IKKα/β Mediated High Glucose-induced Vascular Endothelial Insulin Resistance And Dysfunction Independent Of NF-κB Passway

Objective:Changes of IKKα/βexpression and activity in high glucose-induced vascular endothelial insulin resistance (IR) and dysfunction and the mechanism of IKKα/βmediated high glucose-induced vascular endothelial insulin resistance and dysfunction were investigated in this study.Methods:Cultured HUVECs (CRL-1730) were inoculated in a 6-well culture plate uniformly and were randomly divided into three groups, that is, Control group, HG group, Mannitol group, respectively. The control group were only treated with DMEM containing glucose (5.5 mmol/L), while the HG group was treated with DMEM containing glucose (33 mmol/L), and the Mannitol group was treated with DMEM containing glucose (5.5 mmol/L) and Mannitol (27.5 mmol/L). The HUVECs were cultured for 48 h at an atmosphere of 37℃,5% CO2, and then were stimulated with insulin (100 nmol/L) for 30 min.Ultimately, the cells were collected, and the following parameters were detected,①levels of NO and ET-1 in cell supernatants;②expression levels of IKKα/βmRNA in HUVECs;③expression levels of IKKα/β;④protein expression levels of p-IKKα/β(Ser176/181), p-IRS-1 (Ser312), p-Raf-1 (Ser338); and⑤interactions of IKKα/β-IRS-1 and IKKα/β-Raf-1.Results:①The levels of NO and ET-1 in cell supernatants:HG could significantly induce insulin resistance and dysfunction in HUVECs, manifesting the decreased NO levels, the increased ET-1 levels and significant differences compared with the Control group (P<0.01); while the MNT same osmotic pressure as glucose had no effect (P> 0.05).②The expression levels of IKKα/βmRNA in HUVECs:the elevated expression levels of IKKα/βmRNA were found after the HUVECs were treated with HG for 48h; while no effect was found in the MNT group (P> 0.05).③The expression levels of IKKα/β:the results was similar with the expression levels of IKKα/βmRNA.④The expression levels of p-IKKα/β(Ser176/181), p-IRS-1 (Ser312) and p-Raf-1 (Ser338):HG could significantly increase p-IKKα/β(Ser176/181), p-IRS-1 (Ser312), and p-Raf-1 (Ser338) expression levels; while no effect was found in the MNT group(P>0.05).⑤The interactions of IKKα/β-IRS-1 and IKKα/β-Raf-1:Coimmunoprecipitation assay showed the interactions of IKKα/β-IRS-1 and IKKα/β-Raf-1 HUVECs were present regardless at the atmosphere of an elevated or normal glucose concentration.Conclusion:①HG up-regulated the expression levels of IKKα/βand activated the IKKα/β.②IKKα/βmediated-vascular endothelial insulin resistance and dysfunction might be via the interactions of IKKα/β-IRS一1 and IKKα/β-Raf-1 and the phosphorylations of IRS-1 at Ser312 and Raf-1 at Ser338.