Metabolism of [U^‐13C]Glutamate in Astrocytes Studied by ¹³C NMR Spectroscopy: Incorporation of More Label into Lactate than into Glutamine Demonstrates the Importance of the Tricarboxylic Acid Cycle

Abstract

Primary cultures of cerebral cortical astrocytes were incubated with [U-13C]glutamate (0.5 mM) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify 13C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled 13C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to 13C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [13C]glutamate in the PCA extracts showed that 19% of the glutamate contained 12C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of 12C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

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

“…The presence of isotopomers from subsequent turns also showed that the carbon skeleton stayed in the TCA cycle. This confirms that the glutamate-glutamine cycle does not operate in a stochiometric fashion (McKenna et al, 1993(McKenna et al, , 1994Sonnewald et al, 1993a) and that glutamine is readily oxidized by cerebellar cultures for energy.…”

Generalized Sensory Stimulation of Conscious Rats Increases Labeling of Oxidative Pathways of Glucose Metabolism When the Brain Glucose–Oxygen Uptake Ratio Rises

“…These data suggest either two segregated pyruvate pools or extremely slow pyruvate transamination relative to its conversion to lactate. Synthesis of pyruvate from lower specific activity (Table 4) TCA cycle derivatives (Cerdan et al, 1990;Hassel and Sonnewald, 1995;Sonnewald et al, 1993Sonnewald et al, , 1996 might supply an alanine precursor pool, but metabolism of neurotransmitter-derived glutamate is a less-likely source because labeled glutamate is not incorporated into alanine in cultured astrocytes (McKenna et al, 1996). Thus, use of lactate-or alanine-specific activity (which differs by almost twofold; Tables 3 and 4) to estimate the specific activity of pyruvate for calculation of TCA cycle fluxes might not be appropriate, depending on which pool reflects the true pyruvate precursor pool(s) for the TCA cycle(s) of interest.…”

The Glutamate Transporter GLT1a Is Expressed in Excitatory Axon Terminals of Mature Hippocampal Neurons

“…However, more substantive explanations may also pertain, including the expression of other variant forms of GLT1 not yet recognized, or other transporters, such as EAAC1 (He et al, 2000), post-translational modification, or interacting proteins interfering with antibody binding. In addition, some axon terminals may use alternate pathways to resupply excitatory terminals with glutamate (Fonnum, 1984;Shank et al, 1989;Lehmann et al, 1993;Sonnewald et al, 1993;Westergaard et al, 1995;Hertz et al, 1999;Hassel and Brathe, 2000a,b).…”