Mechanisms of Malnutrition in Cirrhosis with Portosystemic Shunting

在美国，终末期肝硬化及其并发症每年导致27,000人死亡和6,500例肝移植。骨骼肌减少或肌肉减少是肝硬化门静脉系统分流（PSS）的主要并发症之一。骨骼肌减少症对这些患者的发病率和死亡率有重要影响。目前还没有有效的治疗方案，因为潜在的机制尚不清楚，而且迄今为止大多数研究都是描述性的。肝硬化和PSS患者以及动物模型的肌肉蛋白合成减少，但调节这一过程的分子机制目前尚不清楚。高氨血症在肝硬化和PSS中是一致的异常，这是由于肝脏氨解毒功能受损所致。我们在高氨血症动物模型和暴露于氨的肌肉细胞培养中的初步数据显示肌肉生长抑制素的表达增加。肌肉生长抑制素抑制骨骼肌蛋白质合成，导致肌肉质量降低。氨诱导肌生长抑制素升高的机制尚不清楚。我们的初步研究表明，NFkB结合位点在肌生长抑制素启动子上，NFkB可能是高氨血症期间肌生长抑制素的上游调节剂。在我们对成肌细胞的初步研究中，NFkB在氨反应中增加，并且先于肌肉生长抑制素的表达升高。最后，在我们对PCA大鼠的体内研究以及细胞培养中，我们观察到当肌肉生长抑制素表达升高时，细胞能量传感器AMP激酶的激活更大，这些改变通过阻断肌肉生长抑制素在体内被逆转。因此，我们假设高氨血症诱导肌肉生长抑制素表达增加，抑制肌肉蛋白合成和构成肝肌肉轴的肌肉质量。为了确定PSS导致肌肉蛋白合成减少的潜在分子机制，我们提出以下目标：1)确定氨诱导的肌生长抑制素表达在体内构成肝-肌肉轴；2)证明高氨血症介导的肌生长抑制素表达上调机制依赖于NFkB； 3)确定高氨血症中肌生长抑制素对mTOR的调节不依赖于Akt，而由AMP激酶介导。这些研究将使用动物模型和细胞培养系统中的药理学、化学和遗传学方法。提出的研究具有创新性，因为它们将证明高氨血症抑制肌肉蛋白质合成的机制，并确定肌肉生长抑制素和mTOR（一种调节蛋白质合成的关键信号分子）之间的新调控串扰。这些研究具有重要意义，因为它们将为理解门静脉系统分流中肌肉减少的机制和确定潜在的治疗靶点奠定基础。此外，这些研究的结果有可能通过使用药理学方法来降低氨介导的肌生长抑制素表达增加，从而迅速转化为临床应用。

DESCRIPTION (provided by applicant): Abstract End stage cirrhosis and its complications result in 27,000 deaths and 6,500 liver transplants annually in the United States. Reduced skeletal muscle mass or sarcopenia is one of the major complications of portosystemic shunting (PSS) that accompanies cirrhosis. Sarcopenia contributes significantly to the morbidity and mortality of these patients. There are no effective treatment options since the underlying mechanisms are currently unknown and most studies to date have been descriptive. Patients with cirrhosis and PSS as well as animal models have reduced muscle protein synthesis but the molecular mechanisms regulating this process are currently not known. Hyperammonemia is a consistent abnormality in cirrhosis and PSS due to impaired hepatic detoxification of ammonia. Our preliminary data in animal models of hyperammonemia and in muscle cell cultures exposed to ammonia showed an increased expression of myostatin. Myostatin inhibits skeletal muscle protein synthesis and results in lower muscle mass. The mechanism of increased myostatin induced by ammonia is not known. Our preliminary studies have demonstrated NFkB binding sites on the myostatin promoter and NFkB may be an upstream regulator of myostatin during hyperammonemia. In our preliminary studies in myoblast cells, NFkB was increased in response to ammonia and preceded the elevated expression of myostatin. Finally, in both our in vivo studies in PCA rat as well as cell cultures, we observed greater activation of a cellular energy sensor, AMP kinase when myostatin expression was elevated and these alterations were reversed by blocking myostatin in vivo. We therefore hypothesized that hyperammonemia induced increased expression of myostatin inhibits muscle protein synthesis and muscle mass constituting a liver muscle axis. To identify the underlying molecular mechanisms responsible for the reduced muscle protein synthesis with PSS we propose the following aims: 1) Establish that ammonia induced myostatin expression constitutes a liver-muscle axis in vivo, 2) Demonstrate that the mechanism of hyperammonemia mediated upregulation of myostatin expression is NFkB dependent and 3) Establish that the regulation of mTOR by myostatin is independent of Akt and is mediated by AMP kinase in hyperammonemia. Pharmacological, chemical and genetic approaches in animal models and cell culture systems will be used in these studies. The proposed studies are innovative because they will demonstrate the mechanism by which hyperammonemia inhibits muscle protein synthesis and identify a novel regulatory crosstalk between myostatin and mTOR, a critical signaling molecule that regulates protein synthesis. These studies are very significant because they will lay the foundation for understanding the mechanisms of sarcopenia in portosystemic shunting and identify potential therapeutic targets. Furthermore, the results from these studies have the potential to be rapidly translated to clinical application by using pharmacological methods to lower ammonia mediated increased myostatin expression.