Insulin-specific activation of S6 kinase and its desensitization in cultured rat vascular smooth muscle cells Original Research Article

To elucidate the role of hyperinsulinemia in the development of atherosclerosis, we evaluated insulin-specific signaling in cultured vascular smooth muscle cells (SMCs) and its desensitization by continuous exposure to insulin. The concentration of unlabeled insulin that inhibited specific [A14-125I]-insulin binding by 50% (IC50) was 0.33 ± 0.02 nM, which was 100 times less than the IC50 of unlabeled IGF-I. For [125I]-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 insulin. The binding capacities for insulin and IGF-I were found to be 1.28 ± 0.86 and 1200 ± 170 fmol/0.5 mg protein, respectively. Autophosphorylation of the β-subunit of the insulin receptor was stimulated at above 0.17 nM (24 μU/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 [3H]thymidine incorporation into DNA, while both were activated by 38% and 44% with 1 μM 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 capacity for insulin decreased by 34% (P < 0.05), and activation of S6 kinase by insulin almost disappeared, while both IGF-1 binding and the activation 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 that insulin signaling can be desensitized by continuous exposure to 10 nM insulin concentration.