Gnaiger 2020 BEC MitoPathways

Gnaiger E ed (2012) Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. Mitochondr Physiol Network 17.##: ## pp. 3rd ed. ISBN 978-3-9502399-6-6

» MiPNet2007

Gnaiger E (2012)

Abstract: • Keywords: ETS, Q-junction, respiratory states, flux control ratios

• O2k-Network Lab: AT_Innsbruck_Gnaiger E, AT Innsbruck OROBOROS

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Organism: Human, Mouse  Tissue;cell: Skeletal muscle, Fibroblast  Preparation: Permeabilized cells, Permeabilized tissue, Isolated Mitochondria"Isolated Mitochondria" is not in the list (Intact organism, Intact organ, Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, SMP, Chloroplasts, Enzyme, Oxidase;biochemical oxidation, ...) of allowed values for the "Preparation" property. 

Regulation: Mitochondrial Biogenesis; Mitochondrial Density"Mitochondrial Biogenesis; Mitochondrial Density" is not in the list (Aerobic glycolysis, ADP, ATP, ATP production, AMP, Calcium, Coupling efficiency;uncoupling, Cyt c, Flux control, Inhibitor, ...) of allowed values for the "Respiration and regulation" property.  Coupling state: LEAK, ROUTINE, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Theory, MiPNet-Publication"MiPNet-Publication" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

Supplementary online information

• Preface

References Preface

Chapter 2. Mitochondrial pathways to Complex I: Respiratory substrate control with pyruvate, malate and glutamate

Notes - Pitfalls

1. Schwerzmann et al (1989) Proc Natl Acad Sci U S A 86: 1583-1587. “Of the substrates used here, pyruvate/malate activates the chain at complex I, glutamate/malate and succinate at complexes II and III, ..” - This consideration of glutamate+malate requires correction.
2. Ponsot et al (2005) J Cell Physiol 203: 479-486. (a) Respiration (State 3) in permeabilized fibres with malate alone gave 25-50% of the flux with pyruvate+malate. This most likely indicates a large content of endogenous mitochondrial substrates, which interfere to an unknown degree with the kinetics of respiration after addition of exogenous substrates. In such a study, the conventional initial depletion of endogenous substrates would be most important. (b) Maximal respiration rates in muscle should be evaluated at saturating or high Pi, since at a Pi concentration of 3 mM OXPHOS respiration is phosphate limited.
3. Hulbert et al (2006) J Comp Physiol B 176: 93-105. Addition of ‘sparking malate concentrations’. This term can probably be derived from the misconception that tricarboxylic acid cycle intermediates are conserved during respiration of isolated mitochondria. 380 µM malate (instead of mM concentrations) in conjunction with 2.4 mM pyruvate were used, which makes a comparison difficult between different tissues and different species: the low malate concentration may limit PMP flux at various degrees in the different sources of mitochondria, and GMP may support higher fluxes than PMP at tissue- and species-specific degrees.

References Chapter 2

1. Brewer GJ, Jones TT, Wallimann T, Schlattner U (2004) Higher respiratory rates and improved creatine stimulation in brain mitochondria isolated with antioxidants. Mitochondrion 4: 49-57.
2. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217: 409-427. - Substrate depletion in isolated mitochondria is achieved in State 2: ADP is added to induce a transient stimulation of oxygen flux based on oxidation of endogenous substrates.
3. Dawson KD, Baker DJ, Greenhaff PL, Gibala MJ (2005) An accute decrease in TCA cycle intermediates does not affect aerobic energy delivery in contracting rat skeletal muscle. J Physiol 565: 637-643.
4. Duchen MR (2004) Roles of mitochondria in health and disease. Diabetes 53, Suppl 1: S96-S102. - Mitochondrial glutamate dehydrogenase is particularly active in astrocytes, preventing glutamate induced neurotoxicity.
5. Gibala MJ, MacLean DA, Graham TE, Saltin B (1998) Am J Physiol Endocrinol Metab 275: E235-E242. - concentrations of TCA cycle intermediates.
6. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837–1845.
7. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.
8. Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem Soc Trans 30: 252-258.
9. Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000b) Mitochondria in the cold. In: Life in the Cold. (Heldmaier G, Klingenspor M, eds) Springer, Heidelberg, Berlin, New York: pp 431-442. – MiR05 as the basis of MiR06.
10. Gnaiger E, Lassnig B, Kuznetsov AV, Margreiter R (1998) Mitochondrial respiration in the low oxygen environment of the cell: Effect of ADP on oxygen kinetics. Biochim Biophys Acta 1365: 249-254.
11. Gnaiger E, Lassnig B, Kuznetsov AV, Rieger G, Margreiter R (1998) Mitochondrial oxygen affinity, respiratory flux control, and excess capacity of cytochrome c oxidase. J Exp Biol 201: 1129-1139.
12. Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A 97: 11080-11085. - Equilibrium ratio of malate to fumarate is 4.1.
13. Gueguen N, Lefaucheur L, Ecolan P, Fillaut M, Herpin P (2005) Ca2+-activated myosin-ATPases, creatine and adenylate kinases regulate mitochondrial function according to myofibre type in rabbit. J Physiol 564: 723-735.
14. Hildyard JCW, Halestrap AP (2003) Identification of the mitochondrial pyruvate carrier in Saccharomyces cerevidiae. Biochem J 374: 607-611.
15. Johnson G, Roussel D, Dumas JF, Douay O, Malthiery Y, Simard G, Ritz P (2006) Influence of intensity of food restriction on skeletal muscle mitochondrial energy metabolism in rats. Am J Physiol Endocrinol Metab 291: E460-E467. - Uncoupling stimulates coupled OXPHOS respiration, PM_P, by 14%.
16. Kemp RB, Hoare S, Schmalfeldt M, Bridge CM, Evans PM, Gnaiger E (1994) A thermochemical study of the production of lactate by glutaminolysis and glycolysis in mouse macrophage hybridoma cells. In What is Controlling Life? (Gnaiger E, Gellerich FN, Wyss M, eds) Modern Trends in BioThermoKinetics 3, Innsbruck Univ Press: 226-231. - Glutamate derived from hydrolyzation of glutamine is a very important aerobic substrate in cultured cells.
17. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286: H1633–H1641. – Cytochrome ‘’c’’ test.
18. Lemasters JJ (1984) The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. J Biol Chem 259: 13123-13130. - Malonate added to inhibit the succinate-fumarate reaction exerts only a minor effect on liver mitochondrial respiration.
19. Maechler P, Carobbio S, Rubi B (2006) In beta-cells, mitochondria integrate and generate metabolic signals controlling insulin secretion. Int J Biochem Cell Biol 38: 696-709.
20. Messer JI, Jackman MR, Willis WT (2004) Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria. Am J Physiol Cell Physiol 286: C565-572. - With malate alone and saturating [ADP] isolated rat skeletal muscle mitochondria respire at only 1.3% of OXPHOS capacity with pyruvate+malate. Pyruvate alone yields only 2.1% of OXPHOS capacity (State P) with PM.
21. Mootha VK, Arai AE, Balaban RS (1997) Maximum oxidative phosphorylation capacity of the mammalian heart. Am J Physiol 272: H769-H775. – [Pi] <10 mM and [ADP] <0.4 mM limit OXPHOS in isolated heart mitochondria.
22. Nicholls DG, Ferguson SJ (2002) Bioenergetics 3, Academic Press, London. 287 pp. - Carriers
23. O’Donnell JM, Kudej RK, LaNoue KF, Vatner SF, Lewandowski ED (2004) Limited transfer of cytosolic NADH into mitochondria at high cardiac workload. Am J Physiol Heart Circ Physiol 286: H2237-H2242.
24. Owen OE, Kalhan SC, Hanson RW (2002) The key role of anaplerosis and cataplerosis for citric acid cycle function. J Biol Chem 277: 30409-30412.
25. 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. - >90% saturation is reached only >5 mM ADP, yet few studies use such high [ADP] in permeabilized tissues and cells.
26. Puchowicz MA, Varnes ME, Cohen BH, Friedman NR, Kerr DS, Hoppel CL (2004) Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria – case studies. Mitochondrion 4: 377-385. - OXPHOS with glutamate alone is 50% to 85% of respiration with glutamate+malate. Accumulation of fumarate inhibits succinate dehydrogenase and glutamate dehydrogenase (Caughey et al 1957; Dervartanian, Veeger 1964).
27. Rasmussen UF, Rasmussen HN (2000) Human quadriceps muscle mitochondria: A functional characterization. Mol Cell Biochem 208: 37-44. - Uncoupling stimulates coupled OXPHOS respiration, PMP, by 15% in human skeletal muscle. OXPHOS with glutamate alone is 50% to 85% of respiration with glutamate+malate.
28. Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS (1998) Permeabilised cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184: 81-100. - The apparent Km for ADP increases up to 0.5 mM in some permeabilized muscle fibres.
29. Territo PR, Mootha VK, French SA, Balaban RS (2000) Ca²⁺ activation of heart mitochondrial oxidative phosphorylation: role of the F0/F1-ATPase. Am J Physiol Cell Physiol 278: C423-C435.

Chapter 3. Mitochondrial pathways to Complex II. Glycerophosphate dehydrogenase and electrontransferring flavoprotein

References Chapter 3

1. Capel F, Rimbert V, Lioger D, Diot A, Rousset P, Patureau Mirand P, Boirie Y, Morio B, Mosoni L (2005) Due to reverse electron transfer, mitochondrial H2O2 release increases with age in human vastus lateralis muscle although oxidative capacity is preserved. Mech Ageing Develop 126: 505-511. With succinate alone OXPHOS is 30-40% lower than with succinate+rotenone in human skeletal muscle mitochondria.
2. Cecchini G (2003) Function and structure of Complex II of the respiratory chain. Annu Rev Biochem 72: 77-109.
3. Ernster L, Nordenbrand K (1967) Skeletal muscle mitochondria. In: Estabrook RW, Pullman ME (eds) Meth Enzymol: 86-94. – With succinate alone OXPHOS is 30-40% lower than with succinate+rotenone in rat skeletal muscle mitochondria.
4. Jackman MR, Willis WT (1996) Characteristics of mitochondria isolated from type I and type IIb skeletal muscle. Am J Physiol Cell Physiol 270: C673-678. - Glycerophosphate oxidation is 10-fold higher in rabbit gracilis mitochondria compared to soleus.
5. Lehninger AL (1970) Biochemistry. The molecular basis of cell structure and function Worth. 833 pp. - Oxaloacetate is a more potent competitive inhibitor of succinate dehydrogenase than malonate even at small concentration (p. 352).
6. Muller FL, Liu Y, Abdul-Ghani MA, Lustgarten MS, Bhattacharya A, Jang YC, Van Remmen H (2008) High rates of superoxide production in skeletal-muscle mitochondria respiring on both Complex I- and Complex II-linked substrates. Biochem J 409: 491–499. - addition of malate inhibits superoxide production with succinate, probably due to the oxaloacetate inhibition of CII.
7. Rauchova H, Drahota Z, Rauch P, Fato R, Lenaz G (2003) Coenzyme Q releases the inhibitory effect of free fatty acids on mitochondrial glycerophosphate dehydrogenase. Acta Biochim Polonica 50: 405-413. - Glycerophosphate is an important substrate for respiration in brown adipose tissue mitochondria.
8. Rasmussen UF, Rasmussen HN (2000) Human quadriceps muscle mitochondria: A functional characterization. Mol Cell Biochem 208: 37-44. – Glycerophosphate oxidation is relatively slow.
9. Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, Bartlam M, Rao Z (2005) Crystal structure of mitochondrial respiratory membrane protein Complex II. Cell 121: 1043–1057.

Notes - Pitfalls

1. Ponsot et al (2005) J Cell Physiol 203: 479-486. ‘.. the mitochondrial form of GPDH, which produces FADH2 within the mitochondrial matrix and provides electrons to Compoex II of the phosphorylation chain’. – The mitochondrial glycerophosphate dehydrogenase (GpDH), located on the outer side of the inner mitochondrial membrane, does not provide electrons to CII, but feeds electrons into the Q-cycle entirely independent of CII. FADH2 is not produced within the mitochondrial matrix. Electron transfer takes place from the mitochondrial inner membrane flavoprotein-linked glycerophosphate dehydrogenase to CoQ.
2. In the first edition of ‘Mitochondrial Pathways and Respiratory Control’ (2007), the term ‘electron transport’ is used synonymously for ‘electron transfer’.