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The control of glycogen synthesis in the liver
THE
CONTROL
OF GLYCOGEN IN THE
SYNTHESIS
LIVER
H. G. HERS, H. DE WULF, W. STALMANSand G. VAN DEN BERGHE Laboratoiro de ChimiePhysiologique,Universit6de Louvain,Louvain,Belgium INTRODUCTION IN the liver, more than in any other tissue, the concentration of glycogen can vary to a large extent and at a rapid rate; the extreme values in normal conditions can be estimated at 0.01 and 12 ~ and the most rapid rates of synthesis, which are obtained by refeeding a fasted animal or under the action of glucocorticoids, are of the order of 1% of the wet weight per hour. In the normal fed animal, however, both the synthesis and the degradation are slow and the half-life of glycogen has been estimated at 24--36 hr (I, 2). This indicates the existence of important regulatory mechanisms that control the rates of synthesis and of degradation of the polysaccharide. The regulation occurs by the interconversion of two forms of the key enzymes that catalyze the limiting steps of the metabolic transformations; one of these forms, called a, is active, the other, called b is inactive in the ionic conditions which exist in the cell. The key enzymes are glycogen synthetase and glycogen phosphorylase on the pathways of synthesis and of degradation respectively. The interconversion of their a and b forms is achieved by phosphorylation and dephosphorylation and is submitted to regulatory effects that are often very similar; the main difference between the two systems is that phosphorylation causes the activation of the phosphorylase and the inactivation of the synthetase and vice versa. The obvious advantage of this mechanism, which is summarized in Fig. 1, is that synthesis and degradation of glycogen do not occur simultaneously at an important rate. In this paper we will discuss mostly the interconversion of the two forms of glycogen synthetase. THE TWO FORMS OF LIVER GLYCOGEN SYNTHETASE AND THEIR INTERCONVERSION The main properties of the a and b forms of glycogen synthetase are summarized in Table 1. An important point is that, in the presence of 5 mM phosphate, the b enzyme is inactive whatever the concentration of glucose 171
172
H. G. HERS, H. DE WULF, W. STALMANSAND G. VAN DEN BERGHE [ GLYCOGEN DEGRADATION I PHOSPHORYLASE CZ ADP
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KINAS
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H20
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SYNTNETASE g
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PHOSPHATA SE
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SYNTHETASE a.
FIG. 1 TABLE 1
PROPERTIESoF Tim Two FORMSOr LIVERGLYCX~ENSYNTHETASE Glycogen synthetas¢ b Active only in the presence of high, nonphysiological concentrations of glucose 6-phosphate Even under these conditions has a low affinity for UDPG (Km = 0.35 to 1 raM) (3, 4, 5) and is inhibited by 5 mM P, (physiological) or SO,-- (4, 5) Is normally predominant in the liver Is inactive in vivo
Glycogen synthetase a Is activated by P~, SO4-- (4, 5) or glucose 6-phosphate (4) Has a high affinity for UDPG (Kin = 0.07 to 0.2 mM in the presence of glucose 6-phosphate) (4, 5) Is present in the liver after glucose or glucocorticoid treatment Is active in vivo (see Fig. 2).
6-phosphate, whereas the a enzyme is nearly fully active. As has been pointed out by Mersmann and Segal (4) the system has thus the characteristics of an on-off process. As, in vivo, neither form is influenced by the concentration of glucose 6-phosphate, we do not use the D (glucose 6-phosphate dependent) and I (glucose 6-phosphate independent) nomenclature introduced by L a m e r because it may give the false impression that in the liver, as has been shown in certain conditions for the muscle (6), the concentration of glucose 6-phosphate regulates the rate of glycogen synthesis. It is shown in Fig. 2 that this rate is highly correlated to the amount of synthetase a present in the liver.
THE CONTROL
OF GLYCOGEN
SYNTHESIS IN THE LIVER
173
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F[o. 2 Correlation between the activity of liver glycogen synthetase measured in a concentrated homogenate and the rate of the conversion in vivo of glucose to liver glycogen in the same animal. The experiments carded out in vivo lasted 1 rain and were performed by the injection of [6-SH] glucose to mice 5 rain after the injection of glucose (I rag/g) or of 0.15 M NaCl. Half of the mice had been treated with prednisolone. After the death of the animal, one-half of the liver was used for the determination of the tritiated glycogen and the other for the assay of glycogen synthetase at 0 ° in a 50yo homogenate. (©) control mice; ( 0 ) mice injected with glucose; (El) prednisolone treated mice; I prednisolone treated mice injected with glucose. Correlation coefficient: 0.94 (P < 0.001). This Figure is from De Wulf and Hers (7).
The activity of the synthetase a can be measured specifically without interference of b in the presence of 5 mM phosphate. A concentrated (50 ~o) liver homogenate, because of its high content in phosphate, has been used with success for this purpose (8, 9), but one can also utilize more simply a diluted homogenate enriched in phosphate or sulfate (5). By this method, we have followed the variations in the concentration of synthetase a in a liver homogenate incubated at 20 ° (see Fig. 3). In confirmation of the finding of Segal and co-workers (4, 11), it appears that the initial content is usually low (this is however not always true, particularly during the months of June till August; see for instance Fig. 13); after a short lag period, the concentration of synthetase a increases rapidly and reaches a plateau at approximately 60 min. This activation is accelerated by caffeine and inhibited by fluoride and
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FIe. 3 Influence of ATP and cyclic AMP on the activation and inactivation of liver glycogen synthetase. The liver of an untreated mouse was homogenized with 3 vol of a solution containing 150 mM sucrose and 100 mM glycylglycine buffer at pH 7.4. This homogenate, after 0 or 60 rain of preincubation at 20 °, was incubated either alone (O), or in the presence of 4 mM ATP, 8 mM Mg acetate (O), 4 m s ATP, 8 mM Mg acetate, 0.5 mM cyclic AMP (A). This Figure is from De Wulf and Hers (10). E D T A (not shown), p r e s u m a b l y by chelation o f magnesium. A T P - M g c o u n t e r a c t s the activation or causes a reconversion o f synthetase a into b when a d d e d after the synthetase has been activated. As the total activity o f the e n z y m e ( m e a s u r e d in the presence o f s a t u r a t i n g levels o f glucose 6-phosp h a t e a n d U D P G ) does n o t change consistently d u r i n g these t r a n s f o r m a t i o n s , it a p p e a r s t h a t synthetase a is f o r m e d at the expense o f synthetase b a n d vice versa, very p r o b a b l y by d e p h o s p h o r y l a t i o n a n d r e p h o s p h o r y l a t i o n , a c c o r d i n g to the reactions d e p i c t e d in Fig. 1 a n d as previously d e m o n s t r a t e d for the muscle enzyme (12).
THE CONTROL OF GLYC,(~EN SYNTHESIS IN THE LIVER THE CONTROL
BY GLUCOSE
AND
175
GLYCOGEN
The Effect of Glucose in vivo When glucose is administered intravenously to either fed or fasted mice, it induces, after a lag period of 2-3 min, a rapid rise in the rate of glycogen synthesis (Fig. 4) which parallels the conversion of synthetase b into a (see Fig. 2). There is a simultaneous fall in the concentration of glucose 6-phosphate (at least in the fed mice) and of UDPG in the liver to approximately
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FIG. 4 Time sequence of the stimulation of glycogen synthesis by a single glucose load. Glucose was injected intravenously to 24 hr fasted mice in a single dose of I mg/g body weight. At various intervals afterwards, a trace amount of [ U - t ~ "] glucose was administered intravenously and the animals were killed 1 min later. ( © ) glucose concentration in the liver at the end of the experiment; ( O ) amount of glucose converted into glycogen in 1 rain. Vertical bars represent 4- the standard error of the means; the number of experiments is given in parentheses. This Figure is from De Wulf and Hers (8).
60 ~o of their initial value (8). It is clear, therefore, that the stimulation of liver glycogen synthesis by glucose, which has been known for a long time, is not the result of a push given on the whole metabolic pathway by an increased amount of glucose but is entirely explained by the activation of glycogen syntbetase.
176
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BEKGHE
The Effect of Glucose in vitro and the Interference of Salts In order to investigate the effect of glucose on the in vitro interconvcrsion of the two forms of glycogen synthetasc, it has been necessary to remove the endogenous glucose by gel filtration of the liver extract through a column of Sephadex G-25. When a preparation obtained in this way is incubated at 20 °, the conversion of synthetase b into a levels off at values that are only 25 to 50 ~o of the activity that can be attained in the original extract; ff inorganic phosphate or sulfate is added at this moment, at a final concentration of 5-I0 mM the activation starts again and reaches nearly completion (Fig. 5).
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Influence of salt on the activation of glycogen synthetase. Mice were injected with glucagon (0.5 ~,g/g body weight) 6 rain before death in order to cause a complete inactivation of the synthetase. The livers of 7 mice were pooled and a 33 % homogenate, prepared with 50 mM glycylglycine, pH 7.4, was centrifuged at 8,000 x g for 10 rain; the supernatant was filtered through a Sephadex G-25 column and the gel eluate was incubated at 20 °, either alone or with 10 mM (NH0zSO4.
The full activation can also be obtained in one step if the salt is added from the beginning of the experiment. The level reached, but not the time required to complete the reaction, is dependent on the amount of salt added (Fig. 6); the salt effect is unspecific as it has been obtained with 3 mM Mg acetate or 0.1 M KCI. The partially activated enzyme obtained in the absence of salt has the kinetic properties of synthetase a (low Km for U D P G ) but has a lower Vmax than the fully activated preparation. It has some similarity with the partially inactive form of synthetase previously described by Steiner (13) and by Hizukuri and Larner (3). When glucose is added at a final concentration of 5%o it causes an important acceleration of the b to a conversion, in the absence, as well as in the presence
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
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of salts (Fig. 7). This effect appears specific for glucose as it was not obtained with mannose, fructose, galactose, galactosamine, 2-deoxyglucose, arabinose, xylose, mannitol, sorbitol or maltose and is still observed in the presence of caffeine. We tentatively conclude that the synthetase phosphatase is glucose sensitive* and converts the b enzyme into a form which is only partially active * We cannot, however, preclude the possibility that the glucose effect is due to an interaction with glycogen synthetase which is the substrate of the phosphatase.
178
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
in the absence of salt; the second step, presumably non-enzymatic, seems to be an unmasking of active sites by the salt. In agreement with this interpretation, we have observed that the fully activated enzyme loses a part of its activity by passage through a Sephadex column and can recover it by the addition of salt.
The Inhibition by Glycogen and the A~nity for Glucose The inhibitory effect of glycogen on the activation of the synthetase in a liver extract has been previously described (10). After removal of the small molecules by gel filtration, glycogen is no longer an inhibitor but, on the contrary, induces a stimulation; its inhibitory effect reappears as soon as
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phosphate or sulfate are reintroduced. The doses of glycogen required (Fig. 8) to cause an inhibition are however much larger than those reported by Larner (14) to have a similar effect on the muscle synthetase phosphatase; this difference probably explains why the liver can accumulate glycogen at a much higher concentration than the muscle. The glycogen also interferes with the action of glucose by lowering the affinity of the phosphatase for the hexose. It is shown in Fig. 9 that in one experiment in which the effect of the dose of glucose was investigated, the addition of 1 ~ glycogen to the preparation increased the apparent Km from 7 rnM (1.3~) to 11.4 rnM (2.05~/oo); a higher dose o f glycogen did not further increase the Km but reduced the maximal velocity, indicating that the effect
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
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Influence of the concentrations of glucose and glycogen on the activation of glycogen synthetase. Same procedure as in Fig. 5. The gel fihrato was incubated at 20 ~ with 5 mM (NI-ID2SO4in the presence of tho indicated concentrations of glucose, at three different concentrations of particulate glycogen. After 15 min, the amount of synthetase a was measured. of a high amount of glycogen cannot be completely counteracted by an excess of glucose. It must be pointed out however that the apparent K m measured in this type of experiment can depend on the stage of activation at which the glucose effect is estimated and that the kinetics of the reaction are too complex to consider the calculated K m as more than indicative.
The Effect of Glucose on the Phosphorylase Phosphatase Taking into consideration the many resemblances between the enzymatic systems which interconvert glycogen synthetase and glycogen phosphorylase, we have investigated the possibility that glucose would also stimulate phosphorylase phosphatase in the liver. Such an effect was easily demonstrated by passing a liver extract through a Sephadex column and then following the inactivation of phosphorylase; as shown in Fig. 10, the addition of glucose induced a great acceleration of the inactivation with an apparent K m equal to 7 mM (Fig. I 1). This effect was not obtained with mannose, fructose, galactose, 2-deoxyglucose, glucuronolactone, arabinose, xylose, sorbitol or mannitol. The addition of salts did not accelerate the inactivation and glycogen had only a slight inhibitory effect. It is interesting to recall that a stimulation of the muscle phosphorylase phosphatase by glucose or glycogen has been reported by Holmes and Mansour (15).
180
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FIG. 11 Influence of the concentration of glucosc on the inactivation of glycogen phosphorylase. Same p r o c e d u r e a s in Fig. 10, but phosphorylase activity was measured after 5 rain incubation in the presence of ino-easing a m o u n t s of glucose.
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
181
THE CONTROL BY 3",5'-CYCLIC AMP In vivo Experiments When glucagon, epinephrine or cyclic A M P are administered to mice in which the synthetase has been previously activated by glucose or by glucocorticoids (see below), they cause a rapid reconversion of the a enzyme into b. The same effect can be observed in any series of animals that happen to have a high content o f synthetase a in their liver. We show in Fig. 12 the variation in the activity of synthetase a and in the concentration of cyclic AMP in the liver of mice that have received 0.5 ng of glucagon per g body weight. 3
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It is interesting to point out that the variation in the activity o f glycogen synthetase that can be observed in this type of experiment is much greater than the simultaneous effect on the activity of phosphorylase. This is due to the fact that, prior to any treatment, liver phosphorylase already displays a high activity which is only approximately doubled by the administration of glucagon or cyclic AMP (7).
The Stimulation of Synthetase Kinase by Cyclic A M P The hi vitro effect of cyclic AMP on the inactivation of the synthetase in the presence of ATP is illustrated in Fig. 3. This effect is in agreement with the hypothesis that the cyclic nucleotide activates a synthetase kinase that phosphorylates synthetase a into b. In a crude liver extract, a half-maximal effect was obtained with 2 × 10 -7 M cyclic AMP. With a more purified kinase,
182
H.G. HERS, H. DE WULF, W. STALMANSAND G. VAN DEN BERGHE
Bishop and Larner (17) have recently reported an affinity constant equal to 4 × 10-s M. The concentration of the nucleotide in the liver being normally between 5 and 9 x l0 -7 M, it seems that the kinase should always be fully activated. As this is obviously not the case, it appears that an important part of the nucleotide is not available for the stimulation of the kinase; in agreement with this interpretation, Jefferson et al. 0 8 ) have found that 60 % of the nucleotide is in particulate fractions. It is also interesting to mention that many enzymes are present in the tissues at a concentration of 10 -7 to 10-6 M (19) and that therefore, a great part of the nucleotide could be bound to the various enzymes which show affinity for it. The concentration of the free cyclic AMP in the liver is consequently very difficult to evaluate. THE CONTROL BY GLUCOCORTICOIDS
The in vivo Activation of Glycogen Synthetase by Glucocorticoids Since the initial work of Long and co-workers (20) in 1940, it has been repeatedly observed that the administration of glucocorticoids stimulates the deposition of glycogen in the liver of fasted as well as of fed animals. Hornbrook and co-workers (21) were the first to report that the administration of hydrocortisone induces the formation of a glycogen synthetase that is less dependent on glucose 6-phosphate than normally. De Wulf and Hers (9) have shown that, in the animals treated by prednisolone, the rate of glycogen synthesis parallels the activity of glycogen synthetase when the latter is measured in a concentrated liver homogenate; in such a preparation, obtained from control or from treated animals, glucose 6-phosphate is without influence on the activity of the enzyme. This situation has been later clarified by a better knowledge of the properties of the a and b forms of the synthetase, the effect of the hormonal treatment being clearly a conversion of the b form into a (5). It must also be emphasized that in the fasted mice, glucocorticoids stimulate glycogen synthesis without raising the glycemia (9). This indicates that the activation of the synthetase is not mediated by the glucose effect which has been described in the preceding section and is not related to the effect of the hormone on ghiconeogenesis. The stimulation of glycogen synthesis has also been observed in diabetic animals (22) and may therefore be regarded as independent of insulin. The contradictory finding reported by Kreutner and Goldberg (23) is presumably due to a too short duration (2 hr) of their experiment after hydrocortisone administration. The presence of a high amount of synthetase a in the liver of animals treated with prednisolone could result from an increased rate of activation by the phosphatase or from a decreased rate of inactivation by the kinase. We have investigated these two possibilities.
THE CONTROL OF GLYCOGEN SYNTHESIS 1N THE LIVER
183
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Modification of the Properties of Synthetase Phosphatase The administration of glucocorticoids brings about several changes in the properties of the synthetase phosphatase which are conveniently studied in a glucose-free liver extract. As shown in Fig. 13, the activity of the phosphatase has been increased by the treatment but this effect is more apparent in the absence than in the presence of glucose. Furthermore, the activity that is resistant to the inhibition by glycogen is also greater than in the control liver (Fig. 14).
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Interaction of glucose and glycogen in the activation of glycogen synthetas¢ of normal and glucocorti~id-tr~ted mice. Same experiment as in Fig. 13. The effect of increasing amounts of particulate glycogen has been investigated. Synthetase a was assayed after 20 rain incubation.
184
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
From these observations we tentatively conclude that glucocorticoids induce the formation of a synthetase phosphatase that is less sensitive to both glucose and glycogen. Since the normal phosphatase is still present, one can understand why the administration of glucose to animals that have been treated by prednisolone causes a further increase in the rate of glycogen synthesis (see Fig. 2). Being released from the inhibition by glycogen, the new enzyme would allow the activation of the synthetase even when the concentration of the polysaccharide in the liver is already high; not being dependent on glucose, it would also be active in the fasted animal. Mersmann and Segal (24) have reported that the activity of the synthetase phosphatase disappears from the liver of 48-hr-fasted, adrenalectomized rats and is restored by the administration of glucocorticoids. The possible relationship between these observations and those reported above is not clear.
The Properties of the Inactivating System We have conducted a series of experiments in order to investigate the possibility that glucocorticoids could interfere with one of the factors that participate in the inactivation of synthetase a, as represented in the following scheme: Synthetase a
ATP ~
Kinase ((
Glucagon
Cyclic AMP ~
Synthetase b
Cyclase<
Diesterase
AMP
It has been verified that the in vitro sensitivity of the kinase to cyclic AMP is the same whether the synthetase had been previously activated by glucose or by glucocorticoids (10). The concentration of cyclic AMP was slightly but significantly diminished in the liver of mice that had received prednisolone three hours previously (0.63/~t with S.E.M. : 0.03 for 15 determinations) as compared to untreated animals of the same group (0.83 ~.M with S.E.M. = 0.03 for 13 determinations). As discussed above, the significance of such a small change is difficult to appreciate. This decrease in the concentration of cyclic AMP is not the result of a change in the properties of the phosphodiesterase which hydrolyzes cyclic AMP, as neither the total activity of this enzyme nor its affinity for its substrate have been modified by the treatment (7). As shown in Fig. 15, we have also measured the sensitivity of mice to glucagon and observed the same rise in the concentration of cyclic AMP in the liver of glucocorticoid-treated and of control animals. Finally, the half-life of x~I-glucagon has been measured in mice and found to be not modified by the corticoid treatment.
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
PREDNISOLONE CONTROL
25
185
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FIe. 15 Influence of the dose of glucagon on the level of cyclic AMP. Normally fed mice, treated or not with prednisolone 3 hr before, received an intravenous it~ection of glucagon and were killed 1 rain later. Vertical bars represent 4- the standard error of the means (n = 6).
We have therefore no indication that glucocorticoids could have an important influence on the system that inactivates glycogen synthetase in the liver. IS T H E R E
AN INSULIN
EFFECT?
Acute Effect The action of insulin on glycogen synthesis in the liver has for long been a controversial matter. While insulin given alone is glycogenolytic, presumably through glucagon or epinephrine secretion secondary to the hypoglycemia, insulin given with glucose induces a glycogen deposition (25). More recently, Bishop and Lamer (26) have shown that under these conditions the synthetase is in the active form. In their study of the stimulation of glycogen synthesis by glucose, De Wulf and Hers (8) have considered the possibility that the glucose effect could be mediated by the insulin secretion which is known to occur rapidly after glucose administration and they came to a negative conclusion; insulin given alone in short-term experiments (1 rain) was completely ineffective, and given with glucose had the same effect after 3 rain as glucose alone. It is also remarkable that the administration of glucose to animals made acutely diabetic by the administration of anti-insulin serum induces an important activation of glycogen synthesis (Table 2). An induction of glycogen synthesis by glucose in depancreatized (27) and in alloxanized (28) rats has also been reported. As the effect of the simultaneous administration of insulin and glucose can be explained by the action of glucose, we conclude that an acute effect of
186
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
insulin on the activation o f glycogen synthetase in the liver has not been adequately demonstrated.
Long-term Effect W h e n insulin is administered to diabetic animals, it induces in 1-2 hr a rise in the glycogen content o f the liver and an activation o f the synthetase (29). Considering the complexity o f the regulatory processes in diabetes, these TABLE 2 INFLUENCE OF ANTI-INSULIN SERUM ON THE GLUCOSE EFFECT
Treatment
Glucose Glucose incorporated into glycogen concentration rng/g liver /~g/min/gmuscle ~,g/min/g liver
Control (5) Glucose (5) Anti-insulin serum (5) Anti-insulin serum + glucose (5)
1.70 ± 0.05 2.88 4- 0.15 2.27 -4- 0.11 3.92 ± 0.26
4.0 =: 1.1 5.8 2-_ 1.6 i.6 ± 0.3 1.9 ±_ 0.4
7.1 54.1 4.4 36.4
___3.7 -.: 5.0 ± 1.2 ± 4.0
Glucose (1 mg/g body weight) or 0.15 M NaCI was injected intravenously to normally fed mice 20 rain after the administration of anti-insulin serum (10 mU/g body weight); control animals receivod only isotonic saline. Five minutes later, a trace amount of [6-Sill glucose was given intravenously. The mice were killed l min later and the radioactivity of liver and carcass glycogen was determined. Values shown are means 4- standard error of the means with the number of animals in parentheses. effects are difficult to interpret. According to a preliminary report (30) the activity o f synthetase phosphatase is reduced to less than 50 ~o o f the normal value in the liver o f the pancreatectomized dogs.
THE F U T I L E CYCLES I n the metabolism o f glycogen and its regulation, the existence of futile cycles m a y be considered at two levels:
S G ylcogen"~ Glucose-I-p
-~.~
I) The simultaneous operation o f the synthesis and degradation o f glycogen would create a futile cycle in which as much as 60/zmoles o f U T P could be c o n s u m e d per hr and per g o f liver. This waste, however, does not occur at an important rate (1, 2) because the synthesis o f glycogen stops when its degradation is induced and conversely. The main regulators that operate simultaneously in an antagonistic manner on synthesis and degradation are glucose and cyclic A M P .
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
187
2) A second futile cycle exists at the level of phosphorylase as well as of glycogen synthetase, which can both be activated and inactivated simultaneously. At this level, the waste of energy due to the futile cycles is several orders of magnitude smaller than in the case of glycogen and can be considered negligible. Up to now, we have no indication that any of the regulators that act either on the synthetase kinase or on the synthetase phosphatase would have a reverse effect on the antagonistic enzyme. It is therefore reasonable to admit that activation and inactivation occur simultaneously, and that the levels of active synthetase or of active phosphorylase reflect the steady state situation achieved by the activating and inactivating enzymes. It is remarkable, for instance, that, in the animals made acutely diabetic by the administration of anti-insulin serum, the hyperglycemia did not by itself elicit an activation of the synthetase; this can be explained by the fact that, as reported by Jefferson et al. (17), the same treatment causes a large increase in the concentration of cyclic AMP in the liver and that the activation of the phosphatase by glucose is therefore equilibrated by the activation of the kinase by cyclic AMP. A similar situation might exist in chronic diabetes and has also been experimentally produced by the simultaneous administration of glucose and glucagon to mice (7). Such a system offers the advantage of a greater number of regulatory sites; its main interest appears however to be that the dose response for an effector acting on one of the antagonistic enzymes displays a S-shaped curve. If one considers, for instance, the activation of glycogen synthesis by glucose, it appears that, at the normal level of glucose in the blood, the synthetase is still usually mostly inactive and is only activated by a further increase of the glycemia. However, the activation of the synthetase phosphatase by glucose in vitro displays Micha~lis-Menten kinetics. This apparent contradiction is easily understood if one admits that, in vivo, the activity of the synthetase phosphatase reached at normal glycemia is counteracted by a slightly higher activity of the synthetase kinase maintaining the synthetase in the b form and that its activation occurs only when an additional dose of glucose allows the phosphatase to overcome the kinase. The same holds also for the activation produced by glucocorticoids: the increased activity of synthetase phosphatase, brought about by the hormone, need not be large in order to convert an important proportion of the synthetase into the active form. Finally, it is not necessary to assume that the synthetase kinase shows an absolute requirement for cyclic AMP as its activity in the absence of the nucleotide could be balanced by that of the synthetase phosphatase.
188
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE SUMMARY
In the liver, glycogen synthetase exists in two forms, one of them (a) active, the other (b) inactive in the ionic conditions which exist in the cell. These two forms are interconvertible, presumably by phosphorylation and dephosphorylation under the action of a specific kinase and phosphatase respectively. It seems probable that these two enzymes operate simultaneously and that the level of the synthetase a in the liver results from the addition of their antagonistic effects. In normal mice, glycogen synthetase is predominantly in the b form. It is converted into a within 3 to 5 min after the intravenous administration of glucose or within 2 or 3 hr after the administration of glucocorticoids. The a enzyme can then be reconverted into b within 1 to 3 rain after the administration of glucagon, epinephrine or cyclic AMP. The effect of these various effectors has also been demonstrated in vitro, mostly thanks to the use of liver extracts from which glucose had been removed by gel filtration through a Sephadex column. The complete activation of glycogen synthetase in vitro requires the presence of salts. In their absence, synthetase b is converted into a form which has the kinetic properties of synthetase a but which is less active. Glucose markedly enhances the activation, both in the presence and in the absence of salts while glycogen is an inhibitor in the presence of salts only. The affinity of the synthetase phosphatase for glucose is decreased when glycogen is present. A stimulation of the phosphorylase phosphatase by glucose has also been observed. The treatment of mice by glucocorticoids induces the appearance in the liver of a synthetase phosphatase that is less sensitive to glucose stimulation and to glycogen inhibition than normally. No effect of the treatment on the system that inactivates glycogen synthetase could be demonstrated. An effect of cyclic AMP on the synthetase kinase is easily demonstrable in a liver extract in which the synthetase has been previously activated either by glucose or by glucocorticoids; a half-maximal effect has been obtained with a concentration equal to 2 × 10-7 Mcyclic AMP. The two main effectors that act antagonistically on the glycogen synthetase and glycogen phosphorylase appear to be glucose and cyclic AMP; thanks to their action, the synthesis of glycogen is inhibited while its degradation is stimulated and vice versa.
REFERENCES I. D. STE'I-I'EN, JR. and G. E. BOXER, The rate of turnover of liver and carcass glycogen, studied with the aid of deuterium, J. Biol. Chem. 155, 231-236 (1944). 2. K. H. SHUt.Land J. MAYER,The turnover of liver glycogen in obese hyperglycemic mice, J. Biol. Chem. 218, 885-896 (1956).
THE CONTROL OF GLYCOGEN SYN'I'HI~IS IN THE LIVER
189
3. S. HlzLrk'-tn~ and J. LARNm%Studies on U D P G : a-l,4-glucan a--4-glucosyltransferase. VII. Conversion of the enzyme from glucose-6-phosphate-dependent to independent form in liver, Biochemistry 3, 1783-1788 (1964). 4. H. J. MeRSMAt,~ and H. L. St'GAL, An on-off mechanism for liver glycogen synthetase activity, Prec. Natl. Acad. Set., U.S. .58, 1688-1695 (1967). 5. H. DE WULF, W. STAI.MANSand H. (3. HERS, The influence of inorganic phosphate, adenosine triphosphate and glucose 6-phosphate on the activity of liver glycogen synthetase, European J. Biochem. 6, 545-551 (1968). 6. R. PmAs and K. STANELOm, In rive regulation of rat muscle glycogen synthetase activity, Biochemistry 8, 2153-2160 (1969). 7. H. DE WULF and H. G. HERS, The role of glucose, glucagon and glucocorticoids in the regulation of liver glycogen synthesis, European J. Biochem. 6, 558-564 (1968). 8. H. DE WULE and H. G. HERS, The stimulation of glycogen synthesis and of glycogen synthetase in the liver by the administration of glucose, European d. Biochem. 2, 50-56 (1967). 9. H. DE WULF and H. G. HERS, The stimulation of glycogen synthesis and of glycogen synthetase in the liver by glucocorticoids, European d. Bioehem. 2, 57-60 (1967). 10. H. DE WULF and H. G. HERS, The interconversion of liver glycogen synthetase a and b in ~'itro, European .I. Biochem. 6, 552-557 (1968). 11. A. H. GOLD and H. L. SEOAL,Time-dependent increase in rat liver glycogen synthetase activity in vitro, Arch. Biochem. Biophys. 120, 359-364 (1967). 12. D. L. FRIEDMAN and J. LARNER, Studies on UDPG-a-glucan transglucosylase. Ill. Interconversion of two forms of muscle UDPG-a-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence, Biochemistry 2, 669-675 (1963). 13. D. F. STEINER,Reversible inactivation of glycogen synthetase, Biochim. Biophys. Acta .54, 206-209 (1961). 14. J. LARNER,Hormonal and nonhormonal control of glycogen metabolism, Trans. N.Y. Acad. Sci. 29, 192-209 (1966). 15. P.A. HOLMESand T. E. MANSOUR,Glucose as a regulator of glycogen phosphorylase in rat diaphragm. IL Effect of glucose and related compounds on phosphorylase phosphatase, Biochim. Biophys. Acta 156, 275-284 (1968). 16. B. McL. BRECKENRIDGE,The measurement of cyclic adenylate in tissues, Prec. Natl. Acad. Sci., U.S. 52, 1580-1586 (1964). 17. J. S. BISHOP and J. LARNER,Presence in liver of a Y,5'-cyclic AMP stimulated kinase for the I form of UDPG-glycogen glucosyltransferase, Blochim. Biophys. Acta 171, 374--377 (1969). 18. L. S. JEFFERSON,J. H. Ex'roN, R. W. BUTCHER, E. W. SUTHERLANDand C. R. PARK, Role of adenosine 3',5'-monophosphate in the effects of insulin and anti-insulin serum on liver metabolism, d. Biol. Chem. 243, 1031-1038 (1968). 19. P. A. SRER~, Enzyme concentrations in tissues, Science 158, 936-937 (1967). 20. C. N. H. LONe, B. KATZlN and E. G. FRY, The adrenal cortex and carbohydrate metabolism, Endocrinology 26, 309-344 (1940). 21. K. R. HORNBROOK,H. B. BURCH and O. H. LOWRY, The effects of adrenalectomy and hydrocortisone on rat liver metabolites and glycogen synthetase activity, Mol. Pharmacol. 2, 106-116 (1966). 22. W. TARNOWSKI,i . KITTLERand H. HH.Z, Die Wirkung yon Cortisol auf die Umwandlung yon Hexosephosphaten in Glykogen, Biochem. Z. 341, 45-63 (1964). 23. W. KREOTNERand N. D. GOLDBERG, Dependence of insulin of the apparent hydrocortisone activation of hepatic glycogen synthetase, Prec. Natl. Acad. Sci., U.S. 58, 1515-1519 (1967). 24. H. J. MERSMANNand H. L. SEGAL,Glucocorticoid control of the liver glycogen synthetase-activating system, 3. Biol. Chem. 244, 1701-1704 (1969). 25. J. B•RTIIET, P. JACQUES,H. G. HERS and C. De DUVE, Influence de l'insuline et du glucagon sur la syntht~.se du glycog~ne h~patique, Biochim. Biophys. Acta 20, 190-200 (1956).
190
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
26. J. S. BISHOP and J. LARNER, Rapid activation-inactivation of liver uridine diphosphate glucose-glycogen transferase and phosphorylase by insulin and glucagon in vivo, J. Biol. Chem. 242, 1354--1356 0967). 27. R. W. LONOLeY, R. J. BORTNICK and J. H. ROE, Rate of glycogenesis in liver of depancreatized rat after parenteral administration of glucose and fructose, Proc. Soc. ExptL BioL 94, 108-1 I0 (1957). 28. B. FRIEDMANN, E. H. GOODMAN, JR. and S. WEINHOUSE, Effects of glucose feeding, cortisol, and insulin on liver glycogen synthesis in the rat, Endocrinology 81, 486-496 (1967). 29. D. F. STERNERand J. KING, Induced synthesis of hepatic uridine diphosphate glucoseglycogen glucosyl-transfera.se after administration of insulin to alloxan-diabetic rats, J. Biol. Chem. 239, 1292-1298 (1964). 30. J. S. BISHOP, N. D. GOLDBERG, F. GRANDE and J. LARNER, Decreased activity of glycogen synthetase D phosphatase in insulin-insensitive diabetic dog liver, Diabetes 18, 337 (1969). ACKNOWLEDGMENTS This work was supported by the Fonds de la Recherche Seientifique M~dicale and by the U.S. Public Health Service (Grant AM 9235). H. De Wulf and G. Van den Berghe are "Aangesteld Navorser" and W. Stalmans is "Aspirant" of the "Nationaal Fonds Poor Wetenschappelijk Onderzoek".
Note Added ia Proof. It has now been clearly demonstrated that the enhancement of the in vitro activation of glycogen synthetase by glucose or caffeine is due to a shortening of the lag period that precedes the action of the synthetase phosphatase; glycogen (see Fig. 8), fluoride and A M P have an opposite action on the lag period. All these substances (with the exception of glycogen) are indeed ineffective when added after the latency period. From these and other observations we have concluded the existence of an active (a) and of an inactive (b) form of synthetase phosphatase and we have postulated the existence of a "synthetase phosphatase activating enzyme" stimulated by glucose and caffeine and inhibited by glycogen, A M P and fluoride. The livers of animals that have been treated with glucocorticoids contain a larger amount of this activating enzyme and are therefore less sensitive to stimulation by glucose as well as to inhibition by glycogen (De Wulf, Stalmans and Hers, submitted to European J. Biochem.). The same livers contain a larger amount of phosphorylase phosphatase (Stalmans, De Wulf, Lederer and Hers, submitted to European J. Biochem.). As the phosphorylase phosphatase is stimulated by caffeine and inhibited by A M P and fluoride, it has many similarities with the synthetase phosphatase activating enzyme.
CONTROL
OF GLYCOGEN IN THE
SYNTHESIS
LIVER
H. G. HERS, H. DE WULF, W. STALMANSand G. VAN DEN BERGHE Laboratoiro de ChimiePhysiologique,Universit6de Louvain,Louvain,Belgium INTRODUCTION IN the liver, more than in any other tissue, the concentration of glycogen can vary to a large extent and at a rapid rate; the extreme values in normal conditions can be estimated at 0.01 and 12 ~ and the most rapid rates of synthesis, which are obtained by refeeding a fasted animal or under the action of glucocorticoids, are of the order of 1% of the wet weight per hour. In the normal fed animal, however, both the synthesis and the degradation are slow and the half-life of glycogen has been estimated at 24--36 hr (I, 2). This indicates the existence of important regulatory mechanisms that control the rates of synthesis and of degradation of the polysaccharide. The regulation occurs by the interconversion of two forms of the key enzymes that catalyze the limiting steps of the metabolic transformations; one of these forms, called a, is active, the other, called b is inactive in the ionic conditions which exist in the cell. The key enzymes are glycogen synthetase and glycogen phosphorylase on the pathways of synthesis and of degradation respectively. The interconversion of their a and b forms is achieved by phosphorylation and dephosphorylation and is submitted to regulatory effects that are often very similar; the main difference between the two systems is that phosphorylation causes the activation of the phosphorylase and the inactivation of the synthetase and vice versa. The obvious advantage of this mechanism, which is summarized in Fig. 1, is that synthesis and degradation of glycogen do not occur simultaneously at an important rate. In this paper we will discuss mostly the interconversion of the two forms of glycogen synthetase. THE TWO FORMS OF LIVER GLYCOGEN SYNTHETASE AND THEIR INTERCONVERSION The main properties of the a and b forms of glycogen synthetase are summarized in Table 1. An important point is that, in the presence of 5 mM phosphate, the b enzyme is inactive whatever the concentration of glucose 171
172
H. G. HERS, H. DE WULF, W. STALMANSAND G. VAN DEN BERGHE [ GLYCOGEN DEGRADATION I PHOSPHORYLASE CZ ADP
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KINAS
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SYNTNETASE g
f
PHOSPHATA SE
~
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SYNTHETASE a.
FIG. 1 TABLE 1
PROPERTIESoF Tim Two FORMSOr LIVERGLYCX~ENSYNTHETASE Glycogen synthetas¢ b Active only in the presence of high, nonphysiological concentrations of glucose 6-phosphate Even under these conditions has a low affinity for UDPG (Km = 0.35 to 1 raM) (3, 4, 5) and is inhibited by 5 mM P, (physiological) or SO,-- (4, 5) Is normally predominant in the liver Is inactive in vivo
Glycogen synthetase a Is activated by P~, SO4-- (4, 5) or glucose 6-phosphate (4) Has a high affinity for UDPG (Kin = 0.07 to 0.2 mM in the presence of glucose 6-phosphate) (4, 5) Is present in the liver after glucose or glucocorticoid treatment Is active in vivo (see Fig. 2).
6-phosphate, whereas the a enzyme is nearly fully active. As has been pointed out by Mersmann and Segal (4) the system has thus the characteristics of an on-off process. As, in vivo, neither form is influenced by the concentration of glucose 6-phosphate, we do not use the D (glucose 6-phosphate dependent) and I (glucose 6-phosphate independent) nomenclature introduced by L a m e r because it may give the false impression that in the liver, as has been shown in certain conditions for the muscle (6), the concentration of glucose 6-phosphate regulates the rate of glycogen synthesis. It is shown in Fig. 2 that this rate is highly correlated to the amount of synthetase a present in the liver.
THE CONTROL
OF GLYCOGEN
SYNTHESIS IN THE LIVER
173
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F[o. 2 Correlation between the activity of liver glycogen synthetase measured in a concentrated homogenate and the rate of the conversion in vivo of glucose to liver glycogen in the same animal. The experiments carded out in vivo lasted 1 rain and were performed by the injection of [6-SH] glucose to mice 5 rain after the injection of glucose (I rag/g) or of 0.15 M NaCl. Half of the mice had been treated with prednisolone. After the death of the animal, one-half of the liver was used for the determination of the tritiated glycogen and the other for the assay of glycogen synthetase at 0 ° in a 50yo homogenate. (©) control mice; ( 0 ) mice injected with glucose; (El) prednisolone treated mice; I prednisolone treated mice injected with glucose. Correlation coefficient: 0.94 (P < 0.001). This Figure is from De Wulf and Hers (7).
The activity of the synthetase a can be measured specifically without interference of b in the presence of 5 mM phosphate. A concentrated (50 ~o) liver homogenate, because of its high content in phosphate, has been used with success for this purpose (8, 9), but one can also utilize more simply a diluted homogenate enriched in phosphate or sulfate (5). By this method, we have followed the variations in the concentration of synthetase a in a liver homogenate incubated at 20 ° (see Fig. 3). In confirmation of the finding of Segal and co-workers (4, 11), it appears that the initial content is usually low (this is however not always true, particularly during the months of June till August; see for instance Fig. 13); after a short lag period, the concentration of synthetase a increases rapidly and reaches a plateau at approximately 60 min. This activation is accelerated by caffeine and inhibited by fluoride and
174
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FIe. 3 Influence of ATP and cyclic AMP on the activation and inactivation of liver glycogen synthetase. The liver of an untreated mouse was homogenized with 3 vol of a solution containing 150 mM sucrose and 100 mM glycylglycine buffer at pH 7.4. This homogenate, after 0 or 60 rain of preincubation at 20 °, was incubated either alone (O), or in the presence of 4 mM ATP, 8 mM Mg acetate (O), 4 m s ATP, 8 mM Mg acetate, 0.5 mM cyclic AMP (A). This Figure is from De Wulf and Hers (10). E D T A (not shown), p r e s u m a b l y by chelation o f magnesium. A T P - M g c o u n t e r a c t s the activation or causes a reconversion o f synthetase a into b when a d d e d after the synthetase has been activated. As the total activity o f the e n z y m e ( m e a s u r e d in the presence o f s a t u r a t i n g levels o f glucose 6-phosp h a t e a n d U D P G ) does n o t change consistently d u r i n g these t r a n s f o r m a t i o n s , it a p p e a r s t h a t synthetase a is f o r m e d at the expense o f synthetase b a n d vice versa, very p r o b a b l y by d e p h o s p h o r y l a t i o n a n d r e p h o s p h o r y l a t i o n , a c c o r d i n g to the reactions d e p i c t e d in Fig. 1 a n d as previously d e m o n s t r a t e d for the muscle enzyme (12).
THE CONTROL OF GLYC,(~EN SYNTHESIS IN THE LIVER THE CONTROL
BY GLUCOSE
AND
175
GLYCOGEN
The Effect of Glucose in vivo When glucose is administered intravenously to either fed or fasted mice, it induces, after a lag period of 2-3 min, a rapid rise in the rate of glycogen synthesis (Fig. 4) which parallels the conversion of synthetase b into a (see Fig. 2). There is a simultaneous fall in the concentration of glucose 6-phosphate (at least in the fed mice) and of UDPG in the liver to approximately
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FIG. 4 Time sequence of the stimulation of glycogen synthesis by a single glucose load. Glucose was injected intravenously to 24 hr fasted mice in a single dose of I mg/g body weight. At various intervals afterwards, a trace amount of [ U - t ~ "] glucose was administered intravenously and the animals were killed 1 min later. ( © ) glucose concentration in the liver at the end of the experiment; ( O ) amount of glucose converted into glycogen in 1 rain. Vertical bars represent 4- the standard error of the means; the number of experiments is given in parentheses. This Figure is from De Wulf and Hers (8).
60 ~o of their initial value (8). It is clear, therefore, that the stimulation of liver glycogen synthesis by glucose, which has been known for a long time, is not the result of a push given on the whole metabolic pathway by an increased amount of glucose but is entirely explained by the activation of glycogen syntbetase.
176
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BEKGHE
The Effect of Glucose in vitro and the Interference of Salts In order to investigate the effect of glucose on the in vitro interconvcrsion of the two forms of glycogen synthetasc, it has been necessary to remove the endogenous glucose by gel filtration of the liver extract through a column of Sephadex G-25. When a preparation obtained in this way is incubated at 20 °, the conversion of synthetase b into a levels off at values that are only 25 to 50 ~o of the activity that can be attained in the original extract; ff inorganic phosphate or sulfate is added at this moment, at a final concentration of 5-I0 mM the activation starts again and reaches nearly completion (Fig. 5).
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Influence of salt on the activation of glycogen synthetase. Mice were injected with glucagon (0.5 ~,g/g body weight) 6 rain before death in order to cause a complete inactivation of the synthetase. The livers of 7 mice were pooled and a 33 % homogenate, prepared with 50 mM glycylglycine, pH 7.4, was centrifuged at 8,000 x g for 10 rain; the supernatant was filtered through a Sephadex G-25 column and the gel eluate was incubated at 20 °, either alone or with 10 mM (NH0zSO4.
The full activation can also be obtained in one step if the salt is added from the beginning of the experiment. The level reached, but not the time required to complete the reaction, is dependent on the amount of salt added (Fig. 6); the salt effect is unspecific as it has been obtained with 3 mM Mg acetate or 0.1 M KCI. The partially activated enzyme obtained in the absence of salt has the kinetic properties of synthetase a (low Km for U D P G ) but has a lower Vmax than the fully activated preparation. It has some similarity with the partially inactive form of synthetase previously described by Steiner (13) and by Hizukuri and Larner (3). When glucose is added at a final concentration of 5%o it causes an important acceleration of the b to a conversion, in the absence, as well as in the presence
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
i 6
~
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177
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20
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~ 6 O
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TIME(min) FIG. 7 Influence of glucose on the activation of glycogen syntheta..~. Same procedure as in Fig. 5. The gel filtrate was incubated at 20 ° with or without 5~/ooglucose, in the absence or in the presence of 5 rnM P~.
of salts (Fig. 7). This effect appears specific for glucose as it was not obtained with mannose, fructose, galactose, galactosamine, 2-deoxyglucose, arabinose, xylose, mannitol, sorbitol or maltose and is still observed in the presence of caffeine. We tentatively conclude that the synthetase phosphatase is glucose sensitive* and converts the b enzyme into a form which is only partially active * We cannot, however, preclude the possibility that the glucose effect is due to an interaction with glycogen synthetase which is the substrate of the phosphatase.
178
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
in the absence of salt; the second step, presumably non-enzymatic, seems to be an unmasking of active sites by the salt. In agreement with this interpretation, we have observed that the fully activated enzyme loses a part of its activity by passage through a Sephadex column and can recover it by the addition of salt.
The Inhibition by Glycogen and the A~nity for Glucose The inhibitory effect of glycogen on the activation of the synthetase in a liver extract has been previously described (10). After removal of the small molecules by gel filtration, glycogen is no longer an inhibitor but, on the contrary, induces a stimulation; its inhibitory effect reappears as soon as
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TIME(rain) I~O. 8 Influence of the concentration of glycogen on the activation of glycogen synthetase. Same procedure as in Fig. 5. The gel filtrate was incubated at 20 ° in the presence of 5 rnM (NH~)aSO~ and of particulate glycogen at the concentrations indicated.
phosphate or sulfate are reintroduced. The doses of glycogen required (Fig. 8) to cause an inhibition are however much larger than those reported by Larner (14) to have a similar effect on the muscle synthetase phosphatase; this difference probably explains why the liver can accumulate glycogen at a much higher concentration than the muscle. The glycogen also interferes with the action of glucose by lowering the affinity of the phosphatase for the hexose. It is shown in Fig. 9 that in one experiment in which the effect of the dose of glucose was investigated, the addition of 1 ~ glycogen to the preparation increased the apparent Km from 7 rnM (1.3~) to 11.4 rnM (2.05~/oo); a higher dose o f glycogen did not further increase the Km but reduced the maximal velocity, indicating that the effect
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
179
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10
FIG. 9
Influence of the concentrations of glucose and glycogen on the activation of glycogen synthetase. Same procedure as in Fig. 5. The gel fihrato was incubated at 20 ~ with 5 mM (NI-ID2SO4in the presence of tho indicated concentrations of glucose, at three different concentrations of particulate glycogen. After 15 min, the amount of synthetase a was measured. of a high amount of glycogen cannot be completely counteracted by an excess of glucose. It must be pointed out however that the apparent K m measured in this type of experiment can depend on the stage of activation at which the glucose effect is estimated and that the kinetics of the reaction are too complex to consider the calculated K m as more than indicative.
The Effect of Glucose on the Phosphorylase Phosphatase Taking into consideration the many resemblances between the enzymatic systems which interconvert glycogen synthetase and glycogen phosphorylase, we have investigated the possibility that glucose would also stimulate phosphorylase phosphatase in the liver. Such an effect was easily demonstrated by passing a liver extract through a Sephadex column and then following the inactivation of phosphorylase; as shown in Fig. 10, the addition of glucose induced a great acceleration of the inactivation with an apparent K m equal to 7 mM (Fig. I 1). This effect was not obtained with mannose, fructose, galactose, 2-deoxyglucose, glucuronolactone, arabinose, xylose, sorbitol or mannitol. The addition of salts did not accelerate the inactivation and glycogen had only a slight inhibitory effect. It is interesting to recall that a stimulation of the muscle phosphorylase phosphatase by glucose or glycogen has been reported by Holmes and Mansour (15).
180
H . G . HERS, H. DE W ULF, W. STALMANS AND G. VAN DEN BERGHE
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FIG. 10 Influence of glucose on the inactivation o f glycogen phosphorylase. Same proe~lure as in Fig. 5. The gel filtrale was incubated with or without 5%0 glucose. P h o ~ h o r y l a s e was assayed in the absence of added AMP.
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CONCENTRATION OF GLUCOSE (mg/ml}
FIG. 11 Influence of the concentration of glucosc on the inactivation of glycogen phosphorylase. Same p r o c e d u r e a s in Fig. 10, but phosphorylase activity was measured after 5 rain incubation in the presence of ino-easing a m o u n t s of glucose.
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
181
THE CONTROL BY 3",5'-CYCLIC AMP In vivo Experiments When glucagon, epinephrine or cyclic A M P are administered to mice in which the synthetase has been previously activated by glucose or by glucocorticoids (see below), they cause a rapid reconversion of the a enzyme into b. The same effect can be observed in any series of animals that happen to have a high content o f synthetase a in their liver. We show in Fig. 12 the variation in the activity of synthetase a and in the concentration of cyclic AMP in the liver of mice that have received 0.5 ng of glucagon per g body weight. 3
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~o. 12 Influence of a small dose of glucagon on the level of cyclic A M P and of glycogen synthetase a. Glucagon (0.5 ng/g body weight) was injected intravenously to normally fed mice which were killed at various times afterwards. Cyclic A M P was measured by a modification of the method described by Breckenridge (16). Vertical bars represent 4- the standard error of the means (n = 3).
It is interesting to point out that the variation in the activity o f glycogen synthetase that can be observed in this type of experiment is much greater than the simultaneous effect on the activity of phosphorylase. This is due to the fact that, prior to any treatment, liver phosphorylase already displays a high activity which is only approximately doubled by the administration of glucagon or cyclic AMP (7).
The Stimulation of Synthetase Kinase by Cyclic A M P The hi vitro effect of cyclic AMP on the inactivation of the synthetase in the presence of ATP is illustrated in Fig. 3. This effect is in agreement with the hypothesis that the cyclic nucleotide activates a synthetase kinase that phosphorylates synthetase a into b. In a crude liver extract, a half-maximal effect was obtained with 2 × 10 -7 M cyclic AMP. With a more purified kinase,
182
H.G. HERS, H. DE WULF, W. STALMANSAND G. VAN DEN BERGHE
Bishop and Larner (17) have recently reported an affinity constant equal to 4 × 10-s M. The concentration of the nucleotide in the liver being normally between 5 and 9 x l0 -7 M, it seems that the kinase should always be fully activated. As this is obviously not the case, it appears that an important part of the nucleotide is not available for the stimulation of the kinase; in agreement with this interpretation, Jefferson et al. 0 8 ) have found that 60 % of the nucleotide is in particulate fractions. It is also interesting to mention that many enzymes are present in the tissues at a concentration of 10 -7 to 10-6 M (19) and that therefore, a great part of the nucleotide could be bound to the various enzymes which show affinity for it. The concentration of the free cyclic AMP in the liver is consequently very difficult to evaluate. THE CONTROL BY GLUCOCORTICOIDS
The in vivo Activation of Glycogen Synthetase by Glucocorticoids Since the initial work of Long and co-workers (20) in 1940, it has been repeatedly observed that the administration of glucocorticoids stimulates the deposition of glycogen in the liver of fasted as well as of fed animals. Hornbrook and co-workers (21) were the first to report that the administration of hydrocortisone induces the formation of a glycogen synthetase that is less dependent on glucose 6-phosphate than normally. De Wulf and Hers (9) have shown that, in the animals treated by prednisolone, the rate of glycogen synthesis parallels the activity of glycogen synthetase when the latter is measured in a concentrated liver homogenate; in such a preparation, obtained from control or from treated animals, glucose 6-phosphate is without influence on the activity of the enzyme. This situation has been later clarified by a better knowledge of the properties of the a and b forms of the synthetase, the effect of the hormonal treatment being clearly a conversion of the b form into a (5). It must also be emphasized that in the fasted mice, glucocorticoids stimulate glycogen synthesis without raising the glycemia (9). This indicates that the activation of the synthetase is not mediated by the glucose effect which has been described in the preceding section and is not related to the effect of the hormone on ghiconeogenesis. The stimulation of glycogen synthesis has also been observed in diabetic animals (22) and may therefore be regarded as independent of insulin. The contradictory finding reported by Kreutner and Goldberg (23) is presumably due to a too short duration (2 hr) of their experiment after hydrocortisone administration. The presence of a high amount of synthetase a in the liver of animals treated with prednisolone could result from an increased rate of activation by the phosphatase or from a decreased rate of inactivation by the kinase. We have investigated these two possibilities.
THE CONTROL OF GLYCOGEN SYNTHESIS 1N THE LIVER
183
Z
5 0
"~ l.
- PREDNISOLONE - - - CONIROL
~--
I I WITH Gt. UCOSE aO WITHCU" GLUCOSE
/o
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o2
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3 i,.--
// 4: . . . .
1.
I
I0
2'0
30
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TIME(rain) FiG. 13
Effect of prednisolone on the activation of glycogen synthetase. Same procedure as in Fig. 5, except that prednisolone was administered 3 hr before death to one group of animals. The gel filtrates were incubated at 20 ° in the presence of 5 mM (NH4)2SO4 with or without 5%0 glucose.
Modification of the Properties of Synthetase Phosphatase The administration of glucocorticoids brings about several changes in the properties of the synthetase phosphatase which are conveniently studied in a glucose-free liver extract. As shown in Fig. 13, the activity of the phosphatase has been increased by the treatment but this effect is more apparent in the absence than in the presence of glucose. Furthermore, the activity that is resistant to the inhibition by glycogen is also greater than in the control liver (Fig. 14).
---o 110 O0
O ~
~
~.
PREDNISOLONE CONTROL WITH GLUCOSE WITHOUT GLUCOSE
2
I
I
O-
I
I
10 30 50 70 CONCENTRATION OF GLYCOGEN (rng/ml) R G . 14
Interaction of glucose and glycogen in the activation of glycogen synthetas¢ of normal and glucocorti~id-tr~ted mice. Same experiment as in Fig. 13. The effect of increasing amounts of particulate glycogen has been investigated. Synthetase a was assayed after 20 rain incubation.
184
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
From these observations we tentatively conclude that glucocorticoids induce the formation of a synthetase phosphatase that is less sensitive to both glucose and glycogen. Since the normal phosphatase is still present, one can understand why the administration of glucose to animals that have been treated by prednisolone causes a further increase in the rate of glycogen synthesis (see Fig. 2). Being released from the inhibition by glycogen, the new enzyme would allow the activation of the synthetase even when the concentration of the polysaccharide in the liver is already high; not being dependent on glucose, it would also be active in the fasted animal. Mersmann and Segal (24) have reported that the activity of the synthetase phosphatase disappears from the liver of 48-hr-fasted, adrenalectomized rats and is restored by the administration of glucocorticoids. The possible relationship between these observations and those reported above is not clear.
The Properties of the Inactivating System We have conducted a series of experiments in order to investigate the possibility that glucocorticoids could interfere with one of the factors that participate in the inactivation of synthetase a, as represented in the following scheme: Synthetase a
ATP ~
Kinase ((
Glucagon
Cyclic AMP ~
Synthetase b
Cyclase<
Diesterase
AMP
It has been verified that the in vitro sensitivity of the kinase to cyclic AMP is the same whether the synthetase had been previously activated by glucose or by glucocorticoids (10). The concentration of cyclic AMP was slightly but significantly diminished in the liver of mice that had received prednisolone three hours previously (0.63/~t with S.E.M. : 0.03 for 15 determinations) as compared to untreated animals of the same group (0.83 ~.M with S.E.M. = 0.03 for 13 determinations). As discussed above, the significance of such a small change is difficult to appreciate. This decrease in the concentration of cyclic AMP is not the result of a change in the properties of the phosphodiesterase which hydrolyzes cyclic AMP, as neither the total activity of this enzyme nor its affinity for its substrate have been modified by the treatment (7). As shown in Fig. 15, we have also measured the sensitivity of mice to glucagon and observed the same rise in the concentration of cyclic AMP in the liver of glucocorticoid-treated and of control animals. Finally, the half-life of x~I-glucagon has been measured in mice and found to be not modified by the corticoid treatment.
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
PREDNISOLONE CONTROL
25
185
• o
-.& LU
20
0
05 2.5 GLUCAGON (ng/g BODYWEIGHT)
FIe. 15 Influence of the dose of glucagon on the level of cyclic AMP. Normally fed mice, treated or not with prednisolone 3 hr before, received an intravenous it~ection of glucagon and were killed 1 rain later. Vertical bars represent 4- the standard error of the means (n = 6).
We have therefore no indication that glucocorticoids could have an important influence on the system that inactivates glycogen synthetase in the liver. IS T H E R E
AN INSULIN
EFFECT?
Acute Effect The action of insulin on glycogen synthesis in the liver has for long been a controversial matter. While insulin given alone is glycogenolytic, presumably through glucagon or epinephrine secretion secondary to the hypoglycemia, insulin given with glucose induces a glycogen deposition (25). More recently, Bishop and Lamer (26) have shown that under these conditions the synthetase is in the active form. In their study of the stimulation of glycogen synthesis by glucose, De Wulf and Hers (8) have considered the possibility that the glucose effect could be mediated by the insulin secretion which is known to occur rapidly after glucose administration and they came to a negative conclusion; insulin given alone in short-term experiments (1 rain) was completely ineffective, and given with glucose had the same effect after 3 rain as glucose alone. It is also remarkable that the administration of glucose to animals made acutely diabetic by the administration of anti-insulin serum induces an important activation of glycogen synthesis (Table 2). An induction of glycogen synthesis by glucose in depancreatized (27) and in alloxanized (28) rats has also been reported. As the effect of the simultaneous administration of insulin and glucose can be explained by the action of glucose, we conclude that an acute effect of
186
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE
insulin on the activation o f glycogen synthetase in the liver has not been adequately demonstrated.
Long-term Effect W h e n insulin is administered to diabetic animals, it induces in 1-2 hr a rise in the glycogen content o f the liver and an activation o f the synthetase (29). Considering the complexity o f the regulatory processes in diabetes, these TABLE 2 INFLUENCE OF ANTI-INSULIN SERUM ON THE GLUCOSE EFFECT
Treatment
Glucose Glucose incorporated into glycogen concentration rng/g liver /~g/min/gmuscle ~,g/min/g liver
Control (5) Glucose (5) Anti-insulin serum (5) Anti-insulin serum + glucose (5)
1.70 ± 0.05 2.88 4- 0.15 2.27 -4- 0.11 3.92 ± 0.26
4.0 =: 1.1 5.8 2-_ 1.6 i.6 ± 0.3 1.9 ±_ 0.4
7.1 54.1 4.4 36.4
___3.7 -.: 5.0 ± 1.2 ± 4.0
Glucose (1 mg/g body weight) or 0.15 M NaCI was injected intravenously to normally fed mice 20 rain after the administration of anti-insulin serum (10 mU/g body weight); control animals receivod only isotonic saline. Five minutes later, a trace amount of [6-Sill glucose was given intravenously. The mice were killed l min later and the radioactivity of liver and carcass glycogen was determined. Values shown are means 4- standard error of the means with the number of animals in parentheses. effects are difficult to interpret. According to a preliminary report (30) the activity o f synthetase phosphatase is reduced to less than 50 ~o o f the normal value in the liver o f the pancreatectomized dogs.
THE F U T I L E CYCLES I n the metabolism o f glycogen and its regulation, the existence of futile cycles m a y be considered at two levels:
S G ylcogen"~ Glucose-I-p
-~.~
I) The simultaneous operation o f the synthesis and degradation o f glycogen would create a futile cycle in which as much as 60/zmoles o f U T P could be c o n s u m e d per hr and per g o f liver. This waste, however, does not occur at an important rate (1, 2) because the synthesis o f glycogen stops when its degradation is induced and conversely. The main regulators that operate simultaneously in an antagonistic manner on synthesis and degradation are glucose and cyclic A M P .
THE CONTROL OF GLYCOGEN SYNTHESIS IN THE LIVER
187
2) A second futile cycle exists at the level of phosphorylase as well as of glycogen synthetase, which can both be activated and inactivated simultaneously. At this level, the waste of energy due to the futile cycles is several orders of magnitude smaller than in the case of glycogen and can be considered negligible. Up to now, we have no indication that any of the regulators that act either on the synthetase kinase or on the synthetase phosphatase would have a reverse effect on the antagonistic enzyme. It is therefore reasonable to admit that activation and inactivation occur simultaneously, and that the levels of active synthetase or of active phosphorylase reflect the steady state situation achieved by the activating and inactivating enzymes. It is remarkable, for instance, that, in the animals made acutely diabetic by the administration of anti-insulin serum, the hyperglycemia did not by itself elicit an activation of the synthetase; this can be explained by the fact that, as reported by Jefferson et al. (17), the same treatment causes a large increase in the concentration of cyclic AMP in the liver and that the activation of the phosphatase by glucose is therefore equilibrated by the activation of the kinase by cyclic AMP. A similar situation might exist in chronic diabetes and has also been experimentally produced by the simultaneous administration of glucose and glucagon to mice (7). Such a system offers the advantage of a greater number of regulatory sites; its main interest appears however to be that the dose response for an effector acting on one of the antagonistic enzymes displays a S-shaped curve. If one considers, for instance, the activation of glycogen synthesis by glucose, it appears that, at the normal level of glucose in the blood, the synthetase is still usually mostly inactive and is only activated by a further increase of the glycemia. However, the activation of the synthetase phosphatase by glucose in vitro displays Micha~lis-Menten kinetics. This apparent contradiction is easily understood if one admits that, in vivo, the activity of the synthetase phosphatase reached at normal glycemia is counteracted by a slightly higher activity of the synthetase kinase maintaining the synthetase in the b form and that its activation occurs only when an additional dose of glucose allows the phosphatase to overcome the kinase. The same holds also for the activation produced by glucocorticoids: the increased activity of synthetase phosphatase, brought about by the hormone, need not be large in order to convert an important proportion of the synthetase into the active form. Finally, it is not necessary to assume that the synthetase kinase shows an absolute requirement for cyclic AMP as its activity in the absence of the nucleotide could be balanced by that of the synthetase phosphatase.
188
H . G . HERS, H. DE WULF, W. STALMANS AND G. VAN DEN BERGHE SUMMARY
In the liver, glycogen synthetase exists in two forms, one of them (a) active, the other (b) inactive in the ionic conditions which exist in the cell. These two forms are interconvertible, presumably by phosphorylation and dephosphorylation under the action of a specific kinase and phosphatase respectively. It seems probable that these two enzymes operate simultaneously and that the level of the synthetase a in the liver results from the addition of their antagonistic effects. In normal mice, glycogen synthetase is predominantly in the b form. It is converted into a within 3 to 5 min after the intravenous administration of glucose or within 2 or 3 hr after the administration of glucocorticoids. The a enzyme can then be reconverted into b within 1 to 3 rain after the administration of glucagon, epinephrine or cyclic AMP. The effect of these various effectors has also been demonstrated in vitro, mostly thanks to the use of liver extracts from which glucose had been removed by gel filtration through a Sephadex column. The complete activation of glycogen synthetase in vitro requires the presence of salts. In their absence, synthetase b is converted into a form which has the kinetic properties of synthetase a but which is less active. Glucose markedly enhances the activation, both in the presence and in the absence of salts while glycogen is an inhibitor in the presence of salts only. The affinity of the synthetase phosphatase for glucose is decreased when glycogen is present. A stimulation of the phosphorylase phosphatase by glucose has also been observed. The treatment of mice by glucocorticoids induces the appearance in the liver of a synthetase phosphatase that is less sensitive to glucose stimulation and to glycogen inhibition than normally. No effect of the treatment on the system that inactivates glycogen synthetase could be demonstrated. An effect of cyclic AMP on the synthetase kinase is easily demonstrable in a liver extract in which the synthetase has been previously activated either by glucose or by glucocorticoids; a half-maximal effect has been obtained with a concentration equal to 2 × 10-7 Mcyclic AMP. The two main effectors that act antagonistically on the glycogen synthetase and glycogen phosphorylase appear to be glucose and cyclic AMP; thanks to their action, the synthesis of glycogen is inhibited while its degradation is stimulated and vice versa.
REFERENCES I. D. STE'I-I'EN, JR. and G. E. BOXER, The rate of turnover of liver and carcass glycogen, studied with the aid of deuterium, J. Biol. Chem. 155, 231-236 (1944). 2. K. H. SHUt.Land J. MAYER,The turnover of liver glycogen in obese hyperglycemic mice, J. Biol. Chem. 218, 885-896 (1956).
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3. S. HlzLrk'-tn~ and J. LARNm%Studies on U D P G : a-l,4-glucan a--4-glucosyltransferase. VII. Conversion of the enzyme from glucose-6-phosphate-dependent to independent form in liver, Biochemistry 3, 1783-1788 (1964). 4. H. J. MeRSMAt,~ and H. L. St'GAL, An on-off mechanism for liver glycogen synthetase activity, Prec. Natl. Acad. Set., U.S. .58, 1688-1695 (1967). 5. H. DE WULF, W. STAI.MANSand H. (3. HERS, The influence of inorganic phosphate, adenosine triphosphate and glucose 6-phosphate on the activity of liver glycogen synthetase, European J. Biochem. 6, 545-551 (1968). 6. R. PmAs and K. STANELOm, In rive regulation of rat muscle glycogen synthetase activity, Biochemistry 8, 2153-2160 (1969). 7. H. DE WULF and H. G. HERS, The role of glucose, glucagon and glucocorticoids in the regulation of liver glycogen synthesis, European J. Biochem. 6, 558-564 (1968). 8. H. DE WULE and H. G. HERS, The stimulation of glycogen synthesis and of glycogen synthetase in the liver by the administration of glucose, European d. Biochem. 2, 50-56 (1967). 9. H. DE WULF and H. G. HERS, The stimulation of glycogen synthesis and of glycogen synthetase in the liver by glucocorticoids, European d. Bioehem. 2, 57-60 (1967). 10. H. DE WULF and H. G. HERS, The interconversion of liver glycogen synthetase a and b in ~'itro, European .I. Biochem. 6, 552-557 (1968). 11. A. H. GOLD and H. L. SEOAL,Time-dependent increase in rat liver glycogen synthetase activity in vitro, Arch. Biochem. Biophys. 120, 359-364 (1967). 12. D. L. FRIEDMAN and J. LARNER, Studies on UDPG-a-glucan transglucosylase. Ill. Interconversion of two forms of muscle UDPG-a-glucan transglucosylase by a phosphorylation-dephosphorylation reaction sequence, Biochemistry 2, 669-675 (1963). 13. D. F. STEINER,Reversible inactivation of glycogen synthetase, Biochim. Biophys. Acta .54, 206-209 (1961). 14. J. LARNER,Hormonal and nonhormonal control of glycogen metabolism, Trans. N.Y. Acad. Sci. 29, 192-209 (1966). 15. P.A. HOLMESand T. E. MANSOUR,Glucose as a regulator of glycogen phosphorylase in rat diaphragm. IL Effect of glucose and related compounds on phosphorylase phosphatase, Biochim. Biophys. Acta 156, 275-284 (1968). 16. B. McL. BRECKENRIDGE,The measurement of cyclic adenylate in tissues, Prec. Natl. Acad. Sci., U.S. 52, 1580-1586 (1964). 17. J. S. BISHOP and J. LARNER,Presence in liver of a Y,5'-cyclic AMP stimulated kinase for the I form of UDPG-glycogen glucosyltransferase, Blochim. Biophys. Acta 171, 374--377 (1969). 18. L. S. JEFFERSON,J. H. Ex'roN, R. W. BUTCHER, E. W. SUTHERLANDand C. R. PARK, Role of adenosine 3',5'-monophosphate in the effects of insulin and anti-insulin serum on liver metabolism, d. Biol. Chem. 243, 1031-1038 (1968). 19. P. A. SRER~, Enzyme concentrations in tissues, Science 158, 936-937 (1967). 20. C. N. H. LONe, B. KATZlN and E. G. FRY, The adrenal cortex and carbohydrate metabolism, Endocrinology 26, 309-344 (1940). 21. K. R. HORNBROOK,H. B. BURCH and O. H. LOWRY, The effects of adrenalectomy and hydrocortisone on rat liver metabolites and glycogen synthetase activity, Mol. Pharmacol. 2, 106-116 (1966). 22. W. TARNOWSKI,i . KITTLERand H. HH.Z, Die Wirkung yon Cortisol auf die Umwandlung yon Hexosephosphaten in Glykogen, Biochem. Z. 341, 45-63 (1964). 23. W. KREOTNERand N. D. GOLDBERG, Dependence of insulin of the apparent hydrocortisone activation of hepatic glycogen synthetase, Prec. Natl. Acad. Sci., U.S. 58, 1515-1519 (1967). 24. H. J. MERSMANNand H. L. SEGAL,Glucocorticoid control of the liver glycogen synthetase-activating system, 3. Biol. Chem. 244, 1701-1704 (1969). 25. J. B•RTIIET, P. JACQUES,H. G. HERS and C. De DUVE, Influence de l'insuline et du glucagon sur la syntht~.se du glycog~ne h~patique, Biochim. Biophys. Acta 20, 190-200 (1956).
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26. J. S. BISHOP and J. LARNER, Rapid activation-inactivation of liver uridine diphosphate glucose-glycogen transferase and phosphorylase by insulin and glucagon in vivo, J. Biol. Chem. 242, 1354--1356 0967). 27. R. W. LONOLeY, R. J. BORTNICK and J. H. ROE, Rate of glycogenesis in liver of depancreatized rat after parenteral administration of glucose and fructose, Proc. Soc. ExptL BioL 94, 108-1 I0 (1957). 28. B. FRIEDMANN, E. H. GOODMAN, JR. and S. WEINHOUSE, Effects of glucose feeding, cortisol, and insulin on liver glycogen synthesis in the rat, Endocrinology 81, 486-496 (1967). 29. D. F. STERNERand J. KING, Induced synthesis of hepatic uridine diphosphate glucoseglycogen glucosyl-transfera.se after administration of insulin to alloxan-diabetic rats, J. Biol. Chem. 239, 1292-1298 (1964). 30. J. S. BISHOP, N. D. GOLDBERG, F. GRANDE and J. LARNER, Decreased activity of glycogen synthetase D phosphatase in insulin-insensitive diabetic dog liver, Diabetes 18, 337 (1969). ACKNOWLEDGMENTS This work was supported by the Fonds de la Recherche Seientifique M~dicale and by the U.S. Public Health Service (Grant AM 9235). H. De Wulf and G. Van den Berghe are "Aangesteld Navorser" and W. Stalmans is "Aspirant" of the "Nationaal Fonds Poor Wetenschappelijk Onderzoek".
Note Added ia Proof. It has now been clearly demonstrated that the enhancement of the in vitro activation of glycogen synthetase by glucose or caffeine is due to a shortening of the lag period that precedes the action of the synthetase phosphatase; glycogen (see Fig. 8), fluoride and A M P have an opposite action on the lag period. All these substances (with the exception of glycogen) are indeed ineffective when added after the latency period. From these and other observations we have concluded the existence of an active (a) and of an inactive (b) form of synthetase phosphatase and we have postulated the existence of a "synthetase phosphatase activating enzyme" stimulated by glucose and caffeine and inhibited by glycogen, A M P and fluoride. The livers of animals that have been treated with glucocorticoids contain a larger amount of this activating enzyme and are therefore less sensitive to stimulation by glucose as well as to inhibition by glycogen (De Wulf, Stalmans and Hers, submitted to European J. Biochem.). The same livers contain a larger amount of phosphorylase phosphatase (Stalmans, De Wulf, Lederer and Hers, submitted to European J. Biochem.). As the phosphorylase phosphatase is stimulated by caffeine and inhibited by A M P and fluoride, it has many similarities with the synthetase phosphatase activating enzyme.