The Beta-adrenergic agonist, Ractopamine, increases skeletal muscle expression of Asparagine Synthetase as part of an integrated stress response gene program

Sci Rep. 2018 Oct 29;8(1):15915. doi: 10.1038/s41598-018-34315-9.

Abstract

Synthetic beta-adrenergic agonists (BA) have broad biomedical and agricultural application for increasing lean body mass, yet a poor understanding of the biology underpinning these agents is limiting further drug discovery potential. Growing female pigs (77 ± 7 kg) were administered the BA, Ractopamine (20 ppm in feed), or the recombinant growth hormone (GH), Reporcin (10 mg/48 hrs injected) for 1, 3, 7, 13 (n = 10 per treatment, per time point) or 27 days (n = 15 per treatment). Using RNA-sequencing and inferred pathway analysis, we examined temporal changes to the Longissimus Dorsi skeletal muscle transcriptome (n = 3 per treatment, per time point) relative to a feed-only control cohort. Gene expression changes were affirmed by quantitative-PCR on all samples (n = 164). RNA-sequencing analysis revealed that BA treatment had greater effects than GH, and that asparagine synthetase (Asns) was the 5th most significantly increased gene by BA at day 3. ASNS protein expression was dramatically increased by BA treatment at day 7 (p < 0.05). The most significantly increased gene at day 3 was activating transcription factor 5 (Atf5), a transcription factor known to regulate ASNS gene expression. Gene and protein expression of Atf4, another known regulator of Asns expression, was not changed by BA treatment. Expression of more than 20 known Atf4 target genes were increased by BA treatment, suggesting that BA treatment induces an integrated stress response (ISR) in skeletal muscle of pigs. In support of this, mRNA expression of sestrin-2 (Sesn2) and cyclin-dependant kinase 1 alpha (Cdkn1a), two key stress-responsive genes and negative regulators of cellular growth, were also strongly increased from day 3 of BA treatment. Finally, tRNA charging was the most significantly enriched pathway induced by BA treatment, suggesting alterations to the translational capacity/efficiency of the muscle. BA-mediated changes to the skeletal muscle transcriptome are highly indicative of an integrated stress response (ISR), particularly genes relating to amino acid biosynthesis and protein translational capacity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factors / genetics
  • Activating Transcription Factors / metabolism
  • Adrenergic beta-Agonists / pharmacology*
  • Amino Acyl-tRNA Synthetases / genetics
  • Amino Acyl-tRNA Synthetases / metabolism
  • Animals
  • Aspartate-Ammonia Ligase / genetics
  • Aspartate-Ammonia Ligase / metabolism*
  • Cyclin-Dependent Kinase Inhibitor p21 / genetics
  • Cyclin-Dependent Kinase Inhibitor p21 / metabolism
  • Female
  • Gene Expression Regulation / drug effects*
  • Growth Hormone / pharmacology
  • Muscle, Skeletal / metabolism*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phenethylamines / pharmacology*
  • Swine

Substances

  • Activating Transcription Factors
  • Adrenergic beta-Agonists
  • Cyclin-Dependent Kinase Inhibitor p21
  • Nuclear Proteins
  • Phenethylamines
  • ractopamine
  • Growth Hormone
  • Amino Acyl-tRNA Synthetases
  • Aspartate-Ammonia Ligase