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 o...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.展开更多
AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations ...AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a(GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β(IRβ), protein kinase B(also known as Akt), and glycogen synthase kinase-3β(GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay(ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet(HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.展开更多
基金DepartmentofEndocrinology,XieheHospital,TongjiMedicalCollege,Huazhong U niversity ofScienceandTechnology,Wuhan4 30 0 2 2
文摘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.
基金supported by the FundamentalResearch Funds for the Central Universities(No.JKP2011004)
文摘AIM: To investigate the molecular signaling mechanism by which the plant-derived, pentacyclic triterpene maslinic acid(MA) exerts anti-diabetic effects. METHOD: HepG2 cells were stimulated with various concentrations of MA. The effects of MA on glycogen phosphorylase a(GPa) activity and the cellular glycogen content were measured. Western blot analyses were performed with anti-insulin receptor β(IRβ), protein kinase B(also known as Akt), and glycogen synthase kinase-3β(GSK3β) antibodies. Activation status of the insulin pathway was investigated using phospho-IRβ, as well as phospho-Akt, and phospho-GSK3β antibodies. The specific PI3-kinase inhibitor wortmannin was added to the cells to analyze the Akt expression. Enzyme-linked immunosorbent assay(ELISA) was used to measure the effect of MA on IRβ auto-phosphorylation. Furthermore, the effect of MA on glycogen metabolism was investigated in C57BL/6J mice fed with a high-fat diet(HFD). RESULTS: The results showed that MA exerts anti-diabetic effects by increasing glycogen content and inhibiting glycogen phosphorylase activity in HepG2 cells. Furthermore, MA was shown to induce the phosphorylation level of IRβ-subunit, Akt, and GSK3β. The MA-induced activation of Akt appeared to be specific, since it could be blocked by wortmannin. Finally, MA treatment of mice fed with a high-fat diet reduced the model-associated adiposity and insulin resistance, and increased the accumulated hepatic glycogen content. CONCLUSION: The results suggested that maslinic acid modulates glycogen metabolism by enhancing the insulin signaling pathway and inhibiting glycogen phosphorylase.