Effects And Its Mechanisms Of Tautomerase Activity Lacking Of Macrophage Migration Inhibitory Factor(MIF)on High Fat Dietinduced Obesity And Nonalcoholic Fatty Liver Disease In Mice

Background and Objectives:Obesity is a metabolic dysregulation syndrome and it is often accompanied by mild chronic inflammation,which greatly increases the risk of developing diseases such as diabetes,hypertension,cancer,stroke,and Alzheimer's disease.Numerous studies have shown that the interaction between adipose tissue and macrophages which are infiltrated into adipose tissue during obesity is the main cause of mild inflammation in obesity.Macrophage migration inhibitory factor(MIF)is a multifunctional protein containing multiple enzyme activities.There is growing evidence that MIF is involved in the pathogenesis of obesity and other related diseases such as insulin resistance,diabetes and nonalcoholic fatty liver disease(NAFLD)by controlling inflammatory and metabolic processes.Studies showed that MIF possesses the roles of the proinflammatory factor through its tautomerase activity.Therefore,some drugs that currently inhibit the tautomerase activity of MIF have been used to treat some diseases such rheumatism,systemic lupus erythematosus and diabetes.However,the effect of MIF tautomerase activity on obesity and NAFLD is not clear.Therefore,the aim of this study was to investigate the effects and the possible mechanisms of transgenic mice with MIF tautomerase inactivation on high fat diet-induced obesity and NAFLD from both white adipose tissue and liver metabolism,providing the experimental basis for revealing the role of MIF in the development of obesity and NAFLD and discovering new targets of preventing and treating these diseases clinically.Method:1.Establishment of a high fat diet-induced obesity model in mice:4-month-oldMIF^PIG/PIG(tautomerase inactivation),MIF^C60S/C60S(sulfhydryl oxidoreductaseinactivation)and wild-type C57BL/6 male mice with the same genetic backgroundwere fed with a high fat diet(HFD)or a general diet(ND)for 12 weeks.The bodyweights were monitored during feeding.The tissues such as white fat and liverwere isolated and the liver function,glucose tolerance and insulin tolerance weremeasured at the end of the experiment.2.Mechanism analysis:(1)The expressions of the genes involved in lipidmetabolism in WAT from MIF^P1G/P1Gmice and WT mice were detected by Real-time PCR.(2)The expressions of macrophage marker F4/80 were examined byimmunofluorescence and Real-time PCR in WAT of the mice.In addition,we alsoanalyzed the inflammatory factors such as levels were examined in WAT fromMIF^P1G/P1Gmice and WT mice through detecting CD74,TNF-?,MCP-1,IL-1?andIL-6 m RNA expression by Real-time PCR.(3)The expressions of IRS-1,IRS-2,Gl UT-4 and PPAR?were examined by Real-time PCR and Western blotting inWAT from the two groups under ND and HFD.(4)The expressions of apoptosisrelated genes Bax and Bcl-2 were detected by Real-time PCR and Western blotting.And Cleaved caspase-3 expression was examined by immunohistochemistrytechnique.(5)The liver RNA sequencing of biological transcriptome wasperformed and the differentially expressed genes related lipid metabolism wereverified by Real-time PCR.Results:1.The mice with inactivation of MIF tautomerase were significantly resistance to highfat diet-induced obesity and improved insulin resistance:1)The contents of MIF in serum and the expression of MIF protein in adiposetissue were increased in high fat diet-induced obesity of WT mice comparedwith those in normal diet fed mice.2)In HFD condition,the weights of MIF^P1G/P1Gmice were lower than that of WTmice,and the glucose tolerance and insulin tolerance were significantly improved in MIF^P1G/P1G mice compared with WT mice and the mechanism of the resistance might be related with promoting the expressions of IRS-1,PPAR?and GLUT4 in white adipose tissue.However,there was no alternation for these parameters in MIF^C60S/C60Smice fed with HFD.3)The infiltration of macrophage and expression of the inflammatory factors suchas CD74,TNF-?,MCP-1 and IL-1?were markedly increased in WAT fromWT fed with HFD compared to mice fed with ND.However,the expressions ofF4/80,CD74,IL-1?,IL-6 and MCP-1 m RNA were significantly decreased inWAT from MIF^P1G/P1G mice fed with HFD compared with WT mice fed with HFD.4)The protein expression of pro-apoptotic gene Bax was increased,while theexpression of anti-apoptotic gene Bcl-2 and the ratio of Bcl-2/Bax were downregulated in adipose tissue from WT mice fed with HFD compared withmice fed with ND.The Bax protein expression was markedly decreased whilethe ratio Bcl-2/Bax was increased in WAT from MIF^P1G/P1G mice compared with WT mice under HFD condition.In addition,the numbers of the cleaved caspase-3 positive cells were decreased in WAT from MIF^P1G/P1G mice compared with WT mice under HFD condition.2.Inactivation of MIF tautomerase significantly protects against non-alcoholic fattyliver disease in mice induced by high fat diet:1)MIF expression was downregulated in liver tissue from mice under HFD.2)The results from HE,Oil red O staining and biochemistry test showed that theHFD-induced liver lipid accumulation,decreased of liver functions and theincreased contents of lipids in serum and liver tissues were significantlyimproved in MIF^P1G/P1G mice compared with WT mice.3)The transcriptome sequencing showed that there was a difference in theexpressions of genes involved in lipid metabolism in liver tissue between theMIF^P1G/P1G mice and WT mice under HFD.We further demonstrated that theimproved liver lipid deposition might be related the down-regulation of theexpressions of Mogat1,PPAR?,Cidec and Cidea in MIF^P1G/P1G mice.Conclusion:1.The serum content of MIF and the protein expression of MIF in adipose tissue wereincreased from mice fed with HFD.MIF^P1G/P1G mice significantly improved highfat diet induced obesity and insulin resistance.2.MIF^P1G/P1G mice markedly reduced the macrophages infiltration and the expressionsof inflammatory factors in white adipose tissue and reduced adipocyte apoptosis toimprove obesity induced by high fat diet.3.The expression of MIF was decreased in liver tissue from mice fed with HFD.MIF^P1G/P1G mice improved high fat induced NAFLD via down-regulating theexpressions of Mogat1,PPAR?,Cidec and Cidea in liver tissue.