OKHSC COBRE: ACTIVATION OF AMPK PROTECTS AGAINST DIABETIC VASCULAR COMPLICATIONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 众所周知，糖尿病会导致微血管并发症，并加速大血管疾病。糖尿病的一个特征是过度的氧化应激，它损害了一氧化氮(NO)的生物活性，增加了黏附分子的表达，促进了动脉粥样硬化病变的形成。 初步研究表明，高血糖和游离脂肪酸(FFA)给内皮细胞带来氧化应激，导致脂质过氧化、前列环素合成酶(PGIs)酪氨酸硝化和内皮功能障碍；AICAR或过度表达固有活性AMPK的腺病毒治疗细胞可防止O2增加、NO失活、PGIS硝化和内皮功能障碍；缺血预适应(IPC)激活AMPK有效地阻断氧化应激的标志物，可能是通过UCP-2的过度表达。AMPK减轻氧化应激的最确凿证据是，(IPC)未能改变AMPK-KO小鼠的氧化应激标志物和UCP-2的表达。 我们推测，AMPK的激活可以通过增加抗氧化能力(UCP-2和/或超氧化物歧化酶)来保护内皮细胞免受高血糖和FFA的不利影响，从而减少氧化应激，增加NO的生物活性。这一假设将在两个具体目标下进行。1)确定AMPK的激活是否减轻了氧化应激和血管内皮细胞功能障碍 高血糖和游离脂肪酸，并评估其如何发挥作用。将HAEC与葡萄糖和FFA孵育，在AICAR或AICAR改变AMPK活性的条件下，监测氧化应激的标志物(O2.-)、过氧亚硝酸盐、UCP2表达、NO生物活性和黏附分子的表达。 腺病毒结构。2)确定AMPK依赖的氧化应激和内皮功能障碍的减少是否在体内起作用。用野生型和AMPKα1、α2 KO小鼠制作糖尿病动物模型。将主动脉环在高血糖/FFA介质中孵育不同时间后，通过测量功能参数、UCP-2表达和ONOO-前列环素合成酶途径的选定参数来检测或检测主动脉环。这些研究将对糖尿病导致血管疾病的机制提供深入的认识，并可能导致将AMPK激活作为预防和治疗糖尿病血管并发症的重要靶点。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It has been well established that diabetes leads to microvascular complications, and accelerates macrovascular diseases. One feature of diabetes is excessive oxidant stress, which impairs nitric oxide (NO) bioactivity, increase adhesion molecule expression, and promote atherosclerotic lesion formation. Preliminary studies demonstrate that hyperglycemia and free fatty acids (FFA) impart an oxidant stress in endothelial cells, resulting in lipid peroxidation, tyrosine nitration of prostacyclin synthase (PGIS), and endothelial dysfunction; Treatment of cells with either AICAR or with adenoviruse overexpressing constitutively active AMPK prevents the increased O2.-, inactivation of NO, PGIS nitration and endothelial dysfunction; Activation of AMPK by ischemic preconditioning (IPC) effectively blocked the markers of oxidant stress, likely via over expression of UCP-2. The most conclusive evidence that AMPK reduced oxidant stress is that (IPC) failed to alter both the markers of oxidant stress and UCP-2 expression in the AMPK-KO mice. We hypothesize that AMPK activation could protect the endothelial cell against the adverse effects of hyperglycemia and FFA by increasing antioxidant potentials (UCP-2 and/or superoxide dismutase) that lead to a decrease in oxidant stress and increase in NO bioactivity. This hypothesis will be pursued in two specific aims. 1) Determine if activation of AMPK reduces oxidant stress and endothelial dysfunction induced by hyperglycemia and FFA, and evaluate how it works. HAEC will be incubated with glucose and FFA, the markers of oxidant stress such as (O2.-), peroxynitrite, UCP2 expression, NO bioactivity and expression of adhesion molecules will be monitored under conditions in which AMPK activity is altered by using AICAR or adenoviral constructs. 2) Determine if AMPK-dependent reduction in oxidant stress and endothelial dysfunction is operating in vivo. Wild type and AMPK alpha 1 and alpha 2 KO mice will be used to make diabetic animal model. Aortic rings will be assayed or assayed after being incubated for various time in hyperglycemia/FFA media by measurements of functional parameters UCP-2 expression and selected parameters of ONOOprostacyclin synthase pathway. The proposed studies will provide insight into the mechanism by which diabetes leads to vascular diseases and may lead to use activation of AMPK as an important target for the prevention and treatment of diabetic vascular complications.