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A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements

Abstract

Measurement of oxygen consumption is a powerful and uniquely informative experimental technique. It can help identify mitochondrial mechanisms of action following pharmacologic and genetic interventions, and characterize energy metabolism in physiology and disease. The conceptual and practical benefits of respirometry have made it a frontline technique to understand how mitochondrial function can interface with—and in some cases control—cell physiology. Nonetheless, an appreciation of the complexity and challenges involved with such measurements is required to avoid common experimental and analytical pitfalls. Here we provide a practical guide to oxygen consumption measurements covering the selection of experimental models and instrumentation, as well as recommendations for the collection, interpretation and normalization of data. These guidelines are provided with the intention of aiding experimental design and enhancing the overall reputability, transparency and reliability of oxygen consumption measurements.

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Fig. 1: Microplate-based respirometry enables simultaneous measurement of oxygen consumption supported by different substrates.
Fig. 2: Calculation of respiratory rates in common measurement platforms.
Fig. 3: Importance of analysing raw, quantitative rates and potential pitfalls of scaling respiration data.
Fig. 4: Anoxia in the XF microchamber with high respiratory rates causes severe calculation artefacts.
Fig. 5: Common phenotypes observed during intact cell respirometry and their interpretations.

References

  1. Pagliarini, D. J. & Rutter, J. Hallmarks of a new era in mitochondrial biochemistry. Genes Dev. 27, 2615–2627 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. Viale, A. et al. Oncogene ablation-resistant pancreatic cancer cells depend on mitochondrial function. Nature 514, 628–632 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. Huang, S. C. C. et al. Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages. Nat. Immunol. https://doi.org/10.1038/ni.2956 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  4. Tannahill, G. M. et al. Succinate is an inflammatory signal that induces IL-1β through HIF-1α. Nature https://doi.org/10.1038/nature11986 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  5. Choi, S. W., Gerencser, A. A. & Nicholls, D. G. Bioenergetic analysis of isolated cerebrocortical nerve terminals on a microgram scale: spare respiratory capacity and stochastic mitochondrial failure. J. Neurochem. https://doi.org/10.1111/j.1471-4159.2009.06055.x (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Gubser, P. M. et al. Rapid effector function of memory CD8+ T cells requires an immediate-early glycolytic switch. Nat. Immunol. 14, 1064–1072 (2013).

    CAS  PubMed  Article  Google Scholar 

  7. Chandel, N. S. Evolution of mitochondria as signaling organelles. Cell Metab. https://doi.org/10.1016/j.cmet.2015.05.013 (2015).

    Article  PubMed  Google Scholar 

  8. Murphy, M. P. & Hartley, R. C. Mitochondria as a therapeutic target for common pathologies. Nat. Rev. Drug Discov. https://doi.org/10.1038/nrd.2018.174 (2018).

    Article  PubMed  Google Scholar 

  9. Pelletier, M., Billingham, L. K., Ramaswamy, M. & Siegel, R. M. Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption. Methods Enzymol. https://doi.org/10.1016/B978-0-12-416618-9.00007-8 (2014).

    Article  PubMed  Google Scholar 

  10. Mitchell, P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biochim. Biophys. Acta 1807, 1507–1538 (2011).

    CAS  PubMed  Article  Google Scholar 

  11. Nicholls, D. G. & Ferguson, S. J. Bioenergetics 4 (Academic Press, 2013).

  12. Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N. & Jastroch, M. Analysis and interpretation of microplate-based oxygen consumption and pH data. in Methods in Enzymology https://doi.org/10.1016/B978-0-12-801415-8.00016-3 (2014).

  13. Doerrier, C. et al. High-resolution fluorespirometry and oxphos protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. in Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-7831-1_3 (2018).

  14. Will, Y., Hynes, J., Ogurtsov, V. I. & Papkovsky, D. B. Analysis of mitochondrial function using phosphorescent oxygen-sensitive probes. Nat. Protoc. https://doi.org/10.1038/nprot.2006.351 (2007).

    Article  Google Scholar 

  15. Perry, C. G. R., Kane, D. A., Lanza, I. R. & Neufer, P. D. Methods for assessing mitochondrial function in diabetes. Diabetes https://doi.org/10.2337/db12-1219 (2013).

  16. Schmidt, C. A., Fisher-Wellman, K. H. & Neufer, P. D. From OCR and ECAR to energy: perspectives on the design and interpretation of bioenergetics studies. J. Biol. Chem. 297, 101140 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Brand, M. D. & Nicholls, D. G. Assessing mitochondrial dysfunction in cells. Biochem. J. https://doi.org/10.1042/BJ20110162 (2011).

    Article  PubMed  Google Scholar 

  18. Jones, A. E. et al. Forces, fluxes, and fuels: tracking mitochondrial metabolism by integrating measurements of membrane potential, respiration, and metabolites. Am. J. Physiol. 320, C80–C91 (2021).

    Google Scholar 

  19. Connolly, N. M. C. et al. Guidelines on experimental methods to assess mitochondrial dysfunction in cellular models of neurodegenerative diseases. Cell Death Differ. https://doi.org/10.1038/s41418-017-0020-4 (2018).

    Article  PubMed  Google Scholar 

  20. Dranka, B. P., Hill, B. G. & Darley-Usmar, V. M. Mitochondrial reserve capacity in endothelial cells: the impact of nitric oxide and reactive oxygen species. Free Radic. Biol. Med. https://doi.org/10.1016/j.freeradbiomed.2010.01.015 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rogers, G. W. et al. High-throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS ONE https://doi.org/10.1371/journal.pone.0021746 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Divakaruni, A. S., Rogers, G. W. & Murphy, A. N. Measuring mitochondrial function in permeabilized cells using the Seahorse XF analyzer or a Clark-type oxygen electrode. Curr. Protoc. Toxicol. https://doi.org/10.1002/0471140856.tx2502s60 (2014).

    Article  PubMed  Google Scholar 

  23. Hynes, J., Swiss, R. L. & Will, Y. High-throughput analysis of mitochondrial oxygen consumption. in Methods in Molecular Biology https://doi.org/10.1007/978-1-4939-7831-1_4 (2018).

  24. Acin-Perez, R., Benincá, C., Shabane, B., Shirihai, O. S. & Stiles, L. Utilization of human samples for assessment of mitochondrial bioenergetics: gold standards, limitation and future perspectives. Life 11, 949 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. Hill, B. G. et al. Integration of cellular bioenergetics with mitochondrial quality control and autophagy. Biol. Chem. 393, 1485–1512 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nat. Protoc. 2, 287–295 (2007).

    CAS  PubMed  Article  Google Scholar 

  27. Wieckowski, M. R. M. R., Giorgi, C., Lebiedzinska, M., Duszynski, J. & Pinton, P. Isolation of mitochondria-associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4, 1582–1590 (2009).

    CAS  PubMed  Article  Google Scholar 

  28. Kushnareva, Y. E., Wiley, S. E., Ward, M. W., Andreyev, A. Y. & Murphy, A. N. Excitotoxic injury to mitochondria isolated from cultured neurons. J. Biol. Chem. 280, 28894–28902 (2005).

    CAS  PubMed  Article  Google Scholar 

  29. Yang, K., Doan, M. T., Stiles, L. & Divakaruni, A. S. Measuring CPT-1-mediated respiration in permeabilized cells and isolated mitochondria. STAR Protoc. 2, 100687 (2021).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  30. Benador, I. Y. et al. Mitochondria bound to lipid droplets have unique bioenergetics, composition and dynamics that support lipid droplet expansion. Cell Metab. 27, 869–885 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. Divakaruni, A. S. et al. Thiazolidinediones are acute, specific inhibitors of the mitochondrial pyruvate carrier. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.1303360110 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Salabei, J. K., Gibb, A. A. & Hill, B. G. Comprehensive measurement of respiratory activity in permeabilized cells using extracellular flux analysis. Nat. Protoc. https://doi.org/10.1038/nprot.2014.018 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Quintana, A., Kruse, S. E., Kapur, R. P., Sanz, E. & Palmiter, R. D. Complex I deficiency due to loss of Ndufs4 in the brain results in progressive encephalopathy resembling Leigh syndrome. Proc. Natl Acad. Sci. USA 107, 10996–11001 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. Rich, P. R. & Maréchal, A. The mitochondrial respiratory chain. Essays Biochem. https://doi.org/10.1042/BSE0470001 (2010).

    Article  PubMed  Google Scholar 

  35. Kiss, G. et al. The negative impact of α-ketoglutarate dehydrogenase complex deficiency on matrix substrate-level phosphorylation. FASEB J. 27, 2393–2406 (2013).

    Article  CAS  Google Scholar 

  36. Ryan, D. G. et al. Coupling Krebs cycle metabolites to signalling in immunity and cancer. Nat. Metab. https://doi.org/10.1038/s42255-018-0014-7 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Gray, L. R. et al. Hepatic mitochondrial pyruvate carrier 1 is required for efficient regulation of gluconeogenesis and whole-body glucose homeostasis. Cell Metab. 22, 669–681 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. Taylor, E. B. Functional properties of the mitochondrial carrier system. Trends Cell Biol. https://doi.org/10.1016/j.tcb.2017.04.004 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Woodall, B. P. et al. Parkin does not prevent accelerated cardiac aging in mitochondrial DNA mutator mice. JCI Insight 5, e127713 (2019).

    Article  Google Scholar 

  40. Norton, M. et al. ROMO1 is an essential redox-dependent regulator of mitochondrial dynamics. Sci. Signal. 7, ra10 (2014).

    PubMed  Article  CAS  Google Scholar 

  41. Fu, Z. et al. Requirement of mitochondrial transcription factor A in tissue-resident regulatory T cell maintenance and function. Cell Rep. 28, 159–171 (2019).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  42. Pagliarini, D. J. et al. Involvement of a mitochondrial phosphatase in the regulation of ATP production and insulin secretion in pancreatic β cells. Mol. Cell 19, 197–207 (2005).

    CAS  PubMed  Article  Google Scholar 

  43. Wang, G. et al. Modeling the mitochondrial cardiomyopathy of Barth syndrome with iPSC and heart-on-chip technologies. Nat. Med. 20, 616–623 (2014).

    PubMed Central  Google Scholar 

  44. Leonardi, R., Zhang, Y. M., Rock, C. O. & Jackowski, S. Coenzyme A: back in action. Prog. Lipid Res. https://doi.org/10.1016/j.plipres.2005.04.001 (2005).

    Article  PubMed  Google Scholar 

  45. Solmonson, A. & DeBerardinis, R. J. Lipoic acid metabolism and mitochondrial redox regulation. J. Biol. Chem. 293, 7522–7530 (2018).

    PubMed  Article  Google Scholar 

  46. Stefely, J. A. & Pagliarini, D. J. Biochemistry of mitochondrial coenzyme Q biosynthesis. Trends Biochem. Sci. 42, 824–843 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. Daan Westenbrink, B. et al. Mitochondrial reprogramming induced by CaMKIIδ mediates hypertrophy decompensation. Circ. Res. 116, e28–e39 (2015).

    PubMed  PubMed Central  Google Scholar 

  48. Rotig, A. et al. Aconitase and mitochondrial iron–sulphur protein deficiency in Friedreich ataxia. Nat. Genet. 17, 215–217 (1997).

    CAS  PubMed  Article  Google Scholar 

  49. Diers, A. R., Broniowska, K. A., Chang, C. F., Hill, R. B. & Hogg, N. S-nitrosation of monocarboxylate transporter 1: inhibition of pyruvate-fueled respiration and proliferation of breast cancer cells. Free Radic. Biol. Med. 69, 229–238 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  50. CHANCE, B. & WILLIAMS, G. R. Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217, 409–427 (1955).

    CAS  PubMed  Article  Google Scholar 

  51. Estabrook, R. W. Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10, 41–47 (1967).

    CAS  Article  Google Scholar 

  52. Ernster, L. & Schatz, G. Mitochondria: a historical review. J. Cell Biol. 91, 227–255 (1981).

  53. Lopaschuk, G. D., Karwi, Q. G., Tian, R., Wende, A. R. & Abel, E. D. Cardiac energy metabolism in heart failure. Circ. Res. 128, 1487–1513 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  54. Sun, H. & Wang, Y. Branched chain amino acid metabolic reprogramming in heart failure. Biochim. Biophys. Acta 1862, 2270–2275 (2016).

    CAS  PubMed  Article  Google Scholar 

  55. White, P. J. & Newgard, C. B. Branched-chain amino acids in disease. Science 363, 582–583 (2019).

    CAS  PubMed  Article  Google Scholar 

  56. Picard, M. et al. Mitochondrial structure and function are disrupted by standard isolation methods. PLoS ONE 6, e18317 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  57. Arruda, A. P. et al. Chronic enrichment of hepatic endoplasmic reticulum–mitochondria contact leads to mitochondrial dysfunction in obesity. Nat. Med. 20, 1427–1435 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. Nicholls, D. G. Spare respiratory capacity, oxidative stress and excitotoxicity. Biochem. Soc. Trans. https://doi.org/10.1042/BST0371385 (2009).

    Article  PubMed  Google Scholar 

  59. Adhihetty, P. J. et al. The role of PGC-1α on mitochondrial function and apoptotic susceptibility in muscle. Am. J. Physiol. Cell Physiol 297, C217–C225 (2009).

    CAS  PubMed  Article  Google Scholar 

  60. Agier, V. et al. Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations. Biochim. Biophys. Acta 1822, 1570–1580 (2012).

    CAS  PubMed  Article  Google Scholar 

  61. Clerc, P. & Polster, B. M. Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons. PLoS ONE 7, e34465 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  62. Schulz, I. Permeabilizing cells: some methods and applications for the study of intracellular processes. Methods Enzymol. 192, 280–300 (1990).

    CAS  PubMed  Article  Google Scholar 

  63. Buescher, J. M. et al. A roadmap for interpreting 13C metabolite labeling patterns from cells. Curr. Opin. Biotechnol. https://doi.org/10.1016/j.copbio.2015.02.003 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  64. Jang, C., Chen, L. & Rabinowitz, J. D. Metabolomics and isotope tracing. Cell https://doi.org/10.1016/j.cell.2018.03.055 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  65. Antoniewicz, M. R. A guide to 13C metabolic flux analysis for the cancer biologist. Exp. Mol. Med. https://doi.org/10.1038/s12276-018-0060-y (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Divakaruni, A. S. & Brand, M. D. The regulation and physiology of mitochondrial proton leak. Physiology https://doi.org/10.1152/physiol.00046.2010 (2011).

    Article  PubMed  Google Scholar 

  67. Bertholet, A. M. & Kirichok, Y. Mitochondrial H+ leak and thermogenesis. Annu. Rev. Physiol. 84, 381–407 (2022).

    PubMed  Article  CAS  Google Scholar 

  68. Duchen, M. R. Ca2+-dependent changes in the mitochondrial energetics in single dissociated mouse sensory neurons. Biochem. J. 283, 41–50 (1992).

    PubMed  PubMed Central  Article  Google Scholar 

  69. De Stefani, D., Rizzuto, R. & Pozzan, T. Enjoy the trip: calcium in mitochondria back and forth. https://doi.org/10.1146/annurev-biochem-060614-034216 (2016).

  70. Villalobo, A. & Lehninger, A. L. Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+. J. Biol. Chem. 255, 2457–2464 (1980).

    CAS  PubMed  Article  Google Scholar 

  71. Murphy, A. N., Bredesen, D. E., Cortopassi, G., Wang, E. & Fiskum, G. Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria. Proc. Natl Acad. Sci. USA. 93, 9893–9898 (1996).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  72. Veliova, M. et al. Blocking mitochondrial pyruvate import in brown adipocytes induces energy wasting via lipid cycling. EMBO Rep. 21, e49634 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. Gross, M. I. et al. Antitumor activity of the glutaminase inhibitor CB-839 in triple-negative breast cancer. Mol. Cancer Ther. 13, 890–901 (2014).

    CAS  PubMed  Article  Google Scholar 

  74. Leek, R., Grimes, D. R., Harris, A. L. & McIntyre, A. Methods: using three-dimensional culture (spheroids) as an in vitro model of tumour hypoxia. Adv. Exp. Med. Biol. 899, 167–196 (2016).

    CAS  PubMed  Article  Google Scholar 

  75. Simian, M. & Bissell, M. J. Organoids: a historical perspective of thinking in three dimensions. J. Cell Biol. 216, 31–40 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  76. De Graaf, I. A. M. et al. Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies. Nat. Protoc. 5, 1540–1551 (2010).

    PubMed  Article  CAS  Google Scholar 

  77. Lau, A. N. & Vander Heiden, M. G. Metabolism in the tumor microenvironment. Annu. Rev. Cancer Biol. 4, 17–40 (2020).

    Article  Google Scholar 

  78. Kanow, M. A. et al. Biochemical adaptations of the retina and retinal pigment epithelium support a metabolic ecosystem in the vertebrate eye. Elife 6, e28899 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  79. Harada, A. E., Healy, T. M. & Burton, R. S. Variation in thermal tolerance and its relationship to mitochondrial function across populations of Tigriopus californicus. Front. Physiol. 10, 213 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  80. Luz, A. L., Smith, L. L., Rooney, J. P. & Meyer, J. N. Seahorse XFe24 extracellular flux analyzer-based analysis of cellular respiration in Caenorhabditis elegans. Curr. Protoc. Toxicol. 66, 25.7.1–25.7.15 (2015).

    Article  Google Scholar 

  81. Lay, S., Sanislav, O., Annesley, S. J. & Fisher, P. R. Mitochondrial stress tests using seahorse respirometry on intact Dictyostelium discoideum cells. Methods Mol. Biol 1407, 41–61 (2016).

    CAS  PubMed  Article  Google Scholar 

  82. Muthusamy, T. et al. Serine restriction alters sphingolipid diversity to constrain tumour growth. Nature 586, 790–795 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  83. Jiang, L. et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255–258 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  84. Tschöp, M. H. et al. A guide to analysis of mouse energy metabolism. Nat. Methods 9, 57–63 (2012).

    Article  CAS  Google Scholar 

  85. Müller, T. D., Klingenspor, M. & Tschöp, M. H. Revisiting energy expenditure: how to correct mouse metabolic rate for body mass. Nat. Metab. 3, 1134–1136 (2021).

    PubMed  Article  Google Scholar 

  86. Tyrrell, D. J. et al. Blood-cell bioenergetics are associated with physical function and inflammation in overweight/obese older adults. Exp. Gerontol. 70, 84–91 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  87. Kenwood, B. M. et al. Identification of a novel mitochondrial uncoupler that does not depolarize the plasma membrane. Mol. Metab. 3, 114–123 (2013).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  88. Davila, A. et al. Nicotinamide adenine dinucleotide is transported into mammalian mitochondria. Elife 7, e33246 (2018).

    PubMed  PubMed Central  Article  Google Scholar 

  89. Luongo, T. S. et al. SLC25A51 is a mammalian mitochondrial NAD+ transporter. Nature 588, 174–179 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  90. Affourtit, C. & Brand, M. D. Stronger control of ATP/ADP by proton leak in pancreatic beta cells than skeletal muscle mitochondria. Biochem. J. https://doi.org/10.1042/BJ20051280 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  91. Krasnikov, B. F. et al. Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition. Biochim. Biophys. Acta 1708, 375–392 (2005).

    CAS  PubMed  Article  Google Scholar 

  92. Mookerjee, S. A., Goncalves, R. L. S., Gerencser, A. A., Nicholls, D. G. & Brand, M. D. The contributions of respiration and glycolysis to extracellular acid production. Biochim. Biophys. Acta 1847, 171–181 (2015).

    CAS  PubMed  Article  Google Scholar 

  93. Desousa, B. R. et al. Calculating ATP production rates from oxidative phosphorylation and glycolysis during cell activation. Preprint at bioRxiv https://doi.org/10.1101/2022.04.16.488523 (2022).

  94. Mookerjee, S. A., Gerencser, A. A., Nicholls, D. G. & Brand, M. D. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J. Biol. Chem. https://doi.org/10.1074/jbc.M116.774471 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  95. Divakaruni, A. S., Andreyev, A. Y., Rogers, G. W. & Murphy, A. N. In situ measurements of mitochondrial matrix enzyme activities using plasma and mitochondrial membrane permeabilization agents. Anal. Biochem. 552, 60–65 (2018).

    CAS  PubMed  Article  Google Scholar 

  96. Divakaruni, A. S. et al. Inhibition of the mitochondrial pyruvate carrier protects from excitotoxic neuronal death. J. Cell Biol. https://doi.org/10.1083/jcb.201612067 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  97. Grassian, A. R., Metallo, C. M., Coloff, J. L., Stephanopoulos, G. & Brugge, J. S. Erk regulation of pyruvate dehydrogenase flux through PDK4 modulates cell proliferation. Genes Dev. 25, 1716–1733 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  98. Sullivan, W. J. et al. Extracellular matrix remodeling regulates glucose metabolism through TXNIP destabilization. Cell 175, 117–132 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  99. Sessions, A. O. et al. Preserved cardiac function by vinculin enhances glucose oxidation and extends health- and life-span. APL Bioeng. 2, 036101 (2018).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  100. Fan, Y. Y. et al. A bioassay to measure energy metabolism in mouse colonic crypts, organoids, and sorted stem cells. Am. J. Physiol. Gastrointest. Liver Physiol. 309, 1–9 (2015).

    Article  CAS  Google Scholar 

  101. Taddeo, E. P. et al. Individual islet respirometry reveals functional diversity within the islet population of mice and human donors. Mol. Metab. 16, 150–159 (2018).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  102. Kooragayala, K. et al. Quantification of oxygen consumption in retina ex vivo demonstrates limited reserve capacity of photoreceptor mitochondria. Invest. Ophthalmol. Vis. Sci. 56, 8428–8436 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  103. Ludikhuize, M. C., Meerlo, M., Burgering, B. M. T. & Rodríguez Colman, M. J. Protocol to profile the bioenergetics of organoids using Seahorse. STAR Protoc. 2, 100386 (2021).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  104. Gerencser, A. A. et al. Quantitative microplate-based respirometry with correction for oxygen diffusion. Anal. Chem. https://doi.org/10.1021/ac900881z (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  105. Oliver, D. G., Sanders, A. H., Douglas Hogg, R. & Woods Hellman, J. Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay. J. Immunol. Methods 42, 195–201 (1981).

    CAS  PubMed  Article  Google Scholar 

  106. Lundholt, B. K., Scudder, K. M. & Pagliaro, L. A simple technique for reducing edge effect in cell-based assays. J. Biomol. Screen. 8, 566–570 (2003).

    CAS  PubMed  Article  Google Scholar 

  107. Schoonen, W. G. E. J., Stevenson, J. C. R., Westerink, W. M. A. & Horbach, G. J. Cytotoxic effects of 109 reference compounds on rat H4IIE and human HepG2 hepatocytes. III: Mechanistic assays on oxygen consumption with MitoXpress and NAD(P)H production with Alamar Blue. Toxicol. In Vitro 26, 511–525 (2012).

    CAS  PubMed  Article  Google Scholar 

  108. Little, A. C. et al. High-content fluorescence imaging with the metabolic flux assay reveals insights into mitochondrial properties and functions. Commun. Biol. 3, 271 (2020).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  109. Wiley, S. E. et al. Wolfram syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis. EMBO Mol. Med. 5, 904–918 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  110. Wettmarshausen, J. & Perocchi, F. Isolation of functional mitochondria from cultured cells and mouse tissues. Methods Mol. Biol. 1567, 15–32 (2017).

    CAS  PubMed  Article  Google Scholar 

  111. Kirkinezos, I. G. et al. Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice. J. Neurosci. 25, 164–172 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  112. Rolfe, D. F. S. & Brown, G. C. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiol. Rev. 77, 731–758 (1997).

    CAS  PubMed  Article  Google Scholar 

  113. Chacko, B. K. et al. Methods for defining distinct bioenergetic profiles in platelets, lymphocytes, monocytes and neutrophils, and the oxidative burst from human blood. Lab. Invest. 93, 690–700 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  114. van der Windt, G. J. W. et al. Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development. Immunity 36, 68–78 (2012).

    PubMed  Google Scholar 

  115. McMurray, J. et al. Dapagliflozin in patients with heart failure and reduced ejection fraction. N. Engl. J. Med. 381, 1995–2008 (2019).

    CAS  PubMed  Article  Google Scholar 

  116. Molina, J. R. et al. An inhibitor of oxidative phosphorylation exploits cancer vulnerability. Nat. Med. 24, 1036–1046 (2018).

    CAS  PubMed  Article  Google Scholar 

  117. Rath, S. et al. MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res. 49, D1541–D1547 (2021).

    CAS  PubMed  Article  Google Scholar 

  118. Nowinski, S. M. et al. Mitochondrial fatty acid synthesis coordinates oxidative metabolism in mammalian mitochondria. Elife 9, e58041 (2020).

    Article  Google Scholar 

  119. Floyd, B. J. et al. Mitochondrial protein interaction mapping identifies regulators of respiratory chain function. Mol. Cell 63, 621–632 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  120. Brand, M. D. The efficiency and plasticity of mitochondrial energy transduction. Biochem. Soc. Trans. 33, 897–904 (2005).

    CAS  PubMed  Article  Google Scholar 

  121. Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W. & Walker, J. E. Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl Acad. Sci. USA 107, 16823–16827 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  122. Hinkle, P. C. P/O ratios of mitochondrial oxidative phosphorylation. Biochim. Biophys. Acta 1706, 1–11 (2005).

    CAS  PubMed  Article  Google Scholar 

  123. Müller, T. D. et al. P62 links β-adrenergic input to mitochondrial function and thermogenesis. J. Clin. Invest. 123, 469–478 (2013).

    PubMed  Article  CAS  Google Scholar 

  124. Kory, N. et al. MCART1/SLC25A51 is required for mitochondrial NAD transport. Sci. Adv 6, 43 (2020).

    Article  CAS  Google Scholar 

  125. Bricker, D. K. et al. A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila and humans. Science https://doi.org/10.1126/science.1218099 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  126. Herzig, S. et al. Identification and functional expression of the mitochondrial pyruvate carrier. Science https://doi.org/10.1126/science.1218530 (2012).

    Article  PubMed  Google Scholar 

  127. Sharma, A. et al. Impaired skeletal muscle mitochondrial pyruvate uptake rewires glucose metabolism to drive whole-body leanness. Elife 8, e45873 (2019).

    PubMed  PubMed Central  Article  Google Scholar 

  128. Bertholet, A. M. The use of the patch-clamp technique to study the thermogenic capacity of mitochondria. J. Vis. Exp. 171, (2021).

  129. Gerencser, A. A. et al. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria. J. Physiol. https://doi.org/10.1113/jphysiol.2012.228387 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

A.S.D. is supported by National Institutes of Health grants R35GM138003, P30DK063491 and P50CA092131, as well as the W. M. Keck Foundation. M.J. is supported by the Novo Nordisk Research Fonden (NNF20OC0059646). We thank members of both of our laboratories for their helpful discussions during the preparation of this manuscript, as well as L. Stiles (UCLA), B. Desousa (UCSF) and A. Murphy (Cytokinetics) for their critical perspective.

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A.S.D. and M.J. both contributed to the conceptualization, preparation, writing and editing of this manuscript.

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Correspondence to Ajit S. Divakaruni.

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Nature Metabolism thanks Alexander Galkin, David Nicholls and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt, in collaboration with the Nature Metabolism team.

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Divakaruni, A.S., Jastroch, M. A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements. Nat Metab 4, 978–994 (2022). https://doi.org/10.1038/s42255-022-00619-4

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