TMET-61. Glutamate-induced metabolic reprogramming in human glioblastoma cells

Glioma cells form glutamatergic synapses with neurons, and glutamate release at these neuron–glioma junctions is known to promote tumor proliferation via autocrine and paracrine signaling. Glioblastomas are also surrounded by elevated extracellular glutamate due to reduced uptake and active release by tumor cells. However, the direct impact of this excess glutamate on glioma cellular metabolism remains unclear. We employed LC-MS-based metabolomic profiling and tandem mass tag (TMT) proteomic analysis in LN229 human glioblastoma cells to investigate the metabolic effects of glutamate exposure. Exogenous glutamate significantly increased proliferation of LN229 and other patient-derived glioma cell lines in a dose-dependent manner. Metabolomic analysis revealed that glutamate supplementation increased tricarboxylic acid (TCA) cycle intermediates and decreased pentose phosphate pathway metabolites, indicating a rerouting of carbon flux toward energy production and biosynthesis. Isotope tracing with 13C5- and 15N-labeled glutamate confirmed direct uptake of glutamate and its incorporation into TCA cycle intermediates, γ-aminobutyric acid (GABA), and proline. Proteomic profiling showed upregulation of cell cycle regulators and increased thermal stability of select mitochondrial proteins (e.g., COX5A, TIMM8A) in glutamate-treated cells. These findings reveal a previously unrecognized role for extracellular glutamate as a direct metabolic substrate fueling glioma growth, independent of classical glutamate receptor-mediated excitatory signaling. Clinically, this metabolic reliance on glutamate highlights a potential vulnerability that could be targeted to impede glioblastoma progression.