IKKBeta Mediated Impairment of Endothelial Nitric Oxide Production

描述（由申请人提供）：心血管疾病是成人糖尿病患者死亡和残疾的主要原因。内皮功能障碍是糖尿病血管疾病的一个标志，其特征是一氧化氮（NO）生物利用度降低。我们的长期目标是阐明胰岛素抵抗减少内皮细胞 NO 产生的机制，从而促进开发可用于减轻糖尿病和胰岛素抵抗对心血管疾病影响的治疗方法。这项修订后的研究计划的具体假设是，IKKbeta 是调节 NF-κB 炎症通路的关键酶，介导胰岛素抵抗对 NO 产生的影响。该假设基于我们实验室完成的以下实验观察。首先，IKKbeta 在内皮细胞中被游离脂肪酸 (FFA)、高葡萄糖和 TNF-α 激活，所有这些都与胰岛素抵抗的发病机制有关。其次，IKKbeta 的激活与 NO 产生受损有关。第三，使用阿司匹林在药理学上抑制IKKbeta，或使用显性失活IKKbeta构建体在遗传上抑制IKKbeta，从而阻断FFA、TNF-α或葡萄糖损害NO产生的能力。最后，野生型 IKKbeta 的过度表达再现了 FFA、葡萄糖或 TNF-α 损害 NO 产生的作用。这些实验的结果表明，IKKbeta 激活对于导致内皮 NO 产生受损是必要且充分的。这些结果还表明炎症途径在营养过剩和细胞因子介导的内皮胰岛素抵抗中发挥着关键作用。基于这些观察，本提案的实验重点是 IKKbeta 在介导 NO 产生受损中的作用。我们建议在内皮细胞培养模型和胰岛素抵抗体内模型中确定 IKKbeta 的激活如何损害内皮 NO 的产生。接下来我们将讨论胰岛素抵抗的介质如何激活 IKKbeta，并研究先天免疫（Toll 样受体）在介导血管胰岛素抵抗中的潜在作用。提出以下目标： 目标 1. 确定 IKKbeta 激活损害内皮细胞中 NO 产生的机制。目标 2. 在饮食诱导的肥胖和胰岛素抵抗小鼠模型中，确定受损的内皮胰岛素信号传导和 NO 产生是否与体内 IKKbeta 激活相关。目标 3. 确定 Toll 样受体通路在血管胰岛素抵抗的发展过程中是否是 IKKbeta 内皮激活所必需的。

Description (provided by applicant): Cardiovascular disease is the principal cause of death and disability in adults with diabetes. Endothelial dysfunction is a hallmark of diabetic vascular disease and is characterized by decreased bioavailability of nitric oxide (NO). Our long-term goal is to elucidate the mechanism by which insulin resistance reduces NO production in endothelial cells and thereby facilitate the development of therapeutics that can be used to attenuate the impact of diabetes and insulin resistance on cardiovascular disease. The specific hypothesis of this revised research proposal is that IKKbeta a key enzyme in the regulation of the NF-kappaB inflammatory pathway mediates the effect of insulin resistance on NO production. This hypothesis is based on the following experimental observations completed in our laboratory. First, IKKbeta is activated in endothelial cells by free fatty acids (FFA), high glucose, and TNF-alpha all of which have been implicated in the pathogenesis of insulin resistance. Second, activation of IKKbeta is associated with impaired NO production. Third, inhibition of IKKbeta pharmacologically using aspirin or genetically using a dominant negative IKKbeta construct blocks the ability of FFA, TNF-alpha, or glucose to impair NO production. Finally, overexpression of wild-type IKKbeta recapitulates the effect of FFA, glucose, or TNF-alpha to impair NO production. The results of these experiments suggest that IKKbeta activation is both necessary and sufficient to cause impairment of endothelial NO production. These results also indicate that inflammatory pathways play a key role in both nutrient excess and cytokine mediated endothelial insulin resistance. Based on these observations the experimental focus of this proposal is on the role of IKKbeta in mediating impaired NO production. We propose to determine how activation of IKKbeta impairs endothelial NO production in both an endothelial cell culture model and in an in vivo model of insulin resistance. We will next address how mediators of insulin resistance might activate IKKbeta and examine a potential role for innate immunity (Toll Like Receptor) in mediating vascular insulin resistance. The following aims are proposed: Aim 1. To determine the mechanism by which IKKbeta activation impairs NO production in endothelial cells. Aim 2. To determine whether impaired endothelial insulin signaling and NO production are associated with IKKbeta activation in vivo in a mouse model of diet-induced obesity and insulin resistance. Aim 3. To determine whether the Toll Like Receptor pathway is necessary for endothelial activation of IKKbeta during the development of vascular insulin resistance.