Waagepetersen H S, Qu H, Schousboe A, Sonnewald U
Department of Pharmacology, Neuroscience PharmaBiotec Research Center, Royal Danish School of Pharmacy, 2 Universitetsparken, DK-2100 Copenhagen, Denmark.
J Neurosci Res. 2001 Dec 1;66(5):763-70. doi: 10.1002/jnr.10061.
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.
在小脑星形胶质细胞和颗粒神经元中研究了丙酮酸羧化作用。将细胞置于含有[U-(13)C]葡萄糖（2.5 mM）、[U-(13)C]乳酸（1 mM）以及不同量3-硝基丙酸（3-NPA）并添加/不添加天冬氨酸的培养基中培养。单独使用3-NPA时，两种培养类型的三羧酸（TCA）循环活性在琥珀酸脱氢酶步骤均明显停止，这可通过琥珀酸的积累得到证明。在3-NPA存在的情况下，神经元中天冬氨酸和谷氨酸的标记被消除。然而，在星形胶质细胞中，检测到了源自丙酮酸羧化作用的标记谷氨酸和谷氨酰胺。在缺乏正常运转的苹果酸-天冬氨酸穿梭的情况下，葡萄糖和乳酸代谢未发生变化，这表明了3-磷酸甘油穿梭在脑细胞中的重要性。为了补偿在3-NPA存在时草酰乙酸的损失，添加了未标记的天冬氨酸（0.25 mM）。在这种情况下，在神经元中观察到了[1,2-(13)C]天冬氨酸和[3,4-(13)C]天冬氨酸，但在星形胶质细胞中未观察到。天冬氨酸中的这种标记模式发生在TCA循环完整周转之后，因此表明当存在天冬氨酸时，3-NPA在神经元中仅产生部分抑制作用。然而，在星形胶质细胞中，明显观察到了源自均匀标记丙酮酸的天冬氨酸，这清楚地表明了丙酮酸羧化作用。本研究明确证明了星形胶质细胞中丙酮酸羧化作用在数量上具有重要意义，但无法证明神经元中存在这种羧化作用。基于目前的结果，可以有把握地得出结论，神经元丙酮酸羧化作用在数量上不太可能具有重要意义。
