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PubMed1983

The activity of glycogen synthase phosphatase limits hepatic glycogen deposition in the adrenalectomized starved rat.

Hepatocytes from adrenalectomized 48 h-starved rats responded to increasing glucose concentrations with a progressively more complete inactivation of phosphorylase. Yet no activation of glycogen synthase occurred, even in a K+-rich medium. Protein phosphatase activities in crude liver preparations were assayed with purified substrates. Adrenalectomy plus starvation decreased synthase phosphatase activity by about 90%, but hardly affected phosphorylase phosphatase activity. Synthase b present in liver extracts from adrenalectomized starved rats was rapidly and completely converted into the a form on addition of liver extract from a normal fed rat. Glycogen synthesis can be slowly re-induced by administration of either glucose or cortisol to the deficient rats. In these conditions there was a close correspondence between the initial recovery of synthase phosphatase activity and the amount of synthase a present in the liver. The latter parameter was strictly correlated with the measured rate of glycogen synthesis in vivo. The decreased activity of synthase phosphatase emerges thus as the single factor that limits hepatic glycogen deposition in the adrenalectomized starved rat.

Adrenalectomy↗
PubMed1998

Adenovirus-mediated transfer of the acid alpha-glucosidase gene into fibroblasts, myoblasts and myotubes from patients with glycogen storage disease type II leads to high level expression of enzyme and corrects glycogen accumulation.

Glycogen storage disease type II (GSD II) is an autosomal recessive disorder caused by defects in the lysosomal acid alpha-glucosidase (GAA) gene. We investigated the feasibility of using a recombinant adenovirus containing the human GAA gene under the control of the cytomegalovirus promoter (AdCMV-GAA) to correct the enzyme deficiency in different cultured cells from patients with the infantile form of GSD II. In GAA-deficient fibroblasts infected with AdCMV-GAA, transduction and transcription of the human transgene resulted in de novo synthesis of GAA protein. The GAA enzyme activity was corrected from the deficient level to 12 times the activity of normal cells. The transduced cells overexpressed the 110 kDa precursor form of GAA, which was secreted into the culture medium and was taken up by recipient cells. The recombinant GAA protein was correctly processed and was active on both an artificial substrate 4-methylumbelliferyl-alpha-D-glucopyranoside (4MUG) and glycogen. In GAA-deficient muscle cells, a significant increase in cellular enzyme level, approximately 20-fold higher than in normal cells, was also observed after viral treatment. The transduced muscle cells were also able to efficiently secrete the recombinant GAA. Moreover, transfer of the human transgene resulted in normalization of cellular glycogen content with clearance of glycogen from lysosomes, as assessed by electron microscopy, in differentiated myotubes. These results demonstrate phenotypic correction of cultured skeletal muscle from a patient with infantile-onset GSD II using a recombinant adenovirus. We conclude that adenovirus-mediated gene transfer might be a suitable model system for further in vivo studies on delivering GAA to GSD II muscle, not only by direct cell targeting but also by a combination of secretion and uptake mechanisms.

Adenoviridae↗
PubMed1995

Glucose production in glycogen storage disease I is not associated with increased cycling through hepatic glycogen.

Children with glycogen storage disease type I (GSD I) lack the ability to convert glucose 6-phosphate to glucose and yet are able to produce glucose endogenously. To test the hypothesis that the source of this glucose is increased cycling of glucose moieties through hepatic glycogen, six children with GSD I were studied on two occasions during which they received enteral glucose for 6 h at 35 or 50 mumol.kg-1.min-1 along with [6,6-2H2]glucose to measure plasma glucose flux and [1-13C]galactose to label intrahepatic uridyl diphosphate (UDP)-glucose. After 3 h, acetaminophen was given to estimate UDP-glucose flux (reflecting the rate of glycogen synthesis). Mean steady-state plasma glucose concentrations (4.8 +/- 0.2 vs. 5.8 +/- 0.1 mM) and total flux (34.8 +/- 1.7 vs. 47.5 +/- 2.0 mumol.kg-1.min-1) were increased (P < 0.05 or better) on the high-infusion day. Endogenous glucose production was detectable only on the low-infusion day (2.0 +/- 0.5 mumol.kg-1.min-1). UDP-glucose flux was increased (P < 0.05) on the high-infusion day (25.8 +/- 1.6 vs. 34.7 +/- 4.1), ruling out cycling of glucose moieties through glycogen with release of glucose by debrancher enzyme as the source of glucose production.

Blood Glucose↗
PubMed1993

Hepatic lobular patterns of phosphoenolpyruvate carboxykinase, glycogen synthase, and glycogen phosphorylase in fasted and fed rats.

The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase (GS), and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat sections of tissue were collected on slides, fixed by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was confirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed animal but often showed a centrilobular pattern, and fasting did not alter the lobular distribution of either enzyme. Western blot analysis revealed no changes in the amount of these enzymes in the fed or fasted state. The cellular distribution of the three enzymes is similar to that of hepatic glycogen, in that the immunoreactive material has a clumped appearance in the periportal hepatocytes and is more dispersed in the pericentral cells. On fasting the periportal hepatocytes lose the dense compact localization of the enzymes and the protein becomes more homogeneously distributed throughout the cytosol. Further studies are needed to elucidate the functional significance of the regional heterogeneity of the glycogen-metabolizing enzymes and the molecular mechanisms regulating their gene expression.

Animals↗
PubMed1990

[Hepatic glycogen synthetase deficiency or glycogen storage disease-zero. Mild phenotype with partial enzymatic defect].

Since the original description 26 years ago, of the hepatic glycogen synthetase deficiency, only one more case was reported in 1977. We present the studies carried out on an Argentine boy of Italian ancestry who at age 21 months, showed signs of hepatic deficiency with mild clinical symptoms which contrasted with a remarkable fatty liver degeneration. A totally atypic reaction to fructose overload (Table 1, Fig. 1) was the first key to the diagnosis. Glucose levels were not significantly modified by glucagon after 12-hours fasting, but it did increase the glycemia, with decrease of lactate and alanine 3 hours after-meal (Fig. 2a, b). The 24-hours metabolic profile showed fasting hypoglycemia, hyperketonemia, low alanine concentrations and mild lactatemia and hyperglycemia and a net post-prandial increase of lactate (Fig. 3). This profile when reduced to 14 hours, 12-fasting hours and 2-postprandial hours (Fig. 4), revealed similar alterations in an asymptomatic younger brother. The development of the investigation led to a second hepatic biopsy which confirmed hepatic steatosis and to an ultrastructural study, which showed subcellular alterations in the liver and also in muscle (Fig. 5). Moreover low content of hepatic glycogen was observed along with glycogen synthetase activity between 20-25% that of controls, being normal the enzyme activity in muscle and fibroblasts cultured from a skin biopsy (Table 2). The clinical pattern mainly without hypoglycemia, convulsions and/or mental retardation and a normal height and body mass development, allowed us to postulate that this Argentine report would be a mild variant of the disease formerly described and would be correlated with a partial deficiency of the hepatic glycogen synthetase.

Biopsy↗
PubMed2000

Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) is a serine/threonine protein kinase, the activity of which is inhibited by a variety of extracellular stimuli including insulin, growth factors, cell specification factors and cell adhesion. Consequently, inhibition of GSK-3 activity has been proposed to play a role in the regulation of numerous signalling pathways that elicit pleiotropic cellular responses. This report describes the identification and characterisation of potent and selective small molecule inhibitors of GSK-3. RESULTS: SB-216763 and SB-415286 are structurally distinct maleimides that inhibit GSK-3alpha in vitro, with K(i)s of 9 nM and 31 nM respectively, in an ATP competitive manner. These compounds inhibited GSK-3beta with similar potency. However, neither compound significantly inhibited any member of a panel of 24 other protein kinases. Furthermore, treatment of cells with either compound stimulated responses characteristic of extracellular stimuli that are known to inhibit GSK-3 activity. Thus, SB-216763 and SB-415286 stimulated glycogen synthesis in human liver cells and induced expression of a beta-catenin-LEF/TCF regulated reporter gene in HEK293 cells. In both cases, compound treatment was demonstrated to inhibit cellular GSK-3 activity as assessed by activation of glycogen synthase, which is a direct target of this kinase. CONCLUSIONS: SB-216763 and SB-415286 are novel, potent and selective cell permeable inhibitors of GSK-3. Therefore, these compounds represent valuable pharmacological tools with which the role of GSK-3 in cellular signalling can be further elucidated. Furthermore, development of similar compounds may be of use therapeutically in disease states associated with elevated GSK-3 activity such as non-insulin dependent diabetes mellitus and neurodegenerative disease.

Adenosine Triphosphate↗
PubMed1994

A continuous spectrophotometric method for the determination of glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis.

We offer a "continuous" spectrophotometric method for the determination of the glycogen phosphorylase-catalyzed reaction in the direction of glycogen synthesis. This method relies on a coupled enzyme procedure, involving purine nucleoside phosphorylase and its chromophoric substrate, 2-amino-6-mercapto-7-methyl ribonucleoside (7-methyl-6-thioguanosine (MTGuo)), for the estimation of inorganic phosphate (M. R. Webb, Proc. Natl. Acad. Sci. USA 89, 4884-4887, 1992). We have examined the effects of the reaction components on the catalytic activities of both "primary" and "coupling" enzymes. While MTGuo exhibits no effect on the glycogen phosphorylase-catalyzed reaction, glucose 1-phosphate and AMP are partially inhibitory to nucleoside phosphorylase. However, the latter effects pose no problem as long as the coupling enzyme is maintained at a relatively higher concentration in the assay system. The coupled enzyme assay system, standardized for the measurement of glycogen phosphorlase activity, has enabled us to demonstrate explicitly that the rate of the enzyme-catalyzed reaction exhibits sigmoidal dependence on both AMP and glucose 1-phosphate concentrations. We argue that these sigmoidal profiles have been observed due to the sensitivity and precision of the present assay system.

Adenosine Monophosphate↗
PubMed1996

The glucose-induced switch between glycogen phosphorylase and glycogen synthase in the liver: outlines of a theoretical approach.

The glucose-induced switch between glycogen phosphorylase and glycogen synthase in the liver is investigated by means of a theoretical approach based on a minimal, bicyclic cascade model involving the reversible phosphorylation of the two enzymes. The aim of the analysis is to evaluate the contribution of different factors to the sequential changes in the activity of glycogen phosphorylase and glycogen synthase observed following the addition of suprathreshold amounts of glucose.

Animals↗
PubMed1992

Glucose has to be phosphorylated to activate glycogen synthase, but not to inactivate glycogen phosphorylase in hepatocytes.

2-Deoxyglucose and 5-thioglucose, in the same fashion as glucose, cause the inactivation of the rat hepatocyte glycogen phosphorylase and the activation of glycogen synthase. However, 6-deoxyglucose and 1,5-anhydroglucitol inactivate phosphorylase without increasing the activation state of glycogen synthase. With 3-O-methylglucose no changes in the activity of these enzymes occurred. These results prove that while glucose is the molecule that triggers the inactivation of phosphorylase, glucose 6-phosphate is the signal for glucose synthase activation and that a metabolite control of the activation state of glycogen synthase is operative in hepatocytes.

Animals↗
PubMed1993

Glycogen contains phosphodiester groups that can be introduced by UDPglucose: glycogen glucose 1-phosphotransferase.

Rabbit-muscle glycogen contains covalently bound phosphorus, equivalent to 1 phosphate group per 208 glucose residues. This often disputed, minor component was previously thought to represent a phosphomonoester group at C-6 of a glucose residue. Here we show that more than half the phosphorus is present as a phosphodiester, the remainder being monoester. A novel enzyme activity has been found in muscle that can account for the presence of the phosphodiester in glycogen. This is a UDPglucose: glycogen glucose 1-phosphotransferase that positions glucose 1-phosphate on C-6 of glucose residues in glycogen, forming a diester. The phosphomonoester groups present may arise by removal of the glucose residue originally transferred as glucose 1-phosphate.

Animals↗
PubMed1990

A putative neuroendocrine factor that stimulates glycogen mobilization in isolated glycogen cells from the marine mussel Mytilus edulis.

Glycogen synthesized by purified glycogen cells, from the labial palps of Mytilus edulis, was labeled by preincubation in culture medium containing D-[U-14C]glucose. It was stable for at least 4 hr of postincubation in the absence of 14C. Glycogen mobilization was provoked by an acid extract of the cerebral ganglia. The active factor was also found in the hemolymph. The glycogen cells exhibited a dose-dependent response to the cerebral extract. The active cerebral factor is methanol soluble and possesses hydrophobic properties. It is postulated that this factor is a neurohormone.

Animals↗
PubMed1989

Effect of sulfonylureas on hepatic glycogen metabolism: activation of glycogen phosphorylase.

In hepatocytes isolated from fed rats, both tolbutamide and glipizide caused a dose-dependent activation of glycogen phosphorylase, possibly by a Ca2+-mediated mechanism. Maximal effects (about twofold) were already obtained when drugs were used at 0.5 mmol/L, the calculated concentrations of tolbutamide and glipizide responsible for the half-maximal effects being 60 and 30 mumol/L, respectively. The activation of glycogen phosphorylase caused the mobilization of glycogen and increased the cellular concentration of hexose 6-phosphates (glucose 6-phosphate plus fructose 6-phosphate) and that of fructose 2,6-bisphosphate. Under the influence of sulfonylureas, glucose formation was slightly stimulated while the rate of L-lactate production was more markedly incremented, indicating that sulfonylureas canalize the metabolic flux coming from glycogen mainly to the glycolytic pathway. These results suggest that a glycogenolytic action of sulfonylureas could collaborate to raise hepatic fructose 2,6-bisphosphate concentration in the fed animal.

Animals↗
PubMed2003

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1997

Kinetic mechanism of the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis: co-operative interactions of AMP and glucose 1-phosphate during catalysis.

We employed our newly developed, continuous, spectrophotometric method [Sergienko and Srivastava (1994) Anal. Biochem. 221, 348-355] for measuring the glycogen-phosphorylase-catalysed reaction in the direction of glycogen synthesis, utilizing varied concentrations of AMP (2-400 microM) and glucose 1-phosphate (G1P; 4 microM to 41 mM). The experimental data revealed that the enzyme catalysis exhibits sigmoidal dependence on both AMP and G1P concentrations, with Hill coefficient and EC50 values (mutually) affected by the concentrations of the above substrates. A detailed kinetic analysis of the substrate-dependent activation, as well as glucose-inhibition data, lead us to propose the following mechanistic features of the glycogen-phosphorylase-catalysed reaction. (1) The enzyme exhibits catalytic activity when two molecules of AMP and two molecules of G1P are bound to the dimeric unit. (2) The binding of one molecule of glucose (the competitive inhibitor of G1P) per dimeric unit results into a complete loss of the enzyme activity. (3) There is no restriction of binding of AMP or G1P when one of the dimeric subunits is already bound with the other ligand. For example, one or two G1P molecules can bind to the enzyme dimer when zero, one or two molecules of AMP are already bound. The magnitudes of rate and equilibrium constants for the glycogen-phosphorylase-catalysed reaction, derived from analyses of the experimental data in the light of a few selected minimal models, are presented.

Adenosine Monophosphate↗
PubMed1976

Effects of insulin and procaine hydrochloride on glycogen synthetase activation and adipocyte calcium flux: evidence for a role of calcium in insulin activation of glycogen synthetase.

Observations that insulin-induced stimulation of glycogen synthetase occurs without detectable reduction in cyclic AMP suggests an alternative controlling mechanism. The hypothesis that this stimulation might be mediated through calcium was tested using procaine HC1, a drug whose insulin-like action is not associated with reduction in basal or adrenaline-stimulated cyclic AMP levels. Pretreatment of adipose tissue with insulin or porcaine for 30 minutes increased homogenate glucose-6-phosphate independent ("I") form activity to 58% and 48% respectively with no significant change in total activity. Insulin and procaine also had similar effects on calcium efflux from isolated rat adipocytes. Following an initial displacement of calcium from the adipocytes by insulin (but not by procaine) both agents decreased efflux of calcium, measured following 10, 20 and 30 minutes incubation. In adipose tissue homogenates increasing the free calcium concentration from 10(-8) to 10(-3) M increased "I" form glycogen synthetase activity without significantly altering total activity. A complex interrelationship with ATP and calcium was also observed. In the presence of ATP 0.5 mM maximal activation occurred at 10(-6) M calcium concentration. These findings suggest that insulin-induced activation of glycogen synthetase may be effected by alteration of intracellular free calcium with consequent effects on glycogen synthetase-related enzymes in a mechanism independent of or complementary to effects on cyclic AMP levels.

Adipose Tissue↗
PubMed2001

The "glycogen shunt" in exercising muscle: A role for glycogen in muscle energetics and fatigue.

Stimulated by recent (13)C and (31)P NMR studies of exercising muscle, we propose a model of the energetics of contraction. Previous studies of energetics have followed energy consumption. However, the rapidity of contraction, in 10-40 msec, requires that energy be delivered rapidly, so that the muscle has power requirements of rapid energy expenditure that are ultimately met by the slower averaged consumption of carbon and oxygen from blood. We propose that energy is supplied in milliseconds by glycogenolysis and that between contractions, glycogenesis refills the pools. The energy for glycogenesis is supplied by oxidative phosphorylation. This mechanism utilizes the rapid conversion of glycogen phosphorylase, the "fight-or-flight" enzyme, to its active form. Lactate is necessarily generated by this pathway to serve as a time buffer between fast and slow energy needs, which resolves the paradoxical generation of lactate in well oxygenated tissue. Consequences of the glycogen shunt are compatible with numerous biochemical and physiological experiments. The model provides a possible mechanism for muscle fatigue, suggesting that at low but nonzero glycogen concentrations, there is not enough glycogen to supply millisecond energy needs.

Adenosine Triphosphate↗
PubMed1987

Glycogen and glycogen-hydrolysing lysosomal enzyme activity in mouse liver: effects of fasting, adrenoceptor antagonism and insulin-induced hypoglycaemia.

Whereas the phosphorolytic breakdown of liver glycogen is known to be of great physiological importance, the functional role of the hydrolytic glycogenolysis in the lysosomal system is less well understood. In the present study the effects of fasting, alpha- and beta-adrenoceptor antagonism and insulin-induced hypoglycaemia on liver lysosomal glycogen-hydrolysing enzyme activity were investigated in mice. In freely fed mice the glycogen-hydrolysing activity (acid amyloglucosidase) was only 50% of the maltose-hydrolysing activity (acid maltase). Starvation for 24 h reduced the acid amyloglucosidase activity by approximately 30% (P less than 0.001), whereas the activities of acid maltase, acid phosphatase and beta-glucuronidase appeared unaffected. N-acetyl-beta-D-glucosaminidase activity was moderately (20%; P less than 0.01) enhanced by fasting. Thus, liver lysosomal enzyme activities may change independently of each other during fasting. Further, during short-term hypoglycaemic conditions (45 min) induced by endogenous or exogenous insulin, the activity of liver acid amyloglucosidase was found to be moderately reduced (15-20%). Blockade of alpha- and beta-adrenoceptors by phentolamine and propranolol did not result in any apparent influence on acid amyloglucosidase activity except for the indirect effect exerted by the phentolamine-induced hypoglycaemia. A moderate negative correlation (r = -0.51; P less than 0.001) between total liver glycogen concentration and acid amyloglucosidase activity was observed in a series of 43 freely fed NMRI mice. Our data show that in mouse liver the acid maltase activity predominates over the acid amyloglucosidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed1995

Glycogen resynthesis in human muscle fibre types following exercise-induced glycogen depletion.

1. Studies investigating muscle glycogen resynthesis in man have usually examined mixed-fibred biopsies or have used histochemical methods to estimate single fibre resynthesis. Since the accuracy of the latter is open to debate, this study investigated glycogen resynthesis in type I and II fibres using biochemical methods of analysis. 2. Seven subjects performed one-legged cycling exercise to exhaustion. During the initial 2 h of recovery, subjects consumed 3 g of glucose (kg body mass (BM))-1, and a high carbohydrate diet thereafter. Muscle biopsy samples were obtained from both legs at exhaustion, and from the exercised leg after 3, 10 and 24 h of recovery. 3. In the initial 3 h of recovery, there was a 25 +/- 8% higher rate of resynthesis in type I compared with type II fibres (41 +/- 3 and 31 +/- 4 mmol glucosyl units (kg dry mass (DM))-1 h-1, respectively; P < 0.05). Between 3 and 10 h of recovery, resynthesis in type I fibres declined by 60 +/- 13% to 15 +/- 4 mmol glucosyl units (kg DM)-1 h-1 (P < 0.01), whilst the rate in type II fibres was maintained. Good agreement was found when relating the mixed-fibred muscle glycogen concentration to the mean concentration found in type I and type II fibres (r = 0.96). 4. A discrepancy was found to exist with histochemically derived data reported in the literature. The higher initial glycogen resynthesis rate in type I fibres may be attributable to fibre-type differences in glucose uptake and disposal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗