| Diabetes is a metabolic disease characterized by chronic hyperglycemia,according to their differente causes,diabetes can be divided into three types:type ?diabetes,type ? diabetes and gestational diabetes.Among them,Type ? diabetes accounts for over 90%of the overall population with diabetes.Researches demonstrate that there is an inextricably relation between type ? diabetes and obesity.Type ? diabetes often occurs accompanied with abnormal increasement in the number and volume of adipocytes,which play major roles in regulating the insulin resistance by secreting adipokines.Meanwhile,metabolic inflammation is also the main pathological feature of type ? diabetes.PPARy belongs to the nuclear hormone receptor superfamily,which plays a critical role in glucose and lipid metabolism,immune response,and cell differentiation.PPARy dysfunction is closely related to a variety of diseases,such as diabetes,atherosclerosis and cancer,etc.Therefore,PPARy has become an important drug target for treating metabolic related diseases.Recent studies show that the post translational modifications of PPARy,especially phosphorylation,have significant effects on modifying its transcription activity.Our previous work found that several molecules could change the gene expression profile of PPARy,improve insulin sensitivity and reduce metabolic inflammation,in a ligand-independent manner.After the comprehensive analysis,we found that these molecules could not influence the sumoylation,nitrosylation and phosphorylation(Serl12 and Ser273)of PPAR?.Accordingly,we hypothesized that PPAR? might has other unknown modification sites.Therefore,we aimed to discover PPAR? new phosphorylation sites,then discuss the new features of PPAR?.In this thesis,we explored the PPAR?-Ser186 phosphorylation new model,its functions and mechanisms in type ? diabetes.We worked from two aspects:PPAR?-Ser186 phosphorylation regulation in insulin sensitivity and metabolic.The main research contents and results are shown as follows:Identification and analysis of phosphorylation new model in PPAR?1.Bioinformatics analysis:We predicted three new phophorylation sites in PPAR? by using SCANSITE softwares.Then the related site-mutant plasmids were constructed and we finally chose Ser186 as our target site by luciferase reporter gene screen,which could significantly regulate the transcriptional activity of PPAR?.2.Identification of upstream kinases:We predicted that PKC? and PKA might be the upstream kinases of PPARy Ser186 by SCANSITE analysis.After transcriptional activity screening,Western Blot and Co-IP,we found that PKCa could siginificantly increased the phosphorylation of PPARy on Ser186,which obviously regulated the activity of PPARy.Furthermore,there were interactions between PKCa and PPAR?,indicating that PKC? is the upstream kinases of PPARy Ser186.While PKA had no effect on them.3.Kinases activity analysis:Western Blot results showed PKC? had no effect on the phosphorylation of PPAR? Ser273,but could partially improve the phosphorylation of PPAR? Ser112,suggesting that PKC? might also be the upstream kinases of PPAR? Ser112.However,PKA had no significant effect on the phosphorylation of Ser112 and Ser273.Effects of PPAR?-Ser186 phosphorylation on insulin sensitivity1.Analysis of PPAR?-Ser186 phosphorylation in vitro:Through the in vitro differentiation model of adipocytes,we found that the phosphorylation of PPAR? on Ser186 could decrease the secretion of several adipokines,such as Adiponectin and Adipsin.The ChIP assay further demonstrated that the effect was due to the change of PPAR? binding to promoter of Adiponectin.However,PPAR?-Ser186 phosphorylation had no significant effect on the differentiation of adipocytes.2.Analysis of PPAR?-Ser186 phosphorylation in vivo:We used ob/ob mice as the in vivo model,detecting that the phosphorylation of PPAR? on Ser186 was significantly up-regulated in adipocytes.Meanwhile,Fasting model were used and Western Blot analysis showed that PPAR?-Ser186 phosphorylation were reduced in fasting mice.These results suggested that:there is a close correlation between PPAR?-Serl86 phosphorylation and insulin resistance.3.Insulin sensitivity analysis:ob/ob mice were treated with PKC? inhibitor(Ro),then Western Blot,glucose tolerance,insulin tolerance and serum markers(glucose,triglycerides,cholesterol,etc.)were used to observe whether inhibiting upstream kinase activity could regulate PPAR?-Serl86 phosphorylation and Type ? diabetes symptoms.The results indicated that Ro significantly inhibited PPAR?-Ser186 phosphorylation,increased insulin sensitivity,thereby alleviated hyperglycemia,hyperlipidemia and other symptoms of Type ? diabetes.Based on our previous data that metformin(Met)could activate PPAR?,we further analyzed how Met regulated PPAR?.Western Blot,qPCR and ELISA experiments demonstrated that Met could decrease the activity of PKC?,and then inhibit PPAR?-Ser186 phosphorylation,increase the expression of the downstream gene Adiponectin,ultimately improve insulin resistance.Here,we found a new way to improve insulin sensitivity by Met.Effects of PPAR?-Ser186 phosphorylation on metabolic inflammation1.In vivo analyze:ob/ob mice were treated with Met and Ro,the results of HE staining and qPCR detection showed that the infiltration of inflammatory cells(mainly macrophages)were significantly reduced in adipose tissue;the phenotype of adipose tissue macrophages(ATMs)converted from M1 to M2.Indicating that Met and Ro could effectively inhibit metabolism inflammation by regulating the polarization and function of central immune cell-macrophages.2.In vitro analyze:RAW264.7 were used as the cell model,and the phenomenon of PPAR?-Ser186 phosphorylation was confirmed in macrophages by Western Blot.qPCR,flow cytometry and ELISA experiments further validated that this phosphorylation leaded macrophages to the M1 phenotype,while Met and Ro could reverse to M2 phenotype by reducing PPAR?-Ser186 phosphorylation,there fore curbing metabolic inflammation.These results suggested the correlation between PPAR?-Ser186 phosphorylation and metabolic inflammation.Here is the innovation of this thesis:1.Found a new phosphorylation model of PPAR?,and further explored the physiology and pathological significance of this modification;2.Demonstrated that PPAR?-Ser186 phosphorylation changed the secretion of adipokines regulated by PPAR?,and reduced the body's sensitivity to insulin,this process could be reversed by Met and Ro;3.Discovediabetes,atherosclerosis and cancer,etc.Therefore,PPARy has become an important red that PPAR?-Serl86 phosphorylation mediated the polarization and activation of macrophages,hereby leading to the development of metabolic inflammation,the process could also be reversed by Met and Ro;4.Presented a new way of cell metabolism pharmacy effects regulated by Met.Thus,the functional regulation node of PKCa/PPAR? played an important role in the regulation of metabolic disorders caused by diabetes,the results of this study not only proposed a new concept of PPAR?'s biological function,by also provided the experimental basis for the development of new drugs targeting the node of PKC?/PPAR?. |
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