In Vitro Monitoring of the Mitochondrial Beta-Oxidation Flux of Palmitic Acid and Investigation of Its Pharmacological Alteration by Therapeutics

Abstract

Background and Objective

The present study was designed to validate the functional assay that enables rapid screening of therapeutic candidates for their effect on mitochondrial fatty acid oxidation.

Methods

The two whole-cell systems (tissue homogenates and hepatocytes) have been evaluated to monitor the total beta-oxidation flux of physiologically important 3H-palmitic acid by measurement of tritiated water enrichment in incubations using UPLC coupled on-line to radioactivity monitoring and mass spectrometry.

Results

Our results with several known inhibitors of fatty acid oxidation showed that this simple assay could correctly predict a potential in alteration of mitochondrial function by drug candidates. Since the beta-oxidation of palmitic acid takes place almost exclusively in mitochondria of human hepatocytes, this model can be also utilized to distinguish between the mitochondrial and peroxisomal routes of this essential metabolic pathway in some cases.

Conclusions

The present work offers a new in vitro screen of changes in mitochondrial beta-oxidation by xenobiotics as well as a model to study the mechanism of this pathway.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. 1.

    Youssef JA, Song WO, Badr MZ. Mitochondrial, but not peroxisomal, beta-oxidation of fatty acids is conserved in coenzyme A deficient rat liver. Mol Cell Biochem. 1997;175:37–42.

    CAS  Article  Google Scholar 

  2. 2.

    Hettema EH, Henk FT. Transport fatty acids and metabolites across the peroxisomal membrane. Biochim Biophys Acta. 2000;1486:18–27.

    CAS  Article  Google Scholar 

  3. 3.

    Kemp S, Theodoulou FL, Wanders RJA. Mammalian peroxisomal ABC transporters: from endogenous substrates to pathology and clinical significance. Br J Pharmacol. 2011;164(7):1753–66.

    Article  Google Scholar 

  4. 4.

    Mannaerts GP, Van Veldhoven PP. Influence of drugs on liver mitochondria and peroxisomes. Arch Toxicol Suppl. 1988;12:225–32.

    CAS  Article  Google Scholar 

  5. 5.

    Wanders RJA, Ruiter JPN, Lodewijk IJI, Waterham HR, Houten SM. The enzymology of mitochondrial fatty acid beta-oxidation and its application to follow up analyses of positive neonatal screening results. J Inherit Metab Dis. 2010;33(5):479–94.

    CAS  Article  Google Scholar 

  6. 6.

    Poirier Y. Antonenkov, Glumoff, Hiltunen. Peroxisomal beta-oxidation—a metabolic pathway with multiple functions. Biochim Biophys Acta. 2006;1764:1413–26.

    Article  Google Scholar 

  7. 7.

    Fromenty B, Pessayre D. Inhibition of mitochondrial beta-oxidation as a mechanism of hepatotoxicity. Pharmacol Ther. 1995;67(1):101–54.

    CAS  Article  Google Scholar 

  8. 8.

    Begriche K, Massart J, Robin M-A, Borgne-Sanchez A, Fromenty B. Drug-induced toxicity on mitochondria and lipid metabolism: mechanistic diversity and deleterious consequences for the liver. J Hepatol. 2011;54:77–94.

    Article  Google Scholar 

  9. 9.

    Carracedo A, Cantley LC, Pandolfi PP. Cancer metabolism: fatty acid oxidation in the limelight. Nat Rev Cancer. 2013;13(4):227–32.

    CAS  Article  Google Scholar 

  10. 10.

    Vishwanath VA. Fatty acid beta-oxidation disorders: a brief review. Ann Neurosci. 2016;23:51–5.

    Article  Google Scholar 

  11. 11.

    Agius L, Peak M, Sherratt HAS. Difference between human, rat and guinea pig hepatocyte cultures; a comparative study of their rates of beta-oxidation and esterification of palmitate and their sensitivity to R-etomoxir. Biochem Pharmacol. 1991;42(9):1711–5.

    CAS  Article  Google Scholar 

  12. 12.

    Eaton S. Control of mitochondrial beta oxidation flux. Prog Lipid Res. 2002;41:197–239.

    CAS  Article  Google Scholar 

  13. 13.

    Rioux V, Lemarchal P, Legrand P. Myristic acid, unlike palmitic acid is readily metabolized in cultured rat hepatoctyes. J Nutr Biochem. 2000;11:198–207.

    CAS  Article  Google Scholar 

  14. 14.

    Law LK, Nelson LST, Hui J, Ho ChS, Ruiter J, Fok TF, et al. A novel functional assay for simultaneous determination of total fatty acid beta-oxidation flux and acylcarnitine profiling in human skin fibroblast using 2H31-palmitate by isotope ratio mass spectrometry and electrospray tandem mass spectrometry. Clin Chem Acta. 2007;382:25–30.

    CAS  Article  Google Scholar 

  15. 15.

    Seargeant LE, Balachandra K, Dilling LA, Greenberg CR. A simple screening test for fatty acid oxidation defects using whole-blood palmitate oxidation. J Inherit Metab Dis. 1999;22:740–6.

    CAS  Article  Google Scholar 

  16. 16.

    Norden M, Westman O, Venizelos N, Engwall M. Perfluorooctane sulfonate increases beta-oxidation of palmitic acid in chicken liver. Environ Sci Pollut Res. 2012;19:1859–63.

    CAS  Article  Google Scholar 

  17. 17.

    Westman O, Larsson M, Venizelos N, Hollert H, Magnus E. An oxygenated metabolite of benzo[a]pyrene increases hepatic beta-oxidation of fatty acids in chick embryos. Environ Sci Pollut Res. 2014;21(9):6243–51.

    CAS  Article  Google Scholar 

  18. 18.

    Hirschey MD, Verdin E. Measuring fatty acid oxidation in tissue homogenates. In: Nature publishing group. 2010. http://www.nature.com/protocolexchange/protocols/631. Accessed 10 Jan 2018.

  19. 19.

    Moffat C, Bhatia L, Nguyen T, Lynch P, Wang M, Wang D, et al. Acyl-CoA thioesterase-2 facilitates mitochondrial fatty acid oxidation in the liver. J Lipid Res. 2014;12:2458–70.

    Article  Google Scholar 

  20. 20.

    Harris FT, Jamshedur Rahman SM, Hassanein M, Qian J, Hoeksema MD, Chen H, et al. Acyl-coenzyme A-binding protein regulates beta-oxidation required for growth and survival of non-small cell lung cancer. Cancer Prev Res. 2014;7(7):748–57.

    CAS  Article  Google Scholar 

  21. 21.

    Jiang Z, Knudsen NH, Wang G, Qiu W, Chi Naing ZZ, Bai Y, et al. Genetic control of fatty acid beta-oxidation in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol. 2017;56(6):738–48.

    CAS  Article  Google Scholar 

  22. 22.

    Tirado-Velez JM, Jourmady I, Saez-Benito A, Cozar-Casellano I, German P. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation. PloS one. 2012;7(9):e46484.

    CAS  Article  Google Scholar 

  23. 23.

    Schermuly RT, Schultz A, Ghofrani HA, Meidow A, Rose F, Roehl A, et al. Pharmacokinetics and metabolism of infused versus inhaled iloprost in isolated rabbit lungs. J Pharmacol Exp Ther. 2002;303(2):741–5.

    CAS  Article  Google Scholar 

  24. 24.

    Yu XX, Drackley JK, Odle J. Rates of mitochondrial and peroxisomal beta-oxidation of palmitate change during postnatal development and food deprivation in liver, kidney and heart of pigs. J Nutr. 1997;127(9):1814–21.

    CAS  Article  Google Scholar 

  25. 25.

    Kondrup J, Lazarow PB. Flux of palmitate through the peroxisomal and mitochondrial beta-oxidation systems in isolated rat hepatocytes. Biochim Biophys Acta. 1985;835(1):147–53.

    CAS  Article  Google Scholar 

  26. 26.

    Kler RS, Jackson S, Bartlett K, Bindoff L, Eaton S, Pourfarzam M, et al. Quantitation of acyl-CoA and acylcarnitine esters accumulated during abnormal mitochondrial fatty acid oxidation. J Biol Chem. 1991;266(34):22932–8.

    CAS  PubMed  Google Scholar 

  27. 27.

    Fréneaux E, Labbe G, Letteron P, Le Dinh The, Degott C, Genève J, et al. Inhibition of the mitochondrial oxidation of fatty acids by tetracycline in mice and in man: possible role in microvesicular steatosis induced by this antibiotic. Hepatology. 1988;8(5):1056–62.

    Article  Google Scholar 

  28. 28.

    Fromenty B, Fisch C, Labbe G, Degott C, Deschamps D, Berson A, et al. Amiodarone inhibits the mitochondrial beta-oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther. 1990;255(3):1371–6.

    CAS  Google Scholar 

  29. 29.

    Browne GS, Nelson C, Nguyen T, Ellis BA, Day RO, Williams KM. Stereoselective and substrate-dependent inhibition of hepatic mitochondrial beta-oxidation and oxidative phosphorylation by the non-steroidal anti-inflammatory drugs ibuprofen, flurbiprofen and ketorolac. Biochem Pharmacol. 1999;57:837–44.

    CAS  Article  Google Scholar 

  30. 30.

    Amacher DE, Martin BA. Tetracycline-induced steatosis in primary canine hepatocyte cultures. Fundam Appl Toxicol. 1997;40(2):256–63.

    CAS  Article  Google Scholar 

  31. 31.

    Tyni T, Pourfarzam M, Turnbull DM. Analyses of mitochondrial fatty acid oxidation intermediates by tandem mass spectrometry from intact mitochondria prepared from homogenates of cultured fibroblasts, skeletal muscle cells, and fresh muscle. Pediatr Res. 2002;52(1):64–70.

    CAS  Article  Google Scholar 

  32. 32.

    Savir Y, Tu BP, Springer M. Competitive inhibition can linearize dose-response and generate a linear rectifier. Cell Syst. 2015;1(3):238–45.

    CAS  Article  Google Scholar 

  33. 33.

    Gebhardt R, Hengstler JG, Muller D, Glockner R, Buenning P, Laube B, et al. New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operating procedures. Drug Metab Rev. 2003;35:145–213.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Martina Suetterlin-Hachmann (Novartis, Basel, Switzerland) for providing fresh rat lung and liver tissues.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Renata Murgasova.

Ethics declarations

Funding

No source of funding.

Conflict of interest

R. Murgasova, E. Tor Carrera and J. Bourgailh declare no conflict of interest.

Ethics approval

Animal experiments were approved by the local Animal Welfare Committee. All applicable international, national, and/or institutional guidelines for care and use of animals were followed.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Murgasova, R., Tor Carreras, E. & Bourgailh, J. In Vitro Monitoring of the Mitochondrial Beta-Oxidation Flux of Palmitic Acid and Investigation of Its Pharmacological Alteration by Therapeutics. Eur J Drug Metab Pharmacokinet 43, 675–684 (2018). https://doi.org/10.1007/s13318-018-0479-5

Download citation