Regulation of [N-15]urea synthesis from [5-N-15]glutamine - Role of pH, hormones, and pyruvate

We have utilized both [5-N-15]glutamine and [3-C-13] pyruvate as metabolic tracers in order to: (i) examine the effect of pH, glucagon (GLU), or insulin on the precursor-product relationship between (NH3)-N-15, [N-15]citrulline, and, thereby, [N-15]urea synthesis and (ii) elucidate the mechanism(s) by which pyruvate stimulates [N-15] urea synthesis. Hepatocytes isolated from rat were incubated at pH 6.8, 7.4, or 7.6 with 1 mM [5-N-15]glutamine and 0.1 mM (NH4Cl)-N-14 in the presence or the absence of [3-C-13] pyruvate (2 mM). A separate series of experiments was performed at pH 7.4 in the presence of insulin or GLU. (NH3)-N-15 enrichment exceeded or was equal to that of [N-15]citrulline under all conditions except for pH 7.6, when the N-15 enrichment in citrulline exceeded that in ammonia. The formation of [N-15]citrulline (atom % excess) was increased with higher pH. Flux through phosphate-dependent glutaminase (PDG) and [N-15]urea synthesis were stimulated (p < 0.05) at pH 7.6 or with GLU and decreased (p < 0.05) at pH 6.8. Insulin had no significant effect on flux through PDG or on [N-15]urea synthesis, Decreased [N-15]urea production at pH 6.8 was associated with depleted aspartate and glutamate levels, Pyruvate attenuated this decrease in the aspartate and glutamate pools and stimulated [N-15]urea synthesis. Production of Asp from pyruvate was increased with increasing medium pH. Approximately 80% of Asp was derived from [3-C-13]pyruvate regardless of incubation pH or addition of hormone. Furthermore, approxiately 20, 40, and 50% of the mitochondrial N-acetylglutamate (NAG) pool was derived from [3-C-13]pyruvate at pH 6.8, 7.4, and 7.6, respectively. Both the concentration and formation of [C-13]NAG from [3-C-13]pyruvate were increased (p < 0.05) with glucagon and decreased (p < 0.05) with insulin or at pH 6.8. The data suggest a correlation between changes in [N-15]urea synthesis and alterations in the level and synthesis of [C-13]NAG from pyruvate, The current observations suggest that the stimulation of [N-15]urea synthesis in acute alkalosis is mediated via increased flux through PDG and subsequent increased utilization of [5-N-15] Of glutamine for [N-15]citrulline synthesis and/or increased synthesis of NAG from glutamate and pyruvate. The opposite may have occurred in acute acidosis. Glucagon, but not insulin, stimulated [N-15]urea synthesis via increased flux through PDG and synthesis of NAG. Pyruvate stimulated urea synthesis via increased availability of aspartate and/or increased synthesis of NAG, The formation of NAG and aspartate from pyruvate are both pH-sensitive processes.