Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: a stable isotope study

Glutamine's role as an energetic fuel has been extensively studied in the past using ¹⁴C- and ³H-labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N-tracer. We therefore used ¹⁵N-labeled glutamine and glutamate to address the following questions: (1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and (2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: (1) intracellular [¹⁵N]-glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure ¹⁵N-glutamine and no glutamate; in contrast, intracellular ¹⁵N-glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure ¹⁵N-glutamate and no glutamine; (2) the cell-free glutamine content was tightly dependent on extracellular glutamine level, while the cell-free glutamate remained steady irrespective of the extracellular glutamate level; (3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and (4) when cells were confronted with a glutamine-free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: (1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; (2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and (3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.