High-Fat Diet-Induced DeSUMOylation of E4BP4 Promotes Lipid Droplet Biogenesis and Liver Steatosis in Mice.

Dysregulated lipid droplet accumulation has been identified as one of the main contributors to liver steatosis during nonalcoholic fatty liver disease (NAFLD). However, the underlying molecular mechanisms for excessive lipid droplet formation in the liver remain largely unknown. In the current study, hepatic E4 promoter-binding protein 4 (E4BP4) plays a critical role in promoting lipid droplet formation and liver steatosis in a high-fat diet (HFD)-induced NAFLD mouse model. Hepatic E4bp4 deficiency (E4bp4-LKO) protects mice from HFD-induced liver steatosis independently of obesity and insulin resistance. Our microarray study showed a markedly reduced expression of lipid droplet binding genes, such as Fsp27, in the liver of E4bp4-LKO mice. E4BP4 is both necessary and sufficient to activate Fsp27 expression and lipid droplet formation in primary mouse hepatocytes. Overexpression of Fsp27 increased lipid droplets and triglycerides in E4bp4-LKO primary mouse hepatocytes and restored hepatic steatosis in HFD-fed E4bp4-LKO mice. Mechanistically, E4BP4 enhances the transactivation of Fsp27 by CREBH in hepatocytes. Furthermore, E4BP4 is modified by SUMOylation, and HFD feeding induces deSUMOylation of hepatic E4BP4. SUMOylation of five lysine residues of E4BP4 is critical for the downregulation of Fsp27 and lipid droplets by cAMP signaling in hepatocytes. Taken together, this study revealed that E4BP4 drives liver steatosis in HFD-fed mice through its regulation of lipid droplet binding proteins. Our study also highlights the critical role of deSUMOylation of hepatic E4BP4 in promoting NAFLD.

Fructose Protects Against Acetaminophen-Induced Hepatotoxicity Mainly by Activating the Carbohydrate-Response Element-Binding Protein α-Fibroblast Growth Factor 21 Axis in Mice.

Acetaminophen (N-acetyl-para-aminophenol [APAP]) overdose is the most common cause of drug-induced liver injury in the Western world and has limited therapeutic options. As an important dietary component intake, fructose is mainly metabolized in liver, but its impact on APAP-induced liver injury is not well established. We aimed to examine whether fructose supplementation could protect against APAP-induced hepatotoxicity and to determine potential fructose-sensitive intracellular mediators. We found that both high-fructose diet feeding before APAP injection and fructose gavage after APAP injection reduced APAP-induced liver injury with a concomitant induction of the hepatic carbohydrate-response element-binding protein α (ChREBPα)-fibroblast growth factor 21 (FGF21) pathway. In contrast, Chrebpα liver-specific-knockout (Chrebpα-LKO) mice failed to respond to fructose following APAP overdose, suggesting that ChREBPα is the essential intracellular mediator of fructose-induced hepatoprotective action. Primary mouse hepatocytes with deletion of Fgf21 also failed to show fructose protection against APAP hepatotoxicity. Furthermore, overexpression of FGF21 in the liver was sufficient to reverse liver toxicity in APAP-injected Chrebpα-LKO mice. Conclusion: Fructose protects against APAP-induced hepatotoxicity likely through its ability to activate the hepatocyte ChREBPα-FGF21 axis.

miR-3 Encoded by Hepatitis B Virus Downregulates PTEN Protein Expression and Promotes Cell Proliferation.

PURPOSE: Chronic hepatitis B virus (HBV) infection is a key determinant of hepatocellular carcinoma (HCC). However, the mechanism by which HBV contributes to the development of HCC remains to be further explored. HBV-encoded miR-3 (HBV-miR-3) is a newly discovered microRNA that can affect the replication of HBV, but its influence on host genes is unclear. The tumor suppressor phosphatase and tensin homolog (PTEN) is expressed at low levels in most cancer cells. How HBV-miR-3 acts on PTEN to induce tumorigenesis has not been clarified. MATERIALS AND METHODS: PTEN protein expression was evaluated in HBV-miR-3-transfected cells and HBV-related liver cancer and paracancerous tissues. A luciferase reporter assay was employed to identify the HBV-miR-3 binding site on the 3'-untranslated region (3'-UTR) of PTEN. Cell apoptosis was assessed by flow cytometry. Cell proliferation was evaluated by colony formation assays. Transwell assays were used to detect cancer cell invasion. RESULTS: HBV-miR-3 was identified only in HBV-replicating HCC cells and HBV-infected patients. HBV-miR-3 expression in liver cancer tissues was higher than that in paracancerous tissues, and the corresponding PTEN expression was significantly decreased. Wild-type HBV-miR-3 bound to the 3'-UTR of PTEN and downregulated its protein expression in a dose-dependent manner. Moreover, the inhibition of HBV-miR-3 rescued PTEN protein expression. Furthermore, HBV-miR-3 reduced liver cancer cell apoptosis, enhanced cell invasion, and promoted cell proliferation. CONCLUSION: HBV-miR-3 binds to the 3'-UTR of PTEN mRNA and downregulates PTEN protein expression, thereby reducing cell apoptosis and enhancing cell invasion and proliferation. These results indicate that HBV-miR-3 contributes to the development of HBV-related HCC and may be a therapeutic target for this cancer.

Hepatic E4BP4 induction promotes lipid accumulation by suppressing AMPK signaling in response to chemical or diet-induced ER stress.

Prolonged ER stress has been known to be one of the major drivers of impaired lipid homeostasis during the pathogenesis of non-alcoholic liver disease (NAFLD). However, the downstream mediators of ER stress pathway in promoting lipid accumulation remain poorly understood. Here, we present data showing the b-ZIP transcription factor E4BP4 in both the hepatocytes and the mouse liver is potently induced by the chemical ER stress inducer tunicamycin or by high-fat, low-methionine, and choline-deficient (HFLMCD) diet. We showed that such an induction is partially dependent on CHOP, a known mediator of ER stress and requires the E-box element of the E4bp4 promoter. Tunicamycin promotes the lipid droplet formation and alters lipid metabolic gene expression in primary mouse hepatocytes from E4bp4flox/flox but not E4bp4 liver-specific KO (E4bp4-LKO) mice. Compared with E4bp4flox/flox mice, E4bp4-LKO female mice exhibit reduced liver lipid accumulation and partially improved liver function after 10-week HFLMCD diet feeding. Mechanistically, we observed elevated AMPK activity and the AMPKß1 abundance in the liver of E4bp4-LKO mice. We have evidence supporting that E4BP4 may suppress the AMPK activity via promoting the AMPKß1 ubiquitination and degradation. Furthermore, acute depletion of the Ampkß1 subunit restores lipid droplet formation in E4bp4-LKO primary mouse hepatocytes. Our study highlighted hepatic E4BP4 as a key factor linking ER stress and lipid accumulation in the liver. Targeting E4BP4 in the liver may be a novel therapeutic avenue for treating NAFLD.

DDB1 E3 ligase controls dietary fructose-induced ChREBPα stabilization and liver steatosis via CRY1.

Fructose over-consumption contributes to the development of liver steatosis in part by stimulating ChREBPα-driven de novo lipogenesis. However, the mechanisms by which fructose activates ChREBP pathway remain largely undefined. Here we performed affinity purification of ChREBPα followed by mass spectrometry and identified DDB1 as a novel interaction protein of ChREBPα in the presence of fructose. Depletion and overexpression of Ddb1 showed opposite effects on the ChREBPα stability in hepatocytes. We next tested the impact of hepatic Ddb1 deficiency on the fructose-induced ChREBP pathway. After 3-week high-fructose diet feeding, both Ddb1 liver-specific knockout and AAV-TBG-Cre-injected Ddb1flox/flox mice showed significantly reduced ChREBPα, lipogenic enzymes, as well as triglycerides in the liver. Mechanistically, DDB1 stabilizes ChREBPα through CRY1, a known ubiquitination target of DDB1 E3 ligase. Finally, overexpression of a degradation-resistant CRY1 mutant (CRY1-585KA) reduces ChREBPα and its target genes in the mouse liver following high-fructose diet feeding. Our data revealed DDB1 as an intracellular sensor of fructose intake to promote hepatic de novo lipogenesis and liver steatosis by stabilizing ChREBPα in a CRY1-dependent manner.

The hepatic BMAL1/AKT/lipogenesis axis protects against alcoholic liver disease in mice via promoting PPARα pathway.

Alcohol liver disease (ALD) is one of the major chronic liver diseases worldwide, ranging from fatty liver, alcoholic hepatitis, cirrhosis, and potentially, hepatocellular carcinoma. Epidemiological studies suggest a potential link between ALD and impaired circadian rhythms, but the role of hepatic circadian proteins in the pathogenesis of ALD remains unknown. Here we show that the circadian clock protein BMAL1 in hepatocytes is both necessary and sufficient to protect mice from ALD. Ethanol diet-fed mice with liver-specific knockout (Bmal1-LKO) or depletion of Bmal1 develop more severe liver steatosis and injury as well as a simultaneous suppression of both de novo lipogenesis and fatty acid oxidation, which can be rescued by the supplementation of synthetic PPARα ligands. Restoring de novo lipogenesis in the liver of Bmal1-LKO mice by constitutively active AKT not only elevates hepatic fatty acid oxidation but also alleviates ethanol-induced fatty liver and liver injury. Furthermore, hepatic over-expression of lipogenic transcription factor ChREBP, but not SREBP-1c, in the liver of Bmal1-LKO mice also increases fatty acid oxidation and partially reduces ethanol-induced fatty liver and liver injury. Conclusion: we identified a protective role of BMAL1 in hepatocytes against ALD. The protective action of BMAL1 during alcohol consumption depends on its ability to couple ChREBP-induced de novo lipogenesis with PPARα-mediated fatty oxidation. (Hepatology 2018).