AMPK, SIRT1 and mTOR:Mediators of Nutrient Excess

描述（由申请人提供）：已经提出，燃料感应酶AMPK的失调可能是2型糖尿病和其他与代谢综合征相关的疾病的致病因素，也是其治疗的靶点。在培养的大鼠EDL肌肉（高糖或亮氨酸）、培养的HepG2细胞（高糖）和注入葡萄糖（高糖和胰岛素）的大鼠肌肉和肝脏中，营养过剩降低AMPK活性并引起胰岛素抵抗（IR）。在这三种模型中，我们发现这些变化与SIRT1的减少和mTORC1的激活有关，这是另外两种与代谢综合征有关的燃料感应分子。在检查中，AMPK活性的降低似乎是初始事件，AMPK的激活阻止了所有这些变化的发生，包括IR。在单独的研究中，其他人已经表明AMPK可以激活SIRT1，我们发现SIRT1可以激活AMPK，这表明SIRT1/AMPK周期的存在，这些分子共同控制许多关键酶和转录调节因子。在此背景下，研究将以以下具体目的进行：(1)在机制水平上研究AMPK， SIRT1和mTORC1如何在培养肌肉和培养HepG2细胞中相互作用。在此过程中，我们还将测试我们的初步数据提出的假设，即营养过剩的最初影响是通过使AMPK与蛋白质磷酸酶相互作用来下调AMPK。(2)在相同的模型中确定这三种分子的失调如何导致IR，以及IR在什么条件下与炎症、氧化和内质网应激以及线粒体异常相关。在此过程中，我们将通过代谢、药理学和遗传学的方法来评估AMPK、SIRT1和mTORC作为预防和治疗IR的靶点。(3)以葡萄糖输注大鼠和转基因小鼠为模型，测试体外确定的机制是否在肌肉、肝脏和脂肪组织中有效。总的来说，这些研究将为营养过剩导致哺乳动物组织IR的基本机制提供新的信息。它们尤其及时，因为激活AMPK和SIRT1的疗法目前正在用于或正在进行人类疾病预防和治疗的试验。

DESCRIPTION (provided by applicant): It has been proposed that dysregulation of the fuel sensing enzyme AMPK could be both a pathogenic factor for type 2 diabetes and other disorders associated with the metabolic syndrome and a target for their therapy. Nutrient excess reduces AMPK activity and causes insulin resistance (IR) in incubated rat EDL muscle (high glucose or leucine), cultured HepG2 cells (high glucose) and muscle and liver of rats infused with glucose (high glucose and insulin). In all three models, we have found that these changes are associated with a decrease in SIRT1 and activation of mTORC1, two other fuel sensing molecules that have been linked to the metabolic syndrome. Where examined, decreased AMPK activity appeared to be the initial event and activation of AMPK prevented all of these changes from occurring, including the IR. In separate studies, others have shown that AMPK can activate SIRT1 and we have found that SIRT1 can activate AMPK, suggesting the existence of a SIRT1/AMPK cycle in which these molecules jointly govern many key enzymes and transcriptional regulators. Upon this background, studies will be performed with the following specific aims: (1) To examine at a mechanistic level how AMPK, SIRT1 and mTORC1 functionally interact with each other in incubated muscle and cultured HepG2 cells. In doing so, we will also test the hypothesis, suggested by our preliminary data, that the initial effect of a nutrient excess is to downregulate AMPK by causing it to interact with a protein phosphatase. (2) To determine in the same models how dysregulation of the three molecules leads to IR and under what conditions the IR is associated with inflammation, oxidative and ER stress and mitochondrial abnormalities. In the process, we will evaluate AMPK, SIRT1 and mTORC as targets both for preventing and treating IR using metabolic, pharmacological and genetic approaches and (3) To test whether the mechanisms defined in vitro are operative in muscle, liver and adipose tissue in vivo using the glucose- infused rat and genetically modified mice as models. Collectively, these studies will provide novel information about fundamental mechanisms by which nutrient excess leads to IR in mammalian tissues. They are especially timely since therapies that activate AMPK and SIRT1 are presently being used or are undergoing trials for disease prevention and treatment in humans. PUBLIC HEALTH RELEVANCE: The metabolic syndrome underlies such disorders as type 2 diabetes, coronary heart disease, hypertension and Alzheimer's disease and probably contributes to accelerated aging. We have identified three fuel sensing and signaling molecules, AMPK, SIRT1, and mTORC1 whose dysregulation could contribute to these disorders. The proposed studies will explore at a molecular level how these molecules interact with each other and in turn, how their activation (or inhibition) could diminish insulin resistance, a hallmark of the metabolic syndrome, caused by nutrient excess.