Differential effects of calyculin A and okadaic acid on the glucose-induced regulation of glycogen synthease and phosphorylase activities in cultured hepatocytes

Abstract

The effects of the phosphatase inhibitors calyculin A and okadaic acid were investigated to determine the roles of protein phosphatases type 1 and 2A in the regulation of the activities of glycogen synthase and phosphorylase by glucose in a primary culture of hepatocytes. Glycogen synthesis, as measured by the incorporation of labelled glucose into glycogen, was inhibited in a dose-dependent manner by calyculin A (IC 50 = 2.2 nM) and okadaic acid (IC 50 = 14 nM). Glucose-induced activation of glycogen synthase was inhibited by calyculin A and okadaic acid with IC 50 values of 3.7 nM and 90 nM, respectively. Phosphorylase was simultaneously activated by these inhibitors with calyculin A again being more active ( P < 0.001) than okadaic acid. The differing potencies ( P < 0.001) of these inhibitors on the activities of glycogen synthase and phosphorylase were also observed with varying concentrations of glucose (5.6–60 mM) in the medium and at different incubation periods upto 120 min. It has been previously shown that both inhibitors inhibit protein phosphatase-2A with equal potency and calyculin A is a more potent inhibitor of protein phosphatase-1 than okadaic acid. Heat- and proteinase-treated cytosolic fractions from hepatocytes incubated with calyculin A and okadaic acid showed similar differential inhibitory activities towards purified types 1 and 2-A protein phosphatases. Hence, these data provide further evidence that protein phosphatase type-1 plays a major role in the control of glycogen synthesis by regulating the activities of glycogen synthase and phosphorylase.