The Role Of HnRNPA1 In Macrophage M1 Polarization And Insulin Resistance Of Adipose Tissue

The main pathologic changes of metabolic syndrome are systemic insulin resistance and massive production of proinflammatory cytokines.Liver,adipose and muscle are major organs for insulin utilization.In diabetic patients,insulin sensitivity of organs is mostly decreased and the metabolic function is greatly weakened.In adipose tissue,macrophages infiltration and proinflammatory cytokines release arise in the early stage of insulin resistance and always last for the complete progression of diabetes.Deletion of CD11C+inflammatory macrophages can quickly restore the insulin sensitivity of adipose tissue.Macrophages have two polarized phenotypes:the classical Ml and alternative M2 macrophages.M1 macrophages secrete specific inflammatory factors,such as NO,IL-1?,TNF-? and IL-12,IL-23 and aggravate insulin resistance of surrounding adipocyte and muscle cells.While M2 macrophages produce IL-10 to increase insulin sensitivity of surrounding cells.The adipose tissue macrophages(ATM)in lean adipose tissue mainly are M2 macrophages,however,ATM in obese adipose tissue predominantly are M1 macrophages.As M1 and M2 are antagonistic in insulin signals.In the view of macrophage immune regulation,we hope to provide a new experimental support and theoretical basis for treatment of insulin resistance and metabolic syndrome.Based on our previous study on RNA binding proteins(RBP)in immune cells,we generated macrophage-specific hnRNP A1 null mice(hnRNP A1 ?m(?) mice).Under high fat diet(HFD)treatment,obesity and insulin resistance were significantly improved in hnRNP A1?m(?) mice compared with control mice.HnRNP A1 is a multi-functional protein,but its role in macrophages and insulin resistance has not been reported.Owing to the amelioration of obesity and insulin resistance in hnRNP A1?m(?) mice,this paper aims to reveal the function of hnRNP A1 in macrophage-mediated inflammation and insulin resistance.In the second chapter,HFD-induced obesity and insulin resistance model was established to observe pathologic changes after hnRNP A1 knockout.Firstly,in vivo results showed that hnRNP A1 deletion in macrophage significantly improved HFD-induced overweight,body fat ratio,hepatic steatosis,number and size of pancreatic islets in pancreas.Serum total triglyceride and total cholesterol also were significantly reduced in hnRNP A1?m(?) mice compared with control group.GTT/ITT test and insulin signaling pathway analysis indicated that HFD-induced insulin resistance was notably improved in hnRNP A1?m(?) mice.Obesity-associated insulin resistance is a result of imbalance of energy metabolism.Results of metabolic cage showed that hnRNP A1 knockout increased energy expenditure and respiratory exchange rate,while did not impact energy intake.Next,we investigated the effect of hnRNP A1 in adipocytes by using adipocyte-specific hnRNP A1 null mice(hnRNP A1AKO).Results manifested that adipocyte-specific knockout hnRNP A1 had no significant effect in obesity and insulin resistance.It suggested that hnRNP A1 of macrophage may play a more important role in the process of insulin resistance and metabolism syndrome,not adipocyte.Therefore,we mainly focused on the functions of hnRNP A1 in macrophage during obesity and insulin resistance.In the third chapter,we found that quantities of infiltrated Ml macrophages in adipose tissue were decreased in hnRNP A1?m(?) with HFD treatment,while not changed in normal diet treatment.Furthermore,M1 markers were evidently decreased in adipose tissue of hnRNP A1?m(?) mice,and M2 markers were not significantly changed except IL-10.Proinflammatory cytokines,as IL-1?,TNF-? and IL-6 secreted by macrophage,were important regulating factors for insulin signaling pathway of adipocytes.Thus,we established the co-culture system of polarized-macrophages with differentiated 3T3-L1 adipocytes to mimic in vivo environment.Results showed that the insulin resistance of adipocytes was improved by co-cultured with hnRNP A1-knockout macrophages compared with wild type It indicated that prevention of pro-inflammatory cytokines by hnRNP A1 knockout was the key reason for improvement of surrounding insulin resistance.We further confirmed the same result through adoptive transfer experiment in vivo.In the fourth chapter,we explored the molecular mechanism of hnRNP A1 in regulating macrophage polarization.The results showed that expression of hnRNP A1 was not altered but it shuttled from nucleus to cytoplasm during M1 polarization.Through RIP-chip analysis,we found the major target gene of hnRNP A1 in M1 polarizaion was SOCS3.Result of FISH experiment showed that hnRNP A1 protein and Socs3 mRNA were co-localized in cytoplasm during M1 polarization.It has been reported that SOCS3 is involved in regulating macrophage polarization We further found that hnRNP A1 knockout reduced the stability of Socs3 mRNA rather than effect on the translation efficiency of Socs3 mRNA.ARE structure plays an important role in stability regulation of mRNA Interestingly,3'-UTR of Socs3 also has the structure of ARE which is conservative in mouse and human.Luciferase activity of report gene plasmid with 3'-UTR or with ARE structure was significantly increased in hnRNP A1 overexpressioned cells,but the up-regulation was blocked in ARE mutation group.At the same time,results of EMSA and SPR also confirmed the binding of hnRNP A1 and ARE structure of Socs3 mRNA.All above results indicated that hnRNP A1 stabilized Socs3 mRNAby binding with ARE domain of Socs3 mRNA in cytoplasm and regulated Ml macrophage polarization.Phosphorylation of hnRNP A1 has great effects on its mRNA modification procedure.We found that the Ser1 99 phosphorylation of hnRNP A1 was significantly increased and Ser192 phosphorylation was not obviously changed in M1 polarization.Phosphomimitic mutation of Ser199 signif cantly increased the luciferase activity of Socs3 3'-UTR report gene plasmid and mRNA expression of SOCS3.AKT is the executive kinase for Ser199 phosphorylation of hnRNP Al.Our results showed that IGF-1,activator of AKT,upregulated the 3 '-UTR reporter gene luciferase activity,protein expression of SOCS3 and polarized macrophage to M1 phenotye.On the other hand,wortmannin,inhibitor of AKT,inhibited phosphorylation of hnRNP A1 at Ser199 and reduced the reporter gene luciferase activity,protein expression of SOCS3 and inhibited macrophage M1 polarization.These results indicated that Ser199 phosphorylation of hnRNP A1 had an important role in M1 polarization.Meanwhile,we found that the expression of miR-221 was significantly increased when macrophages were polarized to M1.MiR-221 inhibited M1 polarization by promoting Socs3 mRNA degradation.During M1 polarization,hnRNP A1 and miR-221 competitively bond to Socs3 mRNA and regulated its mRNA stability.FISH and RIP analysis showed that hnRNP A1 stabilized Socs3 mRNA by blocking the accumulation of Socs3 mRNA to P body mediated by miR-221.In this thesis,we first revealed the novel mechanisms of hnRNP A1 in macrophage polarization and insulin resistance through regulating Socs3 mRNA stability,which was deeply influenced by Ser199 phosphoration and the competition with miR-221 of hnRNP A1.Our results enriched the regulatory network of macrophage polarization and provided a new potential treatment for insulin resistance and metabolic syndrome by intervention of macrophage-mediated inflammatory response.