The Role Of Estrogen Receptor α In Bisphenol A-induced Lipid Deposition In Hepatocytes

BackgroundNon-alcoholic fatty liver disease(NAFLD)includes a series of pathological processes from lipid deposition to steatohepatitis,cirrhosis in liver.The prevalence of NAFLD is increasing globally.There are significant sex differences in the prevalence of NAFLD.The prevalence of NAFLD in premenopausal women is lower than that in men at the same age group,but postmenopausal women have similar prevalence with men at the same age group.It is speculated that estrogen receptor α(ERα)signaling pathway may play a protective role in liver lipid metabolism.As an ubiquitous endocrine-disrupting chemical in our environmental conditions,Bisphenol A(BPA)is a confirmed risk factor for NAFLD by disturbing estrogen signaling pathways.It has been reported that BPA has different effects on liver glucose and lipid metabolism in males and females.This study aims to investigate the role of ERa in BPA-induced lipid deposition in hepatocytes.MethodsIn our study,pregnant C57BL/6J mice were exposured to BPA(50 μg/kg/day)in water from Gestation day 6(GD6)to Postnatal day 21(PND21).After weaning the offsprings were fed with normal chow diet until PND36.Then the liver tissues from male and female offsprings were collected for transcriptomic sequencing.Based on the results,HepG2,AML 12 and primary hepatocytes were used to investigate the effects of BPA on the expression of genes related to glucose and lipid metabolism in vitro.CCK-8 was used to detected the effect of BPA on HepG2 cell viability.Quantitative Reverse Transcription Polymerase Chain Reaction(qRT-PCR)was used to detect the gene expression.2-NBDG was used to detect cellular glucose uptake in HepG2 and ERα-overexpressed HepG2.Bodipy and flow cytometry were used to analyze intracellular lipid content in HepG2 and ERα overexpression HepG2.QRT-PCR was used to detect the gene expression in ERa overexpression HepG2.The Accl promoter vector was constructed and dual-luciferase reporter genes were used to detect the regulatory mechanism of ERα and Acc1 in HEK-293T and ERα-overexpressed HEK-293T.Western blot were used to dected ACC1 in HepG2,AML12 and ERα-overexpressed HepG2 and AML12.Fifty μg per mL Cycloheximide(CHX)was used to detect the effect of BPA on ACC1 protein stability.ResultsBPA exposure during early life(GD6-PND21)had different effects on the gene expression in liver in males and females.The genes related to liver glucose and lipid metabolism in male were more susceptible to BPA exposure.The effect of BPA on HepG2 cell viability was non-linear.After 10 nM,100 nM,1 μM treatment for 24 h,the cell viability was increasing(P<0.05).After 100 nM,1 μM treatment for 48 h,the cell viability was decreasing(P<0.05).After 10 μM treatment for 72 h,the cell viability was increasing(P<0.05).After 10 nM treatment for 48 h,the SHBG,PPARy increased in HepG2.After 100 nM treatment for 48 h,the ESR1,SHBG,RIP140 increased in HepG2(P<0.05).After 100 nM treatment for 72 h,the ESR1 increased in HepG2(P<0.05).After 10 nM treatment for 48 h,the Chrebp,Glut2,Dgat2 increased in primary hepatocytes.After 100 nM treatment for 48 h,the Pparα,Glut2,Dgat2,Gk,Cd36 increased in in primary hepatocytes(P<0.05).After 10 nM,100 nM treatment for 48 h,the Pim3 increased in AML12(P<0.05).After 10 nM,100 nM treatment for 24 h,the glucose uptake increased by 115%and 105%,respectively in HepG2(P<0.05).After 10 nM,100 nM treatment for 48 h,the glucose uptake increased by 85%and 15%,respectively in HepG2(P<0.05).No effect on glucose uptake was observed in ERα-overexpressed HepG2.After 10 nM treatment for 48 h,the intracellular lipid content increased by 13%in HepG2(P<0.05).After 10 nM,100 nM treatment for 72 h,the intracellular lipid content increased by 27%and 41%,respectively in HepG2(P<0.05).After 10 nM treatment for 48 h,only in ERα-overexpressed HepG2,the intracellular lipid content increased by 16%(P<0.05).After 10 nM,100 nM treatment for 72 h,the intracellular lipid content increased by 29%and 23%,respectively in HepG2 with ERα overexpression(P<0.05).Dual-luciferase reporter genes showed that ERa overexpression directly increased the expression of Accl in HEK-293T(P<0.05).Acc1 was the target gene of ERα.BPA had no effect on Acc1 transcription activity in our study.After 10 nM,100 nM treatment for 48 h,ACC1 was increased in HepG2.And after 100 nM treatment for 48 h,ACC1 increased in AML 12(P<0.05).After treatment for 48 h,ACC1 was not affected by BPA in HepG2,AML 12,and ERα-overexpressed HepG2 and AML12.No changes of the ACC1 degradation were observerd in HepG2 and ERα-overexpressed HepG2.ConclusionBPA exposure during early life had different effects on the gene expression in liver in males and females.The male were more susceptible to BPA exposure.BPA can increase De novo lipogenesis by increasing ACC1 in cells.BPA increases glucose uptake and intracellular lipid content,and the effect of BPA on lipid deposition is reversed,at least in part,by ERa overexpression.