INSULIN-SPECIFIC ACTIVATION OF S6 KINASE AND ITS DESENSITIZATION IN CULTURED RAT VASCULAR SMOOTH-MUSCLE CELLS

To elucidate the role of hyperinsulinemia in the development of athero sclerosis, we evaluated insulin-specific signaling in cultured vascula r smooth muscle cells (SMCs) and its desensitization by continuous exp osure to insulin. The concentration of unlabeled insulin that inhibite d specific [A14-I-125]-insulin binding by 50% (IC50) was 0.33 +/- 0.02 nM, which was 100 times less than the IC50 of unlabeled IGF-I. For [I -125]-IGF-I binding, the IC50 of unlabeled IGF-I was found to be 6.6 /- 0.88 nM, which was 100 times less than the IC50 of unlabeled insuli n. The binding capacities for insulin and IGF-I were found to be 1.28 +/- 0.86 and 100 +/- 170 fmol/0.5 mg protein, respectively. Autophosph orylation of the beta-subunit of the insulin receptor was stimulated a t above 0.17 nM (24 mu U/ml) insulin. Insulin concentrations exceeding 1 nM significantly activated the S6 kinase in a dose-dependent manner . In contrast, 10 nM insulin did not activate MAP kinase nor [H-3]thym idine incorporation into DNA, while both were activated by 38% and 44% with 1 mu M insulin and by 52% and 67% with 10 nM IGF-I, respectively . By pre-exposing cells to 10 nM insulin for 12 h, the binding capacit y for insulin decreased by 34% (P < 0.05), and activation of S6 kinase by insulin almost disappeared, while both IGF-1 binding and the activ ation of S6 kinase by IGF-I were not affected. These results indicate the existence of insulin receptor-specific activation of S6 kinase in SMCs in the presence of physiological concentrations of insulin and th at insulin signaling can be desensitized by continuous exposure to 10 nM insulin concentration.