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Techniques to Investigate Mitochondrial Function in Neurons pp 321–338Cite as

Measuring Mitochondrial Pyruvate Oxidation

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Part of the book series: Neuromethods ((NM,volume 123))

Abstract

Pyruvate is a central metabolic intermediate and plays a prominent role in nervous system function. Neurons are highly reliant on pyruvate oxidation for maintenance of cellular energetics. Disorders in pyruvate metabolism may result in severe neurological defects. Pyruvate is imported into the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) for oxidation by the TCA cycle to support oxidative phosphorylation. Understanding the function of the MPC requires specialized methods for investigating mitochondrial pyruvate metabolism. Multiple instruments are available for measuring respiration of animal tissues, intact cells, permeabilized cells and tissue, and isolated mitochondria. Compared to other platforms, the comparative advantage of the Seahorse extracellular flux analyzer is the ability to sequentially administer treatment compounds and observe the effects on cellular respiration in a multiplexed 96-well format. Here we describe the methods and procedures for: (1) assessing mitochondrial pyruvate oxidation by intact cultured neuronal cells; (2) by mitochondria isolated from HEK293T cells, and (3) by mitochondria isolated from mouse liver, which are useful as a general model of mitochondrial function.

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Acknowledgments

We would like to thank Brett Wagner and the Radiation and Free Radical Research Core Facility for assistance performing the experiments utilizing the Seahorse Bioscience XF96. This research was supported by NIH grants R01 DK104998 (E.B.T.); R00 AR059190 (E.B.T.); F32 DK101183 (L.R.G.); T32 HL007121 to Francois Abboud (L.R.G.); T32 HL007638 to Michael Welsh (A.J.R.); and P30CA086862 to George Weiner, which contributed to support of core facilities utilized for this research.

Author information

Authors and Affiliations

  1. Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA

    Lawrence R. Gray, Lalita Oonthonpan & Adam J. Rauckhorst

  2. Department of Physiology and Molecular Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA

    Alix A. J. Rouault

  3. Department of Physiology and Molecular Biophysics, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Julien A. Sebag

  4. Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Eric B. Taylor

  5. Fraternal Order of the Eagles Diabetes Research Center, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Julien A. Sebag & Eric B. Taylor

  6. Abboud Cardiovascular Research Center, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Julien A. Sebag & Eric B. Taylor

  7. Holden Comprehensive Cancer, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Eric B. Taylor

  8. Pappajohn Biomedical Institute, University of Iowa Carver College of Medicine, Iowa City, IA, 52242, USA

    Julien A. Sebag & Eric B. Taylor

Authors
  1. Lawrence R. Gray
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  2. Alix A. J. Rouault
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  3. Lalita Oonthonpan
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  4. Adam J. Rauckhorst
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  5. Julien A. Sebag
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  6. Eric B. Taylor
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Corresponding author

Correspondence to Eric B. Taylor .

Editor information

Editors and Affiliations

  1. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Stefan Strack

  2. Department of Pharmacology, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA

    Yuriy M. Usachev

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Gray, L.R., Rouault, A.A.J., Oonthonpan, L., Rauckhorst, A.J., Sebag, J.A., Taylor, E.B. (2017). Measuring Mitochondrial Pyruvate Oxidation. In: Strack, S., Usachev, Y. (eds) Techniques to Investigate Mitochondrial Function in Neurons. Neuromethods, vol 123. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6890-9_16

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  • DOI: https://doi.org/10.1007/978-1-4939-6890-9_16

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6888-6

  • Online ISBN: 978-1-4939-6890-9

  • eBook Packages: Springer Protocols

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