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. 2020 Oct 19;10:17599. doi: 10.1038/s41598-020-74718-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Carbon substrate preference and OXPHOS conductance across tissues. (A) Mitochondrial respiratory flux (JO₂) stimulated with 0.5 mM ADP, clamped throughout the experiment via the hexokinase clamp. Substrate and inhibitor additions were as follows: Pyr/M (1 mM/1 mM), UK5099 (pyruvate carrier inhibitor – 0.001 mM), Oct (0.2 mM), Rot (0.0005 mM), G3P (10 mM), Succ (10 mM), Ant (0.0005 mM), TMPD (0.5 mM, plus 2 mM ascorbate). (B) Glycerol-3-phosphate dehydrogenase 2 (GPD2) abundance. (C) Mitochondrial JO₂ in the presence of CaCl₂ (1.5 µM), stimulated by G3P (10 mM) and 0.5 mM ADP via the hexokinase clamp (G3P_ADP). (D) Relationship between JO₂ and ATP free energy (ΔG_ATP) clamped with the CK clamp in mitochondria energized with Pyr/M, G/M, Oct/M, Succ/Rot, and Multi. (E) Respiratory conductance – slope of the relationship between JO₂ and ΔG_ATP. (F) Maximal OXPHOS flux (JO₂ at ΔG_ATP of − 54.16 kJ/mol) in response to various substrate combinations. Data information: (A,D,F) Data normalized to protein corrected for each sample’s MEF. (C) Data normalized to average MEF for that tissue. Figures generated using GraphPad Prism 8 software (Version 8.4.2) Data are Mean ± SEM, N = 5/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.