Glutamate is Preferred over Glutamine for Intermediary Metabolism in Cultured Cerebellar Neurons

Abstract

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-13 C]glutamate or [U-13 C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using highperformance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13 C]glutamate and [U-13 C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13 C]glutamate was higher than that from [U-13 C]glutamine in all metabolites measured. The presence of [U-13 C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13 C]glutamate alone. However, incubation in medium containing [U-13 C]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-13 C]glutamine than from [U-13 C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the c-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.

The Transcriptome and Metabolic Gene Signature of Protoplasmic Astrocytes in the Adult Murine Cortex

“…In contrast, many pathways entering or exiting glycolysis or the TCA cycle, such as those for lactate and glutamate metabolism, were clearly compartmentalized, with some reactions catalyzed by neurons and others catalyzed by astrocytes. These expression-based observations were confirmed by direct metabolic measurements, by exposing acutely isolated GLT1 ϩ astrocytes to [U-13 C]glucose and analyzing the labeled intermediates by mass spectrometry (MS) (Bak et al, 2006;McKenna et al, 2006;Olstad et al, 2007). TCA cycle intermediates and derivates, such as citrate, malate, and glutamate, contained 13 C label from [U-13 C]glucose, confirming that cortical astrocytes have a robust capacity for oxidative metabolism.…”

Limbic Structures Show Altered Glial–Neuronal Metabolism in the Chronic Phase of Kainate Induced Epilepsy

“…3A). A coupling of aspartate and glutamate metabolism has been demonstrated in previous studies [36].…”

Metabolic Remodeling During H9c2 Myoblast Differentiation: Relevance for In Vitro Toxicity Studies

“…This is consistent with a more generalized exchange between lactate and pyruvate/alanine pools in undifferentiated cells than in differentiated ones. This compartmentalized model of cell metabolism has been discussed thoroughly in literature both in cell cultures [33,34] and in isolated perfused organs [35]. The pool of oxidizable pyruvate is more accurately defined by the pool of alanine, and an increase in alanine fractional enrichment in differentiated cells denotes a targeting of [U-13 C]glucose for oxidative metabolism in detriment of lactate production.…”