Objective To study the effect of chronic insulin stimulation on different signal transduction pathways of insulin post-receptor in a human hepatoma cells line (HepG2). Methods HepG2 cells were incubated in serum-free media in either presence or absence of various concentrations of insulin (0-100 nmol/L) for 16 h before the cells were stimulated with 100 nmol/L insulin for 1 min. Protein levels of insulin receptor ?-subunit (IR?), insulin receptor substrate (IRS)-1, IRS-2, P85 subunit of phosphatidylinositol 3-kinase (PI3K), mitogen activated protein kinase (MAPK) and phosphorylated proteins of MAPK were determined in total cell lysates by Western-immunoblot, and determination of phosphorylated proteins IR?, IRS-1, IRS-2 and interaction of PI3K with IRS-1/-2 were performed by immunoprecipitation. Results Insulin stimulation for 1 min rapidly induced tyrosine phosphorylation of IR?, IRS-1 and IRS-2 and phosphorylation of MAPK, which in turn, resulted in binding of P85 with IRS-1/-2 and activation of MAPK. After 100 nmol/L insulin treatment for 16 h, there was a more marked reduction in the phosphorylations of IR?,IRS1andIRS2inHepG2 cells, reaching 22.2% (P

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.
Carcinoma, Hepatocellular ; pathology ; Down-Regulation ; Humans ; Hyperinsulinism ; metabolism ; Insulin Receptor Substrate Proteins ; Insulin Resistance ; Liver Neoplasms ; pathology ; Phosphoproteins ; metabolism ; Phosphorylation ; Receptor, Insulin ; antagonists & inhibitors ; metabolism ; Signal Transduction ; drug effects ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect and mechanism of the antihyperglycemic agent metformin on the expression of phosphoenolpyruvate carboxykinase (PEPCK) gene in hepatocytes and to determine whether the effects of metformin in hepatocytes are transmitted throughout the known insulin signaling pathways.

METHODSConfluent H4IIE rat heptoma cells were cultured for 16 h with 0.1 mmol/L metformin either in absence or presence of 0.1 nmol/L insulin, and then stimulated with various agents. The expression of PEPCK gene was examined by Northern blot analysis.

RESULTSTherapeutic concentrations of metformin significantly inhibited basal PEPCK mRNA expression and also decreased cAMP and dexamethasone induced PEPCK gene expression through interaction with insulin. In the presence of insulin signaling pathway inhibitors wortmannin and UO126, metformin reduced PEPCK mRNA levels, but wortmannin blocked inhibitory regulation of insulin on PEPCK gene expression.

CONCLUSIONMetformin inhibits PEPCK gene expression via either an insulin-independent or an interacting-with-insulin manner. The results suggest that a possible mechanism by which metformin reduces gluconeogenesis could be associated with the inhibition of PEPCK gene expression.

Androstadienes ; pharmacology ; Cells, Cultured ; Gene Expression ; drug effects ; Hepatocytes ; drug effects ; enzymology ; Humans ; Hypoglycemic Agents ; pharmacology ; Metformin ; pharmacology ; Phosphatidylinositol 3-Kinases ; physiology ; Phosphoenolpyruvate Carboxykinase (GTP) ; genetics ; RNA, Messenger ; analysis ; Tumor Cells, Cultured