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

Madelaine C Brearley,John M Brameld,Zoe C T R Daniel,Tim Parr,Paul T Loughna

doi:10.1038/s41598-020-79324-9

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is a gluconeogenic enzyme with a cytosolic (Pck1/PEPCK-C) and mitochondrial (Pck2/PEPCK-M) isoform. Here we investigate the effect of 3-mercaptopicolinic acid (3-MPA), a PEPCK inhibitor, on C2C12 muscle cells. We report that Pck2 mRNA is 50–5000-fold higher than Pck1 during C2C12 myogenesis, indicating Pck2 is the predominant PEPCK isoform. C2C12 cell proliferation was inhibited in a dose-dependent manner following 48 h 3-MPA treatment (0.01–1 mM). C2C12 myogenic differentiation was significantly induced following 3-MPA treatment (0.25, 0.5, 1 mM) from day 0 of differentiation, demonstrated by increased creatine kinase activity, fusion index and myotube diameter; likewise, the myosin heavy chain (MyHC)-IIB isoform (encoded by Myh4) is an indicator of hypertrophy, and both porcine MYH4-promoter activity and endogenous Myh4 mRNA were also significantly induced. High doses (0.5 and/or 1 mM) of 3-MPA reduced mRNA expression of Pck2 and genes associated with serine biosynthesis (Phosphoglycerate dehydrogenase, Phgdh; phosphoserine aminotransferase-1, Psat1) following treatment from days 0 and 4. To conclude, as Pck2/PEPCK-M is the predominant isoform in C2C12 cells, we postulate that 3-MPA promoted myogenic differentiation through the inhibition of PEPCK-M. However, we were unable to confirm that 3-MPA inhibited PEPCK-M enzyme activity as 3-MPA interfered with the PEPCK enzyme assay, particularly at 0.5 and 1 mM.

Highlights

We previously reported that β2-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
C2C12 cells were treated with a range of 3-mercaptopicolinic acid (3-MPA) doses (0–1 mM) and total DNA content quantified as a marker of total cell number
For the first time we report the effects of the gluconeogenic inhibitor, 3-mercaptopicolinic acid (3-MPA), on proliferation and myogenic differentiation of C2C12 cells. 3-MPA treatment (0.01–1 mM) inhibited cell proliferation in a dose-dependent manner and induced myogenic differentiation (CK assay) at 0.5 and 1 mM

Summary

Introduction

We previously reported that β2-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, Psat[1]). We 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 e xpression[3], suggesting that they might be important for myogenic differentiation. 3-Mercaptopicolinic Acid or 3-Mercaptopicolinate (3-MPA), a known inhibitor of gluconeogenesis, has been reported to inhibit both isoforms of PEPCK, but with increased potency for PEPCK-C. in vitro[7,8,9]. The aim of this study was to compare the endogenous expression of Pck[1] and Pck[2] in proliferating and differentiating C2C12 cells and determine the dose-dependent effects of the PEPCK inhibitor, 3-MPA, on proliferation, differentiation and gene expression of C2C12 cells

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