Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations
Ethanol sensitizes skeletal muscle to ammonia-induced molecular perturbations
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DOI：
10.1074/jbc.ra118.005411
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发表时间：
2019-05-03
影响因子：
4.8
通讯作者：
Dasarathy, Srinivasan
中科院分区：
文献类型：
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作者：
Kant, Sashi;Davuluri, Gangarao;Dasarathy, Srinivasan
乙醇引起肌肉蛋白质稳态失调，同时引起肝细胞损伤。由于肝细胞是氨（一种在许多代谢过程中产生的细胞毒性细胞代谢产物）生理处置的主要部位，因此我们确定高氨血症是否会加重乙醇诱导的肌肉损失。分化的小鼠C2 C12肌管，骨骼肌从配对喂养或乙醇处理的小鼠，和人类患者与酒精性肝硬化和健康对照被用来量化蛋白质合成，哺乳动物雷帕霉素复合物1（mTORC 1）的目标信号，和自噬标志物。对小鼠肌肉和肌管中的酒精代谢酶表达和活性以及肝细胞中的尿素生成进行定量。氨转运蛋白，RhBG和RhCG的表达和调节，定量实时PCR，免疫印迹，报告分析，生物素标记的启动子下拉与蛋白质组学，和功能丧失的研究。乙醇和乙醛脱氢酶在肌管中表达并活跃。乙醇暴露损害肝细胞尿素生成，诱导肌肉RhBG表达，并升高肌肉氨浓度。同时乙醇和氨处理损害蛋白质合成和mTORC 1信号传导，并增加自噬，从而降低肌管直径到更大程度上比单独处理。乙醇处理和退出氨暴露导致更大的损害，肌肉信号和蛋白质合成比氨处理乙醇幼稚肌管。在响应于乙醇和氨而与RhBG启动子结合的三个转录因子中，DR 1/NC 2在乙醇和氨处理期间间接调节RhBG的转录。乙醇的直接作用与氨摄取增加协同作用，导致骨骼肌蛋白质稳态失调和信号传导紊乱，并伴有更严重的肌肉减少症表型。
Ethanol causes dysregulated muscle protein homeostasis while simultaneously causing hepatocyte injury. Because hepatocytes are the primary site for physiological disposal of ammonia, a cytotoxic cellular metabolite generated during a number of metabolic processes, we determined whether hyperammonemia aggravates ethanol-induced muscle loss. Differentiated murine C2C12 myotubes, skeletal muscle from pair-fed or ethanol-treated mice, and human patients with alcoholic cirrhosis and healthy controls were used to quantify protein synthesis, mammalian target of rapamycin complex 1 (mTORC1) signaling, and autophagy markers. Alcohol-metabolizing enzyme expression and activity in mouse muscle and myotubes and ureagenesis in hepatocytes were quantified. Expression and regulation of the ammonia transporters, RhBG and RhCG, were quantified by real-time PCR, immunoblots, reporter assays, biotin-tagged promoter pulldown with proteomics, and loss-of-function studies. Alcohol and aldehyde dehydrogenases were expressed and active in myotubes. Ethanol exposure impaired hepatocyte ureagenesis, induced muscle RhBG expression, and elevated muscle ammonia concentrations. Simultaneous ethanol and ammonia treatment impaired protein synthesis and mTORC1 signaling and increased autophagy with a consequent decreased myotube diameter to a greater extent than either treatment alone. Ethanol treatment and withdrawal followed by ammonia exposure resulted in greater impairment in muscle signaling and protein synthesis than ammonia treatment in ethanol-naive myotubes. Of the three transcription factors that were bound to the RhBG promoter in response to ethanol and ammonia, DR1/NC2 indirectly regulated transcription of RhBG during ethanol and ammonia treatment. Direct effects of ethanol were synergistic with increased ammonia uptake in causing dysregulated skeletal muscle proteostasis and signaling perturbations with a more severe sarcopenic phenotype.