloroquine, 184, 188-191 Digestion of carbohydrate, 68
Choline, 157 Dihydroxyacetone, 168
Chromium and insulin response, 119 ethanol and, 80
Chronic alcoholism, see also Ethanol, 80-81, 88 Dihydroxyacetone phosphate (DHAP), 99
Circadian rhythms, 70 Diploid fibroblasts, 157
Citrate, 22, 77, 161 DNA, 158
ethanol and, 78 synthesis of, 162
Citric acid cycle, 76 Double immunofluorescent studies, 13
Clathrin, 11 Down-regulation
Computer simulation studies, 22 of binding, 191
of insulin, 192-193
Contracting muscle, 37-38
Control parameters of enzymes, 20
Copper and insulin response, 119
Cori cycle, see also Glucose-lactate cycle, 34, 55,
E
68
reversal of normal, 55 EDTA, 80
Cornstarch, 60-61 Effectors of binding, 18-20
tolerance for, 64 EGF, see Epidermal growth factor
Corticosterone, 110 EGTA, 41
Cortisol, 153 Electrical stimulation, 15, 36, 38
Creatine kinase, 11 Electron microscopy, 4, 25
Cyclic AMP, 34, 37, 39, 40 Elimination of ethanol, 71
ethanol and, 74, 77, 78, 88 Embryonic chicken fibroblasts, 163
Cyclic AMP-dependent protein kinase, 34, 40, 44 Endometrial glycogen, 35
Cyclic AMP-independent protein kinase, 146, 159 Endoplasmic reticulum (ER), 71
Cystathionine, 157 Energy charge of ethanol, 77
Cytochrome P-450 reductase, 165 Enhanced gluconeogenesis, 61
Cytoplasm, 2 ENOL, see Enolase
Cytoskeleton , 2, 15 Enolase (ENOL), 11, 146, 161, 162, 165, 168
actin in, 11-115, 25 Enzymes, see also specific enzymes
elements of, 15 bound forms of, 23
network of, 25 cluster of, 23
Cytosol, 71, 76, 118 control parameters of, 20
Cytosolic degradation of insulin, 191 distribution of, 13
Cytosolic insulinase, 188 double immunotluorescent studies of, 13
Cytosolic malic dehydrogenase, 159, 164 free forms of, 23
Cytosolic PEP-carboxykinase, 165 gluconeogenic, 81
Cytosol shuttle, 154 glycogen metabolism, 35-36
Volume II 203
glycolytic, 81 lipogenesis and, 107

insulin degrading, 188 in liver, 102
kinetic parameters of, 20 Fructose, 71, 149
lysosomal, 187 absorption of, 97
metabolic dependence of binding of, 15-18 adaptation of diets of, 106-109
organization dynamics of, 15-20 adipose tissue and, 111-112
phosphorylation of, 143 differential effect of, 117
specificity of binding sites of, 5-10 ethanol oxidation and, 106
Enzymopathy, 54 excess, 103-106
Epidermal growth factor (EGF), 164 FFA synthesis and, 117
Epinephrine, 37-41, 44, 74 glycogen metabolism and, 105
Epithelial cells, 23 hyperlipidemia induced by, 109-111, 118
ER, see Endoplasmic reticulum infusion of, 118
Erythrocytes, see Red blood cells intestinal absorption of, 96-97
Estrogen, 44 lipid metabolism and 115-118
Ethanol lipogenesis and, 115-118
actue administration of, 84-86 pathway of metabolism of, 97-100
blood concentration of, 83 pregnancy and, 124
catecholamines and, 74-75 regulation of metabolism of, 101-109
chronic intake of, 80-81
elimination of, 71
energy charge of, 77
D
glUcose and, 73
insulin and, 73 D-Fructose, ethanol and, 80
lipogenesis and, 108 Fructose bisphosphatase, ethanol and, 85,86
metabolism of, 69-73 Fructose-1,6-bisphosphatase (Fru-P2ase), 23, 80,
oxidation of, 71 I65
perfusion of, 81-84 ethanol and, 87, 89
uptake of, 69 Fructose bisphosphate, 18, 20, 21
Ethanolamine, 157 Fructose-I,6-bisphosphate (Fru-P,), 33, 169
Ethanol oxidation, 88 cell proliferation, 159-162
fructose and, 106 ethanol and, 77, 79, 80
redox potential of, 7576 glycolysis, 146, 149
N-Ethylmaleimide, 185 Fructose-2,6-bisphosphate, 22, 23
Eukaryotic cells, 2, 14 Fructose load, 106, 118
Excess fructose, 103-106 Fructose-l-phosphate (F-I-P), 98, 99, 101, 103
Excessive lipolysis, 55 Fructose-6-phosphate (F6P), 22, 69, 77
Exposure to insulin, 191-196 ethanol and, 79
Fru-P.2, see Fructose-I,6-bisphosphate
Fm-P2 ase, see Fructose-1,6-bisphosphatase
F Fumarate, 150
Functional significance of binding, 20-24
F-actin, 4, 5, 20
F-actin-tropomyosin-troponin, 24
FAD-dependent succinate dehydrogenase, 155
G
Fasting, 35, 73
Fasting GSD type I, 55 GA, see D-Glyceraldehyde
Fertility and sucrose, 121, 122 Galactose, 149
Fetal calf brain, 13, 15 ethanol and, 80
Fetal development and sucrose, 121 Gamma subunits
FFA, see Free fatty acids of calmodulin, 37
Fibroblasts, 160 of phosphorylase kinase, 36
diploid, 157 Genetic determinants of binding, 20
embryonic chicken, 163 Gestation, 35
Formyl-FH, 159 GIP, see Glucose- I-phosphate
F-I-P, see Fructose-I-phosphate GIT, see Glutathione-insulin transhydrogenase
F6P, see Fructose-6-phosphate GK, see Glucokinase
Free fatty acids (FFA) Glucagon, 38, 73-74
mobilization of, 112 Glucocorticoids, 96, 115
synthesis of and fructose, 117 Glucokinase (GK), 69, 74, 80, 81, 84, 102, 116
Free forms of enzymes, 23 ethanol and, 85-87
Fructokinase, 97, 101, 103, 121 Gluconeogenesis, 61, 68, 75, 108, 109
enzymes in, 81 type V, 54
Glucose, 35, 81, 84, 164 type VIII, 54
absorption of, 97 Glycogen synthase, 35-36, 40
ethanol and, 73 Glycogen synthase kinase, 39-42, 45
hepatic production of, 54 Glycolysis, 69, 75, 168
homeostasis of, 53 in liver, 41
metabolism of, 68-69, 79-86 in muscle, 39-42
phosphorylation of, 116 Glycolysis, 69, 75, 168
production of, 69 aerobic, 142-143, 150-152, 164, 168
uptake of, 143 anaerobic, 16
Glucose-1 ,6-biphosphate, 22, 146 in cell proliferation, 156
Glucose-lactate cycle, 55 lower part of, 168
Glucose-6-phosphatase (G6Pase), 74, 81, 165 in tumor cells, 143-150
deficiency of, 54 Glycolytic enzymes, 81
ethanol and, 83, 85-87 Golden spiny mice (Acomys russatus), 109
intestinal, 97 Golgi membrane, 184
Glucose-l-phosphate (GIP), 69 Gout, 123
Glucose-6-phosphate (G6P), 2, 18, 34, 35, 39, 69, G6P, see Glucose-6-phosphate
74, 78 G6Pase, see Glucose-6-phosphatase
ethanol and, 79 GPDH, see Glyceraldehyde-3-phosphate
pool modulators of, 73-77 Gracilis, 16
Glucose-6-phosphate (G6P) dehydrogenase, 74, Growth hormones, 162, 163
107, 160, 165 GSD, see Glycogen storage disease
ethanol and, 82 GSK3, 40, 41, 45
Glucose phosphate isomerase, 3, 11 GTP, 100, 124
Glucose-6-phosphate isomerase (PGI), 11 GTT, see Glucose tolerance test
Glucose tolerance test (GTT), 57 Guinea pig, 11
Glucuronate, 165
Glucuronyl transferase, 165
Glutaminase, 150 H
phosphate-dependent, 158
Glutamine, 150, 154 Heart, 18, 22, 40
Glutaminolysis, 143, 150, 152-154, 156, 158, 164 Heat-stable inhibitors of phosphoprotein phospha-
-y-Glutamyl-transferase, 165 tase, 43-46
Glutathione-insulin transhydrogenase (GIT), 185 Hepatocarcinoma, 164 165
Glutathione peroxidase, 154 Hepatocytes, 164-167
Glutathione reductase, 155 binding of insulin to, 182-185
D-Glyceraldehyde (GA), 99 Hexo(gluco)kinase, ethanol and, 82
D-Glyceraldehyde (GA) dehydrogenase, 100 Hexokinase (HK), 3, 4, 11, 74
Glyceraldehyde-3-phosphate dehydrogenase, 149 in brain, 18
Glycerate 3-phosphate, 157, 165 ethanol and, 69, 77, 80, 81, 84-87
Glycerol, 168 fructose and, 100, 116
ethanol and, 80 glycolysis, 146
Glycerol 3-phosphate, 159 Hexose-6-phosphotransferases, 98
Glycerol 3-phosphate oxidase, 112 High-protein feeds, 62
Glycerol phosphate shuttle, 154 1-1K, see Hexokinase
a-Glycerophosphate, 78 HMG CoA, 44
Glycogen, 15, 16, 34-35 HMG CoA reductase kinase, 44
cell transformation, 165 Hormones, see also specific hormones, 39-42
endometrial, 35 balance of, 62
enzymes of metabolism of, 35-36 fructokinase and, 101
ethanol and, 68, 81 growth, 162, 163
fructose and, 105 sex, 35
inborn errors of metabolism of, 54 sucrose diets and, 112-115
in liver, 55 thyroid, 113
metabolism of, 53, 54, 105 Hydrogen peroxide, 154
Glycogenesis, 185 Hyperglycemia, 81
insulin-stimulated, 195, 197 Hyperlipidemia, 109-111, 119-120, 134
Glycogenolysis, 16, 68, 69 fructose and, 118
Glycogen storage disease (GSD) Hypertriglyceridemia, 1 1 I
type I, 54-61, 63, 64 Hyperuricemia, 118
type III, 54, 56-59, 61-62, 64 Hypoglycemia, 54, 68
Volume II 205
Hypoxanthine synthesis, 157 L
I Lactate, 2, 15, 16, 75, 168

ethanol and, 79, 80
production of, 150
I-band, 3-4, 10, 25 Lactate dehydrogenase (LDH), 3, 4, II, 13, 20
ICR/An mice, 38 Lactic acidemia, 55
Immortalization, 168 LDH, see Lactate dehydrogenase
Immunofluorescent studies, 13 Leukocytes, 157
5'IMP, 38 polymorphonuclear, 35
Inborn errors of glycogen metabolism, 54 Lipids, see also specific types
Inhibitors, 37, 40 fructose and, 115-118
dephosphorylation of, 45 synthesis of, 155
heat-stable, 43 synthetic capacity of, 164
phosphoprotein phosphatase, 36, 44-46 Lipogenesis
Inorganic phosphorus, 37, 77 ethanol and, 108
fructokinase and, 107
Inosine, 105, 149
fructose and, 115-118
Insulin, 35, 39-41, 44, 62, 97
Lipolysis, 55
binding of, 182-185, 195, 197
Lipoprotein lipase (LPL), 110, 120
chromium and, 119 Lipoproteins, see also specific lipoproteins, 116
copper and, 119 Liver, 2, 23, 34, 37
cytosolic degradation of, 191 aldolase in, 99
degradation of, 185-191, 193-195 fructokinase in, 102
dissociation of, 183-185 glucose production in, 54
down-regulation of, 192-193 glycogen synthase kinases in, 41
ethanol and, 73 phosphorylase kinase in, 38
exposure to, 191-196 toxicity of sucrose in, 122-123
glycogenesis stimulated by, 195 Liver glycogen, 55
glycogenic action of, 197 Load of fructose, 106, 118
hepatocytes and, 182-185 Loading of ischemic muscle, 16
intralysosomal, 187-188 Longevity and sucrose, 122
Lower part of glycolysis, 168
pH and, 189
LPL, see Lipoprotein lipase
receptors of, 182, 191
Lymphocytes, 157
Insulin degrading enzymes, 188 Lysosomal enzymes, 187
Intact axon, 11 Lysosomes, 187, 197
Intermediate filament systems, 2
Intermediates, intracellular concentrations of, 23
Intestine M
absorption of fructose in, 96-97
G-6-Pase activity in, 97 Macrophages, 26
metabolism of sucrose in, 96-97 Magnesium, 74
Intolerance to sucrose, 96 Malate, 76, 78, 161
Intracellular concentrations of intermediates, 23 Malate-aspartate, 154
Malate-aspartate shuttle, 157
Intralysosomal insulin, 187-188
Malate-citrate shuttle, 154
Ischemia, 18 Malic enzyme, 150
of cardiac muscle, 22 NAD-dependent, 152
loading and, 16 Malignancy, 165-168
tetanized, 18 Malonyl CoA, 60
Isocitrate, 150 Marked heterogeneity of amylo-1,6-glucosidase de-
Isocitrate dehydrogenase, 154 ficiency, 56
Isoenzyme forms of aldolase, 20 McArdle's disease (muscle phosphorylase), 34
Isoenzyme induction, 143-149 Membranes
Isoproterenol, 74 proteins associated with, 14
red cell, 14
ruffling of, 13
K Menstrual cycle, 35
MEOS, see Microsomal ethanol oxidizing system
Metabolic acidosis and ethanol, 85
Ketogenesis, 69 Metabolic channeling, 23
Kinetic parameters of enzymes, 20 Metabolic dependence of enzyme binding, 15-18
Metabolic strategy of tumor cells, 155-156 Nutrition, fructokinase and, 101
Metabolites, 18, 20, 22, 26
specific, 18
Methionine synthesis, 159 0
Methylene-tetrahydrofolate, 159
Methyl trap, 159 OAA, see Oxaloacetate
Mice, fructose and sucrose and, 38, 107, 109, 112, Obesity, 112, 113, 118
119, 122 sucrose and, 114
Microsomal ethanol oxidizing system (MEOS), 71 Onc-genes, 165-168
Microtrabecular system, 11, 25 One step carcinogenesis, 165
Microtubules, 2, 15, 25 Oxaloacetate (OAA), 78, 157, 161
Mitochondrial D-glyceraldehyde dehydrogenase, 100 Oxidation
Mitochondrial glycerol-3-P oxidase, 112 acetyl-CoA, 155, 164
Mitosis, 163 ethanol, 71, 75-76, 88, 106
Modulators of glucose-6-P pool, 73-77 pyruvate, 143, 153-155, 164, 168
Multistep transformation, 165-168 Oxidative pentose phosphate shunt, 160
Muscle, see also specific muscles, 20, 34-35, 37 Oxidative phosphorylation, 23
bovine psoas, 15 Oxoglutarate, 154
cardiac, 22, 23 2-Oxogluturate dehydrogenase, 150
contracting, 37-38 Oxygen, 153
glycogen synthase kinases in, 39-42 in cell culture, 154-155
ischemic cardiac, 22 consumption of, 69
phosphorylase kinase in, 36-37 tension of, 164, 168
protein synthesis in, 62
resting, 36-37
semitendinosus, 15 P
skeletal, 2, 15, 18, 23
uterine, 35 Pancreas, 97
Muscle phosphorylase (McArdle's disease), 34 Pasteur effect, 142, 143
Myofibrils, 5, 11 Pathway of fructose metabolism, 97-100
Myogen, 20 PC, see Pyruvate carboxylase
Myosin, 4, 20 Pentose phosphate shunt, 160
actin interaction with, 8, 25 PEPCK, see Phosphoenolpyruvate-carboxykinase
light chains of, 43 Perfusion of ethanol, 81-84
Perinatal aspects of sucrose, 120-122
PFK, see Phosphofructokinase
N PGI, see Glucose-6-phosphate isomerase
PGK, see Phosphoglycerate kinase
NAD, 75, 150, 163 PGM, see Phosphoglycerate mutase
ethanol and, 78 pH, 18
NADH ratio to, 157, 168 insulin and, 189
NAD-dehydrogenase, 155 Phenylephrine, 38
NAD-dependent malic enzyme, 152 Phosphatase, 37, 40, 45
NADH, 18, 55, 75 inhibitor of, 41
ethanol and, 78, 88 tissue distribution of, 44
NAD ratio to, 157, 168 Phosphate, 35, 160
NADH-NADPH transhydrogenation, 113 glutaminase dependent on, 158
NADH shuttles and ethanol, 88 inorganic, 77
NADP, 115, 150, 161 Phosphate-ribose-PP, 160-161, 163
ethanol and, 78 Phosphate-ribose-PP nucleic acid, 168
NADPH, 113, 161, 162, 165 Phosphate-ribose-PP synthetase, 160
ethanol and, 88 Phosphatidylcholine, 84, 86
NAD-synthesis, 161 Phosphoenolpyruvate, 76, 150, 165
Nerve axons, 11 ethanol and, 79
NIG, see Nocturnal intragastric therapy Phosphoenolpyruvate-carboxykinase (PEPCK), 150,
NMR, see Nuclear magnetic resonance 168
Nocturnal intragastric therapy (NIG), 60, 63 ethanol and, 80, 81, 85-87, 89
Nonmuscle cells, 11-15, 25 Phosphofructokinase (PFK)
Non-oxidative pentose phosphate shunt, 160 ethanol and, 77, 79, 81, 84-87, 89
Nuclear magnetic resonance (NMR), 24 glycolysis, 146, 149
Nucleic acids, 143, 168 Phosphoglucomutase (PGM), 3, 11
Nucleotide catabolism, 105 ethanol and, 82
Volume II 207
Phosphogluconate dehydrogenase, 159 Al-Pyrroline-5-carboxylate, 161

Phosphoglycerate kinase (PGK), 4, 23, 149 Pyrroline-5-carboxylate reductase, 161
Phosphoglycerate mutase (PGM), 11, 69, 75, 81, Pyruvate, 75, 76, 78, 88, 168
161 ethanol and, 79
Phosphoglyceromutase, 146 flow of, 106
Phospholipids, 168 oxidation of, 143, 153-155, 164, 168
synthesis of, 146, 157 Pyruvate carboxylase 80, 81
Phosphoprotein phosphatase, 36, 37, 40-46 ethanol and, 85-87, 89
dephosphorylation of, 45 Pyruvate dehydrogenase, 154, 162
inhibitors of, 36, 44-46 Pyruvate kinase (PK)
Phosphorylase, 3, 34 cell proliferation and, 161, 162, 165
ethanol and, 81, 82 cross linking of, 25
muscle (McArdle's disease), 34 dephosphorylation of, 44
Phosphorylase kinase, 34, 40 distribution of, 13
deficiency of, 38 ethanol and, 77, 78, 80, 81, 84-87
in liver, 38 fructose and, 116
in muscle, 36-37 phosphorylation of, 159
Phosphorylase phosphatase, 35 polymorphism of, 20
Phosphorylation, 23, 44-46 serine and, 158
enzyme, 143 tumor cells and, 146, 149, 164, 168
glucose, 116
pyruvate kinase, 159 R
Phosphotyrosine, 162
Piggy-back binding, 10-11, 23
Rat, 112
PK, see Pyruvate kinase brain of, 11
PKF, 25 skeletal muscle of, 15
Plasma glucose, 73 Red blood cells, 2, 11, 22, 53
Plasma membrane, 182, 185 aerobic glycolysis in, 143
Plasminogen activator, 162 ghosts of, 14, 15
Polymorphonuclear leukocytes, 35 membranes of, 14
Polyol pathway, 100 protein of, 15
Potassium-sodium ratios, 162 Redox potential of ethanol oxidation, 75-76
Pregnancy Refeeding, 35
fructose and, 124 Renal medulla, aerobic glycolysis in, 143, 146
sucrose and, 120-122 Resistance against oxygen tension, 164, 168
Preneoplastic hepatocytes, 164-165 Respiratory chain, 142
PR enzyme, see Phosphoprotein phosphatase Resting muscle, 36-37
Proliferating cells, 142-143 Retina, aerobic glycolysis in, 153, 146
Proline, 161 Ribose, 149
ethanol and, 80 Ribose 5-phosphate, 160, 165
Prosthetic group removing enzyme, see Phosphopro- RNA, 158
tein phosphatase Rous sarcoma virus (RSV), 157, 162-164
Protease, 185 RSV, see Rous sarcoma virus
Protein kinase Ruffling of membranes, 13
cyclic AMP-dependent, 34-40, 44
cyclic AMP-independent, 146, 159 S
Protein phosphatase, 42-45
Proteins, see also specific proteins
actin-associated, 15 Salvage pathway, 161
feeds high in, 62 Sarcomere length, 4
membrane-associated, 14 Sarcoplasmic reticulum, 3
Scatchard plots, 182, 184
muscle, 62
Second messenger theory, 41
red cell, 15
Selenium, 155
synthesis of, 62, 159, 162 Selenous acid, 155
transmembrane, 14 Semimembranosus, 16
Proteolysis, 36 Semitendinosus muscle, 15
Purine, 159 Serine, 146, 157-159, 163, 165, 168
biosynthesis of, 161 activation of, 158
Pyrazole, 71, 79 ethanol and, 80
Pyrimidine synthesis of, 146
biosynthesis of, 161 Serine dehydratase, 165
synthesis of, 159 Serine hydroxymethyltransferase, 159
Serum stimulation, 152 phosphatases distribution in, 44

Serum triiodothyronine, 112 TM, see Tropornyosin
Sex hormones, 35 TN, see Troponin
Sheep, 15, 18 Tocopherols, 155
Simian virus, 40, 157 TPI, see Triosephophate isomerase
Skeletal muscle, 2, IS, 18, 23 Transmembrane protein, 14
rat, 15 Trifluoperazine, 37
SOD, see Superoxide dismutase Triglyceride (TG), 109-112, 116, 117
Sodium-potassium ratios, 162 synthesis of, 146
Sorbitol, 103 Triiodothyronine, 112
Sorbitol dehydrogenase, 100, 103 Triokinase, 100, 121
Specificity Triosephosphate isomerase (TPI), 3, 4, 13, 23
of enzyme binding sites, 5-10 affinity of, 11
of phosphoprotein phosphatase, 42-46 Tropomyosin (TM), 4-5, 7, II, 20, 24
Specific metabolites, 18 isoforms of, 8
Spectrin, 15 Troponin (TN), 4-5, 7, 20, 24, 25, 38
Sphingomyelin, 157 Trypsin, 36, 37, 40
synthesis of, 146 sensitivity to, 40
Spiny mice, sucrose and fructose and, 107, 109, Tumor cells, 26, 142
112, 119, 122 aerobic glycolysis in, 146
Sprague-Dawley rats, 112 formation of, 142-143
Starch, 68 glycolysis in, 143-150
Starvation, 71 metabolic strategy of, 155-156
Stoichiometry, 4, 25 Type I fibers, 8
of binding, 6 Type II fibers, 8
Succinate, 150 Tyrosine, 162, 163
Succinate dehydrogenase, 155
Sucrase, Ill
Sucrose, 97
adaptation of, 106-109
adipose tissue and, 111-112 UDP-glucose (UDPG), 39
deleterious effects of, 118-123 UDP-glucose pyrophosphorylase (UDPGP), 74, 81
diabetes and, 118-119 ethanol and, 82
fertility and, 121, 122 Uric acid, 105, 124
hepatic toxicity of, 122-123 Uridine, 149
hormones and, 112-115 Uterine muscle, 35
hyperlipidemia induced by, 109-111
intestinal metabolism of, 97
intolerance to, 96
V
longevity and, 122
obesity and 114 Vasopressin, 38
perinatal aspects of, 120-122 Very low density lipoproteins (VLDL), 116
pregnancy and, 120-122 VLDL, see Very low density lipoproteins
survival aspects of, 120-122
thyroid hormone and, 113
Superoxide dismutase (SOD), 154, 155
X
Superoxide radicals, 154, 155
Survival aspects of sucrose, 120-122 Xanthine, 105
Sympathetic activity, 38 X-ray diffraction, 25
Synthase phosphatase, 35
Y
T
Yeast, 2
Temperature sensitive mutants, 163
Tetanized ischemia, 18
TG, see Triglyceride
Thyroid hormone and sucrose diets, 113
Tissue, see also specific types Zinc, 80
adipose, 35, Ill- I 12

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Regulation

Rivka Beitner, Ph.D.

CRC Press is an imprint of the

Reissued 2018 by CRC Press

CO 1985 by Taylor & Francis

No claim to original U.S. Government works

ISBN 13: 978-1-315-89717-2 (hbk)

Rivka Beitner, Ph.D., is Associate Professor of Biochemistry in the Department of Life

Ramon Bartrons, Ph.D. Hans Werner Hofer, Prof. Dr.

Nava Bashan, Ph.D. Louis Hue, M.D., Ph.D.

Rivka Beitner, Ph.D. Norman Kalant, Ph.D.

G. E. J. Staal, Ph.D. John Weidemann, B.Sc.

John E. Wilson, Ph.D.

GLYCOLYTIC ENZYME ORGANIZATION VIA THE CYTOSKELETON

Frank Clarke, Petra Stephan, Don Morton, and John Weidemann

II. The Nature of the Adsorbent 3

III. Dynamics of Enzyme Organization 15

IV. Functional Significance of Binding 20

While the classification of the glycolytic enzymes as soluble is an operational definition

Percent enzyme bound

Muscle PFK ALD GPDH PK LDH

Rabbit skeletal (white)

Enzyme binding as determined by Clarke, F. M., Morton, D. J., and

II. THE NATURE OF THE ADSORBENT

A. Muscle: The Role of Actin and the I-Band

B. The Role of Tropomyosin (TM) and Troponin (TN)

FIGURE I . Electron micrograph of aldolase-F-actin-tropomyosin-troponin mixture. The aldolase mol-

formed between aldolase and F-actin-tropomyosin-troponin filaments are quite different in

C. The Specificity of Enzyme Binding Sites

FIGURE 2. (A) Three-dimensional filament bundle formed by aldolase cross-linked F-actin-tropo-

alone binds to isolated myofibrils it does so with an ultimate stoichiometry corresponding

which aldolase may occupy is preferentially a binding site for glyceraldehyde-3-phosphate

considerable polymorphism which could contribute to significant variations of adsorbent in

FIGURE 4. Glyceraldehyde-3-phosphate dehydrogenase binding to myofibrils. Enzyme binding was determined

FIGURE 5. Diagrammatic representation of the binding of aldolase, glyceraldehyde-3-

D. Piggy-Back or Indirect Binding and Glycolytic Enzyme Organization

E. Nonmuscle Cells and Cytoskeletal Actin

FIGURE 6. Binding of triosephosphate isomerase to myofibrils. Binding determined as described in legend to

TM,TN AND ACCESSORY PROTEINS

THIS DETERMINES THEIR

AND PROVIDES THE BLUEPRINT

III. DYNAMICS OF ENZYME ORGANIZATION

A. Metabolic Dependence of Enzyme Binding

HK GPI PFK A LD TPI GPDH PGK PGM ENOL PK LDH

ALDOLASE GPDH GLYCOGEN

PFK ALDOLASE GPDH LACTATE GLYCOGEN

strengthened by experiments involving prior glycogen depletion or by the use of trained or

100 150 200 250 50 100 150 200 250

TIME ( seconds) TIME (seconds)

C. Genetic Determinants of Binding

IV. FUNCTIONAL SIGNIFICANCE OF BINDING

A. Influence on Catalytic Expression

with smaller oligomeric or monomeric forms of structural proteins.' Then modification of

REGULATION OF GLYCOGEN METABOLISM

A. Functions of Glycogen in Specific Tissues

glucose concentration by an insulin induced coordinated increase in glucose uptake' and

B. Enzymes of Glycogen Metabolism — Overview of Recent Developments

II. PHOSPHORYLASE KINASE

A. Muscle Phosphorylase Kinase

2. Activity in Resting Muscle

1. Phosphorylation by cyclic AMP-dependent protein kinase.35.47.4"

The physiologically important mechanism for conversion of phosphorylase b to a is

1. An epinephrine induced increase in sarcoplasmic Ca" concentration to levels which

3. Activity in Contracting Muscle