TAMI-13. MODULATION OF REDUCTIVE CARBOXYLATION FLUX OF GLUTAMINE IN GLIOBLASTOMA CELLS BY USING OSCILLATING MAGNETIC FIELD

Increased cell proliferation in glioblastoma (GBM) leads to hypoxia in the tumor microenvironment. This is a major concern in GBM patients as it promotes tumor invasion. Glutaminolysis is a hallmark of cancer cells and under hypoxic conditions glutamine metabolism proceeds through reductive carboxylation pathway. Recently, we have shown that oscillating magnetic field (OMF) produces oncolytic effects which can influence cellular metabolism. Here, we have explored the effect of OMF on glutamine metabolism in GBM cells. Patient-derived GBM cells were grown in high glucose (25 mM) DMEM supplemented with 20% fetal bovine serum (FBS), 2.0 mM glutamine and 1.0 mM pyruvate at 37 °C under humidified air and 5% CO2. Cells were divided into 2 groups (Test and Sham; n = 4 each group). When reached confluency (~2.0×106 cells/mL), cells in both groups were treated with 4.0 mM of [U-13C]glutamine in DMEM (supplemented with 20% FBS, and 1.0 mM pyruvate). The “Test” group was subjected to OMF for 3 hours, and the “Sham” group was treated similar to the “Test” group but with non-magnetic rods of the same dimensions as the magnets in the Test group. After 3 h, cells were harvested in 50% methanol analyzed by GC-MS. The 13C-isotopomer analysis showed that glutamine metabolism in GBM cells proceeds through reduction carboxylation, confirmed by the higher levels of M+5 citrate (15.42 ± 1.28 % ) than M+4 citrate (2.05 ± 0.28 %). When GBM cells were treated with OMF, a statistically significant decrease in the citrate M+5 was observed, compared to the “Sham” treated group (15.42 ± 1.28 % vs. 8.89 ± 1.30 %; p = 0.0003). This decrease in M+5 citrate upon OMF treatment clearly indicates that the OMF decreases the reductive carboxylation flux of glutamine in GBM cells which would have therapeutic value in treating GBM patients.

CBMT-49. OXALOACETATE ALTERS GLUCOSE METABOLISM IN GLIOBLASTOMA: 13C ISOTOPOMER STUDY

Anhydrous enol-oxaloacetate (AEO) has demonstrated the ability to enhance neuronal cell bioenergetics and activate brain mitochondrial biogenesis. Since oxaloacetate has demonstrated positive effects on brain bioenergetics in neurodegenerative diseases we have begun to investigate whether AEO may also have a positive effect on the altered cellular metabolism found in cancer cells, particularly Glioblastoma multiforme. The “Warburg effect” describes an abnormal metabolic state in cancer, distinct from normal tissue, in which energy is generated through enhanced conversion of pyruvate to lactate even in the presence of oxygen during glycolysis. Oxaloacetate (OAA) is a key anaplerotic substrate that is required to maintain TCA cycle flux. The role of oxaloacetate supplementation on the energy metabolism is not known in cancer cells. Goal of this study is to investigate the changes in metabolic fluxes in glucose metabolism with and without the presence of OAA in patient-derived GBM cells. We use GC-MS based 13C isotopomer analysis for this study. GBM cells are grown in 15mM glucose containing DMEM medium supplemented with 2mM oxaloacetate for 10 days. 6 hours prior to harvesting, [U-13C]glucose is introduced to the medium. 13C isotopomer analysis of GC-MS data showed that OAA supplementation for 10 days drastically decreased Warburg glycolysis by reducing 13C labeling (M+3) by 19.7% and 48.8% in pyruvate and lactate pools respectively in comparison with cells not treated with OAA. M+3 13C labeled pyruvate entered TCA cycle via acetyl-CoA, where we also observed reduced levels of M+2 13C labeled citrate (20.5%) and glutamate (23.9%) isotopomers. Pyruvate can also enter TCA cycle via pyruvate carboxylation pathway and this activity was also found to be slightly decreased in the OAA treated cells. All the differences were statistically significant. These results indicate that OAA can be used to alter bioenergetics of GBM cells, specifically glucose oxidation.

Triidothyronine and epinephrine rapidly modify myocardial substrate selection: a 13C isotopomer analysis

Triiodothyronine (T3) exerts direct action on myocardial oxygen consumption (MV˙o 2), although its immediate effects on substrate metabolism have not been elucidated. The hypothesis, that T3 regulates substrate selection and flux, was tested in isovolumic rat hearts under four conditions: control, T3 (10 nM), epinephrine (Epi), and T3 and Epi (TE). Hearts were perfused with [1,3-13C]acetoacetic acid (AA, 0.17 mM),l-[3-13C]lactic acid (LAC, 1.2 mM), U-13C-labeled long-chain free fatty acids (FFA, 0.35 mM), and unlabeled d-glucose (5.5 mM) for 30 min. Fractional acetyl-CoA contribution to the tricarboxylic acid cycle (Fc) per substrate was determined using 13C NMR and isotopomer analysis. Oxidative fluxes were calculated using Fc, the respiratory quotient, and MV˙o 2. T3increased ( P < 0.05) FcFFA, decreased FcLAC, and increased absolute FFA oxidation from 0.58 ± 0.03 to 0.68 ± 0.03 μmol · min−1 · g dry wt−1( P < 0.05). Epi decreased FcFFA and FcAA, although FFA flux increased from 0.58 ± 0.03 to 0.75 ± 0.09 μmol · min−1 · g dry wt−1. T3 moderated the change in FcFFA induced by Epi. In summary, T3 exerts direct action on substrate pathways and enhances FFA selection and oxidation, although the Epi effect dominates at a high work state.