Chapter Nineteen - 13C Isotope-Assisted Methods for Quantifying Glutamine Metabolism in Cancer Cells
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.
References (46)
- et al.
Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry
Metabolic Engineering
(2011) - et al.
Parallel labeling experiments with [1,2-(13)C]glucose and [U-(13)C]glutamine provide new insights into CHO cell metabolism
Metabolic Engineering
(2013) - et al.
Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements
Metabolic Engineering
(2006) - et al.
Elementary metabolite units (EMU): A novel framework for modeling isotopic distributions
Metabolic Engineering
(2007) - et al.
The mTORC1 pathway stimulates glutamine metabolism and cell proliferation by repressing SIRT4
Cell
(2013) - et al.
A method for measuring both glutamine and glutamate levels and stable isotopic enrichments
Analytical Biochemistry
(1985) - et al.
Metabolic reprogramming in cancer: Unraveling the role of glutamine in tumorigenesis
Seminars in Cell & Developmental Biology
(2012) - et al.
In vivo HIF-mediated reductive carboxylation is regulated by citrate levels and sensitizes VHL-deficient cells to glutamine deprivation
Cell Metabolism
(2013) - et al.
Evaluation of 13C isotopic tracers for metabolic flux analysis in mammalian cells
Journal of Biotechnology
(2009) Metabolic fluxes and metabolic engineering
Metabolic Engineering
(1999)
Analysis of amino acids in human serum by isocratic reversed-phase high-performance liquid chromatography with electrochemical detection
Journal of Chromatography. A
Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells
Metabolic Engineering
Quantifying carbon sources for de novo lipogenesis in wild-type and IRS-1 knockout brown adipocytes
Journal of Lipid Research
The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type
Cell Metabolism
13C metabolic flux analysis in complex systems
Current Opinion in Biotechnology
Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis
Analytical Chemistry
In-vivo measurement of glucose and alanine metabolism with stable isotopic tracers
Diabetes
Q's next: The diverse functions of glutamine in metabolism, cell biology and cancer
Oncogene
Towards a metabolic and isotopic steady state in CHO batch cultures for reliable isotope-based metabolic profiling
Biotechnology Journal
Metformin decreases glucose oxidation and increases the dependency of prostate cancer cells on reductive glutamine metabolism
Cancer Research
Reductive glutamine metabolism is a function of the alpha-ketoglutarate to citrate ratio in cells
Nature Communications
Correction of 13C mass isotopomer distributions for natural stable isotope abundance
Journal of Mass Spectrometry
Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth
Molecular Systems Biology
Cited by (0)
- 1
These authors contributed equally to this paper