Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 inHepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen met abolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on i nsulin concentration in the medium. Insulin caused a maximum of 65% decreas e in glycogen phosphorylase 'a' and 110% increase in glycogen synthase acti vities in 5 min. Although significant changes in enzyme activities were obs erved with as low as 0.5 nM insulin level, the maximum effects were observe d with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R-2 = 0 .66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphoryla se 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' b y 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, insul in decreased glycogen synthase kinase-3 beta activity by 30-50% and activat ed more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 beta and activation of P KB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, o kadaic 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 activ ating PKB and PP-1G and inactivating GSK-3 beta. On the other hand, inactiv ation of phosphorylase by insulin is mediated through the PI-3 kinase pathw ay involving a rapamycin-sensitive p70(s6k) and PP-1G. These experiments de monstrate that insulin regulates glycogen phosphorylase and glycogen syntha se through (i) a common signaling pathway at least up to PI-3 kinase and bi furcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.