Neurochemical Research

, Volume 32, Issue 4, pp 671–680

Complex Glutamate Labeling from [U-13C]glucose or [U-13C]lactate in Co-cultures of Cerebellar Neurons and Astrocytes

  • Lasse K. Bak
  • Helle S. Waagepetersen
  • Torun M. Melø
  • Arne Schousboe
  • Ursula Sonnewald
ORIGINAL PAPER

DOI: 10.1007/s11064-006-9161-4

Cite this article as:
Bak, L.K., Waagepetersen, H.S., Melø, T.M. et al. Neurochem Res (2007) 32: 671. doi:10.1007/s11064-006-9161-4

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.

Keywords

Pyruvate recyclingMass spectrometryNuclear magnetic resonance spectroscopyCo-cultureNeuronAstrocyte

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

Copyright information

© Springer Science+Business Media, LLC 2006

Authors and Affiliations

  • Lasse K. Bak
    • 1
  • Helle S. Waagepetersen
    • 1
  • Torun M. Melø
    • 2
  • Arne Schousboe
    • 1
  • Ursula Sonnewald
    • 2
  1. 1.Department of Pharmacology and PharmacotherapyDanish University of Pharmaceutical SciencesCopenhagenDenmark
  2. 2.Department of NeuroscienceNorwegian University of Science and TechnologyTrondheimNorway