Histone Modifications In FASN Under Insulin Stimulation Are Related To NAFLD

Non-alcoholic fatty liver disease(NAFLD)is defined as lipid droplets accumulation without significant alcohol consumption.Insulin resistance(IR)is the common pathogenesis of NAFLD.Thus,exploring the mechanism between IR and lipid metabolism is critical for elucidating NAFLD pathogenesis and identifying new therapies.Sterol regulatory element-binding transcription factor 1c(SREBP-1c)is the major regulator which control lipid de nove synthase under insulin stimulation.Carbohydrate responsive-element binding protein(Ch REBP),is another key regulator of lipid.It is mainly activated by carbohydrates,but also elevated under insulin stimulation.Both SREBP-1c and ChREBP may regulate Fatty acids synthase(FASN)in a hyperinsulinemic setting.Normally Eukaryotic Chromatin is in a condensed form that inhibit DNA regulatory activity.The signaling molecules binding to the target gene must enter when the local chromatin structure changes to activate or inhibit target gene transcription.These molecules can alter chromatin structure through histone modifications.Nowadays how FASN histone modification is influenced by insulin has not been reported.Understanding histone modification in this setting will clarify NAFLD pathogenesis and new therapeutic method.MethodI.The relation between FASN histone modifications,gene transcription and lipid accumulation under insulin stimulation1.MTT was used to screen the optimal insulin concentration for human hepatocellular carcinoma cell line(HepG2)and primary hepatocyte,the insulin concentration was set as repoted in 0.5?0.75?1.0?1.25 uM.2.After Hep G2 and primary hepatocyte were stimulated by insulin for 0,24 and 48 h,respectively,Western-blot was applied to detect the protein expression level of FASN,and TG assay kit and oil red O stain to determine lipid deposition.3.After Hep G2 and primary hepatocyte were stimulated by insulin for 0,1,2,4,8,12,and 24 h respectively.Insulin stimulation was removed after 12 h insulin stimulation in HepG2 cells and 4h stimulation in primary hepatocyte,the culture medium was changed into normal medium without insulin.The retreat time was 0,1,2,4,8,12,and 24 h.q RT-PCR was utilized to measure the transcriptional time spectrum of FASN mRNA,western-blot was used to measure FASN protein expression.4.According to the transcriptional time spectrum of FASN mRNA after insulin stimulation in hepatocytes,the optimal time point for stimulation was selected,and the histone modification level of transcriptional regulatory region in FASN gene at this time point was measured using the chromatin immunoprecipitation(ChIP)assay.Based on the transcriptional time spectrum of FASN mRNA after the removal of insulin stimulation in hepatocytes,the optimal time point for removal was selected,and the histone modification level of transcriptional regulatory region in FASN gene at this time point was determined by ChIP assay,including 3 regulatory sites: promoter,exon2 and 2000bp(-2kb)at the upstream of the transcription start site.II.The involvement of SREBP-1c or ChREBP in FASN promoter histone modification under insulin stimulation1.After Hep G2 and primary hepatocyte were stimulated by insulin for 0,1,2,4,8,12,16 and 24 h respectively.Western-blot was used to detect the nuclear protein expression level of SREBP-1c or ChREBP.2.After Hep G2 and primary hepatocyte were stimulated by insulin for 0,24 h respectively.Immunofluorescence,was used to measure the location of SREBP-1c and ChREBP.3.After HepG2 was transiently transfected with siRNA-ChREBP or siRNA-SREBP-1c plasmid,primary hepatocyte was transfected with shRNA-ChREBP or shRNA-SREBP-1c lentivirus,SREBP-1c,ChREBP and FASN protein levels were measured by Western-blot.4.HepG2 and primary hepatocyte were divided into 4 groups: normal group,positive control group(insulin stimulation),transfection group(HepG2 was transiently transfected with siRNA-ChREBP or siRNA-SREBP-1c plasmid for 48 h,primary hepatocyte was transfected with shRNA-ChREBP or shRNA-SREBP-1c lentivirus for 72h),and negative control group(high insulin stimulation after 48 h transfection with GV102-scramble plasamid or scramble lentivirus).The histone SREBP-1c-SRE and ChREBP-ChoRE binding level were measured by Ch IP assay.5.HepG2 and primary hepatocyte were divided into 4 groups: normal group,positive control group(insulin stimulation),transfection group(HepG2 was transiently transfected with siRNA-ChREBP or siRNA-SREBP-1c plasmid for 48 h,primary hepatocyte was transfected with shRNA-ChREBP or shRNA-SREBP-1c lentivirus for 72h),and negative control group(high insulin stimulation after 48 h transfection with GV102-scramble plamid or scramble lentivirus);the histone histone modification in FASN promoter region were measured by ChIP assay.III.Effect of histone acetylation affects lipid accumulation in hepatocyteHepG2 cells and primary hepatocytes were divided into three groups: normal control group(cultured in medium),positive control group(insulin stimulated for 12 h in HepG2 8h in primary hepatocyte),and insulin + garcinol group(insulin and garcinol 5 uM stimulated for 12 h in HepG2 8h in primary hepatocyte).The histone acetylation modification level of FASN gene was detected by Ch IP assay,and the protein expression level of SREBP-1c,ChREBP and FASN were measured by Western-blot,respectively;and lipid deposition was determined by oil red O stain.Results:I.The relation between FASN histone modifications,gene transcription and lipid accumulation under insulin stimulation1.MTT analysis revealed that 1.25 uM was the best insulin concentration for HepG2 and primary hepatocyte.2.Western-blot analysis revealed that high insulin stimulation significantly increased FASN protein expression of both Hep G2 and primary hepatocyte in a time-dependent manner.3.After insulin stimulation for 24 h or 48 h,TG contents in both HepG2 and primary hepatocyte increased over time.The lipid drops in these cells were also increased in a time-dependent manner.4.FASN mRNA expression in primary hepatocytes and HepG2 cells rose 1 and 4 h after exposure to insulin and peaked at 4 and 8 h,respectively.The primary hepatocytes and HepG2 cells were incubated with 1.25 uM insulin for 4 or 8 h respectively,before it was removed.FASN mRNA levels in both were decreased at 1 h.All differences were of statistic significance.5.We found significant increases in H3 and H4 acetylation,H3S10 phosphorylation and H3K4 trimethylation and a decrease in H3K9 and H4K20 trimethylation within the TSS regions in cells exposed to high insulin.Notably,these changes were reversed when insulin was removed from the medium.II.The involvement of SREBP-1c or ChREBP in FASN promoter histone modification under insulin stimulation1.Western-blot analysis revealed the expression of SREBP-1c or Ch REBP was distinctly enhanced with insulin incubation.In HepG2 cells,the SREBP-1c protein levels was elevated in 4h and get the highest at 8h,the ChREBP protein levels was elevated in 8h and get the highest at 16 h.In primary hepatocyte,the SREBP-1c protein levels was elevated in 4h and get the highest at 8h,the ChREBP protein levels was elevated in 4h and get the highest at 8h.2.We used siRNA to knock down endogenous SREBP-1c or Ch REBP in HepG2 cells and lentivirus in primary hepatocytes,western blotting confirmed decreased SREBP-1c,ChREBP and FASN levels in these cells compared with the normal and negative groups.3.The locations of SREBP-1c and Ch REBP following insulin treatment for 24 h were detected by immunofluorescence,which showed that high insulin resulted in SREBP-1c and ChREBP nuclear translocation.4.The results show weaker SREBP-SRE or ChREBP-ChORE binding in the siSREBP-1c and siChREBP group,respectively,compared with positive or negative controls in both HepG2 cells and primary hepatocyte.5.SREBP-1c knockdown impaired insulin-induced hyper-H3 acetylation at SRE sites and hyper-H4 acetylation at SRE,TSS sites in HepG2 cells,as well as at TSS and SRE in primary hepatocyte,but it had no effect on H3K4 hypertrimethylation at SRE,or TSS regions.ChREBP knockdown also impaired insulin-induced hyper-H3 acetylation at SRE sites and hyper-H4 acetylation at SRE,TSS sites in HepG2 cells,as well as the SRE,TSS in primary hepatocytes,while it had no effect on H3K4 hypertrimethylation at ChoRE,or TSS regions.The results shows the H3K4 trimethylation in FASN promoter regions not just influenced by SREBP-1c or ChREBP,but H3 and H4 acetylation mainly affected by SREBP-1c or Ch REBP.III.Effect of histone acetylation affects lipid accumulation in hepatocyte1.Garcinol caused H3 and H4 hypo-acetylation at FASN promoter region in HepG2 and primary hepatocyte under insulin stimulation.2.Compared with positive controls,the insulin + garcinol group showed decreased SREBP-SRE and ChREBP-ChORE binding.3.FASN expression was lower in the insulin + garcinol group compared with the positive control groups.There was no significant change in SREBP-1c and ChREBP expression in insulin and insulin + garcinol group.4.The lipid drops was decreased in the insulin + garcinol group compared with the positive control groups.Conclusion:The activation of FASN gene transcription was accompanied by hyper-H3 and H4 acetylation,hyper-H3K4 trimethylation and hypo-H3K9 and H4K20 trimethylation under insulin stimulation.Inulin retreat will decrease FASN gene transcription with hype0-H3 and H4 acetylation,hype0-H3K4 trimethylation and hypr-H3K9 and H4K20 trimethylation in FASN promoter regions.insulin influenced FASN gene transcription by regulating SREBP-1c and ChREBP expression.SREBP-1c and ChREBP can induced histone acetylation and facilitates the SREBP-1c-SRE,Ch REBP-Cho RE combination in FASN promoter region and plays a role of regulation on FASN gene transcriptions.In cases of insulin stimulation and SREBP-1c ChREBP expression increase,inhibition of FASN gene histone acetylation gives rise to the decrease in SREBP-1c-SRE and Ch REBP-Cho RE combinations,which further results in FASN expression decrease.It proves that histone acetylation play an important role of regulating of FASN transcription.