Doerrier 2018 MiP2018b

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Carolina Doerrier
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

COST Action MitoEAGLE

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.


Bioblast editor: Plangger M O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E


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 

Affiliations

Doerrier C(1), Garcia-Souza LF(1,2), Sumbalova Z(3), Krumschnabel G(1), Gnaiger E(1,3)

  1. Oroboros Instruments
  2. Inst Sport Science, Univ Innsbruck
  3. 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

  1. 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.
  2. 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.