Simultaneous Steady-state and Dynamic 13C NMR Can Differentiate Alternative Routes of Pyruvate Metabolism in Living Cancer Cells

Background: C-13 hyperpolarization sensitively and non-destructively detects pyruvate-lactate exchanges in cancer cells. Results: Combining C-13 hyperpolarization with isotopomer analysis allowed many pyruvate-dependent pathways to be quantified simultaneously. Conclusion: Monitoring H[C-13]O-3(-) production from hyperpolarized [1-C-13]pyruvate yielded a quantitative readout of oncogene-regulated pyruvate dehydrogenase activity. Significance: This approach might enable a broader quantitative assessment of metabolic activity in tumors. 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 C-13 and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized C-13 spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined C-13 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-C-13]pyruvate for several hours, then briefly exposed to hyperpolarized [1-C-13]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[C-13]O-3(-) and [1-C-13]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-C-13]pyruvate. Quantitation of hyperpolarized H[C-13]O-3(-) 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[C-13]O-3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-C-13]pyruvate metabolism, enhancing exchanges with [1-C-13]lactate and suppressing H[C-13]O-3(-) 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 C-13 isotopomer analyses and dynamic hyperpolarized C-13 spectroscopy may enable quantitative flux measurements in living tumors.