METABOLISM OF [U-C-13(5)] GLUTAMINE IN CULTURED ASTROCYTES STUDIED BYNMR-SPECTROSCOPY - FIRST EVIDENCE OF ASTROCYTIC PYRUVATE RECYCLING

Metabolism of [U-C-13(5)]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellul ar glutamate. Perchloric acid extracts of the cells as well as redisso lved lyophilized media were subjected to nuclear magnetic resonance an d mass spectrometry to identify C-13-labeled metabolites, Label from g lutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observ ed in aspartate. It could be clearly demonstrated that some [(UC5)-C-1 3]glutamine is metabolized through the tricarboxylic acid cycle, altho ugh to a much smaller extent than previously shown for [U-C-13(5)]glut amate. Lactate formation from tricarboxylic acid cycle intermediates h as previously been demonstrated. It has, however, not been demonstrate d that pyruvate, formed from glutamate or glutamine, may reenter the t ricarboxylic acid cycle after conversion to acetyl-CoA. The present wo rk demonstrates that this pathway is active, because [4,5-C-13(5)]glut amate was observed in astrocytes incubated with [U-C-13(5)]glutamine i n the additional presence of unlabeled glutamate. Furthermore, using m ass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using (CO2)-C-13 produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.