Regulation of Nutrient Sensing and Muscle Wasting by Alcohol

描述（由申请人提供）：酒精滥用是一个主要的公共卫生问题，会导致过早死亡、医院康复受损和多器官系统功能障碍。肌肉萎缩是持续酒精滥用的标志，相关的近端肌肉无力是骨骼肌肌病的一种普遍形式。在过去的资助期内，使用已建立的急性酒精中毒和慢性酒精摄入啮齿动物模型，我们证明酒精不仅损害基础肌肉蛋白质合成，还损害该组织对营养物质（如亮氨酸）刺激的反应性。这种亮氨酸（Leu）抗性源于mTOR激酶活性的抑制，表现为蛋白质合成的抑制，并且似乎在很大程度上与Akt/ tsc无关。我们还报道了类似的机制在酒精培养的肌肉细胞中起作用。我们的长期目标是阐明亮氨酸诱导骨骼肌mTOR活性的Akt/ tsc独立机制，并确定它们在酒精下调这种营养传感器从而产生骨骼肌肌病的能力中的相对重要性。为了解决这一目标中隐含的问题，拟议的研究有以下具体目的：(1)评估酒精诱导的总和/或磷酸化DEPTOR（一种已知的负mtor调节蛋白）的变化作为减少基础和/或亮氨酸刺激的肌肉蛋白合成的机制的重要性；(2)阐明酒精破坏mTOR复合物-1 （mTORC1）的内体运输并损害肌肉中氨基酸感知和蛋白质合成的机制；(3)阐明在基础和营养刺激条件下，MAP4K3信号的改变与酒精诱导的mTOR激酶活性降低的因果关系。我们的应用程序利用了许多创新的方法，使新试剂的可用性成为可能，并得到了令人振奋的初步数据的支持。值得注意的是，提出的慢病毒递送的shRNA在体内电穿孔专门针对骨骼肌，允许进行功能丧失和功能获得的实验，并为观察到的变化分配因果关系。此外，肌肉质量/蛋白质合成的变化将与肌肉力量/收缩性的直接评估相关。这些在体内

DESCRIPTION (provided by applicant): Alcohol abuse is a major public health problem leading to premature death, impaired hospital recovery, and dysfunction of multiple organ systems. Muscle wasting is a hallmark of sustained alcohol abuse and the associated proximal muscle weakness represents a prevalent form of skeletal muscle myopathy. During the past funding period, using established rodent models of acute alcohol intoxication and chronic alcohol ingestion, we demonstrated that alcohol impairs not only basal muscle protein synthesis but also the responsiveness of this tissue to nutrient (e.g., leucine) stimulation. This leucine (Leu) resistance results from suppression of mTOR kinase activity, which is manifested as an inhibition of protein synthesis and which appears to be largely Akt/TSC-independent. We also reported that a similar mechanism is operational in muscle cells cultured with alcohol. Our long-term goal is to elucidate the Akt/TSC-independent mechanisms underpinning Leu-induced mTOR activity in skeletal muscle per se and to determine their relative importance in the ability of alcohol to down-regulate this nutritional sensor thereby producing skeletal muscle myopathy. To address the questions implicit in this goal, the proposed research has the following specific aims: (1) Assess the importance of the alcohol-induced change in total and/or phosphorylated DEPTOR (a known negative mTOR-regulatory protein) as a mechanism for the decrease in basal and/or Leu-stimulated muscle protein synthesis; (2) Delineate the mechanism by which alcohol disrupts endosomal trafficking of mTOR complex-1 (mTORC1) and impairs amino acid sensing and protein synthesis in muscle; and (3) Elucidate the extent to which altered MAP4K3 signaling is causally linked to the alcohol-induced decrease in mTOR kinase activity under basal and nutrient-stimulated conditions. Our application exploits a number of innovative approaches made possible by the availability of novel reagents and is supported by provocative preliminary data. It is noteworthy that the proposed in vivo electroporation of lentiviral-delivered shRNA specifically to skeletal muscle permits loss- and gain-of-function experiments to be performed and to assign causality to the observed changes. Furthermore, changes in muscle mass/protein synthesis will be correlated with direct assessment of muscle strength/contractility. These in vivo methods, used in conjunction with an established model of chronic alcohol ingestion in mice and with the availability of novel phospho-specific antibodies, place us in a unique position to rapidl and significantly advance knowledge pertaining to amino acid regulation of mTORC1. Our focus on state-of-the-art in vivo approaches permits us to definitively assign physiological importance to our observations, while complementary in vitro studies will allow us to define cellular mechanisms and to prioritize future work. The expected research outcomes will have a positive impact by contributing fundamental knowledge concerning nutrient regulation at the molecular level and provide seminal mechanistic insights into the clinically significant pathology of alcohol induced muscle disease.