Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells.
The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R2= 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3beta activity by 30-50% and activated more than 4-fold particulate protein phosphatase- activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3beta and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3beta. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70(s6k) and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.
Syed
N A
NA
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Khandelwal
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RL
D000730:Androstadienes / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000096:biosynthesis / Q000378:metabolism; D006006:Glycogen Synthase / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010080:Oxazoles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D006005:Phosphorylases / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D000077191:Wortmannin
D000730:Androstadienes; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D005419:Flavonoids; D005947:Glucose; D006003:Glycogen; D006006:Glycogen Synthase; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D007328:Insulin; D008387:Marine Toxins; D020928:Mitogen-Activated Protein Kinases; D019319:Okadaic Acid; D010080:Oxazoles; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D006005:Phosphorylases; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D020123:Sirolimus; D000077191:Wortmannin; C059041:calyculin A; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one