Chronic Hyperinsulinism Induced Down-regulation of Insulin Post-Receptor Signaling Transduction in Hep G2 Cells

摘要

To study the regulatory effect of acute and chronic insulin treatment on insulin post-receptor signaling transduction pathway in a human hepatoma cell line (Hep G2), Hep G2 cells were incubated in the presence or absence of insulin with different concentrations in serum free media for 16 h and then stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptor β-subunit (IRβ), insulin receptor substrate-1 (IRS-1) and p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) were determined in total cell lysates by Western-immunoblot. Phosphorylat-ed proteins IRβ, IRS-1 and interaction of PI 3-kinase with IRS-1 were determined by immunopre-cipitation. Results showed that 1-min insulin stimulation rapidly induced tyrosine phosphorylation of IRβ and IRS-1, which in turn, resulting in association of PI 3-kinase with IRS-1. 1 - 100 nmol/ L chronic insulin treatment induced a dose-dependent decrease in the protein level of IRβ and a slight decrease in the protein level of IRS-1. There was, a more marked reduction in the phosphorylation of IRβ, IRS-1, reaching a nadir of 22% (P<0. 01) and 15 % (P<0. 01) of control levels, respectively, after 16 h treatment with 100 nmol/L insulin. The association between IRS-1 and PI 3-kinase was decreased by 66 % (P<0. 01). There was no significant change in PI 3-kinase protein levels. These data suggest that chronic insulin treatment can induce alterations of IRβ, IRS-1 and PI 3-kinase three early steps in insulin action, which contributes significantly to insulin resistance, and may account for desensitization of insulin action.