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.

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

“…Further additional mechanisms contribute to the maintenance of the glutamine-glutamate cycle and the intercellular transfer of ammonia, such as a complementary alanine-lactate-shuttle between neurons and astrocytes (18,27). The present data extend these studies and confirm that ammonia can be indeed detoxified by neuronal synthesis of amino acids including alanine.…”

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

“…Further additional mechanisms contribute to the maintenance of the glutamine-glutamate cycle and the intercellular transfer of ammonia, such as a complementary alanine-lactate-shuttle between neurons and astrocytes (18,27). The present data extend these studies and confirm that ammonia can be indeed detoxified by neuronal synthesis of amino acids including alanine.…”

Alanine metabolism, transport, and cycling in the brain

“…In addition, labeling of Ala C3 correlated strongly with that of lactate C3 and Asp C3, but not Asp C2, supporting previous evidence for an alanine/aspartate metabolon in glial cells (Griffin et al. 1998, 2003) and suggesting that deamination of alanine in the astrocyte largely produces lactate (Zwingmann et al. 2000, 2001).…”

Exercise Alleviates Behavioral Disorders but Shapes Brain Metabolism of APP/PS1 Mice in a Region- and Exercise-Specific Manner

“…These findings imply that both HIIT and MICT corrected the imbalance between excitatory and inhibitory neurotransmitters, one of the possible pathogeneses of AD . It has been proved that the Gln/Glu-GABA cycle cannot proceed without return of ammonia to gliocytes and alanine plays an essential role in ammonia transfer between the astrocyte and neuron. − Herein, our results revealed that the level of alanine was reduced in the brain of AD mice especially in the hypothalamus but increased after both HIIT and MICT. Therefore, we speculate that exercise can maintain a normal Gln/Glu-GABA cycle and alleviate AD-related behavioral disorders.…”

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

“…Further additional mechanisms contribute to the maintenance of the glutamine-glutamate cycle and the intercellular transfer of ammonia, such as a complementary alanine-lactate-shuttle between neurons and astrocytes (18,27). The present data extend these studies and confirm that ammonia can be indeed detoxified by neuronal synthesis of amino acids including alanine.…”

Alanine metabolism, transport, and cycling in the brain

“…In addition, labeling of Ala C3 correlated strongly with that of lactate C3 and Asp C3, but not Asp C2, supporting previous evidence for an alanine/aspartate metabolon in glial cells (Griffin et al. 1998, 2003) and suggesting that deamination of alanine in the astrocyte largely produces lactate (Zwingmann et al. 2000, 2001).…”

Exercise Alleviates Behavioral Disorders but Shapes Brain Metabolism of APP/PS1 Mice in a Region- and Exercise-Specific Manner

“…These findings imply that both HIIT and MICT corrected the imbalance between excitatory and inhibitory neurotransmitters, one of the possible pathogeneses of AD . It has been proved that the Gln/Glu-GABA cycle cannot proceed without return of ammonia to gliocytes and alanine plays an essential role in ammonia transfer between the astrocyte and neuron. − Herein, our results revealed that the level of alanine was reduced in the brain of AD mice especially in the hypothalamus but increased after both HIIT and MICT. Therefore, we speculate that exercise can maintain a normal Gln/Glu-GABA cycle and alleviate AD-related behavioral disorders.…”

Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

“…Further additional mechanisms contribute to the maintenance of the glutamine-glutamate cycle and the intercellular transfer of ammonia, such as a complementary alanine-lactate-shuttle between neurons and astrocytes (18,27). The present data extend these studies and confirm that ammonia can be indeed detoxified by neuronal synthesis of amino acids including alanine.…”

Alanine metabolism, transport, and cycling in the brain

“…In addition, labeling of Ala C3 correlated strongly with that of lactate C3 and Asp C3, but not Asp C2, supporting previous evidence for an alanine/aspartate metabolon in glial cells (Griffin et al. 1998, 2003) and suggesting that deamination of alanine in the astrocyte largely produces lactate (Zwingmann et al. 2000, 2001).…”

Exercise Alleviates Behavioral Disorders but Shapes Brain Metabolism of APP/PS1 Mice in a Region- and Exercise-Specific Manner

“…These findings imply that both HIIT and MICT corrected the imbalance between excitatory and inhibitory neurotransmitters, one of the possible pathogeneses of AD . It has been proved that the Gln/Glu-GABA cycle cannot proceed without return of ammonia to gliocytes and alanine plays an essential role in ammonia transfer between the astrocyte and neuron. − Herein, our results revealed that the level of alanine was reduced in the brain of AD mice especially in the hypothalamus but increased after both HIIT and MICT. Therefore, we speculate that exercise can maintain a normal Gln/Glu-GABA cycle and alleviate AD-related behavioral disorders.…”