AMPK, Metabolic and Inflammatory Stress and the Endothelial Cell

说明申请的广泛、长期目标和具体目标，并提及 ;该项目（即，与原子能机构的使命相关）。简要描述研究设计和实现这些目标的方法。描述 你将用来实现这些目标的基本原理和技术。 i此外，用两三句话，用简单的通俗语言描述这项研究与公共卫生的相关性。如果申请得到资助， 我的描述将成为公开信息。因此，不包括专有/机密信息。不要超过空间 ：已提供。 代谢综合征在临床上被定义为以血脂异常为特征的疾病， 高血压、向心性肥胖和易患过早动脉粥样硬化性心血管疾病， 2型糖尿病它还与肌肉中的毛细血管稀疏和血管生成受损有关。在 在此之前，我们证明AMPK在培养的血管内皮细胞中起作用， （HUVEC，HAEC），并且其活化保护它们免受细胞凋亡，炎症和线粒体损伤。 （在富含葡萄糖、棕榈酸盐或TNF-α的培养基中孵育引起的功能障碍。另外我们 证明了Sirt 1/LKB 1/AMPK信号传导机制在各种培养细胞和大鼠中的存在。 我们已经获得了其存在于培养的内皮细胞中的初步证据。在项目1中， 我们将通过开展具有以下目标的研究来扩展这项研究：1）表征 Sirt 1和LKB 1在通过诸如葡萄糖剥夺等因素介导AMPK活化中的作用， 2）检测高血糖患者是否存在高血糖， 诱导胰岛素抵抗和细胞凋亡并增强TNF-α诱导的粘附分子 通过引起AMPK失调（活性降低或受损）在内皮细胞中的表达 激活）。如果正如预期的那样，我们将确定这种失调是否涉及抑制 Pf Sirt 1/LKB 1机制[可能通过SirT 1的翻译后氧化修饰（项目2）]， 和/或蛋白磷酸酶的活化;和3）检查AMPK介导的事件是否在 血管系统在小鼠体内受损，内皮细胞特异性缺失SirT 1或 LKB 1。研究将在由沃尔什博士（核心B）产生的cre/lox小鼠中进行，他将使用这些小鼠来 研究缺血诱导的血管生成（项目3）和科恩博士（项目2）在主动脉粥样硬化。我们 将与Nathan LeBrasseur博士一起描述运动对这些小鼠和对照小鼠的影响， 啮齿动物运动生理学家特别是，我们将确定SirT 1和LKB 1下调如何影响细胞的生长。 肌毛细血管密度增加和主动脉中通常发生的致动脉粥样硬化变化减少 代谢综合征表型啮齿类动物对运动的反应。 我... ^这些研究将提供关于SirT 1/LKB 1/AMPK信号传导功能的第一个信息。 血管系统中的机制。他们还应该提供新的见解，是否其失调， 是与代谢综合征相关的内皮细胞功能障碍的原因，也是其治疗的靶点。 疗法 ,

State the application's broad, long-term objectives and specific aims, making reference to the health relatedness of ; the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe v the rationale and techniques you will use to pursue these goals. i In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this i description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE : PROVIDED. The metabolic syndrome has been defined clinically as a disorder characterized by dyslipidemia, hypertension, central obesity and a predisposition to premature atherosclerotic cardiovascular disease and type 2 diabetes. It is also associated with capillary rarefaction and impaired angiogenesis in muscle. In the preceding grant periods, we demonstrated that AMPK is operative in cultured vascular endothelial cells '(HUVEC, HAEC) and that its activation protects them against the apoptosis, inflammation and mitochondrial (dysfunction caused by incubation in media enriched in glucose, palmitate or TNF-alpha. In addition, we demonstrated the existence of a Sirt1/LKB1/AMPK signaling mechanism in various cultured cells and in rat liver in vivo and we have obtained preliminary evidence for its presence in cultured endothelium. In project 1, we will extend this research by carrying out studies with the following objectives: 1) To characterize the roles of Sirtl and LKB1 in mediating AMPK activation by such factors as glucose deprivation, phenformin, and resveratrol in cultured endothelial cells; 2) To determine whether hyperglycemia induces insulin resistance and apoptosis and enhances TNF-alpha - induced adhesion molecule expression in endothelial cells by causing AMPK dysregulation (decreased activity or impaired activation). If as anticipated it does, we would then determine whether this dysregulation involves inhibition pf the Sirt1/LKB1 mechanism [possibly by post-translational oxidative modification of SirT1 (with project 2)], and/or activation of protein phosphatases; and 3) To examine whether AMPK-mediated events in the vasculature are impaired in vivo in mice with endothelial-cell specific deletions of either SirT1 or LKB1. Studies will be done in cre/lox mice that will be generated by Dr. Walsh (Core B), which he will use to study ischemia-induced angiogenesis (Project 3), and Dr. Cohen (Project 2) atherogenesis in the aorta. We will characterize the effects of exercise in these and in control mice together with Dr. Nathan LeBrasseur, a rodent exercise physiologist. In particular, we will determine how SirT1 and LKB1 downregulation affect the increase in muscle capillary density and the decrease in atherogenic changes in the aorta that typically occur in response to exercise in rodents with a metabolic syndrome phenotype. j - ¿ ^These studies will provide the first information about the function of the SirT1/LKB1/AMPK signaling mechanism in the vasculature. They should also provide novel insights as to whether its dysregulation could be a cause of the endothelial cell dysfunction associated with the metabolic syndrome and a target for its therapy. ,