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The authors investigated concomitant lactate and glucose metabolism in primary neuronal cultures using ¹³C- and ¹H-NMR spectroscopy. Neurons were incubated in a medium containing either [1-¹³C]glucose and different unlabeled lactate concentrations, or unlabeled glucose and different [3-¹³C]lactate concentrations. Overall, ¹³C-NMR spectra of cellular extracts showed that more ¹³C was incorporated into glutamate when lactate was the enriched substrate. Glutamate ¹³C-enrichment was also found to be much higher in lactate-labeled than in glucose-labeled conditions. When glucose and lactate concentrations were identical (5.5 mmol/L), relative contributions of glucose and lactate to neuronal oxidative metabolism amounted to 21% and 79%, respectively. Results clearly indicate that when neurons are in the presence of both glucose and lactate, they preferentially use lactate as their main oxidative …