Competition between glucose and lactate as oxidative energy substrates was investigated in both primary cultures of astrocytes and neurons using physiological concentrations (1.1 mm for each). Glucose metabolism was distinguished from lactate metabolism by using alternatively labelled substrates in the medium ([1-13C]glucose + lactate or glucose + [3-13C]lactate). After 4 h of incubation, 1H and 13C-NMR spectra were realized on perchloric acid extracts of both cells and culture media. For astrocytic cultures, spectra showed that amino acids (glutamine and alanine) were more labelled in the glucose-labelled condition, indicating that glucose is a better substrate to support oxidative metabolism in these cells. The opposite was observed on spectra from neuronal cultures, glutamate being much more labelled in the lactate-labelled condition, confirming that neurons consume lactate preferentially as an oxidative energy substrate. Analysis of glutamine and glutamate peaks (singlets or multiplets) also suggests that astrocytes have a less active oxidative metabolism than neurons. In contrast, they exhibit a stronger glycolytic metabolism than neurons as indicated by their high lactate production yield. Using a mathematical model, we have estimated the relative contribution of exogenous glucose and lactate to neuronal oxidative metabolism. Under the aforementioned conditions, it represents 25% for glucose and 75% for lactate. Altogether, these results obtained on separate astrocytic and neuronal cultures support the idea that lactate, predominantly produced by astrocytes, is used as a supplementary fuel by neurons in vivo already under resting physiological conditions.

The involvement of brain lactate in neuronal metabolism was analyzed by ex vivo NMR spectroscopy with rats under the effects of pentobarbital, chloralose or morphine, which were infused with a solution of either [1-13 C]glucose þ lactate or glucose þ [3-13 C]lactate for 20 min. Electroencephalogram recordings indicated different brain electrical activity levels under the three drugs with a clear distinction between pentobarbital, on the one hand, and chloralose and morphine on the other. Labeling of metabolites in brain perchloric acid extracts and of blood glucose and lactate was determined by 13 C-and/or 1 H-observed/ 13 C-edited-NMR spectroscopy. The following were found: (i) the ratio between glutamate C3 and C4 13 C-enrichments increased from pentobarbital to chloralose and morphine whatever the labeled precursor, indicating a link between metabolic and electrical activity; (ii) under glucose þ [3-13 C]lactate infusion, alanine C3 and acetyl-CoA C2 enrichments were higher than that of lactate C3, revealing the occurrence of an isotopic dilution of the brain exogenous lactate (arising from blood) by lactate from brain (endogenous lactate); the latter was synthesized from glycolysis in a compartment other than the neurons; (iii) the contributions of labeled glucose and lactate to acetyl-CoA C2 enrichment indicated that the involvement of blood glucose relative to that of blood lactate to brain metabolism was correlated with brain activity. It can therefore be concluded that the brain electrical activity-dependent increase in the contribution of blood glucose relative to that of blood lactate to brain metabolism occurred partly via the increase in the metabolism of lactate generated from astrocytic glycolysis. This conclusion supports the hypothesis of an astrocyte-neuron lactate shuttle component in the coupling mechanism between cerebral activity and energy metabolism.

Brain metabolism of glucose and lactate was analyzed by ex vivo NMR spectroscopy in rats presenting different cerebral activities induced after the administration of pentobarbital, ␣-chloralose, or morphine. The animals were infused with a solution of either [1-13 C]glucose plus lactate or glucose plus [3-13 C]lactate for 20 min. Brain metabolite contents and enrichments were determined from analyses of brain tissue perchloric acid extracts according to their post-mortem evolution kinetics. When amino acid enrichments were compared, both the brain metabolic activity and the contribution of blood glucose relative to that of blood lactate to brain metabolism were linked with cerebral activity. The data also indicated the production in the brain of lactate from glycolysis in a compartment other than the neurons, presumably the astrocytes, and its subsequent oxidative metabolism in neurons. Therefore, a brain electrical activity-dependent increase in the relative contribution of blood glucose to brain metabolism occurred via the increase in the metabolism of lactate generated from brain glycolysis at the expense of that of blood lactate. This result strengthens the hypothesis that brain lactate is involved in the coupling between neuronal activation and metabolism.In the last decade, the idea of the involvement of lactate in the coupling between neuronal activation and energy metabolism has arisen from the results of various experimental investigations. Briefly, in vitro studies have evidenced lactate release from astrocytes after glycolysis stimulation by glutamate uptake (1, 2) and, in the particular case of the mammalian retina, the use of lactate from Mü ller cells as an energy substrate for photoreceptors (3). In vivo studies on brain have demonstrated the uncoupling of oxygen and glucose utilization (4) and the release of lactate into the extracellular fluid in response to neuronal activation (5, 6). On the other hand, brain lactate has been demonstrated to efficiently protect neurons against delayed ischemic damage (7). The metabolic fate of exogenous lactate in whole brain has been investigated by ex vivo NMR spectroscopy. In this way, it has been demonstrated that blood lactate enters the brain and is more specifically metabolized in neurons (8 -10). The proposed astrocyte-neuron lactate shuttle hypothesis (ANLSH) 1 (1) as the coupling model between neuronal activity and energy metabolism requires the involvement of different components, the occurrence and localization of which have been investigated analytically at the molecular level, i.e. glutamate transporter and Na ϩ ,K ϩ -ATPase (11), lactate dehydrogenase isoenzymes (12), and monocarboxylate transporters (13,14). Although brain lactate production and lactate use by neurons as an energy source are widely admitted, the relevance of the coupling model, particularly the neuronal utilization of glia-produced lactate, is a topical issue (15, 16).Brain lactate mostly derives from glycolysis. Therefore, the comparative analysis of the contribution of brain l...