The metabolic and molecular mechanisms of hyperammonaemia and hyperethanolaemia induced protein catabolism in skeletal muscle cells

Crossland, Hannah; Smith, Kenneth; Atherton, Philip J.; Wilkinson, Daniel J.

Abstract

Hyperammonaemia and hyperethanolaemia are thought to be driving factors behind skeletal muscle myopathy in liver disease i.e. cirrhosis. Despite this, the singular and combined impacts of ethanol and ammonia induced protein catabolism are poorly defined. As such, we aimed to dissect out the effects of ammonia and ethanol on muscle catabolism. Murine C2C12 myotubes were treated with ammonium acetate (10 mM) and ethanol (100 mM) either alone or in combination for 4h and/or 24h. Myotube diameter, muscle protein synthesis and anabolic and catabolic signalling pathways were assessed. In separate experiments, cells were co-treated with selected inhibitors of protein breakdown to assess the importance of proteolytic pathways in protein loss with ammonia and ethanol. Ammonia and ethanol in combination resulted in a reduction in myotube width and total protein content, that was greater than the reduction observed with ammonia alone. Both ammonia and ethanol caused reductions in protein synthesis, as assessed by puromycin incorporation. There was also evidence of impairments in regulation of protein translation, and increased protein expression of markers of muscle protein breakdown. Myotube protein loss with ammonia plus ethanol was not affected by autophagy inhibition, but was completely prevented by proteasome inhibition. Thus, combined ammonia and ethanol incubation of C2C12 myotubes exacerbated myotube atrophy and dysregulation of anabolic and catabolic signalling pathways associated with either component individually. Ubiquitin proteasome-mediated protein breakdown appears to play an important role in myotube protein loss with ethanol and ammonia.

Citation

Crossland, H., Smith, K., Atherton, P. J., & Wilkinson, D. J. (2018). The metabolic and molecular mechanisms of hyperammonaemia and hyperethanolaemia induced protein catabolism in skeletal muscle cells. Journal of Cellular Physiology, 233(12), 9663-9673. https://doi.org/10.1002/jcp.26881

• Journal Article Type: Article
• Acceptance Date: May 23, 2018
• Online Publication Date: Aug 24, 2018
• Publication Date: 2018-12
• Deposit Date: Jun 7, 2018
• Publicly Available Date: Sep 5, 2018
• Journal: Journal of Cellular Physiology
• Print ISSN: 0021-9541
• Electronic ISSN: 1097-4652
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 233
• Issue: 12
• Pages: 9663-9673
• DOI: https://doi.org/10.1002/jcp.26881
• Keywords: Skeletal muscle; Protein catabolism; Hyper-ammonaemia
• Public URL: https://nottingham-repository.worktribe.com/output/933803
• Publisher URL: https://onlinelibrary.wiley.com/doi/abs/10.1002/jcp.26881
• Contract Date: Sep 5, 2018