Simultaneous steady-state and dynamic13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells

Chendong Yang, Crystal Harrison, Eunsook S. Jin, David T. Chuang, A. Dean Sherry, Craig R. Malloy, Matthew E. Merritt, Ralph J. DeBerardinis

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Abstract

Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3 - and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]O3 - provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[13C]O3 - appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1- 13C]pyruvate metabolism, enhancing exchanges with [1- 13C]lactate and suppressing H[13C]O3 - formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in living tumors.

Original languageEnglish (US)
Pages (from-to)6212-6224
Number of pages13
JournalJournal of Biological Chemistry
Volume289
Issue number9
DOIs
StatePublished - Feb 28 2014

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Pyruvic Acid
Metabolism
Cells
Nuclear magnetic resonance
Neoplasms
Fluxes
Lactic Acid
Glycolysis
Metabolites
Spectrum Analysis
Oxidoreductases
Carboxylation
Spectroscopy
Decarboxylation
Cell growth
Biomarkers
Tumors
Phosphotransferases
Glucose
Cell Line

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

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title = "Simultaneous steady-state and dynamic13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells",
abstract = "Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3 - and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]O3 - provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[13C]O3 - appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1- 13C]pyruvate metabolism, enhancing exchanges with [1- 13C]lactate and suppressing H[13C]O3 - formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in living tumors.",
author = "Chendong Yang and Crystal Harrison and Jin, {Eunsook S.} and Chuang, {David T.} and Sherry, {A. Dean} and Malloy, {Craig R.} and Merritt, {Matthew E.} and DeBerardinis, {Ralph J.}",
year = "2014",
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doi = "10.1074/jbc.M113.543637",
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journal = "Journal of Biological Chemistry",
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T1 - Simultaneous steady-state and dynamic13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells

AU - Yang, Chendong

AU - Harrison, Crystal

AU - Jin, Eunsook S.

AU - Chuang, David T.

AU - Sherry, A. Dean

AU - Malloy, Craig R.

AU - Merritt, Matthew E.

AU - DeBerardinis, Ralph J.

PY - 2014/2/28

Y1 - 2014/2/28

N2 - Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3 - and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]O3 - provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[13C]O3 - appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1- 13C]pyruvate metabolism, enhancing exchanges with [1- 13C]lactate and suppressing H[13C]O3 - formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in living tumors.

AB - Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3 - and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]O3 - provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[13C]O3 - appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1- 13C]pyruvate metabolism, enhancing exchanges with [1- 13C]lactate and suppressing H[13C]O3 - formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in living tumors.

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