Effects And Mechanisms Of Sumoylation Of PPAR Gamma-1 At The Site Of K77 On Endothelial Cell Insulin Resistance Induced By High Glucose In HUVECs

Objective: The aim of the study is to investigate the role and its potential molecular mechanisms of sumolyation of PPARγ1 at the site of K77 in endothelial insulin resistance by transfecting the Ad-HA-PPARγ,Ad-Myc-SUMO1 and Ad-FlagPIAS1 in human umbilical vein endothelial cells(HUVECs).Methods:(1)The nucleotide sequences of PPARγ,SUMO1 and PIAS1 m RNA were retrieved from Genebank,and then the primers of PPARγ,SUMO1 and PIAS1 were designed and amplified by RT-PCR.Then the adenovirus vectors of AdHA-PPARγ,Ad-Myc-SUMO1 and Ad-Flag-PIAS1 were obtained following the adenovirus packaging kit steps.Subsequently,three adenovirus vectors were transfected into HEK293 T cells to be amplified and purified,and finally the concentrations of the adenovirus vectors were determined.(2)The insulin resistance(IR)model was established by using HG medium(22mmol/L glucose)to treat HUVECs for indicated times(6,12,24,48,72h),then insulin(5m IU/L)was used to incubate cells for another 15 minutes,and finally the levels of NO and AngⅡwere measured.Meanwhile,the levels of e NOS and PPARγ were detected by western blot.(3)IR HUVECs were infected by Ad-HA-PPARγ,Ad-Myc-SUMO1 and Ad-Flag-PIAS1 vectors at indicated concentrations(1.2E+8PFU,1.2E+7PFU,1.2E+6PFU,1.2E+5PFU)for indicated periods(12,24,36,48,72h)to choose the best concentration and treated time.In the later study,four groups were divided,namely,?Control(Ctrl)group;?IR group;?IR+Vehicle group;(4)IR+ total transfection(T-Ad)group.The infection effects were observed by fluorescence microscope and the levels of PPARγ1,SUMO1 and PIAS1 were detected.The AngⅡlevels were detected by enzyme-linked immunosorbent assay kit(ELISA).And the NO levels were measured by the nitrate reductive enzymatic assay kit.Flow flurocytometry was used to test the levels of reactive oxygen species(ROS)and mitochondrial membrance potential(JC-1).The levels of proteins such as e NOS PPARγ,SUMO1,PIAS1,AKT,p-AKT,PI3 K,IKK,and p-IKK were detected by western blotting analysis.Additionally,the SUMO-1 levels of PPAR gamma-1 were assayed by immune co-precipitation(Co-IP)and western blotting.Results:(1)Ad-HA-PPARγ,Ad-Myc-SUMO1 and Ad-Flag-PIAS1 vectors have been successfully contructed according to the result provided by Shanghai Ji Kai technological company.(2)After 48 h of HG-treating HUVECs,the NO level decreased remarkably(P<0.01),on the contrary,the level of AngⅡ and e NOS increased significantly(P<0.01).All the results showed that the insulin resistance model has been established successfully.Interestingly,the PPAR gamma levels were similar between the different groups(P>0.05).(3)The results from the infection efficiency of adenovirus vectors and the levels of PPARγ1,SUMO1 and PIAS1 showed,1.2E+7PFU adenovirus vectors and 48 h were determined to infect HUVECs for 48 h as the premium infected condition.(4)The result of ROS also showed that the levels of ROS in IR group were significantly increased compared with those in control group(P<0.01).Furthermore,the levels of ROS in T-Ad group were further increased compared with those in IR group(P<0.01).However,the results of mitochondrial membrance potential(JC-1)were in sharp contrast with those of ROS levels.(5)After transfected the adenovirus vectors for 48 h,ELISA assay was used to measure the level of AngⅡ,and the NO and AngⅡ level in the supernatant of medium were detected,the result further showed that the level of NO in IR group were significantly decreased compared with those in the control group.Moreover,the levels of NO in T-Ad group were further decreased compared with those in IR group(P<0.01).Nevertheless,the level of AngⅡwas conversed(P<0.01).(6)The result of immune co-precipitation(Co-IP)showed that the PPAR gamma sumolyation levels of IR group of were more than control group(P<0.05),and those in T-Ad group were higher than those in the IR group(P<0.01).(7)The protein’s levels tested such as PI3 K,p-AKT and e NOS in the IR group and T-Ad group were notably decreased compared with control(P<0.01).On the contrary,the p-IKK level of the IR group were significantly elevated than those of the control group(P<0.01).However,there was no great change of the p-IKK level between the IR group and the T-Ad group(P>0.05).Conclusion: The sumolyation of PPAR gamma at the site of K77 may enhance the degree of insulin resistance induced by high glucose in HUVECs,which is responsible for undergoing the transrepression of PI3K/ AKT/e NOS pathway.