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

Abstract

Skeletal muscle is a highly metabolic and dynamic tissue that is formed through the complex and well-organised process of myogenesis. Although there is a good understanding about the role of the Muscle Regulatory Factors during myogenesis, little is known about the potential interplay of other metabolic proteins. The aim of this study was to determine the endogenous mRNA expression profile for a novel group of genes, recently associated with β2-adrenergic agonist (BA) induced muscle hypertrophy in pigs [1], during myogenic differentiation in C2C12 cells and their response to dibutyryl cyclic-AMP (dbcAMP). These genes included mitochondrial phosphoenolpyruvate 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 ASNS mRNA.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 differentiation. 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.

The phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, 3-mercaptopicolinic acid (3-MPA), induces myogenic differentiation in C2C12 cells

“…With increasing doses of 3-MPA, cells became sparser but resulted in more round bulging cells and much larger myotubes. These observations are comparable to those reported previously by our group in C2C12 cells treated with dbcAMP from day 0 of differentiation 3 . Creatine kinase (CK) activity is an established indicator of myogenic differentiation, therefore we next quantified CK activity every 24 h following treatment with 3-MPA from day 0 until day 5.…”

“…There was a significant time × treatment interaction ( P = 0.02) for myogenin (encoded by Myog ) mRNA expression following treatment with 3-MPA (0.25–1 mM) from day 0 of differentiation, but not from day 4 ( P = 0.177). As observed previously 3 , myogenin mRNA peaked at day 2 of differentiation then decreased with differentiation (Fig. 4 a).…”

“…We previously reported that β 2 -adrenergic receptor agonist (BA)-induced muscle hypertrophy in pigs was associated with a coordinated upregulation of a novel group of genes associated with an integrated stress response 1 , 2 , including mitochondrial phosphoenolpyruvate carboxykinase ( Pck2 ) and genes involved in serine biosynthesis (phosphoglycerate dehydrogenase, Phgdh ; phosphoserine aminotransferase-1, Psat1 ). We also reported a coordinated upregulation in mRNA expression of this same group of genes at day 2 of differentiation in C2C12 muscle cells, which coincided with the peak in myogenin expression 3 , suggesting that they might be important for myogenic differentiation.…”

Monitoring and mathematical modeling of mitochondrial ATP in myotubes at single‐cell level reveals two distinct population with different kinetics

“…We found that cluster 1 shows the similar property of slow muscle fiber, and cluster 2 shows the similar property of fast muscle fiber; however, it remains unknown whether both fiber types-like C2C12 myotubes can co-exist in the same dish from a cloned single cell line. Recently, it has been reported that, during the differentiation steps of C2C12 myotubes, sMyHC express in the earlier differentiation step, and fMyHC express in the later differentiation step [21]. If differentiation of C2C12 myotubes progress with inhomogeneous rate, both fiber types-like C2C12 myotubes can co-exist in the same dish.…”

Identification of regulatory networks and hub genes controlling mammary gland development and lactation in dairy goats during the late lactation, dry period, and late gestation stages

“…PYCR1 encodes an enzyme that catalyzes the conversion of NAD(P)H-dependent pyrroline 5-carboxylate to proline [71]. In addition, proteins encoded by PSAT1 and PSPH are involved in serine synthesis [72]. BCAT2 encodes a branched-chain aminotransferase, and the encoded protein forms a dimer.…”