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

Nuclear magnetic resonance (NMR) spectroscopy and biochemical assays were used to study the fate of [3-(13)C]alanine in astrocytes, neurons, and cocultures. (1)H- and (13)C-NMR analysis of the media demonstrated a high and comparable uptake of [3-(13)C]alanine by the cells. Thereafter, alanine is transaminated predominantly to [3-(13)C]pyruvate, from which the (13)C-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 gamma-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-(13)C]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-(13)C]-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.