Project 3 Title: Sarcopenia of ALD: regulation of skeletal muscle autophagy by alcohol

摘要 尽管肌肉减少症在酒精相关肝病中具有很高的临床意义， 治疗，因为机制还不清楚。在当前的融资周期中，我们发现， 骨骼肌中关键信号分子的激酶非依赖性去磷酸化导致 蛋白质合成和增加的自噬介导的蛋白质水解。乙醇提高了蛋白质的活性 磷酸酶2A（PP 2A）导致关键信号分子mTOR和AMPK的靶向失活 调节肌肉蛋白质稳态（蛋白质稳态）。我们还观察到，乙醇抑制了脂质- 磷脂酰肌醇3激酶（PI 3 K β）的γ同种型的蛋白激酶活性，其为已知的PP 2A抑制剂。的 对乙醇介导的PP 2A活性增加的下游信号应答是同时抑制 AMPK和mTOR对蛋白质合成和自噬蛋白水解具有相反的作用， 功能性后果是由于受损的mTORC 1信号传导应答。由于AMPK的丢失发生在 进食状态，而mTORC 1在禁食期间被抑制，我们称之为乙醇介导的信号扰动，以反映 一个伪美联储的国家。由于mTORC 1和AMPK也对细胞能量缺乏和乙醇敏感， 导致线粒体功能障碍，我们评估并报告了乙醇损害线粒体功能， 增加了骨骼肌中自由基的产生，这有助于信号干扰 和肌肉减少表型。具体而言，电子传递链中的复合物IV随着电位的降低而受损。 上游复合物I和III的损伤。乙醇是否直接损害这些复合物或由于游离的 产生的自由基是未知的。确定乙醇损害特定复合物的机制 在电子传递链和功能的后果也是未知的。最后，在正在进行的 研究中，我们正在研究L-亮氨酸对恢复肌肉蛋白质稳态和逆转肌肉减少症的作用， 酒精性肝硬化的人类患者然而，大量的氨基酸负荷增加了氮负荷， 高氨血症和随之而来的副作用。在乙醇处理的肌管和 在乙醇喂养的小鼠中，我们注意到β-羟甲基丁酸酯（HMB），一种具有合成代谢特性的亮氨酸代谢物， 逆转乙醇损害的蛋白质合成和减少线粒体耗氧量。基于这些 根据初步数据，我们假设乙醇诱导的蛋白质稳态和线粒体 功能障碍是肌肉减少症的基础在更新周期中，我们建议剖析 在临床前模型中使用功能丧失和获得研究的PI 3 K β失活和PP 2A活性增加。 我们将结合功能分析和研究来剖析线粒体功能的特定缺陷 以确定电子传递链组分组装成超复合物。最后，我们将测试 通过测试HMB是否可以逆转肌肉扰动， 酒精性肝硬化的患者。

ABSTRACT Despite the high clinical significance of sarcopenia in alcohol-related liver disease, there are no effective therapies because the mechanisms are not well understood. In the ongoing funding cycle, we identified that kinase-independent dephosphorylation of critical signaling molecules in the skeletal muscle resulted in reduced protein synthesis and increased autophagy mediated proteolysis. Ethanol increased the activity of protein phosphatase 2A (PP2A) that caused targeted inactivation of mTOR and AMPK, critical signaling molecules regulating muscle protein homeostasis (proteostasis). We also observed that ethanol inhibited the lipid- and protein-kinase activities of gamma isoform of phosphoinositide 3 kinase (PI3K), a known inhibitor of PP2A. The downstream signaling responses to ethanol-mediated increased PP2A activity was the simultaneous inhibition both AMPK and mTOR, that have opposing effects on protein synthesis and autophagic proteolysis but the functional consequences were due to impaired mTORC1 signaling responses. Since loss of AMPK occurs in a fed state while mTORC1 is inhibited during fasting, we call the ethanol mediated signaling perturbations to reflect a pseudofed state. Since mTORC1 and AMPK are also responsive to cellular energy deficiency and ethanol causes mitochondrial dysfunction, we evaluated and reported that ethanol impaired mitochondrial function and increased the generation of free radicals in the skeletal muscle that contributed to the signaling perturbations and sarcopenic phenotype. Specifically, complex IV in the electron transport chain was impaired with potential impairment of upstream complexes I and III. Whether ethanol impairs these complexes directly or due to the free radicals generated is not known. Determining the mechanism by which ethanol impairs the specific complexes in the electron transport chain and the functional consequences are also not known. Finally, in the ongoing studies, we are studying the effect of L-leucine on restoring muscle proteostasis and reversing sarcopenia in human patients with alcoholic cirrhosis. However, a large amino acid load increases the nitrogen load and cause hyperammonemia and consequent adverse effects. In preclinical studies in ethanol-treated myotubes and ethanol-fed mice, we noted that -hydroxymethyl butyrate (HMB), a leucine metabolite with anabolic properties, reversed ethanol impaired protein synthesis and reduced mitochondrial oxygen consumption. Based on these preliminary data, we hypothesize that ethanol-induced dysregulation in proteostasis and mitochondrial dysfunction underlies sarcopenia. In the renewal cycle, we propose to dissect the molecular mechanisms of inactivation of PI3K and increased PP2A activity using loss and gain of function studies in preclinical models. We will dissect the specific defects in mitochondrial function using a combination of functional assays and studies to determine the assembly of the electron transport chain components into supercomplexes. Finally, we will test the translational significance of our mechanistic studies by testing if HMB can reverse the muscle perturbations in patients with alcoholic cirrhosis.