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

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.