SelenoW Targeting PKM2-HIF-1 ? Pathway Ameliorates HFD-induced Mouse Fatty Liver

Selenium(Se)is an essential trace element in humans and animals,and plays an important role in defense against oxidative stress,thyroid hormone metabolism and inflammation.Se performs its biological function through selenoproteins.Selenoprotein W(SelenoW)is a crucial member of selenoprotein family.Based on SelenoW has ?1-?1-?2 structure and the-Cxx U-motif similar to thioreductin-like folding protein,it is generally believed to have redox activity.SelenoW is widly expressed in various tissues and is involved in skeletal muscle development and differentiation,as well as various cellular processes.At present,non-alcoholic fatty liver disease(NAFLD)is currently considered to be the most common liver disease worldwide,with an incidence of more than 33% in humans,which become one of the major social,economic and public health problems.Recent studies have indicated that high selenium content in serum can increase the risk of NAFLD.However,the role of SelenoW and the effect for NAFLD are still unclear,which is worthy of further study.In this study,the pc DNAFlag-Sleleno W recombinant plasmid was constructed and SelenoW interacting proteins were analyzed by co-immunoprecipitation,LC-MS,confocal co-localization and molecular docking.We constructed HFD-induced NAFLD model in wild-type and SelenoW knockout mice,and AML-12 cell model stimulated by high glucose/2DG.Application of H&E staining,oil red staining,transmission electron microscopy,transcriptome sequencing analysis,immunohistochemistry,TUNEL staining,flow cytometry,Hoechst staining,real-time quantitative fluorescence PCR,western blot and immunofluorescence were used to detect the expression of glycolysis,cell apoptosis and fat metabolism related genes in liver tissue.The purpose of this study is to investigate the mechanism of SelenoW targeting PKM2-HIF-1?signaling in regulating HFD-induced NAFLD in mice.The main results are as follows:(1)Ultrastructural and histopathological results of the liver showed that HFD diet induced a large number of lipid droplets in the liver tissue of wild-type mice,with nuclear shrinkage,mitochondrial enlargement,significant steatosis,hepatic cord disorder and increased cell volume.SelenoW knockout mouse liver cells had a few lipid droplets,but the nucleus was intact and only a few fat vacuoles existed in the liver cells.Oil red staining showed that there were numerous stained triglycerides(TG)in the liver of wild-type mice induced by HFD.While,decreased significantly in SelenoW knockout mice.Therefore,SelenoW knockout can improve liver fat accumulation induced by HFD diet in mice.(2)Antioxidant level test results showed that the activities of SOD,CAT and T-AOC in liver of wild-type mice decreased after HFD diet induction,while the content of MDA increased,while SelenoW deletion significantly increased the activity of antioxidant enzymes and decreased the content of lipid peroxides.These results indicate that SelenoW knockout can reduce liver oxidative stress induced by HFD.(3)Transcriptome analysis explored that there were significant differences in glycolysis metabolism,cell apoptosis,HIF-1 and PPAR pathways between wild-type and SelenoW knockout mice induced by HFD diet.Differential pathway analysis showed that PKM,FBP2,PFKP,PKM,ACSS1,PFKM,PCK2,LDHA,ALDOA,HK2,ENO3 and PGAM2 genes in glycolysis metabolism,The expression of BCL-2,Caspase 3 and Caspase 9 genes in apoptosis,HIF-1? in HIF-1 pathway,and FABP4 and PPAR? genes in PPAR pathway were significantly different.(4)Co-IP and LC-MS results showed that SelenoW interacted with PKM2.Bioinformatics analysis indicated that SelenoW interacting proteins were mainly involved in spliceosome,endoplasmic reticulum protein processing,carbon metabolism and glycolysis/gluconeogenesis signaling pathways.The m RNA and protein levels of PKM2 in liver of SelenoW knockout mice were significantly lower than those of wild-type mice,indicating that SelenoW knockout mice can reduce the expression of PKM2 in liver of mice.(5)m RNA expressions of GLUT1,ENO3,ALDO and PFKFB3 in liver of wild-type mice induced by HFD diet were significantly increased.The m RNA and protein expressions of key ratelimiting enzymes LDHA,HK2 and PFKM were increased,while the expressions of glycoly-related genes and rate-limiting enzymes in liver of SelenoW knockout mice were lower than those of wildtype mice.At the cellular level,SelenoW knockdown can significantly inhibit PKM2 expression,whereas SelenoW overexpression can significantly increase PKM2 expression.These results suggest that SelenoW knockout can reduce glycolysis metabolism in HFD-induced liver of mice.(6)TUNEL staining revealed that the expression of mitochondrial apoptosis-pathway related genes BAX,Cyt-C,Caspase 9 and 3 increased in HFD induced liver apoptosis of wild-type mice,and the expression of anti-apoptotic factor BCL-2 decreased,while the apoptosis in liver of SelenoW knockout mice was less than that of wild-type mice.The expression of genes related to mitochondrial apoptosis pathway decreased,while the expression of anti-apoptotic factors increased.In the AML-12 cell model stimulated by high glucose /2DG,it was found that SelenoW overexpression significantly increased the proportion of apoptosis and the expression of mitochondrial apoptosis related genes,and the addition of 2DG significantly alleviated this effect.In addition,HIF-1? inhibitor BAY87-2243 can reduce the percentage of apoptosis in SelenoW overexpressed cells,reducing the expression of mitochondrial apoptosis related genes under high glucose stimulation.These results indicated that SelenoW knockdown improved hepatocyte apoptosis induced by HFD diet by regulating PKM2-HIF-1? in mice.(7)The expression of HIF-1?,fatty acid transporter FABP4,fatty acid synthetase ACC?,FASN and ACLY increased in liver of wild-type mice induced by HFD diet,while the expression of lipolytic enzymes PPAR? and LIPC decreased.The HIF-1?,fatty acid transporter and synthase in liver of SelenoW knockout mice was lower than that of wild-type mice,but lipolytic enzyme was higher than that of wild-type mice.In AML-12 cell model,overexpression of SelenoW significantly increased HIF-1? and lipid accumulation,and increased the activity of fatty acid transporter and synthase,while decreased the expression of lipolytic enzyme.2DG significantly alleviates these phenomena.The addition of BAY87-2243 in SelenoW overexpressed cells inhibited fat synthesis and promoted fat decomposition by regulating FABP4/PPAR?.These results revealed that SelenoW knockout ameliorates lipid metabolism abnormalities in liver of HFD-induced mice by regulating PKM2-HIF-1?.In conclusion,SelenoW knockout can improve the oxidative stress and lipid accumulation in HFD-induced liver of mice.SelenoW inhibits the expression of HIF-1? via targeting to PKM2 in liver induced by HFD diet,redicing glycolytic metabolism,thereby alleviating mitochondrial pathway apoptosis,inhibiting liposynthase,and promoting the expression of lipolytic enzyme.These results indicate that SelenoW targeting PKM2/HIF-1? signal regulates the formation of HFDinduced atty liver in mice.The results of this study elucidate the new biological functions of SelenoW and provide a new target for the prevention and treatment of NAFLD,providing a reference for further exploration of the function of SelenoW.