Biochemistry and Biophysics Reports 20 (2019) 100694 Contents lists available at ScienceDirect Biochemistry and Biophysics Reports journal homepage: www.elsevier.com/locate/bbrep Changes in expression of serine biosynthesis and integrated stress response T genes during myogenic differentiation of C2C12 cells Madelaine C. Brearleya, Congcong Lic,1, Zoe C.T.R. Daniela, Paul T. Loughnab, Tim Parra,2, ∗ John M. Bramelda,2, a School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK b School of Veterinary Medicine & Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK c Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei, China ARTICLE INFO ABSTRACT Keywords: Skeletal muscle is a highly metabolic and dynamic tissue that is formed through the complex and well-organised C2C12 process of myogenesis. Although there is a good understanding about the role of the Muscle Regulatory Factors dbcAMP during myogenesis, little is known about the potential interplay of other metabolic proteins. The aim of this Hypertrophy study was to determine the endogenous mRNA expression profile for a novel group of genes, recently associated Myogenesis with β2-adrenergic agonist (BA) induced muscle hypertrophy in pigs [1], during myogenic differentiation in PCK2/PEPCK-M C2C12 cells and their response to dibutyryl cyclic-AMP (dbcAMP). These genes included mitochondrial phos- PHGDH phoenolpyruvate carboxykinase (PCK2/PEPCK-M), genes involved in serine biosynthesis (Phosphoglycerate dehydrogenase, PHGDH; Phosphoserine aminotransferase-1, PSAT1; Phosphoserine phosphatase, PSPH) and those involved in an integrated stress response (Asparagine synthetase, ASNS; Sestrin-2, SESN2; and Activating transcription factor-5, ATF5). A coordinated peak in endogenous PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA expression was observed at day 2 of differentiation (P < 0.001) in C2C12 cells, which coincided with the peak in myogenin mRNA. Myotube hypertrophy was induced with dbcAMP (1 mM) treatment from day 0, thereby mimicking the in vivo BA response. Although dbcAMP treatment from day 0 induced larger myotubes and increased both myosin heavy chain-IIB (MyHC-IIB) and pyruvate carboxylase (PC) mRNA, the expression of PCK2, PHGDH, PSAT1 and ASNS mRNA were all unaffected. Treatment with dbcAMP from day 4 increased MyHC-IIB mRNA, however this was less dramatic compared to the response observed following treatment from day 0, but there was no effect on PC mRNA. There was also no effect of dbcAMP treatment from day 4 on PCK2, PHGDH, PSAT1 and ASNSmRNA. To conclude, the coordinated day 2 peak in endogenous expression of PCK2, PHGDH, PSAT1, PSPH, ASNS, ATF5 and SESN2 mRNA may relate to a shift in biosynthetic demand required to initiate myogenic differ- entiation. However, dbcAMP had no effect on the expression of these genes in vitro suggesting that the effects observed in BA-treated pigs might be via other signalling pathways from the activation of the β2-adrenergic receptor, but independent of cAMP, or that there are species differences in the response. 1. Introduction We previously compared the effects of Ractopamine (BA) and Reporcin (GH) administered to growing gilts for up to 27 days on porcine skeletal Growth promoters, including β2-adrenergic receptor agonists (BA) muscle transcriptomes to identify mechanistic differences in the re- and recombinant porcine growth hormone (GH), are used commercially sponses [5,6]. GH had a whole-body growth effect and increased liver in Australia, India and the USA to improve livestock feed efficiency [1]. weight, whereas BA induced greater increases in muscle weights. Both BA and GH are repartitioning agents that increase weight gain by re- BA and GH induced a coordinated upregulation of a novel group of directing nutrients to muscle growth and reducing fat deposition, but genes in muscle at day 3, but only BA induced a sustained effect (> 7 via different mechanisms, which have yet to be fully elucidated [2–4]. days). This novel group of genes included those encoding proteins ∗ Corresponding author. E-mail address: [email protected] (J.M. Brameld). 1 Present Address: College of Animal Science and Technology, Henan University of Animal Husbandry and Economy, Zhengzhou, Henan, China. 2 These authors contributed equally to this work. https://doi.org/10.1016/j.bbrep.2019.100694 Received 6 February 2019; Received in revised form 5 August 2019; Accepted 2 October 2019 2405-5808/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). M.C. Brearley, et al. Biochemistry and Biophysics Reports 20 (2019) 100694 involved in serine biosynthesis (phosphoglycerate dehydrogenase, closely by MyoD [20], and both are essential for proliferation, specifi- PHGDH; phosphoserine aminotransferase-1, PSAT1; phosphoserine cation and commitment to a muscle cell fate; whereas Myogenin and phosphatase, PSPH), a mitochondrial gluconeogenic enzyme (phos- MRF4 are expressed later [20], associated with their role in the in- phoenolpyruvate carboxykinase, PCK2/PEPCK-M) [5] and proteins in- itiation of myogenic differentiation, leading to upregulation of various volved in an integrated stress response (asparagine synthetase, ASNS; muscle-specific genes and contractile proteins, such as Myosin Heavy sestrin-2, SESN2; activating transcription factor-5, ATF5; and CCAAT/ Chain (MyHC) isoforms. Therefore selected myogenesis associated enhancer binding protein-γ, CEBPG/C/EBP-γ) [6]. This demonstrated, genes were measured alongside the novel group of genes. The mouse for the first time, that coordinated upregulation of this novel groupof myoblast C2C12 cell line is often used to model skeletal muscle differ- genes was associated with hypertrophic growth of terminally differ- entiation in vitro as it provides a robust system that has the capacity to entiated muscle, whereas previous studies had associated upregulation generate well-established myotube networks [21]. Therefore, we uti- of PHGDH and PEPCK-M with cancer. lised C2C12 cells to perform comprehensive time-course experiments to There has been increased interest in the genes involved in the serine study the endogenous mRNA expression profiles for this novel group of biosynthesis pathway, since PHGDH was observed to be upregulated in genes, as well as genes associated with myogenesis and skeletal muscle certain breast cancers and reported to be key for cancer cell metabolism metabolism. Despite expressing β2-adrenergic receptors (β2-AR), it has and survival [7–10]. Serine is synthesised from 3-phosphoglycerate, an been reported that C2C12 cells do not display a hypertrophic response intermediate of glycolysis, through a three-step enzymatic process in- to BA treatment [22]. Hence, C2C12 cells were treated with dibutyryl volving PHGDH, PSAT1 and PSPH. Serine plays an important role in cyclic AMP (dbcAMP) from day 0 (as differentiation was initiated) or growth and metabolism and can subsequently be used for the synthesis day 4 of differentiation (terminally differentiated myotubes) to induce of amino acids, phospholipids, and nucleotides [11]. The upregulation down-stream signalling from the β2-AR and determine the effects on of PHGDH indicates that serine biosynthesis is important during times mRNA expression for the same group of genes. of high metabolic activity and tumour growth, however little is known about the role of serine biosynthesis in muscle cell metabolism. Simi- 2. Materials and methods larly there has been increased interest in the mitochondrial isoform of phosphoenolpyruvate carboxykinase (PEPCK-M) in recent years as it 2.1. Cell culture materials and compounds too is upregulated in certain cancers [12,13]. Phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme involved in the re- High glucose Dulbecco's modified eagle's medium (DMEM), generation of phosphoenolpyruvate from oxaloacetate, but has also dbcAMP and cell culture grade water were purchased from Sigma- been identified to play a role in glyceroneogenesis, amino acid synthesis Aldrich (Poole, UK) and other cell culture reagents were purchased and cataplerosis [14]. There are two genes that encode for the two from Fisher Scientific (Loughborough, UK). Cell culture grade water PEPCK isoforms: PCK1 encodes for cytosolic PEPCK (or PEPCK-C) and was used to resuspend dbcAMP sodium salt (CAS #: 16980-89-5; LOT PCK2 for PEPCK-M. For many years, studies of PEPCK function focussed #: SLBJ2610V), filter-sterilised through a 0.2 μm filter and stored in on the role of PEPCK-C and the specific role of PEPCK-M was over- aliquots at −20 °C. dbcAMP was freshly diluted (1:100) into differ- looked, however PEPCK-M has been recently identified as a key reg- entiation media (DM) on each day of treatment. ulator of cancer cell metabolism. Upregulation of PEPCK-M has been associated with the rewiring of energy metabolism to reroute glycolytic 2.2. Cell culture intermediates into serine biosynthesis in order to sustain cancer cell growth and survival through the activation of an endoplasmic re- C2C12 cells were seeded onto 6-well plates in growth medium ticulum stress response [12,15]. PCK2, but not PCK1, was identified in (DMEM supplemented with 10% foetal bovine serum and 1% peni- the same gene cluster as PHGDH, showing the same