OKHSC COBRE: ACTIVATION OF AMPK PROTECTS AGAINST DIABETIC VASCULAR COMPLICATIONS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 糖尿病可导致微血管并发症，并加速大血管疾病的发生。糖尿病的一个特征是过度氧化应激，其损害一氧化氮（NO）生物活性，增加粘附分子表达，并促进动脉粥样硬化病变形成。 初步研究表明，高血糖症和游离脂肪酸（FFA）在内皮细胞中产生氧化应激，导致脂质过氧化、前列环素合酶（PGIS）的酪氨酸硝化和内皮功能障碍;用AICAR或用过表达组成型活性AMPK的腺病毒处理细胞可防止增加的O2-，缺血预处理（IPC）激活AMPK有效地阻断了氧化应激的标志物，可能是通过UCP-2的过度表达。AMPK降低氧化应激的最确凿证据是（IPC）未能改变AMPK-KO小鼠中氧化应激标志物和UCP-2表达。 我们假设AMPK激活可以通过增加抗氧化潜力（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，从而导致氧化应激的减少和NO生物活性的增加。这一假设将在两个具体目标下进行。1)确定AMPK的激活是否减少氧化应激和内皮功能障碍诱导的 高血糖和FFA，并评估它是如何工作的。HAEC将与葡萄糖和FFA一起孵育，FFA是氧化应激的标志物，例如（O2.-），在AMPK活性通过使用AICAR或AMPK抑制剂改变的条件下，将监测过氧亚硝酸盐、UCP 2表达、NO生物活性和粘附分子的表达。 腺病毒构建体。2)确定氧化应激和内皮功能障碍的AMPK依赖性减少是否在体内起作用。野生型和AMPK α 1和α 2 KO小鼠将用于制备糖尿病动物模型。将通过测量功能参数UCP-2表达和ONOO的选定参数来测定或在高血糖/FFA培养基中孵育不同时间后测定主动脉环前列环素合成酶途径。拟议的研究将深入了解糖尿病导致血管疾病的机制，并可能导致使用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.