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

  • Sci Rep. 2020 Dec 17;10(1):22177. doi: 10.1038/s41598-020-79324-9.
Madelaine C Brearley  1  2 ,  Zoe C T R Daniel  1 ,  Paul T Loughna  3 ,  Tim Parr  1 ,  John M Brameld  4
Affiliations
  • 1. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK.
  • 2. Department of Medicine-Cardiology, University of California, Los Angeles, CA, USA.
  • 3. School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK.
  • 4. School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, UK. [email protected].
PMID: 33335245 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.

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