Comparative study of nuclear and cytoplasmic glycogen isolated from mutant HD33 ascites cells

Mutant cells of the HD33 subline of the Ehrlich-Lettré ascites tumor synthesize and store glycogen mainly intranuclearly, when growing in vivo, and exclusively in the cytoplasm, when permanently cultivated as a suspension cell strain. To investigate whether there exist differences between glycogen of nuclear and cytoplasmic origin, the ultrastructure and the biophysical and biochemical properties of glycogen from in vivo and in vitro grown HD33 ascites cells were compared. Pronounced heterogeneity and differences in glycogen particle ultrastructure were evident in situ and after isolation of the native, high-molecular polysaccharide. Nuclear glycogen contains a fraction of heavier molecules (up to 2 X 10(9)) and larger particles (up to 340 nm) which could not be found in the cytoplasmic preparations, which contained only particles smaller than 140 nm. The subparticles of beta-type are similar in both nuclear and cytoplasmic glycogen. The absorption spectra and glucose analysis after degradation with phosphorylase and debranching enzyme indicate that nuclear glycogen has a higher degree of branching, associated with a decrease in the average chain length between the branching points, and shorter external polyglucosidic chains than cytoplasmic glycogen. This is the first report about the analysis and properties of isolated nuclear glycogen.     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Purification and steady state kinetic mechanism of glycogen synthase-D from human polymorpho-nuclear leukocytes

Summary The authors' work on the purification and steady state kinetic investigation of the enzyme glycogen synthase D (UDP-glucose: glycogen 4--glucosyl-transferase, EC 2.4.1.11) from human polymorphonuclear leukocytes is reviewed. The main features of the kinetic mechanism for catalysis of the reaction UDPG + glycogen_n UDP + glycogen_(n+1) are: (i) Lineweaver-Burk plots in both substrates are linear, exhibiting intersecting patterns; (ii) UDP is a competitive, respectively noncompetitive, inhibitor towards the substrates UDPG and glycogen; (iii) the essential activator glucose-6-phosphate (G-6-P) showed an intersecting pattern towards glycogen and an equilibrium ordered pattern towards UDPG. These features identify in this case the mechanism as a rapid equilibrium random bi-bi mechanism, with G-6-P adding to the enzyme prior to the substrate UDPG. New results on the influence of the modifiers NaCl, Ca⁺⁺, Mn⁺⁺, Mg⁺⁺, HPO₄ ^–-, SO₄ ^–-, and ATP on the enzyme are reported. Interpreting the observations in terms of the established mechanism, the following results are obtained: The effect of salt (NaCl) is nonspecific and fairly small, probably reflecting a general action of the electrolyte medium on the conformation of the enzyme. Divalent cations affect only the rate limiting step, i.e. the interconversion of the quaternary enzyme-substrate-activator complexes. The anions interact exclusively with the G-6-P binding site of the enzyme. The dissociation constants for the enzyme-modifier complexes are determined, and a kinetic mechanism for the action of the anions is proposed, leading to activation or inhibition, depending on the concentration of G-6-P.An invited article     

Kinetic properties of glycogen synthase from skeletal muscle after phosphorylation by glycogen synthase kinase 4

Glycogen synthase I (EC 2.4.1.11) from rat and from rabbit skeletal muscle was phosphorylated in vitro by glycogen synthase kinase 4 (EC 2.7.1.37) to the extent of 0.8 phosphates/subunit. For both phosphorylated enzymes, the activity ratio (activity without glucose 6-P divided by activity with 8 mM glucose 6-P) was 0.8 when determined with low concentrations of glycogen synthase and/or short incubation times. However, the activity ratio was 0.5 with high enzyme concentrations and longer incubation times. It was found that the lower activity ratios result largely from UDP inhibition of activity measured in the absence of glucose 6-P. Inhibition by UDP was much less pronounced for glycogen synthase I, indicating that a major consequence of phosphorylation by glycogen synthase kinase 4 is an increased sensitivity to UDP inhibition.     

Glucose 6-phosphate plays a central role in the regulation of glycogen synthesis in a glycogen-storing liver cell line

Two substrains of the epithelial liver cell line C1I, one storing large amounts of glycogen, the other one being very poor in glycogen were used as a model for studying glycogen synthesis. The glycogen content of glycogen-rich cells doubled during the proliferative phase and remained high in plateau phase although glycogen synthase I activity was not significantly altered during growth cycle and was too low to account for the increase in glycogen. However, the activity of the glucose 6-phosphate (Glc6-P)-dependent synthase rose continuously during growth cycle, and intracellular Glc6-P-concentration increased about 10-fold in log phase cells to 0.72 mumol g-1 wet weight. A0.5 of synthase for Glc6-P was 0.79 mM. It was also found that in contrast to the enzyme from normal liver, glycogen phosphorylase a from C1I cells was inhibited by Glc6-P, the apparent Ki being 0.45 mM. It was concluded that glycogen accumulation in C1I cells was due to stimulation of synthase and inhibition of phosphorylase by Glc6-P. Findings from the glycogen-poor cell line which revealed similar specific activities of synthase and phosphorylase but only low Glc6-P (0.056 mumol g-1 wet weight) supported this conclusion. Addition of glucose to starved cells resulted in a transient activation of synthase in both cell lines. Net glycogen synthesis, was, however, only observed in the cells with a high Glc6-P-content. Thus, modulation of synthase and phosphorylase by Glc6-P and not activation/inactivation of the enzymes seems to play a predominant role in glycogen accumulation in this cell line.     

Regulation of renal glycogen synthase interconversion of two forms in vitro

Glycogen synthase (UDP glucose: glycogen α-4-glucosyltransferase, EC 2.4. 1.11) from rat kidney was stimulated 4- to 5-fold by glucose 6-phosphate. The for glucose 6-phosphate stimulation was about 0.45 mM. Glycogen synthase was not evenly distributed throughout the kidney. Total synthase activity was greatest in the outer cortex and cortico-medullary junction and least in the inner medulla. Glucose 6-phosphate stimulation was greatest in the outer cortex and least in the inner medulla. Glycogen synthase in crude homogenates was not complexed with glycogen and eluted from Sepharose 6-B with an apparent molecular weight of about 390 000.Renal glycogen synthase appeared to exist in two interconvertible forms, synthase I (activity in the absence of glucose 6-phosphate) and synthase D (requires glucose 6-phosphate for activity). The conversion of synthase D to I (synthase D phosphatase) was inhibited by F⁻, glycogen, ATP, Mn²⁺, and Co²⁺. The conversion was not altered by mercaptoethanol, AMP, Mg²⁺, or Ca²⁺. The conversion of synthase I to D (synthase I kinase) required ATP-Mg and was stimulated by cyclic AMP.It was suggested that the interconversion of renal glycogen synthase involved a phosphorylation-dephosphorylation. The significance of glycogen synthase interconversion to the regulation of renal glycogen synthesis is discussed.     

On the process of activation and inactivation of dog skeletal muscle glycogen synthase.

The presence of additional forms of glycogen synthase (UDPG: alpha-1,4-glucan alpha-4-glucosyltransferase) besides the I form (independent on glucose-6-P for activity) and the D form (dependent on glucose-6-P for activity) long ago described, is inferred from patterns of their interconversions obtained by processes of phosphorylation and dephosphorylation. An intermediate form more phosphorylated than the I form and less than the D form, which is completely inactive in these assay conditions, and a superphosphorylated form, more phosphorylated than the D form and also inactive even in presence of 0.01 M glucose-6-P are described.     

Insulin promotes glycogen synthesis in the absence of GSK3 phosphorylation in skeletal muscle

Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating glycogen synthase kinase (GSK) 3 through phosphorylation. Insulin also promotes glucose uptake and glucose 6-phosphate (G-6-P) production, which allosterically activates GS. The relative importance of these two regulatory mechanisms in the activation of GS in vivo is unknown. The aim of this study was to investigate if dephosphorylation of GS mediated via GSK3 is required for normal glycogen synthesis in skeletal muscle with insulin. We employed GSK3 knockin mice in which wild-type GSK3 alpha and -beta genes are replaced with mutant forms (GSK3 alpha/beta S21A/S21A/S9A/S9A), which are nonresponsive to insulin. Although insulin failed to promote dephosphorylation and activation of GS in GSK3 alpha/beta S21A/S21A/S9A/S9A mice, glycogen content in different muscles from these mice was similar compared with wild-type mice. Basal and epinephrine-stimulated activity of muscle glycogen phosphorylase was comparable between wild-type and GSK3 knockin mice. Incubation of isolated soleus muscle in Krebs buffer containing 5.5 mM glucose in the presence or absence of insulin revealed that the levels of G-6-P, the rate of [14C]glucose incorporation into glycogen, and an increase in total glycogen content were similar between wild-type and GSK3 knockin mice. Injection of glucose containing 2-deoxy-[3H]glucose and [14C]glucose also resulted in similar rates of muscle glucose uptake and glycogen synthesis in vivo between wild-type and GSK3 knockin mice. These results suggest that insulin-mediated inhibition of GSK3 is not a rate-limiting step in muscle glycogen synthesis in mice. This suggests that allosteric regulation of GS by G-6-P may play a key role in insulin-stimulated muscle glycogen synthesis in vivo.     

Sucrose biosynthesis in Dunaliella

Sucrose phosphate synthetase (EC 2.4.1.14) is the key enzyme for sucrose synthesis in Dunaliella tertiolecta. It has been partially purified and characterized. The enzyme contains one binding site for uridine diphosphoglucose and two binding sites for fructose-6-phosphate; it is allosterically controlled by fructose-6-phosphate. Inorganic phosphate stimulates the enzymic activity, particularly in the presence of higher concentrations of fructose-6-phosphate. Sucrose phosphate synthetase is not halophilic or halotolerant. The temperature dependence of the enzymic activity cannot fully explain the observed increase in sucrose synthesis in Dunaliella by elevated temperature.Abbreviations F-6-P fructose 6-phosphate - UDP uridine biphosphate - UDPG uridine biphosphoglucose     

Interconversion between multiple glucose 6-phosphate-dependent forms of glycogen synthase in intact adipose tissue.

We have tested the hypothesis that interconversion between multiple glucose-6-P-dependent forms of glycogen synthase helps regulate glycogen synthesis in adipose tissue. Our results indicate that interconversion of glycogen synthase in adipose tissue involves primarily dependent forms and that these interconversions were measured better by monitoring the activation constant (A0.5) for glucose-6-P than measuring the -: + glucose-6-P activity ratio. Insulin decreased and epinephrine increased the A0.5 for glucose-6-P without significant change in the activity ratio. Insulin consistently decreased the A0.5 in either the presence or absence of glucose, indicating that the insulin-promoted interconversion did not require increased hexose transport. Isoproterenol increased the A0.5 for glucose-6-P, while methoxamine was without effect, indicating beta receptors mediate adrenergic control of interconversion between glucose-6-P-dependent forms. The changes in the A0.5 produced by incubations with insulin or epinephrine were mutually reversible. We conclude that 1) glycogen synthesis in adipose tissue is catalyzed by multiple glucose-6-P-dependent forms of glycogen synthase, 2) hormones regulate glycogen metabolism by promoting reversible interconversions between these forms, and 3) there is no evidence that a glucose-6-P-independent form of glycogen synthase exists in intact adipose tissue.     

Effect of fructose 1-phosphate on the activation of liver glycogen synthase.

The activation (dephosphorylation) of glycogen synthase and the inactivation (dephosphorylation) of phosphorylase in rat liver extracts on the administration of fructose were examined. The lag in the conversion of synthase b into a was cancelled, owing to the accumulation of fructose 1-phosphate. A decrease in the rate of dephosphorylation of phosphorylase a was also observed. The latency re-appeared in gel-filtered liver extracts. Similar latency was demonstrated in extracts from glucagon-treated rats. Addition of fructose 1-phosphate to the extract was able to abolish the latency, and the activation of glycogen synthase and the inactivation of phosphorylase occurred simultaneously. Fructose 1-phosphate increased the activity of glycogen synthase b measured in the presence of 0.2-0.4 mM-glucose 6-phosphate. According to kinetic investigations, fructose 1-phosphate increased the affinity of synthase b for its substrate, UDP-glucose. The accumulation of fructose 1-phosphate resulted in glycogen synthesis in the liver by inducing the enzymic activity of glycogen synthase b in the presence of glucose 6-phosphate in vivo and by promoting the activation of glycogen synthase.     

Muscle glycogen inharmoniously regulates glycogen synthase activity, glucose uptake, and proximal insulin signaling

Insulin-stimulated glucose uptake and incorporation of glucose into skeletal muscle glycogen contribute to physiological regulation of blood glucose concentration. In the present study, glucose handling and insulin signaling in isolated rat muscles with low glycogen (LG, 24-h fasting) and high glycogen (HG, refed for 24 h) content were compared with muscles with normal glycogen (NG, rats kept on their normal diet). In LG, basal and insulin-stimulated glycogen synthesis and glycogen synthase activation were higher and glycogen synthase phosphorylation (Ser(645), Ser(649), Ser(653), Ser(657)) lower than in NG. GLUT4 expression, insulin-stimulated glucose uptake, and PKB phosphorylation were higher in LG than in NG, whereas insulin receptor tyrosyl phosphorylation, insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, and GSK-3 phosphorylation were unchanged. Muscles with HG showed lower insulin-stimulated glycogen synthesis and glycogen synthase activation than NG despite similar dephosphorylation. Insulin signaling, glucose uptake, and GLUT4 expression were similar in HG and NG. This discordant regulation of glucose uptake and glycogen synthesis in HG resulted in higher insulin-stimulated glucose 6-phosphate concentration, higher glycolytic flux, and intracellular accumulation of nonphosphorylated 2-deoxyglucose. In conclusion, elevated glycogen synthase activation, glucose uptake, and GLUT4 expression enhance glycogen resynthesis in muscles with low glycogen. High glycogen concentration per se does not impair proximal insulin signaling or glucose uptake. "Insulin resistance" is observed at the level of glycogen synthase, and the reduced glycogen synthesis leads to increased levels of glucose 6-phosphate, glycolytic flux, and accumulation of nonphosphorylated 2-deoxyglucose.     

Liver glycogen synthase in the developing foetal rat

Kinetic constants for liver glycogen synthase (UDPglucose: glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) with respect to UDPglucose have been measured in foetal liver homogenates from samples taken during late gestation (days 17-22) and the first hours after birth. The V of the inactive form of glycogen synthase increased markedly in this period and there was a significant increase in V of the active enzyme to a maximum at day 20 of gestation. The Km for UDPglucose measured in the presence of glucose-6-P (total activity) did not vary greatly, mean values of 0.51 +/- 0.04 mM. Values derived for the inactive enzyme were almost identical. In contrast, Km values for active glycogen synthase in foetal livers during gestation were significantly higher than those for adult liver. Highest values were seen at day 19 of gestation (1.84 +/- 0.08 mM) followed by a steady fall to 0.55 +/- 0.05 mM in the newborn compared with a mean value of 0.48 +/- 0.04 mM for adult liver. Existence of a reduced affinity of active glycogen synthase for UDPglucose must be recognized when assaying the enzyme in foetal liver, particularly when extrapolating values to rates of glycogen synthesis in vivo. Data were obtained only after removal of an amylase-like contaminant from foetal liver samples which invalidated the radioassay of glycogen synthase. This work illustrates the care needed in the analysis of foetal tissue and the interpretation of resulting data when utilizing methods developed for adult tissue.     

Acute inhibition of hepatic glucose-6-phosphatase does not affect gluconeogenesis but directs gluconeogenic flux toward glycogen in fasted rats. A pharmacological study with the chlorogenic acid derivative S4048

Effects of acute inhibition of glucose-6-phosphatase activity by the chlorogenic acid derivative S4048 on hepatic carbohydrate fluxes were examined in isolated rat hepatocytes and in vivo in rats. Fluxes were calculated using tracer dilution techniques and mass isotopomer distribution analysis in plasma glucose and urinary paracetamol-glucuronide after infusion of [U-(13)C]glucose, [2-(13)C]glycerol, [1-(2)H]galactose, and paracetamol. In hepatocytes, glucose-6-phosphate (Glc-6-P) content, net glycogen synthesis, and lactate production from glucose and dihydroxyacetone increased strongly in the presence of S4048 (10 microm). In livers of S4048-treated rats (0.5 mg kg(-1)min(-)); 8 h) Glc-6-P content increased strongly (+440%), and massive glycogen accumulation (+1260%) was observed in periportal areas. Total glucose production was diminished by 50%. The gluconeogenic flux to Glc-6-P was unaffected (i.e. 33.3 +/- 2.0 versus 33.2 +/- 2.9 micromol kg(-1)min(-1)in control and S4048-treated rats, respectively). Newly synthesized Glc-6-P was redistributed from glucose production (62 +/- 1 versus 38 +/- 1%; p < 0.001) to glycogen synthesis (35 +/- 5% versus 65 +/- 5%; p < 0.005) by S4048. This was associated with a strong inhibition (-82%) of the flux through glucokinase and an increase (+83%) of the flux through glycogen synthase, while the flux through glycogen phosphorylase remained unaffected. In livers from S4048-treated rats, mRNA levels of genes encoding Glc-6-P hydrolase (approximately 9-fold), Glc-6-P translocase (approximately 4-fold), glycogen synthase (approximately 7-fold) and L-type pyruvate kinase (approximately 4-fold) were increased, whereas glucokinase expression was almost abolished. In accordance with unaltered gluconeogenic flux, expression of the gene encoding phosphoenolpyruvate carboxykinase was unaffected in the S4048-treated rats. Thus, acute inhibition of glucose-6-phosphatase activity by S4048 elicited 1) a repartitioning of newly synthesized Glc-6-P from glucose production into glycogen synthesis without affecting the gluconeogenic flux to Glc-6-P and 2) a cellular response aimed at maintaining cellular Glc-6-P homeostasis.     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay     

Effect of fructose on glycogen synthesis in the perfused rat liver

The effect of fructose on glycogen synthesis was examined in the perfused liver of starved rats. With increasing fructose concentration in the perfusate, glycogen synthesis and the % a form of glycogen synthase increased to a maximum at 2 mM and then decreased, progressively. The glucose 6-P level increased with the increase in fructose concentration. On the other hand, the ATP content was unchanged at a concentration of 2 mM or less and decreased at 3 mM or more. We also showed that the stimulation of glycogen synthesis by fructose at a concentration of 2 mM or less was due to activation of glycogen synthase by accumulated glucose 6-P and that ATP depletion at a concentration of 3 mM or more caused an increase in phosphorylase a and a decrease in glycogen synthase activity even in the presence of a high concentration of glucose 6-P.     

Glycogen synthesis from glucose and fructose in hepatocytes from diabetic rats

In rat hepatocytes, the basal glycogen synthase activation state is decreased in the fed and diabetic states, whereas glycogen phosphorylase a activity decreases only in diabetes. Diabetes practically abolishes the time- and dose-dependent activation of glycogen synthase to glucose especially in the fed state. Fructose, however, is still able to activate this enzyme. Glycogen phosphorylase response to both sugars is operative in all cases. Cell incubation with the combination of 20 mM glucose plus 3 mM fructose produces a great activation of glycogen synthase and a potentiated glycogen deposition in both normal and diabetic conditions. Using radiolabeled sugars, we demonstrate that this enhanced glycogen synthesis is achieved from both glucose and fructose even in the diabetic state. Therefore, the presence of fructose plays a permissive role in glycogen synthesis from glucose in diabetic animals. Glucose and fructose increase the intracellular concentration of glucose 6-phosphate and fructose reduces the concentration of ATP. There is a close correlation between the ratio of the intracellular concentrations of glucose 6-phosphate and ATP (G6-P/ATP) and the activation state of glycogen synthase in hepatocytes from both normal and diabetic animals. However, for any given value of the G6-P/ATP ratio, the activation state of glycogen synthase in diabetic animals is always lower than that of normal animals. This suggests that the system that activates glycogen synthase (synthase phosphatase activity) is impaired in the diabetic state. The permissive effect of fructose is probably exerted through its capacity to increase the G6-P/ATP ratio which may partially increase synthase phosphatase activity, rendering glycogen synthase active.     

Flux control in the rat gastrocnemius glycogen synthesis pathway by in vivo 13C/31P NMR spectroscopy

To determine the relative contributions of glucose transport/hexokinase, glycogen synthase (GSase), and glycolysis to the control of insulin-stimulated muscle glycogen synthesis, we combined 13C and 31P NMR to quantitate the glycogen synthesis rate and glucose 6-phosphate (G-6-P) levels in rat (Sprague-Dawley) gastrocnemius muscle during hyperinsulinemia at euglycemic (E) and hyperglycemic (H) glucose concentrations under thiopental anesthesia. Flux control was calculated using metabolic control analysis. The combined control coefficient of glucose transport/hexokinase (GT/Hk) for glycogen synthesis was 1.1 +/- 0.03 (direct measure) and 1.14-1.16 (calculated for a range of glycolytic fluxes), whereas the control coefficient for GSase was much lower (0.011-0.448). We also observed that the increase in in vivo [G-6-P] from E to H (0.22 +/- 0.03 to 0.40 +/- 0.03 mM) effects a supralinear increase in the in vitro velocity of GSase, from 14.6 to 26.1 mU. kg(-1). min(-1) (1.8-fold). All measurements suggest that the majority of the flux control of muscle glycogen synthesis is at the GT/Hk step.