The Role Of S6k1 In Pathogenesis Of Hepatic Insulin Resistance And Non-alcoholic Fatty Liver Disease

Objective To construct S6K1 shRNA gene recombinant adenovirus (S6KlAx) and evaluate its gene silencing effects on AML12 cell and mice. We explored the role and mechanism of S6K.1 gene silencing in the pathogenesis of hepatic insulin resistance and non-alcoholic fatty liver disease(NAFLD) after it was injected into db/db mice vein tail.Methods Three S6K1 shRNA gene sequence were designed and transferred from pcPUR plasmid, pcDNA plasmid to cosmid plasmid and finally transfected into adenovirus. S6K1Ax which had the best gene silencing effect was selected and proliferated in 293 cell. The silencing effect of high titer adenovirus was checked on mice AML12 cell lines and in C57BL/6J mice by Western blot or/and reverse transcriptase-PCR. S6K1Ax were injected into tail vein of db/db mice and pU6Ax injection group was treated as a control. There were six mice in each group.Mice were killed in the random fed state and food deprivation in the 6th day. Hepatic weight and triglycerol content were determined after Liver was obtained. Liver morphology was observed by HE stain.Hepatic protein were determined for protein expressions of IRS1,IRS2 and phosphotyrosine-IRS 1,IRS2 by western blot and immunoprecipitation, Hepatic total RNAs were extracted to analyze gluconeogenic genes of PGCla,PEPCK,G6Pase mRNA and lipid metabolic genes of PPARα,CPT-1,SREBP1c,FAS,SCD-1 mRNA by reverse transcription PCR. Serum blood glucose,insulin,FFA,TG, et al were checked before and after S6K1 Ax injection.Results S6KlAx can silence S6K1 expression in mouce AML12 cell lines, In the livers of C57BL/6J mice obtained six days after S6KlAx were injected into its tail vein, S6K1 protein of mice liver was restrained and 54.7% mS6Kl of S6K1Ax group were inhibited (P<0.01). Alanine aminotransferase (ALT) levels which indicate of mice hepatic function was not changed after S6KlAx injected. Compared with the pU6Ax control group, hepatic S6K1 protein expression of IRS1,IRS2 and tyrosine-IRS1,IRS2 in random fed state and food deprivation increased as measured by Western blot. But only in the fasting state,phosphotyrosine-IRS1,2 showed more obvious expression and gluconeogenic genes of PGCla,PEPCK,G6Pase mRNA descented(P<0.01). So did the serum FFA(P<0.05).Serum insulin didn't show difference and cholesterol reduced in both fed and starved state(P<0.05).Fasting blood glucose declined but there was no significant difference (P>0.05). Fatty liver were improved after hepatic gene silencing. Fat drop in the liver cell lowered comparing with the control group on HE stain.Hepatic weight and triglycerol content declined (P<0.01), Lipogenic genes of SREBPlc,FAS,SCD1 mRNA reduced, fatty acid oxidation genes of PPARa,CPTl mRNA lowered,too.Conclusion Construction of S6K1Ax can knockdown S6K1 gene on mouse AML12 cells and in the liver of C57BL/6J mice, it iprovides a good tool to study the function of S6K1. S6K1 silencing can ameliorate hepatic insulin resistance and reduce hepatic gluconeogenesis in the fasting state. Meanwhole S6K1 silencing ameliorate fatty liver by inhibiting fatty synthesis not increasing beta-oxidation, This gives it potential to become a gene target for the treatment of hepatic insulin resistance and NAFLD.