Valproate-induced Epigenetic Upregulation Of Hypothalamic Fto Expression Linked With Increases Of Body Weight And Food Intake

IntroductionValproate（VPA）is a commonly-used drug for treatment of several neurological and neuropsychiatric disorders including epilepsy,chronic pain and bipolar disorder.Both clinical and animal models strudies suggest that VPA leads to weight gain and metabolic changes accompanied by fatty liver.Accumulating evidences support that VPA can alter the levels of serum metabolites and hormones such as glucose,insulin and leptin,and increase the risk of developing insulin resistance which are believed to be associated with the VPA-associated obesity.Other studies suggest that VPA results in alterations in the levels of several neurotransmitters in the hypothalamus,implicating an important role of VPA in the central control of food intake and body weight.VPA,a histone deacetylase（HDAC）inhibitor for inducing histone acetylation and DNA demethylation,is involved in the epigenetic regulation of gene expression.In the hypothalamus,VPA has been demonstrated to regulate the expression of multiple genes,which may be related to hypothalamic dysfunction and weight gain.However,its detailed mechanism remains to be further studied.Recent studies found that the intronic single nucleotide polymorphisms（SNPs）of fat mass and obesity-associated（FTO）gene are strong associated with appetite and body mass regulation,and the direct evidences from the genetic animal models that overexpression of FTO leads to increased appetite and obesity and loss of this protein results in lean phenotypes.Our recent evidence demonstrate an important role of the homeostatic expression of hypothalamic FTO in the regulation of weight balance.Our another study found that VPA could induce upregulation of FTO in the hippocampus.Thus,we propose that VPA should also promote FTO expression in hypothalamus,which may be associated with VPA-induced weight gain.ObjectiveThe purpose of this study is to analyze the epigenetic regulatory mechanism of VPA-induced FTO up-regulation in the hypothalamic cells and tissues by promoting histone H3/H4 acetylation and impairing the auto-negative feedback loop of Fto gene,which increase the binding ability of transcription factor TAF1 and FTO gene promoter to enhance the transcription activity of Fto gene.This study will reveal the epigenetic regulatory mechanism of VPA-induced hypothalamic FTO up-regulation,and analyze its relationship with increases in body weight and food intake.Methods1.In vitro analyses:（1）qRT-PCR and Western blot were used to determine the Fto mRNA and protein levels in hypothalamic GT1-7 cells treated with VPA.（2）Western blot was used to detect the status of histone H3/H4 acetylation in GT1-7 cells after VPA treatment and/or histone acetyltransferase P300 inhibitor C646.（3）Chromatin immunoprecipitation（ChIP）analysis was used to analyze the bindings of TAF1 and FTO to the Fto gene promoter in GT1-7 cells after VPA treatment.2.In vivo analyses:（1）Assessments of body weight and food intake:C57BL/6 mice were fed with normal diet with or without VPA for 13 weeks;or with VPA and/or C646 by intraperitoneal injection with for 2 weeks.Fto knockout and wild-type mice were fed with normal diet with or without VPA for 2 weeks,their body weight and food intake were recorded daily.（2）qRT-PCR and Western blot were used to detect the Fto mRNA and protein levels in hypothalamus of the mice treated described above.（3）Western blot was used to detect the status of histone H3/H4 acetylation in hypothalamus.（4）ChIP-qPCR analysis was used to determine the bindings of TAF1 and FTO to the Fto gene promoter using hypothalamic immunoprecipitate.Results1.The qRT-PCR and Western blot results showed that the Fto mRNA and protein levels in the VPA-treated GT1-7 cells gradually increased with the VPA titrated from 0 to 8 mM,suggesting that VPA could induce upregulation of the Fto gene expression in the GT1-7 cells.2.Compared with the control cells,the proportions of Ac-H3（Ac-H3/H3）and Ac-H4（Ac-H4/H4）were gradually increased along with the VPA titrated from 0 to 8mM.Furthermore,C646,an inhibitor of histone acetyltransferase P300,could reverse the effect of VPA on histone H3/H4 acetylation and Fto expression.These data demonstrate that VPA could up-regulated the expression of Fto gene by promoting the acetylation of histone H3/H4.3.The ChIP PCR showed that VPA could weaken the binding of FTO and enhance the binding of transcription factor TAF1 to Fto gene promoter,which up-regulated the Fto expression.4.The body weight and food intake of the VPA treated mice were significantly higher than those of the control mice from the 2^nd week to the 13^th week,indicating that VPA could promote the weight gain by increasing food intake.5.The weight and food intake of the mice treated with VPA plus C646significantly decreased compared with that of the mice treated with VPA alone,suggesting that C646 could reverse the effect of VPA on body weight and food intake.6.Further studies showed that VPA could significantly enhance the expression of FTO in hypothalamus,promote the acetylation of histone H3/H4,weaken the binding of FTO to its own gene promoter,and enhance the binding of TAF1;while VPA plus C646 could reverse the effects of VPA described above.These data demonstrate that VPA up-regulates the expression of hypothalamic FTO by blocking the auto-feedback loop of Fto gene through the epigenetic regulatory pathway.7.Compared with the Fto knockout mice without VPA,no significant change in the body weight and food intake of the Fto knockout mice treated with VPA,indicating that FTO was indeed involved in the VPA-induced increase of body weight and food intake.ConclusionTaken together,these data demonstrate that VPA impairs the auto feedback loop of the Fto gene by promoting the acetylation of histone H3/H4,and then increases the hypothalamic FTO levels which lead to the increase of body weight and foodintake.Thus this study reveals an epigenetic regulatory mechanism of VPA’s actionon the up-regulation of the Fto gene,which should play an important role in the central control of body weight and food intake.