Abstract
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.
Highlights
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
To determine how ethanol exposure and hyperammonemia cause an increase in muscle ammonia concentrations, we determined the expression of ammonia transporters, RhBG and RhCG mRNA and protein in myotubes and skeletal muscle from mice and human subjects
Summary
Ethanol impairs ureagenesis in hepatocytes and results in more severe muscle hyperammonemia In murine primary hepatocytes, ethanol exposure resulted in decreased urea synthesis measured by urea concentrations in the medium (Fig. 1A). Based on the putative binding sites of the two transcription factors identified to bind to the RhBG promoter, we used our reporter constructs to determine the response to ethanol, ammonia, and a combination of ethanol and ammonia (Fig. 4D). Compared with untreated control cells, the full-length reporter showed increased luciferase activity in response to hyperammonemia, ethanol, or a combination of ethanol and ammonia. Depletion of FoxP1 did not affect either baseline response in untreated cells or the response to interventions and was similar to that observed with myotubes transfected with a random construct These data suggested that DR1/NC2 regulated RhBG promoter activity. Our observations of an increased RhBG expression and reporter activity of the TATA-less promoter suggested that ethanol and ammonia regulate RhBG potentially by inhibiting RhBG repressors This interpretation is consistent with the responses to ethanol and ammonia in the deletional constructs. Even though ethanol-induced perturbations were identified in multiple systems, whether ethanol is metabolized in the myotubes is currently unknown
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