Involvement Of Peroxisome Proliferator-activated Receptor Gamma 1 Sumoylation In Insulin Resistance In Endothelial Cells Induced By High Glucose And Palmitic Acid

Objective: The aim of the study is to confirm the model of endothelial IR in HUVECs using high glucose and palmitic acid to investigate the change of sumoylated PPAR?,and whether sumolyation of PPAR?1 is involved in endothelial insulin resistance by transfecting the Ad-PIAS1-RNAi in human umbilical vein endothelial cells(HUVECs).Methods:(1)We obtained the nucleotide sequences of PIAS1 mRNA from Genebank,after that we designed and amplified the primers of PIAS1 by using RT-PCR.The adenovirus vectors of Ad-PIAS1-RNAi was obtained following the steps of adenovirus packaging kit.The following steps were the amplification and purification of adenovirus by transfecting into HEK293 T cells,and eventually the determination of the concentrations of the adenovirus vectors.(2)Briefly,the insulin resistance(IR)model was established by using HG and PA medium(22mmol/L glucose,0.25mmol/L palmitic acid)to treat HUVECs for 24 h,then insulin(100nmol/L)was used to incubate cells for another 30 minutes,after that the levels of NO ?Ang?and p-Akt were measured.After the establishment of IR model,flow flurocytometry was used to test the levels of reactive oxygen species(ROS),the levels of p-IKK,IKK,NcoR were detected by Western blotting,and the SUMO-1 levels of PPAR gamma-1 were assayed by immunoprecipitation(IP)and Western blotting.The combination of NcoR with PPAR gamma 1 were detected by Confocal.(3)HUVECs were infected by Ad-PIAS1-RNAi vectors at indicated concentrations.In the later study,different groups were divided on the basis of experiment needs,and then measured the levels of NO?Ang?and p-Akt.Results:(1)Ad-PIAS1-RNAi vectors have been successfully contructed according to the result provided by Shanghai JiKai technological company.(2)After24h of HG and PA-treating HUVECs,the level of NO and p-Akt decreased significantly,on the contrary,the level of Ang? increased remarkably.All the results showed that the model of HUVECs insulin resistance was successfully established.(3)The levels of ROS and p-IKK,NcoR in IR group were significantly elevatedcompared with those in control group;the levels of sumolyated PPAR gamma in IR group of were higher than those control group.Moreover,NcoR combined more PPAR? in IR group than control group.(4)More interestingly,after transfected the Ad-Flag-siPIAS1,IR degree was improved.Conclusion: PPAR? 1 sumoylation is involved in endothelial cell IR induced by HG and PA,and knock-down of PIAS1 may improve endothelial insulin resistance.The mechanism may be through the PPAR? 1 sumoylation,which was induced by the increase of ROS,activation of IKK and interaction between IKK and PIAS1.