Glycogen synthase 2 knock-out zebrafish exhibit enhanced glucose metabolism in the liver.

Conversion to glycogen is a major fate of ingested glucose in the body. A rate-limiting enzyme in the synthesis of glycogen is glycogen synthase encoded by two genes, GYS1, expressed in muscle and other tissues, and GYS2, primarily expressed in liver (liver glycogen synthase). Defects in GYS2 cause the inherited monogenic disease glycogen storage disease 0. We have generated mice with a liver-specific disruption of the Gys2 gene (liver glycogen synthase knock-out (LGSKO) mice), using Lox-P/Cre technology. Conditional mice carrying floxed Gys2 were crossed with mice expressing Cre recombinase under the albumin promoter. The resulting LGSKO mice are viable, develop liver glycogen synthase deficiency, and have a 95% reduction in fed liver glycogen content. They have mild hypoglycemia but dispose glucose less well in a glucose tolerance test. Fed, LGSKO mice also have a reduced capacity for exhaustive exercise compared with mice carrying floxed alleles, but the difference disappears after an overnight fast. Upon fasting, LGSKO mice reach within 4 h decreased blood glucose levels attained by control floxed mice only after 24 h of food deprivation. The LGSKO mice maintain this low blood glucose for at least 24 h. Basal gluconeogenesis is increased in LGSKO mice, and insulin suppression of endogenous glucose production is impaired as assessed by euglycemic-hyperinsulinemic clamp. This observation correlates with an increase in the liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase expression and activity. This mouse model mimics the pathophysiology of glycogen storage disease 0 patients and highlights the importance of liver glycogen stores in whole body glucose homeostasis.

Glycogen metabolism has been the subject of extensive research, but the mechanisms by which it is regulated are still not fully understood. It is well accepted that the rate-limiting enzymes in glycogenesis and glycogenolysis are glycogen synthase (GS) and glycogen phosphorylase (GPh), respectively. Both enzymes are regulated by reversible phosphorylation and by allosteric effectors. However, evidence in the literature indicates that changes in muscle GS and GPh intracellular distribution may constitute a new regulatory mechanism of glycogen metabolism. Already in the 1960s, it was proposed that glycogen was present in dynamic cellular organelles that were termed glycosomas but no such cellular entities have ever been demonstrated. The aim of this study was to characterize muscle GS and GPh intracellular distribution and to identify possible translocation processes of both enzymes. Using in situ stimulation of rabbit tibialis anterior muscle, we show GS and GPh intracellular redistribution at the beginning of glycogen resynthesis after contraction-induced glycogen depletion. We identify a new "player," a new intracellular compartment involved in skeletal muscle glycogen metabolism. They are spherical structures that were not present in basal muscle, and we present evidence that indicate that they are products of actin cytoskeleton remodeling. Furthermore, for the first time, we show a phosphorylation-dependent intracellular distribution of GS. Here, we present evidence of a new regulatory mechanism of skeletal muscle glycogen metabolism based on glycogen enzyme intracellular compartmentalization.

Catecholamines, acting through adrenergic receptors, play an important role in modulating the effects of insulin on glucose metabolism. Insulin activation of glycogen synthesis is mediated in part by the inhibitory phosphorylation of glycogen synthase kinase-3 (GSK-3). In this study, catecholamine regulation of GSK-3beta was investigated in Rat-1 fibroblasts stably expressing the alpha1A-adrenergic receptor. Treatment of these cells with either insulin or phenylephrine (PE), an alpha1-adrenergic receptor agonist, induced Ser-9 phosphorylation of GSK-3beta and inhibited GSK-3beta activity. Insulin-induced GSK-3beta phosphorylation is mediated by the phosphatidylinositol 3-kinase/Akt signaling pathway. PE treatment does not activate phosphatidylinositol 3-kinase or Akt (Ballou, L. M., Cross, M. E., Huang, S., McReynolds, E. M., Zhang, B. X., and Lin, R. Z. (2000) J. Biol. Chem. 275, 4803-4809), but instead inhibits insulin-induced Akt activation and GSK-3beta phosphorylation. Experiments using protein kinase C (PKC) inhibitors suggest that phorbol ester-sensitive novel PKC and Gö 6983-sensitive atypical PKC isoforms are involved in the PE-induced phosphorylation of GSK-3beta. Indeed, PE treatment of Rat-1 cells increased the activity of atypical PKCzeta, and expression of PKCzeta in COS-7 cells stimulated GSK-3beta Ser-9 phosphorylation. In addition, PE-induced GSK-3beta phosphorylation was reduced in Rat-1 cells treated with a cell-permeable PKCzeta pseudosubstrate peptide inhibitor. These results suggest that the alpha1A-adrenergic receptor regulates GSK-3beta through two signaling pathways. One pathway inhibits insulin-induced GSK-3beta phosphorylation by blocking insulin activation of Akt. The second pathway stimulates Ser-9 phosphorylation of GSK-3beta, probably via PKC.

Defects in liver and muscle glycogen synthesis are major factors contributing to postprandrial hyperglycemia in patients with type 2 diabetes. Therefore, activation of glycogen synthase through inhibition of glycogen synthase kinase (GSK)-3 represents a potential new therapeutic target. To examine this possibility, we performed oral glucose tolerance tests (OGTTs) and euglycemic-insulinemic clamp studies in Zucker diabetic fatty (fa/fa) rats before and after treatment with novel GSK-3 inhibitors. GSK-3 inhibition caused a 41 +/- 2% (P < 0.001) and 26 +/- 4% (P < 0.05) reduction in the area under the glucose and insulin concentration curves, respectively, during the OGTT. This improvement in glucose disposal could mostly be attributed to an approximate twofold increase in liver glycogen synthesis. In contrast, there was no significant increase in muscle glycogen synthesis despite an approximate threefold activation of muscle glycogen synthase activity. GSK-3 inhibitor treatment increased liver glycogen synthesis about threefold independent of insulin concentration during the clamp studies. In contrast, muscle glucose uptake and muscle glycogen synthesis were independent of drug treatment. GSK-3 inhibitor treatment lowered fasting hyperglycemia in diabetic rats by 6.0 +/- 1.3 mmol/l but had no significant effect on glucose disposal during the clamp. In conclusion, GSK-3 inhibition significantly improved oral glucose disposal, mostly by increasing liver glycogen synthesis. These studies suggest that GSK-3 inhibition may represent an important new therapeutic target for treatment of patients with type 2 diabetes.

The pathway for hepatic glycogen synthesis in the postprandial state was studied in meal-fed rats chronically cannulated in the portal vein. Plasma glucose concentration in the portal vein was found to be 4.50 +/- 1.01 mM (mean +/- SE; n = 3) before a meal and 11.54 +/- 0.70 mM (mean +/- SE; n = 4) after a meal in rats meal-fed a diet consisting of 100% commercial rat chow for 7 d. The hepatic-portal difference of plasma glucose concentration showed that liver released glucose in the fasted state and either extracted or released glucose after feeding depending on plasma glucose concentration in the portal vein. The concentration of portal vein glucose at which liver changes from glucose releasing to glucose uptake was 8 mM, the Km of glucokinase [E.C. 2.7.1.12]. The rate of glycogen synthesis in liver during meal-feeding was found to be approximately 1 mumol glucosyl U/g wet wt/min in rats meal-fed a 50% glucose supplemented chow diet. The relative importance of the direct vs. indirect pathway for the replenishment of hepatic glycogen was determined by the incorporation of [3-3H,U-14C]glucose into liver glycogen. Labeled glucose was injected into the portal vein at the end of meal-feeding. The ratio of 3H/14C in the glucosyl units of glycogen was found to be 83-92% of the ratio in liver free glucose six minutes after the injection, indicating that the majority of exogenous glucose incorporated into glycogen did not go through glycolysis. The percent contribution of the direct versus indirect pathway was quantitated from the difference in the relative specific activity (RSA) of [3H] and [14C]-glycogen in rats infused with [3-3H,U-14C]glucose. No significant difference was found between the RSA of [3H]glycogen and [14C]glycogen, indicating further that the pathway for glycogen synthesis in liver from exogenous glucose is from the direct pathway. Our results do not support the thesis that the majority of liver glycogen is synthesized from glucose-6-phosphate derived from gluconeogenesis. Reasons for the discrepancy between current findings and other reports supporting the indirect pathway for glycogen synthesis in the liver are discussed.

Pharmacological activation of peroxisome proliferator-activated receptor δ/β (PPARδ/β) improves glucose handling and insulin sensitivity. The target tissues of drug actions remain unclear. We demonstrate here that adenovirus-mediated liver-restricted PPARδ activation reduces fasting glucose levels in chow- and high fat-fed mice. This effect is accompanied by hepatic glycogen and lipid deposition as well as up-regulation of glucose utilization and de novo lipogenesis pathways. Promoter analyses indicate that PPARδ regulates hepatic metabolic programs through both direct and indirect transcriptional mechanisms partly mediated by its co-activator, PPARγ co-activator-1β. Assessment of the lipid composition reveals that PPARδ increases the production of monounsaturated fatty acids, which are PPAR activators, and reduces that of saturated FAs. Despite the increased lipid accumulation, adeno-PPARδ-infected livers exhibit less damage and show a reduction in JNK stress signaling, suggesting that PPARδ-regulated lipogenic program may protect against lipotoxicity. The altered substrate utilization by PPARδ also results in a secondary effect on AMP-activated protein kinase activation, which likely contributes to the glucose-lowering activity. Collectively, our data suggest that PPARδ controls hepatic energy substrate homeostasis by coordinated regulation of glucose and fatty acid metabolism, which provide a molecular basis for developing PPARδ agonists to manage hyperglycemia and insulin resistance.

With the cessation of exercise, glycogen repletion begins to take place rapidly in skeletal muscle and can result in glycogen levels higher than those present before exercise. Understanding the rate-limiting steps that regulate glycogen synthesis will provide us with strategies to increase the resynthesis of glycogen during recovery from exercise, and thus improve performance. Given the importance of muscle glycogen to endurance performance, various factors which may optimise glycogen resynthesis rate and insure complete restoration have been of interest to both the scientist and athlete. The time required for complete muscle glycogen resynthesis after prolonged moderate intensity exercise is generally considered to be 24 hours provided approximately 500 to 700g of carbohydrate is ingested. Muscle glycogen synthesis rate is highest during the first 2 hours after exercise. Ingestion of 0.70g glucose/kg bodyweight every 2 hours appears to maximise glycogen resynthesis rate at approximately 5 to 6 mumol/g wet weight/h during the first 4 to 6 hours after exhaustive exercise. Further enhancement of glycogen resynthesis rate with ingestion of greater than 0.70g glucose/kg bodyweight appears to be limited by the constraints imposed by gastric emptying. Ingestion of glucose or sucrose results in similar muscle glycogen resynthesis rates while glycogen synthesis in liver is better served with the ingestion of fructose. Also, increases in muscle glycogen content during the first 4 to 6 hours after exercise are greater with ingestion of simple as compared with complex carbohydrate. Glycogen synthase activity is a key component in the regulation of glycogen resynthesis. Glycogen synthase enzyme exists in 2 states: the less active, more phosphorylated (D) form which is under allosteric control of glucose-6-phosphate, and the more active, less phosphorylated (I) form which is independent of glucose-6-phosphate. There is generally an inverse relationship between glycogen content in muscle and the percentage synthase in the activated (I) form. Exercise and insulin by themselves activate glycogen synthase by conversion to glycogen synthase I. Although small changes in the activity ratio (% I form) can lead to large changes in the rate of glycogen synthesis, glycogen synthase I appears to increase very little (approximately 25%) in response to glycogen depletion and returns to pre-exercise levels as glycogen levels return to normal. Thus glycogen resynthesis, which may increase 3- to 5-fold, may also be influenced by glucose-6-phosphate, which can activate glycogen synthase in the D form.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Growth factor stimulation and oncogenic transformation lead to increased glucose metabolism that may provide resistance to cell death. We have previously demonstrated that elevated glucose metabolism characteristic of stimulated or cancerous cells can stabilize the anti-apoptotic Bcl-2 family protein Mcl-1 through inhibition of GSK-3. Here we show that the pro-apoptotic Bcl-2 family protein, Puma, is also metabolically regulated. Growth factor deprivation led to the loss of glucose uptake and induction of Puma. Maintenance of glucose uptake after growth factor withdrawal by expression of the glucose transporter, Glut1, however, suppressed Puma up-regulation and attenuated growth factor withdrawal-induced activation of Bax, DNA fragmentation, and cell death. Conversely, glucose deprivation led to Puma induction even in the presence of growth factor. This regulation of Puma expression was a central component in cell death as a consequence of growth factor or glucose deprivation because Puma deficiency suppressed both of these cell death pathways. Puma induction in growth factor or glucose withdrawal was dependent on p53 in cell lines and in activated primary T lymphocytes because p53 deficiency suppressed Puma induction and delayed Bax and caspase activation, DNA fragmentation, and loss of clonogenic survival. Importantly, although p53 levels did not change or were slightly reduced, p53 activity was suppressed by elevated glucose metabolism to inhibit Puma induction after growth factor withdrawal. These data show that p53 is metabolically regulated and that glucose metabolism initiates a signaling mechanism to inhibit p53 activation and suppress Puma induction, thus promoting an anti-apoptotic balance to Bcl-2 family protein expression that supports cell survival.

Blunt snout bream (Megalobrama amblycephala) were randomly assigned into three diets: normal-carbohydrate diet (NCD, 30% carbohydrate, w/w), high-carbohydrate diet (HCD, 43% carbohydrate), and HCB (HCD supplemented with 50 mg/kg berberine (BBR)). After 10 weeks' feeding trial, the results showed that higher levels of plasma glucose, triglyceride, and total cholesterol were observed in HCD-fed fish than in NCD-fed fish, while HCB feeding significantly ameliorated this effect. Moreover, HCB feeding remarkably reversed HCD-induced hepatic glycogen and lipid contents. In insulin signaling, BBR inclusion restored HCD-induced suppression of insulin receptor substrate mRNA expression and elevation of forkhead transcription factor 1 mRNA expression. In glucose metabolism, upregulated glucose transporter 2 and glycogen synthase mRNA expressions in the HCD group were observed compared to the NCD group. However, BBR adding reduced the mRNA expressions of glycogen synthase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphatase and increased the transcriptional levels of glucose transporter 2 and pyruvate kinase. In lipid metabolism, BBR supplementation could reverse downregulated hepatic carnitine palmitoyl transferase I mRNA expression and upregulated hepatic acetyl-CoA carboxylase and fatty acid synthetase mRNA expressions in the HCD group. Taken together, it demonstrates that BBR could improve glucose metabolism of this species via enhancing liver's glycolysis and insulin signaling, while inhibiting liver's glycogen synthesis and gluconeogenesis. It also indicates that BBR could reduce the metabolic burden of the liver by inhibiting fat synthesis and promoting lipid decomposition, and then enhance fat uptake in peripheral tissues.

Chemical coding of the hypothalamic neurones in metabolic control. I. Acetylcholine-sensitive neurones and glycogen synthesis in liver

Lafora disease (LD) is caused by mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of polyglucosan inclusions called Lafora Bodies (LBs). Malin knockout (KO) mice present polyglucosan accumulations in several brain areas, as do patients of LD. These structures are abundant in the cerebellum and hippocampus. Here, we report a large increase in glycogen synthase (GS) in these mice, in which the enzyme accumulates in LBs. Our study focused on the hippocampus where, under physiological conditions, astrocytes and parvalbumin-positive (PV+) interneurons expressed GS and malin. Although LBs have been described only in neurons, we found this polyglucosan accumulation in the astrocytes of the KO mice. They also had LBs in the soma and some processes of PV+ interneurons. This phenomenon was accompanied by the progressive loss of these neuronal cells and, importantly, neurophysiological alterations potentially related to impairment of hippocampal function. Our results emphasize the relevance of the laforin–malin complex in the control of glycogen metabolism and highlight altered glycogen accumulation as a key contributor to neurodegeneration in LD.

In fetal rat liver in utero, an increase in glycogen and the glycogen synthetic enzyme glycogen synthetase (EC 2.4.1.11) occurs between gestational days 17 and 19. We used an organ culture system for finding the stimulus for increased enzyme activity and defining the relationship of this increase to glycogen synthesis. In fetal-liver explants from an earlier period (gestational day 16), hydrocortisone causes an elevation in total glycogen synthetase activity. This effect, which can be blocked by actinomycin D, is strikingly similar in time course and magnitude to the normal increase in utero. However, in order for glycogen synthesis to proceed after hydrocortisone increases glycogen synthetase levels, insulin is required. Unlike hydrocortisone, insulin does not increase total glycogen synthetase; it appears to act by converting the b or phospho form of synthetase to the a or dephospho form. Insulin alone does not stimulate glycogen synthesis in explants from 16-day fetal liver, although no defect in insulin binding was demonstrable. These findings support the hypothesis that the increase in liver-glycogen synthesis during the last trimester requires glucocorticoids for the developmental induction of glycogen synthetase and insulin for activation of the enzyme.

12 Liver Glycogen Synthase

The hexosamine biosynthesis pathway has been hypothesized to mediate some of the regulatory as well as the deleterious effects of glucose. We have stably overexpressed the cDNA for human glutamine:fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the hexosamine biosynthesis pathway, in rat-1 fibroblasts. Two cell lines expressing the human RNA were selected by Northern analysis, and they exhibited 51-95% increases in GFA activity. Insulin-stimulated glycogen synthase (GS) activity and net glycogen synthesis were assayed, and GFA cells revealed decreased insulin sensitivity for both GS and net glycogen synthesis. The ED50 for insulin stimulation of GS was 2.45 +/- 0.4 nmol/l insulin in controls and 5.29 +/- 1.01 nmol/l in GFA cells (P < 0.005). For insulin-stimulated glycogen synthesis, the ED50 was 3.43 +/- 0.88 nmol/l in controls and 5.54 +/- 0.98 nmol/l in GFA cells (P < 0.005). There were no significant differences in maximally insulin-stimulated or total GS activities, insulin binding or receptor number, or glucose uptake between GFA and control cells. We also examined the effects of glucose on GS activity. GFA cells had a twofold increase in GS activity at low glucose (0.5 mmol/l) when compared with controls (P < 0.025). Both GFA and control cells had an approximately 75-80% decrease in GS activity as glucose concentration was increased from 0.5 to 20 mmol/l. This change in GS activity was not observed until after 12 h in culture. GFA cells were more sensitive to the effects of glucose.(ABSTRACT TRUNCATED AT 250 WORDS)