Gnaiger 2013 Abstract MiP2013

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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.
Erich Gnaiger, Greenland
MiP2013, Book of Abstracts Open Access, Gnaiger 2015 Scand J Med Sci Sports

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: MiPNet18.08_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.

Keywords: Flux control factor

O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros, AT Innsbruck MitoCom, DK Copenhagen Christiansen M, NL Maastricht Schrauwen P, SE Stockholm Boushel RC, CA Vancouver Boushel RC

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

Organism: Human  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue  Enzyme: Uncoupling protein  Regulation: Coupling efficiency;uncoupling, Flux control  Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, NS  HRR: Oxygraph-2k, Theory 


Biochemical coupling efficiency

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 a consistent expression of biochemical coupling efficiency, ΔjE-L, ranging from 0.0 at zero coupling to 1.0 at the limit of a completely coupled system.

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 factors express the control of respiration by a specific metabolic variable, X, as a dimensionless (normalized) fractional change of flux, Δj. Z is the reference state with high (stimulated or un-inhibited) flux; Y is the background state at low flux, upon which X acts (jY=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 factor of X upon background Y is expressed as the change of flux from Y to Z, normalized for the reference state Z:

ΔjZ-Y = (Z-Y)/Z = 1-jY

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

With ETF-linked or CII-linked substrates, ET-capacity (E) was not in excess of phosphorylation system capacity, such that P=E or P/E=1.0. In this case, the biochemical coupling efficiency is simply related to the respiratory acceptor control ratio, ΔjE-L = (RCR-1)/RCR = (P-L)/P. In the present example, LEAK respiration is the background state (Y=L) and P=E is the reference state (Z).

Biochemical coupling efficiencies measured with ETF-linked and CII-linked substrates were identical in Inuit and Europeans, arm and leg, and before and after the 42 days adaptation to life in the cold.


  1. Mishmar D, Ruiz-Pesini E, Golik P, Macaulay V, Clark AG, Hosseini S, Brandon M, Easley K, Chen E, Brown MD, Sukernik RI, Olckers A, Wallace DC (2003) Natural selection shaped regional mtDNA variation in humans. Proc Natl Acad Sci U S A 100: 171-176.
  2. Ruiz-Pesini E, Mishmar D, Brandon M, Procaccio V, Wallace DC (2004) Effects of purifying and adaptive selection on regional variation in human mtDNA. Science 303: 223-226.
  3. 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.
  4. Pesta D, Gnaiger E (2012) High-resolution respirometry. OXPHOS protocols for human cells and permeabilized fibres from small biopisies of human muscle. Methods Mol Biol 810: 25-58.


Gnaiger Erich1,2, Søndergaard H3, Munch-Andersen T4, Damsgaard R5, Hagen C4, Díez-Sánchez C5, Ara I3,6, Wright-Paradis C3,7, Schrauwen P8, Hesselink M8, Calbet J3,9, Christiansen M4, Helge JW3,10, Saltin B3, Boushel R3,11

1D. Swarovski Research Lab, Dept. Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck; 2Oroboros Instruments, Innsbruck, Austria; 3Copenhagen Muscle Research Centre, Denmark; 4National Serum Institute, Copenhagen, Denmark; 5Dept. Biochem. Molec. Cell Biol., Univ. Zaragoza, Spain; 6GENUD Toledo Research Group, Univ. Castilla-La Mancha, Spain; 7Dept. Exercise Sci., Concordia Univ., Montreal, Canada; 8NUTRIM - School Nutrition, Toxicol. Metabolism, Dept. Human Biol., Maastricht Univ. Med. Center, The Netherlands; 9Dept. Physical Education, Univ. Las Palmas, Gran Canaria, Spain; 10X-Lab, Dept. Biomed. Sci., Univ. Copenhagen, Denmark; 11Dept. Biomed. Sci., Heart Circulatory Section, Univ. Copenhagen, Denmark.


Author contributions and acknowledgments

E Gnaiger designed the mitochondrial respiratory protocols and analysis of flux control capacities, conducted experiments, performed data analysis; H. Søndergaard was expedition manager, conducted exercise testing, supervised all subjects throughout the study; T. Munch-Andersen conducted exercise testing, data analysis, co-supervised all subjects throughout the study; C Hagen, C Díez-Sánchez and M Christiansen performed mitochondrial haplogroup analysis; P Schrauwen and M Hesselink performed UCP3 analysis; I Ara undertook muscle fibre type analyses; R Damsgaard and J Calbet were medical monitors of the study, took muscle biopsies; JW Helge co-organized and planned the expedition; B Saltin was leader of all aspects of the study; R Boushel conducted experiments, performed data analysis.

We thank the volunteers who participated in the study. This work was sponsored by Crown Prince Frederick and supported by the John and Birthe Meyer Foundation, the Danish National Research Foundation (504-14), Fonds de le Recherche en Sante Quebec (FRSQ), and The Natural Science and Engineering Research Council of Canada (NSERC). Support was provided from the Hunter Society in Qaanaaq and Hans Jensen at Hotel Qaanaaq. The Danish and American staff at the Thule Base and the staff at the hospital in Qaanaaq and at Thule Base kindly provided technical support. Jaya Rosenmeier also provided medical support. EG was supported by K-Regio project MitoCom Tyrol.