Whole-brain glutamate metabolism evaluated by steady-state kinetics using a double-isotope procedure: Effects of gabapentin

Cerebral rates of anaplerosis are known to be significant, yet the rates measured in vivo have been debated. In order to track glutamate metabolism in brain glutamatergic neurons and brain glia, for the first time unrestrained awake rats were continuously infused with a combination of H¹⁴CO ₃^- and [1-¹³C]glucose in over 50 infusions ranging from 5 to 60 min. In whole-brain extracts from these animals, the appearance of ¹⁴C in brain glutamate and glutamine and appearance of ¹³C in the C-4 position of glutamate and glutamine were measured as a function of time. The rate of total neuronal glutamate turnover, the anaplerotic rate of synthesis of glutamine and glutamate from H ¹⁴CO₃^-, flux through the glutamate/glutamine cycle, and a minimum estimate of whole-brain anaplerosis was obtained. The rate of synthesis of ¹⁴C-glutamate from H¹⁴CO₃ ^- was 1.29 ± 0.11 nmoles/min/mg protein, whereas the rate of synthesis of ¹⁴C-glutamine was 1.48 ± 0.10 nmoles/min/mg protein compared to total glutamate turnover of 9.39 ± 0.73 nmoles/min/mg protein. From the turnover rate of glutamine, an upper limit for flux through the glutamate/glutamine cycle was estimated at 4.6 nmoles/min/mg protein. Synthesis of glutamine from H¹⁴CO₃^- was substantial, amounting to 32% of the glutamate/glutamine cycle. These rates were not significantly affected by a single injection of 100 mg/kg of the antiepileptic drug gabapentin. In contrast, acute administration of gabapentin significantly lowered incorporation of H¹⁴CO₃^- into glutamate and glutamine in excised rat retinas, suggesting metabolic effects of gabapentin may require chronic treatment and/or are restricted to brain areas enriched in target enzymes such as the cytosolic branched chain aminotransferase. We conclude that the brain has a high anaplerotic activity and that the combination of two tracers with different precursors affords unique insights into the compartmentation of cerebral metabolism.