13C isotope-assisted methods for quantifying glutamine metabolism in cancer cells - PubMed (original) (raw)

13C isotope-assisted methods for quantifying glutamine metabolism in cancer cells

Jie Zhang et al. Methods Enzymol. 2014.

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.

Keywords: Cancer cell; Glutamine metabolism; Metabolic flux; Reductive carboxylation; Stable isotopic tracer; Tricarboxylic acid cycle.

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Figures

Figure 19.1. Glutamine metabolism in mammalian cells

The figure shows the two major routes for glutamine metabolism in mammalian cells, glutaminolysis and reductive carboxylation. In either pathway, glutamine is first deaminated to glutamate, which is then converted to α-ketoglutarate via glutamate dehydrogenase (GDH) or transamination. In glutaminolysis, α-ketoglutarate is oxidized to malate via the TCA cycle and subsequently decarboxylated to pyruvate via malic enzyme (ME) or oxidized to oxaloacetate via malate dehydrogenase (MDH). In reductive carboxylation, α-ketoglutarate is reductively converted via isocitrate dehydrogenase (IDH) to isocitrate, which is then isomerized to citrate. Abbreviations: LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; CS, citrate synthase; ACL, ATP citrate lyase; ACO, aconitase; OGDH, oxoglutarate dehydrogenase; SDH, succinate dehydrogenase; FH, fumarase; MDH, malate dehydrogenase; GLS, glutaminase.

Figure 19.2. Typical flow of isotopic tracer experiments to study cancer metabolism

The scheme summarizes the main steps in an isotopic tracer-assisted study of cancer metabolism and common factors need to be considered, which are discussed in this section.

Figure 19.3. Carbon atom transition map for [U-13C5]glutamine

The map illustrates the fate of [U-13C5]glutamine in the TCA cycle and palmitate (12C atoms are represented by empty circles and 13C-atoms are represented by filled circles). Mass isotopomers generated by RC include M5 citrate (Cit), M3 oxaloacetate (Oac), M3 aspartate (Asp), M3 malate (Mal), and M3 fumarate (Fum). Mass isotopomers generated by oxidative metabolism include M4 Cit, M4 succinate (Suc), M4 Fum, and M4 Oac. Both RC and glutaminolysis (not shown) generate fully labeled acetyl-CoA (M2 AcCoA). For simplicity, labeling patterns arising from molecular symmetry and cellular compartments are not shown. The first round of the TCA cycle is illustrated, and positional labeling is depicted for relevant metabolites.

Figure 19.4. Carbon atom transition map for [1-13C]glutamine and [5-13C]glutamine

The map illustrates the fate of [1-13C] and [5-13C]glutamine used to trace the reductive TCA cycle (carbon atoms are represented by circles). The [1-13C]glutamine-derived isotopic label (gray circle) is lost during oxidation of α-ketoglutarate, but it is retained during the reductive TCA cycle and transferred to citrate and downstream metabolites. [5-13C] glutamine-derived isotopic label (black circle) is incorporated into acetyl-CoA and fatty acids through RC only; isotopic label from [5-13C]glutamine cannot be transferred to fatty acids through oxidative TCA cycle. Metabolites containing the acetyl-CoA carbon skeleton are highlighted by dashed circles. For simplicity, labeling patterns arising from molecular symmetry and cellular compartments are not shown. Positional labeling is depicted for relevant metabolites only.

Figure 19.5. The isotopomer spectral analysis (ISA) method

13C-labeled glucose tracer is used to trace the glucose-to-lipid flux. The glucose-derived 13C atoms and endogenous sources (e.g., acetate and glutamine) contribute to the pool of lipogenic acetyl-CoA. The D parameter indicates the unknown fractional contribution (or relative flux) of the glucose tracer to lipogenic acetyl-CoA. During the incubation with the tracer, palmitate is synthesized from eight molecules of acetyl-CoA. The newly synthesized palmitate mixes with an existing pool of palmitate (prior to tracer addition) and the fraction of de novo palmitate synthesis after a period of time, t is g(t). Each bar graph represents the palmitate MID. Preexisting palmitate has a significant M1 enrichment due to natural abundance. The parameters D and g(t) are estimated by fitting the predicted to the measured MID in the sampled palmitate.

Figure 19.6. Hypoxia case study data

Representative data based on authors’ previous study on RC and hypoxia. (A) Cell-specific uptake and secretion rates of glutamine and glutamate, respectively, in A549 cells under normoxic and hypoxic conditions. (B) Fractional contribution of total glutamine and RC-metabolized glutamine to lipogenic acetyl-CoA for multiple cancer cell lines, as determined by ISA. (C) Fractional contribution of glucose oxidation and glutamine-derived αKG reduction toward lipid synthesis in A549 cells under normoxic and hypoxic conditions. (D) Fraction of TCA cycle metabolites containing M1 label when cultured in [1-13C]glutamine in A549 cells under normoxic and hypoxic conditions. (E) Small-hairpin RNA-induced knockdown of IDH1 but not IDH2 expression reduces citrate M1 labeling in various cancer cell lines when cultured in [1-13C]glutamine.

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13C isotope-assisted methods for quantifying glutamine metabolism in cancer cells - PubMed (original) (raw)