Elsevier

Methods in Enzymology

Volume 542, 2014, Pages 369-389
Methods in Enzymology

Chapter Nineteen - 13C Isotope-Assisted Methods for Quantifying Glutamine Metabolism in Cancer Cells

https://doi.org/10.1016/B978-0-12-416618-9.00019-4Get rights and content

Abstract

Glutamine has recently emerged as a key substrate to support cancer cell proliferation, and the quantification of its metabolic flux is essential to understand the mechanisms by which this amino acid participates in the metabolic rewiring that sustains the survival and growth of neoplastic cells. Glutamine metabolism involves two major routes, glutaminolysis and reductive carboxylation, both of which begin with the deamination of glutamine to glutamate and the conversion of glutamate into α-ketoglutarate. In glutaminolysis, α-ketoglutarate is oxidized via the tricarboxylic acid cycle and decarboxylated to pyruvate. In reductive carboxylation, α-ketoglutarate is reductively converted into isocitrate, which is isomerized to citrate to supply acetyl-CoA for de novo lipogenesis. Here, we describe methods to quantify the metabolic flux of glutamine through these two routes, as well as the contribution of glutamine to lipid synthesis. Examples of how these methods can be applied to study metabolic pathways of oncological relevance are provided.

Introduction

In recent years, glutamine has emerged as a central precursor in the metabolism of cancer cells. Not only does glutamine, a nonessential amino acid, serve as the major mechanism of nitrogen transport into cells, but it also supplements glucose as a substantial carbon source via anaplerosis into the tricarboxylic acid (TCA) cycle (Daye and Wellen, 2012, DeBerardinis and Cheng, 2010). Given the necessity of transformed cells to perform elevated macromolecular biosynthesis to continue their growth and invasion within the body, targeting glutamine metabolism represents a promising opportunity for disrupting tumor proliferation (Vander Heiden, 2011).

As has been the case with glucose, mutated genes and malfunctioned signaling pathways in cancers have been found to influence the regulation of glutamine metabolism, including K-Ras (Gaglio et al., 2011, Son et al., 2013), p53 (Hu et al., 2010, Suzuki et al., 2010), and mTOR (Csibi et al., 2013). Most strikingly, c-Myc has been found to elicit “addiction” to the amino acid by inducing the expression of genes involved in glutamine metabolism, such as the glutamine transporter ASCT2 and glutaminase (GLS) (Gao et al., 2009, Wise et al., 2008).

Once taken up by the cell, glutamine is directed toward protein synthesis or deaminated, typically by GLS; nonproteinogenic glutamate is then converted to α-ketoglutarate via either glutamate dehydrogenase or transamination. After reaching this step, glutamine-derived α-ketoglutarate can be further metabolized along the TCA cycle through two different routes: The first, glutaminolysis, traditionally refers to oxidation of this α-ketoglutarate to malate and subsequent decarboxylation to pyruvate by malic enzyme (ME) or further oxidation to oxaloacetate by malate dehydrogenase. This progression contributes to ATP production through generation of substrates for oxidation in aerobic respiration and enables redox control from NADPH production through ME, formation of precursors for macromolecular biosynthesis such as alanine and pyruvate, or excretion of carbon as lactate by lactate dehydrogenase in Fig. 19.1 (DeBerardinis & Cheng, 2010). The second major route of glutamine metabolism, RC, has been shown to dominate in cell lines under hypoxic stress or disrupted mitochondrial functioning; in these situations, glutamine-derived α-ketoglutarate has been found to preferentially undergo reductive metabolism through the TCA cycle to isocitrate and then citrate, where it can then be converted to acetyl-CoA for lipid synthesis (Metallo et al., 2012, Mullen et al., 2012, Wise et al., 2011). Induction of this pathway has been shown to be controlled by mass action via conditions that perturb the citrate-to-α-ketoglutarate ratio, such as stabilization of the HIF-2α oncogene and/or oxidative energetic stress, and its activity has been demonstrated both in vitro and in vivo; targeting glutamine metabolism via GLS inhibition holds promise as a potential therapeutic strategy under these conditions, especially in combination with other anticancer drugs (Fendt, Bell, Keibler, Davidson, et al., 2013, Fendt, Bell, Keibler, Olenchock, et al., 2013, Gameiro et al., 2013).

In recognizing the significance of glutamine anaplerosis and its potentially divergent fates toward meeting the demands of either energy and combating oxidative stress or synthesizing macromolecules, it is necessary to have a means of quantifying these fates experimentally. Stable isotope labeling provides a direct readout of intracellular metabolism, and it can be combined with the known stoichiometry of biochemical pathways to estimate the activity of corresponding enzyme fluxes (Keibler, Fendt, & Stephanopoulos, 2012). We describe here how stable isotope tracers can be used to assess the use of glutamine by cancer cells for survival and proliferation.

Section snippets

Methods

The typical flow of a stable isotopic tracer-assisted study in cancer cells is schemed as in Fig. 19.2. Depending on the goals of each experiment, various factors need to be considered in each of these steps. In this section, we describe and discuss some of the common considerations to be taken during labeling experiments using cultured cancer cells. Although we limit our discussion to in vitro cell culture systems, many principles, such as the selection of tracers and analyses of intracellular

Choice of tracers and MID analysis

Various glutamine tracers are available, and the choice depends on which specific pathway or reaction needs to be monitored. Uniformly 13C-labeled glutamine ([U-13C5]glutamine), [1-13C]glutamine, and [5-13C]glutamine are good isotopic tracers to analyze the major pathways of glutamine metabolism in mammalian cells.

To trace glutamine catabolism in the TCA cycle, we can use [U-13C5]glutamine and [1-13C]glutamine. The [U-13C5]glutamine tracer transfers four 13C atoms to TCA cycle intermediates

Summary

Glutamine has been well known as a central precursor for protein and nucleotide synthesis in proliferating cells. However, in an anaplerotic pathway upregulated in many cancer cells, it can also be converted to α-ketoglutarate and incorporated in the TCA cycle, where it can serve as a supplementary carbon. Strikingly, under conditions of hypoxia or defective mitochondrial function, glutamine can become the major source of lipogenic acetyl-CoA through reductive carboxylation. Given the fact that

Acknowledgments

Research on cancer metabolism in Stephanopoulos Lab is funded by NIH grants 1R01DK075850-01 and 1R01CA160458-01A1. J. Z. is supported by a fellowship from Luxembourg Centre for Systems Biomedicine, University of Luxembourg. M. A. K. is funded by the David H. Koch Graduate Fellowship Fund and the Ludwig Fund for Cancer Research.

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