To study the regulatory effect of acute and chronic insulin treatmenton insulin post- re- ceptor signaling transduction pathway in a human hepatom a cell line (Hep G2 ) ,Hep G2 cells were incubated in the presence or absence of insulin with different concentrations in serum free m edia for16 h and then stim ulated with10 0 nmol/ L insulin for1m in.Protein levels of insulin receptor β- subunit(IRβ) ,insulin receptor substrate- 1(IRS- 1) and p85 subunit of phosphatidylinositol3- kinase(PI3- kinase) were determined in total cell lysates by Western- im munoblot.Phosphorylat- ed proteins IRβ,IRS- 1and interaction of PI3- kinase with IRS- 1were determ ined by im munopre- 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- 10 0 nm ol/ 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 m ore marked reduction in the phospho- rylation of IRβ,IRS- 1,reaching a nadir of2 2 % (P<0 .0 1) and15 % (P<0 .0 1) of control lev- els,respectively,after16 h treatment with 10 0 nm ol/ L insulin.The association between IRS- 1 and PI3- kinase was decreased by6 6 % (P<0 .0 1) .There was no significant change in PI3- ki- nase protein levels. These data suggest that chronic insulin treatm ent can induce alterations of IRβ,IRS- 1and PI 3- kinase three early steps in insulin action,which contributes significantly to insulin resistance,and may account for desensitization of insulin action. To study the regulatory effect of acute and chronic insulin treatment on insulin post-re- ceptor signaling transduction pathway in a human hepatom a cell line (Hep G2), Hep G2 cells were incubated in the presence or absence of insulin with different concentrations in serum free m edia for 16 h and then stimulated with 100 nmol / L insulin for 1 m in. Protein levels of insulin receptor β-subunit (IRβ), insulin receptor substrate- 1 (IRS- 1) and p85 subunit of phosphatidylinositol 3-kinase ) were determined in total cell lysates by Western-munoblot. Phosphorylat-ed proteins IRβ, IRS-1 and interaction of PI3-kinase with IRS-1 were determined by im munopre- 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-1.1-10-10 nm ol / 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 marked reduction in the phospho-rylation of IRβ, IRS-1, reaching a nadir of 2% (P <0.01) and 15% (P < ) of control lev-els, respectively, after 16 h treatment with 10 0 nm ol / L insulin. The association between IRS-1 and PI3-kinase was decreased by 6% (P <0.01). There was no significant change in PI3-ki- nase protein levels. These data suggest that chronic insulin treatm ent 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.