Abstracts

Rationale: Many gliomas are known to be epileptogenic; however, the underlying mechanism(s) are not well understood. The glutamatergic hypothesis attributes excitotoxicity and seizures to high extracellular glutamate levels. Glioma cell lines do not express the normal glial glutamate transporters GLT-1 and GLAST (EAA1 and 2) and therefore fail to effectively clear glutamate from the extracellular fluid. Moreover, astrocytoma cells extrude glutamate through the system X/c- co-transporter (glutamate-cystine exchange). The poorly developed blood brain barrier characteristic of neovascularization facilitates the influx of glutamate from the blood. Thus, the source of the high extracellular glutamate is unknown. Methods: We studied neuronal-glial metabolism in brain slices prepared from biopsies of primary human gliomas; comparison material was obtained from the temporal neocortex overlying epileptogenic hippocampi. Using the brain slice preparation avoided the complications of systemic metabolism and the variable functionality of the blood-brain barrier inherent with tumor tissue, thus providing more uniform conditions. The slices were incubated with 10 mM 13C-glucose or 2 mM 13C-acetate and unlabeled glucose; after two hours the tissue and incubation fluid were frozen, extracted and the isotopic enrichments (IE) of glutamate (13C-glu) and glutamine (13C-gln) measured using tandem mass spectrometry. All patients provided informed consent; the studies were approved by the Yale HIC. Results: Glucose oxidation by tumor tissue was reduced to 42% of control values, whereas acetate oxidation was 75% of control. Acetate, which is primarily metabolized by glia, comprised 22% of label utilization by the tricarboxylic acid (TCA) cycle in control slices, which increased to 33% in tumor tissue. However, glutamine synthesis was reduced by 38% in tumor tissue and the release of freshly synthesized glutamine into the extracellular fluid was reduced by ~50% in tumor tissue, when 13C-acetate was used as a label source. In addition, the tumor slices released a greater proportion of labeled glutamate into the extracellular fluid than control tissues, independent of label source. Notably, we found that there was a significant negative relationship between the Ki67 percentage, a marker of tumor growth, and the isotopic enrichment of glutamine. Conclusions: Our data demonstrate that there is a significant alteration in glutamate and glutamine metabolism in epileptogenic tumors and that this altered glutamine metabolism is significantly correlated with tumor grade. Mitochondrial glucose oxidation is reduced (the Warburg effect). Our data indicate that there is a preferential release of mitochondrially derived glutamate into the bath along with a significant decrease in labeled glutamine, mediated by reduced GS activity. We hypothesize that these epileptogenic tumors are able to generate TCA cycle intermediates thus allowing them to siphon off TCA cycle-generated glutamate to support System Xc- glutamate efflux without inducing metabolic failure. Targeting this anepleurotic pathway may be a promising therapeutic avenue.