Suppression of E4BP4 SUMOylation sensitize mice to HFD‐induced fatty liver disease by promoting lipid droplet formation

Abstract

Background As the most common chronic liver disease, non-alcoholic fatty liver disease (NAFLD) affects 10-30% of the general population. Characterized by excessive lipid droplet formation in hepatocytes, fatty liver may progress to nonalcoholic steatohepatitis (NASH), eventually leading to cirrhosis and liver failure. Currently, there have been no effective therapeutic or preventive measures for NAFLD. Thus, there is an urgent need to identify hepatic mediators that promote lipid droplet accumulation as well as the underlying molecular mechanisms in the liver during the pathogenesis of NAFLD. We previously reported that E4 promoter-binding protein 4 (E4BP4) is an insulin-induced stabilizer of the lipogenic factor SREBP-1c and promotes the SREBP-1c-mediated lipogenesis in hepatocytes. Our most recent mass spectrometry data revealed that E4BP4 may undergo SUMOylation, a reversible post-translational modification that was shown to regulate E4BP4-dependent NK cell development. In this study, we set out to determine how hepatic E4BP4 regulates lipid droplet formation and liver steatosis a high-fat diet-induced NAFLD mouse model and investigate how E4BP4 SUMOylation impacts the process. Results Compared with E4bp4flox/flox mice, E4bp4 liver-specific KO (E4bp4-LKO) mice exhibit decreased lipid accumulation in the liver despite similar body weight after 12 weeks of high-fat diet feeding. Our microarray analysis showed a reduction in the expression of lipid droplet biding genes such as Cidea, Cidec (Fsp27), Plin4 in the liver of E4bp4-LKO mice. Restoring E4bp4 expression in E4bp4-LKO primary hepatocytes was sufficient to induce lipid droplet formation and Fsp27β expression, while overexpression of Fsp27β increased lipid droplets and triglycerides in E4bp4-LKO primary mouse hepatocytes and promoted lipid accumulation HFD-fed E4bp4-LKO mice. Meanwhile, we generated an E4BP4 mutant, namely E4BP4-5KR, which contains five lysine to arginine (5KR) substitutions in the five SUMOylation motifs. Our in vitro SUMOylation assay confirmed the SUMOylation of E4BP4-WT but not E4BP4-5KR mutant in the presence of E1/2, SUMO2/3, and ATP. We detected more lipid droplet formation in hepa1 cells transfected with E4bp4-5KR vs. E4bp4-WT. Lastly, the abundance of SUMOylated E4BP4 was reduced in the liver of high-fat diet-fed mice vs. regular chow-fed mice. Conclusion All together, our data demonstrated that E4bp4 drives lipid droplet formation and live steatosis in high-fat diet-fed mice likely through its regulation of lipid-droplet binding genes. Our study also highlights the critical role of deSUMOylation of hepatic E4BP4 in promoting NAFLD and suggests that E4BP4 SUMOylation in the liver could be targeted for treating NAFLD.