Hif-2α Regulates Lipid Metabolism Of Alcoholic Fatty Liver Through Mitophagy

Drinking and ALD is a major public health problem in the United States and worldwide.AFLD is the earliest stage of ALD.It characterized by lipid accumulation and could further develop towards more severe lesions such as alcoholic steatohepatitis(ASH),alcoholic cirrhosis(AC)and hepatocellular carcinoma(HCC)in hepatocytes.So it is critical to find effective targets and treatments for lipid accumulation and block ALD at the initial stage.Studies have showed that oxidative damage of fatty acids has been demonstrated to be involved in the etiology of AFLD.Hypoxia-inducible factor-2a(Hif-2α)played a vital role in lipid accumulation by regulating fatty acid β-oxidation.This study is aim to further study the effect of Hif-2α in the development of AFLD and the possible molecular mechanism of Hif-2α in regulating hepatic lipid metabolism.The research contents are as follows:1.Expression of Hif-2α in ethanol-feeding mice and ethanol-induced AML-12cellsIn order to create a AFLD model in mice,we adopted the Gao’s modeling method.As expected,ethanol feeding significantly increased liver lipid droplets accumulation,the liver-to-body ratio,serum AST and ALT activities,T-CHO and TG levels,as well as enhanced liver damage in H&E staining.The result of Oil Red O staining showed that there were lipid deposition in AML-12 cells after treatment with100 m M ethanol for 48 h in vitro.On the other hand,the expression level of CPT-1α and MCAD,two key enzymes involved in the regulation of fatty acids β-oxidation were down-regulated in ethanol-feeding mice and ethanol-induced AML-12 cells.Next,the IHC staining、m RNA and Western blotting results consistently showed that Hif-2α was highly expressed in ethanol-feeding mice and ethanol-induced AML-12 cells.In addition,IF staining uncovered that Hif-2α was significantly elevated in the nucleus after treatment with Et OH for 48h in AML-12 cells,this founding was further demonstrated by Western blotting analysis.2.Effect of Hif-2α on fatty acid β-oxidation of ethanol-induced AML-12 cells and ethanol-feeding miceIn AML-12 cells,Hif-2α-siRNA was used to establish a silencing Hif-2α cell model.Then,we treated cells with 100 m M ethanol.The MCAD and CPT-1α proteins were distinctly up-regulated by Hif-2α-siRNA,compared with the negative control‐transfected cells.TG analysis in the cell supernatant also showed the diminution of lipid deposition by Hif-2α-siRNA.Meanwhile,when we treated ethanol-induced AML-12 cells with 2.0 u M PT2399 for 48 h,a highly effective and selective Hif-2α antagonist,the expression levels of fatty acid β-oxidation-related proteins: CPT-1α and MCAD were enhanced.Oil red O staining and TG analysis also showed the reduction of lipid droplets accumulation in the PT2399 group.To further identify a potential relationship between fatty acid β-oxidation and Hif-2α,PT2399 was daily intragastric administrated in mice at 10mg/kg for one week on the 10 nd day of chronic-plus-binge feeding mice.The Oil Red O and H&E staining showed the decreased liver lipid droplets accumulation and liver damage,respectively.The liver-to-body ratio,serum AST and ALT activities,T-CHO and TG levels were significantly reduced by PT2399.Apart from these observations,we also examined the expression level of MCAD,CPT-1α proteins in the same liver tissues,which were up-regulated by PT2399.3.Effect of Hif-2α on BNIP3-dependent mitophagy of ethanol-induced AML-12 cells and ethanol-feeding miceIn AML-12 cells,we found that ethanol inhibited the expression level of mitophagy-related proteins,including BNIP3,Beclin1 and LC3II/LC3.Likewise,IF analysis showed the co-localization of LC3 and Mito tracker Red(MTR)was decreased in ethanol-induced AML-12 cells,as well as a reduced fluorescence punctum of BNIP3 and LC3.To determine the effect of Hif-2α on ethanol-induced mitophagy,Hif-2α-siRNA was transfected into AML-12 cells.Western blotting analysis showed that the expression level of mitophagy-related proteins was boosted in the Hif-2α-siRNA group,which was consistent with the PT2399 group.Simultaneously,IF analysis revealed the co-localization of LC3 and mitochondria and the fluorescence punctum of BNIP3 and LC3 were increased in the Hif-2α-siRNA or PT2399 group.However,the expression level of mitophagy-related proteins was inhibited in the Hif-2α-siRNA and BNIP3-shRNA co-treatment group.In vivo,compared to the control-fed group,we also found ethanol inhibited the expression levels of mitophagy-related genes and proteins,which were reversed by PT2399.Furthermore,the ultra-structures of hepatic tissues were analyzed by TEM.Mitochondria displayed normal shape and active fission in the control-fed mice liver,while displayed swollen without fission shape in the Et OH-fed mice liver.Besides,the number of lipid vacuoles were increased but the autophagosomes were decreased in the Et OH-fed mice liver.More importantly,PT2399 treatment recovered a large proportion of damaged mitochondria to nearly intact mitochondrial morphology and mitochondrial fission.Concurrently,the lipid vacuoles were decreased and the autophagosomes were increased in the PT2399 group,compared to vehicle group.4.The regulatory mechanism of Hif-2α on fatty acid β-oxidation in ethanol-induced AML-12 cells and ethanol-feeding miceIn order to further explore the mechanism of Hif-2α regulating fatty acidβ-oxidation,we detected the expression level of PPAR-α/PGC-1α signaling pathway-related proteins.As shown in Western blotting results,both Hif-2α-siRNA and PT2399 apparently promoted the expression level of PPAR-α/PGC-1α signaling pathway-related proteins.In addition,we transfected pc-DNA3.1-BNIP3 into ethanol-induced AML-12 cells to establish overexpression cell models,the expression level of BNIP3-dependent mitophagy-related proteins was greatly enhanced.IF analysis also revealed the co-localization of LC3 and mitochondria and the fluorescence punctum of BNIP3 and LC3 were improved by pc-DNA3.1-BNIP3,compared to the negative control‐transfected cells.Beyond these,the expression level of PGC-1α and PPAR-α,MCAD and CPT-1α proteins were increased after pc-DNA3.1-BNIP3 treatment.Afterwards,the expression level of PGC-1α and PPAR-α,MCAD and CPT-1α proteins were noticeably declined in the Hif-2α-siRNA and BNIP3-shRNA co-treatment group.In general,these observations displayed that Hif-2α negative regulates hepatic fatty acid β-oxidation through PPAR-α/PGC-1α signaling pathway in BNIP3-dependent mitophagy manner.