Insulin receptor phosphorylation in intact adipocytes and in a cell-free system

Abstract

The effect of insulin on the phosphorylation of the insulin receptor was studied using isolated rat adipocytes. Rat adipocytes were labeled with [ 32 p] orthophosphate, treated with insulin for 15 min and solubilized in 1% Triton. The insulin receptor was then enriched by affinity chromatography on wheat germ lectin and identified by immunoprecipitation and NaDodSO 4 /acrylamide gel electrophoresis. Insulin (10 −8 M, 10 −7 M) stimulated the phosphorylation of the 95K subunit of the receptor 2 to 5-fold. This effect of insulin could also be shown in a cell-free system, i.e., if insulin was added after solubilization and wheat germ purification. In this cell-free system, insulin (3×10 −10 M to 10 −7 M) increased the incorporation of [ 32 p] from [γ- 32 p]-ATP into the 95K subunit of the receptor up to 16-fold and caused also a minor phosphorylation of the 135K subunit. By contrast, cyclic AMP (5×10 −6 M) and dibutyryl-cyclic AMP (10 −4 M) had no effect on basal or insulin stimulated receptor phosphorylation in the broken cell. These data suggest that insulin stimulation of phosphorylation of the insulin receptor is an early step in insulin action in adipocytes and is controlled by a non-cyclic AMP-dependent kinase.