Kane 2014 Abstract MiP2014

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Assessing mitochondrial lactate oxidation in permeabilized skeletal muscle fibers highlights important experimental considerations: malate concentration and the cytochrome c effect.
Link:
Kane DA
Mitochondr Physiol Network 19.13 - MiP2014

Kane DA, Kane C, George M (2014)

Event: MiP2014

Lactate serves as an important metabolic intermediate for many tissues including skeletal and cardiac muscles, liver and brain. It is thought that a primary purpose of cytosolic lactate production from pyruvate by lactate dehydrogenase (LDH) is to regenerate NAD+ for continued glycolytic ATP production [1]. Considered in isolation, the NAD+ recycling afforded by cytosolic lactate production does not reconcile with the traditional view of aerobic glycolysis in which pyruvate, generated by glycolysis, enters the mitochondria directly for subsequent oxidation in the TCA cycle. The intracellular lactate shuttle hypothesis posits that lactate, generated in the cytosol, is oxidized by mitochondrial LDH of the same cell [2]. The details of the shuttle, however, are not entirely clear. Evidence is presented which supports that in skeletal muscle, extra-matrix LDH is strategically positioned within the cell to functionally interact with mitochondria [3]. A model incorporating mitochondrial lactate oxidation makes sense of aerobic glycolysis by permitting, among other things, cytosolic NAD+ regeneration, locally. However, experimental support requires attention to methodological detail. Important experimental conditions for assessing mitochondrial lactate oxidation in permeabilized fibers are discussed. Proper malate concentration [4] is necessary for robust NAD+-dependent lactate oxidation, suggesting that a functional malate-aspartate shuttle is essential to the assay. The cytochrome c test, a convenient means of confirming the integrity of the mitochondrial outer membrane [5], may not accurately reflect the integrity of mitochondrial preparations when assessing lactate oxidation. Indeed, parallel experiments in high-resolution respirometry reveal that in permeabilized rat skeletal muscle fibers, exogenous cytochrome c stimulates respiration with lactate but not with pyruvate as substrate.

These findings highlight the importance of optimizing seemingly trivial experimental variables.


O2k-Network Lab: CA Antigonish Kane DA


Labels: MiParea: Respiration 


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

Regulation: Aerobic glycolysis, ATP production, Cyt c, Redox state, Substrate 


HRR: Oxygraph-2k  Event: C2, Oral  MiP2014 

Affiliation

Dep Human Kinetics, St. Francis Xavier Univ, Antigonish, Canada. – dkane@stfx.ca

References

  1. Voet D, Voet JG, Pratt CW (2012) Fundamentals of Biochemistry. 4th ed John Wiley & Sons.
  2. Brooks GA (1998) Mammalian fuel utilization during sustained exercise. Comp Biochem Phys B 120:89-107.
  3. Elustondo PA, White AE, Hughes ME, Brebner K, Pavlov E, Kane DA (2013) Physical and functional association of lactate dehydrogenase (LDH) with skeletal muscle mitochondria. J Biol Chem 288:25309-17.
  4. Sumbalova Z, Vancova O, Krumschnabel G, Gnaiger E (2014) Optimization of malate concentration for high-resolution respirometry: mitochondria from rat liver and brain. In: Mitochondrial Physiology MiP2014. Mitochondr Physiol Network 19.13.
  5. 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:H1633–41.