Study On The Role And Molecular Mechanism Of Deacetylase SIRT6 In Hepatic Lipid Metabolism

Background and aim: Non-alcoholic fatty liver disease(NAFLD)has emerged as the most prevalent chronic liver disease which affects nearly a quarter of the world’s population.And often coexist with other metabolic diseases,such as: obesity,type 2 diabetes,hyperlipidemia,hypertension.An in-depth study of its pathogenesis will help us understand the disease more clearly and find new targets for prevention and treatment.The deacetylase SIRT6plays an important role in glucose and lipid metabolism.Here,we aimed to explore the role and related mechanisms of SIRT6 on hepatic glucose and lipid metabolism on the basis of SIRT6 liver-specific knockout mouse model.Methods: 1.SIRT6-LKO mice model was generated by using the Cre-Lox P recombinase system verified by agarose gel electrophoresis,western blot and immunohistochemistry(IHC).2.Using Streptozocin(STZ)injection and high fat diet(HFD)diet induces the NAFLD model;detecting body weight,fasting blood glucose to evaluate the influence of SIRT6 deletion.3.When the model was completed,the influence of SIRT6-LKO on mice liver was evaluated by observing various index,including liver morphology,ratio of liver and weight,hematoxylin-eosin and oil red O dyeing,serum biochemistry.4.To investigate the role and mechanism of SIRT6 in lipid metabolism with SIRT6 knock-out and overexpressing L02 cell line,and to observe lipid droplet formation by Bodipay staining after stimulating with oleic acid(OA),to explore the role of SIRT6 in regulating C/EBPα’s expression.5.To construct C/EBPα knocking-down and overexpressing cell line and identify the role of C/EBPα on lipid metabolism by the oil red and Nile red stanining and the expression of enzymes involved in lipid metabolism.6.To investigate the mechanism that SIRT6 regulated the expression of C/EBPα by way of chip and muting SIRT6 deacetylation site.Results:1.SIRT6-LKO mice model was established successfully.2.There was no significant difference in body weight between SIRT6-LKO and WT mice under STZ-HFD condition.But SIRT6-LKO aggravated mice glucose disturbances induced by STZ-HFD.3.The liver of SIRT6-LKO mice was more bulky than the same litter of WT mice.After STZ-HFD treatment,the liver/body weight ratio of SIRT6-LKO mice wassignificantly higher than that of wild-type mice.The H&E stain and oil O red dying of liver tissues showed that the hepatic statuses of SIRT6-LKO mice was heavier than that of the WT mice and with apparent inflammatory infiltration.Sirius red staining showed that the fibrous collagen outside the portal area in SIRT6-LKO group was significantly increased.Feeding a HFD diet also led SIRT6-LKO mice to a significant increase in TG,TC,AST,ALT,DBIL and TBIL.4.Lipid drop in SIRT6-KO cells after oleic acid stimulation was significantly increased and lipid droplets were relatively large,indicating that knockout of SIRT6 in human liver cells promoted lipid formation.We also find that SIRT6 can regulate the expression of C/EBPα.5.We constructed C/EBPα high-and low-expression hepatic cell lines successfully.Knocking down C/EBPα promoted the formation of lipid droplets by Oil red O staining and increased the expression of Acetyl-Co A carboxylase(ACC1)and Fatty Acid Synthase(FASN)involved lipid metabolism.6.We demonstrate that SIRT6 can bind to the promoter region of C/EBPα by Chromatin immunoprecipitation,and the deacetylase activity of SIRT6 affects the expression level of C/EBPα.Conclusion: SIRT6 plays a protective role in hepatic glucose and lipid metabolism.The absence of SIRT6 causes severe hyperglycemia and hepatic steatosis in NAFLD model induced by STZ-HFD.The deletion of SIRT6 and its down-stream regulatory molecular C/EBPα promotes the production of cellular lipids.This finding provides new targets for the prevention and treatment of metabolic disorders.