Abstract
Glutamate metabolism was studied in co-cultures of mouse cerebellar neurons (predominantly glutamatergic) and astrocytes. One set of cultures was superfused (90 min) in the presence of either [U-13C]glucose (2.5 mM) and lactate (1 mM) or [U-13C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-13C]lactate (1 mM) and glucose (2.5 mM) for 4 h. Regardless of the experimental conditions cell extracts were analyzed using mass spectrometry and nuclear magnetic resonance spectroscopy. 13C labeling of glutamate was much higher than that of glutamine under all experimental conditions indicating that acetyl-CoA from both lactate and glucose was preferentially metabolized in the neurons. Aspartate labeling was similar to that of glutamate, especially when [U-13C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-13C]lactate. The first part of the pyruvate recycling pathway, pyruvate formation, was detected in singlet and doublet labeling of alanine under all experimental conditions. However, full recycling, detectable in singlet labeling of glutamate in the C-4 position was only quantifiable in the superfused cells both from [U-13C]glucose and [U-13C]lactate. Lactate and alanine were mostly uniformly labeled and labeling of alanine was the same regardless of the labeled substrate present and higher than that of lactate when superfused in the presence of [U-13C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.
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Abbreviations
- GABA:
-
γ-Aminobutyric acid
- GC-MS:
-
Gas chromatography-mass spectrometry
- Hepes:
-
N-2-Hydroxyethyl-piperazine-N′-2-ethanesulfonic acid
- HBS:
-
Hepes-buffered saline
- LC-MS:
-
Liquid chromatography-mass spectrometry
- ME:
-
Malic enzyme
- MRS:
-
Nuclear magnetic resonance spectroscopy
- TCA:
-
Tricarboxylic acid
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Acknowledgments
The expert technical assistance by Mrs. Kirsten Thuesen (Danish University of Pharmaceutical Sciences, Copenhagen, Denmark) as well as Dr. Turid Nilsen and Mr. Lars Evje, M.Sc. (both Norwegian University of Science and Technology), is hereby cordially acknowledged. The Danish Medical Research Council (22-03-0250; 22-4-0314), the Alfred Benzon, Hørslev, Lundbeck, Norwegian Epilepsy and Novo Nordisk Foundations are acknowledged for generous financial support.
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Special issue dedicated to John P. Blass.
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Bak, L.K., Waagepetersen, H.S., Melø, T.M. et al. Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes. Neurochem Res 32, 671–680 (2007). https://doi.org/10.1007/s11064-006-9161-4
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DOI: https://doi.org/10.1007/s11064-006-9161-4