Chronic hyperinsulinism induced down-regulation of insulin post-receptor signaling transduction in Hep G2 cells.
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 beta-subunit (IR beta), 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. Phosphorylated proteins IR beta, IRS-1 and interaction of PI 3-kinase with IRS-1 were determined by immunoprecipitation. Results showed that 1-min insulin stimulation rapidly induced tyrosine phosphorylation of IR beta 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 IR beta and a slight decrease in the protein level of IRS-1. There was a more marked reduction in the phosphorylation of IR beta, 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 IR beta, 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.
Yuan
Li
L
Department of Endocrinology and Metabolism, University-Hospital Heidelberg, Heidelberg 69115, Germany.
Ziegler
Reinhard
R
Hamann
Andreas
A
D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D015536:Down-Regulation; D006801:Humans; D006946:Hyperinsulinism / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D008113:Liver Neoplasms / Q000473:pathology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured
C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D011972:Receptor, Insulin