NMR spectroscopic study on the metabolic fate of [3-13C]alanine in astrocytes, neurons, and cocultures: Implications for glia-neuron interactions in neurotransmitter metabolism

Abstract

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3‐13C]alanine in astrocytes, neurons, and cocultures. 1H‐ and 13C‐NMR analysis of the media demonstrated a high and comparable uptake of [3‐13C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3‐13C]pyruvate, from which the 13C‐label undergoes different metabolic pathways in astrocytes and neurons: Lactate is almost exclusively synthesized in astrocytes, while in neurons and cocultures labeled neurotransmitter amino acids are formed, i.e., glutamate and γ‐aminobutyric acid (GABA). A considerable contribution of the anaplerotic pathway is observed in cocultures, as concluded from the ratio (C‐2–C‐3)/C‐4 of labeled glutamine. Analysis of the multiplet pattern of glutamate isotopomers indicates carbon scrambling through the TCA cycle and the use of alanine also as energy substrate in neurons. In cocultures, astrocyte‐deduced lactate and unlabeled exogenous carbon substrates contribute to glutamate synthesis and dilute the [2‐13C]acetyl‐CoA pool by 30%. The coupling of neuronal activity with shuttling of tricarboxylic acid (TCA) cycle‐derived metabolites between astrocytes and neurons is concluded from the use of [4‐13C]‐monolabeled glutamate leaving the first TCA cycle turn already for glutamine and GABA synthesis, as well as from the labeling pattern of extracellular glutamine. Further evidence of a metabolic interaction between astrocytes and neurons is obtained, as alanine serves as a carbon and nitrogen carrier through the synthesis and regulated release of lactate from astrocytes for use by neurons. Complementary to the glutamine‐glutamate cycle in the brain, a lactate‐alanine shuttle between astrocytes and neurons would account for the nitrogen exchange of the glutamatergic neurotransmitter cycle in mammalian brain. GLIA 32:286–303, 2000. © 2000 Wiley‐Liss, Inc.

Energy Metabolism in Astrocytes and Neurons Treated with Manganese: Relation among Cell-Specific Energy Failure, Glucose Metabolism, and Intercellular Trafficking Using Multinuclear NMR-Spectroscopic Analysis

“…Primary cell cultures of cortical astrocytes, cortical neurons, and cocultures of both cell types were obtained as described previously (Booher and Sensenbrenner, 1972;Zwingmann et al, 2000). Astrocytes were prepared from the brains of 1-or 2-day-old Sprague-Dawley rats and cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum for 3 weeks.…”

“…P-NMR spectra were analyzed with reference to the C-enrichments in individual carbons of amino acids are derived from 13 C-NMR spectra using the known 13 C-enrichment in lactate as described previously (Zwingmann et al, 2000):…”

Ex Vivo Analysis of Lactate and Glucose Metabolism in the Rat Brain under Different States of Depressed Activity

“…Indeed, these authors evidenced that the two pyruvate species, the glycolytic pyruvate and the pyruvate synthesized from exogenous lactate by lactate dehydrogenase, were compartmentalized and that the production of the first was inhibited when the second was used preferentially as an oxidative substrate. Moreover, the fact that alanine was synthesized in neurons suggested that it could be involved in the alanine-lactate shuttle between neurones and astrocytes (34,35), which might contribute to regenerating the cytoplasmic NAD ϩ in astrocytes through NADH oxidation by the respiratory chain in neurons.…”

The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells

“…Thus, cells may have increased their uptake of carbohydrates from the medium to account for higher energy metabolism. Neurons also have the ability to utilise alanine as an alternative energy source, which could explain the reduction in the level of alanine (Tables 3, 4) (Zwingmann et al 2000).…”