Michel Merle

Glucose and glutamine metabolism in C6 glioma cells studied by carbon 13 NMR

The question as to whether glutamine and glucose are both required for optimal growth of glioma cells is studied through the role of these substrates on the metabolism of the cells. C6 rat glioma cells grow only very slowly when glutamine is omitted from the culture medium. The rates of glucose consumption and lactate production on confluent cells in glutamine-free medium were 0.88 +/- 0.09 and 1.06 +/- 0.25 mumol/h/mg protein, respectively. In the presence of 4 mM glutamine, glucose utilization increase to 60% leading to a 45% increase of lactate production. We have studied the kinetics of enrichment of intracellular glutamate at C2, C3 and C4 positions on cells incubated with 5 mM 99% enriched [1-(13)C]glucose in the presence or the absence of glutamine in the incubation medium. The specific enrichments at metabolic steady state of all carbon positions were the same under both conditions, but we observed a significantly reduced rate of 13C incorporation in the presence of glutamine, showing an isotopic dilution of tricarboxylic acid cycle intermediates and indicating the use of this amino acid as an anaplerotic substrate. The fact that no dilution occurred at the level of pyruvate suggests strongly the lack of glutaminolysis in these cells. The main conclusion from this work is that glutamine metabolism in C6 cells appears complementary to that of glucose as far as energy production and carbon sources for the growing of the cells are concerned: glutamine is mainly utilized for anaplerosis as carbon donor to replenish the tricarboxylic acid cycle; it is not a substrate for energy metabolism. In contrast, glucose is poorly anaplerotic and is essentially used as energetic fuel by the C6 cells.

Microglia in Close Vicinity of Glioma Cells: Correlation Between Phenotype and Metabolic Alterations

Microglia are immune cells within the central nervous system. In brain-developing tumors, gliomas are able to silence the defense and immune functions of microglia, a phenomenon which strongly contributes to tumor progression and treatment resistance. Being activated and highly motile, microglia infiltrate tumors and secrete macrophagic chemoattractant factors. Thereafter, the tumor cells shut down their immune properties and stimulate the microglia to release tumor growth-promoting factors. The result of such modulation is that a kind of symbiosis occurs between microglia and tumor cells, in favor of tumor growth. However, little is known about microglial phenotype and metabolic modifications in a tumoral environment. Co-cultures were performed using CHME5 microglia cells grown on collagen beads or on coverslips and placed on monolayer of C6 cells, limiting cell/cell contacts. Phagocytic behavior and expression of macrophagic and cytoskeleton markers were monitored. Respiratory properties and energetic metabolism were also studied with regard to the activated phenotype of microglia. In co-cultures, transitory modifications of microglial morphology and metabolism were observed linked to a concomitant transitory increase of phagocytic properties. Therefore, after 1u2009h of co-culture, microglia were activated but when longer in contact with tumor cells, phagocytic properties appear silenced. Like the behavior of the phenotype, microglial respiration showed a transitory readjustment although the mitochondria maintained their perinuclear relocation. Nevertheless, the energetic metabolism of the microglia was altered, suggesting a new energetic steady state. The results clearly indicate that like the depressed immune properties, the macrophagic and metabolic status of the microglia is quickly driven by the glioma environment, despite short initial phagocytic activation. Such findings question the possible contribution of diffusible tumor factors to the microglial metabolism.

[1-13C]Glucose Metabolism in Brain Cells: Isotopomer Analysis of Glutamine from Cerebellar Astrocytes and Glutamate from Granule Cells

We analyzed the glutamine isotopomers released into the extracellular medium by cerebellar astrocytes incubated with [1-13C]glucose. We developed a mathematical model of the tricarboxylic acid (TCA) cycle to determine the relative flux of molecules through the anaplerotic versus oxidative pathways and the relative pyruvate carboxylase versus pyruvate dehydrogenase activities. As glutamine C2 and C3 exhibited unequivalent enrichments, we examined the possibility of: (1) both the entry of the label into the TCA cycle from pyruvate and the conversion of the oxaloacetate into citrate before equilibration with fumarate, and (2) the occurrence of an orientation-conserved transfer of the symmetrical 4-carbon intermediates. The best fit required partial cycling between oxaloacetate and fumarate, whereas the occurrence of any orientation-conserved transfer was rejected. On the other hand, the analysis of glutamate isotopomers from perchloric acid extracts of granule cells incubated with [1-13C]glucose indicated that total cycling of oxaloacetate into fumarate occurred in these cells.

Glutathione, but not glutamine, is detected in 13C-NMR spectra of perchloric acid extracts from C6 glioma cells

Glutamine, which is expected to be produced by C6 glioma cells, is not detected in both amino‐acid analyses and 13C‐NMR spectra of perchloric acid extracts of cells incubated for 4 h with [1‐13C]glucose in the absence of extracellular glutamine. However, the resonances of a glutamate‐linked product are observed in these spectra. The analysis of the pH dependence of chemical shifts from various glutamate‐derived compounds shows that the observed resonances came from glutathione. Glutamine and glutathione signals are in close proximity on the frequency scale, leading to possible misinterpretation of the spectra.

Comparative 31P and 1H NMR studies on rat astrocytes and C6 glioma cells in culture

Rat astroglial cells in primary culture (95% enrichment) and C6 glioma cells were adapted to grow on microcarrier beads. In vivo 31P NMR spectra were collected from cell-covered beads perfused in the NMR tube. The NMR-visible phosphorylated metabolite contents of both cell types were determined using saturation factors calculated from the values of longitudinal relaxation times determined for C6 cells using progressive saturation experiments. On the other hand, the amounts of phosphorylated metabolites in cells were determined from proton decoupled 31P NMR spectra of cell perchloric acid extracts. The results indicate that the NTP and Pi contents of the normal and tumoral cells were similar, whereas the PCr level was higher in C6 cells and the NDP and phosphomonoester levels higher in astrocytes. The comparison of 1H NMR spectra of cell perchloric acid extracts evidenced larger inositol and alanine contents in C6 cells, whereas larger taurine and choline (and choline derivatives) contents were found in astrocytes. The Glu/Gln ratio was very different, 3.5 and 1 in C6 cells and astrocytes, respectively. In both cases, the more intense resonance in the 1H NMR spectrum was assigned to glycine. Based on the comparison of the metabolite content of a tumoral and a normal cell of glial origin, this work emphasizes the usefulness of a multinuclear NMR study in characterizing intrinsic differences between normal and tumoral cells.

Calculation of Metabolic Fluxes by Mathematical Modeling of Carbon-13 Distribution in Metabolites. II. Application to the Study of C6 Cell Metabolism

The metabolism of the rat C6 cell line has been extensively studied1–4, because these cells constitute a model of malignant glioma. As neoplastic cells preferentially utilize the relatively inefficient Embden-Meyerhof pathway for satisfying their energy requirements, the carbohydrate metabolism, up to now, was in the center of interest. Besides glycolysis, C6 cells have been shown to possess non-negligible activities of the hexose monophosphate shunt (HMPS), and glycogen synthesis.