H., Brindle K. 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 usually 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. detection of cancer and for monitoring response to therapy (15, 21). However, malignancy cells also oxidize pyruvate in the mitochondria, generating both energy and macromolecular precursors for cell growth (23). This is of particular interest because lung tumors, gliomas, and metastatic brain tumors have all been demonstrated to oxidize pyruvate in humans and Rabbit Polyclonal to HBP1 mice (24,C28). Therefore, assessment of both pyruvate/lactate exchanges and pyruvate oxidation in the mitochondria would provide a much more comprehensive view of malignancy cell metabolism than Methoxamine HCl lactate formation Methoxamine HCl alone. We previously used standard 13C NMR spectroscopy to evaluate fluxes through competing metabolic pathways supplied by pyruvate, including LDH and the TCA cycle, in cultured malignancy cells (29, 30). These same activities were detected in mouse and human tumors by infusing 13C-enriched glucose before surgery, extracting metabolites from surgically resected tumor tissue, and analyzing 13C enrichment patterns by NMR (26, 28). We also used hyperpolarized [1-13C]pyruvate to quantify flux into lactate (31). Here, we combined these methods to study two metabolically unique malignancy cell lines. First, we incubated malignancy cells with thermally polarized [3-13C]pyruvate for several hours Methoxamine HCl to produce steady-state labeling of metabolic intermediates. Next, using a selective excitation pulse to maximize Methoxamine HCl detection of H[13C]O3? and [1-13C]lactate, we subjected cells to hyperpolarized [1-13C]pyruvate to measure flux into lactate and the TCA cycle. Combining the rate of pyruvate decarboxylation with steady-state isotopomer data provided a method to evaluate absolute flux rates through a variety of reactions associated with the TCA cycle. EXPERIMENTAL PROCEDURES Cell Culture Reagents and Basic Metabolism Experiments Two cell lines, SF188-derived glioblastoma cells overexpressing human Bcl-xL (SFxL) and Huh-7 hepatocellular carcinoma cells were maintained in culture as explained previously (30, 32, 33). Metabolic experiments were performed in Dulbecco’s altered Eagle’s medium (DMEM) prepared from powder lacking glucose, glutamine, phenol reddish, sodium pyruvate, and sodium bicarbonate. This basal medium was supplemented with 4 mmol/liter l-glutamine, 10% dialyzed fetal calf serum, 42.5 mmol/liter sodium bicarbonate, 25 mmol/liter HEPES, 10 units/ml penicillin, and 10 g/ml streptomycin. Glucose and pyruvate were added as indicated for each experiment. To measure the rates of metabolite consumption/excretion in the medium, glucose, lactate, glutamine, and glutamate were measured using a BioProfile Basic 4 analyzer (NOVA Biomedical), and ammonia was measured using a spectrophotometric assay (Megazyme). For oxygen consumption assays, cells were harvested by trypsinization, suspended in fresh medium at a concentration of 108 cells/ml, and transferred to an Oxygraph water-jacketed oxygen electrode (Hansatech). The Akt inhibitor was Akt Inhibitor VIII (Calbiochem). Pyruvate Decarboxylation Assay Decarboxylation of [1-14C]pyruvate was measured essentially as explained (34). Micro-bridges (Hampton Research) were placed into wells Methoxamine HCl of a 24-well plate with one piece of 0.6 1 cm2 chromatography paper in each. Assay medium was prepared by supplementing DMEM (made up of 10% fetal calf serum, 4 mm glutamine, and 6 mm sodium pyruvate) with 2.2 Ci of [1-14C]pyruvate. This medium was warmed to 37 C and incubated for 2 h to remove any 14CO2 produced from spontaneous decarboxylation, then an aliquot was.