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

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

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 48h 3-MPA treatment (0.01–1mM). C2C12 myogenic differentiation was significantly induced following 3-MPA treatment (0.25, 0.5, 1mM) 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 1mM) 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 1mM.

Citation

Brearley, M. C., Daniel, Z. C., Loughna, P. T., Parr, T., & Brameld, J. M. (2020). The phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, 3-mercaptopicolinic acid (3-MPA), induces myogenic differentiation in C2C12 cells. Scientific Reports, 10, Article 22177. https://doi.org/10.1038/s41598-020-79324-9

• Journal Article Type: Article
• Acceptance Date: Dec 2, 2020
• Online Publication Date: Dec 17, 2020
• Publication Date: Dec 17, 2020
• Deposit Date: Dec 9, 2020
• Publicly Available Date: Jan 5, 2021
• Journal: Scientific Reports
• Print ISSN: 2045-2322
• Electronic ISSN: 2045-2322
• Publisher: Nature Publishing Group
• Peer Reviewed: Peer Reviewed
• Volume: 10
• Article Number: 22177
• DOI: https://doi.org/10.1038/s41598-020-79324-9
• Keywords: Multidisciplinary
• Public URL: https://nottingham-repository.worktribe.com/output/5129246
• Publisher URL: https://www.nature.com/articles/s41598-020-79324-9
• Additional Information: Received: 11 February 2020; Accepted: 2 December 2020; First Online: 17 December 2020; : The authors declare no competing interests.