The Role And Mechanism Of SIRT2 In The Regulation Of Non-alcoholic Fatty Liver Disease

?Objective?NAFLD is one of the most common chronic liver diseases worldwide,with a high prevalence and unclarified mechanisms.The NAD+-dependent deacetylase SIRT2 is an important regulator of several physiological processes,including aging,metabolic deseases,inflamation,stress response,and apoptosis through multiple substrates.However,the role of SIRT2 in NAFLD is unknown.In this part we aim to identify the effect of SIRT2 on NAFLD.?Methods?Firstly,we analized the GEO data of NAFLD to explore the role of SIRT2 in NAFLD.Then,our research group established PA induced cellular model,HFD-induced animal model,and ob/ob mouse model of NAFLD,and detected SIRT2 protein expression in these models.Additionally,we constructed hepatocyte-specific SIRT2 overexpress or knockout mice through tail vein injection to explore the association between SIRT2 and NAFLD.In vitro experiment,SIRT2 was overexpressed or scilenced by plasmid or si RNA transfected in Hep G2 cells,and the effects of SIRT2 on hepatic lipid accumulation was observed.Finally,to explore the effect of deacetylases activity of SIRT2 on hepatic steatosis.We constructed deacetylation mutation plasmids of SIRT2(SIRT2-H168 A,SIRT2-H187Y),and and observed their effects on hepatic steatosis.?Results?The microarray analysis showed that the SIRT2 expression was decreased in HFD-induced mice model and associated with the severity of NAFLD.In cellular model of NAFLD,SIRT2 expression decreased in time and dose dependent.In livers of HFD-induced mice model and ob/ob mice,SIRT2 expression decreased gradually.Compared with wild-type mice,insulin resistance,hepatic lipid accumulation and concomitant inflammatory responses,were significantly ameliorated by hepatocyte-specific SIRT2 overexpression HFD-induced or ob/ob mouse models.By contrast,specific SIRT2 deficiency in liver aggravated these pathological alterations.Additionally,deacetylation inactivation of SIRT2 was not able to improved hepatic steatosis.?Conclusion?The decrease in SIRT2 expression observed in the fatty livers of NAFLD patients,and mouse and cellular models suggests that SIRT2 is involved in the pathopathological process of NAFLD.SIRT2 is required for maintaining hepatic metabolic homeostasis and for suppressing inflammation.?Objective?In the first part of the study,we found that SIRT2 was invoved in the pathological process of NAFLD.Hepatic SIRT2 overexpression alleviated hepatic steatosis,insulin resistance and inflammatory response.Conversely,hepatic SIRT2 defiency notably exacerbated the pathological alterations in NAFLD.However,the underlying mechanism of hepatic SIRT2 actions remains largely unknown.Thus,we further investigated the downstream molecular events underlying the effects of SIRT2.?Methods?Fisrtly,we used bioinformatics technique to explore the potential target protein of SIRT2.Co-immunoprecipitation(Co-IP)and immunofluorescence(IF)technical were used to examine if SIRT2 physically interacts with HNF4?.We then determined the regulatory role of SIRT2 in HNF4? and its downstream genes by using Western blot and RT-q PCR.To identify the regulation of SIRT2 on HNF4? proteion expression,Hep G2 cells transfected with si RNA-SIRT2 were subjected to MG132 and CHX.Furtherly,to evaluate whether the suppression HNF4? contributed to the beneficial role of SIRT2 in the current model,Hep G2 cells were transfected plasmid/ si RNA for HNF4? ectopic expression or silencing,and were subjected to PA.Based on the knowledge that SIRT2 is a deacetylation enzyme,we further investigated the effect of SIRT2 on HNF4? deacetylation in hepatocytes.Subsequently,to further identify the site required for SIRT2/ HNF4? interaction,a series of acetylation inactive mutants of HNF4? were constructed,and site-mapping experiments were performed.Then,we examed the impact of deacetylation mutation of HNF4? on its protein stability by using MG132 and CHX.?Results?The IP and IF results indicated that SIRT2 interacted with HNF4? and vice versa.Overexpression of SIRT2 could promot the protein expression of HNF4? and the m RNA expression of the downstream genes,and inhibition of SIRT2 had the opposite results.Additionally,SIRT2 regulated HNF4? on its proteion level.The intervation of HNF4? expression could reverse the effect of SIRT2 on hepatic fat steatosis.Co-IP results suggested that the K458 amino acid(aa)site of HNF4? were required for SIRT2/ HNF4? interaction and HNF4? protein stability.?Conclusion? SIRT2-HNF4? interaction and subsequent HNF4? deacetylation were required for the amelioration of hepatic steatosis and the HNF4? protein stability.