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.     

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.     

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.     

Liver glycogen metabolism in endotoxin shock. I. Endotoxin administration decreases glycogen synthase activities in dog livers

The effects of E. coli endotoxin administration on hepatic glycogen content and glycogen synthase activities in dogs were studied. Liver glycogen content was decreased by 80% 2 hr after endotoxin injection. When enzyme preparations were preincubated at 25 degrees C for 3 hr prior to their assays, 75% of total glycogen synthase was in I form in control dogs. Under such conditions, endotoxin administration decreased the percentage I activity from 75 to 37%; decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 62.2 and 35.3%, respectively; decreased the Vmax and Km for UDP-glucose for glycogen synthase I by 75.6 and 15.6%, respectively; increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 126% at high (10 mM) and by 18-fold at low (1 mM) UDP-glucose concentration; increased the percentage D activity from 24 to 72%; decreased the I50 for ATP for the inhibition of total glycogen synthase by 49.7%; decreased the I50 for ATP for the inhibition of glycogen synthase I by 26.4%; and decreased the percentage I activity from 78 to 33% at ATP concentrations below 6 mM. When enzyme preparations were not preincubated prior to their assays, 90% of total glycogen synthase was in D form in control dogs. Under such conditions, endotoxin administration decreased the Vmax and Km for UDP-glucose for total glycogen synthase by 47.1 and 33.3%, respectively, and increased the A0.5 for glucose-6-P for the activation of glycogen synthase D by 24.2% at high (10 mM) and by 106% at low (1 mM) UDP-glucose concentration. From these results, it is clear that endotoxin administration greatly impaired hepatic glycogenesis by decreasing the activity of glycogen synthase; this impairment is at least in part responsible for the depletion of liver glycogen content in endotoxin shock. Kinetic analyses revealed that the decrease in the activity of glycogen synthase in endotoxic shock is a result of a decrease in the interconversion of this enzyme from inactive to active form and an increase in the interconversion from active to inactive form.     

Purification and properties of the glucose-6-phosphate-dependent form of human placental glycogen synthase.

Glycogen synthase in the glucose-6-phosphate (glucose-6-P)-dependent form was purified over 10,000-fold from an extract of term human placenta. The purified enzyme shows a single protein band on polyacry1amide-gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme activity in the presence of glucose-6-P is increased by the single addition of Mg²⁺, Ca²⁺, or Mn²⁺ and is reduced by the addition of either sulfate or phosphate. Addition of either Mg²⁺, Ca²⁺, or Mn²⁺ relieves the inhibition by sulfate or phosphate. The enzyme activity in the absence of glucose 6-P is greatly increased by the addition of MnSO₄, CoSO₄, and NiSO₄ and is increased to a lesser extent by MgSO₄, CaSO₄, and FeSO₄. The activation of the glucose-6-P-dependent form of the enzyme by these metal sulfates in the absence of glucose-6-P has never been reported. MnSO₄, which shows homotropic cooperativity, is the best activator among the various metal sulfates tested. The human placental glucose-6-P-dependent form of glycogen synthase (D form) can be converted to the glucose-6-P-independent form (I form) of the enzyme by incubating the partially purified glycogen synthase, which is copurified with synthase phosphatase, with Mn²⁺. This conversion can be reversed by the addition of cyclic AMP-dependent protein kinase. The synthase D to synthase I converting system from human placenta is unique in its stringent requirement for Mn²⁺.     

Epinephrine regulation of skeletal muscle glycogen metabolism. Studies utilizing the perfused rat hindlimb preparation

Studies of rat skeletal glycogen metabolism carried out in a perfused hindlimb system indicated that epinephrine activates phosphorylase via the cascade of phosphorylation reactions classically linked to the beta-adrenergic receptor/adenylate cyclase system. The beta blocker propranolol completely blocked the effects of epinephrine on cAMP, cAMP-dependent protein kinase, phosphorylase, and glucose-6-P, whereas the alpha blocker phentolamine was totally ineffective. Omission of glucose from the perfusion medium did not modify the effects of epinephrine. Glycogen synthase activity in control perfused and nonperfused muscle was largely glucose-6-P-dependent (-glucose-6-P/+glucose-6-P activity ratios of 0.1 and 0.2, respectively). Epinephrine perfusion caused a small decrease in the enzyme's activity ratio (0.1 to 0.05) and a large increase in its Ka for glucose-6-P (0.3 to 1.5 mM). This increase in glucose-6-P dependency correlated in time with protein kinase activation and was totally blocked by propranolol and unaffected by phentolamine. Comparison of the kinetics of glycogen synthase in extracts of control and epinephrine-perfused muscle with the kinetics of purified rat skeletal muscle glycogen synthase a phosphorylated to various degrees by cAMP-dependent protein kinase indicated that the enzyme was already substantially phosphorylated in control muscle and that epinephrine treatment caused further phosphorylation of synthase, presumably via cAMP-dependent protein kinase. These data provide a basis for speculation about in vivo regulation of the enzyme.     

Purification and some regulatory properties of glycogen synthase I from rabbit renal medulla

1. Glycogen synthase I (activity ratio approximately equal to 1) was purified over 10,000-fold from rabbit renal medulla. 2. The purified synthase was stimulated about 1.5-fold by glucose-6-P and other divalent anions when assayed at pH 7.7 and near saturating UDPGlc. When assayed at physiological UDPGlc (75-100 microM), the enzyme was stimulated about 5-fold by glucose-6-P. 3. At pH 7.7 the activation by either Na2SO4 or glucose-6-P was due to an increase in V and a decrease in S0.5 for UDPGlc. At pH. 6.9, activation was due to a decrease in S0.5. 4. At low UDPGlc, synthase activity was inhibited by adenine nucleotides and the inhibition was partially relieved by glucose-6-P, UDP inhibited in a competitive manner with respect to UDPGlc. 5. These results suggest that the activity of renal medullary synthase I may be regulated by cellular metabolites.     

Purification and characterization of a cAMP- and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex

We recently reported the partial purification of a cAMP-independent and Ca2+-calmodulin-independent glycogen synthase kinase from porcine renal cortex (Schlender, K. K., Beebe, S. J., and Reimann, E. M. (1981) Cold Spring Harbor Conf. Cell Proliferation, 389-400). Subsequent purification indicated that the enzyme preparation consisted of at least three forms of glycogen synthase kinase which could be resolved by ATP gradient elution from aminoethylphosphate-agarose (AEP-agarose). The predominant form of glycogen synthase kinase, which eluted from AEP-agarose between 2 and 6 mM ATP, was purified approximately 800-fold and is designated GSK-A1. It had a molecular weight of 45,000-50,000 as determined by gel filtration and sucrose density gradient centrifugation. It catalyzed the transfer of 1 mol of 32P/mol of synthase subunit into a low molecular weight (10,000) CNBr peptide which was tentatively identified as Ser-7 (site 2) by high performance liquid chromatography. This phosphorylation decreased the activity ratio (activity in the absence of glucose-6-P divided by activity in the presence of 7.2 mM glucose-6-P) from 0.95 to about 0.55. GSK-A1 appeared to be specific for and had low s0.5 values for both substrates, ATP (13 microM) and glycogen synthase (0.3-0.4 microM). The enzyme could not use GTP as the phosphate donor. GSK-A1 was not affected by the protein kinase inhibitor, cAMP, cGMP, Ca2+-calmodulin, EGTA, or trifluoperazine and had a broad pH optimum (pH 7.0-8.5). A second form, GSK-A2, was eluted from AEP-agarose between 7 and 9 mM ATP. GSK-A2 could transfer a 2nd mol of 32P/mol of synthase subunit and decreased the activity ratio to 0.30. The interrelation among these multiple forms is not clear, but the data suggest that multiple kinases are required to form the highly inactivated glycogen synthase in renal tissues.     

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.     

Purification and properties of cyclic AMP-independent glycogen synthase kinase 1 from rabbit skeletal muscle.

A cyclic AMP-independent casein (phosvitin) kinase eluted from a phosphocellulose column with 0.35 M KCl also possesses glycogen synthase kinase activity. This kinase, designated synthase kinase 1, is separable from other cyclic AMP-independent protein kinases, which also contain glycogen synthase kinase activity, by chromatography on a phosphocellulose column. This kinase was purified 15,000-fold from the crude extract. Synthase kinase activity co-purifies with casein and phosvitin kinase activities. Heat inactivation of these three kinase activities follow similar kinetics. It is suggested that these three kinase activities reside in a single protein. This kinase has a molecular weight of approximately 34,000 as determined by glycerol density gradient centrifugation and by gel filtration. The Km values for the synthase kinase-catalyzed reaction are 0.12 mg/ml (0.35 micronM) for synthase, 12 micronM for ATP, and 0.15 mM for Mg2+. The phosphorylation of glycogen synthase by the kinase results in the incorporation of 4 mol of phosphate/85,000 subunit; however, only two of the phosphate sites predominantly determine the glucose-6-P dependency of the synthase. Synthase kinase activity is sensitive to inhibition by NaCl or KCl at concentrations encountered during purification. Synthase kinase activity is insensitive to the allosteric effector (glucose-6-P) or substrate (UDP-glucose) of glycogen synthase at concentrations usually found under physiological condition.     

Last Step in the Conversion of Trehalose to Glycogen: A MYCOBACTERIAL ENZYME THAT TRANSFERS MALTOSE FROM MALTOSE 1-PHOSPHATE TO GLYCOGEN

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [¹⁴C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [¹⁴C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the ¹⁴C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 μmol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named α1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [¹⁴C]maltose-1-P, but not [¹⁴C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.     

Effect of manganese(ous) and sulfate on activity of human placental glucose 6-phosphate-dependent form of glycogen synthase.

The human placental glucose-6-P-dependent form of glycogen synthase, in the absence of glucose-6-P, can be activated by MnSO4. Separately, Mn2+ and SO4(2-) have no significant effect. In the presence of glucose-6-P, Mn2+ activates the enzyme, but SO4(2-) inhibits; MnSO4 synergetically increases the enzyme activity. Mn2+ reduces the Ka for glucose-6-P to one-tenth of the control value; SO4(2-) increases the Ka 5-fold; however, MnSO4 has no effect on Ka. MnSO4, like glucose-6-P, increases the Vmax of the enzyme in the presence of its substrate, UDP-glucose; it slightly increases the Km for UDP-glucose. In the presence of glucose-6-P, Mn2+ increases and SO4(2-) decreases the Vmax of the enzyme, but neither has an effect on the Km for UDP-glucose. At physiological concentrations of UDP-glucose and glucose-6-P, either Mn2+ or MnSO4 at concentrations less than 1 mM increases the enzyme activity as much as 8 mM glucose-6-P does. At physiological concentrations of UDP-glucose and glucose-6-P, Mn2+ or MnSO4 reverses the inhibition of the enzyme by ATP.     

Phosphorylation of glycogen synthase by the Ca2+- and phospholipid-activated protein kinase (protein kinase C)

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been found to phosphorylate and inactivate glycogen synthase. With muscle glycogen synthase as a substrate, the reaction was stimulated by Ca2+ and by phosphatidylserine. The tumor-promoting phorbol esters 12-O-tetradecanoyl phorbol 13-acetate was also a positive effector, half-maximal activation occurring at 6 nM. Phosphorylation of glycogen synthase, but not histone, was partially inhibited by glycogen, half-maximally at 0.05 mg/ml, probably via a substrate-directed mechanism. The rate of glycogen synthase phosphorylation was approximately half that for histone; the apparent Km for glycogen synthase was 0.25 mg/ml. Protein kinase C also phosphorylated casein, the preferred substrate among the individual caseins being alpha s1-casein. Glycogen synthase was phosphorylated to greater than 1 phosphate/subunit with an accompanying reduction in the -glucose-6-P/+glucose-6-P activity ratio from 0.9 to 0.5. Phosphate was introduced into serine residues in both the NH2-terminal and COOH-terminal CNBr fragments of the enzyme subunit. The two main tryptic phosphopeptides mapped in correspondence with the peptides that contain site 1a and site 2. Lesser phosphorylation in an unidentified peptide was also observed. Rabbit liver and muscle glycogen synthases were phosphorylated at similar rates by protein kinase C. The above results are compatible with a role for protein kinase C in the regulation of glycogen synthase as was suggested by a recent study of intact hepatocytes.     

Biosynthesis of glycogen in Neurospora crassa. Purification and properties of the UDPglucose:glycogen 4-alpha-glucosyltransferase.

The Neurospora crassa glycogen synthase (UDPglucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) was purified to electrophoretic homogeneity by a procedure involving ultracentrifugation, DEAE-cellulose column chromatography, (NH4)2SO4 fractionation and 3-aminopropyl-Sepharose column chromatography. The final purified enzyme preparation was almost entirely dependent on glucose-6-P and had a specific activity of 6.9 units per mg of protein. The subunit molecular weight of the glycogen synthase was determined by electrophoresis in sodium dodecyl sulfate-polyacrylamide gel to be 88 000--90 000. The native enzyme was shown to have a molecular weight of 270 000 as determined by sucrose density gradient centrifugation. Thus, the glucose-6-P-dependent form of the N. crassa glycogen synthase can exist as trimer of the subunit. Limited proteolysis with trypsin or chymotrypsin converted the glucose-6-P-dependent form of the enzyme into an apparent glucose-6-P-independent form. The enzyme was shown to catalyze transfer of glucose from UDPglucose to glycogen as well as to its phosphorylase limit dextrin, but not to its beta-amylase limit dextrin. Moreover, glucose, maltose and maltotriose were not active as acceptors.     

Mutations of muscle glycogen synthase that disable activation by glucose 6-phosphate.

Glycogen synthase, an enzyme of historical importance in the field of reversible protein modification, is inactivated by phosphorylation and allosterically activated by glucose 6-phosphate (glucose-6-P). Previous analysis of yeast glycogen synthase had identified a conserved and highly basic 13-amino-acid segment in which mutation of Arg residues resulted in loss of activation by glucose-6-P. The equivalent mutations R578R579R581A (all three of the indicated Arg residues mutated to Ala) and R585R587R590A were introduced into rabbit muscle glycogen synthase. Whether expressed transiently in COS-1 cells or produced in and purified from Escherichia coli, both mutant enzymes were insensitive to activation by glucose-6-P. The effect of phosphorylation was studied in two ways. Purified, recombinant glycogen synthase was directly phosphorylated by casein kinase 2 and glycogen synthase kinase 3, under conditions that inactivate the wild-type enzyme. In addition, phosphorylation sites were converted to Ala by mutagenesis in wild-type and in the glucose-6-P desensitized mutants expressed in COS-1 cells. Phosphorylation inactivated the R578R579R581A mutant but had little effect on the R585R587R590A. This result was surprising since phosphorylation had the opposite effects on the corresponding yeast enzyme mutants. The results confirm that the region of glycogen synthase, Arg-578-Arg-590, is required for activation by glucose-6-P and suggest that it is part of a sensitive and critical switch involved in transitions between different conformational states. However, the role must differ subtly between the mammalian and the yeast enzymes.     

Purification and characterization of glycogen synthase from a glycogen-deficient strain of Saccharomyces cerevisiae

Chromatography of wild-type yeast extracts on DEAE-cellulose columns resolves two populations of glycogen synthase I (glucose-6-P-independent) and D (glucose-6-P-dependent) (Huang, K. P., Cabib, E. (1974) J. Biol. Chem. 249, 3851-3857). Extracts from a glycogen-deficient mutant strain, 22R1 (glc7), yielded only the D form of glycogen synthase. Glycogen synthase D purified from either wild-type yeast or from this glycogen-deficient mutant displayed two polypeptides with molecular masses of 76 and 83 kDa on sodium dodecyl sulfate-gel electrophoresis in a protein ratio of about 4:1. Phosphate analysis showed that glycogen synthase D from either strain of yeast contained approximately 3 phosphates/subunit. The 76- and 83-kDa bands of the mutant strain copurified through a variety of procedures including nondenaturing gel electrophoresis. These two polypeptides showed immunological cross-reactivity and similar peptide maps indicating that they are structurally related. The relative amounts of these two forms remained constant during purification and storage of the enzyme and after treatment with cAMP-dependent protein kinase or with protein phosphatases. The two polypeptides were phosphorylated to similar extent in vitro by the catalytic subunit of mammalian cyclic AMP-dependent protein kinase. Phosphorylation of the enzyme in the presence of labeled ATP followed by tryptic digestion and reversed phase high performance liquid chromatography yielded two labeled peptides from each of the 76- and 83-kDa subunits. Treatment of wild-type yeast with Li+ increased the glycogen synthase activity, measured in the absence of glucose-6-P, by approximately 2-fold, whereas similar treatment of the glc7 mutant had no effect. The results of this study indicate that the GLC7 gene is involved in a pathway that regulates the phosphorylation state of glycogen synthase.     

Stimulation of autophosphorylation of rabbit skeletal muscle phosphorylase kinase by glycogen synthase on glycogen particles

Glycogen synthase stimulated the autophosphorylation and autoactivation of phosphorylase kinase from rabbit skeletal muscle. This stimulation was additive to that by glycogen and the reaction was dependent on Ca2+. The effect by glycogen synthase was maximum within the activity ratio (the activity of enzyme without glucose-6-P divided by the activity with 10 mM glucose-6-P) of 0.3 and over 0.3 it was rather inhibitory. The results suggest that autophosphorylation of phosphorylase kinase in the presence of glycogen synthase on glycogen particles may be an important regulatory mechanism of glycogen metabolism in skeletal muscle.     

The control of hepatic glycogen metabolism in an in vitro model of sepsis

Culturing hepatocytes with a combination of LPS, TNF-α, IL-1β and IFN-γ resulted in an inhibition of glucose output from glycogen and prevented the repletion of glycogen in freshly cultured cells. The reduced glycogen mobilisation correlated with the lower cell glycogen content and reduced rate of glycogen synthesis from [U-¹⁴C]glucose rather than alterations in either total phosphorylase or phosphorylase a activity. There was no change in the percentage of glycogen exported as glucose nor the production of lactate plus pyruvate indicating that redistribution of the Gluc-6-P cannot explain the failure of the liver to export glucose. Although changes in glycogen mobilisation correlated with NO production, inhibition of NO synthase by inclusion of L-NMMA in the culture medium failed to prevent the inhibition of either glycogen accumulation or mobilisation by the proinflammatory cytokines, precluding the involvement of NO in this response. LPS plus cytokine treatment had no effect on total glycogen synthase activity although the activity ratio was lowered, indicative of increased phosphorylation. The inhibition of glycogen synthesis correlated with a fall in the intracellular concentrations of Gluc-6-P and UDP-glucose and in the absence of measured changes in kinase activity, it is suggested that the fall in Gluc-6-P reduces both substrate supply and glycogen synthase phosphatase activity. The fall in Gluc-6-P coincided with a reduction in total glucokinase and hexokinase activity within the cells, but no significant change in either the translocation of glucokinase or glucose-6-phosphatase activity. This demonstrates direct cytokine effects on glycogen metabolism independent of changes in glucoregulatory hormones.     

Mechanism of palmityl coenzyme A inhibition of liver glycogen synthase.

Palmityl-CoA inhibits free liver glycogen synthase; the concentration required for half-maximum inhibition is 3 to 4 micrometer. Almost complete inhibition was observed at 50 micrometer. Palmityl-CoA inhibition is associated with dissociation of the tetrameric enzyme into monomers, and binding of palmityl-CoA to the monomers. Glycogen-bound enzyme is also inhibited by palmityl-CoA, resulting in dissociation of the enzyme into monomers and concomitant release of the enzyme from the primer glycogen. Palmityl-CoA inhibition of the enzyme is partially reversed by the glycogen synthase activator, glucose-6-P, whereas sodium lauryl sulfate-inhibited enzyme is not reactivated by glucose-6-P. Sodium lauryl sulfate inhibition results in the dissociation of the tetramer into the monomers. Bovine serum albumin and cyclodextrin can prevent palmityl-CoA inhibition only when they are added prior to palmityl-CoA addition. The possible physiological role of palmityl-CoA in glucose homeostasis is discussed.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.