Doerrier 2018 MiP2018b
Evaluation of anaplerotic pathways to avoid artefacts in respirometric measurement of fatty acid oxidation. |
Link: MiP2018
Doerrier C, Garcia-Souza LF, Sumbalova Z, Krumschnabel G, Gnaiger E (2018)
Event: MiP2018
In recent years, research of mitochondrial (mt) function received an increasing interest in the context of many pathophysiological conditions. Identification of metabolic reprogramming and specific defects in mt-pathways is critical for diagnosis and selection of targeted treatments. Fatty acid oxidation (FAO, F-pathway) plays an essential role in many pathologies (e.g. insulin resistance, heart failure, ischemia-reperfusion, cancer) and must therefore be studied carefully. Here we show that respirometric measurements can significantly overestimate F-pathway capacities due to activation of anaplerotic pathways (e.g. mt-malic enzyme) [1,2]. F-pathway respiration requires the combined use of fatty acid(s) (FA) and malate to avoid acetyl-CoA accumulation which inhibits mt-respiration. In the presence of enzymes supporting malate- and pyruvate-linked anaplerotic pathways the widely used malate concentration of 2 mM supports NADH-linked (N-) respiration in addition to the F-pathway, resulting in overestimation of F-OXPHOS capacity. We developed the substrate-uncoupler-inhibitor titration (SUIT) protocol RP2 for reliable FAO evaluation by high-resolution respirometry (HRR). By comparison, we quantified the degree of FAO overestimation in several mouse and human mt-preparations. In conclusion, HRR allows the accurate study of mt-pathways, including F-pathway capacity, which is required for comprehensive OXPHOS analysis in diagnostic studies of mt-function.
β’ Bioblast editor: Plangger M
β’ O2k-Network Lab: AT Innsbruck Oroboros
Affiliations
Doerrier C(1), Garcia-Souza LF(1,2), Sumbalova Z(3), Krumschnabel G(1), Gnaiger E(1,3)
- Oroboros Instruments
- Inst Sport Science, Univ Innsbruck
- Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck; Innsbruck, Austria. - carolina.doerrier@oroboros.at
Support
- Supported by K-Regio project MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF). Contributiuon to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
References
- Sauer LA, Dauchy RT, Nagel WO, Morris HP (1980) Mitochondrial malic enzymes. Mitochondrial NAD(P)+-dependent malic enzyme activity and malate-dependent pyruvate formation are progression-linked in Morris hepatomas. J Biol Chem 255:3844-8.
- Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80 pp.
Labels: MiParea: Respiration, Instruments;methods
Stress:Cryopreservation Organism: Human, Mouse Tissue;cell: Heart, Nervous system, Blood cells, HEK, Platelet Preparation: Permeabilized cells, Homogenate, Isolated mitochondria
Regulation: Fatty acid Coupling state: OXPHOS Pathway: F, N HRR: Oxygraph-2k Event: Oral MitoEAGLE