Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Sp
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 perturbati
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roduction Living cells require metabolic activity to generate energy, synthesize biomass, and execute specific biochemical functions. Alterations in nutrient availability, gene expression/function, exposure to toxins, or other stresses contribute to cellular dysfunction and disease, which often results in metabolic reprogramming. Many pharmaceuticals target enzymes that play direct or indirect roles in metabolic pathways; therefore, identifying quantitative differences in pathway activity or flux is critically important for elucidating Angelo D’Alessandro (ed.), High-Throughput Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1978, https://doi.org/10.1007/978-1-4939-9236-2_14, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Thekla Cordes and Christian M. Metallo
echanisms that can be exploited therapeutically. As such, interest m in metabolic flux analysis (MFA) has increased significantly within the biomedical research community in the past several decades. This approach and related methods require a command of cell biology, analytical chemistry, and biochemical engineering principles to resolve fluxes in a given system [1]. The dynamic nature of metabolism can be quantitatively assessed with stable, nonradioactive isotope tracers in conjunction with a wide variety of analytical platforms [2, 3]. For example, mass spectrometry is typically combined with gas chromatography (GC-MS), liquid chromatography (LC-MS), or capillary electrophoresis (CE-MS) to quantify abundance and isotope enrichment in specific metabolites [4, 5]. Exposure of living cell cultures to stable isotopic tracer leads to an incorporation of isotopes into molecules within downstream biochemical pathways, providing quantitative information on the origin and turnover of a given metabolite. Tracer choice (e.g., substrates labeled with 13C, 15N, and/or 2H) directly impacts how and if a given metabolite will be labeled and can also impact flux resolution. Since mammalian cells consume diverse nutrients and various tracers are often available, the specific tracer(s) used will focus the information provided and hypothesis to be tested [6–8]. Detailed consideration prior to executing studies is recommended. Here we provide a detailed workflow for quantifying substrate utilization associated with TCA metabolism and de novo lipogenesis in mammalian cell cultures. As a case study, we apply uniformly labeled [U-13C6]glucose and [U-13C5]glutamine to the A549 non- small cell lung carcinoma cell line to quantify carbon utilization for TCA cycle metabolism and lipogenesis. We include protocols for media preparation, metabolite extraction, and GC-MS-based quantitation of isotope enrichment of intracellular metabolites and those present in culture media. The latter allows for quantification of cellular uptake and secretion fluxes. Finally, we provide an overview of tools available for data analysis and visualization. We have applied this workflow to various cell systems, including cancer cell lines [9–12],
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