| Background and Aim：Endothelial dysfunction is a key point of cardiovascular disease, includinghypertension, atherosclerosis, and coronary artery disease. Recently, insulin resistance asa prominent feature of metabolic disorders, is recognized as a common basis for a varietyof metabolic disease and cardiovascular disease, scholars pay more attention to insulinresistance on glucose and lipid metabolism disorders. Endothelial cells as insulin targetcells, have potential contact with insulin resistance. The relationship between insulinresistance and endothelial dysfunction leading to a vicious cycle between metabolicdisorders and cardiovascular disease, the mechanisms of interaction between those is notvery clear. Atorvastatin, an endothelium protective drug, is commonly used to controldyslipidemia, have been proven to be able toreduce cardiovascular events in patients byprotecting endothelial function, exhibiting anti-inflammatory action, and decreasing thelevels of cholesterol. We designed this study to evaluate the ability of atorvastatin toameliorate insulin resistance-induced endothelial dysfunction. Currently, there are somecell models of insulin resistance were established in fat cell and liver cell, these studies,the main focus is on glucose and lipid metabolism. However, the endothelial cells modelof insulin resistance is still uncommon. In this study, we established a reproducible andhighly stable vascular endothelium cell model. Using this model, we demonstrated thathigh concentration of glucose in combination with superphysiological concentrations ofinsulin impair insulin signaling and endothelium dysfuncion in HUVECs. In addition, weshowed that atorvastatin decreased this effect by increasing insulin-inducedIRS1/phosphatidylinositol3-kinase (PI3K) signaling pathway.Methods:HUVECs were incubated in culture medium containing5,15or30mM D-glucose.The cells were maintained at37°C in humidified air (5%CO2) with or without10-5M insulin for24h, then cell morphology was observed by invert microscope and cellviability by MTT, and then through the detection phosphorylation of insulin receptorand its substrate to evaluate insulin receptor signaling pathway if it is inhibited, andthen detection of eNOS phosphorylation and activity, NO generation and expression ofET-1mRNA to evalute endothelial function. Then, HUVECs cultured in mediumcontaining30mM glucose and10-5M insulin for24h were defined as insulin-resistantHUVEC (IR vec). Both Nor vec and IR vec were treated with a series dose ofatorvastatin (0,10-6,10-5,10-4M) for24h or treated with10-4M atorvastatin for differenttime.Cell proliferation by MTS to evaluate the toxicity of the drug. Test these index thatabove-mentioned to evaluate the function of atorvastatin on vascular endothelialspecific insulin. Finally, in order to explore the possible role of the mechanism ofatorvastatin, LY24002, PI3k inhibitors are only or in combination with atorvastatintreated cells were observed in the expression of these indicators to determineatorvastatin improves endothelial Insulin resistance may be signaling pathway andendothelial dysfunction.Results:1．There were no difference of cell proliferation and cell morphology between cellscultured in different concentration of glucose with or without10-5M insulin.2．30mM glucose significantly decreased the phosphorylation levels of IR and IRS1to60%and40%respectivley, compared to lower dose of glucose (5mM). When thecells were treated with h i g h e r d o s e (30mM) glucose and10-5M insulin, there was afurther decrease of both p(tyr)IR and p(tyr)IRS1(p <0.05) compared with samples thatwere treated with30mM glucose only.3.30mM glucose or10-5M insulin significantly decreased the phosphorylationlevels of Akt to50%and30%，When the cells were treated with h i g h e r d o s e (30mM)glucose and10-5M insulin, there was a further decrease of Akt phosphorylationlevel(p<0.01).4. As for the nitric oxide production, compared to the5mM glucose,30mM glucoseinduced a significant reduction (p<0.01), while30mM glucose combined with10-5M insulin caused a further reduction of nitric oxide production (p<0.05) Glucose increasedthe mRNA expression level of ET-1in a dose dependent manner, which was furtherinducted by insulin. The combination treatment of30mM glucose and10-5M insulininduced about13times fold increase of ET-1mRNA, compared to5mM glucose only.5. Glucose and insulin treatment did not change the expression level of eNOS, buthigher dose of glucose decreased its phosphorylation level, which was further enhancedby the presence of insulin. In addition, the activity of eNOS changed in parallel with thephosphorylation level.6. There were significant differences in the protein of p(tyr)IR between non-treatedIR vec and IR vec treated with a increased dose of atorvastatin The expression trend ofp(tyr)IRS1was similar to p(tyr)IR. It is worth notice that increasing dose of atorvastatin didnot change the phosphorylation levels of either IR or IRS1.7. There were significant differences in the phosphorylation level of Akt betweennon-treated IR vec and IR vec treated with a increased dose of atorvastatin.8. Atorvastatin significantly increased the NO production compared to not-treat IRvec in a dose dependent manner In contrast, atorvastatin reduced the expression of ET-1mRNA compared to non-treat IR vec. Among the4treatment levels, the maximalinhibition rate occurred at the dose of10-4M (76%vs non-treat IR vec),9. Atorvastatin significantly increase phosphorylation levels of eNOS on Ser1177and activity of eNOS in a dose-dependent manner, compared to non-treat IR vec. Forp(ser1177)eNOS expression,10. In IR VEC, atorvastatin induced the production of NO, whose highest level occurredafter9hours and maintained its level during the test time. In contrast, there was nosignificant change in Nor vec cells. Atorvastatin induced deduction of ET-1mRNA level inboth Nor vec and IR vec cells.11. Atorvastatin increased the eNOS phosphorylation level in a time dependent manner.Further，the eNOS activity in IR vec also increased after atorvastatin treatment， butremained the same level in Nor vec cells in24hours.12. The induction of atorvastatin on both phosphorylation level of eNOS and activity of eNOS were also blocked by LY294002in Nor vec and IR vec cells. However, thededuction effect on ET-1mRNA expression was not affected by LY294002.Conclusions:1. In the early stage of insulin resistance, it doesn’t affect the cell morphology, justcause endothelial dysfunction.2. Impaired insulin signaling pathway cause endothelial dysfunction, including thereduction of phosphorylation of eNOS on ser1177sites and the activity of eNOS; NOproductiod, increased expression of ET-mRNA indirectly.3. Impaired insulin signaling pathway cause insulin tyrosine-phosphorylation of (IR),(IRS1), and phosphorylation of protein kinase B (Akt) expression.4. Atorvastatin not only improves the normal endothelial function, but also improveinsulin resistance induced endothelial dysfunction resulting in a dose and time-dependentmanner.5. Atorvastatin intervention of impaired insulin signaling pathways, increase insulintyrosine-phosphorylation of (IR),(IRS1), and phosphorylation of protein kinase B (Akt)expression.6. Atorvastatin has an independent protective effect of its outer lipid effects ofendothelial cells, a major role in PI3k/Akt/eNOS pathway... |
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