Project 3: Sarcopenia of ALD: Regulation of Skeletal Muscle Autophagy by Alcohol

摘要 酒精性肝病的患病率和发病率正在增加。骨骼肌块丢失或 骨质疏松症是酒精性肝病最常见的并发症，对患者的生存、生活质量有不利影响。 生活和发展的其他并发症的肝病。骨质疏松症更严重，进展更快 酒精中毒的患者比非酒精中毒的患者更快。这表明除了其他因素外，乙醇还有直接的作用。 肝脏疾病在调节石棺减少中的后果。众所周知，与酒精相关的肌肉损失和 肌病的发病率是肝病的五倍。尽管认识到了高度的临床意义， 目前还没有治疗方案，主要是因为酒精性肝病的肌肉量减少的机制是 不知道。酒精导致的骨骼肌蛋白质合成减少在过去已经被描述过了。然而， 由于蛋白质合成受损本身不足以导致肌肉损失，因此蛋白质分解的增加是 也是酒精性肝病发生石棺减少所必需的。泛素-蛋白酶体途径 和自噬是骨骼肌蛋白质分解的两种公认的机制。我们有 先前报道，乙醇不改变或减少增加骨骼肌介导的自噬 蛋白酶体活性。本研究的目的是确定导致这些疾病的分子机制 乙醇介导的骨骼肌自噬增加，以确定潜在的治疗靶点并执行 临床转化性研究。将使用一系列全面的模型进行分子和代谢研究 在患有酒精性肝硬化的人类受试者中，转基因小鼠和C2C12肌肉细胞暴露于 酒精。我们的初步研究表明，酒精抑制mTOR的激活，mTOR是一种已知的 自噬和AMPK，由于这些反调节因子的去磷酸化而激活自噬 信号分子。我们还观察到，关键的去磷酸化酶蛋白磷酸酶2A的活性， 乙醇使其上游抑制因子PI3Kγ的活性降低。最后，活性氧 线粒体和微粒体醇代谢产生的物种抑制PI3Kγ。因此，我们 假设PI3Kγ-PP2A轴受损在酒精性肝病中介导骨骼肌自噬。 我们将通过两个特定的目标来检验这一假说，以证明酒精介导的机制 骨骼肌PI3Kγ-PP2A轴受损是由于酒精代谢产生ROS和 去磷酸化增加损害下游标准mTOR靶点，从而增加 自噬和石棺减少症。PI3Kγ基因敲除小鼠的骨骼肌 基因敲除，体内电穿孔与成分活性的mTOR，暴露于酒精和 酒精性肝硬变患者服用含有过量亮氨酸的必需氨基酸直接激活 将使用mTOR。这些对一系列模型系统的全面研究将为 开发新的、基于机制的靶向疗法来逆转酒精性肝病的肌钙减少。

ABSTRACT The prevalence and incidence of alcoholic liver disease is increasing. Loss of skeletal muscle mass or sarcopenia is the most frequent complication of alcoholic liver disease and adversely affects survival, quality of life and development of other complications of liver disease. Sarcopenia is more severe and progresses more rapidly in alcoholic than in non-alcoholic patients. This suggests a direct effect of ethanol in addition to other consequences of liver disease in mediating sarcopenia. It is also known that alcohol related muscle loss and myopathy are five times more frequent than liver disease. Despite recognition of the high clinical significance, there are no therapeutic options primarily because the mechanisms of sarcopenia in alcoholic liver disease are not known. Reduced skeletal muscle protein synthesis by alcohol has been described in the past. However, since impaired protein synthesis alone is not enough to cause muscle loss, an increase in protein breakdown is also necessary for the development of sarcopenia in alcoholic liver disease. The ubiquitin-proteasome pathway and autophagy are two well-recognized mechanisms of skeletal muscle protein breakdown. We have previously reported that ethanol increases skeletal muscle mediated autophagy with unaltered or decreased proteasome activity. The goal of the present studies is to determine the molecular mechanisms responsible for ethanol mediated increase in skeletal muscle autophagy, to identify potential therapeutic targets and to perform clinical translational studies. Molecular and metabolic studies in a comprehensive array of models will be used in human subjects with alcoholic cirrhosis, and genetically modified mice and C2C12 muscle cells exposed to alcohol. Our preliminary studies showed that alcohol inhibits activation of mTOR, a known inhibitor of autophagy, and AMPK, that activates autophagy due to dephosphorylation of these counter-regulatory signaling molecules. We also observed that the activity of protein phosphatase 2A, a critical dephosphorylase, increased while the activity of its upstream inhibitor, PI3Kγ was reduced by alcohol. Finally, reactive oxygen species, generated by mitochondrial and microsomal alcohol metabolism, inhibits PI3Kγ. We therefore hypothesize that impaired PI3Kγ-PP2A axis mediates skeletal muscle autophagy in alcoholic liver disease. We will test this hypothesis by 2 specific aims to demonstrate that the mechanism of alcohol mediated impairment of the skeletal muscle PI3Kγ-PP2A axis is due to ROS generated from alcohol metabolism and increased dephosphorylation impairs downstream canonical mTOR targets with consequent increased autophagy and sarcopenia. Skeletal muscle from genetically modified mice with PI3Kγ knockout, CYP2E1 knockout, in-vivo electroporation with constitutively active mTOR, murine myotubes exposed to alcohol and patients with alcoholic cirrhosis administered essential amino acids with excess leucine to directly activate mTOR will be used. These studies in a comprehensive array of model systems will lay the foundation for developing novel, mechanism based targeted therapies to reverse sarcopenia in alcoholic liver disease.