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

高氨血症通过 β-连环蛋白-cMyc 介导的核糖体生物合成受损减少骨骼肌蛋白合成

• 批准号: 9533467
• 负责人: Srinivasan Dasarathy
• 金额: $ 21.24万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-24 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9533467
• 关键词: Affect; Ammonia; Animal Model; Animals; Biogenesis; Cell Culture Techniques; Cessation of life; Chronic; Chronic Disease; Cirrhosis; Clinical; Complement; Complex; Complication; Data; Development; Down-Regulation; Foundations; Genetic Transcription; Glycogen (Starch) Synthase; Goals; Heart failure; Human; Hyperammonemia; Immunoprecipitation; Impairment; In Vitro; Incidence; Intervention; Lung; Mass Spectrum Analysis; Mediating; Modeling; Molecular; Morbidity - disease rate; Mus; Muscle; Muscle Fibers; Muscle Proteins; Muscular Atrophy; Outcome; Pathway interactions; Patients; Phenotype; Phosphorylation; Post-Translational Protein Processing; Protein Biosynthesis; Proteins; Quality of life; Rattus; Regulation; Reporting; Ribosomal Biogenesis Pathway; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Rodent; Signal Transduction; Skeletal Muscle; Surgical Portacaval Shunt; Surgical Portosystemic Shunt; Testing; Tissues; Transplantation; alpha catenin; ammonium acetate; base; beta catenin; c-myc Genes; clinically significant; effective therapy; gain of function; hepatic ureagenesis; in vivo; mortality; multicatalytic endopeptidase complex; muscle hypertrophy; new therapeutic target; novel; novel therapeutics; skeletal muscle wasting; targeted treatment; therapeutic target; tibialis anterior muscle

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.

抽象的 肝硬化患者超过250万，每年40,000例，死亡约27,000例 每年。大多数患者没有被移植，并发症的管理仍然是 治疗肝硬化。 骨骼肌损失是肝硬化中最常见的并发症，导致减少 生活质量，发病率提高和死亡率。 尽管肌肉损失具有很高的临床意义 肝硬化，由于尚不清楚潜在的机制，因此没有建立的疗法。识别 因此，肝硬化中骨骼肌丧失的机制具有很高的临床意义。高症血症 由于肝尿素发生和门移植分流，是肝硬化的一致异常。 我们以前曾报道过氨会降低肌肉蛋白的合成。在初步研究中，我们 表明高氨比会导致β-catenin信号受损并改善其靶标C-的表达 妈妈。 GSK3β介导的磷酸化介导的β-catenin的规范调节。有趣的是，我们 指出氨会激活IKKβ并降低β-catenin的表达和转录活性 独立于GSK3β。我们还表明，氨可以通过一种新型的非典型IKKβ抑制β-catenin 依赖机制。在肌肉中，CMYC通过 核糖体生物发生的激活。但是，较低的β-catenin是否并因此减少了CMYC 表达和活性导致肌肉丧失尚不清楚。本应用程序中提出的研究将旨在 确定氨损害β-catenin信号传导和扰动的分子机制 核糖体生物发生途径。基于在综合数组中生成的引人入胜的初步数据 肌肉高氨血血症的模型，包括人肝硬化，portacaval吻合（PCA）大鼠和 C2C12肌管培养物，我们假设减少了骨骼肌核糖体生物发生和蛋白质 高氨血症期间的合成是由非典型IKKβ依赖性β-catenin介导的 信号。我们将通过啮齿动物和细胞培养的功能研究的丧失和功能研究来检查这一假设 以下目标：首先，我们将确定高症损害β-的机制 通过非典型IKKβ介导的机制信号传导。 PCA大鼠中IKKβ的体内沉默和 在肌管中的分子研究将用于剖析β-catenin失活的机制。第二，我们 将确定高氨比降低核糖体生物发生的机制，这是关键的步骤 蛋白质合成，通过核糖体蛋白的C-MYC转录复合物。我们将确定 氨抑制鼠肌管中的β-catenin-cmyc-核糖体生物发生的机制， C2C12在功能研究中因损失和增益而获得的肌管。我们的研究将确定分子机制 负责受损的肌肉蛋白质合成，并为开发新的干预措施提供基础 肝硬化和其他慢性疾病的反向肌肉丧失，包括心力衰竭。