Journal Article

Bioenergetic profiles and respiratory control in mitochondrial physiology: Precision analysis of oxidative phosphorylation

Doerrier, Carolina, Timón‐Gómez, Alba, Cardoso, Luiza H. D., Gnaiger, Erich, Baglivo, Eleonora, Sumbalová, Zuzana, Garcia‐Souza, Luiz F.

2026

Overview

Oxidative phosphorylation (OXPHOS) is fundamental to mitochondrial function. Respirometry with living cells provides limited information compared to precision OXPHOS analysis with mitochondrial preparations, including isolated mitochondria, tissue homogenates, permeabilized tissues, and permeabilized cells. We studied mouse mitochondria from brain, a glucose‐dependent tissue, and from heart, which relies highly on fatty acid oxidation (FAO). HEK 293T cells were analysed as a widely used experimental model. Human peripheral blood mononuclear cells (PBMCs) and platelets were obtained from non‐invasive liquid biopsies, considering their potential as mitochondrial biomarkers. Twenty respiratory states were interrogated applying two substrate–uncoupler–inhibitor titration (SUIT) reference protocols in parallel. Convergent electron transfer (ET) into the coenzyme Q junction increased OXPHOS and ET capacities compared to separately stimulated pathways. In mouse heart and human PBMCs, OXPHOS capacities were identical to ET capacities in every pathway state. While this equivalence applied to the NADH‐linked pathway in platelets, ET capacity exceeded OXPHOS capacity supported by NADH‐linked substrates plus succinate. Surprisingly, mouse brain exhibited the highest excess ET capacity in the NADH‐linked pathway. In contrast, ET capacity of different batches of HEK 293T cells varied at constant OXPHOS capacity. Precision OXPHOS analysis enables attribution of respiratory performance to nutrient‐specific pathways. In studies ranging from exercise physiology to mitochondrial diseases, metabolic adjustments must be distinguished from functional defects. Bioenergetic profiles obtained by precision OXPHOS analysis gain perspective in the context of comparative mitochondrial physiology. What is the central question of this study? OXPHOS is fundamental for mitochondrial function and cellular metabolism: can the physiological relevance of mitochondrial function be effectively addressed using precision OXPHOS analysis? What is the main finding and its importance? Bioenergetic profiles revealed mitochondrial functional diversity and tissue‐ and cell‐specific metabolic patterns. Precision OXPHOS analysis is essential for understanding mitochondrial function, formulating robust working hypotheses, selecting appropriate models, and explaining tissue‐specific differences. Bioenergetic profiling unravels the complexities of mitochondrial respiratory control on the basis of a consistent comparative database.

• Publisher: John Wiley & Sons, Inc,Wiley
• Subject: Animals / Bioenergetics / Biopsy / Blood Platelets - metabolism / Brain - metabolism / Cell Respiration - physiology / Coenzyme Q / coupling control / Electron transfer / electron transfer system (ETS) / Electron Transport - physiology / Energy Metabolism - physiology / HEK293 Cells / high‐resolution respirometry (HRR) / Humans / Leukocytes (mononuclear) / Leukocytes, Mononuclear - metabolism / Male / Mice / Mice, Inbred C57BL / Mitochondria / Mitochondria - metabolism / Mitochondria - physiology / Mitochondria, Heart - metabolism / NAD - metabolism / NADH / Oxidation-Reduction / Oxidative Phosphorylation / OXPHOS / pathway control / Peripheral blood mononuclear cells / Phosphorylation / Physiology / Platelets / Respiration / substrate‐uncoupler‐inhibitor‐titration (SUIT) protocol / Titration