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

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-(13)C]glucose (2.5 mM) and lactate (1 mM) or [U-(13)C]lactate (1 mM) and glucose (2.5 mM). Other sets of cultures were incubated in medium containing [U-(13)C]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. (13)C 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-(13)C]glucose was the substrate. Labeling of glutamate, aspartate and glutamine was lower in the cells incubated with [U-(13)C]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-(13)C]glucose and [U-(13)C]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-(13)C]glucose. These results show that metabolism of pyruvate, the precursor for lactate, alanine and acetyl-CoA is highly compartmentalized.

Metabolism of [U-13C]Glutamine and [U-13C]Glutamate in Isolated Rat Brain Mitochondria Suggests Functional Phosphate-Activated Glutaminase Activity in Matrix

“…Note that triple labeled aspartate (not shown in Fig. 1) may only be formed if labeled malate leaves the TCA cycle and re-enters as labeled acetyl-CoA in condensation with labeled oxaloacetate, a process known as pyruvate recycling [19].…”

Effects of Adrenergic Agents on Intracellular Ca2+ Homeostasis and Metabolism of Glucose in Astrocytes with an Emphasis on Pyruvate Carboxylation, Oxidative Decarboxylation and Recycling: Implications for Glutamate Neurotransmission and Excitotoxicity

“…The present study allows such comparison to be performed since the formation of the different isotopomers of the analyzed metabolites depends on the activity of these two metabolic pathways of pyruvate (Bak et al 2007;Dadsetan et al 2011;Waagepetersen et al 2001a). The finding that malate and aspartate both of which originate from OAA had considerably more relative prevalence of the M ?…”

“…2 alanine appears enigmatic as only the M ? 2 isotopomer can be formed from pyruvate originating from malate generated from both backflux of OAA produced by pyruvate carboxylation and the forward reactions of the TCA cycle (Bak et al 2007;Dadsetan et al 2011;Waagepetersen et al 2001a). The M ?…”

“…The M ? 1 isotopomer, on the other hand, can occur only as a product of OAA produced by the forward reactions of the TCA cycle which allow scrambling due to the symmetrical succinate molecule which subsequently is converted to malate and pyruvate (Bak et al 2007;Dadsetan et al 2011). Since succinate M ?…”

Metabolomics and fluxomics in biotechnology: current trends

“…C tracer is introduced into cell cultures, and later, the biological material is gathered and analyzed in the contents of the set of "isotopically enriched 13 C" intracellular or extracellular metabolites (Burgess et al, 2004;Bak et al, 2007;Yoo et al, 2008;Toya et al, 2010;Jain et al, 2012). The next necessary step is to determine the labeled metabolites within their topoisomers and calculate their levels.…”