| Insulin resistance usually appears together with endothelial dysfunction. They possessthe same pathogenesis such as high blood glucose, high blood lipid, high insulin, inflammation,coagulation disorders, and hormone etc. They affect each other, aggravate each other,eventually will lead to diabetes, obesity, and the metabolic syndrome and other metabolicdiseases and systemic vascular diseases. Insulin resistance and endothelial dysfunction are theimportant cornerstone and decisive factor of the dieases aboved.Insulin resistance is defined as the diminishing of sensitivity and/or metabolism ofcells to respond to the action of insulin in transporting glucose. It is an imbalance amongenergy homeostasis, cell growth and tissue repair. After binding with the insulin receptor onendothelial cells to activate IRS (insulin receptor substrate), insulin successively activatePI3K-Akt through specific cellular signal pathway, and further stimulate endothelial cells toproduce eNOS. Eventually lead to the formation of nitric oxide, dilation of blood vessels,increasing of blood flow, enhancement of the glucose uptake in skeletal muscle. However,under the condition of high glucose, oxidative stress and other incentives, insulin signal mayalso act on vascular endothelial cells through MAPk (mitogen activated protein kinase)-ET-1cell signal pathway to stimulate the discharge of vascular endothelial (ET-1), which will resultin vasoconstriction. Additionally, insulin can continue to stimulate the adhesion molecules(AM) by ET-1to achieve the opposite physiological and pathological response from the firstpathway. Therefore, the normal functioning of physiological response of insulin andendothelial cells play important roles on maintaining the equilibrium between the healthymetabolism and hemodynamics. However, the balance will be broken while in pathologicalconditions. The appearance and aggravation of insulin resistance and endothelial dysfunctionwill lead to glucose and lipid metabolism disorders, lipid accumulation, oxidative stress, inflammation reaction, chemokine and cytokine release, further aggravation of insulinresistance and endothelial dysfunction caused by vascular injury, eventually will cause avariety of chronic diseases and lead to the irreversible stage.Thus before the changes in vascular structure, insulin impaired and compensatoryincrease, insulin target organs losing sensitivity and insulin secretion function can becompensated, it is very necessary and urgent to seek a treatment which can improve vascularendothelia cell function and/or increase insulin sensitivity, and apply the treatment to clinicalas soon as possible.As one of thiazolidine ketone family, pioglitazone is the insulin sensitizing agent. Ithas been wildly used in clinical treatment of diabetes. It will improve the sensitivity of tissuesto insulin, promote glucose uptake and to reduce blood sugar content. Some recentexperiments, investigations and researches shown pioglitazone may improve the impairedendothelia function, but if it can protect endothelia cells and the mechanism of the protectionare still unclear. This study applied pioglitazone on insulin resistance in vascular endotheliacells, investigated if pioglitazone will protect insulin resistance on endothelia cells and itsprotection mechanism.Methods:1. The human vein endothelial cells were cultured in concentrated glucose medium plusglucocorticoid and different concentrations of insulin. The insulin resistance model wasestablished by simulating the common environment formed by human insulin resistance. Thesugar consumption was calculated by conducting glucose depletion experiment, and which isused to determine the optimal time and dosage to establish the model of insulin resistance onvascular endothelial cells. mRNA and the protein expression level of insulin resisting onendothelial cell IRS-A and PPAR-were observed using RR-PCR and western blot method,respectively. The effect of insulin resistance on IRS-1and PPAR-was also investigated. 2. The effect of pioglitazone on insulin resistance in endothelial cells was determined byadding different concentrations of pioglitazone into each insulin resistance endothelial cellgroup. Cell viability was measured by MTT method. Cell death rate was measured by flowcytometry. Caspase-3was determined by spectrophotometry, and also the cell death waschecked in each group. Nitric oxide content was determined by nitro reductase method. Theexpr ession of eNOS protein was detected by western blot method.3. RT-PCR and western blot methods were employed to detect the expression of IRS-1,PPAR-, PI3K, Akt Mrna and protein in each pioglitazone group as well as the control groups.The protection mechanism of pioglitazone on insulin resisting vascular endothelial cells wasalso discussed.Results:1.The glucose consumption results shown that10-4mM insulin/30mM glucose/1ìMdexamethasone medium had the least glucose consumption after48hours (P<0.05), which isthe best dosage for the model. Comparing to the control group, the IRS-1and PPAR-in theinsulin resistance model was decreased.2. The results of MTT experiment shown the cell survival rate of pioglitazone group wassignificantly higher than that of the insulin resistance group. The survival rate was related todosage and50ng/mL was the optimal dosage. Comparing to the insulin resistance group,pioglitazone could reduce cell death rate and caspase-3activity effectively. The eNOS proteinexpression level could be increased at the dosage of50ng/mL in pioglitazone group.3.The protein expression and mRNA ofIRS-1〠PI3K and Akt were increased afterpioglitazone applying to insulin to resist vascular endothelial cells. Pioglitazone was able toprotect insulin resistance on vascular endothelial by adjusting PI3K-Akt signal pathway. Conclusions:1.The model of insulin resistance in vascular endothelial cells was establishdedsuccessfully.2.Pioglitazone may protect insulin resistance on vascular endothelial cells, reduce celldamage. and the cell mechanism achieves its protection by the activation of PI3K-Akt signalpathways.Innovation points: From the point of view of cell signal pathway, discussing theprotective effects of pioglitazone on insulin resistance will provide experimental andtheoretical support for clinical application. |
