Gnaiger 2013 Abstract MiP2013

Gnaiger E, Søndergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Díez-Sánchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet J, Christiansen M, Helge JW, Saltin B, Boushel R (2013) Biochemical coupling efficiency in permeabilized fibres from arm and leg muscle in Inuit versus Caucasians: A functional test of the uncoupling hypothesis in Greenland. Mitochondr Physiol Network 18.08.

Link: Flux control capacity

Gnaiger E, Soendergaard H, Munch-Andersen T, Damsgaard R, Hagen C, Diez-Sanchez C, Ara I, Wright-Paradis C, Schrauwen P, Hesselink M, Calbet J, Christiansen M, Helge JW, Saltin B, Boushel RC (2013)

Event: MiP2013

Flux control ratios, j, are oxygen fluxes normalized relative to a common respiratory reference state [2]. For a given protocol or set of respiratory protocols, flux control ratios provide a fingerprint of coupling and substrate control independent of (i) mt-content of cells or tissues, (ii) purification in preparations of isolated mitochondria, and (iii) assay conditions for determination of tissue mass or mt-markers external to a respiratory protocol (CS, protein, stereology, etc.).

Complementary to the concept of flux control ratios and analogous to elasticities of metabolic control analysis [3], flux control capacities express the control of respiration by a specific metabolic variable, X, as a dimensionless (normalized) fractional change of flux, Δj. Z is the reference sate with high (stimulated or un-inhibited) flux; Y is the background state at low flux, upon which X acts (j_Y=Y/Z); X is either added (stimulation, activation) or removed (reversal of inhibition) to yield a flux Z from background Y. Note that X, Y and Z denote both, the metabolic control variable (X) or respiratory state (Y, Z) and the corresponding respiratory flux, X=Z-Y. Experimentally, inhibitors are added rather than removed; then Z is the reference state and Y the background state in the presence of the inhibitor. The flux control capacity of X upon background Y is expressed as the change of flux from Y to Z, normalized for the reference state Z:

Δj_Z-Y = (Z-Y)/Z = 1-j_Y

Substrate control capacities express the relative change of oxygen flux in response to a transition of substrate availability in a defined coupling state. Coupling and phosphorylation control capacities are determined in an ETS-competent substrate state.

In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 °C [4]. Phosphorylation control capacities, i.e. LEAK (Y=L) to OXPHOS state (Z=P), with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. Normalization of respiration in terms of flux control capacities is generally applicable to mitochondrial preparations and intact cells, eliminating any errors in separate measurements of mitochondrial markers.

• O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom, DK Copenhagen Boushel RC

Labels: MiParea: Respiration, mtDNA;mt-genetics, Comparative MiP;environmental MiP, Exercise physiology;nutrition;life style 

Organism: Human  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue 

Regulation: Coupling efficiency;uncoupling, Flux control  Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Oxygraph-2k, Theory 

MiP2013 

10 years ago the uncoupling hypothesis was presented for mitochondrial haplogroups of arctic populations suggesting that lower coupling of mitochondrial respiration to ATP production was selected for in favor of higher heat dissipation as an adaptation to cold climates [1,2]. Up to date no actual tests have been published to compare mitochondrial coupling in tissues obtained from human populations with regional mtDNA variations. Analysis of oxidative phosphorylation (OXPHOS) is a major component of mitochondrial phenotyping [3]. We studied mitochondrial coupling in small biopsies of arm and leg muscle of Inuit of the Thule and Dorset haplogroups in northern Greenland compared to Danes from western Europe haplogroups. Inuit had a higher capacity to oxidize fat substrate in leg and arm muscle, yet mitochondrial respiration compensating for proton leak was proportionate with OXPHOS capacity. Biochemical coupling efficiency was preserved across variations in muscle fibre type and uncoupling protein-3 content. After 42 days of skiing on the sea ice in northern Greenland, Danes demonstrated adaptive substrate control through an increase in fatty acid oxidation approaching the level of the Inuit, yet coupling control of oxidative phosphorylation was conserved. Our findings reveal that coupled ATP production is of primary evolutionary significance for muscle tissue independent of adaptations to the cold. Our functional test of the uncoupling hypothesis was based on a substrate-uncoupler-inhibitor titration (SUIT) protocol using high-resolution respirometry designed to interrogate coupling control in different substrate states (ETF-linked octanoylcarnitine+malate and CII-linked succinate) and compare OXPHOS capacities for ETF-, CI-, CI+II and CII-linked substrate states [3,4]. Various coupling control ratios can be derived from such experiments (respiratory acceptor control ratio, RCR=P/L; which is the inverse of the OXPHOS control ratio, L/P) which are related to but do not directly express biochemical coupling efficiency [3]. A general concept of normalization of mt-respiration is presented here, which provides an expression of biochemical coupling efficiency ranging from 0.0 at zero coupling control capacity to 1.0 at the limit of a completely coupled system.

Affiliations, acknowledgements and author contributions

Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck;

OROBOROS INSTRUMENTS, Schöpfstr. 18, Innsbruck, Austria

Email: [email protected]

Supported by K-Regio project MitoCom Tyrol. I thank Anna Draxl for excellent experimental assistance.

References

1. Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.
2. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837–1845.
3. Fell D (1997) Understanding the control of metabolism. Portland Press, London.
4. Doerrier CV, Draxl A, Wiethüchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.

Note on respiratory states

1. Which question or concept provides the basis for studying a particular respiratory state? Example: OXPHOS capacity.
2. How is a particular respiratory state obtained experimentally? Example: Addition of reduced carbon-substrate after ADP to obtain ‘State 3’, and evaluation of saturation of flux by experimental concentrations of ADP, Pi, and O2 to verify that OXPHOS capacity rather than a substrate-limited flux is measured.