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.

摘要 肝硬化患者超过250万人，年发病率为4万人，死亡约2.7万人 每一年。大多数患者不接受移植，并发症的管理仍然是主要的治疗方法。 治疗肝硬化。 骨骼肌丧失是肝硬化最常见的并发症， 生活质量下降，发病率和死亡率上升。 尽管肌肉损失的临床意义很高， 对于肝硬化，没有确定的治疗方法，因为其潜在机制尚不清楚。识别 因此，肝硬化中骨骼肌损失的机制具有高度的临床意义。血氨 由于肝尿素生成减少和门体分流减少，因此在肝硬化中是一致的异常。 我们以前曾报道氨会降低肌肉蛋白质的合成。在初步研究中，我们 表明高氨血症导致β-连环蛋白信号传导受损，其靶蛋白c- MYC。β-catenin的典型调节由GSK 3 β介导的磷酸化介导。有趣的是，我们 注意到氨激活IKKβ并降低β-连环蛋白的表达和转录活性 不依赖于GSK 3 β。我们还发现氨通过一种新的、非经典的IKKβ抑制β-catenin。 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 核糖体生物合成的激活。然而，是否降低β-连环蛋白和随之而来的cMYC减少， 表达和活性导致肌肉损失的原因尚不清楚。本申请中提出的研究旨在 确定氨损害β-catenin信号传导的分子机制，以及氨对β-catenin信号传导的干扰。 核糖体生物合成途径根据一系列全面的 肌肉高氨血症模型，包括人肝硬化、门腔静脉吻合（PCA）大鼠和 C2 C12肌管培养，我们假设减少骨骼肌核糖体生物合成和蛋白质 高氨血症期间的合成由非经典IKKβ依赖性受损β-连环蛋白介导 信号我们将通过啮齿动物和细胞培养的功能丧失和获得研究来检验这一假设 模型的目的如下：首先，我们将确定高氨血症损害β- 通过非经典IKKβ介导机制的连环蛋白信号传导。IKKβ在PCA大鼠体内的沉默和 在肌管中的分子研究将用于剖析β-连环蛋白失活的机制。二是 将确定高氨血症减少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转录复合物。康贝特人将以 氨抑制鼠肌管中β-连环蛋白-cMYC-核糖体生物合成的机制， C2 C12肌管功能研究中的损失和增益。我们的研究将确定 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， 逆转肝硬化和其他慢性高氨血症疾病（包括心力衰竭）的肌肉损失。