Pyruvate carboxylation was studied in cerebellar astrocytes and granule neurons. The cells were incubated in medium containing [U-(13)C]glucose (2.5 mM) and [U-(13)C]lactate (1 mM) and varying amounts of 3-nitropropionic acid (3-NPA) plus/minus aspartate. 3-NPA alone clearly stopped tricarboxylic acid (TCA) cycle activity at the succinate dehydrogenase step in both culture types as evidenced by a buildup of succinate. Labeling of aspartate and glutamate was abolished in neurons in the presence of 3-NPA. In astrocytes, however, labeled glutamate and glutamine derived from pyruvate carboxylation was detected. Unchanged glucose and lactate metabolism in the absence of a functioning malate aspartate shuttle indicates the importance of the glycerol-3-phosphate shuttle in brain cells. To compensate for the loss of oxaloacetate in the presence of 3-NPA, unlabeled aspartate (0.25 mM) was added. In this case [1,2-(13)C] and [3,4-(13)C]aspartate were observed in neurons but not in astrocytes. This labeling pattern in aspartate occurs after a full turn of the TCA cycle and thus indicates only partial inhibition by 3-NPA in the neurons when aspartate is present. In astrocytes, however, aspartate derived from uniformly labeled pyruvate was observed clearly indicating pyruvate carboxylation. The present study has unequivocally demonstrated a quantitatively important pyruvate carboxylation in astrocytes but it was not possible to demonstrate the presence of such carboxylation in neurons. Based on the present results it may be safely concluded that neuronal pyruvate carboxylation is unlikely to be of quantitative significance.
Copyright 2001 Wiley-Liss, Inc.