Effects And Mechanisms Of Peroxisome Proliferator-Activated Receptor Gamma1 DeSUMOylation On Insulin Resistance Induced By High Glucose And Palmitic Acid In HUVECs

Objective: In this study,we sought to investigate the effect of SENP1-mediated PPAR?1 deSUMOylation on insulin resistance induced by high glucose and palmitic acid in endothelial cells and finally probe its underlying mechanisms.Methods:(1)HUVECs were transfected with vehicle vectors in various concentrations(2E+8PFU,2E+7PFU,2E+6PFU)for 48 h to select an optimal titer.Then,Ad-SENP1 vectors were introduced into endothelial cells with different multiplicities of infection(2.5,5,10,20)for 48 h.The expression levels of SENP1 were measured by western blotting.(2)In this section,four groups were divided,namely Control group,IR group,Vehicle group and Ad-SENP1 group.Briefly,when HUVECs were grown to 60% confluence,Vehicle group and Ad-SENP1 group were infected with the corresponding adenovirus vectors respectively.Then,all groups except Control were replaced with glucose and PA medium(22mmol/L glucose,0.25mmol/L palmitic acid)for 24 h in order to establish the insulin resistance(IR)model.After that,all groups were substituted with fresh L-DMEM containing insulin(100nmol/L)for 30 minutes.Immunocoprecipitation(Co-IP)and western blotting assays were used to detect the SUMO-1 levels of PPAR?1.The levels of NO and AngII were measured to validate the improvement of SENP1-mediated PPAR?1deSUMOylation on insulin resistance induced by high glucose and palmitic acid in endothelial cell.(3)To probe its underlying mechanisms,fluorescence microscope and flow flurocytometry were utilized to test the levels of reactive oxygen species(ROS)and mitochondrial membrane potential.Besides,IKK and IKK-pS176 were detected by western blotting analysis.Co-IP and western blotting assays were used to detect the interactions between IKK and PIAS1.Finally,target proteins of PPAR?1including PI3 K,AKT,AKT-pS473 and eNOS were detected by western blotting analysis.Results:(1)Higher infection efficiency and better cell shape were shown in HUVECs transfected with vehicle vectors at 2E+7PFU concentration than those at other concentrations.The over-expression of SENP1 by western blotting analysis demonstrated that Ad-SENP1 vectors was constructed successfully.When themultiplicity of infection was 10,the effect was optimal.(2)SENP1-mediated PPAR?1 deSUMOylation can improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.After the infection of adenovirus vectors and establishment of the insulin resistance(IR)model,the results were as follows.Compared with Vehicle group,the levels of SUMOylated PPAR?1 were reduced in Ad-SENP1 group.Besides,the level of NO increased significantly,yet the level of AngII decreased remarkably in Ad-SENP1 group.Together,overexpression of SENP1 can mediate PPAR?1 deSUMOylation and improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.(3)Compared with Vehicle group,the ROS production was significantly reduced and the mitochondrial membrane potential showed attenuated collapse in Ad-SENP1 group.In addition,the interaction between IKK and PIAS1 was weakened as well.Moreover,the results of western blotting revealed that the expression of IKK-pS176 decreased notably while the levels of PI3 K,AKT-pS473 and eNOS were significantly increased.Conclusion: Overexpression of SENP1 can mediate PPAR?1 deSUMOylation and improve the insulin resistance induced by high glucose and palmitic acid in endothelial cells.This may be due to reducing the levels of ROS,inhibiting the activity of IKK and interaction between IKK and PIAS1 and resultantly activating the PI3K/Akt/eNOS pathway.