Overexpression of glycogen synthase in mouse muscle results in less branched glycogen

Glycogen, a branched polymer of glucose, serves as an energy reserve in many organisms. The degree of branching likely reflects the balance between the activities of glycogen synthase and branching enzyme. Mice overexpressing constitutively active glycogen synthase in skeletal muscle (GSL30) have elevated muscle glycogen. To test whether excess glycogen synthase activity affected glycogen branching, we examined the glycogen from skeletal muscle of GSL30 mice. The absorption spectrum of muscle glycogen determined in the presence of iodine was shifted to higher wavelengths in the GSL30 animals, consistent with a decrease in the degree of branching. As judged by Western blotting, the levels of glycogenin and the branching enzyme were also elevated. Branching enzyme activity also increased approximately threefold. However, this compared with an increase in glycogen synthase of some 50-fold, so that the increase in branching enzyme in response to overexpression of glycogen synthase was insufficient to synthesize normally branched glycogen.     

Mechanisms of monomeric and dimeric glycogenin autoglucosylation

Initiation of glucose polymerization by glycogenin autoglucosylation at Tyr-194 is required to prime de novo biosynthesis of glycogen. It has been proposed that the synthesis of the primer proceeds by intersubunit glucosylation of dimeric glycogenin, even though it has not been demonstrated that this mechanism is responsible for the described polymerization extent of 12 glucoses produced by the dimer. We reported previously the intramonomer glucosylation capability of glycogenin without determining the extent of autoglucopolymerization. Here, we show that the maximum specific autoglucosylation extent (MSAE) produced by the non-glucosylated glycogenin monomer is 13.3 ± 1.9 glucose units, similar to the 12.5 ± 1.4 glucose units measured for the dimer. The mechanism and capacity of the dimeric enzyme to carry out full glucopolymerization were also evaluated by construction of heterodimers able to glucosylate exclusively by intrasubunit or intersubunit reaction mechanisms. The MSAE of non-glucosylated glycogenin produced by dimer intrasubunit glucosylation was 16% of that produced by the monomer. However, partially glucosylated glycogenin was able to almost complete its autoglucosylation by the dimer intrasubunit mechanism. The MSAE produced by heterodimer intersubunit glucosylation was 60% of that produced by the wild-type dimer. We conclude that both intrasubunit and intersubunit reaction mechanisms are necessary for the dimeric enzyme to acquire maximum autoglucosylation. The full glucopolymerization capacity of monomeric glycogenin indicates that the enzyme is able to synthesize the glycogen primer without the need for prior dimerization.     

Regulation of glycogen concentration and glycogen synthase activity in skeletal muscle of insulin-resistant rats

The aim of this study was to investigate the effect of insulin resistance on glycogen concentration and glycogen synthase activity in the red and white gastrocnemius muscles and to determine whether the inverse relationship existing between glycogen concentration and enzyme activity is maintained in insulin resistant state. These questions were addressed using 3 models that induce various degrees of insulin resistance: sucrose feeding, dexamethasone administration, and a combination of both treatments (dex+sucrose). Sucrose feeding raised triglyceride levels without affecting plasma glucose or insulin concentrations whereas dexamethasone and dex+sucrose provoked severe hyperinsulinemia, hyperglycemia and hypertriglyceridemia. Sucrose feeding did not alter muscle glycogen concentration but provoked a small reduction in the glycogen synthase activity ratio (-/+ glucose-6-phosphate) in red but not in white gastrocnemius. Dexamethasone administration augmented glycogen concentration and reduced glycogen synthase activity ratio in both muscle fiber types. In contrast, dex+sucrose animals showed decreased muscle glycogen concentration compared to dexamethasone group, leading to levels similar to those of control animals. This was associated with lower glycogen synthase activity compared to control animals leading to levels comparable to those of dexamethasone-treated animals. Thus, in dex+sucrose animals, the inverse relationship observed between glycogen levels and glycogen synthase activity was not maintained, suggesting that factors other than the glycogen concentration modulate the enzyme's activity. In conclusion, while insulin resistance was associated with a reduced glycogen synthase activity ratio, we found no correlation between muscle glycogen concentration and insulin resistance. Furthermore, our results suggest that sucrose treatment may modulate dexamethasone action in skeletal muscle.     

Continuous assay method for glycogen synthase activity

An assay method for glycogen synthase (EC 2.4.1.11) has been developed based on the continuous measurement of the change of pH accompanying the glycogen synthesis reaction. The use of low buffer concentrations and an amplifier with variable gain and offset voltage allow us to register changes in the pH of the system small enough to ignore the significant pH dependence of the enzyme activity. A theoretical approach has been used to correlate the pH measurements with the progres of the reaction in terms of glucose incorporated into glycogen. The method offers the advantages of being continuous and of low cost.     

C-chain-bound glycogenin is released from proteoglycogen by isoamylase and is able to autoglucosylate

Proteoglycogen glycogenin is linked to the glucose residue of the C-chain reducing end of glycogen. We describe for the first time the release by isoamylase and isolation of C-chain-bound glycogenin (C-glycogenin) from proteoglycogen. The treatment of proteoglycogen with alpha-amylase releases monoglucosylated and diglucosylated glycogenin (a-glycogenin) which is able to autoglucosylate. It had been described that isoamylase splits the glucose-glycogenin linkage of fully autoglucosylated glycogenin previously digested with trypsin, releasing the maltosaccharide moiety. It was also described that carbohydrate-free apo-glycogenin shows higher mobility in SDS-PAGE and twice the autoglucosylation capacity of partly glucosylated glycogenin. On the contrary, we found that the C-glycogenin released from proteoglycogen by isoamylolysis shows lower mobility in SDS-PAGE and about half the autoglucosylation acceptor capacity of the partly glucosylated a-glycogenin. This behavior is consistent with the release of maltosaccharide-bound glycogenin instead of apo-glycogenin. No label was split from auto-[14C]glucosylated C-glycogenin or fully auto-[14C]glucosylated a-glycogenin subjected to isoamylolysis without previous trypsinolysis, thus proving no hydrolysis of the maltosaccharide-tyrosine linkage. The ability of C-glycogenin for autoglucosylation would indicate that the size of the C-chain is lower than the average length of the other glycogen chains.     

Glycogen synthesis in the absence of glycogenin in the yeast Saccharomyces cerevisiae

In eukaryotic cells, glycogenin is a self-glucosylating protein that primes glycogen synthesis. In yeast, the loss of function of GLG1 and GLG2, which encode glycogenin, normally leads to the inability of cells to synthesize glycogen. In this report, we show that a small fraction of colonies from glg1glg2 mutants can switch on glycogen synthesis to levels comparable to wild-type strain. The occurrence of glycogen positive glg1glg2 colonies is strongly enhanced by the presence of a hyperactive glycogen synthase and increased even more upon deletion of TPS1. In all cases, this phenotype is reversible, indicating the stochastic nature of this synthesis, which is furthermore illustrated by colour-sectoring of colonies upon iodine-staining. Altogether, these data suggest that glycogen synthesis in the absence of glycogenin relies on a combination of several factors, including an activated glycogen synthase and as yet unknown alternative primers whose synthesis and/or distribution may be controlled by TPS1 or under epigenetic silencing.     

A gel filtration assay to determine glycogen synthase activity

We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.     

Direct detection of glycogenin reaction products during glycogen initiation

Glycogenin initiates glycogen synthesis in an autocatalytic reaction in which individual glucose residues are covalently linked to Tyrosine 194 in order to form a short priming chain of glucose residues that is a substrate for glycogen synthase which, combined with the branching enzyme, catalyzes the bulk synthesis of glycogen. We sought to develop a new enzymatic assay to better characterize both the chemical and enzymatic characteristics of this unusual reaction. By directly detecting the reaction products using electrospray mass spectrometry this procedure permits both the visualization of the intact individual reaction species produced as a function of time and quantitation of the levels of each of species. The quantitation of the reaction agrees well with previous measurements of both catalytic rate and the change in rate as a function of average glucosylation. The results from this assay provide new insight into the mechanism by which glycogenin catalyzes the initiation reaction.     

Phosphorylation of glycogen synthase in isolated rabbit hepatocytes

Specific antibodies were used to purify glycogen synthase from isolated rabbit hepatocytes that had been incubated in a medium containing [³²P]phosphate. The enzyme gave rise to two main ³²P-labeled CNBr fragments of electrophoretic mobilities similar to those obtained after phosphorylation of the enzyme by individual protein kinases in vitro.     

Different effects of glucagon and epinephrine on the kinetic properties of liver glycogen synthase

Kinetic constants of glycogen synthase (M0.5 for glucose-6-P and S0.5 for UDP-glucose) were determined after hepatocytes isolated from starved rats were incubated with either glucagon or epinephrine. Incubation with these hormones resulted in an increase in both S0.5 and M0.5. However, the action of glucagon resulted in great modifications on S0.5 whereas epinephrine affected mainly M0.5. Therefore, glucagon and epinephrine alter the kinetic properties of glycogen synthase provoke the phosphorylation of glycogen synthase at different site(s) acting through different mechanisms.     

Frog oocyte glycogen synthase: enzyme regulation under in vitro and in vivo conditions

Frog oocyte glycogen synthase properties differ significantly under in vitro or in vivo conditions. The K(mapp) for UDP-glucose in vivo was 1.4mM (in the presence or absence of glucose-6-P). The in vitro value was 6mM and was reduced by glucose-6-P to 0.8mM. Under both conditions (in vitro and in vivo) V(max) was 0.2 m Units per oocyte in the absence of glucose-6-P. V(max) in vivo was stimulated 2-fold by glucose-6-P, whereas, in vitro, a 10-fold increase was obtained. Glucose-6-P required for 50% activation in vivo was 15 microM and, depending on substrate concentrations, 50-100 microM in vitro. The prevailing enzyme obtained in vitro was the glucose-6-P-dependent form, which may be converted to the independent species by dephosphorylation. This transformation could not be observed in vivo. We suggest that enzyme activation by glucose-6-P in vivo is due to allosteric effects rather than to dephosphorylation of the enzyme. Regulatory mechanisms other than allosteric activation and covalent phosphorylation are discussed.     

Lithium ions increase hepatic glycogen synthase stability through a proteasome-related mechanism

Incubation of rat hepatocytes with LiCl resulted in an overall increase in the activity ratio of glycogen synthase (GS), concomitantly with a decrease in active GS kinase-3 levels. GS total activity was also increased in a dose- and time-dependent manner. This latter effect correlated with the amount of immunoreactive enzyme determined by immunoblotting. Cycloheximide and actinomycin-D did not modify LiCl action on GS activity. Lithium ions did not induce any changes in GS mRNA levels. Furthermore, the increase in the total amount of GS induced by LiCl was further augmented after addition of a specific, calpain and proteasome inhibitor. Our results indicate that LiCl increases hepatocyte GS activity through increasing both the activation state of the enzyme and its cellular content. This latter increase is mediated through a modification of the proteasome-regulated proteolytic pathway of the enzyme.     

Expression and regulation of glycogen synthase kinase 3 in human neutrophils

Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in the regulation of cellular processes ranging from glycogen metabolism to cell cycle regulation. Its two known isoforms, α and β, are differentially expressed in tissues throughout the body and exert distinct but often overlapping functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3α) or Ser9 (GSK-3β) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system function, yet little is known about the role of this enzyme in neutrophils, the most abundant leukocyte type. In the present study, we examined GSK-3 expression and regulation in human neutrophils. GSK-3α was found to be the predominant isoform, it was constitutively expressed and cell stimulation with different agonists did not alter its expression. Stimulation by fMLP, LPS, GM-CSF, Fcγ receptor engagement, or adenosine A_2A receptor engagement all resulted in phosphorylation of Ser21. The use of metabolic inhibitors revealed that combinations of Src kinase, PKC, PI3K/AKT, ERK/RSK and PKA signaling pathways could mediate phosphorylation, depending on the agonist. Neither PLC nor p38 were involved. We conclude that GSK-3α is the main isoform expressed in neutrophils and that many different pathways can converge to inhibit GSK-3α activity via Ser21-phosphorylation. GSK-3α thus might be a hub of cellular regulation.     

Glucose activation of liver glycogen synthase. Insulin-mediated restoration of glucose effect in diabetic rate is blocked by protein synthesis inhibitor

The loss of glucose regulation of glycogen synthase in perfused livers from diabetic rats was associated with a substantial reduction in synthase phosphatase activity. Treatment of diabetic rats with insulin alone resulted in total restoration of the glucose effect and synthase phosphatase activity, while simultaneous treatment with cycloheximide severely reduced the hormonal effect. Although treatment of normal rats with cycloheximide had no effect on glucose activation of synthase, it did result in severe depletion of liver glycogen increased liver glycogen phosphorylase activity, and elevation of liver adenosine 3′,5′-monosphosphate (cyclic AMP), but without elevation of liver protein kinase activity. Simultaneous treatment of alloxan-diabetic rats with insulin and cycloheximide resulted in reduction of total liver glycogen, increased phosphorylase activity, a reduction in the ability of insulin to lower hepatic cyclic AMP, and a further reduction of protein kinase activity.In summary, the effect of insulin treatment of diabetic rats to restore glucose regulation of hepatic glycogen synthase probably involves synthesis of new protein, and the data remain consistent with the hypothesis that the defect may be due to a diabetes-related deficiency in a specific synthase phosphatase and/or alteration of the synthase molecule itself.     

The ADP-glucose binding site of the Escherichia coli glycogen synthase

Bacterial glycogen/starch synthases are retaining GT-B glycosyltransferases that transfer glucosyl units from ADP-Glc to the non-reducing end of glycogen or starch. We modeled the Escherichia coli glycogen synthase based on the coordinates of the inactive form of the Agrobacterium tumefaciens glycogen synthase and the active form of the maltodextrin phosphorylase, a retaining GT-B glycosyltransferase belonging to a different family. In this model, we identified a set of conserved residues surrounding the sugar nucleotide substrate, and we replaced them with different amino acids by means of site-directed mutagenesis. Kinetic analysis of the mutants revealed the involvement of these residues in ADP-Glc binding. Replacement of Asp21, Asn246 or Tyr355 for Ala decreased the apparent affinity for ADP-Glc 18-, 45-, and 31-fold, respectively. Comparison with other crystallized retaining GT-B glycosyltransferases confirmed the striking similarities among this group of enzymes even though they use different substrates.     

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle: Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3

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     

The primary defect in glycogen synthase activity is not based on increased glycogen synthase kinase-3alpha activity in diabetic myotubes

The mechanism responsible for the diminished activation of glycogen synthase (GS) in diabetic myotubes remains unclear, but may involve increased activity and/or expression of glycogen synthase kinase-3 (GSK-3). In myotubes established from type 2 diabetic and healthy control subjects we determined GS activity ratio, protein expression, and activity of GSK-3alpha and beta under basal and insulin-stimulated conditions when precultured in increasing insulin concentrations. In myotubes precultured at low insulin concentrations acute insulin stimulation increased GS activity more in control than in diabetic subjects, whereas the corresponding GSK-3alpha but not GSK-3beta activity was significantly reduced by acute insulin treatment in both groups. However, in myotubes precultured at high insulin concentrations the effect of insulin on GS and GSK-3alpha activity was blunted in both groups. The protein expression of GSK-3alpha or beta was unaffected. In conclusion, myotubes with a primary defect in GS activity express insulin responsive GSK-3alpha, suggesting that failure of insulin to decrease GS phosphorylation involves abnormal activity of another kinase or phosphatase.     

Aberrant spindle dynamics and cytokinesis in Dictyostelium discoideum cells that lack glycogen synthase kinase 3

Eukaryotic cell division requires the co-ordinated assembly and disassembly of the mitotic spindle, accurate chromosome segregation and temporal control of cytokinesis to generate two daughter cells. While the absolute details of these processes differ between organisms, there are evolutionarily conserved core components common to all eukaryotic cells, whose identification will reveal the key processes that control cell division. Glycogen synthase kinase 3 (GSK-3) is a major protein kinase found throughout the eukaryotes and regulates many processes, including cell differentiation, growth, motility and apoptosis. In animals, GSK-3 associates with mitotic spindles and its inhibition causes mis-regulation of chromosome segregation. Two suppressor screens in yeast point to a more general effect of GSK-3 on cell division, however the direct role of GSK-3 in control of mitosis has not been explored outside the animal kingdom. Here we report that the Dictyostelium discoideum GSK-3 orthologue, GskA, associates with the mitotic spindle during cell division, as seen for its mammalian counterparts. Dictyostelium possesses only a single GSK-3 gene that can be deleted to eliminate all GSK-3 activity. We found that gskA-null mutants failed to elongate their mitotic spindle and were unable to divide in shaking culture, but have no chromosome segregation defect. These results suggest further conservation for the role of GSK-3 in the regulation of spindle dynamics during mitosis, but also reveal differences in the mechanisms ensuring accurate chromosome segregation.