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

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.

Effect of adeno-associated virus (AAV)-mediated overexpression of PEPCK-M (Pck2) on Clenbuterol-induced muscle growth

“…It is also likely that the biology underpinning skeletal muscle growth in pigs and mice differs mechanistically, as we have observed clear differences in the response to beta-adrenergic agonists in the two species. In pigs we observed significant changes in the expression of genes associated with an integrated stress response [4, 19], whilst we saw no induction of the ISR gene program in mice [16]. It is possible that mice are already metabolically stressed (due to their high metabolic rate), so respond to beta adrenergic agonists without inducing the ISR gene program.…”

“…These agents increase muscle weights with an associated increase in fast MyHC isoform expression and decrease in slow MyHC, reflecting a characteristic shift to a faster fibre type [4, 18]. In addition, we have recently shown that beta-adrenergic agonist stimulated muscle growth in pigs is associated with increased expression of genes associated with serine/one-carbon/glycine (SOG) biosynthesis [4], amino acid biosynthesis, protein translational capacity and an integrated stress response [19]. It has been postulated that PEPCK-M is an enzyme capable of redirecting TCA cycle intermediates into the SOG biosynthesis pathway [9].…”

Changes in expression of serine biosynthesis and integrated stress response genes during myogenic differentiation of C2C12 cells

“…Upregulation of PEPCK-M has been associated with the rewiring of energy metabolism to reroute glycolytic intermediates into serine biosynthesis in order to sustain cancer cell growth and survival through the activation of an endoplasmic reticulum stress response [12,15]. PCK2, but not PCK1, was identified in the same gene cluster as PHGDH, showing the same response profile to BA treatment in pigs [5]; and a similar mRNA expression profile was reported for ASNS, ATF5, CEBPG and SESN2 [6], which have all been linked to an integrated stress response [16,17]. The apparent co-ordinated upregulation of this novel group of genes suggests the possible transcriptional regulation by a common transcription factor or factors.…”

“…BA and GH are repartitioning agents that increase weight gain by redirecting nutrients to muscle growth and reducing fat deposition, but via different mechanisms, which have yet to be fully elucidated [[2], [3], [4]]. We previously compared the effects of Ractopamine (BA) and Reporcin (GH) administered to growing gilts for up to 27 days on porcine skeletal muscle transcriptomes to identify mechanistic differences in the responses [5,6]. GH had a whole-body growth effect and increased liver weight, whereas BA induced greater increases in muscle weights.…”

“…Both BA and GH induced a coordinated upregulation of a novel group of genes in muscle at day 3, but only BA induced a sustained effect (>7 days). This novel group of genes included those encoding proteins involved in serine biosynthesis (phosphoglycerate dehydrogenase, PHGDH; phosphoserine aminotransferase-1, PSAT1; phosphoserine phosphatase, PSPH), a mitochondrial gluconeogenic enzyme (phosphoenolpyruvate carboxykinase, PCK2/PEPCK-M) [5] and proteins involved in an integrated stress response (asparagine synthetase, ASNS; sestrin-2, SESN2; activating transcription factor-5, ATF5; and CCAAT/enhancer binding protein-γ, CEBPG/C/EBP-γ) [6]. This demonstrated, for the first time, that coordinated upregulation of this novel group of genes was associated with hypertrophic growth of terminally differentiated muscle, whereas previous studies had associated upregulation of PHGDH and PEPCK-M with cancer.…”