Erythropoietin Alleviates Hepatic Steatosis And Surpresses Endoplasmic Reticulum Stress

Chapter 1 Erythropoietin alleviates hepatic steatosis by activating autophagyAims:Erythropoietin(EPO),besides its stimulatory effect on erythropoiesis,is beneficial to hepatic steatosis.However,its role in hepatic steatosis remains unexplored.Activating autophagy seems a promising mechanism for improving fatty liver disease.The aim of our study is to investigate whether its function is mediated by the activation of autophagy.Methods:Lipid content and expression levels of proteins related to autopaghy were determined in the liver of ob/ob mice with or without EPO treatment.Electron microscope was employed to observe the autophagosome accumulation.In palmitate(PA)-treated hepatocytes,the induction of autophagy after EPO treatment was indicated by western blot assay,transmission electron microscopy,and confocal microscopy.Moreover,co-localization of lipids and autophagosomes was evaluated by immunofluorescence double staining to evluate the effect of EPO on lipophagy.After suppressing autophagy by an inhibitor or small interfering RNA,the lipid accumulation was measured in EPO-treated hepatocytes.Results:Compared with that in control group,the lipid content in the liver of mice in EPO group was decreased.Moreover,EPO treatment induced autophagy as indicated by elevating expression levels of autopaghy related gene(LC3-II,beclin-1,ATG5 and ATG7),and increasing autophagosome formation and autophagic flux.Next,as supportive evidence,co-localization of lipids and autophagosomes was increased by EPO intervention in hepatocytes.Furthermore,block autophagy largely abrogated the TG-lowering effect of EPO in HepG2 cells.Conclusions:Taken together,these results suggest that EPO-induced alleviation of hepatic steatosis involves autophagic machinery.Chapter 2 SIRT1-mediated autophagy is required for EPOinduced alleviation of hepatic steatosisAims:Evidence showed that the role of erythropoietin(EPO)in alleviating hepatic steatosis by inducing autophagy.Sirtuinl(SIRT1),a vital regulator in hepatic metabolism,has emerged as a potent autophagy modulator by deacetylating auto phagyrelated genes(ATGs)and autophagy mediators.The present study is to investigate whether SIRT1-mediated autophagy is required for EPO-induced alleviation of hepatic steatosis.Methods:The protein expression level and activity of SIRT1 were determined in ob/ob mice and palmitate(PA)-treated HepG2 cells with EPO intervention.Immunocoprecipitation was employed to measure the deacetylation levels of microtubule-associated protein light chain 3(LC3-II).The protein expression levels of autophagy markers and lipid content were determined in HepG2 cells with siRNAmediated SIRT1 silencing or SIRT1 inhibitor EX-527.In liver-specific Sirt1 knockout(SIRT1-LKO)and its wild type(WT)mice,body weight,lipid content and protein expression levels of autophagy markers in liver tissues were measured after EPO treatment.Results:The protein expression level and activity of SIRT1 were up-regulated in ob/ob mice and PA-incubated HepG2 cells with EPO treatment.In addition,the result of immunocoprecipitation showed that the acetylation level of LC3 was decreased in EPOtreated hepatocytes.SIRT1 inhibition abrogated the effects of EPO on activating autophagy and alleviatiing steatosis in HepG2 cells.Compared with that in WT mice,the lipid-lowering effect of EPO was diminished in SIRT1-LKO mice.Moreover,the protein expression level of LC3-Ⅱ was comparable in SIRT1-LKO mice with or without EPO treatment.Conclusions:SIRT1-mediated autophagy is required for EPO-induced alleviation of hepatic steatosis,which might provide theoretical basis for investigating the role of EPO in metabolism.Chapter 3 EPO surpresses hepatic endoplasmic reticulum stress by SIRT1Aims:To investigate the role of EPO in inhibiting hepatic endoplasmic reticulum stress(ERS)and sought to determine whether its function is mediated by the activation of SIRT1.Methods:Palmitate(PA)or thapsigargin(Tg)-incubated primary hepatocytes were treated with EPO.The protein expression levels of SIRT1，GRP78,p-PERK/PERK and p-eIF2α/eIF2α were determined by western blot assay.Triglyceride kit was employed to detect the lipid content.Additionaly,the protein expression levels of these ERS markers were determined in hepatocytes with siRNA-mediated SIRT1 silencing.In high-fed diet liver-specific Sirt1 knockout mice(SIRT1-LKO)and its wild type(WT)mice,immunofluorescence staining and western blot assay were employed to determine the effect of EPO on ERS.Results:The lipid content,and protein expression levels of GRP78,p-PERK/PERK and p-eIF2α/eIF2α were increased in PA or Tg-incubated primary hepatocytes.However,these effects induced by PA or Tg were abrogated by EPO treatment.Furthermore,SIRT1 inhibition abrogated the role of EPO in inhibiting ERS in PA or Tg-incubated hepatocytes.Compared with that in WT mice,the protein expression levels of GRP78,p-PERK/PERK and p-eIF2α/eIF2α and were comparable in SIRT1LKO mice with EPO treatment.Conclusions:EPO alleviates ERS in hepatocytes through SIRT1,which might extend knowledge of the beneficial role of EPO in hepatic metabolism.Chapter 4 Decreased expression of protein disulfide isomerase is associated with impaired glucose metabolism in nonalcoholic fatty liver diseaseAims:To investigate the epigenetic modification and altered gene expression of protein disulfide isomerase Al(PDI)contribute to the development of type 2 diabetes mellitus(T2DM)in nonalcoholic fatty liver disease(NAFLD)patients and explore the potential underlying mechanism.Methods:66 biopsy-proven NAFLD subjects were enrolled and divided into T2DM group(n=28)and non-T2DM group(n=38).The expression level of PDI was determined by real time-PCR,western-blots and Immunohistochemistry.In addition,the mRNA levels of genes related to hepatic glucose metabolism were measured in NAFLD subjects.Moreover,pyrosequencing was employed to evaluate the methylation level of PDI gene.The associations between PDI expression level and clinical paramiters were performed by spearman analysis.Results:The mRNA and protein expression levels were lower in biopsy-proven NAFLD subjects with T2DM than those with non-T2DM.Spearman analysis showed that mRNA level of PDI was negatively correlated with FBG(r=-0.440),PBG(r=-0.381)and HbA1c(r=-0.261)in NAFLD subjects(all P<0.05).Meanwhile,the expression level of hepatic PDI was negatively correlated with genes related to hepatic gluconeogenesis(G6P,r=-0.539;PEPCK,r=-0.262,all P<0.05),and positively correlated with glycogen synthesis(r=0.308,P<0.05).Furthermore,hypermethylation at cg09221994 site in PDI was observed in the liver from NAFLD subjects with T2DM,as compared to those without T2DM.Conclusions:In the liver of subjects with co-exist NAFLD and T2DM,the decreased expression of PDI may contribute to the disturbance of glucose metabolism.