STIMULATION OF PROTEIN PHOSPHATASE-1 ACTIVITY BY INSULIN IN RAT ADIPOCYTES - EVALUATION OF THE ROLE OF MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY

In this study, we examined the distribution of protein serine/threonine phosphatase-1 (PP-1) and analyzed the effect of insulin on PP-1 and its mechanism of activation in freshly isolated rat adipocytes. The adipocyte particulate fraction (PF) constituted approximate to 80% of cellular PP-1 activity, while PP-2A was entirely cytosolic. Insulin rapidly stimulated PF PP-1 in a time- and dose-dependent manner (maximum stimulation at 5 min with 4 nM insulin). Immunoprecipitation of PF with an antibody against the site-1 sequence of rabbit skeletal muscle glycogen-associated PP-1 (PP-1G) subunit indicated that approximate to 40% of adipocyte PP-1 activity was due to PP-1G form of the enzyme, Insulin stimulated PP-1G (120% over basal levels) without affecting the other forms of PP-1 in the PF. Insulin activation of PP-1 was accompanied by > 2-fold increase in the phosphorylation state of the 160-kDa regulatory subunit of PP-1. Stimulation of p21(Ras)/mitogen-activated protein kinase pathway (MAP) with GTP analogues also resulted in stimulation of PP-1 similar to insulin, The insulin effect on MAP kinase and PP-1 activation was blocked by a GTP antagonist, guanyl-5'-yl thiophosphate. The inhibitors of MAP kinase activation (viz. cAMP agonists, SpcAMP and ML-9) also blocked PP-1 stimulation by insulin. The time course of MAP kinase activation preceded the phosphorylation of PP-1 regulatory subunit and PP-1 activation, We conclude that insulin rapidly activates a membrane-associated PP-1 in adipocytes, which may be similar to rabbit skeletal muscle PP-1G, and the activation is mediated by p21(Ras)/MAP kinase pathway.