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Deuterium Tracing to Interrogate Compartment-Specific NAD(P)H Metabolism in Cultured Mammalian Cells

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Metabolic Flux Analysis in Eukaryotic Cells

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2088))

Abstract

Oxidation–reduction (redox) reactions are ubiquitous in biology and typically occur in specific subcellular compartments. In cells, the electron transfer between molecules and organelles is commonly facilitated by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). While often taken for granted, these metabolic reactions are critically important for maintaining redox homeostasis and biochemical potentials across membranes. While 13C tracing and metabolic flux analysis (MFA) have emerged as powerful tools to study intracellular metabolism, this approach is limited when applied to pathways catalyzed in multiple cellular compartments. To address this issue, we and others have applied 2H (deuterium) tracers to observe transfer of labeled hydride anions, which accompanies electron transfer. Furthermore, we have developed a reporter system for indirectly quantifying NADPH enrichment in specific subcellular compartments. Here, we provide a detailed description of 2H tracing applications and the interrogation of mitochondrial versus cytosolic NAD(P)H metabolism in cultured mammalian cells. Specifically, we describe the generation of reporter cell lines that express epitope-tagged R132H-IDH1 or R172K-IDH2 and produce (d)2-hydroxyglutarate in a doxycycline-dependent manner. These tools and methods allow for quantitation of reducing equivalent turnover rates, the directionality of pathways present in multiple compartments, and the estimation of pathway contributions to NADPH pools.

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Acknowledgments

We thank members of the Metallo Lab for their feedback and discussion. C.M.M. is funded by NIH (R01 CA188652) and NSF CAREER (#145442) grants. E.W.L. is supported by a training grant from the NIH (T32 EB009380).

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Authors and Affiliations

  1. Department of Bioengineering, University of California San Diego, La Jolla, CA, USA

    Esther W. Lim & Christian M. Metallo

  2. Department of Radiation Oncology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA

    Seth J. Parker

  3. Moores Cancer Center, University of California San Diego, La Jolla, CA, USA

    Christian M. Metallo

  4. Diabetes and Endocrinology Research Center, University of California San Diego, La Jolla, CA, USA

    Christian M. Metallo

  5. Institute of Engineering in Medicine, University of California San Diego, La Jolla, CA, USA

    Christian M. Metallo

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  1. Esther W. Lim
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  2. Seth J. Parker
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  3. Christian M. Metallo
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Correspondence to Christian M. Metallo .

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Editors and Affiliations

  1. Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA

    Deepak Nagrath

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Lim, E.W., Parker, S.J., Metallo, C.M. (2020). Deuterium Tracing to Interrogate Compartment-Specific NAD(P)H Metabolism in Cultured Mammalian Cells. In: Nagrath, D. (eds) Metabolic Flux Analysis in Eukaryotic Cells. Methods in Molecular Biology, vol 2088. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0159-4_4

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  • DOI: https://doi.org/10.1007/978-1-0716-0159-4_4

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  • Print ISBN: 978-1-0716-0158-7

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