GLS2 reshapes glycolytic metabolism and bioenergetic homeostasis in glioblastoma cells

BACKGROUND: Glioblastoma (GBM) exhibits extensive metabolic reprogramming, including glucose and glutamine (Gln) addiction, with Gln being converted into glutamate by glutaminase, encoded by the GLS and GLS2 genes. GLS2 acts as a context-dependent tumor suppressor, although the underlying mechanisms remain poorly understood. GLS2 is silenced in GBM, and we previously showed that its re-expression modulates pyruvate dehydrogenase (PDH) activity and tricarboxylic acid cycle (TCAC) metabolism, suggesting a distinct metabolic rewiring associated with its tumor-suppressive function. METHODS: LN229 and T98G GBM cell lines were used as experimental models. Metabolomic profiling by liquid chromatography-mass spectrometry was performed to compare changes in central metabolic pathways following GLS2 or GLS overexpression. Glutaminase and PDH enzymatic activities were measured, and Western blotting was performed to assess GLS, GLS2, and PDHA1 protein levels and PDHA1 phosphorylation. A Seahorse analyzer was employed to quantify real-time ATP production derived from glycolysis and oxidative phosphorylation. RESULTS: Overexpression of GLS2, but not GLS, in LN229 cells caused the accumulation of TCAC-related metabolites, except for citrate, whose depletion is attributed to GLS2's effect on PDH phosphorylation, causing reduced PDH activity. This was accompanied by decreased levels of several acyl-carnitines. Furthermore, GLS2 induced the accumulation of glucose/fructose, while reducing levels of glucose-6-phosphate and pentose-phosphates, consistent with altered glycolysis. GLS2 also diminished the ATP/AMP ratio, confirmed by a reduced ATP synthesis rate, particularly glycolytic ATP, whereas mitochondrial ATP production remained unaltered. CONCLUSIONS: GLS2 exerts an isoenzyme-specific effect on Gln and glucose metabolism in GBM, likely modulating the glycolysis/pentose-phosphate pathway (PPP)-TCAC-axis.