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 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 beta-subunit of the insulin receptor was stimulated at above 0.17 nM (24 microU/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 microM 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-I binding and the activation of S6 kinase by IGF-I were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)
Takagi
Y
Y
Third Department of Medicine, Shiga University of Medical Science, Japan.
Kashiwagi
A
A
Tanaka
Y
Y
Maegawa
H
H
Shigeta
Y
Y
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002455:Cell Division; D002478:Cells, Cultured; D015536:Down-Regulation; D004789:Enzyme Activation; D007328:Insulin / Q000378:metabolism / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases
D007328:Insulin; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases