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）的生物利用度降低。我们的长期目标是阐明胰岛素抵抗减少内皮细胞NO生成的机制，从而促进治疗方法的发展，可用于减轻糖尿病和胰岛素抵抗对心血管疾病的影响。本次修订的研究方案的具体假设是：IKKbeta是调节NF-kappaB炎症通路的关键酶，可介导胰岛素抵抗对NO生成的影响。这个假设是基于我们实验室完成的以下实验观察。首先，ikkβ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，ikkβ的激活与一氧化氮生成受损有关。第三，在药理学上使用阿司匹林或基因上使用IKKbeta显性阴性构建体抑制IKKbeta，可阻断FFA、tnf - α或葡萄糖损害NO生成的能力。最后，野生型IKKbeta的过表达概括了FFA、葡萄糖或tnf - α对NO生成的影响。这些实验结果表明，ikkβ激活是导致内皮细胞NO生成损伤的必要和充分条件。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都起着关键作用。基于这些观察结果，本提案的实验重点是IKKbeta在介导受损NO生成中的作用。我们建议在内皮细胞培养模型和体内胰岛素抵抗模型中确定ikkβ的激活如何损害内皮细胞NO的产生。接下来，我们将探讨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。提议的目标如下：目标1。确定ikkβ激活损害内皮细胞NO生成的机制。目标2。在饮食诱导的肥胖和胰岛素抵抗小鼠模型中，确定内皮胰岛素信号和NO生成受损是否与体内ikkβ激活相关。目标3。为了确定在血管胰岛素抵抗的发展过程中，Toll样受体通路对于IKKbeta的内皮活化是否必要。

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.