Endothelial lipid droplet turnover and regulation of metabolic function

项目摘要 越来越多的证据表明，血管内皮细胞在动脉粥样硬化中起着重要作用。 代谢稳态的调节。相应地，内皮功能障碍有助于新陈代谢 与肥胖和胰岛素抵抗相关的精神错乱。我们的实验室提供了第一个证据 血管内皮细胞(ECs)在体内具有形成脂滴(LDs)的能力，并证明了LD的周转 (即合成和降解)是维持EC静止和功能所必需的。具体来说， 二酰甘油乙酰转移酶(DGAT)对LD合成途径中关键酶的药理抑制作用 1和2，导致内质网应激。或者，内皮细胞特异性的脂肪缺失 甘油三酯脂肪酶(ATGL)是LD水解过程中的限速酶，导致血管内皮细胞显著减少。 一氧化氮合酶(ENOS)蛋白水平和一氧化氮(NO)在标准饲料喂养小鼠中的生物利用度。 事实上，这些血管紊乱与全身新陈代谢紊乱有关，但其作用 EC LD周转率与血管和代谢功能的控制有关，目前尚不清楚。因此，在这个 提案中，我将定义内皮LD周转在维持血管和代谢方面的具体作用 在正常生理、DIO以及对急性和慢性运动的反应中发挥作用。要实现这些目标 结束后，我将产生可诱导的内皮特异性ATGL基因敲除(IECKO)、DGAT1-iECKO和DGAT2-iECKO 以小鼠为工具剖析LD周转控制血管和全身的分子机制 代谢动态平衡。在目标1中，我将剖析LD周转中断导致的细胞机制 在正常生理和DIO下通过严格和细致的分子和代谢来治疗内皮功能障碍 人物刻画。在目标2中，我将破译内皮细胞LD周转对维持胰岛素的重要性 通过全面的体内代谢研究和体外组织分析，对正常生理和DIO具有敏感性。 最后，在目标3中，我将确定内皮LD周转在调节适应中的作用 使用与目标2类似的方法进行的一场剧烈的运动比赛和慢性耐力训练。 这些研究将解决迄今未知的EC LD周转在全身新陈代谢中的作用。结果来自 这些研究将更好地理解血管和代谢功能之间的相互关系。 在正常生理期间以及在肥胖和II型糖尿病的背景下。除了这些研究目标外， 本提案描述了一项为期五年的强化导师培训计划，其目标是发展校长 调查员(PI，Nabil Boutagy，博士)成为一位独立而有影响力的血管领域的科学家 在他的主要导师威廉·塞萨博士和共同导师威廉·塞萨博士的指导下，生物和新陈代谢。 杰拉尔德·舒尔曼。此外，还组建了一支由世界级科学家组成的团队，提供监督和 指导实现拟议研究的所有方面和PI的职业发展。

Project Summary Accumulating evidence demonstrates that the vascular endothelium plays an important role in the regulation of metabolic homeostasis. In accordance, endothelial dysfunction contributes to metabolic derangements associated with obesity and insulin resistance. Our laboratory provided the first evidence that endothelial cells (ECs) have the capacity to form lipid droplets (LDs) in vivo and demonstrated that LD turnover (i.e., synthesis and degradation) is necessary for maintaining EC quiescence and function. Specifically, pharmacological inhibition of key enzymes in the LD synthesis pathway, diacylglycerol acetyl transferase (DGAT) 1 and 2, led to endoplasmic reticulum (ER) stress. Alternatively, endothelial specific deletion of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in LD hydrolysis, led to a profound reduction in endothelial nitric oxide synthase (eNOS) protein levels and nitric oxide (NO) bioavailability in standard chow fed mice. Indeed, these vascular perturbations have been linked to derangements in whole-body metabolism, but the role of EC LD turnover as it relates to the control of vascular and metabolic function is unknown. Therefore, in this proposal, I will define the specific role of endothelial LD turnover in maintaining vascular and metabolic function during normal physiology, DIO and in response to acute and chronic exercise. To achieve these ends, I will generate inducible, endothelial specific ATGL knockout (iECKO), DGAT1-iECKO and DGAT2-iECKO mice as tools to dissect the molecular mechanisms by which LD turnover controls vascular and whole-body metabolic homeostasis. In aim 1, I will dissect the cellular mechanisms by which disruption in LD turnover leads to endothelial dysfunction under normal physiology and DIO via rigorous and meticulous molecular and metabolic characterization. In aim 2, I will decipher the importance of endothelial LD turnover in maintaining insulin sensitivity in normal physiology and DIO via comprehensive in vivo metabolic studies and ex vivo tissue analysis. Lastly, in aim 3, I will establish the role of endothelial LD turnover in the regulation of adaptations in response to an acute bout of exercise bout and chronic endurance training using a similar approach as aim 2. Collectively, these studies will address a heretofore unknown role of EC LD turnover in whole-body metabolism. Results from these studies will provide a better understanding of the interrelationship between vascular and metabolic function during normal physiology and in the setting of obesity and type II diabetes. In addition to these research aims, this proposal describes a five-year intensive mentored training program with the goal of developing the Principal Investigator (PI, Nabil Boutagy, Ph.D.) into an independent and high impact, scientist in the fields of vascular biology and metabolism under the supervision of his primary mentor, Dr. William Sessa, and co-mentor, Dr. Gerald Shulman. Furthermore, a team of world-class scientists has been assembled to provide oversight and guidance in achieving all aspects of the proposed research and the PI’s career development.