IKKBeta Mediated Impairment of Endothelial Nitric Oxide Production

描述(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血管内皮细胞产生NO的机制，从而促进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药物的开发。这项修订的研究方案的特定假设是，作为调节核因子-kappaB炎症途径的关键酶，IKKbeta介导了胰岛素抵抗对NO产生的影响。这一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)、高糖和肿瘤坏死因子-α激活，所有这些都与胰岛素抵抗的发病机制有关。第二，IKKβ的激活与NO的产生受损有关。第三，使用阿司匹林或从基因上使用显性负的IKKβ结构抑制IKKβ，可阻断FFA、肿瘤坏死因子-α或葡萄糖损害NO产生的能力。最后，野生型IKKbeta的过度表达概括了FFA、葡萄糖或肿瘤坏死因子-α对NO产生的影响。这些实验结果表明，IKKβ的激活是导致内皮NO产生受损的必要条件和充分条件。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起着关键作用。基于这些观察，这项提议的实验重点是IKKbeta在调节受损的NO产生中的作用。我们建议在胰岛素抵抗的内皮细胞培养模型和体内模型中确定IKKβ的激活如何损害内皮细胞NO的产生。接下来，我们将讨论胰岛素抵抗的介体如何激活IKKβ，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。目的：1.确定IKKβ激活对内皮细胞产生NO的影响机制。目的2.在饮食诱导肥胖和胰岛素抵抗的小鼠模型中，确定内皮胰岛素信号转导受损和NO产生是否与体内IKKβ激活有关。目的3.探讨在血管胰岛素抵抗形成过程中，Toll样受体通路是否是血管内皮细胞激活IKKβ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.