Insulin Resistance in Vascular Endothelial Cells and Foxo

项目3的中心主题是研究生物化学、遗传学、细胞生物学和综合 2型患者动脉粥样硬化性心血管疾病易感性的生理学基础 糖尿病。在这个更大的背景下。项目3开始确定胰岛素信号受损的机制 在血管内皮细胞中-重点是FoxO转录因子在这一过程中的作用。我们 假设胰岛素信号通过转录因子FoxO的异构体在 内皮细胞功能及其改变促进内皮功能障碍。为了解决这一假设， 在目标1中，我们建议表征功能丧失的小鼠对动脉粥样硬化的易感性 三种FOXO基因在内皮细胞中的突变。三种FOXO基因敲除小鼠的初步数据显示， FoxO消融的动脉粥样硬化保护作用，与增加一氧化氮合成有关，减少ROS 代，并减少单核细胞募集。我们建议将其机制描述为 内皮细胞特异性三氧氟沙星对动脉粥样硬化的保护作用及血管功能表型 淘汰赛。了解胰岛素抵抗和高血糖是如何改变内皮细胞的 在AIM 2中，我们将在血管内皮细胞中产生两种不同的FoxOI功能增益突变体 转基因小鼠。胰岛素抵抗导致FoxOI核滞留。我们在过去的周期中已经表明， FoxOI核滞留与ROS生成和单核细胞募集增加有关。我们会 通过建立FoxOI功能增益模型来测试这些体外观察的生理学影响 这分别模拟了胰岛素抵抗和高血糖对内皮功能的影响。我们 预计这项工作将产生以下创新：(I)建立FoxO蛋白质作为 动脉粥样硬化中易导致内皮功能障碍的细胞生物学过程的关键，包括 炎症、细胞存活、NO生成、ROS产生和细胞黏附，(Ii)识别效应基因 通过三者的功能分析探讨胰岛素抵抗中血管内皮功能障碍的机制 血管内皮细胞FOXO基因敲除和FOXO功能增强模型：(III)一种新基因的鉴定 调节FOXO-Akt前馈环，从而FOXO功能控制内皮细胞的胰岛素敏感性， (4)建立和分析小鼠模型，以了解高血糖对血管的影响 功能。鉴于内皮功能障碍在糖尿病动脉粥样硬化中的核心作用，缺乏合适的 简化论者的实验模型，以及动脉粥样硬化治疗必须在 高血糖，对目标2中描述的靶向小鼠突变的分析将提供独特的机制 和治疗洞察力，以确定生物化学和细胞生物学途径，调节 高血糖和内皮功能障碍之间的重要相互作用。 相关性(请参阅说明)： 拟议的研究将揭示紊乱的胰岛素作用之间相互作用的新维度， 血管细胞生物学和动脉粥样硬化，并扩展目前可用的靶点库 2型糖尿病的动脉粥样硬化治疗。我们将根据过去筹资周期的经验教训，探索新的 血管疾病的机制，有可能为治疗提供可操作的靶点。驾驶 主题是定义可用于临床对抗动脉粥样硬化和胰岛素抵抗的途径。

The central theme of Project 3 is to investigate the biochemical, genetic, cell biological and integrated physiological basis of the predisposition to atherosclerotic cardiovascular disease in patients with type 2 diabetes. Within this larger context. Project 3 sets out to determine mechanisms of impaired insulin signaling in vascular endothelial cells-with a focus on the contribution of FoxO transcription factors to this process. We hypothesize that insulin signaling through isoforms of transcription factor FoxO plays an important role in endothelial cell function and that its alteration promotes endothelial dysfunction. To address this hypothesis, in AIM 1 we propose to characterize the susceptibility to atherosclerosis in mice with loss-of-function mutations of the three Foxo in endothelial cells. Preliminary data in triple Foxo knockout mice show a striking atheroprotective effect of FoxO ablation, associated with increased nitric oxide synthesis, reduced ROS generation, and reduced monocyte recruitment. We propose to characterize the mechanism of atherosclerosis protection and the vascular function phenotype in endothelial cell-specific triple Foxo knockouts. To understand how insulin resistance and hyperglycemia contribute to alter endothelial cell function, in AIM 2 we will generate two different FoxOI gain-of-function mutants in endothelial cells of transgenic mice. Insulin resistance results in FoxOI nuclear retention. We have shown in the past cycle that FoxOI nuclear retention is associated with increased ROS generation and monocyte recruitment. We will test the physiologic impact of these in vitro observations by developing models of FoxOI gain-of-function that mimic the effects of insulin resistance and hyperglycemia, respectively, on endothelial function. We envision that the following innovations will arise from this work: (i) Establishing FoxO proteins as the linchpin of cell biological processes that predispose to endothelial dysfunction in atherosclerosis, including inflammation, cell survival, NO generation, ROS production, and cell adhesion, (ii) Identifying effector genes and mechanisms of endothelial dysfunction in insulin resistance through the functional analysis of the triple Foxo knockout and Foxo gain-of-function models in vascular endothelial cells, (iii) Identification of a novel regulatory Foxo-Akt feed-forward loop, whereby Foxo function controls insulin sensitivity in endothelial cells, (iv) Development and analysis of mouse models to understand the effects of hyperglycemia on vascular function. Given the central role of endothelial dysfunction in diabetic atherosclerosis, the dearth of suitable reductionist experimental models, and the fact that atherosclerosis treatment must occur in the context of hyperglycemia, analyses of the targeted mouse mutants described in Aim 2 will provide unique mechanistic and treatment insight into the identification of biochemical and cell biological pathways that regulate the important interaction between hyperglycemia and endothelial dysfunction. RELEVANCE (See instructions): The proposed studies will reveal new dimensions to the interaction between disordered insulin action, vascular cell biology and atherosclerosis, and expand the repertoire of currently available targets for atherosclerosis therapy in type 2 diabetes. Building on lessons of the past funding cycle, we will explore new mechanisms of vascular disease with the potential to provide actionable targets for therapy. The driving theme is to define pathways that can be enlisted in the clinic against atherosclerosis and insulin resistance.