Hyperammonemia reduces skeletal muscle protein synthesis via a beta-catenin-cMyc mediated impaired ribosomal biogenesis

摘要 中国有250多万名肝硬变患者，每年发病4万人，死亡约2.7万人 每年。大多数患者没有接受移植，并发症的处理仍然是主要的 治疗肝硬变。 骨骼肌丢失是肝硬变最常见的并发症，其结果是 生活质量、发病率和死亡率的增加。 尽管肌肉损失具有很高的临床意义 对于肝硬变，目前还没有既定的治疗方法，因为其潜在的机制尚不清楚。识别 因此，研究肝硬变时骨骼肌丢失的机制具有重要的临床意义。高氨血症 在肝硬变中是一种一贯的异常，因为肝尿素生成减少和门体分流减少。 我们之前已经报道过氨会减少肌肉蛋白质的合成。在初步研究中，我们 结果表明，高氨血症导致β-连环蛋白信号转导受损，其靶基因c-连环蛋白表达减少。 MYC。β-连环蛋白的规范调节是由β介导的磷酸化介导的。有趣的是，我们 注意到氨能激活IKKβ并降低β-连环蛋白的表达和转录活性 独立于Gsk3β。我们还表明，氨通过一种新的、非规范的IKKβ来抑制IkA-连环蛋白。 依赖机制。在肌肉中，cMYC通过以下途径增加蛋白质合成和肌肉肥大 核糖体生物发生的激活。然而，β-连环蛋白的降低是否会降低cmyc 表达和活动导致肌肉损失的原因尚不清楚。本申请书中提出的研究将旨在 确定氨损害β-连环蛋白信号转导的分子机制及其在 核糖体生物发生途径。基于一系列综合的、令人信服的初步数据 肌肉高氨血症模型包括人肝硬变、门腔静脉吻合术(PCA)大鼠和 C2C12肌管培养，我们假设减少了骨骼肌核糖体的生物合成和蛋白质 高氨血症时的合成是由非典型的IKKβ依赖受损的β-连环蛋白介导的 发信号。我们将通过在啮齿动物和细胞培养中的功能研究的得失来检验这一假说。 通过以下目的建立模型：首先，我们将确定高氨血症损害β的机制。 非典型IKKβ介导的连环蛋白信号转导机制。IKKβ在前列腺癌大鼠体内的沉默作用 肌管中的分子研究将被用来剖析β-连环蛋白失活的机制。第二，我们 将决定高氨血症降低核糖体生物合成的机制，这是 蛋白质合成，通过核糖体蛋白的c-myc转录复合体。我们将确定 氨抑制小鼠肌管β-连环蛋白-cMYC-核糖体生物发生的机制 C2C12肌管在功能研究中的损失和增益。我们的研究将确定分子机制 对肌肉蛋白质合成受损负责，并为开发新的干预措施提供基础 逆转肝硬变和包括心力衰竭在内的高氨血症慢性疾病的肌肉损失。

ABSTRACT There are over 2.5 million patients with cirrhosis, with an annual incidence of 40,000 and about 27,000 deaths per year. Most patients do not get transplanted and management of complications remains the mainstay of therapy for cirrhosis. Skeletal muscle loss is the most frequent complication in cirrhosis and results in reduced quality of life, increased morbidity and mortality. Despite the high clinical significance of muscle loss in cirrhosis, there are no established therapies because the underlying mechanisms are not known. Identifying the mechanisms of skeletal muscle loss in cirrhosis is therefore of high clinical significance. Hyperammonemia is a consistent abnormality in cirrhosis because of reduced hepatic ureagenesis and portosystemic shunting. We have previously reported that ammonia decreases muscle protein synthesis. In preliminary studies, we show that hyperammonemia results in impaired β-catenin signaling and decreased expression of its target, c- MYC. Canonical regulation of β-catenin is mediated by GSK3β mediated phosphorylation. Interestingly, we noted that ammonia activates IKKβ and decreases β-catenin expression and transcriptional activity independent of GSK3β. We also showed that ammonia inhibits β-catenin by a novel, non-canonical IKKβ dependent mechanism. In the muscle, cMYC increases protein synthesis and muscle hypertrophy via activation of ribosomal biogenesis. However, whether lower β-catenin and consequent reduced cMYC expression and activity result in muscle loss is not known. The studies proposed in this application will aim to identify the molecular mechanisms by which ammonia impairs β-catenin signaling and the perturbations in the ribosomal biogenesis pathways. Based on compelling preliminary data generated in a comprehensive array of models with muscle hyperammonemia including human cirrhosis, portacaval anastamosis (PCA) rat and C2C12 myotube cultures, we hypothesize that reduced skeletal muscle ribosomal biogenesis and protein synthesis during hyperammonemia are mediated by a non-canonical IKKβ dependent impaired β-catenin signaling. We will examine this hypothesis by loss and gain of function studies in rodent and cell culture models by the following aims: First we will identify the mechanism by which hyperammonemia impairs β- catenin signaling by a non-canonical IKKβ-mediated mechanism. In-vivo silencing of IKKβ in the PCA rat and molecular studies in myotubes will be used to dissect the mechanisms of inactivation of β-catenin. Second, we will determine the mechanism by which hyperammonemia decreases ribosomal biogenesis, the critical step in protein synthesis, via the c-MYC transcriptional complex of ribosomal proteins. We will determine the mechanism by which ammonia inhibits the β-catenin-cMYC-ribosome biogenesis in murine myotubes and C2C12 myotubes by loss and gain in function studies. Our studies will determine the molecular mechanisms responsible for impaired muscle protein synthesis and provide the basis for developing novel interventions to reverse muscle loss in cirrhosis and other chronic diseases with hyperammonemia including heart failure.