STUDIES ON INSULIN-STIMULATED PHOSPHORYLATION OF ACETYL-COA CARBOXYLASE, ATP CITRATE LYASE AND OTHER PROTEINS IN RAT EPIDIDYMAL ADIPOSE-TISSUE - EVIDENCE FOR ACTIVATION OF A CYCLIC AMP-INDEPENDENT PROTEIN-KINASE

Protein kinase activity in high speed supernatant fractions prepared from rat epididymal adipose tissue previously incubated in the absence or presence of insulin was investigated by following the incorporation of 32P from [.gamma.-32P]ATP into phosphoproteins separated by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis. Incorporation of 32P into several endogenous proteins in the supernatant fractions from insulin-treated tissue was significantly increased. These included acetyl CoA carboxylase and ATP citrate lyase (which exhibit increased phosphorylation within fat cells exposed to insulin), together with 2 unknown proteins of subunit MW 78,000 and 43,000. The protein kinase activity increased by insulin was distinct from cAMP-dependent protein kinase, was not dependent on Ca2+ and was not appreciably affected by dialysis or gel filtration. The rate of phosphorylation of added purified fat cell acetyl CoA carboxylase and ATP citrate lyase was also increased by 60-90% in high speed supernatant fractions prepared from insulin-treated tissue. No evidence for any persistent changes in phosphoprotein phosphatase activity was found. Insulin action on acetyl-CoA carboxylase, ATP citrate lyase and other intracellular proteins exhibiting increased phosphorylation involves an increase in cAMP-independent protein kinase activity in the cytoplasm. The possibility that the increase reflects translocation from the plasma membrane, perhaps after phosphorylation by the protein tyrosine kinase associated with insulin receptors, is discussed.