Regulation of [15N]urea synthesis from [5-15N]glutamine. Role of pH, hormones, and pyruvate.
We have utilized both [5-15N]glutamine and [3-13C] pyruvate as metabolic tracers in order to: (i) examine the effect of pH, glucagon (GLU), or insulin on the precursor-product relationship between 15NH3, [15N]citrulline, and, thereby, [15N]urea synthesis and (ii) elucidate the mechanism(s) by which pyruvate stimulates [15N] urea synthesis. Hepatocytes isolated from rat were incubated at pH 6.8, 7.4, or 7.6 with 1 mM [5-15N]glutamine and 0.1 mM 14NH4Cl in the presence or the absence of [3-13C] pyruvate (2 mM). A separate series of experiments was performed at pH 7.4 in the presence of insulin or GLU. 15NH3 enrichment exceeded or was equal to that of [15N]citrulline under all conditions except for pH 7.6, when the 15N enrichment in citrulline exceeded that in ammonia. The formation of [15N]citrulline (atom % excess) was increased with higher pH. Flux through phosphate-dependent glutaminase (PDG) and [15N]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 [15N]urea synthesis. Decreased [15N]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 [15N]urea synthesis. Production of Asp from pyruvate was increased with increasing medium pH. Approximately 80% of Asp was derived from [3-13C]pyruvate regardless of incubation pH or addition of hormone. Furthermore, approximately 20, 40, and 50% of the mitochondrial N-acetylglutamate (NAG) pool was derived from [3-13C]pyruvate at pH 6.8, 7.4, and 7.6, respectively. Both the concentration and formation of [13C]NAG from [3-13C]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 [15N]urea synthesis and alterations in the level and synthesis of [13C]NAG from pyruvate. The current observations suggest that the stimulation of [15N]urea synthesis in acute alkalosis is mediated via increased flux through PDG and subsequent increased utilization of [5-15N] of glutamine for [15N]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 [15N]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.
Nissim
I
I
Division of Biochemical Development, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Yudkoff
M
M
Brosnan
J T
JT
D000596:Amino Acids / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D002952:Citric Acid Cycle; D002956:Citrulline / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005934:Glucagon / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000378:metabolism*; D008297:Male; D019289:Pyruvic Acid / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014508:Urea / Q000378:metabolism*
D000596:Amino Acids; D005971:Glutamates; D007328:Insulin; D005973:Glutamine; D002956:Citrulline; D000641:Ammonia; D019289:Pyruvic Acid; D014508:Urea; D005934:Glucagon; D005972:Glutaminase; C016195:N-acetylglutamic acid