Mechanisms of Malnutrition in Cirrhosis with Portosystemic Shunting

描述(申请人提供)：摘要在美国，终末期肝硬变及其并发症每年导致27,000人死亡和6,500例肝脏移植。骨骼肌块减少或骨质疏松症是门体分流(PSS)合并肝硬变的主要并发症之一。骨质疏松症极大地增加了这些患者的发病率和死亡率。目前还没有有效的治疗选择，因为其潜在机制目前尚不清楚，而且到目前为止大多数研究都是描述性的。患有肝硬变和PSS的患者以及动物模型都减少了肌肉蛋白质的合成，但调控这一过程的分子机制目前尚不清楚。高氨血症在肝硬变和PSS中是一种一贯的异常现象，这是由于肝组织氨排毒受损所致。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，导致肌肉重量减少。氨诱导肌肉生长抑制素升高的机制尚不清楚。我们的初步研究表明，NFkB结合在myostatin启动子上，NFkB可能是高氨血症时myostatin的上游调节因子。在我们对成肌细胞的初步研究中，NFkB对氨的反应增加，并且先于Myostatin的表达增加。最后，在我们对PCA大鼠和细胞培养的体内研究中，我们观察到当myostatin的表达增加时，细胞能量感受器AMP激酶的激活程度更高，这些变化可以通过体内阻断myostatin来逆转。因此，我们假设，高氨血症诱导的肌肉生长抑制素的表达增加，抑制了肌肉蛋白质的合成和构成肝脏肌轴的肌肉质量。为了确定PSS导致肌肉蛋白质合成减少的潜在分子机制，我们提出了以下目标：1)在体内建立氨诱导的Myostatin表达构成肝脏-肌肉轴；2)证明高氨血症诱导myostatin表达上调的机制是NFkB依赖的；3)证实myostatin对mTOR的调节不依赖Akt，而是由高氨血症中的AMP激酶介导的。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。这些研究具有创新性，因为它们将证明高氨血症抑制肌肉蛋白质合成的机制，并确定肌肉抑制素和mTOR之间的新的调节串扰，mTOR是调节蛋白质合成的关键信号分子。这些研究具有非常重要的意义，因为它们将为了解门体分流术中肌质疏松症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诱导的肌肉生长抑素表达的增加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.