Glycogen Synthase Phosphatase Research Articles

A Whole-Body Model for Glycogen Regulation Reveals a Critical Role for Substrate Cycling in Maintaining Blood Glucose Homeostasis

Timely, and sometimes rapid, metabolic adaptation to changes in food supply is critical for survival as an organism moves from the fasted to the fed state, and vice versa. These transitions necessitate major metabolic changes to maintain energy homeostasis as the source of blood glucose moves away from ingested carbohydrates, through hepatic glycogen stores, towards gluconeogenesis. The integration of hepatic glycogen regulation with extra-hepatic energetics is a key aspect of these adaptive mechanisms. Here we use computational modeling to explore hepatic glycogen regulation under fed and fasting conditions in the context of a whole-body model. The model was validated against previous experimental results concerning glycogen phosphorylase a (active) and glycogen synthase a dynamics. The model qualitatively reproduced physiological changes that occur during transition from the fed to the fasted state. Analysis of the model reveals a critical role for the inhibition of glycogen synthase phosphatase by glycogen phosphorylase a. This negative regulation leads to high levels of glycogen synthase activity during fasting conditions, which in turn increases substrate (futile) cycling, priming the system for a rapid response once an external source of glucose is restored. This work demonstrates that a mechanistic understanding of the design principles used by metabolic control circuits to maintain homeostasis can benefit from the incorporation of mathematical descriptions of these networks into “whole-body” contextual models that mimic in vivo conditions.

Endometrial secretions: creating a stimulatory microenvironment within the human early placenta and implications for the aetiopathogenesis of preeclampsia

Endometrial glands represent an important source of nutrients for the conceptus during the first trimester. Their secretions are enriched with carbohydrates, and glycogen accumulates within the syncytiotrophoblast of the placenta. It has been assumed that fetal and placental metabolism follow adult pathways, although it is now appreciated that early development occurs in a low-oxygen environment. In past decades, a novel family of putative insulin mediators, inositol phosphoglycans (IPGs), was discovered. These molecules act as allosteric activators and/or inhibitors of enzymes and transduction proteins involved in the control of cell signalling and metabolic pathways, and determine the specificity of responses after activation of the insulin receptor. One member, IPG P-type, activates pyruvate dehydrogenase phosphatase (PDH-Pase), glycogen synthase phosphatase, and glycerol-3-phosphate acyltransferase. Activation of key phosphatases play a major role in the regulation of glucose disposal by oxidative metabolism via PDH, and the non-oxidative storage by glycogen synthesis, both pathways classically known to be regulated by insulin. High concentrations of IPG P-type in amniotic fluid suggest a role in the regulation of carbohydrate metabolism in the fetal-placental unit. Glycogen accumulation in the syncytiotrophoblast also occurs in preeclamptic pregnancies, and is consistently associated with higher placental levels of IPG P-type. Here, we explore the relationship between nutrients provided by the endometrial glands during early pregnancy, IPG P-type and fetal metabolic requirements. We also discuss whether a disconnect between the placental/fetal metabolic state and oxygen tension could lead to a preeclamptic-type syndrome via leakage of Warburg/IPG mediators into the maternal circulation.

Glucokinase and molecular aspects of liver glycogen metabolism

Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.

The control of hepatic glycogen metabolism in an in vitro model of sepsis

Culturing hepatocytes with a combination of LPS, TNF-alpha, IL-1beta and IFN-gamma resulted in an inhibition of glucose output from glycogen and prevented the repletion of glycogen in freshly cultured cells. The reduced glycogen mobilisation correlated with the lower cell glycogen content and reduced rate of glycogen synthesis from [U-(14)C]glucose rather than alterations in either total phosphorylase or phosphorylase a activity. There was no change in the percentage of glycogen exported as glucose nor the production of lactate plus pyruvate indicating that redistribution of the Gluc-6-P cannot explain the failure of the liver to export glucose. Although changes in glycogen mobilisation correlated with NO production, inhibition of NO synthase by inclusion of L-NMMA in the culture medium failed to prevent the inhibition of either glycogen accumulation or mobilisation by the proinflammatory cytokines, precluding the involvement of NO in this response. LPS plus cytokine treatment had no effect on total glycogen synthase activity although the activity ratio was lowered, indicative of increased phosphorylation. The inhibition of glycogen synthesis correlated with a fall in the intracellular concentrations of Gluc-6-P and UDP-glucose and in the absence of measured changes in kinase activity, it is suggested that the fall in Gluc-6-P reduces both substrate supply and glycogen synthase phosphatase activity. The fall in Gluc-6-P coincided with a reduction in total glucokinase and hexokinase activity within the cells, but no significant change in either the translocation of glucokinase or glucose-6-phosphatase activity. This demonstrates direct cytokine effects on glycogen metabolism independent of changes in glucoregulatory hormones.

Expression of Human Protein Phosphatase-1 in Saccharomyces cerevisiae Highlights the Role of Phosphatase Isoforms in Regulating Eukaryotic Functions

Human (PP1) isoforms, PP1alpha, PP1beta, PP1gamma1, and PP1gamma2, differ in primary sequences at N and C termini that potentially bind cellular regulators and define their physiological functions. The GLC7 gene encodes the PP1 catalytic subunit with >80% sequence identity to human PP1 and is essential for viability of Saccharomyces cerevisiae. In yeast, Glc7p regulates glycogen and protein synthesis, actin cytoskeleton, gene expression, and cell division. We substituted human PP1 for Glc7p in yeast to investigate the ability of individual isoforms to catalyze Glc7p functions. S. cerevisiae expressing human PP1 isoforms were viable. PP1alpha-expressing yeast grew more rapidly than strains expressing other isoforms. On the other hand, PP1alpha-expressing yeast accumulated less glycogen than PP1beta-or PP1gamma1-expressing yeast. Yeast expressing human PP1 were indistinguishable from WT yeast in glucose derepression. However, unlike WT yeast, strains expressing human PP1 failed to sporulate. Analysis of chimeric PP1alpha/beta subunits highlighted a critical role for their unique N termini in defining PP1alpha and PP1beta functions in yeast. Biochemical studies established that the differing association of PP1 isoforms with the yeast glycogen-targeting subunit, Gac1p, accounted for their differences in glycogen synthesis. In contrast to human PP1 expressed in Escherichia coli, enzymes expressed in yeast displayed in vitro biochemical properties closely resembling PP1 from mammalian tissues. Thus, PP1 expression in yeast should facilitate future structure-function studies of this protein serine/threonine phosphatase.

Quantification of the glycogen cascade system: the ultrasensitive responses of liver glycogen synthase and muscle phosphorylase are due to distinctive regulatory designs

BackgroundSignaling pathways include intricate networks of reversible covalent modification cycles. Such multicyclic enzyme cascades amplify the input stimulus, cause integration of multiple signals and exhibit sensitive output responses. Regulation of glycogen synthase and phosphorylase by reversible covalent modification cycles exemplifies signal transduction by enzyme cascades. Although this system for regulating glycogen synthesis and breakdown appears similar in all tissues, subtle differences have been identified. For example, phosphatase-1, a dephosphorylating enzyme of the system, is regulated quite differently in muscle and liver. Do these small differences in regulatory architecture affect the overall performance of the glycogen cascade in a specific tissue? We address this question by analyzing the regulatory structure of the glycogen cascade system in liver and muscle cells at steady state.ResultsThe glycogen cascade system in liver and muscle cells was analyzed at steady state and the results were compared with literature data. We found that the cascade system exhibits highly sensitive switch-like responses to changes in cyclic AMP concentration and the outputs are surprisingly different in the two tissues. In muscle, glycogen phosphorylase is more sensitive than glycogen synthase to cyclic AMP, while the opposite is observed in liver. Furthermore, when the liver undergoes a transition from starved to fed-state, the futile cycle of simultaneous glycogen synthesis and degradation switches to reciprocal regulation. Under such a transition, different proportions of active glycogen synthase and phosphorylase can coexist due to the varying inhibition of glycogen-synthase phosphatase by active phosphorylase.ConclusionThe highly sensitive responses of glycogen synthase in liver and phosphorylase in muscle to primary stimuli can be attributed to distinctive regulatory designs in the glycogen cascade system. The different sensitivities of these two enzymes may exemplify the adaptive strategies employed by liver and muscle cells to meet specific cellular demands.

Glucose 6-Phosphate Produced by Gluconeogenesis and by Glucokinase Is Equally Effective in Activating Hepatic Glycogen Synthase

Glucose 6-phosphate (Glc-6-P) produced in cultured hepatocytes by direct phosphorylation of glucose or by gluconeogenesis from dihydroxyacetone (DHA) was equally effective in activating glycogen synthase (GS). However, glycogen accumulation was higher in hepatocytes incubated with glucose than in those treated with DHA. This difference was attributed to decreased futile cycling through GS and glycogen phosphorylase (GP) in the glucose-treated hepatocytes, owing to the partial inactivation of GP induced by glucose. Our results indicate that the gluconeogenic pathway and the glucokinase-mediated phosphorylation of glucose deliver their common product to the same Glc-6-P pool, which is accessible to liver GS. As observed in the treatment with glucose, incubation of cultured hepatocytes with DHA caused the translocation of GS from a uniform cytoplasmic distribution to the hepatocyte periphery and a similar pattern of glycogen deposition. We hypothesize that Glc-6-P has a major role in glycogen metabolism not only by determining the activation state of GS but also by controlling its subcellular distribution in the hepatocyte.

Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes.

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase), induced by cell swelling, have been proposed as underlying mechanisms. Because PI 3-kinase controls autophagic proteolysis, we examined the possibility that PI 3-kinase inhibitors interfere with glycogen production due to their anti-proteolytic action. The PI 3-kinase inhibitor wortmannin inhibited endogenous proteolysis, the production of glycogen from glucose and the activity of active (dephosphorylated) GS (GS a ) in the absence of added amino acids. The stimulation by amino acids of glycogen production and of GS a was only slightly affected by wortmannin. These effects of wortmannin could be mimicked by proteinase inhibitors. A combination of leucine, phenylalanine and tyrosine, which we showed previously to stimulate PI 3-kinase-dependent phosphorylation of ribosomal protein S6, did not stimulate glycogen production from glucose. In contrast with wortmannin, LY294002, another PI 3-kinase inhibitor, strongly inhibited both glycogen synthesis and GS a activity, irrespective of the presence of amino acids. Inhibition of glycogen synthesis by LY294002 could be ascribed in part to increased glycogenolysis and glycolysis. It is concluded that, in hepatocytes, activation of PI 3-kinase may not be responsible for the stimulation of glycogen synthesis by amino acids; LY294002 inhibits glycogen synthesis and stimulates glycogen breakdown by a mechanism that is unrelated to its action as an inhibitor of PI 3-kinase.

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, G(L), R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, G(L)-PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and G(L) complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and G(L) were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6-PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and G(L) and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for G(L). The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of G(L)-PP1c, suggesting that R5-PP1c may function as a hepatic phosphorylase phosphatase, whereas G(L)-PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and G(L)

The level of the glycogen targetting regulatory subunit R5 of protein phosphatase 1 is decreased in the livers of insulin-dependent diabetic rats and starved rats

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats owing to defective activation of glycogen synthase by glycogen-bound protein phosphatase 1 (PP1). The identification of three glycogen-targetting subunits in liver, GL, R5/PTG and R6, which form complexes with the catalytic subunit of PP1 (PP1c), raises the question of whether some or all of these PP1c complexes are subject to regulation by insulin. In liver lysates of control rats, R5 and R6 complexes with PP1c were found to contribute significantly (16 and 21% respectively) to the phosphorylase phosphatase activity associated with the glycogen-targetting subunits, GL–PP1c accounting for the remainder (63%). In liver lysates of insulin-dependent diabetic and of starved rats, the phosphorylase phosphatase activities of the R5 and GL complexes with PP1c were shown by specific immunoadsorption assays to be substantially decreased, and the levels of R5 and GL were shown by immunoblotting to be much lower than those in control extracts. The phosphorylase phosphatase activity of R6–PP1c and the concentration of R6 protein were unaffected by these treatments. Insulin administration to diabetic rats restored the levels of R5 and GL and their associated activities. The regulation of R5 protein levels by insulin was shown to correspond to changes in the level of the mRNA, as has been found for GL. The in vitro glycogen synthase phosphatase/phosphorylase phosphatase activity ratio of R5-PP1c was lower than that of GL–PP1c, suggesting that R5–PP1c may function as a hepatic phosphorylase phosphatase, whereas GL–PP1c may be the major hepatic glycogen synthase phosphatase. In hepatic lysates, more than half the R6 was present in the glycogen-free supernatant, suggesting that R6 may have lower affinity for glycogen than R5 and GL

Sphingomyelinase treatment of rat hepatocytes inhibits cell-swelling-stimulated glycogen synthesis by causing cell shrinkage.

Breakdown of plasma-membrane sphingomyelin caused by TNF-alpha is known to inhibit glucose metabolism and insulin signalling in muscle and fat cells. In hepatocytes, conversion of glucose to glycogen is strongly activated by amino acid-induced cell swelling. In order to find out whether breakdown of plasma-membrane sphingomyelin also inhibits this insulin-independent process, the effect of addition of sphingomyelinase was studied in rat hepatocytes. Sphingomyelinase (but not ceramide) inhibited glycogen synthesis, caused cell shrinkage, decreased the activity of glycogen synthase a, but had no effect on phosphorylase a. Cell integrity was not affected by sphingomyelinase addition as gluconeogenesis and the intracellular concentration of ATP were unchanged. As a control, glycogen synthesis was studied in HepG2 cells. In these cells, the basal rate of glycogen production was high, could not be stimulated by amino acids, nor be inhibited by sphingomyelinase. Regarding the mechanism responsible for the inhibition of glycogen synthase a, sphingomyelinase did not affect amino acid-induced, PtdIns 3-kinase-dependent, phosphorylation of p70S6 kinase, but caused an increase in intracellular chloride, which is known to inhibit glycogen synthase phosphatase. It is concluded that the decrease in cell volume, following the breakdown of sphingomyelin in the plasma membrane of the hepatocyte, may contribute to the abnormal metabolism of glucose when TNF-alpha levels are high.

Glycogen synthase phosphatase interacts with heat shock factor to activate CUP1 gene transcription in Saccharomyces cerevisiae.

Upon heat shock, transcription of many stress-inducible genes is rapidly and dramatically stimulated by heat shock factor (HSF). A central region of the yeast HSF (designated HSFrr for "repression region") was previously identified and proposed to be involved in repressing the activation domain under non-heat-shock conditions. Here, we used the phage display system to isolate proteins that interact with HSFrr. This should identify factors that modulate HSF activity or directly participate in HSF-mediated transcriptional activation. We constructed a randomly sheared yeast genomic library to express yeast proteins on the surface of lambda phage. HSFrr binding phages were selected by cycles of affinity chromatography. DNA sequencing identified an HSFrr-interacting phage that contains the GAC1 gene. The GAC1 gene encodes the regulatory subunit for a type 1 serine/threonine phosphoprotein phosphatase, Glc7. Both gac1 and glc7 mutations had little effect on HSF activation of gene transcription of two heat shock genes, SSA4 and HSP82. In contrast, heat shock induction of CUP1 gene expression was completely abolished in a glc7 mutant and reduced in a gac1 mutant. The results demonstrate that the Glc7 phosphatase and its Gac1 regulatory subunit play positive roles in HSF activation of CUP1 transcription.

Loss of the hepatic glycogen-binding subunit (GL) of protein phosphatase 1 underlies deficient glycogen synthesis in insulin-dependent diabetic rats and in adrenalectomized starved rats.

Hepatic glycogen synthesis is impaired in insulin-dependent diabetic rats and in adrenalectomized starved rats, and although this is known to be due to defective activation of glycogen synthase by glycogen synthase phosphatase, the underlying molecular mechanism has not been delineated. Glycogen synthase phosphatase comprises the catalytic subunit of protein phosphatase 1 (PP1) complexed with the hepatic glycogen-binding subunit, termed GL. In liver extracts of insulin-dependent diabetic and adrenalectomized starved rats, the level of GL was shown by immunoblotting to be substantially reduced compared with that in control extracts, whereas the level of PP1 catalytic subunit was not affected by these treatments. Insulin administration to diabetic rats restored the level of GL and prolonged administration raised it above the control levels, whereas re-feeding partially restored the GL level in adrenalectomized starved rats. The regulation of GL protein levels by insulin and starvation/feeding was shown to correlate with changes in the level of the GL mRNA, indicating that the long-term regulation of the hepatic glycogen-associated form of PP1 by insulin, and hence the activity of hepatic glycogen synthase, is predominantly mediated through changes in the level of the GL mRNA.

Effects of Ionic Strength and Chloride Ion on Activities of the Glucose-6-phosphatase System: Regulation of the Biosynthetic Activity of Glucose-6-phosphatase by Chloride Ion Inhibition/Deinhibition

Certain amino acids stimulate glycogenesis from glucose. The regulatory volume decrease mechanism explaining these effects was defined by Meijeret al.(1992,J. Biol. Chem.267, 5823–5828). It involves amino acid-induced swelling of hepatocytes resulting in loss of chloride ions which leads to deinhibition of glycogen synthase phosphatase. This results in enhanced conversion of the inactive to active form of glycogen synthase and thus enhanced glycogen synthesis. We have studied the effects of amino acids and chloride ion on the glucose-6-phosphatase system (Glc-6-Pase) with rat liver microsomal preparations, and correlated our results with those reported by others with glycogen synthase. Glc-6-Pase activities are increased by elevated ionic strength varied by increasing theconcentration of various buffers or charged aminoacids but are not affected by changes in osmolarity, varied with disaccharides or uncharged amino acids. With undisrupted microsomes, chloride ion competitively inhibits carbamyl phosphate: glucose phosphotransferase (KCP,t,UMi,Cl−= 19 mM) more extensively than Glc-6-P phosphohydrolase (KG6P,h,UMi,Cl−= 117 mM). Inhibition by chloride ion and activation due to ionic strength may be important considerations when assessing in vitro Glc-6-Pase activities where an attempt is made to replicate physiologic conditions. Further we propose that amino acids may play a role in increasing biosynthetic activity of Glc-6-Pase, as well as previously characterized glycogen synthase (Meijeret al.,op. cit.), via the regulatory volume decrease mechanism through diminished chloride ion inhibition. Reduced concentration of chloride ion will (1) deinhibit the biosynthetic activity of Glc-6-Pase, while still inhibiting Glc-6-P hydrolysis, leading to an increased cellular concentration of Glc-6-P (an important glycogenic intermediate as well as allosteric activator of glycogen synthase) and (2) increase the active form of glycogen synthase by deinhibiting glycogen synthase phosphatase both through the previously defined mechanism (see above) and via Glc-6-P-enhanced conversion of glycogen synthase from its inactive to active form. We propose that the biosynthetic activity of Glc-6-Pase may act in concert with glycogen synthase during amino acid-induced glycogenesis from glucose.

New insights into the role and mechanism of glycogen synthase activation by insulin.

The metabolism of the storage polysaccharide glycogen is intimately linked with insulin action and blood glucose homeostasis. Insulin activates both glucose transport and glycogen synthase in skeletal muscle. The central issue of a long-standing debate is which of these two effects determines the rate of glycogen synthesis in response to insulin. Recent studies with transgenic animals indicate that, under appropriate conditions, each process can contribute to determining the extent of glycogen accumulation. Insulin causes stable activation of glycogen synthase by promoting dephosphorylation of multiple sites in the enzyme. A model linking this action to the mitogen-activated protein kinase signaling pathway via the phosphorylation of the regulatory subunit of glycogen synthase phosphatase gained widespread acceptance. However, the most recent evidence argues strongly against this mechanism. A newer model, in which insulin inactivates the enzyme glycogen synthase kinase-3 via the protein kinase B pathway, has emerged. Though promising, this model still does not completely explain the molecular basis for the insulin-mediated activation of glycogen synthase, which remains one of the many unknowns of insulin action.

Glucose-induced glycogenesis in the liver involves the glucose-6-phosphate-dependent dephosphorylation of glycogen synthase.

Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-alpha-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1-0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.

Time-dependent pseudo-activation of hepatic glycogen synthase b by glucose 6-phosphate without involvement of protein phosphatases

During a 30 min incubation at 25 degrees C in the presence of 5-10 mM glucose 6-phosphate, pure glycogen-bound glycogen synthase b from dog liver was progressively converted into a form that was fully catalytically active in the presence of 10 mM Na2SO4 plus 0.5 mM glucose 6-phosphate. The latter enzyme was unlike synthase a (which does not require glucose 6-phosphate for activity), and unlike synthase b (which is strongly inhibited by sulphate). The conversion was insensitive to various inhibitors of Ser/Thr-protein phosphatases and alkaline phosphatases, and was therefore termed 'pseudo-activation'. Kinetically, pseudo-activation increased the V(max) 4-fold without affecting the K(m) for the substrate UDP-glucose. Pseudo-activation appeared to be an irreversible process, but several lines of evidence argue against a limited proteolysis. Pseudo-activation of glycogen synthase occurred also readily in a rat liver cytosol, but it was not observed with purified synthase from skeletal muscle. These observations have important implications for the assay of liver gycogen-synthase phosphatase; the possible physiological implications remain to be explored.

Effect of glucose phosphorylation on the activation by insulin of skeletal muscle glycogen synthase

The effect of insulin injection on skeletal muscle glycogen synthase activation was studied in anesthetized, normal, fed rats. Insulin stimulated the conversion of glycogen synthase to the active I form, increased the concentration of glucose 6-phosphate, and activated glycogen synthase phosphatase. A close correlation between glucose 6-phosphate concentrations, per cent glycogen synthase in the active I form, and phosphatase activity was found. When boiled extracts of muscle from control and insulin-injected animals were added to glycogen pellets containing phosphatase 1 G, the difference in phosphatase activity between muscle extracts from insulin-injected and control rats was restored, indicating that the phosphatase was activated by heat-stable factors. Deproteinized muscle extracts from control and insulin-injected rats, at concentrations equivalent to those present in muscle, were tested for the activation of glycogen synthase by purified protein phosphatases 1 and 2A. The activation with the insulin extracts was four-fold larger than with the control extracts. When the extracts from insulin-injected rats were treated with glucose 6-phosphatase, the difference in activation with the control rat extracts was canceled. It would appear that, as in other insulin sensitive tissues, in skeletal muscle the increase in glucose 6-phosphate subsequent to the activation of glucose transport by insulin contributes to the activation of glycogen synthase.

Transient ischemia induces regional myocardial glycogen synthase activation and glycogen synthesis in vivo

Glycogen is consumed during ischemic preconditioning and synthesized during the subsequent period of ischemic tolerance. To better understand this sequence, we examined the effect of brief coronary artery occlusions on regional myocardial glycogen metabolism in intact, anesthetized rats. Sequential 2-min periods of left coronary artery occlusion reduced the glycogen concentration of the anterior left ventricle approximately 30% relative to the posterior region. During subsequent reperfusion, the activity of the physiologically active glycogen synthase I form of glycogen synthase increased threefold in the anterior region (0.58 +/- 0.11 vs. 0.18 +/- 0.08 mumol.g-1.min-1, P < 0.01), stimulating a similar regional increase in glycogen synthesis rate (0.24 +/- 0.04 vs. 0.08 +/- 0.03 mumol.g-1.min-1, P < 0.01). These events were preceded by a rise in regional glucose 6-phosphate concentration, which increased the activity of a myocardial glycogen synthase phosphatase. In diabetic rats glycogen synthase phosphatase activity was significantly lower, and postischemic glycogen synthase activation was significantly impaired. These data suggest the operation of a feedback loop in which transient ischemia leads to a glucose 6-phosphate-mediated increase in the activity of a phosphoprotein phosphatase active toward glycogen synthase. This suggests phospho-protein phosphatase activation may be a feature of the preconditioned myocardium.

Factors Affecting the Activation of Glycogen Synthase in Primary Culture Cardiomyocytes

The ability of insulin, IGF-1 and IGF-2 to stimulate the activation of glycogen synthase in the heart was compared under completely defined conditions using primary culture cardiomyocytes. Both insulin and IGF-1 produced similar time-and concentration-dependent activation of glycogen synthase with the maximum stimulation observed at 10-15 min following hormone administration and at ⩾ 10 nM insulin or IGF-1. IGF-2 was largely ineffective at physiological concentrations. When primary culture cardiomyocytes were incubated with 100 μM palmitate for 2 h and then challenged with various concentrations of insulin or IGF-1, there was a significant decrease in the ability of the cells to activate glycogen synthase. In addition, maintaining cardiomyocytes in hormone deficient culture conditions for 24 or 48 h also resulted in a reduced ability to activate glycogen synthase in response to these hormones. These results suggest that (1) both insulin and IGF-1 are potent regulators of glycogen synthesis in the heart, (2) the enzymes involved in the dephosphorylation (activation) of glycogen synthase are closely linked to both insulin and IGF-1, but not IGF-2 receptor signaling pathways, (3) glycogen synthase activation is adversely affected by the maintenance of cardiomyocytes in the presence of palmitate or for ⩾ 24 h in hormone deficient media which results in insulin and IGF-1 resistance, and (4) this resistance, like that found in cells from diabetic rats, is due at least in part to a decrease in glycogen synthase phosphatase activity.