Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

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Biochimica et Biophysica Acta, 500 (1977) 267- 276 © Elsevier/North-Holland Biomedical Press

BBA 28363

HORMONE-INDUCED CHANGES IN P Y R U V A T E KINASE ACTIVITY IN ISOLATED HEPATOCYTES II. R E L A T I O N TO THE H O R M O N A L R E G U L A T I O N OF GLUCONEOGENESIS

THEO J.C. VAN BERKEL, JOHAN K. K R U I J T and JOHAN F. KOSTER

Department of Biochemistry I, Faculty of Medicine, Erasmus University, Rotterdam (The Netherlands) (Received March 16th, 1977)

Summary 1. Incubation of isolated hepatocytes with glucagon (10 -6 M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM M"2÷~free- The magnitude of the decrease in activity is n o t influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP. 2. The extent of inhibition of pyruvate kinase induced b y incubation of hepatocytes with glucagon or d i b u t y t y l cyclic AMP is n o t influenced by the extracellular Ca 2÷ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca :÷ (2.6 mM) as compared to a low concentration of extracellular Ca 2+ (0.26 mM). 3. Incubation of hepatocytes in a Na+-free, high K÷-concentration medium does n o t influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 raM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition b y dibutyryl cyclic GMP is n o t influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 raM). 5. The relative insensitivity of the pyruvate kinase inhibition induced by

Abbreviation: Bt2-cAMP, dibutyryl cyclic AMP; Bt2-cGMP, dibutyryl cyclic GMP.

268 glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellulal environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted b y changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofruetokinase-fruetose1,6-phosphatase cycle.

Introduction The regulation by hormones of the dynamic balance between glycolysis and gluconeogenesis in liver has been studied extensively [ 1--4]. Although the overall stimulation of gluconeogenesis by glucagon is well-characterized, the site and mechanism of hormone action on both gluconeogenic and glycolytic enzymes is still uncertain [1--5]. Recent studies with isotopes have focussed attention upon the phosphofructokinase-fructose diphosphatase cycle [6,7] and the so-called phosphoenolpyruvate cycle, involving pyruvate carboxylase, phosphoenolpyruvate carboxykinase and pyruvate kinase [8,9], as primary regulatory sites. The relative contribution of both potentially wasteful cycles to the glucagon stimulation of gluconeogenesis is under discussion. Clark [10] suggests that the primary site of action of glucagon is at the level of the fructose 6-phosphate cycle and that regulation of the phosphoenolpyruvate cycle is the consequence of an alteration in the fructose 1,6-diphosphate concentration, excluding acute hormonal control at the pyruvate kinase site. This view is opposed by Rognstad [8], Feliu et al. [11] and Pilkis et al. [12] who state that acute hormonal control of pyruvate kinase accounts for the action of glucagon on the rate of gluconeogenesis. This is an attractive possibility because inhibition of pyruvate kinase flux [8] can be related to molecular changes in the enzyme [11,13--15] probably involving phosphorylation and dephosphorylation [16,34]. Our current interest in pyruvate kinase regulation [17] focussed our attention upon the question of to what extent the hormonal regulation of pyruvate kinase could be responsible for the hormonal control of gluconeogenesis. To approach this question, we incubated liver cells under various conditions which are known to influence the hormonal stimulation of gluconeogenesis and determined under these different conditions the effect of glucagon and cyclic nucleotides upon pyruvate kinase. Materials and Methods

Isolation and incubation o f liver cells Parenchymal cells were isolated from fed animals as described previously [18]. The purity and integrity of every cell preparation was tested as reported [18]. The cells were incubated at 37°C in 50-ml Erlenmeyer flasks stoppered with rubber caps in either a Na ÷ or a Na*-free, high-K + medium. The Na * medium consisted of 120 mM NaC1, 4.8 mM KC1, 0.26 mM CaC12, 1.2 mM

269 KH2PO4, 1.2 mM MgSO4 and 24 mM NaHCO3. In the Na*-free, high-K* medium all the Na ÷ salts were replaced by the corresponding K ÷ salts. The last two washings of the cells were p e r f o r m e d with the applied incubation medium. The viability of cells incubated in the Na + or Na+-free, high-K* media did n o t differ from each other. The incubations were started by addition of cells to the different incubation mixtures stored on ice. Zero-time samples were withdrawn and the cells were incubated at 37°C. At the indicated times samples were withdrawn, homogenized immediately in a tightly-fitting Potter-Elvehjem h o m o g e n i z e r with a Teflon pestle (clearance 0.003 inch). To the homogenates m e r c a p t o e t h a n o l (1 mM) was added and the homogenates were subsequently centrifuged for 10 min at 20 000 × g in a cooled centrifuge. The supernatants were immediately tested for pyruvate kinase activity by adding 0.5 ml supernatant to a cuvette containing 24 mM Tris • HC1, pH 7.5, 200 mM KC1, 1 mM ADP, 0.1 mg lactate dehydrogenase, 0.4 mM NADH and 1 mM free Mg 2+ with a total assay volume o f 3.0 ml. The free Mg 2÷ c o n c e n t r a t i o n was calculated as described earlier [19]. The reaction t e m p e r a t u r e was 23°C. The reaction was started by adding 2.5 mM phosphoenolpyruvate after 5 min preincubation. This sequence of additions is i m p o r t a n t because b o u n d fructose 1,6-diphosphate will be split off under the applied high salt c o n c e n t r a t i o n in the absence of phosphoenolpyruvate [18]. Such a procedure is essential to discriminate between h o r m o n a l effects on the fructose 1,6-diphosphate c o n c e n t r a t i o n and on pyruvate kinase itself [20]. With our procedure linear time courses were obtained for at least 10 min. A m m o n i u m sulphate precipitation of the e n z y m e did not change the described effects. Pyruvate kinase activity did n o t change with time up to 3 h after termination o f the cell incubations and h o m o g e n i z a t i o n of the cells when stored at 0 °C. However, overnight storage or deep-freezing does lower the activity in the absence of fructose 1,6-diphosphate (100% value). However, the h o r m o n a l effect u p o n the e n z y m e remains detectable, although to a lower extent. Storage at 23°C does minimize the h o r m o n a l effect to a b o u t 50% within 30 min. Fructose 1,6-diphosphate (0.5 mM) was always i nt roduced as second addition to reveal V values. These V values were n o t influenced by the different additions [13]. F o r the sake of clarity these data are n o t pl ot t ed in the figures, but for the experiments shown these values remained within 10% of the zero time values during the indicated incubation times. T he data pl ot t ed in the figures are expressed as percentage o f the pyruvate kinase activity at 2.5 mM phosphoenolpyruvate and 1 mM Mgfree 2+ of the zero times (100%). For the different experiments the zero time, V values were 136 + 4 n m o l . min -1. mg -1 protein (+ S.E.) with n = 16 and the 100% values pl ot t ed in the figures 79 -+ 3 nm ol • min -1 • mg -~ protein (+ S.E.) with n = 16. T he data p l o t t e d in the figures are the mean percentages o f two experiments with different cell batches. The duplicates differed maximally 10% f r om each other e x c e p t for the experiments with fructose as the substrate (Fig. 3), which showed greater differences especially at the longer incubation times. T he r efore in Fig. 3 the data are pl ot t ed + standard deviation (S.E.) of f our i n d e p e n d e n t experiments. Glucagon, dibut y r y l cyclic AMP and d i b u t y r y l cyclic GMP, when indicated, were present respectively at 10 -6 M, 1 0 - 4 M and 10 -4 M. Initial studies indicated that at

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these concentrations optimal effects on pyruvate kinase were exerted (unpublished results). Collagenase (Type I) was obtained from Sigma (St. Louis, Mo, U.S.A.). Other chemicals used were obtained from Boehringer (Mannheim, G.F.R.). Results

Incubation o f cells with different substrates Several groups of investigators have shown that the stimulation of glucogeogenesis by glucagon is dependent upon the gluconeogenic substrate concentration [1--7] and the nature of the substrate [1--7]. In general, higher activation factors are found at suboptimal substrate concentrations. We reported previously [ 13] that addition of glucagon or Bt2-cAMP to isolated hepatocytes in the presence of 1 mM pyruvate causes a decrease in pyruvate kinase activity, measured at a suboptimal phosphoenolpyruvate concentration, of about 50%. Fig. 1 shows that with 1 mM lactate as substrate for gluconeogenesis addition of Bt2cAMP (0.1 mM) results in a b o u t the same degree of inhibition. An increase in lactate concentration to 5 mM does not influence the magnitude of the inhibition by Bt:-cAMP. It is further shown in Fig. 1 that lactate itself does not influence the pyruvate kinase activity. Earlier we showed that with pyruvate (1 mM) a temporary stimulation of the pyruvate kinase activity is found after 10 min of incubation of 25%. We reported earlier [13] that incubation of hepatocytes with 10 mM glucose did n o t influence the pyruvate kinase activity although it has been shown that glucose inhibits gluconeogenesis [4]. Feliu et al. [ 11] observed the same phenomenon. If regulation of gluconeogenesis is exerted at the level of pyruvate kinase it should be expected that the presence of glucose increases pyruvate kinase activity. Fig. 2 shows that incubation of hepatocytes with either a physiological glucose concentration (5 mM) or a high glucose concentration (30

% activity 10|

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time (minutes) F i g . 1 . I n f l u e n c e o f l a c t a t e a n d l a c t a t e + B t 2 - c A M P o n t h e p y r u v a t e kinase a c t i v i t y o f i s o l a t e d h e p a t o c y t e s . T h e a d d i t i o n s to i n c u b a t i o n s o f i n t a c t cells a r e : Q o 1 mM lactate; ~ ~, 1 m M l a c t a t e + 0.1 raM Bt2-cAMP; n ~, 5 m M l a c t a t e ; v ~, 5 r a M l a c t a t e + 0 . 1 m M B t 2 - c A M P . F o r f u r t h e r c o n d i t i o n s see M a t e r i a l s a n d M e t h o d s .

27] % actlvlty 5 rnM glucose 100-

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Fig. 2. I n f l u e n c e o f g l u c o s e a n d g l u c o s e + Bt2-cAMP o n t h e p y r u v a t e k i n a s e a c t i v i t y o f i s o l a t e d h e p a t o c y t e s . T h e a d d i t i o n s t o i n c u b a t i o n s o f i n t a c t cells are: o o 5 mM glucose; • •, 5 mM g l u c o s e + 0.1 mM Bt2-cAMP; z~ -% 30 mM glucose; • A 30 mM glucose + 0.1 mM Bt2-cAMP. F o r furt h e r c o n d i t i o n s s e e Materials a n d M e t h o d s . Fig. 3. I n f l u e n c e o f f r u c t o s e a n d f r u c t o s e + g l u c a g o n o n t h e p y r u v a t e k i n a s e a c t i v i t y o f i s o l a t e d h e p a t o c y t e s . T h e a d d i t i o n s t o i n c u b a t i o n s o f i n t a c t cells are: D D 10 mM fructose; • •, 10 mM fruct o s e + 10 -6 M glucagon; ~ v, 25 mM fructose; v v 25 mM fructose + 10-6 M g l u c a g o n . F o r f u r t h e r c o n d i t i o n s s e e Materials a n d M e t h o d s .

mM) does not influence the pyruvate kinase activity. Addition of Bt2-cAMP (0.1 mM) results in an inhibition of pyruvate kinase activity, indicating that the response of pyruvate kinase is not blocked by the presence of glucose. Effects of fructose on perfused liver [21] and isolated hepatocytes [1,6] have been studies extensively. Clark [10] showed that especially at high fructose concentrations, glucagon is not able to inhibit the pyruvate + lactate formation from this substrate. This has been used as evidence for the statement that glucagon does not mediate glycolytic flux at the level of pyruvate kinase. Fig. 3 shows that the presence of fructose itself induces a decrease in pyruvate kinase activity. However, irrespective of the fructose concentration, addition of glucagon results in an inhibition of pyruvate kinase activity. This effect is clear even with 25 mM fructose when lowering of the ATP level may be expected [ 22].

Effect of extracellular Ca 2+ and EGTA Ca 2÷ has been shown to stimulate gluconeogenesis in perfused liver [23] and isolated hepatocytes [2,24,25]. The interest in the effects of Ca2÷ is currently activated by its possible rSle as second messenger for hormone action [26]. Pilkis et al. [24] showed that Ca 2+ or the presence of EGTA did not affect the basal gluconeogenic activity. However, Ca 2+ promoted the glucagon-induced stimulation while in the presence of EGTA no stimulatory effect of glucagon is detected. Fig. 4 shows that the effect of glucagon on pyruvate kinase is independent of the extracellular Ca 2+ concentration and is also not affected by the presence of 2 mM EGTA. With 1 mM pyruvate (not with lactate, compare Fig. 11 as substrate for the cells and 0.26 mM extracellular Ca 2+ already after 20

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time (mlnutes/ Fig. 4. I n f l u e n c e o f d i f f e r e n t e x t r a c e l l u l a r Ca 2+ c o n c e n t r a t i o n s a n d 2 m M E G T A o n t h e p y r u v a t e kinase a c t i v i t y o f i s o l a t e d h e p a t o c y t e s i n c u b a t e d w i t h 1 m M p y r u v a t e + g l u e a g o n ( 1 0 -6 M). T h e a d d i t i o n s t o i n c u b a t i o n s o f i n t a c t cells are i n d i c a t e d in t h e figures. F o r f u r t h e r c o n d i t i o n s see Materials a n d M e t h o d s .

min incubation, the pyruvate kinase activity starts to become reactivated. It is shown in Fig. 4 that this process, in contrast to the absolute amount of inhibition, is influenced by the extracellular Ca 2÷ concentration. At high Ca 2+ (2.6 mM) the activity at 20 min is lower than after 10 min incubation. This obser% activity 1 mM pyr. + 2 mM EGTA

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time (minutes) Fig. 5. I n f l u e n c e o f d i f f e r e n t e x t r a c e U u l a r Ca 2~ c o n c e n t r a t i o n s a n d 2 m M E G T A o n t h e p y r u v a t e kinasc a c t i v i t y o f i s o l a t e d h e p a t o c y t e s i n c u b a t e d w i t h 1 m M p y r u v a t e + B t 2 - c A M P ( 0 . 1 raM). T h e a d d i t i o n s t o i n c u b a t i o n s o f i n t a c t cells are i n d i c a t e d in t h e figures. F o r f u r t h e r c o n d i t i o n s see Materials a n d M e t h o d s .

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vation might be related to the observed inhibition of the pyruvate kinase phosphatase by high [Ca 2÷] [34]. Earlier it was stated that the glucagon effect on pyruvate kinase is likely to be mediated by cyclic AMP [13] as is also the case with the glucagon effect on gluconeogenesis [2]. Fig. 5 raises the possibility of a direct comparison of the Bt2-cAMP and glucagon effects upon pyruvate kinase because here the pyruvate kinase activity is plotted after incubation of the cells with 0.1 mM Bt2cAMP at different extracellular Ca 2÷ concentrations. It can be seen that the time-dependent influence of Bt2-cAMP upon pyruvate kinase activity is identical with the effect of glucagon. Also with Bt2-cAMP the extracellular Ca 2÷ concentration or the presence of EGTA does not influence the cyclic nucleotide effect.

Effect o f Na÷-free, high K ÷medium Friedman et al. [23,26,27] investigated extensively the molecular nature of the glucagon and cyclic AMP stimulation of gluconeogenesis and the role of the activated protein kinase in this process. They were able to block the stimulation of gluconeogenesis by cyclic AMP by replacing Na ÷ by K ÷ in the rat liver perfusion medium. These observations made it of interest to look at the Bt2cAMP induced changes in pyruvate kinase under the above mentioned incubation conditions. Fig. 6 shows the Bt2-cAMP effect upon pyruvate kinase in a Na+-free, high-K ÷ medium with two different substrates. It can be noticed that in the Na+-free, high-K ÷ medium the magnitude of inhibition after 10 min of incubation is about equal as compared to the normal medium [13]. However, it must be emphasized that the reactivation process especially with pyruvate as the substrate proceeds faster. Incubation of the cells with 1 mM pyruvate alone in this medium leads to a temporary (at 10 min) increase in pyruvate kinase activity of 15%, as is also noticed in the normal medium. % activity 10 mM glucose

1 mM pyr.

1004

50 ÷ Bt 2-c AMP + Bt 2 - c AMP

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0 o

20

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Fig. 6. Influence of pyruvate, glucose of isolated hepatocytes incubated in cells are: © o, 1 m M p y r u v a t e ; glucose; ~ ~, 1 0 m M g l u c o s e + ods.

and pyruvate or glucose + Bt 2-cAMP on the pyruvate kinase activity a Na+-free, high-K + medium. The additions to incubations of intact ~ ~, 1 m M p y r u v a t e + 0 . 1 m M B t 2 - c A M P ; o ~, 1 0 m M 0 . 1 m M B t 2 - c A M P . F o r f u r t h e r c o n d i t i o n s see Materials a n d M e t h -

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% activity

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o

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o

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time (minutes) Fig. 7. I n f l u e n c e o f Bt 2 - c G M P (0.1 m M ) o n t h e p y r u v a t e kinase a c t i v i t y of isolated h e p a t o c y t e s i n c u b a t e d w i t h 1 m M p y r u v a t e , 5 m M glucose, 30 m M glucose a n d n o a d d e d s u b s t r a t e . T h e a d d i t i o n s to i n c u b a t i o n s of i n t a c t cells are i n d i c a t e d in t h e figures. F o r f u r t h e r c o n d i t i o n s see Materials a n d M e t h o d s .

Effect of Bt2-cGMP The rSle of cyclic GMP as second messenger of hormone action is widely discussed [2,26,28]. Earlier we reported that incubation of hepatocytes with Bt2cGMP (0.1 mM) resulted in an inhibition of pyruvate kinase although to a lesser extent than with glucagon or Bt2-cAMP. Glinsmann et al. [29] and Exton et al. [30] showed that cyclic GMP is able to stimulate gluconeogenesis. Subsequently Kneer et al. [28] indicated that this stimulation by Bt2-cGMP is inhibited by high concentrations of glucose (27.8 mM). Fig. 7 shows the effect of Bt2-cGMP (0.1 mM) upon pyruvate kinase. In agreement with the earlier report the pyruvate kinase inhibition is less than with Bt2-cAMP or glucagon. It can be seen that the Bt2-cGMP effect upon pyruvate kinase is not influenced by increasing the glucose concentration from 5 to 30 mM nor by the absence or presence of 1 mM pyruvate. Discussion The presented experiments show that the magnitude of the decrease in pyruvate kinase activity induced by glucagon or Bt2-cAMP is n o t influenced by the applied substrate (lactate, glucose or fructose) nor by the substrate concentration. This is in contrast to the magnitude of the effect of glucagon or Bt2-cAMP upon gluconeogenesis [1--7]. Furthermore it is observed that the extracellular Ca 2÷ concentration does not influence the glucagon or Bt2-cAMP induced inhibition of pyruvate kinase. It is generally noticed that the hormonal stimulation of gluconeogenesis is stimulated by the presence of Ca 2÷ [23--25], while a total

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disappearance of the hormonal effect occurs upon addition of excess EGTA [24]. Figs. 5 and 6 show that even in the presence of 2 mM EGTA the glucagon or Bt2-cAMP-induced inhibition occurs. The cyclic nucleotide-induced activation of gluconeogenesis can also be blocked by replacing the Na ÷ salts in the Krebs-Ringer bicarbonate buffer by K* [26,27]. However, even under these extreme circumstances the pyruvate kinase activity becomes inhibited by the addition of Bt2-cAMP. Reactivation of pyruvate kinase is however p r o m o t e d by this medium. The stimulation of the reactivation in Na÷-free, high-K* medium may be related to the reactivation introduced by insulin as reported by Feliu et al. [11]. Recently Hue et al. [31] noticed that a high K* medium increased the inactivation of glycogen phosphorylase and increased the activation of glycogen synthetase, an effect that would be expected from insulin. The present experiments with pyruvate kinase support this view and might serve to extend the hypothesis that insulin and high K * concentrations influence the same process. The meaning of the stimulation of gluconeogenesis by cyclic GMP is not clear. Kneer et al. [28] suggested that cyclic GMP could be a second messenger of epinephrine action, a conclusion based u p o n the similar disappearance of the stimulatory effect of cyclic GMP and epinephrine upon gluconeogenesis by the presence of high glucose concentrations. However, the absence of any increase of cyclic GMP after epinephrine administration is n o t in favour of such a hypothesis [32]. Up till now a cyclic GMP dependent phosphorylation has been shown [33] only in rat small intestine. Our present experiments show that Bt2cGMP lowers the pyruvate kinase activity, although to a lesser extent than Bt2cAMP or glucagon. The magnitude of the Bt2-cGMP effect upon pyruvate kinase is, in contrast to the effect u p o n gluconeogenesis, independent of the presence of a low (5 mM) or high (30 mM) glucose concentration. At least to our knowledge, this is the first time that an effect of Bt2-cGMP is found upon a single enzyme. It remains to be determined however, whether cyclic AMP and cyclic GMP influence the same process. The glucagon and cyclic nucleotide effect on pyruvate kinase seems rather insensitive to the extracellular environment as compared to the effects on gluconeogenesis. This leads to the conclusion that the phosphorylation-dephosphorylation reaction of pyruvate kinase cannot be the only decisive step for the hormonal regulation of gluconeogenesis. This is in agreement with the earlier found change in kinetic properties of pyruvate kinase, induced by glucagon or Bt2-cAMP which showed that the hormonal effects axe overcome by relatively low fructose 1,6-diphosphate concentrations [13,34]. Therefore it can be concluded that hormonal control of pyruvate kinase activity is not only exerted by changes in the degree of phosphorylation state of the enzyme but also in a more remote sense, in that hormones influence the balance of fructose-l,6-diphosphatase to phosphofructokinase activities leading to a change in the concentration of fructose 1,6-diphosphate, the allosteric activator of pyruvate kinase [17]. This implies that the balance between glycolysis and gluconeogenesis is regulated by a complex coordination between several points of control including both covalent and allosteric modulation of pyruvate kinase activity.

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Acknowledgements Professor Dr. W.C. Hiilsmann is thanked for critical reading of the manuscript and Miss A.C. Hanson for the preparation of it. The Netherlands Foundation for Fundamental Medical Research (FUNGO) is acknowledged for partial financial support (grant 13-39-18). References 1 Z a h l t e n , R . N . , S t r a t m a n , F.W. a n d L a r d y , H . A . ( 1 9 7 3 ) P r o c . N a t l . A c a d . Sci. U.S. 7 0 , 3 2 1 3 - - 3 2 1 8 2 F a i n , J . N . , T o l b e r t , M.E.M., P o i n t e r , R . H . , B u t c h e r , F . R . a n d A r n o l d , A. ( 1 9 7 5 ) M e t a b o l i s m 2 4 , 3 9 5 - 407 3 G a r r i s o n , J.C. a n d H a y n e s , R . C . ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 5 3 3 3 - - 5 3 4 3 4 Claus, T . H . , Pilkis, S.J. a n d P a r k , C . R . ( 1 9 7 5 ) B i o c h i m . B i o p h y s . A c t a 4 0 4 , 1 1 0 - - 1 2 3 5 Claus, T . H . a n d Pilkis, S.J. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 1 , 2 4 6 - - 2 6 2 6 C l a r k , M . G . , K n e e r , N.M., B o s c h , A . L . a n d L a r d y , H . A . ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 5 6 9 5 - - 5 7 0 3 7 C l a r k , M . G . , B l o x h a m , D.P., H o l l a n d , P.C. a n d L a r d y , H . A . ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 2 7 9 - - 2 9 0 8 R o g n s t a d , R. ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 3 , 9 0 0 - - 9 0 5 9 K a t z , J. a n d R o g n s t a d , R. ( 1 9 7 6 ) in C u r r e n t T o p i c s in C e l l u l a r R e g u l a t i o n ( H o r e c k e r , B.L. a n d S t a d t m a n , E . A . , eds.), p p . 2 3 7 - - 2 8 9 , A c a d e m i c Press, N e w Y o r k 1 0 C l a r k , M.G. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 8 , 1 2 0 - - 1 2 6 11 F e l i u , J . E . , H u e , L. a n d H e r s , H . G . ( 1 9 7 6 ) P r o c . N a t l . A c a d . Sci. U.S. 7 3 , 2 7 6 2 - - 2 7 6 6 12 Pilkis, S.J., R i o u , J.P. a n d C l a u s , T . H . ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 7 8 4 1 - - 7 8 5 2 13 V a n B e r k e l , T . J . C . , K r u i j t , J , K . , K o s t e r , J . F . a n d H f i l s m a n n , W.C. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. Commun. 72,917--925 1 4 Blair, J . B . , C i m b a l a , M . A . , F o s t e r , J . L . a n d M o r g a n , R . A . ( 1 9 7 6 ) J. Biol. C h e m . 2 5 1 , 3 7 5 6 - - 3 7 6 2 1 5 F r i e d r i c h s , D. ( 1 9 7 6 ) i n Use o f I s o l a t e d L i v e r Cells a n d K i d n e y T u b u l e s in M e t a b o l i c S t u d i e s (Tager, J . M . , SSling, H . D . a n d W i l l i a m s o n , J . R . , eds.) p p . 4 4 4 - - 4 4 7 , Elsevier, A m s t e r d a m 16 E k m a n , P., D a h l q v i s t , U., H u m b l e , E. a n d E n g s t r D m , L. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 9 , 3 7 4 - - 3 8 2 17 V a n B e r k e l , T . J . C . , K o s t e r , J . F . , K r u i j t , J . K . a n d H i i l s m a n n , W.C. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 7 0 , 450--458 18 V a n B e r k e l , T . J . C . , K r u i j t , J . K . a n d K o s t e r , J . F . ( 1 9 7 5 ) E u r . J. B i o c h e m . 5 8 , 1 4 5 - - 1 5 2 19 V a n B e r k e l , T.J.C. ( 1 9 7 4 ) B i o c h i r n . B i o p h y s . A c t a 3 7 0 , 1 4 0 - - 1 5 2 2 0 R i o u , J.P., Claus, T . H . a n d Pilkis, S.J. ( 1 9 7 6 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 7 3 , 5 9 1 - - 5 9 9 21 E x t o n , J . H . a n d P a r k , C . R . ( 1 9 6 9 ) J. Biol. C h e m . 2 4 4 , 1 4 2 4 - - 1 4 3 3 2 2 S e s t o f t , L. a n d F l e r o n , P. ( 1 9 7 4 ) in R e g u l a t i o n o f H e p a t i c M e t a b o l i s m ( L u n d q v i s t , F. a n d T y g s t r u p , N., eds.) p p . 7 6 5 - - 7 7 7 , M u n k s g a a r d , C o p e n h a g e n 23 F r i e d m a n , N. a n d R a s m u s s e n , H. ( 1 9 7 0 ) B i o c h i m . B i o p h y s . A c t a 2 2 2 , 4 1 - - 5 2 2 4 Pilkis, S.J., C l a u s , T . H . , J o h n s o n , R . A . a n d P a r k , C . R . ( 1 9 7 5 ) J. Biol. C h e m . 2 5 0 , 6 3 2 8 - - 6 3 3 6 25 Elliott, K.R.F. (1976) FEBS Lett. 64, 62--64 26 F r i e d m a n , N. ( 1 9 7 6 ) B i o c h i m . B i o p h y s . A c t a 4 2 8 , 4 9 5 - - 5 0 8 27 D a m b a c h , G. a n d F r i e d m a n , N. ( 1 9 7 4 ) B i o c h i m . B i o p h y s . A c t a 3 3 2 , 3 7 4 - - 3 8 6 28 K n e e r , N.M., B o s c h , A . L . , C l a r k , M.G. a n d L a r d y , H . A . ( 1 9 7 4 ) P r o c . N a t l . A c a d . Sci. U.S. 7 1 , 4 5 2 3 - 4527 29 G l i n s m a n n , W . H . , H e m , E.P., L i n a r e l l i , L . G . a n d F a r e s e , R . V . ( 1 9 6 9 ) E n d o c r i n o l o g y 8 5 , 7 1 1 - - 7 1 9 3 0 E x t o n , J . H . , H a r d m a n , J . G . , Williams, T . F . , S u t h e r l a n d , E.W. a n d P a r k , C . R . ( 1 9 7 1 ) J. Biol. C h e m . 246, 2658--2664 31 H u e , L., B o n t e m p s , F. a n d H e r s , H . G . ( 1 9 7 5 ) B i o c h e m . J. 1 5 2 , 1 0 5 - - 1 1 4 3 2 B o s c h , A., K n e e r , N. a n d L a r d y , H. in Use o f I s o l a t e d L i v e r Cells a n d K i d n e y T u b u l e s in M e t a b o l i c S t u d i e s ( T a g e r , J . M . , SSling, H . D . a n d w i n i a m s o n , J . R . , eds.) p p . 2 8 5 - - 2 9 2 , Elsevier, A m s t e r d a m 33 De J o n g e , H . R . ( 1 9 7 6 ) N a t u r e 2 6 2 , 5 9 0 - - 5 9 3 3 4 V a n Berkel, T . J . C . , K r u i j t , J . K . a n d K o s t e r , J . F . ( 1 9 7 7 ) Eur. J. l~lOCllvm, m ~ne press