Activation of glycogen synthase by insulin in rat adipocytes. Evidence of hormonal stimulation of multisite dephosphorylation by glucose transport-dependent and -independent pathways.

Adipocytes were incubated with [32P]phosphate to achieve steady state labeling of glycogen synthase. The enzyme was then rapidly immunoprecipitated and subjected to electrophoresis on polyacrylamide slab gels in the presence of sodium dodecyl sulfate. The 32P-labeled glycogen synthase had an apparent molecular weight ( Mapp ) equal to 90,000. All of the [32P]phosphate could be recovered in two cyanogen bromide fragments. The larger fragment, CB-2 ( Mapp = 28,000), contained about five times more [32P]phosphate than the smaller fragment, CB-1 ( Mapp = 15,500). Insulin increased the activity ratio (-glucose-6-P/+glucose-6-P) of glycogen synthase from 0.12 to 0.26, but did not decrease the amount of [32P]phosphate in the enzyme. However, insulin promoted the formation of species of CB-2 of lower Mapp , suggesting dephosphorylation of sites that affected the electrophoretic mobility of the fragment. Glucose did not affect the mobility of CB-2, but slightly increased the activity ratio and decreased the [32P] phosphate by approximately 20%. With insulin plus glucose, the increase in activity ratio was much greater than the additive effects of either agent alone. The combination decreased the [32P]phosphate in each cyanogen bromide fragment by approximately 60%, indicating that the synergistic activation was due to enhanced dephosphorylation of multiple sites. 2-Deoxyglucose also promoted dephosphorylation of glycogen synthase, decreasing the 32P content of CB-1 and CB-2 by approximately 40% each. 3-O-Methylglucose was without effect. The results presented suggest that the activation of glycogen synthase by insulin via a glucose transport-dependent pathway may involve increased intracellular glucose-6-P which promotes dephosphorylation of sites in both CB-1 and CB-2. Activation by a glucose transport-independent pathway appears to be confined to sites located in CB-2.