AMPK, SIRT1 and mTOR:Mediators of Nutrient Excess

描述（由申请人提供）：已经提出，燃料传感酶AMPK的失调可能是2型糖尿病和与代谢综合征相关的其他疾病的致病因素，也可能是其治疗的靶点。营养过剩降低AMPK活性，并导致培养的大鼠EDL肌肉（高葡萄糖或亮氨酸），培养的HepG 2细胞（高葡萄糖）和肌肉和肝脏输注葡萄糖（高葡萄糖和胰岛素）的大鼠胰岛素抵抗（IR）。在所有三种模型中，我们发现这些变化与SIRT 1的减少和mTORC 1的激活有关，这两种燃料传感分子与代谢综合征有关。在研究中，AMPK活性降低似乎是最初的事件，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SIRT 1，我们发现SIRT 1可以激活AMPK，这表明存在SIRT 1/AMPK循环，其中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，将进行以下具体目的的研究：（1）在机制水平上检查AMPK、SIRT 1和mTORC 1在孵育的肌肉和培养的HepG 2细胞中如何功能性地相互作用。在这样做的过程中，我们还将测试我们的初步数据所提出的假设，即营养过剩的最初影响是通过使AMPK与蛋白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，以及在何种条件下IR与炎症、氧化和ER应激以及线粒体异常相关。在此过程中，我们将使用代谢、药理学和遗传学方法评估AMPK、SIRT 1和mTORC作为预防和治疗IR的靶点;（3）使用葡萄糖输注大鼠和遗传修饰小鼠作为模型，测试体外定义的机制是否在体内肌肉、肝脏和脂肪组织中有效。总的来说，这些研究将提供新的信息，营养过剩导致哺乳动物组织IR的基本机制。它们特别及时，因为激活AMPK和SIRT 1的疗法目前正在使用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.