Study On Mechanisms By Which E4BP4 Promote The Development And Progression Of Non-alcoholic Fatty Liver Disease

Background:Non-alcoholic fatty liver disease（NAFLD）is the most common chronic liver disease worldwide,which was first described in the 1980s.The prevalence rates of NAFLD,in most studies,are now increasing in parallel with the increasing numbers with obesity and other components of metabolic syndrome,sometimes,the patients could be lean.According to the histology,NAFLD is divided into 1)simple steatosis without inflammation and 2)non-alcoholic steatohepatitis（NASH）with the presence of inflammation,hepatocyte ballooning,which may result in advanced fibrosis or even cirrhosis and liver failure.Among patients with NASH,cardiovascular diseases and malignancy attribute about half of the deaths,and the third leading cause is cirrhosis.Although clinicians and researchers put lots of effort into and made some progressions on the understanding of NAFLD in recent years,the mechanisms of NAFLD pathogenesis and progression remain largely unknown,which means no effective clinic therapeutic strategies.Dysregulated lipid droplet accumulation is reported to be one of the critical triggers to liver steatosis during the development of NAFLD,usually due to excessive intake of high-calorie food.Unrealized stimulus often lead to NASH,which is characterized by the presence of inflammation and hepatocellular ballooning.Persistent activation of hepatic stellate cells is the key point of the onset of hepatic fibrosis,which is the main determining factor of outcomes of NAFLD.Although the reverse of fibrosis is possible after eliminating the stimulus,unchecking chronic injury can lead to the distortion of hepatic architecture and cirrhosis can be developed eventually.However,it remains largely unclear why superabundant lipid droplets accumulated,and hepatic stellate cells got continuously activated in the liver.B-zip transcription factor（nuclear factor interleukin,Nfil3;also known as E-box binding protein4,E4bp4）plays a critical role in immunoregulation,circadian rhythm and de novo lipogenesis.We found that E4BP4 augments the SREBP-1c-mediated de novo lipogenesis via enhancing nuclear SREBP-1c acetylation.We also reported that hepatic E4bp4 deficiency lowered liver steatosis induced by high-fat,low-methionine,and choline-deficiency diet（HFLMCD diet）and partially improved liver function.Besides,compared to the E4bp4^flox/floxmice,E4bp4-LKO mice showed higher levels of AMPK activity and AMPKβ1abundance.Our study highlighted hepatic E4BP4 might promote hepatic lipid accumulation via enhancing AMPKβ1 ubiquitination and degradation and inhibiting AMPK activity.Altogether,the evidence shown above suggests that E4BP4 might take part in the development of NAFLD via enhancing hepatic lipid formation and accumulation.Thus,this study will focus on the detailed mechanisms by which E4BP4 participates in NAFLD and provide some inspirations for the clinical treatment of NAFLD.Objectives:To explore the potential effects of E4BP4 on the occurrence and progression of NAFLD and underlying mechanisms.Methods:1.The homologous gene targeting technique generated E4bp4^flox/floxmice and E4bp4-LKO mice were applied for high-fat diet-induced NAFLD model.Insulin-tolerance test and glucose tolerance test were performed to check the effect of the hepatic E4bp4 deficiency on the glucose metabolism.We also measured serum Alanine transaminase（ALT）and lipids levels in the serum and liver,which was verified with histology evidence.Microarray analysis was performed to locate the potential downstream target genes of E4bp4 associated with liver injury,lipid metabolism,hepatic fibrosis,and immunoreaction.2.Primary mouse hepatocytes（PMHs）isolated from E4bp4^flox/flox mice and E4bp4-LKO mice were transduced with Ad-Lac Z or Ad-E4bp4 before oleic acid incubation overnight,then BODIPY staining were performed to stain the lipid droplets and RT-q PCR was applied to check the potential downstream target genes of E4bp4.The expression level of Fsp27βwas also measured following E4bp4 knockdown by Ad-sh E4bp4 with or without palmitate acid and TNFαtreatment.Also,the abundance of CREBH,a potent transcription activator of Fsp27β,was measured following E4bp4overexpression in hepatocytes using western blotting,of which the activity was verified by luciferase assay.Besides,the lipids contents were measured to evaluate the effect of restoring Fsp27βin the liver of E4bp4-LKO mice after HFD feeding.3.Mass spectrometry was used to detect potential protein interactions or post-translation modifications of E4bp4 with the specific bands showed only in the E4bp4 overexpression cells on Coomassie blue staining.The effect of nutritional stress on the level of E4BP4 SUMOylation was determined with immunoprecipitation.The effect of E4BP4 SUMOylation deficiency was studied by treating hepatocytes with rapamycin,AICAR,forskolin and nor epinephrine,and its effect on the lipid formation and accumulation was evaluated with BODIPY staining.4.NASH diet was used to induce chronic hepatic fibrosis on E4bp4^flox/floxmice and E4bp4-LKO mice,and serum ALT,histology analysis,RNA-seq and RT-q PCR were applied to evaluate the hepatic fibrosis.Also,the activation and migration of hepatic stellate cells were also monitored following conditional medium treatment from hepatocytes with or without E4bp4 overexpression.Besides,we measured the effect of E4bp4 deficiency in the liver on hepatic stellate cells activation in vivo with acute fibrosis model using CCl₄ injection.Results:1.Loss of hepatic E4bp4 reduced the lipid accumulation and hepatic fibrosis in the HFD-induced liver steatosis model but is largely dispensable for the glucose metabolism body weight gain.Also,Fsp27βis a major downstream target of E4BP4.2.Overexpression of E4bp4 or Fsp27βrescued the induction of lipid droplets by oleic acid in the E4bp4-LKO originated PMHs.E4BP4 enhanced lipid droplet formation and Fsp27βexpression via interacting with CREBH in hepatocytes.Meanwhile,restoring Fsp27 expression in the liver of mice rescued liver steatosis induced by HFD feeding.3.E4BP4 can be SUMOylated in an ATP-dependent way.Nutritional stress reduced E4BP4 SUMOylation,while de-SUMOylation of E4BP4reduces the sensitivity of E4BP4 to the activation of c AMP-PKA pathway activation in hepatocytes and enhanced lipid droplet formation.4.Hepatic E4bp4 deficiency reduced liver injury and hepatic fibrosis result from the NASH diet feeding.Hepatic E4bp4 overexpression enhances hepatic stellate cells activation in vitro,and loss of hepatic protects mice against CCl₄-induced stellate cells activation.Conclusions:1.Hepatic E4BP4 drives NAFLD via enhancing the expression of lipid-droplet binding gene Fsp27βvia interacting with CREBH.2.Hepatic E4BP4 SUMOylation deficiency enhances its ability to induce lipid droplet formation,and nutritional stress-induced de SUMOylation of E4BP4 promotes NAFLD.3.Hepatic E4BP4 exacerbates liver steatohepatitis by enhancing the activation of stellate cells.There are 35 figures,19 tables,119 references in this paper.