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Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry

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High-Throughput Metabolomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1978))

Abstract

Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply 13C isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.

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Acknowledgments

We thank members of the Metallo Lab for helpful discussions. Figure 2 was adapted with permission from unpublished work by Jamey D. Young (Vanderbilt University). This work was supported, in part, by NIH grant R01CA188652, a Searle Scholar Award, a NSF CAREER Award (1454425), a Camille Dreyfus Teacher Scholar Award (all to C.M.M.), and Deutsche Forschungsgesellschaft (German Research Foundation) (CO1488/1-1 to T.C.).

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Authors and Affiliations

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

    Thekla Cordes & Christian M. Metallo

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

    Christian M. Metallo

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

    Christian M. Metallo

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

    Christian M. Metallo

Authors
  1. Thekla Cordes
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  2. Christian M. Metallo
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Corresponding author

Correspondence to Christian M. Metallo .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO, USA

    Angelo D'Alessandro

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Cordes, T., Metallo, C.M. (2019). Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry. In: D'Alessandro, A. (eds) High-Throughput Metabolomics. Methods in Molecular Biology, vol 1978. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9236-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9236-2_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9235-5

  • Online ISBN: 978-1-4939-9236-2

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