Glycogen Concentration and Regulation of Synthase Activity in Rat Liver in Vivo
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Glycogen debranching enzyme
Glycogen branching enzyme
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About 90% of the glycogenin in skeleta muscle extracts prepared from fed, 24‐h starved or alloxan‐diabetic rabbits sedimented at 140 000xg with the glycogen/sarcovesicular fraction, from which it was released by glycogenolysis, but not by 1% SDS. Glycogenin in the glycogen/sarcovesicular fraction is therefore bound covalently to glycogen, and not associated (covalently or non‐covalently) with the sarcoplasmic reticulum. The same proportion of glycogen synthase was also recovered in the glycogen/sarcovesicle fraction, was solubilised by glycogenolysis, and copurified with glycogenin to yield a heterodimer composed of a 1:1 complex between these proteins. Glycogen synthase and glycogenin are therefore present in equimolar amounts in skeletal muscle and there is an average of one glycogen synthase catalytic subunit associated with each molecule of glycogen in vivo . Glycogenin and glycogen synthase released into the muscle cytosol by degradation of glycogen did not form a complex initially, and only 50% reassociation took place after storage for several hours or overnight dialysis. This suggests that the muscle cytosol may contain a factor(s) which regulates glycogen biogenesis by modulating the association of glycogenin and glycogen synthase. Only glycogen synthase that was complexed to glycogenin was capable of elongating the primer formed by incubation of glycogenin with Mn 2+ and micromolar concentration of UDP‐glucose, demonstrating the critical importance of this complex for glycogen biogenesis.
Glycogen debranching enzyme
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Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counter-acted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.
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Changes in the glucosylation state of the glycogen primer, glycogenin, or its association with glycogen synthase are potential sites for regulation of glycogen synthesis. In this study we found no evidence for hormonal control of the glucosylation state of glycogenin in hepatocytes. However, using a modified glycogen synthase assay that separates the product into acid‐soluble (glycogen) and acid‐insoluble (proteoglycogen) fractions we found that insulin and glucagon increase and decrease, respectively, the association of glycogen synthase with an acid‐insoluble substrate. The latter fraction had a higher affinity for UDP‐glucose and accounted for between 5 and 21% of total activity depending on hormonal conditions. Phosphorylase overexpression mimicked the effect of glucagon. It is concluded that phosphorylase activation or overexpression causes dissociation of glycogen synthase from proteoglycogen causing inhibition of initiation of glycogen synthesis.
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In these studies we expressed and characterized wild-type (WT) GSK-3 (glycogen synthase kinase-3) and its mutants, and examined their physiological effect on glycogen synthase activity. The GSK-3 mutants included mutation at serine-9 either to alanine (S9A) or glutamic acid (S9E) and an inactive mutant, K85,86MA. Expression of WT and the various mutants in a cell-free system indicated that S9A and S9E exhibit increased kinase activity as compared with WT. Subsequently, 293 cells were transiently transfected with WT GSK-3 and mutants. Cells expressing the S9A mutant exhibited higher kinase activity (2.6-fold of control cells) as compared with cells expressing WT and S9E (1.8- and 2.0-fold, respectively, of control cells). Combined, these results suggest serine-9 as a key regulatory site of GSK-3 inactivation, and indicate that glutamic acid cannot mimic the function of the phosphorylated residue. The GSK-3-expressing cell system enabled us to examine whether GSK-3 can induce changes in the endogenous glycogen synthase activity. A decrease in glycogen synthase activity (50%) was observed in cells expressing the S9A mutant. Similarly, glycogen synthase activity was suppressed in cells expressing WT and the S9E mutant (20-30%, respectively). These studies indicate that activation of GSK-3 is sufficient to inhibit glycogen synthase in intact cells, and provide evidence supporting a physiological role for GSK-3 in regulating glycogen synthase and glycogen metabolism.
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Abstract The sections in this article are: Glycogen Structure Function Pathways of Glycogen Metabolism Overview Glycogen Biosynthesis Glycogen Synthase and Branching Enzyme Glycogenolysis and Debranching Enzyme Glycogen Particles and Physical Interactions among Glycogen‐Metabolizing Proteins Hormonal Regulation of Glycogen Synthesis in Muscle Insulin Mechanisms of Insulin Action Epinephrine Regulation of Glycogen Metabolism in Liver Overview Two Pathways of Glycogen Synthesis in Liver Glucose Transport Is Not Regulated in Liver Regulation of Glucokinase Activity Regulation of Glycogen Synthase Activity Regulation of Glucose‐6‐Phosphatase Activity Regulation of Glycogen Phosphorylase Activity Conclusion
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The astrocyte of the newborn rat brain has proven to be a versatile system in which to study glycogen biogenesis. We have taken advantage of the rapid stimulation of glycogen synthesis that occurs when glucose is fed to astrocytes, and the marked limitation on this synthesis that occurs in astrocytes previously exposed to ammonium ions. These observations have been related to our earlier reports of the initiation of glycogen synthesis on a protein primer, glycogenin, and the discovery of a low-molecular-weight form of glycogen, proglycogen. The following conclusions have been drawn: 1) In the ammonia-treated astrocytes starved of glucose, free glycogenin is present. 2) When these astrocytes are fed with glucose, proglycogen is synthesized from the glycogenin primer by a glycogen-synthase-like UDPglucose transglucosylase activity (proglycogen synthase) distinct from the well-recognized glycogen synthase, and synthesis stops at this point. 3) Proglycogen is the precursor of macromolecular glycogen, which is synthesized from proglycogen by glycogen synthase when glucose is fed to untreated astrocytes, accounting for the much greater accumulation of total glycogen. 4) The stimulus to proglycogen and macroglycogen synthesis that occurs on feeding glucose to untreated or ammonia-treated astrocytes is the result of the activation of proglycogen synthase, not of glycogen synthase. 5) Therefore, in the synthesis of macromolecular glycogen from glycogenin via proglycogen, the step between glycogenin and proglycogen is rate-limiting. 6) The discovery of additional potential control points in glycogen synthesis, now emerging, may assist the identification of so-far-unexplained aberrations of glycogen metabolism.—Lomako, J., Lomako, W. M., Whelan, W. J., Dombro, R. S., Neary, J. T., and Norenberg, M. D. Glycogen synthesis in the astrocyte: from glycogen to proglycogen to glycogen. FASEB J. 7: 1386-1393; 1993.
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Glycogen synthase kinase‐5 (casein kinase‐II) phosphorylates glycogen synthase on a serine termed site 5. This residue is just C‐terminal to the 3 serines phosphorylated by glycogen synthase kinase‐3, which are critical for the hormonal regulation of glycogen synthase in vivo. Although phosphorylation of site 5 does not affect the catalytic activity, it is demonstrated that this modification is a prerequisite for phosphorylation by glycogen synthase kinase‐3. Since site 5 is almost fully phosphorylated in vivo under all conditions, the role of glycogen synthase kinase‐5 would appear to be a novel one in forming the recognition site for another protein kinase
Glycogen branching enzyme
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Glycogen branching enzyme
Glycogen debranching enzyme
Glycogenesis
GSK3B
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Abstract Glycogen synthase (GS) is the rate limiting enzyme for glycogen production and together with glycogenin (GN) and glycogen branching enzyme (GBE), can generate glycogen particles containing up to 50,000 glucose units. Dysregulation of glycogen synthesis, for example overproduction or accumulation of misshapen glycogen, is the source of many glycogen storage diseases affecting glucose homeostasis and muscle and neuronal cell function. As such, GS is an attractive candidate enzyme for therapeutic targeting, which until recently, was hampered by difficulties in producing active human GS enzyme preparations. Here, we describe the large-scale production of GS in complex with GN, and assay conditions to measure enzyme activity in the absence and presence of the allosteric activator glucose-6-phosphate (G6P). These protocols, together with assays for quality control assessment of enzyme preparations, provide a useful resource for studying the biochemical, biophysical and structural properties of the GS-GN complex, and facilitate drug discovery pipelines to develop therapeutics for glycogen storage diseases.
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