Hepatic Glycogen Synthesis Is Highly Sensitive to Phosphorylase Activity
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m101454200
ISSN1083-351X
AutoresSusan Aiston, Laura J. Hampson, Anna M. Gómèz‐Foix, Joan J. Guinovart, Loranne Agius,
Tópico(s)Glycogen Storage Diseases and Myoclonus
ResumoWe used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S0.5) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states. We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S0.5) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states. glucose 6-phosphate The liver maintains blood glucose homeostasis by uptake of glucose in the absorptive state, which is converted to glycogen and triacylglycerol, and by production of glucose from glycogenolysis and gluconeogenesis in the postabsorptive state. Glycogen phosphorylase catalyzes the first step in glycogen degradation (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar). It is regulated by allosteric mechanisms and by phosphorylation of Ser-14 by phosphorylase kinase. The dephosphorylated form (phosphorylase b) is less active than the phosphorylated form (phosphorylase a) (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar). Hormones that raise cAMP or cytoplasmic Ca2+ favor the formation of phosphorylase a, whereas insulin and leptin have the converse effect (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar, 2Aiston S. Agius L. Diabetes. 1999; 48: 15-20Crossref PubMed Scopus (99) Google Scholar). Phosphorylase is a dimer and exists in two conformations, an inactive T-state (tight) and an active R-state (relaxed). The R-state is favored by substrate, phosphorylation, and allosteric activators (AMP), whereas the T-state is favored by dephosphorylation and by inhibitors (glucose, glucose 6-P,1 and caffeine) (3Browner M.F. Fletterick R.J. Trends Biochem. Sci. 1992; 17: 66-71Abstract Full Text PDF PubMed Scopus (67) Google Scholar, 4Johnson L.N. FASEB J. 1992; 6: 2274-2282Crossref PubMed Scopus (256) Google Scholar). The liver isoform, unlike the muscle isoform, is more tightly controlled by phosphorylation than by allosteric regulation (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar).Two sets of evidence suggest that liver phosphorylase is a candidate pharmacological target for controlling hyperglycemia in diabetes. Firstly, a potent inhibitor of liver phosphorylase a that acts synergistically with glucose lowers blood glucose in the leptin-deficient ob/ob mouse (5Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.R. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1776-1781Crossref PubMed Scopus (213) Google Scholar). Secondly, the activity of phosphorylase is elevated in the leptin receptor-defective db/db mouse (6Board M. Hadwen M. Johnson L.N. Eur. J. Biochem. 1995; 228: 753-761Crossref PubMed Scopus (34) Google Scholar) and Zucker fa/fa rat (7Aiston S. Peak M. Agius L. Diabetologia. 2000; 43: 589-597Crossref PubMed Scopus (26) Google Scholar), which are widely used as animal models for human type 2 diabetes and insulin resistance.Recent studies have applied metabolic control analysis (8Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar) to determine how control of glycogen synthesis is shared between diverse sites. In skeletal muscle, a high degree of control resides at the glucose transport/phosphorylation sites (9Shulman R.G. Bloch G. Rothman D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8535-8542Crossref PubMed Scopus (142) Google Scholar, 10Jucker B.M. Barucci N. Shulman G.I. Am. J. Physiol. 1999; 277: E505-E512PubMed Google Scholar, 11Schulz A.R. Arch. Biochem. Biophys. 1998; 353: 172-180Crossref PubMed Scopus (14) Google Scholar), whereas in liver, a high degree of control is exerted by glucokinase in conjunction with its regulatory protein (12Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 13Aiston S. Trinh K. Lange A.J. Newgard C.B. Agius L. J. Biol. Chem. 1999; 274: 24559-24566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14De la Iglesia N. Mukhtar M. Seoane J. Guinovart J.J. Agius L. J. Biol. Chem. 2000; 275: 10597-10603Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Schulz (11Schulz A.R. Arch. Biochem. Biophys. 1998; 353: 172-180Crossref PubMed Scopus (14) Google Scholar) developed a minimal model for glycogen synthesis in muscle that demonstrates that as glycogenolysis increases, the distribution of control shifts to the terminal enzymes in the glycogen synthesis pathway. Mathematical models enable identification of conditions that alter the distribution of control but do not allow a quantitative estimate of the degree of control exerted by specific sites. There has been no experimental analysis of the control exerted by phosphorylase on glycogen synthesis. In this study, we used a specific inhibitor of phosphorylase (5Martin W.H. Hoover D.J. Armento S.J. Stock I.A. McPherson R.R. Danley D.E. Stevenson R.W. Barrett E.J. Treadway J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1776-1781Crossref PubMed Scopus (213) Google Scholar) and titrated phosphorylase overexpression by adenovirus-mediated gene transfer of the muscle isoform of phosphorylase (15Gomez-Foix A.M. Coats W.S. Baque S. Alam T. Gerard R.D. Newgard C.B. J. Biol. Chem. 1992; 267: 25129-25134Abstract Full Text PDF PubMed Google Scholar) to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes. Muscle phosphorylase b, unlike liver phosphorylase b (16Stalmans W. Gevers G. Biochem. J. 1981; 200: 327-336Crossref PubMed Scopus (33) Google Scholar), is very sensitive to activation by AMP. We took advantage of this property of muscle phosphorylase to increase phosphorylase catalytic activity in hepatocytes independently of the phosphorylation state of the hepatocyte.DISCUSSIONMetabolic control analysis is a powerful analytical approach to describe how control of flux through a metabolic pathway is distributed among enzymes that have direct or indirect effects on pathway flux (8Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar). The flux control coefficient of an enzyme is a measure of the sensitivity of metabolic flux to small changes in enzyme activity or concentration. Heinrich and Rapoport (25Heinrich R. Rapoport T.A. Eur. J. Biochem. 1974; 42: 89-95Crossref PubMed Scopus (993) Google Scholar) defined the control strength in terms of the fractional change in flux that results from a fractional change in enzyme activity, whereas Kacser and Burns (26Kacser H. Burns J.A. Symp. Soc. Exp. Biol. 1973; 27: 65-104PubMed Google Scholar) defined the sensitivity coefficient in terms of the fractional change in flux that results from a fractional change in enzyme concentration. If the enzyme rate is proportional to the enzyme concentration, then the two definitions are equivalent. However, when an enzyme is regulated by covalent modification, as in the case of liver phosphorylase (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar), then the relevant coefficient for determining the degree of control on pathway flux is expressed in terms of the activity of the enzyme. The control coefficient usually has a value between zero (minimum control) and unity (high control) and is either positive or negative, depending on whether the enzyme stimulates or inhibits pathway flux. Enzymes with control coefficients greater than unity are considered to be rare (23Fell D.A. Biochem. J. 1992; 286: 313-330Crossref PubMed Scopus (632) Google Scholar). In muscle, a high degree of control of glucose utilization lies at or before glucose phosphorylation (9Shulman R.G. Bloch G. Rothman D.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8535-8542Crossref PubMed Scopus (142) Google Scholar, 10Jucker B.M. Barucci N. Shulman G.I. Am. J. Physiol. 1999; 277: E505-E512PubMed Google Scholar, 11Schulz A.R. Arch. Biochem. Biophys. 1998; 353: 172-180Crossref PubMed Scopus (14) Google Scholar), and in liver high degree of control lies at glucose phosphorylation (12Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 13Aiston S. Trinh K. Lange A.J. Newgard C.B. Agius L. J. Biol. Chem. 1999; 274: 24559-24566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14De la Iglesia N. Mukhtar M. Seoane J. Guinovart J.J. Agius L. J. Biol. Chem. 2000; 275: 10597-10603Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In hepatocytes, glucokinase has a high control coefficient on glycogen synthesis, which is glucose-dependent. This is explained by the unique compartmentation of glucokinase that involves glucose-dependent partitioning of the enzyme between a free active state and an inactive state bound to its regulatory protein (12Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar,14De la Iglesia N. Mukhtar M. Seoane J. Guinovart J.J. Agius L. J. Biol. Chem. 2000; 275: 10597-10603Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The stimulation of glycogenic flux by an increase in glucokinase activity by either translocation (19Agius L. Biochem. J. 1997; 325: 667-673Crossref PubMed Scopus (21) Google Scholar) or enzyme overexpression (27Seoane J. Gomez-Foix A.M. O'Doherty M. Gomez-Ara C. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1996; 271: 23756-23760Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) is at least in part explained by the increase in glucose 6-P concentration (11Schulz A.R. Arch. Biochem. Biophys. 1998; 353: 172-180Crossref PubMed Scopus (14) Google Scholar), the product of the glucokinase-catalyzed reaction, which is a potent activator of glycogen synthase (28Guinovart J.J. Gómez-Foix A.M. Seoane J. Fernandez-Novell J.M. Bellido D. Vilaro S. Biochem. Soc. Trans. 1997; 25: 157-160Crossref PubMed Scopus (17) Google Scholar). Glucose 6-phosphatase, which lowers the concentration of glucose 6-P in hepatocytes, has a negative control coefficient on glycogen synthesis (13Aiston S. Trinh K. Lange A.J. Newgard C.B. Agius L. J. Biol. Chem. 1999; 274: 24559-24566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, in contrast with glucokinase, the control coefficient of glucose 6-phosphatase is much lower than unity and is glucose-independent, confirming that the high control coefficient of glucokinase and its glucose dependence are best explained by the subcellular compartmentation of glucokinase and its association with its regulatory protein (14De la Iglesia N. Mukhtar M. Seoane J. Guinovart J.J. Agius L. J. Biol. Chem. 2000; 275: 10597-10603Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The recent findings that stimulation of glycogen synthesis by leptin is associated with inactivation of phosphorylase (2Aiston S. Agius L. Diabetes. 1999; 48: 15-20Crossref PubMed Scopus (99) Google Scholar) and that impaired glycogen synthesis in hepatocytes from fa/fa rats is associated with elevated phosphorylase activity (7Aiston S. Peak M. Agius L. Diabetologia. 2000; 43: 589-597Crossref PubMed Scopus (26) Google Scholar) raised the question of the degree of control of glycogen synthesis by phosphorylase activity. In this study, we demonstrate that phosphorylase has a very high negative flux control coefficient on glycogen synthesis, based on two independent approaches (titration with a specific phosphorylase inhibitor and expression of the muscle isoform of phosphorylase).Three key findings emerged from the studies with phosphorylase inhibitor CP-91149. First, it caused time-dependent inactivation of phosphorylase a and sequential activation of glycogen synthase. This is analogous to the mechanism proposed by Stalmans et al. (22Stalmans W. De Wulf H. Hue L. Hers H.-G. Eur. J. Biochem. 1974; 41: 127-134Crossref PubMed Scopus (147) Google Scholar) for the glucose-induced inactivation of phosphorylase and sequential activation of glycogen synthase. Binding of glucose to phosphorylase a causes a conformational change (R-state to T-state) that renders the enzyme a better substrate for dephosphorylation by protein phosphatase-1. Glucose thus favors the conversion of phosphorylase a to phosphorylase b. Because phosphorylase a is a potent inhibitor of glycogen synthase phosphatase by binding to the C terminus of the liver-specific glycogen-targeting subunit (GL) of protein phosphatase-1 (29Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T.W. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (139) Google Scholar, 30Armstrong C.G. Doherty M.J. Cohen P.T.W. Biochem. J. 1998; 336: 699-704Crossref PubMed Scopus (80) Google Scholar), the decrease in phosphorylase a alleviates the inhibition of synthase phosphatase. This results in a delayed activation of glycogen synthase relative to the inactivation of phosphorylase, which has been described as the "sequential activation of synthase" (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar, 22Stalmans W. De Wulf H. Hue L. Hers H.-G. Eur. J. Biochem. 1974; 41: 127-134Crossref PubMed Scopus (147) Google Scholar). The present results support a model whereby CP-91149 favors the T-conformation of phosphorylase and thereby causes the inactivation of phosphorylase and sequential activation of synthase. Second, the phosphorylase inhibitor markedly increases the sensitivity of glycogen synthesis to glucose (S0.5, 19versus 26 mm) by causing a greater fold stimulation of glycogen synthesis at 5 mm glucose than at 20 mm glucose. This is consistent with the higher activity of phosphorylase a at low glucose and the greater fractional inactivation by the inhibitor at low glucose. The rate of glucose phosphorylation in hepatocytes is a sigmoidal function with respect to [glucose] but with a higher S0.5 for glucose than can be explained by glucokinase kinetics (20 versus 9 mm) (31Bontemps F. Hue L. Hers H.-G. Biochem. J. 1978; 174: 603-611Crossref PubMed Scopus (143) Google Scholar). This higher S0.5 for glucose phosphorylation in the intact cell is explained by the glucokinase regulatory protein, which functions as a competitive inhibitor with respect to glucose and as a nuclear receptor for the enzyme (32Van Schaftingen E. Eur. J. Biochem. 1989; 179: 179-184Crossref PubMed Scopus (160) Google Scholar, 33Agius L. Adv. Enzyme Regul. 1998; 38: 303-311Crossref PubMed Scopus (61) Google Scholar). Glycogen synthesis has a higher S0.5 for glucose than glucose phosphorylation in intact cells (33Agius L. Adv. Enzyme Regul. 1998; 38: 303-311Crossref PubMed Scopus (61) Google Scholar). The inhibitor studies show that phosphorylase is a major component of the mechanism that accounts for the difference in glucose saturation curves of glycogen synthesis and glucose phosphorylation. Third, the titrations with increasing concentration of inhibitor show a large fractional increase in glycogen synthesis for a corresponding inactivation of phosphorylase, with a control coefficient greater than unity.We show in this study that muscle phosphorylase expressed using recombinant adenovirus is a powerful tool to alter the catalytic activity of phosphorylase in hepatocytes independently of the cAMP status and/or the phosphorylation state of the cell. In the absence of glucagon, muscle phosphorylase is expressed mainly in the unphosphorylated (b) form. However, it is partially catalytically active at physiological concentrations of AMP and ATP, unlike liver phosphorylase b (16Stalmans W. Gevers G. Biochem. J. 1981; 200: 327-336Crossref PubMed Scopus (33) Google Scholar), and this enables determination of the flux control coefficient of phosphorylase in defined substrate and hormone conditions. By using low titers of adenovirus that result in small fractional changes in phosphorylase activity and a short culture time after treatment with the adenovirus (<20 h), secondary changes in gene expression are minimized. Two metabolic effects of muscle phosphorylase expression were noted, inactivation of glycogen synthase, which was progressive with enzyme expression, and a decrease in glycogen synthesis, which reached a plateau at low levels of phosphorylase expression. The lack of effect of AdCMV-MGP treatment on ATP, glucose 6-P, glucokinase activity, glycolysis, or conversion of glucose to triacylglycerol indicates that at the viral titers and incubation times used, the effects of muscle phosphorylase expression are confined to glycogen metabolism. The inactivation of synthase and lack of effect on triacylglycerol metabolism contrast with findings on phosphorylase overexpression in muscle cultures (34Baqué S. Guinovart J.J. Gómez-Foix A.M. J. Biol. Chem. 1996; 271: 2594-2598Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). This may represent a tissue difference, or it may be related to the lower levels of phosphorylase overexpression used in the present study.The inhibition of glycogen synthase by muscle phosphorylase overexpression is of interest because it was not associated with either a change in glucose 6-P, an activator of synthase phosphatase (28Guinovart J.J. Gómez-Foix A.M. Seoane J. Fernandez-Novell J.M. Bellido D. Vilaro S. Biochem. Soc. Trans. 1997; 25: 157-160Crossref PubMed Scopus (17) Google Scholar), or an increase in phosphorylase a, a potent allosteric inhibitor of synthase phosphatase (1Bollen M. Keppens S. Stalmans W. Biochem. J. 1998; 336: 19-31Crossref PubMed Scopus (320) Google Scholar). Two types of mechanism can be considered, involving either catalytic activity of phosphorylase or an effect of the protein independent of catalytic activity. Catalytic activity of muscle phosphorylase may cause dissociation of glycogen synthase from glycogen, an allosteric effector of the enzyme (35Solling H. Eur. J. Biochem. 1979; 94: 231-242Crossref PubMed Scopus (16) Google Scholar), or from glycogenin, which is also a substrate for phosphorylase (36Cao Y. Skurat A.V. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1993; 268: 21717-21721Abstract Full Text PDF PubMed Google Scholar), or it may cause dissociation of a glycogen-targeting subunit of protein phosphatase-1 such as GL or PTG (37Newgard C.B. Brady M.J. O'Doherty R.M. Saltiel A.R. Diabetes. 2000; 49: 1967-1977Crossref PubMed Scopus (148) Google Scholar) from glycogen. Muscle phosphorylase b may bind to a glycogen-targeting unit and cause inactivation of synthase phosphatase activity either through an allosteric effect (29Doherty M.J. Moorhead G. Morrice N. Cohen P. Cohen P.T.W. FEBS Lett. 1995; 375: 294-298Crossref PubMed Scopus (139) Google Scholar, 30Armstrong C.G. Doherty M.J. Cohen P.T.W. Biochem. J. 1998; 336: 699-704Crossref PubMed Scopus (80) Google Scholar) or by competitive binding with glycogen synthase (38Fong N.M. Jensen T.C. Shah A.S. Parekh N.N. Saltiel A.R. Brady M.J. J. Biol. Chem. 2000; 275: 35034-35039Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). GL has a high-affinity site for phosphorylase a as well as a substrate site, whereas PTG has a single binding site for glycogen synthase and phosphorylase (38Fong N.M. Jensen T.C. Shah A.S. Parekh N.N. Saltiel A.R. Brady M.J. J. Biol. Chem. 2000; 275: 35034-35039Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Inhibition of synthase phosphatase by phosphorylase b has been demonstrated, but with much lower affinity than that for phosphorylase a (39Alemany S. Cohen P. FEBS Lett. 1986; 198: 194-202Crossref PubMed Scopus (69) Google Scholar). The experiments with CP-91149, which counteracted the inhibitory effects of low levels of muscle phosphorylase overexpression on glycogen synthesis and glycogen synthase, suggest that the catalytic activity of phosphorylase accounts for the inhibition of synthase by low levels of phosphorylase expression. However, an additional protein effect independent of catalytic activity at higher levels of muscle phosphorylase expression cannot be ruled out.Two points are of interest with regard to the inhibition of glycogen synthesis by phosphorylase overexpression. First, the high control coefficient is observed at both 10 and 25 mm glucose and is also observed in the presence of insulin. This contrasts with the strong glucose dependence of the control coefficient of glucokinase (12Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 13Aiston S. Trinh K. Lange A.J. Newgard C.B. Agius L. J. Biol. Chem. 1999; 274: 24559-24566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Second, unlike the control coefficients of glucokinase or its regulatory protein (positive and negative, respectively), which are sustained over a wide range of protein overexpression (2–3-fold above endogenous activity (13Aiston S. Trinh K. Lange A.J. Newgard C.B. Agius L. J. Biol. Chem. 1999; 274: 24559-24566Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar)), inhibition of glycogen synthesis by muscle phosphorylase reaches a plateau (50% inhibition) at low levels of phosphorylase overexpression (<30% above endogenous activity), with no further inhibition at higher protein expression. If the inhibition of [14C]glucose incorporation into glycogen were due to increased degradation of glycogen rather than inhibition of synthesis, then a progressive or a linear response as a function of phosphorylase activity would be expected, as is observed for inactivation of synthase. A more plausible explanation for the sharp inhibition of glycogen synthesis that reaches a plateau at fairly low activities of phosphorylase is that it represents inhibition of synthesis and that there are two compartments of glycogen synthesis, only one of which is sensitive to inhibition by phosphorylase.The lack of correlation between the rate of glycogen synthesis and the activity of glycogen synthase at high phosphorylase overexpression could be explained by compartmentation of glycogen synthase (40Fernandez-Novell J.M. Bellido D. Vilaro S. Guinovart J.J. Biochem. J. 1997; 321: 227-231Crossref PubMed Scopus (66) Google Scholar). The rate of glycogen synthesis in hepatocytes may depend on the fraction of glycogen synthase that is associated with glycogen or the protein primer, glycogenin. An increase in phosphorylase activity may cause dissociation of synthase from glycogenin, which is a substrate for phosphorylase (36Cao Y. Skurat A.V. DePaoli-Roach A.A. Roach P.J. J. Biol. Chem. 1993; 268: 21717-21721Abstract Full Text PDF PubMed Google Scholar). By analogy with the high flux control coefficient of glucokinase on glycogen synthesis, which is explained by the subcellular compartmentation of glucokinase (12Agius L. Peak M. Newgard C.B. Gomez-Foix A.M. Guinovart J.J. J. Biol. Chem. 1996; 271: 30479-30486Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), the high flux control coefficient of phosphorylase on glycogen synthesis could be due in part to an effect of phosphorylase catalytic activity on the subcellular compartmentation of glycogen synthase. This hypothesis is consistent with both the higher flux control coefficient of phosphorylase on glycogen synthesis relative to the control coefficient on glycogen synthase and the effect of glucokinase overexpression, which lowers the flux control coefficient of phosphorylase on glycogen synthesis but not on synthase. Overexpression of glucokinase increases the hepatocyte glucose 6-P content (27Seoane J. Gomez-Foix A.M. O'Doherty M. Gomez-Ara C. Newgard C.B. Guinovart J.J. J. Biol. Chem. 1996; 271: 23756-23760Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and glucose 6-P affects the translocation of glycogen synthase (41Fernandez-Novell J.M. Arino J. Vilaro S. Bellido D. Guinovart J.J. Biochem. J. 1992; 288: 497-501Crossref PubMed Scopus (46) Google Scholar). A difference in subcellular compartmentation of glycogen synthase in cells overexpressing glucokinase could therefore explain the lower control coefficient and could also explain the lower fractional inhibition of glycogen synthesis by phosphorylase overexpression in cells with elevated glucokinase activity.One of the advantages of metabolic control analysis is that by providing a quantitative estimate for the degree of control exerted by an enzyme, it enables the study of how this control changes in different physiological or pathological states (8Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar). This study shows that glucokinase overexpression by 60% above endogenous activity lowers the control coefficient of phosphorylase on glycogen synthesis by 50%. 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Biol. 2000; 7: 677-682Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar) could be of therapeutic benefit for inhibiting hepatic glycogenolysis in type 2 diabetes. The high control coefficient of phosphorylase on glycogen synthesis suggests that phosphorylase inhibitors would also be highly effective in promoting hepatic glycogen synthesis in the absorptive state and that the
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