Garcia-Souza 2016 Abstract Mito Xmas Meeting Innsbruck

From Bioblast
Assessment of fatty acid oxidation in mouse brain and liver mitochondria.


Garcia-Souza LF, Sumbalova Z, Gnaiger E (2016)

Event: Mito Xmas Meeting 2016 Innsbruck AT

Mitochondrial (mt) respiratory control by substrates and inhibitors represents a key aspect of bioenergetics research. Substrate-uncoupler-inhibitor titration (SUIT) protocols are applied for determining respiratory capacities of selected mt-pathways, with fatty acid oxidation (FAO) becoming a particularly hot topic for mt-fitness [1,2]. FAO measurement in mt-preparations requires addition of fatty acids and a NADH-linked substrate (malate) to prevent inhibition by accumulating acetyl-CoA. Malate, however, may stimulate respiration above the level of FAO-OXPHOS capacity mainly due to the presence of mt-malic enzyme (mtME) [3]. Therefore, conventional SUIT protocols require adjustments for accurate determination of FAO capacity.

In the present study we investigated FAO in liver and brain isolated mitochondria (imt) and homogenate (thom) from C57BL/6 mice. Malate concentration was varied in the range of 0.05 to 10 mM. Titration of octanoylcarnitine alone resulted in a modest increase of oxygen consumption in liver imt and thom, suggesting the presence of endogenous substrates. In liver and brain thom, malate titration stimulated respiration in the presence of ADP. FAO was saturated by malate at 0.1 mM (M.1), whereas mtME required 2 mM. FAO capacity is obtained accurately as the increase of respiration when titrating fatty acid after ADP and M.05.

Complex II is not required for the FAO pathway [3]. Respiration of brain mt with octanoylcarnitine and malate was inhibited by 50% by malonate (inhibitor of CII). In liver mt, however, FAO-linked respiration was paradoxically increased by malonate, which then was inhibited by rotenone. This may be explained by mt-malonyl-CoA synthase activity [4] in liver. These results illustrate the requirement of strict quality control of SUIT protocols and critical evaluation of metabolic assumptions [5] made for mitochondria studied in different tissues and species.

β€’ O2k-Network Lab: AT Innsbruck MitoFit, AT Innsbruck Oroboros, BR Rio de Janeiro Oliveira MF, SK Bratislava Sumbalova Z


Garcia-Souza LF(1,2,3), Sumbalova Z(2), Gnaiger E(2,3)
  1. Dept Sport Sc, Univ Innsbruck, Austria
  2. Oroboros Instruments, Innsbruck, Austria
  3. D Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria

References and Support

  1. Bienholz A, et al. (2014) Substrate modulation of fatty acid effects on energization and respiration of kidney proximal tubules during hypoxia/reoxygenation. PloS One 9.4:e94584.
  2. Ojuka E, et al. (2016) Measurement of Ξ²-oxidation capacity of biological samples by respirometry: a review of principles and substrates. Am J Physiol Endocrinol Metabol 310.9:E715-23.
  3. Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. Oroboros MiPNet Publications, Innsbruck:80pp.
  4. Witkowski A, Thweatt J, Smith S (2011) Mammalian ACSF3 protein is a malonyl-CoA synthetase that supplies the chain extender units for mitochondrial fatty acid synthesis. J Biol Chem 286.39:33729-36.
  5. Malonate
Supported by K-Regio K-Regio MitoFit. Contribution to COST Action MitoEAGLE.

Labels: MiParea: Respiration, Exercise physiology;nutrition;life style 

Tissue;cell: Nervous system, Liver  Preparation: Homogenate, Isolated mitochondria 

Regulation: Substrate  Coupling state: OXPHOS  Pathway: F, N  HRR: Oxygraph-2k  Event: Poster 

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