Allosteric Activation Of Glycogen Synthase Research Articles

Liver ChREBP Protects Against Fructose-Induced Glycogenic Hepatotoxicity by Regulating L-Type Pyruvate Kinase.

Excessive fructose consumption is closely linked to the pathogenesis of metabolic disease. Carbohydrate response element-binding protein (ChREBP) is a transcription factor essential for fructose tolerance in mice. However, the functional significance of liver ChREBP in fructose metabolism remains unclear. Here, we show that liver ChREBP protects mice against fructose-induced hepatotoxicity by regulating liver glycogen metabolism and ATP homeostasis. Liver-specific ablation of ChREBP did not compromise fructose tolerance, but rather caused severe transaminitis and hepatomegaly with massive glycogen overload in mice fed a high-fructose diet, while no obvious inflammation, cell death, or fibrosis was detected in the liver. In addition, liver ATP contents were significantly decreased by ChREBP deficiency in the fed state, which was rendered more pronounced by fructose feeding. Mechanistically, liver contents of glucose-6-phosphate (G6P), an allosteric activator of glycogen synthase, were markedly increased in the absence of liver ChREBP, while fasting-induced glycogen breakdown was not compromised. Furthermore, hepatic overexpression of LPK, a ChREBP target gene in glycolysis, could effectively rescue glycogen overload and ATP reduction, as well as mitigate fructose-induced hepatotoxicity in ChREBP-deficient mice. Taken together, our findings establish a critical role of liver ChREBP in coping with hepatic fructose stress and protecting from hepatotoxicity by regulating LPK.

Alcohol-induced ketonemia is associated with lowering of blood glucose, downregulation of gluconeogenic genes, and depletion of hepatic glycogen in type 2 diabetic db/db mice

Alcoholic ketoacidosis and diabetic ketoacidosis are life-threatening complications that share the characteristic features of high anion gap metabolic acidosis. Ketoacidosis is attributed in part to the massive release of ketone bodies (e.g., β-hydroxybutyrate; βOHB) from the liver into the systemic circulation. To date, the impact of ethanol consumption on systemic ketone concentration, glycemic control, and hepatic gluconeogenesis and glycogenesis remains largely unknown, especially in the context of type 2 diabetes. In the present study, ethanol intake (36% ethanol- and 36% fat-derived calories) by type 2 diabetic db/db mice for 9 days resulted in significant decreases in weight gain (∼19.5% ↓) and caloric intake (∼30% ↓). This was accompanied by a transition from macrovesicular-to-microvesicular hepatic steatosis with a modest increase in hepatic TG (∼37% ↑). Importantly, ethanol increased systemic βOHB concentration (∼8-fold ↑) with significant decreases in blood glucose (∼4-fold ↓) and plasma insulin and HOMA-IR index (∼3-fold ↓). In addition, ethanol enhanced hepatic βOHB content (∼5-fold ↑) and hmgcs2 mRNA expression (∼3.7-fold ↑), downregulated key gluconeogenic mRNAs (e.g., Pcx, Pck1, and G6pc), and depleted hepatic glycogen (∼4-fold ↓). Furthermore, ethanol intake led to significant decreases in the mRNA/protein expression and allosteric activation of glycogen synthase (GS) in liver tissues regardless of changes in the phosphorylation of GS, GSK-3β, or Akt. Together, our findings suggest that ethanol-induced ketonemia may occur in concomitance with significant lowering of blood glucose concentration, which may be attributed to suppression of gluconeogenesis in the setting of glycogen depletion in type 2 diabetes.

Dysregulation of Multiple Facets of Glycogen Metabolism in a Murine Model of Pompe Disease

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

Exercise training‐induced adaptations associated with increases in skeletal muscle glycogen content

Chronic exercise training results in numerous skeletal muscle adaptations, including increases in insulin sensitivity and glycogen content. To understand the mechanism leading to increased muscle glycogen, we studied the effects of exercise training on glycogen regulatory proteins in rat skeletal muscle. Female Sprague Dawley rats performed voluntary wheel running for 1, 4 or 7 weeks. After 7 weeks of training, insulin-stimulated glucose uptake was increased in epitrochlearis muscle. As compared with sedentary control rats, muscle glycogen did not change after 1 week of training, but increased significantly after 4 and 7 weeks. The increases in muscle glycogen were accompanied by elevated glycogen synthase activity and protein expression. To assess the regulation of glycogen synthase, we examined its major activator, protein phosphatase 1 (PP1), and its major deactivator, glycogen synthase kinase (GSK)-3. Consistent with glycogen synthase activity, PP1 activity was unchanged after 1 week of training but significantly increased after 4 and 7 weeks of training. Protein expression of R(GL)(G(M)), another regulatory PP1 subunit, significantly decreased after 4 and 7 weeks of training. Unlike PP1 activity, GSK-3 phosphorylation did not follow the pattern of glycogen synthase activity. The ~ 40% decrease in GSK-3α phosphorylation after 1 week of exercise training persisted until 7 weeks, and may function as a negative feedback mechanism in response to elevated glycogen. Our findings suggest that exercise training-induced increases in muscle glycogen content could be regulated by multiple mechanisms, including enhanced insulin sensitivity, glycogen synthase expression, allosteric activation of glycogen synthase, and PP1 activity.

Control of Glycogen Content in Retina: Allosteric Regulation of Glycogen Synthase

Retinal tissue is exceptional because it shows a high level of energy metabolism. Glycogen content represents the only energy reserve in retina, but its levels are limited. Therefore, elucidation of the mechanisms controlling glycogen content in retina will allow us to understand retina response under local energy demands that can occur under normal and pathological conditions. Thus, we studied retina glycogen levels under different experimental conditions and correlated them with glucose-6-phosphate (G-6-P) content and glycogen synthase (GS) activity.Glycogen and G-6-P content were studied in ex vivo retinas from normal, fasted, streptozotocin-treated, and insulin-induced hypoglycemic rats. Expression levels of GS and its phosphorylated form were also analyzed. Ex vivo retina from normal rats showed low G-6-P (14±2 pmol/mg protein) and glycogen levels (43±3 nmol glycosyl residues/mg protein), which were increased 6 and 3 times, respectively, in streptozotocin diabetic rats. While no changes in phosphorylated GS levels were observed in any condition tested, a positive correlation was found between G-6-P levels with GS activity and glycogen content. The results indicated that in vivo, retina glycogen may act as an immediately accessible energy reserve and that its content was controlled primarily by G-6-P allosteric activation of GS. Therefore, under hypoglycemic situations retina energy supply is strongly compromised and could lead to the alterations observed in type 1 diabetes.

Liver-specific Inducible Nitric-oxide Synthase Expression Is Sufficient to Cause Hepatic Insulin Resistance and Mild Hyperglycemia in Mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.

Molecular Mechanism by Which AMP-Activated Protein Kinase Activation Promotes Glycogen Accumulation in Muscle

OBJECTIVEDuring energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation.RESEARCH DESIGN AND METHODSWe recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed.RESULTSEven though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal.CONCLUSIONSWe provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake and subsequent rise in cellular [G6P].

Allosteric Regulation of Glycogen Synthase Controls Glycogen Synthesis in Muscle

Glycogen synthase (GS), a key enzyme in glycogen synthesis, is activated by the allosteric stimulator glucose-6-phosphate (G6P) and by dephosphorylation through inactivation of GS kinase-3 with insulin. The relative importance of these two regulatory mechanisms in controlling GS is not established, mainly due to the complex interplay between multiple phosphorylation sites and allosteric effectors. Here we identify a residue that plays an important role in the allosteric activation of GS by G6P. We generated knockin mice in which wild-type muscle GS was replaced by a mutant that could not be activated by G6P but could still be activated normally by dephosphorylation. We demonstrate that knockin mice expressing the G6P-insensitive mutant display an ∼80% reduced muscle glycogen synthesis by insulin and markedly reduced glycogen levels. Our study provides genetic evidence that allosteric activation of GS is the primary mechanism by which insulin promotes muscle glycogen accumulation invivo.

Allosteric Trumps Covalent in the Control of Glycogen Synthesis

Glycogen metabolism serves as a critical regulator of energy storage and maintenance of blood glucose levels in a physiological range. In this issue of Cell Metabolism, Bouskila etal. (2010) unexpectedly find that skeletal muscle glycogen accumulation is principally controlled by the allosteric activation of glycogen synthase, opening new avenues for investigation.

Control of glycogen synthase through ADIPOR1-AMPK pathway in renal distal tubules of normal and diabetic rats

Diabetic nephropathies are characterized by glycogen accumulation in distal tubular cells, which eventually leads to their apoptosis. The present study aims to determine whether adiponectin and AMPK are involved in the regulation of glycogen synthase (GS) in these structures. Western blots of isolated distal tubules revealed the presence of adiponectin receptor ADIPOR1, catalytic AMPK subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding AMPK subunit beta(2). ADIPOR2 was not detected. Expression levels of ADIPOR1, AMPKalpha(1), AMPKalpha(2), and AMPKbeta(2) were increased in streptozotocin-treated diabetic rats, whereas phosphorylated active AMPK levels were strongly decreased. Immunohistochemistry revealed the presence of ADIPOR1 on the luminal portion of distal tubules and thick ascending limb cells. Catalytic subunits alpha(1) and alpha(2), their phosphorylated active forms, and the glycogen-binding subunit beta(2) were also found in the same cells, confirming immunoblot results. In vitro, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR; 2 mM) and globular adiponectin (10 mug/ml) activated catalytic AMPK in distal tubules isolated from kidneys of normal rats but much more weakly in those from diabetic rats. GS inhibition paralleled AMPK activation in both groups of animals: active GS levels were low in control animals and elevated in diabetic ones. Finally, glucose-6-phosphate, an allosteric activator of GS, was also increased in diabetic rats. These results demonstrate that in distal tubular cells, adiponectin through luminal ADIPOR1 activates AMPK, leading to the inhibition of GS. During hyperglycemia, this regulation is altered, which may explain, at least in part, the accumulation of large glycogen deposits.

Kei on GSK: a contribution by the 2007 recipient of the Young Scientist Award

EDITORIAL FOCUSKei on GSK: a contribution by the 2007 recipient of the Young Scientist AwardAmira KlipAmira KlipPublished Online:01 Jan 2008https://doi.org/10.1152/ajpendo.00731.2007This is the final version - click for previous versionMoreSectionsPDF (29 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat from time to time, we like to highlight articles authored by winners of the awards offered by the American Physiological Society. The Endocrinology and Metabolism section of APS conferred the 2007 Young Scientist Award to Kei Sakamoto, PhD. Kei is a prolific young scientist who has contributed to the American Journal of Physiology-Endocrinology and Metabolism with numerous publications and by serving on its Editorial Board since January 2007.Kei began his doctoral training at Yokohama City University in his native Japan, but soon transferred to the Joslin Diabetes Center/Harvard Medical School to complete it under the supervision of Dr. Laurie Goodyear. In 2003 he joined the group led by Dr. Dario Alessi at the MRC Protein Phosphorylation Unit of the University of Dundee for further training as a postdoctoral fellow. Since January 2006 he has been a Principal Investigator in the same Unit, and this year he was appointed Head of Molecular Physiology.What is Kei focusing on now? These days it is glycogen metabolism.Muscle glycogen synthesis is stimulated by insulin, and this depends on increased glucose transport and phosphorylation to glucose 6-phosphate, an allosteric activator of glycogen synthase, along with the activation of glycogen synthase by insulin-promoted dephosphorylation of the enzyme on sites Ser641 and Ser645. Insulin achieves the latter by inactivating glycogen synthase kinase-3 (GSK-3). Insulin-stimulated Akt phosphorylates and inactivates both the α- and β-isoforms of GSK-3, thus allowing for a net decrease in glycogen synthase phosphorylation at Ser641/645. Hence, it has been thought that insulin stimulates glycogen synthesis largely by dephosphorylating glycgen synthase through the PI3K-PKB-GSK-3 pathway. The study by Sakamoto and colleagues (1), highlighted below, provides genetic evidence that the insulin-stimulated muscle can synthesize glycogen normally in the absence of this system.The story began with a previous study in which Kei participated as a postodoctoral fellow in the group of Dr. Dario Alessi. McManus, Sakamoto, Alessi, and colleagues created a genetically modified homozygous mouse model in which the two endogenous GSK-3 genes had been eliminated and replaced, through gene knockin (KI), by corresponding genes encoding mutations at the NH2-terminal phosphorylation sites [from serine to alanine residues (2)]. The clear advantage of such a model is the complete replacement of the wild-type kinases with constitutively active forms whose expression is transcriptionally regulated by the wild-type GSK-3 gene promoters.Sakamoto and colleagues [Bouskila et al. (1)] now took advantage of that mouse model to show that, compared with wild-type littermates, muscles from these KI mice have similar glycogen content in the fasted and fed state or following a bolus injection of glucose, despite lack of insulin-stimulated glycogen synthase activity. The muscles from the KI mice also had similar rates of glycogen accumulation compared with controls 1 or 2 h after exposure to insulin in vitro. As well, insulin increased normally both glucose transport and glucose 6-phosphate levels in KI mice. Furthermore, KI mice did not have compensatory alterations in glycogen metabolism muscle phosphorylase activity and responded normally to epinephrine by inhibition of glycogen synthase and stimulation of glycogen phosphorylase. The authors suggest, by extrapolation of the results, that glucose 6-phosphate may be the major regulator of insulin-stimulated glycogen accumulation. This is an intriguing proposition that will likely elicit further direct analysis, a challenge that Kei Sakamoto contemplates, probably along with many of our reader experts in this area.We are pleased to highlight the work of this young scientist and hope that it will inspire young investigators in pursuit of the intricacies of endocrinology and metabolism.REFERENCES1 Bouskila M, Hirshman M, Jensen J, Goodyear L, Sakamoto K. Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle. Am J Physiol Endocrinol Metab (November 6, 2007). doi:10.1152/ajpendo.0407.2007.Google Scholar2 McManus EJ, Sakamoto K, Armit LJ, Ronaldson L, Shapiro N, Marquez R, Alessi DR. Role that phosphorylation of GSK3 plays in insulin and Wnt signalling defined by knockin analysis. EMBO J 24: 1571–1583, 2005.Crossref | PubMed | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: A. Klip, Division of Cell Biology, The Hospital for Sick Children, Toronto, ON, M5G 1X8 Canada (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 294Issue 1January 2008Pages E27-E27 Copyright & PermissionsCopyright © 2008 by American Physiological Societyhttps://doi.org/10.1152/ajpendo.00731.2007PubMed18042667History Published online 1 January 2008 Published in print 1 January 2008 Metrics

Role of the PDK1–PKB–GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1–PKB–GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1 −/−), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3α (GSK3α) (Ser21) and GSK3β (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1 −/− or double GSK3α/GSK3β knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3β playing a more prominent role. This contrasts with skeletal muscle where GSK3β is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1 −/− or double GSK3α/GSK3β knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin’s ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1 −/− hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.

Glucosamine-induced Activation of Glycogen Biosynthesis in Isolated Adipocytes: EVIDENCE FOR A RAPID ALLOSTERIC CONTROL MECHANISM WITHIN THE HEXOSAMINE BIOSYNTHESIS PATHWAY

Enhanced flux through the hexosamine biosynthesis pathway (HBP) induces insulin resistance and facilitates lipid storage through the up-regulation of enzyme mRNA levels. Both actions occur over several hours and require gene expression. We now identify a regulatory arm of the HBP that involves rapid allosteric activation of glycogen synthase (GS) and stimulation of glycogen biosynthesis (GBS). When insulin-pretreated adipocytes were exposed to 2 mM GlcN, incorporation of [14C]glucose into glycogen doubled by 10 min (t(1/2) of <5 min), whereas UDP-glucose levels were concomitantly decreased during this time (t(1/2) of 1.4 min; >90% depletion). Stimulation of GBS and depletion of UDP-glucose both correlated with an early and rapid rise in the levels of glucosamine-6-phosphate (GlcN-6-P), a known activator of GS. The lowering of GlcN-6-P levels by removing extracellular GlcN (>80% reduction by 45 min) was accompanied by the restoration of UDP-glucose levels. Prolonged GlcN treatment (20 min to 2 h) inhibited GBS, which corresponded to a massive intracellular accumulation of GlcN-6-P (t(1/2) of approximately 32 min; >1,400 nmol/g). From these data, we conclude the following. 1) GlcN treatment elevated intracellular GlcN-6-P levels within minutes, resulting in allosteric activation of GS, stimulation of GBS, and a reduction in steady-state levels of UDP-glucose due to increased precursor utilization. 2) Prolonged treatment with high concentrations of GlcN caused massive accumulation of GlcN-6-P that adversely affected cellular metabolism and reduced GBS. 3) The biphasic actions of GlcN on GBS may explain many of the discrepant reports on the role of the HBP in glycogen metabolism.

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.

Glycogen synthase sensitivity to insulin and glucose-6-phosphate is mediated by both NH2- and COOH-terminal phosphorylation sites.

In skeletal muscle, insulin activates glycogen synthase by reducing phosphorylation at both NH2- and COOH-terminal sites of the enzyme and by elevating the levels of glucose-6-phosphate, an allosteric activator of glycogen synthase. To study the mechanism of regulation of glycogen synthase by insulin and glucose-6-phosphate, we generated stable Rat-1 fibroblast clones expressing rabbit muscle glycogen synthase with Ser-->Ala substitutions at key phosphorylation sites. We found that 1) elimination of the phosphorylation of either NH2- or COOH-terminal sites did not abolish insulin stimulation of glycogen synthase; 2) mutations at both Ser-7 and Ser-640 were necessary to bypass insulin activation; 3) mutation at Ser-7, coupled with the disruption of the motif for recognition by glycogen synthase kinase-3 (GSK-3), did not eliminate the insulin effect; and 4) mutation of either Ser-7 or Ser-640 increased the sensitivity of glycogen synthase to glucose 6-phosphate >10-fold. We conclude that Ser-7 and Ser-640 are both involved in mediating the response of glycogen synthase to insulin and activation by glucose 6-phosphate. In Rat-1 fibroblasts, GSK-3 action is not essential for glycogen synthase activation by insulin, and GSK-3-independent mechanisms also operate.

Allosteric regulation of glycogen synthase and hexokinase by glucosamine-6-phosphate during glucosamine-induced insulin resistance in skeletal muscle and heart.

Glucosamine infusion induces insulin resistance in vivo, but the effect of glucosamine on intracellular metabolites of the hexosamine pathway, especially glucosamine-6-phosphate (GlcN6P) is unknown. Because of the structural similarity of glucose-6-phosphate (G-6-P) and GlcN6P, we hypothesized that accumulation of this metabolite might alter the activities of enzymes such as glycogen synthase and hexokinase. We infused glucosamine (30 micromol x kg(-1) x min(-1)) to induce insulin resistance in rats during a euglycemic-hyperinsulinemic clamp. Glucosamine induced whole-body insulin resistance, which was apparent after 90 min and continued progressively for 360 min. Despite inducing severe whole-body insulin resistance and decrease in glycogen synthase fractional activity in rectus abdominis muscle (69+/-3 vs. 83+/-1%, P<0.01) and heart (7+/-1 vs. 32+/-4%, P<0.001), glucosamine did not change the glycogen content in rectus and even increased it in the heart (209+/-13 vs. 117+/-9 mmol/kg dry wt, P<0.001). Glucosamine increased tissue concentrations of UDP-GlcNAc 4.4- and 4.6-fold in rectus abdominis and heart, respectively. However, GlcN6P concentrations increased 500- and 700-fold in glucosamine-infused animals in rectus abdominis (590+/-80 vs. 1.2+/-0.1 micromol/kg wet wt, P<0.001) and heart (7,703+/-993 vs. 11.2+/-2.3 micromol/kg wet wt, P<0.001). To assess the possible significance of GlcN6P accumulation, we measured the effect of GlcN6P on glycogen synthase and hexokinase activity in vitro. At the GlcN6P concentrations measured in rectus abdominis and heart in vivo, glycogen synthase was activated by 21 and 542%, while similar concentrations inhibited hexokinase activity by 5 and 46%, respectively. This study demonstrates that infusion of glucosamine during a euglycemic-hyperinsulinemic clamp results in marked accumulation of intracellular GlcN6P. The GlcN6P concentrations in the heart and rectus abdominis muscle reach levels sufficient to cause allosteric activation of glycogen synthase and inhibition of hexokinase.

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.

Inhibition by glucose 6-phosphate of cyclic AMP-dependent protein kinase phosphorylation of glycogen synthase

Cyclic AMP-dependent protein kinase phosphorylates and inactivates glycogen synthase. In the absence of cyclic AMP, glycogen synthase is able to partially activate cyclic AMP-dependent protein kinase, probably by inducing the dissociation of the catalytic and regulatory subunits. The activation of cyclic AMP-dependent protein kinase by glycogen synthase is greatly reduced by the addition of low, physiological concentrations of the allosteric activator of glycogen synthase, glucose 6-phosphate. This effect appears to be specific for both glycogen synthase as substrate of the kinase and for cyclic AMP-dependent protein kinase as glycogen synthase phosphorylating enzyme. The result is an apparent, although not real effect of glucose 6-phosphate as an inhibitor competing with cyclic AMP. The reported inhibition by insulin of the activity of cyclic AMP-dependent protein kinase in skeletal muscle may be explained by the increased intracellular levels of glucose 6-phosphate resulting from the action of the hormone on glucose transport.

Effect of Acute Hyperglycemia on Glucose Metabolism in Skeletal Muscles in IDDM Patients

The effect of acute hyperglycemia on glucose metabolism in skeletal muscles was assessed during replacement insulin infusion in 11 patients with insulin-dependent diabetes mellitus (IDDM). With a primed continuous [3-3H]glucose infusion and indirect calorimetry, glucose metabolism was assessed during a basal period (plasma glucose [PG] 5 mM) and during a hyperglycemic period (4-h i.v. glucose infusion, PG 12.1 mM). Biopsies were taken from the vastus lateralis muscle during both periods. On a control day, glucose metabolism was assessed in 10 patients during a basal period (PG 5.2 mM) and after 4 h with no glucose infusion (PG 4.2 mM). Nonoxidative glucose disposal increased during hyperglycemia (32 +/- 7 vs. 51 +/- 9 mg.m-2.min-1, P less than 0.05), whereas glucose oxidation remained constant. On the control day, nonoxidative glucose disposal decreased from the basal to the second (control) period (33 +/- 7 vs. 22 +/- 6 mg.m-2.min-1, P less than 0.05), and glucose oxidation remained constant. The activity of glycogen synthase in muscle biopsies (fractional velocities [0.1 and 10 mM glucose 6-phosphate (G6P)]) decreased slightly during hyperglycemia (18 +/- 2 vs. 12 +/- 2%, P less than 0.05) and on the control day (26 +/- 4 vs. 20 +/- 3%, P less than 0.05). Hyperglycemia increased the intracellular concentration of free glucose, corrected for estimated extracellular glucose (0.56 +/- 0.11 vs. 1.43 +/- 0.19 mM, P less than 0.01), G6P (0.14 +/- 0.04 vs. 0.23 +/- 0.08 mM, P less than 0.02), and lactate (2.88 +/- 0.33 vs. 4.46 +/- 0.61 mM, P less than 0.05), whereas these substrate concentrations remained constant on the control day.(ABSTRACT TRUNCATED AT 250 WORDS)