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On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat
225
Biochimica et Biophysiea Acta, 544 (1978) 225--233 © Elsevier/North-Holland Biomedical Press
BBA 28692
ON THE ACTIVITIES OF GLYCOGEN PHOSPHORYLASE AND GLYCOGEN SYNTHASE IN THE LIVER OF THE RAT
JULIE D. NEWMAN and J. McD. ARMSTRONG
Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia) (Received April 3rd, 1978)
Summary A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.
Introduction
The critical enzymes in glycogen synthesis and degradation in the liver are glycogen synthase (UDPglucose--glycogen glucosyltransferase, EC 2.4.1.11) and glycogen phosphorylase (1,4-~-D-glucan : orthophosphate a-giucosyltransferase, EC 2.4.1.1). These enzymes are regulated by interconversion between active and inactive forms by enzyme-catalysed covalent modification [1,2]. The effects of various modulators on the interconversion processes are difficult to measure in vivo, because the regulatory system is sensitive to neural stimulation [3,4] or to humoral products resulting from surgical trauma and from other manipulations [5,6]. The reported activities of these two enzymes in liver show quite wide variation particularly for the active forms of the enzymes [5,9, 16--19]. Furthermore, satisfactory methods for the estimation of total phos-
226 phorylase activity in liver have been available only since 1975 [7,8]. Prior to this, estimates of total hepatic phosphorylase relied on activation by epinephrine or glucagon [9,10], or on activation by added phosphorylase kinase [11]. In the course of an investigation of the in vivo regulation of tissue glycogen levels by peptides related to growth hormone, a need arose for establishing a suitable technique for the measurement of the activities of glycogen synthase and phosphorylase in intact animals, with the requirements that the values obtained in control animals were consistent on a long-term basis, and approximated to the values pertaining in vivo. A study was made, based on the observations of Hornbrook and Brody [5] and of Stalmans et al. [6], of procedures for obtaining liver samples, to find conditions under which stimulation of the regulatory system, and the consequent changes in the activities of glycogen synthase and phosphorylase, could be minimised. Various tests of the method finally selected have been made to establish whether the system responded in the anticipated physiological manner to appropriate stimuli. The results obtained are discussed in terms of the control of hepatic glycogen metabolism by the alteration in the balance of a cycle between glycogen and glucose 1-phosphate. The model proposes that net synthesis or degradation of glycogen is achieved by modulating the relative activities of the active forms of glycogen synthase and phosphorylase, which are present at high and equivalent activities in the resting, glycostatic condition. This provides the necessary basis on which studies of the in vivo regulation of glycogen metabolism can be made and integrated with in vitro data. Materials and Methods
Animals. Unless otherwise specified, male rats from an outbred colony of a hooded Lister strain, weighing between 160 and 190 g, were used. All animals were fed ad libitum on a standard diet. On the afternoon of the day preceding an experiment, food was removed from the cages, although the animals were allowed free access to water. They were then used between approx. 9.30 a.m. and 11 a.m. on the following day, giving a nominal fasting period of 18--20 h. Intraperitoneal injections were made in each animal's lower left quadrant, while intravenous injections used the femoral vein, which was exposed by cutting the skin on the front of the thigh. Chemicals. Rabbit liver glycogen, UDPglucose, glucose 6-phosphate and 5'-AMP were obtained from Sigma Chemical Co. Glucose 1-phosphate (potassium salt) was from Calbiochem. UDP-[U-14C]glucose was obtained from the Radiochemical Centre, Amersham, and was diluted for use to a specific activity of 0.08 gCi/pmol. Insulin, free of glucagon, was from Eli Lilly and Co. All other chemicals were of the highest purity available and were used without further purification. Liver homogenates were prepared in a Potter homogeniser using three downand-up strokes at 3000 rev./min. Liver samples of approx. 1 g wet weight of liver were homogenised in 2 ml of cold buffer (50 mM triethanolamine-HC1, 5 mM EDTA, 25 mM sodium fluoride and 5 mM 2-mercaptoethanol, adjusted to pH 7.5 with sodium hydroxide). The homogenate was centrifuged for 15 min at 10 000 × g and 0°C and the supernatant was used for enzyme assay.
227
Stability and storage of homogenates. Phosphorylase and synthase activities were determined for freshly prepared homogenates and again after storage under various conditions. The phosphorylase activity remained unchanged for at least 6 h at 0°C, and also when stored overnight at --20°C after being quickfrozen in an acetone/solid COs bath. Synthase activity did not change appreciably over approx. 4 h at 0°C, but a marked loss of activity was observed for quick-frozen homogenates stored at --20°C overnight. Therefore, both phosphorylase and synthase activities were determined on freshly prepared homogenates held at 0°C for not more than 4 h. For large numbers of samples the synthase activities were determined immediately after the preparation of the homogenates, which were then quick-frozen and stored at --20°C overnight, after which phosphorylase activities were determined. Enzyme assays. The active and total forms of both synthase and phosphorylase were determined. Phosphorylase was assayed according to Stalmans et al. [7]. Final concentrations of reagents in the assay were 50 mM glucose 1-phosphate, pH 6.5, 1% glycogen and 0.15 M sodium fluoride. Phosphorylase a activity was measured in the presence of 0.5 mM caffeine, which suppresses the activity of liver phosphorylase b. Total liver phosphorylase was measured in the presence of 1 mM 5'-AMP and 0.5 M sodium sulphate. Activity was measured as the rate of release of inorganic phosphate, which was determined as described by Fiske and Subbarow [12] on aliquots of the incubation mixture taken at 5, 10 and 15 min after the initiation of the reaction. Glycogen synthase activity was determined as described by Thomas, Schlender and Lamer [13], using the incubation conditions for liver homogenates described by de Wulf et al. [14]. Final concentrations in the assay mixture were 0.05 M triethanolamine-HC1, 5 mM EDTA, pH 7.4, 5 mM UDPglucose, 1% glycogen and either 10 mM sodium sulphate (synthase a) * or 6.7 mM glucose 6-phosphate (total synthase). Aliquots of the incubation mixture were taken at 2, 4 and 6 min, and the rate of incorporation of labelled glucose into glycogen was determined. The results of both assays were calculated as units of enzyme per g of wet liver (1 unit--- 1 ~mol of substrate transformed per min) and as the activity ratio, that is, the fraction of enzyme present in the active form. Blood glucose was determined on samples of blood from the tall of the animal, using "Dextrostix" and an Ames reflectance meter (Miles Laboratories Inc., Elkhart, Indiana). Radioactivity was determined by liquid scintillation counting in a Packard Tricarb counter. Results
Traumatic effects in taking liver samples Preliminary studies ( T a b l e d confirmed earlier observations [5,6] that decapitation of conscious rats prior to taking liver samples resulted in very high levels of phosphorylase a activity, with little improvement when anaesthetised * T h e n o m e n c l a t u r e for t h e a c t i v e (a) and inactive (b) f o r m s o f p h o s p h o r y l a s e and s y n t h a s e is that sugg e s t e d b y M e r s m a n n and Segal [ 2 6 ] .
228 TABLE I EFFECTS OF VARIOUS SAMPLING PROCEDURES GLYCOGEN METABOLISM IN RAT LIVER
ON THE ACTIVITY
OF THE ENZYMES
OF
R a t s w e r e f a s t e d o v e r n i g h t a n d g i v e n a n i n t r a v e n o u s i n j e c t i o n o f p h y s i o l o g i c a l saline or of g l u c o s e ( 0 . 5 g/ kg b o d y weight). Where anaesthetised animals were used, these injections were given after the animal had b e c o m e u n c o n s c i o u s . L i v e r s a m p l e s w e r e t a k e n b y t h e p r o c e d u r e s d e s c r i b e d 15 m i n a f t e r t h e i n j e c t i o n , a n d t h e a c t i v i t y r a t i o s o f g l y c o g e n p h o s p h o r y l a s e a n d s y n t h a s e w e r e d e t e r m i n e d . B o t h h o o d e d a n d Wistarstrain rats were tested. Procedure
Strain
Percentage phosphorylase a
Percentage synthase a
Saline
Glucose
Saline
Glucose
a
C o n s c i o u s rats, i n j e c t e d , stunned and decapitated
Wistar Hooded
90.0±4.0 97.1±1.1
85.0±5.0 94.8±3.8
6.7±1.2 31.0±1.2
7.0±1.6 28.0±0.6
b
Anaesthetised rats, injected, decapitated
Wistar Hooded
-97.8±0.6
-88.3±2.4
7.1±1.6 27.5±2.7
5.2±0.5 37.7±3.5
c
Anaesthetised rats, a b d o m e n o p e n e d , i n j e c t e d , liver l o b e c u t off with scissors
Wistar Hooded
53.9±2.3
60.5±9.1
49.2±7.5
57.1±3.0
d
As in c, b u t i n j e c t e d w i t h p r o p r a n o l o l (1 m g / k g ) p r i o r to further treatment
Wistar Hooded
69.0±4.1 59.5±0.8
64.7±1.8 61.5±4.2
25.5±2.4 44.6±3.2
25.2±1.8 69.7±0.7
e
Anaesthetised, abdomen opened, injected, portal vein clamped b e f o r e c u t t i n g o f f liver l o b e w i t h scissors
Wista~ Hooded
53.0±1.8 47.8+2.3
-22.2±3.1
56.0±2.0 52.2±4.5
64.6±4.3 68.0±5.9
f
Anaesthetised, abdomen opened, i n j e c t e d , liver l o b e f r e e z e c l a m p e d w i t h liquid-nitrogen*cooled
Wistar Hooded
51.6±2.2 48.0±2.6
-20.5±0.5
46.2±1.0 54.0±3.4
-79.0±4.0
Wonenberge clamp
rats were used. When liver samples were excised directly from animals prepared surgically as described below, the phosphorylase a levels were reduced considerably, as previously observed by Stalmans et al. [6]. However, it was not clear whether traumatic effects on enzyme activity had been eliminated by this procedure, and it was decided to use the response of the activity ratios of phosphorylase and glycogen synthase to an intravenous glucose load (0.5 g/kg body weight) as a test for effects of trauma. The rationale of this test is that glucose has been shown to decrease phosphorylase a activity and to increase synthase a activity in the liver [6]. Traumatic effects occurring subsequent to the administration of glucose should tend to obscure this response. It may be seen that liver samples obtained after decapitation or by direct excision showed activity ratios for the two enzymes which were essentially similar whether or n o t glucose was administered (Table I). This suggested that the trauma produced by these procedures acted on the glycogen regulatory system to promote extensive glycogenolysis, thereby masking any earlier modulatory effects on the activities of glycogen synthase and phosphorylase. In rats which were injected with the beta blocking agent, propranolol, prior to excising the liver sample (Table I), there was a marked increase in the glycogen synthase activity ratio in response to glucose in hooded rats but not in Wistar-strain animals. However, the phosphorylase activity ratio did n o t change
229 significantly with glucose administration. This would suggest that cutting the liver lobe stimulates glycogenolysis, presumably by stimulation of the sympathetic nervous system. The involvement of humoral agents in the effects of excision on enzyme activities was demonstrated in the following experiment. Just before the liver sample was taken, the portal vein was clamped with an artery clamp. It may be seen that this resulted in an increase in the activity ratio of the synthase, which was increased further by the glucose load. Similarly, the phosphorylase activity ratio decreased, and was lowered considerably more in animals given a glucose load (Table I). As a result of these findings, the following procedure was adopted for obtaining liver samples. It is essentially that described by Stalmans et al. [6] for obtaining serial liver samples from individual animals.
Recommended method for obtaining liver samples Animals are prepared surgically as follows. Rats are anaesthetised by intraperitoneal injection of sodium pentobarbitone {60 mg/kg b o d y weight) and kept warm in still air. This dose maintains deep anaesthesia for at least 90 min. The abdominal cavity is opened by a midline incision from the sternum to the pelvis. A lateral cut of the abdominal wall from the midline incision to the animal's right at the lower level of the rib cage produces a flap which may be used to expose the liver. The a b d o m e n is then covered with a gauze moistened with warm isotonic saline. If blood glucose is monitored during this procedure, it should remain essentially constant during surgery. When a liver sample is to be taken the liver is exposed, raised gently, and a portion of the liver is frozen in situ using Wollenberger tongs [15] cooled in liquid nitrogen *. The frozen sample is broken off and stored in liquid nitrogen until it can be h0mogenised. Care must be taken in raising the liver, since excessive manipulation or the use of forceps stimulates changes in the activity ratios of the enzymes. The liver lobe is gently lifted with the broad flat end of a scalpel handle, and then with a finger. As may be seen in Table I, the results for this procedure are similar to those obtained by clamping the portal vein, with the activity ratios of both enzymes responding appropriately to a glucose load. This freeze-clamping procedure was used in all subsequent experiments. Synthase and phosphorylase activities in the normal fasted rat Fig. 1 shows that there was a marked positive correlation between phosphorylase and synthase activities in the liver of the normal fasted rat, whether expressed as the total activities of each enzyme or as the amounts of the active forms. The group represents various normal animals used as controls in different experiments over approx. 2 years. It is clear that the absolute activities showed considerable variation from one animal to another. In contrast, the activity ratios of the two enzymes remained essentially constant over the group. This is reflected in the magnitude of the standard error of the mean, expressed as a percentage of the mean. It may be seen (Table II) that there is a 4-fold (approx.) reduction in the variability of the activity ratios of synthase and phosphorylase, as compared to the absolute activities. The change in the * C o o l i n g w i t h d r y ice is u n s a t i s f a c t o r y , and results in v e r y l o w p h o s p h o r y l a s e activities.
230
.•25
A
B
• ° "~w ~1o!
~2C +
: i : .o
~° 15 .J >- IC
5
o
i i
I &.
/
I
1
I
2 3 SYNTHASE ( a + b ) ( u n i t s / g )
J
:
0"6 1'2 SYNTHASE a ( u n i t s / g )
1.8
F i g . 1. C o r r e l a t i o n o f P h o s p h o r y l a s e and glycogen synthase activities in the liver of normal fasted rats. L i v e r s a m p l e s w e r e o b t a i n e d b y f r e e z e - c l a m p i n g i n s i t u . O t h e r d e t a i l s a r e as i n t h e l e g e n d t o T a b l e n . I n A , t o t a l p h o s p h o r y l a s e a c t i v i t y (a + b ) is p l o t t e d a g a i n s t t o t a l s y n t h a s e a c t i v i t y (a + b ) , w h i l e i n B o n l y t h e a c t i v i t i e s o f t h e a c t i v e f o r m s a r e p l o t t e d . I n e a c h c a s e t h e s o l i d l i n e is f o r t h e r e g r e s s i o n e q u a t i o n o f y = fix, as d e s c r i b e d i n T a b l e I I . S t a t i s t i c a l t e s t i n g s h o w e d t h a t t h i s l i n e is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e l i n e y = a + ~ x , a n d t h a t (~ w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o .
activities of the two enzymes in response to various stimuli is important in studies on the regulation of glycogen metabolism, and the results in Table II show that activity ratios are satisfactory for comparing such changes in different animals. The variability in absolute activities has generally restricted such experiments to comparison of serial tissue samples from individual animals [6]. It also may be seen that approximately one half of either enzyme was present in its active form in normal fasted rats. From the regression analysis in TABLE
II
PHOSPHORYLASE FASTED RAT
AND
GLYCOGEN
SYNTHASE
ACTIVITIES
IN THE
LIVER
OF THE NORMAL,
Activities of the two enzymes, as active forms and total activities, were determined on liver samples from 72 rats after injection of physiological saline. The measurements have been made in small groups of animals, over a period of approx. 20 months. Activities are reported as units of enzyme per g of liver; the a c t i v e f o r m i s r e p o r t e d as a a n d t o t a l a c t i v i t y a s a + b. T h e r a t i o o f a c t i v e t o t o t a l e n z y m e is s h o w n a s % a . Phosphorylase
Mean tS.E. S . E . as % o f m e a n
Synthase
a
a + b
%a
a
a + b
%a
5.76 0.25 4.3
12.44 0.56 4.5
46.87 0.49 1.04
0.998 0.036 3.6
1.929 0.070 3.6
52.08 0.56 1.07
Total enzyme
Active forms
Correlation coefficient
0.839
0.748
Regression coefficient of phosphorylase on synthase *
6 . 4 6 -+ 0 . 1 5
5.72 ± 0.16
Difference between regression coefficients
0.73 (0.4 > P > 0.3)
* F o r t h e l i n e y = bx, b o t h v a r i a b l e s s u b j e c t t o e r r o r , b u t e a c h o f u n i f o r m y l a s e , g - 1 x ~ u n i t s o f g l y c o g e n s y n t h a s e , g-1.
weight, y ~ units of phosphor-
231 TABLE In EFFECTS OF H O R M O N E S A N D F E E D I N G ON G L Y C O G E N S Y N T H A S E AND P H O S P H O R Y L A S E ACTIVITES IN RAT LIVER R a t s w e r e a n a e s t h e t i s e d a n d t h e i r a b d o m e n s w e r e o p e n e d . Saline o r h o r m o n e s w e r e i n j e c t e d i n t o t h e f e m o r a l vein. A f t e r 1 5 rain liver s a m p l e s w e r e t a k e n b y f r e e z e - c l a m p i n g in situ. T h e rats used w e r e f a s t e d o v e r n i g h t , e x c e p t f o r t h e f e d g r o u p , a n d all g r o u p s w e r e used o n t h e m o r n i n g o f t h e e x p e r i m e n t . E a c h g r o u p c o n t a i n e d s e v e n rats.
Condition
B l o o d glucose ( m g / 1 0 0 ml)
A c t i v i t y r a t i o (%) Synthase
Phosphorylase
F a s t e d rats Saline Insulin (0.07 unit/kg) Glucagon (0.03 mg/kg)
74 ± 4 68 ± 3 120 ± 3
54.3 -+ 1.7 6 2 . 4 ± 3.4 1 8 . 5 -+ 1.2
4 7 . 6 ± 2.2 3 2 . 2 ± 1.0 8 2 . 0 ± 3.3
F e d rats Saline
105 ± 6
6 4 . 8 ± 4.7
4 1 . 5 ± 3.8
Table II, the ratio of phosphorylase activity to synthase activity in the liver was 6 : 1 (approx.) under the conditions of assay. This proportionality was observed for both the total activities and the active forms of the two enzymes, and implies some balance between the activities of glycogen synthase and phosphorylase in the liver.
Modulation of enzyme activity If the results obtained by the freeze-clamping procedure are a valid approximation of the activities of the two enzymes in vivo for normal animals, then the glycogen metabolising system should respond in the anticipated manner to appropriate stimuli. It may be seen (Table III) that insulin increased the activity ratio of synthase and decreased the activity ratio of phosphorylase, consistent with the giycogenic effects of this hormone, whereas the activity ratio increased for phosphorylase and decreased for synthase in animals treated with the glycogenolytic agent, glucagon. When fed animals were compared with fasted animals the changes were qualitatively similar to those for insulin, although rather smaller. The effects of insulin and feeding were similar to those reported for a glucose load in Table I. Discussion The results in Tables I and II confirm and extend the observations of Hornbrook and Brody [5] and Stalmans et al. [6] that trauma can markedly increase the extent of phosphorylase activation in the liver. It is further shown that trauma causes extensive conversion of glycogen synthase to its inactive form. It is our impression that liver glycogen synthase a activity is a more sensitive indicator of trauma than phosphorylase a activity. Of the procedures for obtaining liver samples which were tested, there are two methods which seem to give reliable results and which may be a fair app]coximation of the conditions existing in vivo prior to taking the sample. These are by freeze-clamping the liver in situ, and by clamping the portal vein and restricting blood flow in the
232 liver prior to excising a liver sample. Both methods give essentially similar results for phosphorylase and synthase activity ratios, and these ratios respond as expected to a glucose load [6]. From Fig. 1 it may be seen that the amounts of phosphorylase and synthase per g of liver may vary widely, whether as total enzyme or the active form. However, the correlation coefficients indicate that the amount of phosphorylase is proportional to the amount of synthase present. This variability may present difficulties when units of enzyme activity are compared between animals. When the results are calculated as activity ratios the variability between animals is reduced approx. 4-fold, and there is a remarkable constancy in the activity ratios of phosphorylase and synthase over the entire group of animals used. The total activities of phosphorylase and synthase reported in Table II lie within the range of values obtained by other workers in studies on intact animals [9,6,17--19]. There is poor agreement between our results and published data for the activity ratios of the two enzymes; this may reflect traumatic effects arising from sampling procedures. The most notable difference is the activity ratio of 0.55 for glycogen synthase for normal fasted rats, as compared with values of between 0.02 and 0.2 obtained by others. The only comparable values (approx. 0.4) were obtained for perfused liver [20,21]. This raises the question of the in vivo activity ratios of the two enzymes. If it is accepted that trauma produced by tissue sampling is a major factor in the observed values, then the higher the activity ratio for synthase and the lower the activity ratio of phosphorylase, the more likely are the values to approximate to the in vivo condition. Other criteria which may be applied rely on the response of the activity ratios to modulatory effects, such as fasting, feeding, glucose loads and the actions of injected hormones. Such responses must be consistent with the expected changes in biochemical and physiological status. The directions of change in the activity ratios (Tables I, III) fulfil these expectations, being consistent with net synthesis, degradation or stasis of liver glycogen. The qualitative effects resemble other reported data [6,16,17,22]. On the basis of fulfilling the above criteria, and the consistency of the activity ratios obtained with many animals over a long time, the results reported here are probably a reasonable approximation of the activity ratios of phosphorylase and synthase in the liver of the fasted rat. If this is so, then some conclusions on the control of glycogen metabolism may be drawn. Firstly, it was found that a b o u t half the amount of each enzyme was present in the active form. Secondly, the marked correlations between the activities of the two enzymes indicates some balance in their action in the normal rat. The measured activities of phosphorylase and synthase reported here are very different from one another. However, it must be remembered that these values refer to the activities under the conditions of assay rather than those existing in the cell. While the assay conditions for synthase correspond to saturating substrate concentration at physiological pH in the appropriate direction of synthesis, this is n o t so for phosphorylase. This enzyme was assayed at pH 6.5, also in the direction of synthesis. Therefore, the two activity measurements cannot be compared directly, and it is necessary to correct the results for phosphorylase to the equivalent activity at pH 7.4 in the direction of gly-
233 cogen degradation. From published data on the pH vs. activity profiles and relative rates of the synthetic and degradative reactions catalysed by phosphorylase [23,24], the corrected activity of phosphorylase becomes one-sixth of the activity measured by the assay procedure. The measured activities of synthase and phosphorylase were in the ratio approx. 1 : 6 (Table II), so that the maximum activities of the two enzymes for the synthesis and degradation of glycogen at pH 7.4 are probably essentially equal. This would suggest that the two activities are in balance in the normal fasted rat, and that glycostasis is maintained by a balanced futile cycle of glycogen synthesis and degradation. Such a cycle has been postulated previously [25]. Furthermore, our results on the effects of insulin, of glucagon and of a glucose load are consistent with a change from glycostasis towards net synthesis or degradation of glycogen, by altering the activities of the two enzymes so that the futile cycle becomes unbalanced. Thus, changes in the amount of liver glycogen would occur as the net difference in the rates of its synthesis and degradation. None of the conditions tested resulted in the complete conversion of either of the enzymes to its inactive form. Consequently, futile cycling between glycogen and glucose 1-phosphate would occur at all times, even though the amount of tissue glycogen was changing. Acknowledgements This work was supported in part by a grant from the Australian Research Grant Committee. J.D.N. was in receipt of an Australian Government Postgraduate Award. We wish to thank Professor J. Bornstein for his helpful discussions of this work. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Segal, H.L. (1975) Am. Chem. Soe. Symp. Set. 15, 235--243 Hers, H.G. (1976) Annu. Rev. Biochem. 45, 167--187 Shimazu, T. (1971) B i o c h i m . B i o p h y s . A c t a 252, 28--38 Shimazu, T. and Amakawa, A. (1975) Biochim. B i o p h y s . A c t a 385, 242--256 Hornbrook, K.R. and B r o d y , T.M. (1963) B i o e h e m . Pharmacol. 12, 1407--1415 Stalmans, W., de Wulf, H., Hue, L. and Hers, H.G. (1974) Eur. J. Biochem, 4 1 , 1 2 7 - - 1 3 4 Stalmans, W. and Hers, H.G. (1975) Eur. J. Biochem. 54, 341--350 Tan, A.W.H. and Nuttall, F.Q. (1975) B i o e h i m . B i o p h y s . A c t a 410, 45--60 Maddaiah, V.T. and Madsen, N.B. (1966) Bioehim. B i o p h y s . A c t a 1 2 1 , 2 6 1 - - 2 6 8 Glinsmann, W., Pauk, G. and Hem, E. (1970) B i o c h e m . B i o p h y s . Res. C ommun. 39, 774--782 Shimazu, T. and Amakawa, A. (1968) B i o c h i m . B i o p h y s . A c t a 165, 335--348 Fiske, C.H. and S u b b a r o w , Y. (1925) J. Biol. Chem. 66,375---400 Thomas, J.A., Scb.lender, K.K. and Lamer, J. (1973) Biochirn. B i o p h y s . A c t a 293, 84--93 De Wulf, H., Staimans, W. and Hers, H.G. (1968) Eur. J. Biochem. 6, 545--551 Wollenberger, A., Ristau, O. and S c h o f f a , G. (1960) Pfliiger's Arch. Gesamte Physiol. Menschen Tiere 270, 399--412 Kreutner, W. and Goldberg, N.D. (1967) Proc. Natl. A c a d . Sci. U.S. 58, 1515--1519 Gold, A.H. (1970) J. Biol. Chem. 2 4 5 , 9 0 3 - - 9 0 5 Curnow, R.T. and Nuttall, F.Q. (1972) J. Biol. Chem. 247, 1892--1898 Tan, A.W.H. and Nuttall, F.Q. (1976) Biochim. B i o p h y s . A c t a 445, 118--130 Buschiazzo, H., E x t o n , J.H. and Park, C.R. (1970) Proe. Natl. A c a d . Sei. U.S. 65, 383--387 Whitton, P.D. and Hems, D.A. (1975) Biochem. J. 150, 153--165 Ishikawa, K. and Shimazu, T. (1976) Life Sci. 19, 1873--1878 Graves, D.J. and Wang, J.H. (1972) in T h e E n z y m e s (Boyer, P.D., ed.), 3rd edn. Vol. 7, pp. 435--82, A c a d e m i c Press, N e w Y o r k Schliselfeld, L.H., Davis, C.H. and Krebs, E.G. (1970) Biochemistry 9, 4959--4965 Hers, H.G. (1976) Biochem. Soc. Trans. 4 , 9 8 5 - - 9 8 8 Mersmann, H.J. and Segal, H.L. (1967) Proc. Natl. A c a d . Sci. U.S. 58, 1688
Biochimica et Biophysiea Acta, 544 (1978) 225--233 © Elsevier/North-Holland Biomedical Press
BBA 28692
ON THE ACTIVITIES OF GLYCOGEN PHOSPHORYLASE AND GLYCOGEN SYNTHASE IN THE LIVER OF THE RAT
JULIE D. NEWMAN and J. McD. ARMSTRONG
Department of Biochemistry, Monash University, Clayton, Victoria 3168 (Australia) (Received April 3rd, 1978)
Summary A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.
Introduction
The critical enzymes in glycogen synthesis and degradation in the liver are glycogen synthase (UDPglucose--glycogen glucosyltransferase, EC 2.4.1.11) and glycogen phosphorylase (1,4-~-D-glucan : orthophosphate a-giucosyltransferase, EC 2.4.1.1). These enzymes are regulated by interconversion between active and inactive forms by enzyme-catalysed covalent modification [1,2]. The effects of various modulators on the interconversion processes are difficult to measure in vivo, because the regulatory system is sensitive to neural stimulation [3,4] or to humoral products resulting from surgical trauma and from other manipulations [5,6]. The reported activities of these two enzymes in liver show quite wide variation particularly for the active forms of the enzymes [5,9, 16--19]. Furthermore, satisfactory methods for the estimation of total phos-
226 phorylase activity in liver have been available only since 1975 [7,8]. Prior to this, estimates of total hepatic phosphorylase relied on activation by epinephrine or glucagon [9,10], or on activation by added phosphorylase kinase [11]. In the course of an investigation of the in vivo regulation of tissue glycogen levels by peptides related to growth hormone, a need arose for establishing a suitable technique for the measurement of the activities of glycogen synthase and phosphorylase in intact animals, with the requirements that the values obtained in control animals were consistent on a long-term basis, and approximated to the values pertaining in vivo. A study was made, based on the observations of Hornbrook and Brody [5] and of Stalmans et al. [6], of procedures for obtaining liver samples, to find conditions under which stimulation of the regulatory system, and the consequent changes in the activities of glycogen synthase and phosphorylase, could be minimised. Various tests of the method finally selected have been made to establish whether the system responded in the anticipated physiological manner to appropriate stimuli. The results obtained are discussed in terms of the control of hepatic glycogen metabolism by the alteration in the balance of a cycle between glycogen and glucose 1-phosphate. The model proposes that net synthesis or degradation of glycogen is achieved by modulating the relative activities of the active forms of glycogen synthase and phosphorylase, which are present at high and equivalent activities in the resting, glycostatic condition. This provides the necessary basis on which studies of the in vivo regulation of glycogen metabolism can be made and integrated with in vitro data. Materials and Methods
Animals. Unless otherwise specified, male rats from an outbred colony of a hooded Lister strain, weighing between 160 and 190 g, were used. All animals were fed ad libitum on a standard diet. On the afternoon of the day preceding an experiment, food was removed from the cages, although the animals were allowed free access to water. They were then used between approx. 9.30 a.m. and 11 a.m. on the following day, giving a nominal fasting period of 18--20 h. Intraperitoneal injections were made in each animal's lower left quadrant, while intravenous injections used the femoral vein, which was exposed by cutting the skin on the front of the thigh. Chemicals. Rabbit liver glycogen, UDPglucose, glucose 6-phosphate and 5'-AMP were obtained from Sigma Chemical Co. Glucose 1-phosphate (potassium salt) was from Calbiochem. UDP-[U-14C]glucose was obtained from the Radiochemical Centre, Amersham, and was diluted for use to a specific activity of 0.08 gCi/pmol. Insulin, free of glucagon, was from Eli Lilly and Co. All other chemicals were of the highest purity available and were used without further purification. Liver homogenates were prepared in a Potter homogeniser using three downand-up strokes at 3000 rev./min. Liver samples of approx. 1 g wet weight of liver were homogenised in 2 ml of cold buffer (50 mM triethanolamine-HC1, 5 mM EDTA, 25 mM sodium fluoride and 5 mM 2-mercaptoethanol, adjusted to pH 7.5 with sodium hydroxide). The homogenate was centrifuged for 15 min at 10 000 × g and 0°C and the supernatant was used for enzyme assay.
227
Stability and storage of homogenates. Phosphorylase and synthase activities were determined for freshly prepared homogenates and again after storage under various conditions. The phosphorylase activity remained unchanged for at least 6 h at 0°C, and also when stored overnight at --20°C after being quickfrozen in an acetone/solid COs bath. Synthase activity did not change appreciably over approx. 4 h at 0°C, but a marked loss of activity was observed for quick-frozen homogenates stored at --20°C overnight. Therefore, both phosphorylase and synthase activities were determined on freshly prepared homogenates held at 0°C for not more than 4 h. For large numbers of samples the synthase activities were determined immediately after the preparation of the homogenates, which were then quick-frozen and stored at --20°C overnight, after which phosphorylase activities were determined. Enzyme assays. The active and total forms of both synthase and phosphorylase were determined. Phosphorylase was assayed according to Stalmans et al. [7]. Final concentrations of reagents in the assay were 50 mM glucose 1-phosphate, pH 6.5, 1% glycogen and 0.15 M sodium fluoride. Phosphorylase a activity was measured in the presence of 0.5 mM caffeine, which suppresses the activity of liver phosphorylase b. Total liver phosphorylase was measured in the presence of 1 mM 5'-AMP and 0.5 M sodium sulphate. Activity was measured as the rate of release of inorganic phosphate, which was determined as described by Fiske and Subbarow [12] on aliquots of the incubation mixture taken at 5, 10 and 15 min after the initiation of the reaction. Glycogen synthase activity was determined as described by Thomas, Schlender and Lamer [13], using the incubation conditions for liver homogenates described by de Wulf et al. [14]. Final concentrations in the assay mixture were 0.05 M triethanolamine-HC1, 5 mM EDTA, pH 7.4, 5 mM UDPglucose, 1% glycogen and either 10 mM sodium sulphate (synthase a) * or 6.7 mM glucose 6-phosphate (total synthase). Aliquots of the incubation mixture were taken at 2, 4 and 6 min, and the rate of incorporation of labelled glucose into glycogen was determined. The results of both assays were calculated as units of enzyme per g of wet liver (1 unit--- 1 ~mol of substrate transformed per min) and as the activity ratio, that is, the fraction of enzyme present in the active form. Blood glucose was determined on samples of blood from the tall of the animal, using "Dextrostix" and an Ames reflectance meter (Miles Laboratories Inc., Elkhart, Indiana). Radioactivity was determined by liquid scintillation counting in a Packard Tricarb counter. Results
Traumatic effects in taking liver samples Preliminary studies ( T a b l e d confirmed earlier observations [5,6] that decapitation of conscious rats prior to taking liver samples resulted in very high levels of phosphorylase a activity, with little improvement when anaesthetised * T h e n o m e n c l a t u r e for t h e a c t i v e (a) and inactive (b) f o r m s o f p h o s p h o r y l a s e and s y n t h a s e is that sugg e s t e d b y M e r s m a n n and Segal [ 2 6 ] .
228 TABLE I EFFECTS OF VARIOUS SAMPLING PROCEDURES GLYCOGEN METABOLISM IN RAT LIVER
ON THE ACTIVITY
OF THE ENZYMES
OF
R a t s w e r e f a s t e d o v e r n i g h t a n d g i v e n a n i n t r a v e n o u s i n j e c t i o n o f p h y s i o l o g i c a l saline or of g l u c o s e ( 0 . 5 g/ kg b o d y weight). Where anaesthetised animals were used, these injections were given after the animal had b e c o m e u n c o n s c i o u s . L i v e r s a m p l e s w e r e t a k e n b y t h e p r o c e d u r e s d e s c r i b e d 15 m i n a f t e r t h e i n j e c t i o n , a n d t h e a c t i v i t y r a t i o s o f g l y c o g e n p h o s p h o r y l a s e a n d s y n t h a s e w e r e d e t e r m i n e d . B o t h h o o d e d a n d Wistarstrain rats were tested. Procedure
Strain
Percentage phosphorylase a
Percentage synthase a
Saline
Glucose
Saline
Glucose
a
C o n s c i o u s rats, i n j e c t e d , stunned and decapitated
Wistar Hooded
90.0±4.0 97.1±1.1
85.0±5.0 94.8±3.8
6.7±1.2 31.0±1.2
7.0±1.6 28.0±0.6
b
Anaesthetised rats, injected, decapitated
Wistar Hooded
-97.8±0.6
-88.3±2.4
7.1±1.6 27.5±2.7
5.2±0.5 37.7±3.5
c
Anaesthetised rats, a b d o m e n o p e n e d , i n j e c t e d , liver l o b e c u t off with scissors
Wistar Hooded
53.9±2.3
60.5±9.1
49.2±7.5
57.1±3.0
d
As in c, b u t i n j e c t e d w i t h p r o p r a n o l o l (1 m g / k g ) p r i o r to further treatment
Wistar Hooded
69.0±4.1 59.5±0.8
64.7±1.8 61.5±4.2
25.5±2.4 44.6±3.2
25.2±1.8 69.7±0.7
e
Anaesthetised, abdomen opened, injected, portal vein clamped b e f o r e c u t t i n g o f f liver l o b e w i t h scissors
Wista~ Hooded
53.0±1.8 47.8+2.3
-22.2±3.1
56.0±2.0 52.2±4.5
64.6±4.3 68.0±5.9
f
Anaesthetised, abdomen opened, i n j e c t e d , liver l o b e f r e e z e c l a m p e d w i t h liquid-nitrogen*cooled
Wistar Hooded
51.6±2.2 48.0±2.6
-20.5±0.5
46.2±1.0 54.0±3.4
-79.0±4.0
Wonenberge clamp
rats were used. When liver samples were excised directly from animals prepared surgically as described below, the phosphorylase a levels were reduced considerably, as previously observed by Stalmans et al. [6]. However, it was not clear whether traumatic effects on enzyme activity had been eliminated by this procedure, and it was decided to use the response of the activity ratios of phosphorylase and glycogen synthase to an intravenous glucose load (0.5 g/kg body weight) as a test for effects of trauma. The rationale of this test is that glucose has been shown to decrease phosphorylase a activity and to increase synthase a activity in the liver [6]. Traumatic effects occurring subsequent to the administration of glucose should tend to obscure this response. It may be seen that liver samples obtained after decapitation or by direct excision showed activity ratios for the two enzymes which were essentially similar whether or n o t glucose was administered (Table I). This suggested that the trauma produced by these procedures acted on the glycogen regulatory system to promote extensive glycogenolysis, thereby masking any earlier modulatory effects on the activities of glycogen synthase and phosphorylase. In rats which were injected with the beta blocking agent, propranolol, prior to excising the liver sample (Table I), there was a marked increase in the glycogen synthase activity ratio in response to glucose in hooded rats but not in Wistar-strain animals. However, the phosphorylase activity ratio did n o t change
229 significantly with glucose administration. This would suggest that cutting the liver lobe stimulates glycogenolysis, presumably by stimulation of the sympathetic nervous system. The involvement of humoral agents in the effects of excision on enzyme activities was demonstrated in the following experiment. Just before the liver sample was taken, the portal vein was clamped with an artery clamp. It may be seen that this resulted in an increase in the activity ratio of the synthase, which was increased further by the glucose load. Similarly, the phosphorylase activity ratio decreased, and was lowered considerably more in animals given a glucose load (Table I). As a result of these findings, the following procedure was adopted for obtaining liver samples. It is essentially that described by Stalmans et al. [6] for obtaining serial liver samples from individual animals.
Recommended method for obtaining liver samples Animals are prepared surgically as follows. Rats are anaesthetised by intraperitoneal injection of sodium pentobarbitone {60 mg/kg b o d y weight) and kept warm in still air. This dose maintains deep anaesthesia for at least 90 min. The abdominal cavity is opened by a midline incision from the sternum to the pelvis. A lateral cut of the abdominal wall from the midline incision to the animal's right at the lower level of the rib cage produces a flap which may be used to expose the liver. The a b d o m e n is then covered with a gauze moistened with warm isotonic saline. If blood glucose is monitored during this procedure, it should remain essentially constant during surgery. When a liver sample is to be taken the liver is exposed, raised gently, and a portion of the liver is frozen in situ using Wollenberger tongs [15] cooled in liquid nitrogen *. The frozen sample is broken off and stored in liquid nitrogen until it can be h0mogenised. Care must be taken in raising the liver, since excessive manipulation or the use of forceps stimulates changes in the activity ratios of the enzymes. The liver lobe is gently lifted with the broad flat end of a scalpel handle, and then with a finger. As may be seen in Table I, the results for this procedure are similar to those obtained by clamping the portal vein, with the activity ratios of both enzymes responding appropriately to a glucose load. This freeze-clamping procedure was used in all subsequent experiments. Synthase and phosphorylase activities in the normal fasted rat Fig. 1 shows that there was a marked positive correlation between phosphorylase and synthase activities in the liver of the normal fasted rat, whether expressed as the total activities of each enzyme or as the amounts of the active forms. The group represents various normal animals used as controls in different experiments over approx. 2 years. It is clear that the absolute activities showed considerable variation from one animal to another. In contrast, the activity ratios of the two enzymes remained essentially constant over the group. This is reflected in the magnitude of the standard error of the mean, expressed as a percentage of the mean. It may be seen (Table II) that there is a 4-fold (approx.) reduction in the variability of the activity ratios of synthase and phosphorylase, as compared to the absolute activities. The change in the * C o o l i n g w i t h d r y ice is u n s a t i s f a c t o r y , and results in v e r y l o w p h o s p h o r y l a s e activities.
230
.•25
A
B
• ° "~w ~1o!
~2C +
: i : .o
~° 15 .J >- IC
5
o
i i
I &.
/
I
1
I
2 3 SYNTHASE ( a + b ) ( u n i t s / g )
J
:
0"6 1'2 SYNTHASE a ( u n i t s / g )
1.8
F i g . 1. C o r r e l a t i o n o f P h o s p h o r y l a s e and glycogen synthase activities in the liver of normal fasted rats. L i v e r s a m p l e s w e r e o b t a i n e d b y f r e e z e - c l a m p i n g i n s i t u . O t h e r d e t a i l s a r e as i n t h e l e g e n d t o T a b l e n . I n A , t o t a l p h o s p h o r y l a s e a c t i v i t y (a + b ) is p l o t t e d a g a i n s t t o t a l s y n t h a s e a c t i v i t y (a + b ) , w h i l e i n B o n l y t h e a c t i v i t i e s o f t h e a c t i v e f o r m s a r e p l o t t e d . I n e a c h c a s e t h e s o l i d l i n e is f o r t h e r e g r e s s i o n e q u a t i o n o f y = fix, as d e s c r i b e d i n T a b l e I I . S t a t i s t i c a l t e s t i n g s h o w e d t h a t t h i s l i n e is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e l i n e y = a + ~ x , a n d t h a t (~ w a s n o t s i g n i f i c a n t l y d i f f e r e n t f r o m z e r o .
activities of the two enzymes in response to various stimuli is important in studies on the regulation of glycogen metabolism, and the results in Table II show that activity ratios are satisfactory for comparing such changes in different animals. The variability in absolute activities has generally restricted such experiments to comparison of serial tissue samples from individual animals [6]. It also may be seen that approximately one half of either enzyme was present in its active form in normal fasted rats. From the regression analysis in TABLE
II
PHOSPHORYLASE FASTED RAT
AND
GLYCOGEN
SYNTHASE
ACTIVITIES
IN THE
LIVER
OF THE NORMAL,
Activities of the two enzymes, as active forms and total activities, were determined on liver samples from 72 rats after injection of physiological saline. The measurements have been made in small groups of animals, over a period of approx. 20 months. Activities are reported as units of enzyme per g of liver; the a c t i v e f o r m i s r e p o r t e d as a a n d t o t a l a c t i v i t y a s a + b. T h e r a t i o o f a c t i v e t o t o t a l e n z y m e is s h o w n a s % a . Phosphorylase
Mean tS.E. S . E . as % o f m e a n
Synthase
a
a + b
%a
a
a + b
%a
5.76 0.25 4.3
12.44 0.56 4.5
46.87 0.49 1.04
0.998 0.036 3.6
1.929 0.070 3.6
52.08 0.56 1.07
Total enzyme
Active forms
Correlation coefficient
0.839
0.748
Regression coefficient of phosphorylase on synthase *
6 . 4 6 -+ 0 . 1 5
5.72 ± 0.16
Difference between regression coefficients
0.73 (0.4 > P > 0.3)
* F o r t h e l i n e y = bx, b o t h v a r i a b l e s s u b j e c t t o e r r o r , b u t e a c h o f u n i f o r m y l a s e , g - 1 x ~ u n i t s o f g l y c o g e n s y n t h a s e , g-1.
weight, y ~ units of phosphor-
231 TABLE In EFFECTS OF H O R M O N E S A N D F E E D I N G ON G L Y C O G E N S Y N T H A S E AND P H O S P H O R Y L A S E ACTIVITES IN RAT LIVER R a t s w e r e a n a e s t h e t i s e d a n d t h e i r a b d o m e n s w e r e o p e n e d . Saline o r h o r m o n e s w e r e i n j e c t e d i n t o t h e f e m o r a l vein. A f t e r 1 5 rain liver s a m p l e s w e r e t a k e n b y f r e e z e - c l a m p i n g in situ. T h e rats used w e r e f a s t e d o v e r n i g h t , e x c e p t f o r t h e f e d g r o u p , a n d all g r o u p s w e r e used o n t h e m o r n i n g o f t h e e x p e r i m e n t . E a c h g r o u p c o n t a i n e d s e v e n rats.
Condition
B l o o d glucose ( m g / 1 0 0 ml)
A c t i v i t y r a t i o (%) Synthase
Phosphorylase
F a s t e d rats Saline Insulin (0.07 unit/kg) Glucagon (0.03 mg/kg)
74 ± 4 68 ± 3 120 ± 3
54.3 -+ 1.7 6 2 . 4 ± 3.4 1 8 . 5 -+ 1.2
4 7 . 6 ± 2.2 3 2 . 2 ± 1.0 8 2 . 0 ± 3.3
F e d rats Saline
105 ± 6
6 4 . 8 ± 4.7
4 1 . 5 ± 3.8
Table II, the ratio of phosphorylase activity to synthase activity in the liver was 6 : 1 (approx.) under the conditions of assay. This proportionality was observed for both the total activities and the active forms of the two enzymes, and implies some balance between the activities of glycogen synthase and phosphorylase in the liver.
Modulation of enzyme activity If the results obtained by the freeze-clamping procedure are a valid approximation of the activities of the two enzymes in vivo for normal animals, then the glycogen metabolising system should respond in the anticipated manner to appropriate stimuli. It may be seen (Table III) that insulin increased the activity ratio of synthase and decreased the activity ratio of phosphorylase, consistent with the giycogenic effects of this hormone, whereas the activity ratio increased for phosphorylase and decreased for synthase in animals treated with the glycogenolytic agent, glucagon. When fed animals were compared with fasted animals the changes were qualitatively similar to those for insulin, although rather smaller. The effects of insulin and feeding were similar to those reported for a glucose load in Table I. Discussion The results in Tables I and II confirm and extend the observations of Hornbrook and Brody [5] and Stalmans et al. [6] that trauma can markedly increase the extent of phosphorylase activation in the liver. It is further shown that trauma causes extensive conversion of glycogen synthase to its inactive form. It is our impression that liver glycogen synthase a activity is a more sensitive indicator of trauma than phosphorylase a activity. Of the procedures for obtaining liver samples which were tested, there are two methods which seem to give reliable results and which may be a fair app]coximation of the conditions existing in vivo prior to taking the sample. These are by freeze-clamping the liver in situ, and by clamping the portal vein and restricting blood flow in the
232 liver prior to excising a liver sample. Both methods give essentially similar results for phosphorylase and synthase activity ratios, and these ratios respond as expected to a glucose load [6]. From Fig. 1 it may be seen that the amounts of phosphorylase and synthase per g of liver may vary widely, whether as total enzyme or the active form. However, the correlation coefficients indicate that the amount of phosphorylase is proportional to the amount of synthase present. This variability may present difficulties when units of enzyme activity are compared between animals. When the results are calculated as activity ratios the variability between animals is reduced approx. 4-fold, and there is a remarkable constancy in the activity ratios of phosphorylase and synthase over the entire group of animals used. The total activities of phosphorylase and synthase reported in Table II lie within the range of values obtained by other workers in studies on intact animals [9,6,17--19]. There is poor agreement between our results and published data for the activity ratios of the two enzymes; this may reflect traumatic effects arising from sampling procedures. The most notable difference is the activity ratio of 0.55 for glycogen synthase for normal fasted rats, as compared with values of between 0.02 and 0.2 obtained by others. The only comparable values (approx. 0.4) were obtained for perfused liver [20,21]. This raises the question of the in vivo activity ratios of the two enzymes. If it is accepted that trauma produced by tissue sampling is a major factor in the observed values, then the higher the activity ratio for synthase and the lower the activity ratio of phosphorylase, the more likely are the values to approximate to the in vivo condition. Other criteria which may be applied rely on the response of the activity ratios to modulatory effects, such as fasting, feeding, glucose loads and the actions of injected hormones. Such responses must be consistent with the expected changes in biochemical and physiological status. The directions of change in the activity ratios (Tables I, III) fulfil these expectations, being consistent with net synthesis, degradation or stasis of liver glycogen. The qualitative effects resemble other reported data [6,16,17,22]. On the basis of fulfilling the above criteria, and the consistency of the activity ratios obtained with many animals over a long time, the results reported here are probably a reasonable approximation of the activity ratios of phosphorylase and synthase in the liver of the fasted rat. If this is so, then some conclusions on the control of glycogen metabolism may be drawn. Firstly, it was found that a b o u t half the amount of each enzyme was present in the active form. Secondly, the marked correlations between the activities of the two enzymes indicates some balance in their action in the normal rat. The measured activities of phosphorylase and synthase reported here are very different from one another. However, it must be remembered that these values refer to the activities under the conditions of assay rather than those existing in the cell. While the assay conditions for synthase correspond to saturating substrate concentration at physiological pH in the appropriate direction of synthesis, this is n o t so for phosphorylase. This enzyme was assayed at pH 6.5, also in the direction of synthesis. Therefore, the two activity measurements cannot be compared directly, and it is necessary to correct the results for phosphorylase to the equivalent activity at pH 7.4 in the direction of gly-
233 cogen degradation. From published data on the pH vs. activity profiles and relative rates of the synthetic and degradative reactions catalysed by phosphorylase [23,24], the corrected activity of phosphorylase becomes one-sixth of the activity measured by the assay procedure. The measured activities of synthase and phosphorylase were in the ratio approx. 1 : 6 (Table II), so that the maximum activities of the two enzymes for the synthesis and degradation of glycogen at pH 7.4 are probably essentially equal. This would suggest that the two activities are in balance in the normal fasted rat, and that glycostasis is maintained by a balanced futile cycle of glycogen synthesis and degradation. Such a cycle has been postulated previously [25]. Furthermore, our results on the effects of insulin, of glucagon and of a glucose load are consistent with a change from glycostasis towards net synthesis or degradation of glycogen, by altering the activities of the two enzymes so that the futile cycle becomes unbalanced. Thus, changes in the amount of liver glycogen would occur as the net difference in the rates of its synthesis and degradation. None of the conditions tested resulted in the complete conversion of either of the enzymes to its inactive form. Consequently, futile cycling between glycogen and glucose 1-phosphate would occur at all times, even though the amount of tissue glycogen was changing. Acknowledgements This work was supported in part by a grant from the Australian Research Grant Committee. J.D.N. was in receipt of an Australian Government Postgraduate Award. We wish to thank Professor J. Bornstein for his helpful discussions of this work. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
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