Effects Of ChREBP And Histone Modifications Of FASN Gene On NAFLD And Its Underlying Molecular Mechanism

Background and Objective:Non-alcoholic fatty liver disease(NAFLD)is one of the most prevalent metabolic diseases,yet its pathogenesis remains elusive.The aberrant lipid deposition in hepatocytes,so called hepatic steatosis,is the pathological basis of NAFLD.Diabetes is clsoely associated with NAFLD.Therefore,the investigation of molecular mechanisms concerning the abnormity of glucolipid metabolism is required for the development of new targeted treatments against NAFLD.Carbohydrate responsive element binding protein(Ch REBP)has been recognized as a transcription factor that plays a vital role in glycolysis and lipogenesis,which leads excessive activation of FASN gene(a key enzyme of de novo lipogenesis)to promote hepatic steatosis and also the progression of NAFLD.Given the high stability of the gene,signaling molecules generally influence gene alternative expression via mechanisms that do not alter the DNA sequence.Herein,epigenetic mechanisms are inherited through generations and cell divisions without any DNA sequence change due to the variation in structure and modification of chromatin.Epigenetics includes DNA methylation,histone modifications,mi RNAs,and chromatin remodeling.Through histone modifications,interaction between histones and DNA,as well as between the promoter region of the target genes and transcription factors can be affected,so that the condensation status of the chromatin can be altered to facilitate or inhibit transcription.Ch REBP induces FASN expression by directly binding to carbohydrate response elements(Cho RE)found in their promoters.However,histone modification in FASN gene modulated by Ch REBP is still unclear.In the current study,we examined the relation between histone modifications and FASN gene transcription.In additon,we determined effect of high glucose-Ch REBP signaling pathway to epigenetic modification in FASN gene.This study aims to provide a deeper insight into the pathophysiology of hepatic steatosis induced by high glucose stimuli,as well as to identify the patterns of FASN gene histone modifications as a novel strategy for NAFLD therapy based on epigenetic mechanisms.Methods:I.The relation between FASN histone modifications and gene transcription in hepatic steatosis.1.CCK8 was used to screen the optimal glucose concentration for human hepatocellular carcinoma cell line(Hep G2)and normal human cell line(L02).2.After Hep G2 and L02 cells were stimulated by glucose for 0,24 and48 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 L02 cells were stimulated by glucose for 0,1,2,4,8,12,24 and 48 h,respectively,q RT-PCR was utilized to measure the transcriptional time spectrum of FASN mRNA.4.Glucose stimulation was removed after 48 h glucose stimulation in Hep G2 and L02 cells;namely,the culture medium containing high glucose was replaced with common culture medium,which was followed by the measurement of the transcriptional time spectrum of FASN m RNA using q RT-PCR at 0,1,2,4,8,12,24 and 48 h later.5.According to the transcriptional time spectrum of FASN mRNA after glucose 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(Ch IP)assay.6.Based on the transcriptional time spectrum of FASN mRNA after the removal of glucose 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 Ch IP assay,including 4 regulatory sites: Cho RE,promoter,exon2 and 2000bp(-2kb)at the upstream of the transcription start site.II.Effect of high glucose-Ch REBP signal pathway on the histone modification of FASN gene in hepatocytes.1.After Hep G2 and L02 cells were stimulated by glucose for 0,24 and 48h,respectively.Western-blot was used to detect the nuclear protein expression level of Ch REBP.2.After Hep G2 and L02 cells were transiently transfected withsi RNA-Ch REBP plasmid or with Ch REBP-overexpression plasmid,protein level of FASN were measured by Western-blot.3.Hep G2 and L02 cells were divided into 4 groups:normal group(common culture medium),positive control group(high glucose stimulation),transfection group(high glucose stimulation after 48 h transfection with si RNA-Ch REBP plasmid or 48 h transfection with Ch REBP-overexpression plasmid),and negative control group(high glucose stimulation after 48 h transfection with GV102-scramble plamid or 48 h transfection with p EX3-scramble plasmid);the histone Ch REBP-Cho RE modification level of transcriptional regulatory region in FASN gene was measured by Ch IP assay.III.Effect ofhistone acetylationto lipid synthesis in hepatocytes.Hep G2 and L02 cells were divided into 3 groups: normal group(treated with common culture medium),glucose + inhibitor group(treated with garcinol + glucose for 24h),and glucose group(treated with glucose for24h);the histone acetylation modification level of FASN gene was detected by Ch IP assay,and the m RNA and protein expression level of FASN were measured by q RT-PCR and Western-blot,respectively;and lipid deposition was determined with TG assay kit and oil red O stain.Results:I.The relation between FASN histone modifications and gene transcription in hepatic steatosis.1.CCK8 analysis revealed that 50 mM and 33 mM were the best glucose concentration for Hep G2 and L02,respectively.2.Western-blot analysis revealed that high glucose significantly increased FASN protein expression of both Hep G2 and L02 cells in a time-dependent manner.Similarly,TG contents in both Hep G2 and L02 cells increased over time.Fat droplets of both Hep G2 and L02 cells measured by oil o red was in the same pattern as western-blot and thehepatic TG content assay have shown.Compared with 0h,the levels of lipogenesis significantly increased after high glucose incubation for24 or 48 h.3.Compared with 0h,FASN m RNA expressions were gradually increased with time of 12 h and 8h in the presence of glucose for both Hep G2 and L02 cells.After high glucose removal,the increasing range of FASN m RNA was significantly declined with time in both Hep G2 and L02 cells,respectively.These experiments were used to determine suitable time points for the following experiments.4.In the high glucose situation,we observed significant increases in H3 and H4 acetylation,H3K4 trimethylation and H3S10 phosphorylation and a decrease in H3K9 and H4K20 trimethylation within the Cho RE,promoter and exon2 regions in Hep G2(12h exposure of high glucose)and L02(8h exposure of high glucose)cells compared with controls(0h exposure of high glucose).In this section,whether in the presence of high glucose after its removal,on change of epigenetic marks was detectedin-2kb in both hepatocytes compared with controls.5.In contrast,when high glucose was removed for 1h,we observed the increasing range of H3 and H4 acetylation,H3S10 phosphorylation,H3K4 trimethylation was decreased,and H3K9 and H4K20 trimethylation was increased in FASN regulatory regions in Hep G2 and L02 cells compared with controls(0h: time point of the removal of high glucose).In this section,whether in the presence of high glucose after its removal,no change of epigenetic marks was detectedin-2kb in both hepatocytes compared with controls.II.Effect of high glucose-Ch REBP signal pathway on the histone modification of FASN gene in hepatocytes.1.Western-blot analysis revealed the expression of nuclear Ch REBP was distinctly enhanced as glucose incubation time went on,which was accompanied by elevated intracelluar lipogenesis.2.When stimulated by high glucose,the Ch REBP-Cho RE binding in both Hep G2 and L02 cells increased almost linearly,as did the FASN expression.3.After high glucose removal,we found that the increasing range of Ch REBP-Cho RE binding decreased linearly,which was consistent with the change of FASN m RNA expression levels.4.After 48 h transfection of si RNA-Ch REBP only,FASN protein expression was reduced in both Hep G2 and L02 cells compared with normal group.And after 48 h transfection of Ch REBP-overexpression,FASN protein expression was greatly increased in both Hep G2 and L02 cells compared with normal group.5.we examined the effects of Ch REBP knockdown on histone modifications of FASN.The Ch REBP-Cho RE binding of FASN was weaker in the transfection group than in the positive and negative controls in both cell lines.FASN histone hypo-acetylation,H3S10hypo-phosphorylation,H3K4 hypo-trimethylation and H3K9 and H4K20 hyper-trimethylation at Cho RE,promoter and exon2 regions were observed in the transfection group compared to the positive and negative controls.6.we examined the effects of Ch REBP overexpression on histone modifications of FASN.The Ch REBP-Cho RE binding,histone acetylation,H3S10 phosphorylation,and H3K4 trimethylation of FASN regulatory regions were increased in the transfection group compared with positive control.In contrast,H3K9 and H4K20 trimethylation dropped off in the transfection group compared with normal,positive and negative controls.In this section,whether the transfection of si RNA-Ch REBP or Ch REBP overexpression plasmid,there was on change of epigenetic marks at-2kb among the four groups.III.Effect of histone acetylation to lipid synthesis in hepatocytes.Garcinol/high glucose caused histone hypo-acetylation of FASN compared with glucose group,but still resulted in a increase compared with normal group.Cells in glucose + garcinol group failed to promote FASN protein and m RNA expression relative to glucose group.The results of TG content assay and Oil red O staining were consistent with FASN expression.All the evidence mentioned above indicated that garcinol could alleviate TG biosynthesis and lipid accumulation in both Hep G2 and L02 cells in response to garcinol/ high glucose cocktail treatment.Conclusion:The activation of FASN gene transcription was caused by the modification level increased in H3 and H4 acetylation and H3K4 trimethylation while decreased in histone H3K9 and H4K20 trimethylation.Glucose exerts an influence on FASN gene transcription expression by regulating the expression level and the transcription activity of Ch REBP,and the possible mechanism for it can be described as follows: Ch REBP inhibits trimethylation of H3K9 and H4K20,facilitates the Ch REBP-Cho RE combination in a promoter region and plays a role of regulation on FASN gene transcriptions by inducing H3 and H4 acetylation,H3K4 trimethylation and H3S10 phosphorylation for the FASN gene.In cases of glucose stimulation and Ch REBP expression increase,inhibition of FASN gene histone acetylation gives rise to the decrease in Ch REBP-Cho RE combinations,which further results in FASN expression decrease and hepatocyte fatty degeneration degree lessening.It proves that the combination between Ch REBP and Cho RE of FASN gene relies on histone acetylation;and the transcriptional activity of Ch REBP must be exerted on the premise that histone acetylation has given rise to conformational changes in FASN chromatin and finally plays a role of regulating FASN gene transcriptions.