Over-SUMOylation Of PPARγ Facilitates Endothelial Insulin Resistance In A Regulatory Loop Of ROS-IKK-PIAS1-SUMO1

Objective:The aim of this study was to construct virus Ad-HA-PPARγ,Ad-Myc-SUMO1,Ad-Flag-PIAS1 and transfected them into normal cultured human umbilical vein endothelial cells(HUVECs).To observed whether PPARγK77 over SUMO can mimic high glucose and high fat(HG/HF)stress to produce vascular endothelium and explore its molecular mechanism.Methods(1)HUVECs were seeded in a 96-well plate at a density of 2×10~4 per well.When growing to 80%confluence,HUVECs were transfected with 2×10~9plaque forming unit(abbreviated as 2E+9 PFU),2E+8 PFU,2E+7 PFU and 2E+6PFU respectively for various times including 24 h,36 h and 48 h,respectively.In this prosess,we need to choose the best concentration and treated time.After that the viruses of Ad-HA-PPARγ,Ad-Myc-SUMO1 and Ad-Flag-PIAS1 were first mixed at the ratio of 1:1:1 and the titers of the mixed virus were measured.ransfection efficiency was assessed by fluorescence intensity under a fluorescence microscope and cell viability was measured.Protein expression was detected by western blot.(2)The experiment was divided into 4 groups,namely,normal control(Ctrl),HG/HF group,Vehicle and combined viral transfection(Over-SUMO)group.The cells were evenly cultured in 10 cm dishes.When the cells grew to approximately80%confluence,the cells in Over-SUMO group and Vehicle group were respectively transfected with the mixed viruses of Ad-HA-PPARγ,Ad-Myc-SUMO1 and Ad-Flag-PIAS1 as well as negative control viruses at a titer of 2E+7 PFU for 36 h.Besides,the cells in HG/HF group were treated with HG/HF(22 m M of glucose and0.25 m M of PA)for 36 h and the cells neither transfected nor treated with HG/HF were considered as Ctrl group.We use Immunocoprecipitation(Co-IP)and western blot to detect the SUMO-1 levels of PPARγ1.The levels of NO and AngⅡwere detected by ELISA and Nitrate reduction.(3)Fluorescence microscope and flow flurocytometry were used to test the levels of reactive oxygen species(ROS)and mitochondrial membrane potential.Meanwhile,We used western blot to detect IKK and IKK-p S176.CO-IP and western blot to analyzed the interactions between IKK and PIAS1.In the end,we used western blotting assays to tested target proteins of PPARγ1 including PI3K,AKT,AKT-p S473and e NOS.Result:(1)2E+7PFU titer transfection for 36 hours was the best transfection condition.Western blotting showed overexpression of HA,Myc and Flag,suggesting successful transfection.(2)The data of co-immunoprecipitation further displayed that the SUMO1 levels in both HG/HF and Over-SUMO group were higher than those in Ctrl or Vehicle group,illustrating that the combined adenoviral transfection,like HG/HF,resulted in the over-SUMOylation of PPARγ.Moreover,similar to HG/HF,the combined adenoviral transfection noticeably lowered the levels of NO whereas elevated the levels of Ang II,compared with Ctrl or Vehicle group,suggesting that over-SUMOylation of PPARγsimulates HG/HF to induce the endothelial IR.(3)Compared with Ctrl group,the expression levels of PI3K,AKT-p S473 and e NOS in HG/HF group and Over-SUMO group were notably decreased,but no significant difference between the Ctrl and Vehicle group was found.Compared with Vehicle group,the expression levels of PI3K,AKT-p S473 and e NOS were also significantly decreased in Over-SUMO group.The results indicated that over-SUMOylation of PPARγ,like HG/HF,induced the endothelial IR by inhibiting the PI3K-AKT-e NOS pathway.(4)Compared with Ctrl group,the ROS levels in HG/HF group and Over-SUMO group were markedly increased,but no significant difference between the Ctrl and Vehicle group was found.Over-SUMOylation of PPARγ,like HG/HF,substantially increased the ROS levels,suggesting that over-SUMOylation of PPARγmight result in cellular oxidative stress.The mitochondrial membrane potentials in HG/HF group and Over-SUMO group were lower than those in Ctrl group or the Vehicle group(P<0.01),but no significant difference between the Ctrl and Vehicle group was found.The results indicated that over-SUMOylation of PPARγ,similar to HG/HF,could cause the collapse of mitochondrial membrane potential,giving rise to the cell damage。(5)Similar to HG/HF,while the over-SUMOylation of PPARγfailed to increase the IKK expression levels,it significantly elevated the IKK-p S176levels,indicating that the over-SUMOylation of PPARγinduces the activation of IKK like HG/HF(Fig.3.6a).Furthermore,the results from the co-immunoprecipitation exhibited that the over-SUMOylation of PPARγalso promoted the interaction between the IKK and PIAS1 like HG/HF。Conclusion:PPARγover-SUMO at normal cellular levels mimics insulin resistance in vascular endothelial cells induced by high glucose and high lipid stress.Furthermore,we found that PPARγover-SUMOization facilitates endothelial cell insulin resistance triggered by HG/HF in the form of the ROS-IKK-PIAS1-SUMO1loop.