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

ABSTRACT: 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.

SUBMITTER: Wera S

PROVIDER: S-EPMC1217201 | biostudies-other | 1996 Apr

REPOSITORIES: biostudies-other

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Glucose-6-phosphate-mediated activation of liver glycogen synthase plays a key role in hepatic glycogen synthesis.

Project description:The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg(582)) required for activation of GYS2 by G6P. We then used GYS2 Arg(582)Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.

| S-EPMC3837029 | biostudies-literature

Structural basis for glucose-6-phosphate activation of glycogen synthase.

Project description:Regulation of the storage of glycogen, one of the major energy reserves, is of utmost metabolic importance. In eukaryotes, this regulation is accomplished through glucose-6-phosphate levels and protein phosphorylation. Glycogen synthase homologs in bacteria and archaea lack regulation, while the eukaryotic enzymes are inhibited by protein kinase mediated phosphorylation and activated by protein phosphatases and glucose-6-phosphate binding. We determined the crystal structures corresponding to the basal activity state and glucose-6-phosphate activated state of yeast glycogen synthase-2. The enzyme is assembled into an unusual tetramer by an insertion unique to the eukaryotic enzymes, and this subunit interface is rearranged by the binding of glucose-6-phosphate, which frees the active site cleft and facilitates catalysis. Using both mutagenesis and intein-mediated phospho-peptide ligation experiments, we demonstrate that the enzyme's response to glucose-6-phosphate is controlled by Arg583 and Arg587, while four additional arginine residues present within the same regulatory helix regulate the response to phosphorylation.

| S-EPMC2955087 | biostudies-literature

Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate.

Project description:Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0-4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.

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Structural basis for 2'-phosphate incorporation into glycogen by glycogen synthase.

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| S-EPMC3876246 | biostudies-literature

Brain insulin action augments hepatic glycogen synthesis without suppressing glucose production or gluconeogenesis in dogs.

Project description:In rodents, acute brain insulin action reduces blood glucose levels by suppressing the expression of enzymes in the hepatic gluconeogenic pathway, thereby reducing gluconeogenesis and endogenous glucose production (EGP). Whether a similar mechanism is functional in large animals, including humans, is unknown. Here, we demonstrated that in canines, physiologic brain hyperinsulinemia brought about by infusion of insulin into the head arteries (during a pancreatic clamp to maintain basal hepatic insulin and glucagon levels) activated hypothalamic Akt, altered STAT3 signaling in the liver, and suppressed hepatic gluconeogenic gene expression without altering EGP or gluconeogenesis. Rather, brain hyperinsulinemia slowly caused a modest reduction in net hepatic glucose output (NHGO) that was attributable to increased net hepatic glucose uptake and glycogen synthesis. This was associated with decreased levels of glycogen synthase kinase 3β (GSK3β) protein and mRNA and with decreased glycogen synthase phosphorylation, changes that were blocked by hypothalamic PI3K inhibition. Therefore, we conclude that the canine brain senses physiologic elevations in plasma insulin, and that this in turn regulates genetic events in the liver. In the context of basal insulin and glucagon levels at the liver, this input augments hepatic glucose uptake and glycogen synthesis, reducing NHGO without altering EGP.

| S-EPMC3163950 | biostudies-literature

Characterization of recombinant UDP- and ADP-glucose pyrophosphorylases and glycogen synthase to elucidate glucose-1-phosphate partitioning into oligo- and polysaccharides in Streptomyces coelicolor.

Project description:Streptomyces coelicolor exhibits a major secondary metabolism, deriving important amounts of glucose to synthesize pigmented antibiotics. Understanding the pathways occurring in the bacterium with respect to synthesis of oligo- and polysaccharides is of relevance to determine a plausible scenario for the partitioning of glucose-1-phosphate into different metabolic fates. We report the molecular cloning of the genes coding for UDP- and ADP-glucose pyrophosphorylases as well as for glycogen synthase from genomic DNA of S. coelicolor A3(2). Each gene was heterologously expressed in Escherichia coli cells to produce and purify to electrophoretic homogeneity the respective enzymes. UDP-glucose pyrophosphorylase (UDP-Glc PPase) was characterized as a dimer exhibiting a relatively high V(max) in catalyzing UDP-glucose synthesis (270 units/mg) and with respect to dTDP-glucose (94 units/mg). ADP-glucose pyrophosphorylase (ADP-Glc PPase) was found to be tetrameric in structure and specific in utilizing ATP as a substrate, reaching similar activities in the directions of ADP-glucose synthesis or pyrophosphorolysis (V(max) of 0.15 and 0.27 units/mg, respectively). Glycogen synthase was arranged as a dimer and exhibited specificity in the use of ADP-glucose to elongate α-1,4-glucan chains in the polysaccharide. ADP-Glc PPase was the only of the three enzymes exhibiting sensitivity to allosteric regulation by different metabolites. Mannose-6-phosphate, phosphoenolpyruvate, fructose-6-phosphate, and glucose-6-phosphate behaved as major activators, whereas NADPH was a main inhibitor of ADP-Glc PPase. The results support a metabolic picture where glycogen synthesis occurs via ADP-glucose in S. coelicolor, with the pathway being strictly regulated in connection with other routes involved with oligo- and polysaccharides, as well as with antibiotic synthesis in the bacterium.

| S-EPMC3294831 | biostudies-literature

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

Project description: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.

| S-EPMC1218250 | biostudies-other

Role of glucose 6-phosphate in the translocation of glycogen synthase in rat hepatocytes.

Project description:Incubation of rat hepatocytes with glucose induces the translocation of glycogen synthase from soluble fractions to fractions which sediment at 10,000 g. Incubation of the cells with fructose, galactose, 2-deoxyglucose or 5-thioglucose, which activate glycogen synthase, also resulted in the translocation of the enzyme, whereas 3-O-methylglucose, 6-deoxyglucose and 1,5-anhydroglucitol, which do not cause the activation of the enzyme, were ineffective. Adenosine and carbonyl cyanide m-chlorophenylhydrazone, although activating glycogen synthase, did not induce its translocation. Mannoheptulose, which inhibits glucose phosphorylation, impaired the translocation of glycogen synthase induced by glucose. Furthermore, the extent of the translocation of the enzyme triggered by glucose and other sugars showed a high positive correlation with the intracellular concentration of glucose 6-phosphate. Microcystin, which blocks the activation of glycogen synthase by glucose, but not the accumulation of glucose 6-phosphate, did not affect the translocation of the enzyme. These results indicate that glucose 6-phosphate plays a role in the translocation of glycogen synthase in rat hepatocytes.

| S-EPMC1132038 | biostudies-other

Hepatic glycogen synthase (GYS2) deficiency: seven novel patients and seven novel variants.

Project description:Glycogen storage disease type 0 (GSD 0) is an autosomal recessive disorder of glycogen metabolism caused by mutations in the GYS2 gene manifesting in infancy or early childhood and characterized by ketotic hypoglycemia after prolonged fasting, and postprandial hyperglycemia and hyperlactatemia. GSD 0 is a rare form of hepatic glycogen storage disease with less than 30 reported patients in the literature so far.DNA samples of 93 Russian patients with clinical diagnoses of hepatic GSDs were collected and analyzed by next-generation sequencing custom target panel and by direct sequencing. Seven new GSD 0 patients with variable phenotypes were found showing 10 variants. Seven variants are novel.We present seven new GSD 0 patients with variable phenotypes. Overall, 10 different mutant alleles of the GYS2 gene were found. Seven of them are novel: c.214delC, c.845delT, c.1644C>A, c.205T>A, c.929G>T, c.1169G>C and c.1703C>A. Three of the novel variants were annotated as pathogenic and likely pathogenic; four other variants have an uncertain significance.The current results expand the spectrum of known mutations in GYS2 and suggest that phenotypes of GSD 0 are more variable and less specific than the reported ones.SynopsisSeven new patients with glycogen storage disease type 0 were found using next-generation sequencing and seven novel variants of GYS2 gene were annotated.

| S-EPMC7203652 | biostudies-literature

Shared control of hepatic glycogen synthesis by glycogen synthase and glucokinase.

Project description:We have used recombinant adenoviruses (AdCMV-RLGS and AdCMV-GK) to overexpress the liver isoforms of glycogen synthase (GS) and glucokinase (GK) in primary cultured rat hepatocytes. Glucose activated overexpressed GS in a dose-dependent manner and caused the accumulation of larger amounts of glycogen in the AdCMV-RLGS-treated hepatocytes. The concentration of intermediate metabolites of the glycogenic pathway, such as glucose 6-phosphate (Glc-6-P) and UDP-glucose, were not significantly altered. GK overexpression also conferred on the hepatocyte an enhanced capacity to synthesize glycogen in response to glucose, as described previously [Seoane, Gómez-Foix, O'Doherty, Gómez-Ara, Newgard and Guinovart (1996) J. Biol. Chem. 271, 23756-23760], although, in this case, they accumulated Glc-6-P. When GS and GK were simultaneously overexpressed, the accumulation of glycogen was enhanced in comparison with cells overexpressing either GS or GK. Our results are consistent with the hypothesis that liver GS catalyses the rate-limiting step of hepatic glycogen synthesis. However, hepatic glycogen deposition from glucose is submitted to a system of shared control in which the 'controller', GS, is, in turn, controlled by GK. This control is indirectly exerted through Glc-6-P, which 'switches on' GS dephosphorylation and activation.

| S-EPMC1221423 | biostudies-other

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