The Mechanism Of SIRT1 Regulating Sulfotransferase SULT1E1 And SULT2A1 In Liver Lipid Metabolism

BackgroundWith the increasing pandemic of obesity and metabolic syndrome,the prevalence of nonalcoholic fatty liver disease?NAFLD?increased significantly in the past decade.NAFLD has become the most common liver disorder in the world,which is emerging into a new significant health problem.To date,our understanding of the mechanisms involved in the pathophysiology of NAFLD is insufficient to pinpoint the major determinants involved in the development and progression of the disease and to develop therapeutic strategies for NAFLD.Both“the two hits hypothesis”and“the multiple-hit hypothesis”think that excessive triglyceride?TG?accumulation of the hepatocytes is one of the core initial factors which lead to NAFLD.Therefore,a pressing need for clarifying the mechanisms of hepatocytes lipid metabolism involved in the pathophysiology of NAFLD and developing new novel pharmacological treatments is very important.Recent studies show that protein lysine acetylation has emerged as a key posttranslational modification in cellular metabolism regulation.Silent information regulator 1?SIRT1?,a highly conserved NAD+-dependent protein deacetylase,has emerged as a key metabolic sensor that directly links environmental nutrient signals to metabolic homeostasis.SIRT1 is responsible for the regulation of protein activation by means of deacetylating a variety of proteins that play important roles in the process of aging,cellular metabolism,tumorigenesis and circadian clocks.Previous studies show that the activity and expression of SIRT1 are decreased in the liver of NAFLD mice models and NAFLD patients,and SIRT1 activation by small molecules ameliorates fatty liver in NAFLD mice models,which suggest that SIRT1 plays an important role in liver lipid metabolism.However,the detailed mechanisms of SIRT1 regulating liver lipid metabolism are still unclear.With the rapid development and maturity of omics and bioinformatics technology,this study will demonstrate the mechanisms of SIRT1 regulation of hepatic lipid metabolism involved in the occurrence and development of NAFLD by means of proteomics and genomic techniques.This study will also help us understand the mechanisms of NAFLD and find therapeutic targets for the treatment of NAFLD.Aims1.To screen SIRT1 associated key molecules involved in non-alcoholic fatty liver disease by means of proteomics and genomic techniques;2.To screen and identify SIRT1 interacting proteins;3.To determine the role of candidate molecules SULT1E1 and SULT2A1 in regulating liver lipid metabolism;4.To demonstrate the mechanisms of SIRT1 regulating sulfotransferase SULT1E1 and SULT2A1 in liver lipid metabolismMethods1.Liver specific SIRT1 knockout mice(SIRT1^LKO)and control mice(SIRT1^flox)were fed ad libitum either normal chow diet or a high-fat diet providing 60%Kcal fat for 12weeks to induce NAFLD mouse model;quantitative real-time PCR,Western blot and immunohistochemical staining were performed to determine the expression of SIRT1 in the liver of SIRT1^LKO mice.The body weight,blood glucose,serum free fatty acid?FFA?,glutamic-pyruvic transaminase?AST?,glutamic oxalacetic transaminase?AST?,insulin tolerance test?IPITT?and glucose tolerance test?IPGTT?were performed to observe the glucose and lipid metabolism and insulin resistance of SIRT1^LKOKO and SIRT1^flox mice upon high fat diet.Hematoxylin-eosin staining?H&E?and oil red O staining were used to observe hepatic steatosis of every group mice.We also developed a transgenic mouse model?FASN-GLuc?that allows non-invasive and longitudinal measurement of FASN expression.2.Based on the combinational use of label-free LC-MS/MS proteomic approach and cDNA microarrays analysis,the liver differential genes of each group mice were screened by means of high throughput methods in both the transcriptional and post-transcriptional levels.KEGG signal pathway of differential genes was also analyzed by bioinformatics analysis and the key lipid metabolism associated genes were screened.Q-PCR and Western blot were used to identify the expression of candidate genes in vivo and in vitro.The effects of SULT2A1 on lipid metabolism in vitro were analyzed3.The hepatocyte steatosis model in vitro induced by Oleic acid?OA?or Palmitic acid?PA?was established,high content cellular analysis system was used to screen the proper concentration and the best treatment duration of OA or PA by BODOPY staining.SULT1E1 and SULT2A1 overexpression lentiviral vectors were also constructed.Via combinational use of high content cellular analysis system and BODOPY or Nile red staining,the effects of overexpression SULT1E1 and SULT2A1 on liver lipid metabolism were determined.Q-PCR and Western blot were used to detect the expression of lipogenesis genes in SULT1E1 and SULT2A1 overexpression cell line upon OA or PA stimulation.4.L02 Flag-SIRT1 cell line was established,Immunoprecipitation?IP?was used for enrichment of SIRT1 interacting proteins,SIRT1 interacting proteins were screened and identified by means of label-free LC-MS/MS proteomic approach.The GO analysis was performed using DAVID Bioinformatics Resources 6.7.Co-immunoprecipitation?CO-IP?was used to verify the SIRT1-HLTF interaction.The upstream transcription factor binding sites in the SULT1E1 and SULT2A1 promoter region were predicted by means of bioinformatics analysis.L02 HA-HLTF cell line was established and the effect of HLTF on the transcription of SULT1E1 and SULT2A1 was observed.Results1.Q-PCR,Western blot and IHC results demonstrated that SIRT1 was specific knockout in the liver of SIRT1^LKO mice.Compared with SIRT1flox mice upon normal chow diet,the body weight,blood glucose,serum FFA,ALT and AST were no changed in SIRT1^LKOKO mice upon normal chow diet,while compared with SIRT1flox mice upon high fat diet,the body weight,blood glucose,serum FFA,ALT and AST were increased in SIRT1^LKOKO mice upon high fat diet.IPGTT and IPITT results revealed that SIRT1^LKO mice upon high fat diet displayed greater glucose intolerance and insulin resistance than any other groups.H&E staining and Oil Red O staining of the liver sections in each group mice showed that hepatic steatosis of SIRT1^flox upon high fat diet was greater than any other group mice.2.Based on the combinational use of proteomics and genomics technology and bioinformatics analysis,10 molecules that were significantly downregulated and 21molecules that were significantly upregulated were identified by label-free LC-MS/MS proteomic approach,70 molecules that were significantly downregulated and 45molecules that were significantly upregulated were identified by cDNA microarrays.The GO analysis showed that most of the differential molecules involved in the process of cellular metabolism,liver development and endoplasmic reticulum stress.We mainly focused on the molecules involved in lipid metabolism such as GSTM3,SULT1E1,SULT2A1,Lipin.Q-PCR and Western blot results were consistent with the proteomics and genomics data.Finally,we selected SULT1E1and SULT2A1 for further study.Then,we demonstrated that the expression of SULT1E1 and SULT2A1 were downregulated by overexpression of SIRT1,while CO-IP results showed that SULT1E1 and SULT2A1 were not the direct target of SIRT1.3.L02 Flag-SULT1E1 and Flag-SULT2A1 cell lines were established.The hepatocytes steatosis model was successfully established,the proper concentration?100?M OA,200?M PA?and the best treatment duration?12h?of OA or PA by BODOPY staining were confirmed with the help of high content cellular analyze system.Under this condition,overexpression SULT1E1 and SULT2A1 promoted the fat synthesis in L02cell line by means of oil red O staining and BODIPY staining and Nile red staining.Q-PCR and Western blot results revealed that overexpression SULT1E1 and SULT2A1promoted the lipogenesis gene expression including FASN and ChREBP.4.51 proteins were identified as SIRT1 interacting proteins by combinational use of CO-IP and label-free LC-MS/MS proteomic approach,30 proteins were previously reported,21 proteins were newly characterized in this study.The GO analysis results showed that SIRT1 interacting proteins mainly involved in cellular metabolism and oxidative stress.We mainly focused on the transcription factor HLTF because HLTF can bind to the promoter region of SULT1E1 and SULT2A1 that predicted by means of bioinformatics analysis.L02 HA-HLTF cell line was established and SIRT1-HLTF interaction was confirmed by CO-IP.We found that the expression of SULT1E1 and SULT2A1 was transcriptionally regulated by HLTF.Conclusions1.Based on approaches of proteomics and functional genomics,using spontaneous occurrence of fatty liver SIRT1^LKO animal model,we got a group of non-alcoholic fatty liver disease associated molecules including sulfotransferase SULT2A1,SULT1E1 and Lipin.This stufy demonstrates the transcription factors profile regulated by SIRT1 and the network control system formed by the NAFLD associated transcription factors and their regulatory proteins in liver lipid metabolism.2.This study demonstrated that SULT1E1 and SULT2A1 promotes fat synthesis through regulation of lipogenesis genes including ChREBP and FASN.3.This study identified previous unknown SIRT1 interacting proteins and CO-IP verified the SIRT1-HLTF interaction.We also demonstrated that the expression of SULT1E1 and SULT2A1 was transcriptionally regulated by HLTF...