Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay
The regulation of cellular energetics is a complex process that requires the coordinated function of multiple organelles. Historically, studies focused on understanding cellular energy utilization and production have been overwhelmingly concentrated on the mitochondria. While mitochondria account for the majority of intracellular energy production, they alone are incapable of maintaining the variable energetic demands of the cell. The peroxisome has recently emerged as a secondary metabolic organelle that complements and improves mitochondrial performance. Although mitochondria and peroxisomes are structurally distinct organelles, they share key functional similarities that allows for the potential to repurpose readily available tools initially developed for mitochondrial assessment to interrogate peroxisomal metabolic function in a novel manner. To this end, we report here on procedures for the isolation, purification and real-time metabolic assessment of peroxisomal β-oxidation using the Agilent Seahorse® system. When used together, these protocols provide a straightforward, reproducible and highly quantifiable method for measuring the contributions of peroxisomes to cellular and organismal metabolism.
1. Reddy JK, Hashimoto T. Peroxisomal β-oxidation and peroxisome proliferator-activated receptor α: an adaptive metabolic system. Annu Rev Nutr. 2001;21(1):193–230. https://doi.org/10.1146/annurev.nutr.21.1.193 PMID:11375435
2. Osellame LD, Blacker TS, Duchen MR. Cellular and molecular mechanisms of mitochondrial function. Best Pract Res Clin Endocrinol Metab. 2012 Dec;26(6):711–23. https://doi.org/10.1016/j.beem.2012.05.003 PMID:23168274
3. Erdmann R. Assembly, maintenance and dynamics of peroxisomes. Biochim Biophys Acta. 2016 May;1863(5):787–9. https://doi.org/10.1016/j.bbamcr.2016.01.020 PMID:26851075
4. Dimitrov L, Lam SK, Schekman R. The role of the endoplasmic reticulum in peroxisome biogenesis. Cold Spring Harb Perspect Biol. 2013 May;5(5):a013243. https://doi.org/10.1101/cshperspect.a013243 PMID:23637287
5. Flis VV, Daum G. Lipid transport between the endoplasmic reticulum and mitochondria. Cold Spring Harb Perspect Biol. 2013 Jun;5(6):a013235. https://doi.org/10.1101/cshperspect.a013235 PMID:23732475
6. Lodhi IJ, Semenkovich CF. Peroxisomes: a nexus for lipid metabolism and cellular signaling. Cell Metab. 2014 Mar;19(3):380–92. https://doi.org/10.1016/j.cmet.2014.01.002 PMID:24508507
7. Chang CL, Weigel AV, Ioannou MS, Pasolli HA, Xu CS, Peale DR, et al. Spastin tethers lipid droplets to peroxisomes and directs fatty acid trafficking through ESCRT-III. J Cell Biol. 2019 Aug;218(8):2583–99. https://doi.org/10.1083/jcb.201902061 PMID:31227594
8. Joshi AS, Cohen S. Lipid droplet and peroxisome biogenesis: do they go hand-in-hand? Front Cell Dev Biol. 2019 May;7:92. https://doi.org/10.3389/fcell.2019.00092 PMID:31214588
9. Fransen M, Nordgren M, Wang B, Apanasets O. Role of peroxisomes in ROS/RNS-metabolism: implications for human disease. Biochim Biophys Acta. 2012 Sep;1822(9):1363–73. https://doi.org/10.1016/j.bbadis.2011.12.001 PMID:22178243
10. Bonekamp NA, Völkl A, Fahimi HD, Schrader M. Reactive oxygen species and peroxisomes: struggling for balance. Biofactors. 2009 Jul-Aug;35(4):346–55. https://doi.org/10.1002/biof.48 PMID:19459143
11. Hashimoto T. Individual peroxisomal β-oxidation enzymes. Ann N Y Acad Sci. 1982;386 1 Peroxisomes a:5–12. https://doi.org/10.1111/j.1749-6632.1982.tb21403.x PMID:6953852
12. Violante S, Achetib N, van Roermund CW, Hagen J, Dodatko T, Vaz FM, et al. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4. FASEB J. 2019 Mar;33(3):4355–64. https://doi.org/10.1096/fj.201801498R PMID:30540494
13. Wicks SE, Vandanmagsar B, Haynie KR, Fuller SE, Warfel JD, Stephens JM, et al. Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism. Proc Natl Acad Sci USA. 2015 Jun;112(25):E3300–9. https://doi.org/10.1073/pnas.1418560112 PMID:26056297
14. Violante S, Ijlst L, Te Brinke H, Koster J, Tavares de Almeida I, Wanders RJ, et al. Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient. Biochim Biophys Acta. 2013 Sep;1831(9):1467–74. https://doi.org/10.1016/j.bbalip.2013.06.007 PMID:23850792
15. Hunt MC, Siponen MI, Alexson SE. The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism. Biochim Biophys Acta. 2012 Sep;1822(9):1397–410. https://doi.org/10.1016/j.bbadis.2012.03.009 PMID:22465940
16. Wanders RJ, Komen J, Kemp S. Fatty acid omega-oxidation as a rescue pathway for fatty acid oxidation disorders in humans. FEBS J. 2011 Jan;278(2):182–94. https://doi.org/10.1111/j.1742-4658.2010.07947.x PMID:21156023
17. Abdel-Aleem S, Youssef J, Frangakis C, Badr M. Selective inhibition of hepatic peroxisomal fatty acid beta-oxidation by enoximone. Life Sci. 1992;51(1):53–7. https://doi.org/10.1016/0024-3205(92)90218-E PMID:1535408
18. Lazarow PB, De Duve C. A fatty acyl-CoA oxidizing system in rat liver peroxisomes; enhancement by clofibrate, a hypolipidemic drug. Proc Natl Acad Sci USA. 1976 Jun;73(6):2043–6. https://doi.org/10.1073/pnas.73.6.2043 PMID:180535
19. Horan MP, Pichaud N, Ballard JWO. Review: Quantifying mitochondrial dysfunction in complex diseases of aging.
J Gerontol A Biol Sci Med Sci. 2012 Oct;67(10):1022-35. https://doi.org/10.1093/gerona/glr263 PMID:22459622
20. Meng H, Gonzales NM, Lonard DM, Putluri N, Zhu B, Dacso CC, et al. XBP1 links the 12-hour clock to NAFLD and regulation of membrane fluidity and lipid homeostasis. Nat Commun. 2020 Dec;11(1):6215. https://doi.org/10.1038/s41467-020-20028-z PMID:33277471
21. Leighton F, Poole B, Beaufay H, Baudhuin P, Coffey JW, Fowler S, et al. The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J Cell Biol. 1968 May;37(2):482–513. https://doi.org/10.1083/jcb.37.2.482 PMID:4297786
22. Bartlett K, Eaton S. Mitochondrial β-oxidation. Eur J Biochem. 2004 Feb;271(3):462–9. https://doi.org/10.1046/j.1432-1033.2003.03947.x PMID:14728673
23. Perry RJ, Peng L, Cline GW, Petersen KF, Shulman GI. A Non-Invasive Method to Assess Hepatic Acetyl-CoA in Vivo. Cell Metab. 2017 Mar 7;25(3):749-756. https://doi.org/10.1016/j.cmet.2016.12.017 PMID:28111213
24. Wanders RJ. Metabolic functions of peroxisomes in health and disease. Biochimie. 2014 Mar;98:36–44. https://doi.org/10.1016/j.biochi.2013.08.022 PMID:24012550
25. Islinger M, Voelkl A, Fahimi HD, Schrader M. The peroxisome: an update on mysteries 2.0. Histochem Cell Biol. 2018 Nov;150(5):443–71. https://doi.org/10.1007/s00418-018-1722-5 PMID:30219925
26. Houten SM, Wanders RJ, Ranea-Robles P. Metabolic interactions between peroxisomes and mitochondria with a special focus on acylcarnitine metabolism. Biochim Biophys Acta Mol Basis Dis. 2020 May;1866(5):165720. https://doi.org/10.1016/j.bbadis.2020.165720 PMID:32057943
27. Sakamuri SS, Sperling JA, Sure VN, Dholakia MH, Peterson NR, Rutkai I, et al. Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research. Geroscience. 2018 Jun;40(3):347–56. https://doi.org/10.1007/s11357-018-0021-3 PMID:29860557
28. Marcelo KL, Lin F, Rajapakshe K, Dean A, Gonzales N, Coarfa C, et al. Deciphering hepatocellular responses to metabolic and oncogenic stress. J Biol Methods. 2015;2(3):28. https://doi.org/10.14440/jbm.2015.77 PMID:26504887