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Effects of extracellular orotic acid on acute contraction-induced adaptation patterns in C2C12 cells

  • Thomas Beiter
  • Jens Hudemann
  • Christof Burgstahler
  • Andreas M. Nieß
  • Barbara Munz
Article
  • 113 Downloads

Abstract

Dietary administration of orotic acid (OA), an intermediate in the pyrimidine biosynthetic pathway, is considered to provide a wide range of beneficial effects, including cardioprotection and exercise adaptation. Its mechanisms of action, when applied extracellularly, however, are barely understood. In this study, we evaluated potential effects of OA on skeletal muscle using an in vitro contraction model of electrically pulse-stimulated (EPS) C2C12 myotubes. By analyzing a subset of genes representing inflammatory, metabolic, and structural adaptation pathways, we could show that OA supplementation diminishes the EPS-provoked expression of inflammatory transcripts (interleukin 6, Il6; chemokine (C-X-C Motif) ligand 5, Cxcl5), and attenuated transcript levels of nuclear receptor subfamily 4 group A member 3 (Nr4A3), early growth response 1 (Egr1), activating transcription factor 3 (Atf3), and fast-oxidative MyHC-IIA isoform (Myh2). By contrast, OA had no suppressive effect on the pathogen-provoked inflammatory gene response in skeletal muscle cells, as demonstrated by stimulation of C2C12 myotubes with bacterial LPS. In addition, we observed a suppressive effect of OA on EPS-induced phosphorylation of AMP-activated protein kinase (AMPK), whereas EPS-triggered phosphorylation/activation of the mammalian target of rapamycin (mTOR) was not affected. Finally, we demonstrate that OA positively influences glycogen levels in EP-stimulated myotubes. Taken together, our results suggest that in skeletal muscle cells, OA modulates both the inflammatory and the metabolic reaction provoked by acute contraction. These results might have important clinical implications, specifically in cardiovascular and exercise medicine.

Keywords

Orotic acid Electrical pulse stimulation Skeletal muscle contraction 

Notes

Acknowledgements

Work in the authors’ laboratory was furthermore supported by grants from the Monika-Kutzner-Stiftung, Berlin, Germany, and from the Else-Übelmesser-Stiftung, Tübingen, Germany.

Funding

This work was supported by a grant from Wörwag Pharma, Böblingen, Germany.

Supplementary material

11010_2018_3330_MOESM1_ESM.mov (2.6 mb)
Supplementary material 1 (MOV 2652 KB)
11010_2018_3330_MOESM2_ESM.mov (4.6 mb)
Supplementary material 2 (MOV 4718 KB)

References

  1. 1.
    Geiss KR, Stergiou N, Jester I, Neuenfeld HU, Jester HG (1998) Effects of magnesium orotate on exercise tolerance in patients with coronary heart disease. Cardiovasc Drugs Ther 12(Suppl 2):153–156CrossRefPubMedGoogle Scholar
  2. 2.
    Löffler M, Carrey EA, Zameitat E (2015) Orotic acid, more than just an intermediate of pyrimidine de novo synthesis. J Genet Genomics 42:207–219CrossRefPubMedGoogle Scholar
  3. 3.
    Muntean DM, Fira-Mladinescu O, Mirica NS, Duicu OM, Trancota SL, Sturza A (2010) Metabolic therapy: cardioprotective effects of orotic acid and its derivatives. Biomed Rev 21:47–55CrossRefGoogle Scholar
  4. 4.
    Rai M, Demontis F (2016) Systemic nutrient and stress signaling via myokines and myometabolites. Annu Rev Physiol 78:85–107CrossRefPubMedGoogle Scholar
  5. 5.
    Rosenfeldt FL (1998) Metabolic supplementation with orotic acid and magnesium orotate. Cardiovasc Drugs Ther 12(Suppl 2):147–152CrossRefPubMedGoogle Scholar
  6. 6.
    Rosenfeldt FL, Richards SM, Lin Z, Pepe S, Conyers RA (1998) Mechanism of cardioprotective effect of orotic acid. Cardiovasc Drugs Ther 12(Suppl 2):159–170CrossRefPubMedGoogle Scholar
  7. 7.
    Stepura OB, Martynow AI (2009) Magnesium orotate in severe congestive heart failure (MACH). Int J Cardiol 131:293–295CrossRefPubMedGoogle Scholar
  8. 8.
    Evans DR, Guy HI (2004) Mammalian pyrimidine biosynthesis: fresh insights into an ancient pathway. J Biol Chem 279:33035–33038CrossRefPubMedGoogle Scholar
  9. 9.
    Desler C, Lykke A, Rasmussen LJ (2010) The effect of mitochondrial dysfunction on cytosolic nucleotide metabolism. J Nucleic Acids 24:701518Google Scholar
  10. 10.
    Griffin JL, Bonney SA, Mann C, Hebbachi AM, Gibbons GF, Nicholson JK, Shoulders CC, Scott J (2004) An integrated reverse functional genomic and metabolic approach to understanding orotic acid-induced fatty liver. Physiol Genomics 17:140–149CrossRefPubMedGoogle Scholar
  11. 11.
    Jung EJ, Kwon SW, Jung BH, Oh SH, Lee BH (2011) Role of the AMPK/SREBP-1 pathway in the development of orotic acid-induced fatty liver. J Lipid Res 52:1617–1625CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Jung EJ, Lee KY, Lee BH (2012) Proliferating effect of orotic acid through mTORC1 activation mediated by negative regulation of AMPK in SK-Hep1 hepatocellular carcinoma cells. J Toxicol Sci 37:813–821CrossRefPubMedGoogle Scholar
  13. 13.
    Durschlag RP, Robinson JL (1980) Species specificity in the metabolic consequences of orotic acid consumption. J Nutr 110:822–828CrossRefPubMedGoogle Scholar
  14. 14.
    Donohoe JA, Rosenfeldt FL, Munsch CM, Williams JF (1993) The effect of orotic acid treatment on the energy and carbohydrate metabolism of the hypertrophying rat heart. Int J Biochem 25:163–182CrossRefPubMedGoogle Scholar
  15. 15.
    Porto LC, de Castro CH, Savergnini SS, Santos SH, Ferreira AV, Cordeiro LM, Sobrinho DB, Santos RA, de Almeida AP, Botion LM (2012) Improvement of the energy supply and contractile function in normal and ischemic rat hearts by dietary orotic acid. Life Sci 90:476–483CrossRefPubMedGoogle Scholar
  16. 16.
    Choi YJ, Yoon Y, Lee KY, Kang YP, Lim DK, Kwon SW, Kang KW, Lee SM, Lee BH (2015) Orotic acid induces hypertension associated with impaired endothelial nitric oxide synthesis. Toxicol Sci 144:307–317CrossRefPubMedGoogle Scholar
  17. 17.
    Evers-van Gogh IJ, Alex S, Stienstra R, Brenkman AB, Kersten S, Kalkhoven E (2015) Electric pulse stimulation of myotubes as an in vitro exercise model: cell-mediated and non-cell-mediated effects. Sci Rep 5:10944CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Manabe Y, Miyatake S, Takagi M, Nakamura M, Okeda A, Nakano T, Hirshman MF, Goodyear LJ, Fujii NL (2012) Characterization of an acute muscle contraction model using cultured C2C12 myotubes. PLoS ONE 7:e52592CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Nieuwoudt S, Mulya A, Fealy CE, Martelli E, Dasarathy S, Naga Prasad SV, Kirwan JP (2017) In vitro contraction protects against palmitate-induced insulin resistance in C2C12 myotubes. Am J Physiol Cell Physiol 313:C575-C583CrossRefPubMedGoogle Scholar
  20. 20.
    Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Fernández-Verdejo R, Vanwynsberghe AM, Essaghir A, Demoulin JB, Hai T, Deldicque L, Francaux M (2017) Activating transcription factor 3 attenuates chemokine and cytokine expression in mouse skeletal muscle after exercise and facilitates molecular adaptation to endurance training. FASEB J 31:840–851CrossRefPubMedGoogle Scholar
  22. 22.
    Pearen MA, Muscat GE (2010) Minireview: nuclear hormone receptor 4A signaling: implications for metabolic disease. Mol Endocrinol 24:1891–1903CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Beiter T, Hoene M, Prenzler F, Mooren FC, Steinacker JM, Weigert C, Niess AM, Munz B (2015) Exercise, skeletal muscle and inflammation: ARE-binding proteins as key regulators in inflammatory and adaptive networks. Exerc Immunol Rev 21:42–57PubMedGoogle Scholar
  24. 24.
    Pagel JI, Deindl E (2011) Early growth response 1—a transcription factor in the crossfire of signal transduction cascades. Indian J Biochem Biophys 48:226–235PubMedGoogle Scholar
  25. 25.
    Shirwany NA, Zou MH (2014) AMPK: a cellular metabolic and redox sensor. A minireview. Front Biosci 19:447–474CrossRefGoogle Scholar
  26. 26.
    Nikolic N, Bakke SS, Kase ET, Rudberg I, Flo Halle I, Rustan AC, Thoresen GH, Aas V (2012) Electrical pulse stimulation of cultured human skeletal muscle cells as an in vitro model of exercise. PLoS ONE 7:e33203CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Aas V, Torbla S, Andersen MH, Jensen J, Rustan AC (2002) Electrical stimulation improves insulin responses in a human skeletal muscle cell model of hyperglycemia. Ann N Y Acad Sci 967:506–515CrossRefPubMedGoogle Scholar
  28. 28.
    Lai YC, Zarrinpashneh E, Jensen J (2010) Additive effect of contraction and insulin on glucose uptake and glycogen synthase in muscle with different glycogen contents. J Appl Physiol 108:1106–1115CrossRefPubMedGoogle Scholar
  29. 29.
    Löffler M, Carrey EA, Zameitat E (2016) Orotate (orotic acid): an essential and versatile molecule. Nucleosides Nucleotides Nucleic Acids 35:566–577CrossRefPubMedGoogle Scholar
  30. 30.
    Farmawati A, Kitajima Y, Nedachi T, Sato M, Kanzaki M, Nagatomi R (2013) Characterization of contraction-induced IL-6 up-regulation using contractile C2C12 myotubes. Endocr J 60:137–147CrossRefPubMedGoogle Scholar
  31. 31.
    Nedachi T, Fujita H, Kanzaki M (2008) Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle. Am J Physiol Endocrinol Metab 295:E1191-E1204CrossRefGoogle Scholar
  32. 32.
    Whitham M, Chan MH, Pal M, Matthews VB, Prelovsek O, Lunke S, El-Osta A, Broenneke H, Alber J, Brüning JC, Wunderlich FT, Lancaster GI, Febbraio MA (2012) Contraction-induced interleukin-6 gene transcription in skeletal muscle is regulated by c-Jun terminal kinase/activator protein-1. J Biol Chem 287:10771–10779CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Burch N, Arnold AS, Item F, Summermatter S, Brochmann Santana Santos G, Christe M, Boutellier U, Toigo M, Handschin C (2010) Electric pulse stimulation of cultured murine muscle cells reproduces gene expression changes of trained mouse muscle. PLoS ONE 5:e10970CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Pattamaprapanont P, Garde C, Fabre O, Barrès R (2016) Muscle contraction induces acute hydroxymethylation of the exercise-responsive gene Nr4a3. Front Endocrinol 7:165CrossRefGoogle Scholar
  35. 35.
    Hoffman NJ, Parker BL, Chaudhuri R, Fisher-Wellman KH, Kleinert M, Humphrey SJ, Yang P, Holliday M, Trefely S, Fazakerley DJ, Stöckli J, Burchfield JG, Jensen TE, Jothi R, Kiens B, Wojtaszewski JF, Richter EA, James DE (2015) Global phosphoproteomic analysis of human skeletal muscle reveals a network of exercise-regulated kinases and AMPK substrates. Cell Metab 22:922–935CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Catoire M, Mensink M, Boekschoten MV, Hangelbroek R, Müller M, Schrauwen P, Kersten S (2012) Pronounced effects of acute endurance exercise on gene expression in resting and exercising human skeletal muscle. PLoS ONE 7:e51066CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Vissing K, Schjerling P (2014) Simplified data access on human skeletal muscle transcriptome responses to differentiated exercise. Sci Data 1:140041CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Marino M, Scuderi F, Provenzano C, Bartoccioni E (2011) Skeletal muscle cells: from local inflammatory response to active immunity. Gene Ther 18:109–116CrossRefPubMedGoogle Scholar
  39. 39.
    Pillon NJ, Bilan PJ, Fink LN, Klip A (2013) Cross-talk between skeletal muscle and immune cells: muscle-derived mediators and metabolic implications. Am J Physiol Endocrinol Metab 304:E453-E465CrossRefGoogle Scholar
  40. 40.
    Delaney NF, Sharma R, Tadvalkar L, Clish CB, Haller RG, Mootha VK (2017) Metabolic profiles of exercise in patients with McArdle disease or mitochondrial myopathy. Proc Natl Acad Sci USA 114:8402–8407CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Nielsen JN, Wojtaszewski JF, Haller RG, Hardie DG, Kemp BE, Richter EA, Vissing J (2002) Role of 5′AMP-activated protein kinase in glycogen synthase activity and glucose utilization: insights from patients with McArdle’s disease. J Physiol 541:979–989CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Ha J, Guan KL, Kim J (2015) AMPK and autophagy in glucose/glycogen metabolism. Mol Aspects Med 46:46–62CrossRefPubMedGoogle Scholar
  43. 43.
    Sylow L, Kleinert M, Richter EA, Jensen TE (2017) Exercise-stimulated glucose uptake- regulation and implications for glycaemic control. Nat Rev Endocrinol 13:133–148CrossRefPubMedGoogle Scholar
  44. 44.
    Jensen TE, Richter EA (2012) Regulation of glucose and glycogen metabolism during and after exercise. J Physiol 590:1069–1076CrossRefPubMedGoogle Scholar
  45. 45.
    Philp A, Hargreaves M, Baar K (2012) More than a store: regulatory roles for glycogen in skeletal muscle adaptation to exercise. Am J Physiol Endocrinol Metab 302:E1343-E1351Google Scholar
  46. 46.
    Sarma DS, Sidransky H (1969) Studies on orotic acid fatty liver in rats: factors influencing the induction of fatty liver. J Nutr 98:33–40CrossRefPubMedGoogle Scholar
  47. 47.
    Ferdinandy P, Fazekas T, Kadar E (1998) Effects of orotic acid on ischaemic/reperfused myocardial function and glycogen content in isolated working rat hearts. Pharmacol Res 37:111–114CrossRefPubMedGoogle Scholar
  48. 48.
    McCarthy MF, DiNicolantonio JJ (2014) β-Alanine and orotate as supplements for cardiac protection. Open Heart 1:e000119CrossRefGoogle Scholar
  49. 49.
    Fork C, Bauer T, Golz S, Geerts A, Weiland J, Del Turco D, Schömig E, Gründemann D (2011) OAT2 catalyses efflux of glutamate and uptake of orotic acid. Biochem J 436:305–312CrossRefPubMedGoogle Scholar
  50. 50.
    Miura D, Anzai N, Jutabha P, Chanluang S, He X, Fukutomi T, Endou H (2011) Human urate transporter 1 (hURAT1) mediates the transport of orotate. J Physiol Sci 61:253–257CrossRefPubMedGoogle Scholar
  51. 51.
    Hey-Mogensen M, Goncalves RL, Orr AL, Brand MD (2014) Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria. Free Radic Biol Med 72:149–155CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Thomas Beiter
    • 1
  • Jens Hudemann
    • 1
  • Christof Burgstahler
    • 1
  • Andreas M. Nieß
    • 1
  • Barbara Munz
    • 1
  1. 1.Medical Clinic, Department of Sports MedicineUniversity Hospital TübingenTübingenGermany

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