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.
Highlights
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
Sequencing analysis revealed that BA treatment had greater effects than growth hormone (GH) and inferred pathway enrichment analysis (IPA) revealed that four of the top ten enriched pathways for BA were involved in tRNA charging, serine biosynthesis or arginine metabolism (Fig. 1A)
The serine biosynthesis pathway genes (Phgdh, Psat[1], Psph) formed the second most significantly enriched pathway, which again were evident from day 3 onwards (Fig. 1A,B) and agreed with our previous microarray analyses[14] where we saw increases in Phgdh and Pck[2] mRNA and protein in these same animals
Summary
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. Expression of more than 20 known Atf[4] target genes were increased by BA treatment, suggesting that BA treatment induces an integrated stress response (ISR) in skeletal muscle of pigs. Two previous studies examined time-dependant responses of the skeletal muscle transcriptome to BA administration following 1 and 4 hours[10] or 1 and 10 days[9] of administration These studies revealed impressive alterations in gene expression by BA, but the strength of the data set was limited by the use of only 2 time points. In that study[14], we identified coordinate increases in expression of mitochondrial phosphoenolpyruvate carboxykinase 2 (PEPCK-M) and 3-phosphoglycerate dehydrogenase (PHGDH) in response to BA These enzymes are both involved in amino acid biosynthesis and their function in adult skeletal muscle is currently being explored. Given the increasing use of the pig as a biomedical model for human research[16], this study provides biologically relevant information to both biomedical and agricultural fields
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