title: Metformin modulates insulin post-receptor signaling transduction in chronically insulin-treated Hep G2 cells.
abstract: OBJECTIVE
  To study the effect of chronic insulin treatment on insulin post-receptor signaling transduction and whether the effects of metformin are transmitted throughout the cascade of insulin signaling intermediates in a human hepatoma cell line (Hep G2).
  
  
  METHODS
  Hep G2 cells were incubated in serum free media containing either insulin 100 nmol/L or insulin 100 nmol/L plus different concentrations (0.01-10 mmol/L) of metformin for 16 h and then were stimulated with insulin 100 nmol/L for 1 min.
  
  
  RESULTS
  Chronic treatment of insulin 100 nmol/L induced a significant reduction in the phosphorylation and protein expression of IR?, IRS1 and IRS2, which therefore resulted in a downregulation of association of PI3K with IRS. Therapeutic concentrations (0.01-0.1 mmol/L) of metformin prevented the changes induced by chronic insulin treatment in these post-receptor components of insulin signaling pathway. Tyrosine phosphorylation of IR?, IRS1, and IRS2 was increased by 2.7 fold (P < 0.01), 6.8 fold (P < 0.01), and 2.3 fold (P <0.01) of chronically insulin-treated cells alone, respectively, after metformin 0.1 mmol/L was added. The association of p85 with IRS1 and IRS2 was also increased from 34 % to 86 % (P <0.01) and from 30 % to 92 % (P <0.01), respectively. In contrast, metformin in pharmacological concentration (1-10 mmol/L) further inhibited tyrosine phosphorylation of IR?, IRS1, IRS2 and the interaction of PI3K with IRS. The association of IRS1 with p85 was further decreased by 58 % (P >0.05) and of IRS2 by 30 % (P <0.05).
  
  
  CONCLUSIONS
  Chronic insulin exposure of Hep G2 cells induces the downregulation of insulin signal transduction via PI3K pathway. The effect of metformin on insulin signaling transduction represent a primary mechanism of metformin action in insulin resistant state.
authors.affiliation: Department of Endocrinology and Metabolism in Hospital of University Heidelberg, Heidelberg 69115, Germany. yuanli18cn@yahoo.com.cn
authors.forename: Li
authors.initials: L
authors.lastname: Yuan
authors.forename: Reinhard
authors.initials: R
authors.lastname: Ziegler
authors.forename: Andreas
authors.initials: A
authors.lastname: Hamann
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000473:pathology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007333:Insulin Resistance; D008113:Liver Neoplasms / Q000473:pathology; D008687:Metformin / Q000494:pharmacology*; D011972:Receptor, Insulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D014407:Tumor Cells, Cultured
chemical_list: D007004:Hypoglycemic Agents; D008687:Metformin; D011972:Receptor, Insulin