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，每年发病4万例，死亡约2.7万人 大多数患者没有接受移植，并发症的治疗仍然是主要的治疗方法。 治疗肝硬化。 骨骼肌损失是肝硬化最常见的并发症，导致 生活质量、发病率和死亡率增加。 尽管肌肉损失具有很高的临床意义 肝硬化目前尚无成熟的治疗方法，因为其潜在机制尚不清楚。 因此，肝硬化中骨骼肌损失的机制具有很高的临床意义。 由于肝脏尿素生成减少和门体分流，这是肝硬化的一致异常。 我们之前曾报道过，在初步研究中，氨会降低肌肉蛋白质的合成。 研究表明，高氨血症会导致 β-连环蛋白信号传导受损，并降低其靶标 c- 的表达 MYC。β-连环蛋白的典型调节是由 GSK3β 介导的磷酸化介导的。 指出氨会激活 IKKβ 并降低 β-catenin 表达和转录活性 我们还证明氨通过一种新型的非典型 IKKβ 抑制 β-连环蛋白。 在肌肉中，cMYC 通过增加蛋白质合成和肌肉肥大。 然而，β-catenin 的降低是否会导致 cMYC 的降低。 表达和活动导致肌肉损失尚不清楚。本申请中提出的研究旨在 确定氨损害 β-连环蛋白信号传导的分子机制以及 核糖体生物发生途径基于一系列全面的令人信服的初步数据。 肌肉高氨血症模型，包括人肝硬化、门腔静脉吻合 (PCA) 大鼠和 C2C12 肌管培养物，我们追求减少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 依赖性受损 β-catenin 介导的 我们将通过啮齿动物和细胞培养中的功能丧失和获得研究来检验这一假设。 模型的目标如下：首先，我们将确定高氨血症损害 β- 的机制 PCA 大鼠体内 IKKβ 沉默通过非经典 IKKβ 介导的连环蛋白信号传导。 肌管的分子研究将用于剖析 β-连环蛋白失活的机制。 将确定高氨血症降低核糖体生物合成的机制，这是核糖体生物发生的关键步骤 通过核糖体蛋白的 c-MYC 转录复合物进行蛋白质合成。 氨抑制小鼠肌管中β-连环蛋白-cMYC-核糖体生物合成的机制 我们的研究将通过功能研究中的 C2C12 肌管损失和增益来确定其分子机制。 负责肌肉蛋白质合成受损，并为开发新的干预措施提供基础 逆转肝硬化和其他伴有高氨血症的慢性疾病（包括心力衰竭）引起的肌肉损失。