Regulation of muscle glycogen synthase phosphorylation and kinetic properties by insulin, exercise, adrenaline and role in insulin resistance.
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In mammals, excess carbohydrate is stored as glycogen and glycogen synthase is the enzyme that incorporates glucose units into the glycogen particle. Glycogen synthase activity is regulated by phosphorylation and allosterically activated by glucose 6-phosphate. Phosphorylation of nine serines by different kinases regulates glycogen synthase affinity for glucose 6-phosphate and its substrate UDP-glucose. Glucose 6-phosphate increases both enzyme activity and substrate affinity. Insulin and exercise increase glycogen synthase affinity for glucose 6-phosphate and activity whereas high glycogen content and adrenaline decrease affinity for glucose 6-phosphate and activity. However, insulin, exercise and adrenaline also regulate intracellular concentration of glucose 6-phosphate which will influence in vivo glycogen synthase activity. Importantly, type 2 diabetes is associated with reduced insulin-stimulated glycogen synthase activation. The nine phosphorylation sites theoretically allow 512 combinations of phosphorylation configurations of glycogen synthase with different kinetic properties. However, due to hierarchal phosphorylation, the number of configurations in vivo is most likely much lower. Unfortunately, many studies only report data on glycogen synthase activity measured with high concentration of UDP-glucose which holds back information about changes in substrate affinity. In this paper we discuss the physiological regulation of glycogen synthase phosphorylation and how the phosphorylation pattern regulates glycogen synthase kinetic properties.Keywords:
Glycogen branching enzyme
Glycogen debranching enzyme
Glucose 6-phosphate
GSK3B
Glycogenesis
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Glycogen branching enzyme
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The effects of several inhibitors (ATP, ADP, AMP, UDP, and P1) and activators (Mg2+, glucose-6-P) of rabbit muscle glycogen synthase (UDP-glucose:glycogen 4-alpha-glucosyltransferase, EC 2.4.1.11) were studied in relation to the phosphorylation state of the purified enzyme. All the modifiers had increasing effects with enzyme of increasing alkali-labile phosphate content. In experiments where combinations of effectors were present, it was apparent that (a) concentrations of modifiers in the physiological range could be significant in determining enzymic activity and (b) the sensitivity of the reaction rate to changes in phosphorylation state was critically dependent on the concentration of the small molecules. Changes in the phosphorylation of the enzyme corresponding to changes in the %I activity reported in the literature for studies in vivo were capable of producing large alterations in glycogen synthase activity. Because the magnitudes of such changes were dependent on the effector concentrations, there may be an integration of local cellular control, through small molecule effects, with hormonal control, through the phosphorylation state of glycogen synthase.
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Liver glycogen is synthesized in the postprandial state in response to elevated concentrations of glucose and insulin or by activation of neuroendocrine signals and it is degraded in the postabsorptive state in response to changes in the concentrations of insulin and counter-regulatory hormones. Dysregulation of either glycogen degradation or synthesis through changes in allosteric control or covalent modification of glycogen phosphorylase and glycogen synthase leads to disturbance of blood glucose homeostasis. Liver glycogen phosphorylase has a dual role in the control of glycogen metabolism by regulation of both glycogen degradation and synthesis. The phosphorylated form (GPa) is the active form and determines the rate of degradation of glycogen and it is also a potent allosteric inhibitor of the protein complex, involving the glycogen targeting protein GL and protein phosphatase-1, which catalyses dephosphorylation (activation) of glycogen synthase. Drug discovery programmes exploring the validity of glycogen phosphorylase as a therapeutic target for type 2 diabetes have generated a wide array of selective phosphorylase ligands that modulate the catalytic activity and / or the phosphorylation state (interconversion of GPa and GPb) as well as the binding of GPa to the allosteric site of GL. Glycogen phosphorylase inhibitors that act in hepatocytes either exclusively by dephosphorylating GPa (e.g. indole carboxamides) or by allosteric inhibition of GPa (1,4-dideoxy-1,4-D-arabinitol) are very powerful experimental tools to determine the relative roles of covalent modification of glycogen phosphorylase and / or cycling between glycogen synthesis and degradation in the mechanism(s) by which insulin and neurotransmitters regulate hepatic glycogen metabolism.
Glycogen debranching enzyme
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The dephosphorylation of glycogen synthase is a key step in the stimulation of glycogen synthesis by insulin. To further investigate the hormonal regulation of glycogen synthase activity, enzymatic localization in 3T3-L1 adipocytes was determined by immunocytochemistry and confocal microscopy. In basal cells, glycogen synthase and the protein phosphatase-1-glycogen-targeting subunit, protein targeting to glycogen (PTG), were diffusely distributed throughout the cell. Insulin treatment had no effect on PTG distribution but resulted in a reorganization of glycogen synthase into punctate clusters. Glycogen synthase aggregation was restricted to discrete cellular sites, presumably where glycogen synthesis occurred. Omission of extracellular glucose or substitution with 2-deoxy-glucose blocked the insulin-induced redistribution of glycogen synthase. Addition of the glycogenolytic agent forskolin after insulin stimulation disrupted the clusters of glycogen synthase protein, restoring the immunostaining pattern to the basal state. Conversely, adenoviral-mediated overexpression of PTG resulted in the insulin-independent dephosphorylation of glycogen synthase and a redistribution of the enzyme from the cytosolic- to glycogen-containing fractions. The effects of PTG on glycogen synthase activity were mediated by multisite dephosphorylation, which was enhanced by insulin and 2-deoxy-glucose, and required a functional glycogen synthase-binding domain on PTG. However, PTG overexpression did not induce distinct glycogen synthase clustering in fixed cells, presumably because cellular glycogen levels were increased more than 7-fold under these conditions, resulting in a diffusion of sites where glycogen elongation occurred. Cumulatively, these data indicate that the hormonal regulation of glycogen synthesis rates in 3T3-L1 adipocytes is mediated in part through changes in the subcellular localization of glycogen synthase.
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Glycogen branching enzyme
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Glycogen synthase catalyzes the transfer of the glucosyl moiety from UDP-glucose to the terminal branch of the glycogen molecule and is considered to be the rate-limiting enzyme for glycogen synthesis. However, under ideal assay conditions, i.e. 37 degrees C with saturating concentrations of UDP-glucose and the activator, glucose-6-P, the maximal catalytic activity of glycogen synthase was only 78% of the in vivo glycogen synthetic rate. Using concentrations of UDP-glucose and glucose-6-P likely to be present in vivo, the rate was only approximately 30%. This prompted us to reassess a possible role of allosteric effectors on synthase activity. Glycogen synthase was assayed at 37 degrees C using dilute, pH 7.0, buffered extracts, initial rate conditions, and UDP-glucose and glucose-6-P concentrations, which approximate those calculated to be present in total liver cell water. Several allosteric effectors were tested. Magnesium and AMP had little effect on activity. Pi, ADP, ATP, and UTP inhibited activity. When a combination of effectors were added at concentrations approximating those present in cell water, synthase activity could account for only 2% of the glycogen synthetic rate. Thus, although allosteric effectors are likely to be playing a major role in regulating synthase enzymic activity in liver cells, to date, a metabolite that can stimulate activity and/or overcome nucleotide inhibition has yet to be identified. If such a metabolite cannot be identified, an additional or alternative pathway for glycogen synthesis must be considered.
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Non-metabolized glucose derivatives may cause inactivation of phosphorylase but, unlike glucose, they are unable to elicit activation of glycogen synthase in isolated hepatocytes. We report here that, after the previous inactivation of phosphorylase by one of these glucose derivatives (2-deoxy-2-fluoro-alpha-glucosyl fluoride), glycogen synthase was progressively activated by addition of increasing concentrations of glucose. Under these conditions, the degree of activation of glycogen synthase was linearly correlated with the intracellular glucose-6-phosphate (Glc-6-P) concentration. Addition of glucosamine, an inhibitor of glucokinase, decreased both parameters in parallel. Further experiments using an inhibitor of either protein kinases (5-iodotubercidin) or protein phosphatases (microcystin) in isolated hepatocytes indicated that Glc-6-P does not affect glycogen-synthase kinase activity but enhances the glycogen-synthase phosphatase reaction. Experiments in vitro showed that the synthase phosphatase activity of glycogen-bound type-1 protein phosphatase was increased by physiological concentrations of Glc-6-P (0.1-0.5 mM), but not by 2.5 mM fructose-6-P, fructose-1-P or glucose-1-P. At physiological ionic strength, the glycogen-associated synthase phosphatase activity was nearly entirely Glc-6-P-dependent, but Glc-6-P did not relieve the strong inhibitory effect of phosphorylase a. The large stimulatory effects of 2.5 mM Glc-6-P, with glycogen synthase b and phosphorylase a as substrates, appeared to be mostly substrate-directed, while the modest effects observed with casein and histone IIA pointed to an additional stimulation of glycogen-bound protein phosphatase-1 by Glc-6-P. We conclude that glucose elicits hepatic synthase phosphatase activity both by removal of the inhibitor, phosphorylase a, and by generation of the stimulator, Glc-6-P.
Glycogen branching enzyme
Glycogenesis
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In mammals, excess carbohydrate is stored as glycogen and glycogen synthase is the enzyme that incorporates glucose units into the glycogen particle. Glycogen synthase activity is regulated by phosphorylation and allosterically activated by glucose 6-phosphate. Phosphorylation of nine serines by different kinases regulates glycogen synthase affinity for glucose 6-phosphate and its substrate UDP-glucose. Glucose 6-phosphate increases both enzyme activity and substrate affinity. Insulin and exercise increase glycogen synthase affinity for glucose 6-phosphate and activity whereas high glycogen content and adrenaline decrease affinity for glucose 6-phosphate and activity. However, insulin, exercise and adrenaline also regulate intracellular concentration of glucose 6-phosphate which will influence in vivo glycogen synthase activity. Importantly, type 2 diabetes is associated with reduced insulin-stimulated glycogen synthase activation. The nine phosphorylation sites theoretically allow 512 combinations of phosphorylation configurations of glycogen synthase with different kinetic properties. However, due to hierarchal phosphorylation, the number of configurations in vivo is most likely much lower. Unfortunately, many studies only report data on glycogen synthase activity measured with high concentration of UDP-glucose which holds back information about changes in substrate affinity. In this paper we discuss the physiological regulation of glycogen synthase phosphorylation and how the phosphorylation pattern regulates glycogen synthase kinetic properties.
Glycogen branching enzyme
Glycogen debranching enzyme
Glucose 6-phosphate
GSK3B
Glycogenesis
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Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M r = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle. Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M r = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle. Insulin lowers blood glucose by inhibiting hepatic glucose output and by increasing the uptake of glucose by various target tissues. In lean individuals, skeletal muscle represents the largest mass of insulin-sensitive tissue, and the majority of the glucose taken up in response to insulin following a meal is converted to muscle glycogen (1Jue T. Rothman D.L. Tavitian B.A. Shulman R.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1439-1442Crossref PubMed Scopus (84) Google Scholar, 2Shulman G.I. Rothman D.L. Jue T. Stein P. DeFronzo R.A. Shulman R.G. N. Engl. J. Med. 1990; 322: 223-228Crossref PubMed Scopus (1056) Google Scholar). Thus, the stimulation of glycogen deposition in skeletal muscle is of particular importance in the maintenance of glucose homeostasis. Insulin promotes glycogen synthesis both by increasing glucose entry into muscle fibers and by increasing conversion of the intracellular glucose into glycogen. This response involves activation of glucose transport and glycogen synthase. Glycogen synthase catalyzes the reaction in which glucose from UDP-glucose is incorporated into glycogen. The enzyme is subject to complex control by both allosteric and covalent mechanisms (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). Skeletal muscle glycogen synthase may be phosphorylated in 10 or more sites, which are clustered in regions near the NH2 and COOH termini (4Cohen P. Boyer P. Krebs E.G. The Enzymes. Academic Press, Orlando, FL1986: 461-497Google Scholar). Insulin activates the enzyme by promoting dephosphorylation of sites in both regions (5Skurat A.V. Wang Y. Roach P.J. J. Biol. Chem. 1994; 269: 25534-25542Abstract Full Text PDF PubMed Google Scholar, 6Skurat A.V. Roach P.J. J. Biol. Chem. 1995; 270: 12491-12497Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In general, phosphorylation decreases glycogen synthase activity (3Lawrence Jr., J.C. Roach P.J. Diabetes. 1997; 46: 541-547Crossref PubMed Google Scholar). However, the allosteric effector, glucose-6-phosphate (G6P), 1The abbreviations used are: G6P, glucose 6-phosphate; DARPP-32, dopamine- and cAMP-regulated phosphoprotein,M r = 32,000; EDL, extensor digitorum longus; G1P, glucose 1-phosphate; MAP, mitogen-activated protein; PP1, type 1 protein phosphatase; PP1G, a form of PP1 containing a catalytic subunit bound to RGL; PTG, a PP1-targeting subunit; RGL, muscle glycogen-binding regulatory subunit of PP1G. is able to activate fully even highly phosphorylated forms of the enzyme. When provided with sufficient substrate, nonphosphorylated glycogen synthase is fully active even in the absence of G6P (7Roach P.J. Larner J. Trends Biochem. Sci. 1976; 1: 110-112Abstract Full Text PDF Scopus (6) Google Scholar). Consequently, the activation of glycogen synthase may be monitored by the increase in the activity ratio (−G6P/+G6P). Despite several decades of investigation, the mechanism by which insulin activates glycogen synthase is still not clear. The multisite dephosphorylation of glycogen synthase is suggestive of phosphatase activation. Indeed, insulin has been shown to increase the activity of PP1 (8Cohen P. Adv. Second Messenger Phosphoprotein Res. 1990; 24: 230-235PubMed Google Scholar), an enzyme that is capable of dephosphorylating both the NH2- and COOH-terminal sites in glycogen synthase. The action of PP1 in cells is dependent on its subcellular localization, which is determined by different regulatory/targeting subunits. RGL (also known as the G subunit or GM) and PTG are two glycogen-binding subunits that target PP1 to glycogen particles in skeletal muscle (9Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (243) Google Scholar, 10Tang P.M. Bondor J.A. Swiderek K.M. DePaoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar). These glycogen-bound forms of PP1 are believed to be responsible for the dephosphorylation of glycogen synthase, which is also bound to glycogen. PP1 activity is controlled by two related heat-stable proteins, Inhibitor-1 (11Huang F.L. Glinsmann W.H. Eur. J. Biochem. 1976; 70: 419-426Crossref PubMed Scopus (309) Google Scholar, 12Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 341-351Crossref PubMed Scopus (209) Google Scholar) and DARPP-32 (13Strålfors P. Hemmings Jr., H.C. Greengard P. Eur. J. Biochem. 1989; 180: 143-148Crossref PubMed Scopus (18) Google Scholar). Inhibitor-1 (I-1) is expressed in a wide variety of tissues, including skeletal muscle (12Nimmo G.A. Cohen P. Eur. J. Biochem. 1978; 87: 341-351Crossref PubMed Scopus (209) Google Scholar). DARPP-32 is expressed in certain neurons (14Hemmings Jr., H.C. Nairn A.C. Aswad D.W. Greengard P. J. Neurosci. 1984; 4: 99-110Crossref PubMed Google Scholar), kidney, and adipocytes (15Meister B. Fried G. Hökfelt T. Hemmings Jr., H.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8713-8716Crossref PubMed Scopus (22) Google Scholar). The nonphosphorylated forms of I-1 and DARPP-32 are essentially devoid of PP1 inhibitory activity, but both proteins become potent inhibitors of PP1 after phosphorylation by cAMP-dependent protein kinase (13Strålfors P. Hemmings Jr., H.C. Greengard P. Eur. J. Biochem. 1989; 180: 143-148Crossref PubMed Scopus (18) Google Scholar). It was recently proposed that the activation of glycogen synthase by insulin in 3T3-L1 adipocytes is mediated by activation of PP1 via a mechanism involving decreased susceptibility of the PTG-bound form of the phosphatase to inhibition by DARPP-32 (16Brady M.J. Printen J.A. Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20198-20204Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 17Mastick C.C. Brady M.J. Printen J.A. Ribon V. Saltiel A.R. Mol. Cell. Biol. 1998; 182: 65-71Google Scholar). If this mechanism were to apply to skeletal muscle, the most important site of insulin-stimulated glycogen deposition, then the effect of insulin would presumably depend on I-1, as skeletal muscle does not express DARPP-32 (15Meister B. Fried G. Hökfelt T. Hemmings Jr., H.C. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8713-8716Crossref PubMed Scopus (22) Google Scholar). To investigate this possibility, we have compared the effects of insulin on glycogen synthase and glycogen synthesis in muscles from wild-type mice and mice lacking I-1 and DARPP-32 as a result of targeted-disruption of the genes encoding the two proteins. Mice lacking I-1 were generated by targeted disruption of the I-1 gene. The preparation of these animals and additional phenotypic characterization will be described in detail elsewhere. 2P. B. Allen, Ø. Hvalby, V. Jensen, M. L. Errington, M. Ramsay, F. A. Chaudhry, T. V. P. Bliss, J. Storm-Mathisen, R. G. M. Morris, and P. Greengard, manuscript in preparation. Briefly, the I-1 gene was disrupted in the E14 embryonic stem cell line (18Thompson S. Clarke A.R. Pow A.M. Hooper M.L. Melton D.W. Cell. 1989; 56: 313-321Abstract Full Text PDF PubMed Scopus (312) Google Scholar) by using a targeting vector containing 1.5 kilobases (5′) and 5.5 kilobases (3′) of I-1 locus genomic DNA flanking a neomycin resistance gene(PGK-neo). Homologous recombination at the endogenous locus resulted in replacement of a 400-bp genomic fragment withPGK-neo. The deleted genomic fragment contains the I-1 exon encoding the initiation of translation. Correctly targeted clones were identified by the shift of a genomic restriction fragment from 10.1 to 11.5 kilobases, as determined by Southern (DNA) blot analysis. After C57BL/6J blastocyst injection and embryo transfer, chimeric offspring were crossed to C57BL/6J females, and those mice carrying the mutation were further backcrossed to C57BL/6J for five generations. Male I-1 knockout mice and wild-type littermates 5–8 months of age were selected from the offspring of heterozygous breeding pairs. To generate mice lacking both I-1 and DARPP-32, homozygous I-1 knockout mice were bred with homozygous DARPP-32 knockout mice (19Fienberg A.A. Hiroi N. Mermelstein P.G. Song W.J. Snyder G.L. Nishi A. Cheramy A. O'Callaghan J.P. Miller D.B. Cole D.G. Corbett R. Haile C.N. Cooper D.C. Onn S.P. Grace A.A. Ouimet C.C. White F.J. Hyman S.E. Surmeier D.J. Girault J.A. Nestler E.J. Greengard P. Science. 1998; 281: 838-842Crossref PubMed Scopus (396) Google Scholar). The F-1 offspring were intercrossed to generate wild-type and double knockout lines (129/Ola-C57BL/6J, backcrossed to C57BL/6J for five generations). Male mice from these lines were age-matched and studied at 4–8 months. For all mice used, genotype was determined by Southern blot or polymerase chain reaction analysis of tail DNA. To confirm the presence/absence of the protein(s), skeletal muscle extracts were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, which were then probed with polyclonal antibodies to I-1 (20Gustafson E.L. Girault J.A. Hemmings Jr., H.C. Nairn A.C. Greengard P. J. Compar. Neurol. 1991; 310: 170-188Crossref PubMed Scopus (43) Google Scholar) and glycogen synthase (21Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar), or monoclonal ascites to DARPP-32 (22Hemmings Jr., H.C. Greengard P. J. Neurosci. 1986; 6: 1469-1481Crossref PubMed Google Scholar). Media used for muscle incubations were continuously gassed by bubbling with a 19:1 mixture of O2:CO2. Hemidiaphragm and extensor digitorum (EDL) muscles were incubated at 30 °C in Dulbecco's modified Eagle's medium (30 ml/muscle) for 30 min to remove endogenous hormones. The muscles were then incubated without or with either 250 milliunits/ml insulin (Humulin, Eli Lilly Co.) or 10 μmepinephrine at 37 °C in Krebs-Henseleit buffer (118 mmNaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm potassium phosphate, 1.2 mmMgSO4, 25 mm NaHCO3) containing 5 mm glucose. To terminate the incubations, the muscles were blotted on tissue paper and immediately frozen in liquid nitrogen. The frozen tissues were manually ground with a porcelain mortar and pestle that had been chilled in liquid nitrogen. Samples (approximately 25 mg) of powdered muscle were homogenized at 0 °C using a tissue grinder (Teflon-glass) in 500 μl of Homogenization Buffer (100 mm KF, 10 mm EDTA, 2 mmEGTA, 2 mm potassium phosphate, 0.1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, 20 μm leupeptin, 1.5 μm aprotinin, and 50 mm Tris-HCl, pH 7.8, at 30 °C). The homogenates were centrifuged for 30 min at 10,000 × g, and the supernatants were retained for analyses. The protein content of the extracts was measured and adjusted to a concentration of 1 mg/ml by adding Homogenization Buffer. Glycogen synthase activity was measured by the method of Thomas et al. (23Thomas J.A. Schlender K.K. Larner J. Anal. Biochem. 1968; 25: 486-499Crossref PubMed Scopus (949) Google Scholar). Briefly, samples (30 μl) were added to solutions (60 μl) containing 10 mg/ml rabbit liver glycogen, 20 mm EDTA, 25 mm KF, 10 mm UDP-[1-14C]glucose (100,000 cpm, Amersham Pharmacia Biotech), 50 mm Tris-HCl (pH 7.8 at 30 °C) and incubated without or with 10 mm G6P at 30 °C for 20 min. The glycogen synthase activity ratio was determined by dividing the activity measured without added G6P by the activity measured in the presence of 10 mm G6P (total activity). Phosphorylase activity was measured in the direction of glycogen synthesis from [U-14C]G1P by using the method of Gilboe et al. (24Gilboe D.P. Larson K.L. Nuttall F.Q. Anal. Biochem. 1972; 47: 20-27Crossref PubMed Scopus (289) Google Scholar). Samples (30 μl) of extracts were added to solutions (60 μl) containing 10 mg/ml rabbit liver glycogen, 200 mmKF, 100 mm [U-14C]G1P (50,000 cpm, NEN Life Science Products), and incubated without or with 5 mm5′-AMP at 30 °C for 20 min. The activity ratio was determined by dividing the activity measured without 5′-AMP by the activity measured in the presence of 5′-AMP. The amount of glucose incorporated into glycogen was measured as described previously (25Smith R.L. Lawrence Jr., J.C. J. Biol. Chem. 1984; 259: 2201-2207Abstract Full Text PDF PubMed Google Scholar). Following the incubation to remove endogenous hormones, muscles were incubated in medium containing 5 mmd-[U-14C]glucose (ICN, Irvine, CA). Samples of muscles were weighed, then dissolved by heating for 45 min at 100 °C in 30% KOH (1 ml/100 mg of tissue). Ethanol was added to a final concentration of 70%, and the glycogen was allowed to precipitate at −20 °C. After 8 h, the samples were centrifuged at 2000 × g for 20 min to pellet the glycogen. The glycogen pellets were washed four times with 66% ethanol before the amount of 14C-labeled glycogen was determined by liquid scintillation counting. Relative levels of I-1 and DARPP-32 in diaphragm muscles of wild-type and I-1 knock-out mice were assessed by immunoblotting (Fig.1). I-1 from wild-type mice was readily detected as a protein of apparent M r = 29,000. As expected, I-1 protein was absent in muscles from mice in which the I-1 gene had been disrupted by targeted gene disruption. No DARPP-32 was observed in the wild-type muscle extracts (Fig. 1, middle blot), although the protein was readily detected in mouse brain extract (BE). The amount of glycogen synthase was unaffected by the I-1 gene deletion in muscles from I-1 knockout mice (Fig.1, lower blot). As skeletal muscle does not express DARPP-32, it is clear that the activation of glycogen synthase by insulin in this tissue cannot be explained by a decrease in the susceptibility of the PTG-bound form of PP1 to DARPP-32, as has been suggested to occur in 3T3-L1 adipocytes (17Mastick C.C. Brady M.J. Printen J.A. Ribon V. Saltiel A.R. Mol. Cell. Biol. 1998; 182: 65-71Google Scholar). To investigate the possibility that the effect of insulin in skeletal muscle is mediated by the DARPP-32-homologue, I-1, the effect of insulin on the glycogen synthase activity ratio was assessed in diaphragms from I-1 knockout mice (Fig.2). Insulin increased the activity ratio in these muscles from 0.22 to 0.41. In muscles from wild-type mice, insulin increased the activity ratio from 0.20 to 0.44. Thus, the effects of insulin on glycogen synthase were almost identical in I-1 knockout mice and their wild-type littermates. The stimulation of glycogen synthesis by insulin involves multiple steps. To determine whether I-1 was essential for the overall process of glycogen synthesis, we assessed the rates of [U-14C]glucose incorporation into glycogen in diaphragm muscles from wild-type and I-1 knockout mice (Fig.3). Basal rates of14C-labeled glycogen synthesis were not significantly different in muscles from wild-type and I-1 knockout animals. Insulin increased 14C-labeled glycogen accumulation by approximately 9-fold in muscles from wild-type mice. Insulin was equally effective in increasing 14C-labeled glycogen synthesis in muscles from the I-1 knockout animals. Thus, I-1 is not an essential component of signal transduction pathway leading to the stimulation of glycogen synthesis. A potential problem in interpreting results from knockout animals is that the absence of a gene product during development may result in compensatory expression of another functionally related protein. For this reason, we considered the possibility that expression of DARPP-32 might be induced in the I-1 knockout animals. This did not appear to be the case, as DARPP-32 was not detected in muscles from the I-1 knockout animals (Fig. 1, middle blot). Nevertheless, to be certain that the activation of glycogen synthase did not depend on either I-1 or DARPP-32, experiments were performed using muscles from animals that lacked both I-1 and DARPP-32. The activation of glycogen synthase in muscles from the double knockout animals was indistinguishable from that observed in muscles from the wild-type control animals (Fig. 4). The effect of insulin on incorporation of glucose into glycogen was assessed in EDL muscles from the same animals (Fig. 5). Basal rates of [U-14C]glucose into glycogen were almost identical in muscles from wild-type and double knockout mice. Insulin increased [U-14C]glucose into glycogen by approximately 2-fold in muscles from both groups of animals. The stimulation of [14C]glycogen synthesis by insulin was lower in the EDL muscles than in diaphragm (Fig. 3). This difference is most likely due to the different fiber type composition of the two muscles. Diaphragm is composed primarily of oxidative fiber types, which are more responsive to insulin than fast-glycolytic fibers, which comprise approximately half of the fibers in the EDL (26Ariano M.A. Armstrong R.B. Edgerton V.R. J. Histochem. 1973; 21: 51-55Crossref PubMed Scopus (788) Google Scholar).Figure 5Effect of insulin on synthesis of [14C]glycogen in I-1/DARPP-32 knockout mice. EDL muscles from I-1/DARPP-32 knockout and wild-type mice were incubated at 37 °C for 30 min without (CONTROL) or with (INSULIN) 20 milliunits/ml insulin in medium containing [14C]glucose. The amount of [14C]glycogen synthesized (in nmol/min/g wet weight) was determined by liquid scintillation counting. The results are mean values ± S.E. for muscles obtained from five experiments performed on different days (*,p < 0.05, control versusinsulin-stimulated).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The possibility that insulin might control glycogen metabolism by decreasing the phosphorylation of I-1 in skeletal muscle has been investigated previously. Khatra et al. (27Khatra B.S. Soderling T.R. Arch. Biochem. Biophys. 1983; 227: 39-51Crossref PubMed Scopus (14) Google Scholar) observed no effect of insulin on I-1 phosphorylation in perfused rat hindlimb. In contrast, Foulkes et al. (28Foulkes J.G. Jefferson L.S. Cohen P. FEBS Lett. 1980; 112: 21-24Crossref PubMed Scopus (58) Google Scholar) reported that insulin decreased the phosphorylation of I-1 in hindlimb muscles in a perfused hemicorpus model. Later, it was found that I-1 phosphorylation was elevated in the hemicorpus model due to circulating epinephrine and that the effect of insulin to decrease I-1 phosphorylation was dependent on the presence of the β-adrenergic agonist (29Dietz M.R. Chiasson J.L. Soderling T.R. Exton J.H. J. Biol. Chem. 1980; 255: 2301-2307Abstract Full Text PDF PubMed Google Scholar). Unfortunately, we were unable to use the I-1 knockout mice to investigate the role of I-1 in the inactivation of glycogen synthase in response to epinephrine because epinephrine was without effect on the glycogen synthase activity in either the wild-type or I-1 knockout muscles. Epinephrine did activate phosphorylase and the response was essentially identical in muscles from the wild-type and I-1 knockout mice (Fig. 6). It is not clear why glycogen synthase was not inactivated by epinephrine in the mouse muscles. Nevertheless, it is well established that insulin activates glycogen synthase in isolated muscles incubated without epinephrine (30Lawrence Jr., J.C. Annu. Rev. Physiol. 1992; 54: 177-193Crossref PubMed Scopus (55) Google Scholar, 31Villar-Palasi C. Larner J. Biochem. Biophys. Acta. 1960; 39: 171-173Crossref PubMed Scopus (96) Google Scholar), and that the effect of insulin is not inhibited by the β-adrenergic receptor antagonist, propranolol (29Dietz M.R. Chiasson J.L. 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Glycogen debranching enzyme
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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 or 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.
Glycogen branching enzyme
Phosphorylase kinase
Glycogen debranching enzyme
Glycogenesis
Glucose 6-phosphate
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