Glutamate is Preferred over Glutamine for Intermediary Metabolism in Cultured Cerebellar Neurons
Glutamate is Preferred over Glutamine for Intermediary Metabolism in Cultured Cerebellar Neurons
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DOI：
10.1038/sj.jcbfm.9600400
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发表时间：
2007-04
影响因子：
6.3
通讯作者：
E. Olstad;H. Qu;U. Sonnewald
中科院分区：
文献类型：
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作者：
E. Olstad;H. Qu;U. Sonnewald
谷氨酸-谷氨酰胺循环被认为对于成熟的大脑至关重要。然而，其重要性可能会随着星形胶质细胞和突触之间距离的区域差异而变化。本研究旨在评估该循环在小脑神经元（主要由谷氨酸能颗粒细胞组成）培养物中的作用。将细胞在含有[U-13C]谷氨酸或[U-13C]谷氨酰胺的培养基中分别在存在和不存在未标记的谷氨酰胺和谷氨酸的情况下孵育。使用高效液相色谱 (HPLC) 和气相色谱结合质谱 (GC/MS) 分析细胞提取物和培养基。 [U-13C]谷氨酸和[U-13C]谷氨酰胺均被证明是合成神经活性氨基酸和三羧酸（TCA）循环中间体的优异前体。在所有测量的代谢物中，[U-13C]谷氨酸的标记高于[U-13C]谷氨酰胺的标记。实验培养基中存在[U-13C]谷氨酸和未标记的谷氨酰胺导致标记与单独的[U-13C]谷氨酸非常相似。然而，在未标记谷氨酸存在的情况下，在含有[U-13C]谷氨酰胺的培养基中孵育几乎消除了代谢物的标记。因此，可以证明谷氨酸是小脑神经元中间代谢的首选底物。指示 TCA 循环活性的标签分布显示，[U-13C]谷氨酰胺的循环比 [U-13C]谷氨酸的循环更显着。琥珀酸的标记低于其他 TCA 循环中间体，表明 γ-氨基丁酸分流在这些培养物中的积极作用。可以得出结论，小脑神经元更多地依赖于谷氨酸的再摄取，而不是星形胶质细胞提供的谷氨酰胺来维持谷氨酸稳态。
The glutamate—glutamine cycle is thought to be of paramount importance in the mature brain; however, its significance is likely to vary with regional differences in distance between astrocyte and synapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellar neurons, mainly consisting of glutamatergic granule cells. Cells were incubated in medium containing [U-13C]glutamate or [U-13C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13C]glutamate and [U-13C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13C]glutamate was higher than that from [U-13C]glutamine in all metabolites measured. The presence of [U-13C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13C]glutamate alone. However, incubation in medium containing [U-13C]glutamine in the presence of unlabeled glutamate almost abolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substrate for intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activity showed more prominent cycling from [U-13C]glutamine than from [U-13C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the γ-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons rely more on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.