Studies of Hepatic Glutamine Metabolism in the Perfused Rat

This study examines the role of glucagon and insulin in the incorporation of N derived from N-labeled glutamine into aspartate, citrulline and, thereby, [N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mM NH4Cl and either 2Nor 5-N-labeled glutamine (1 mM). The isotopic enrichment of the two nitrogenous precursor pools (ammonia and aspartate) involved in urea synthesis as well as the production of [N]urea isotopomers were determined using gas chromatography-mass spectrometry. This information was used to examine the hypothesis that 5-N of glutamine is directly channeled to carbamyl phosphate (CP) synthesis. The results indicate that the predominant metabolic fate of [2-N] and [5-N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form Um11 and Um12 (urea containing one or two atoms of N). However, insulin had little effect compared with control. The [5-N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-N]glutamine was predominantly incorporated via aspartate. This is evident from the relative enrichments of aspartate and of citrulline generated from each substrate. Furthermore, the data indicate that the NH3 that was generated in the mitochondria by either PDG (from 5-N) or glutamate dehydrogenase (from 2-N) enjoys the same partition between incorporation into CP or exit from the mitochondria. Thus, there is no evidence for preferential access for ammonia that arises by the action of PDG to carbamyl-phosphate synthetase. To the contrary, we provide strong evidence that such ammonia is metabolized without any such metabolic channeling. The glucagoninduced increase in [N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis of N-acetylglutamate (an obligatory activator of CP). The current study may provide the theoretical and methodological foundations for in vivo investigations of the relationship between the hepatic urea cycle enzyme activities, the flux of N-labeled glutamine into the urea cycle, and the production of urea isotopomers. We have previously demonstrated that glutamine is the chief precursor for urea-N (1–3), following its metabolism via the phosphate-dependent glutaminase (PDG) pathway to provide NH3 and glutamate (1–3). A smaller fraction of ammonia may be derived via the glutamate dehydrogenase (GDH) reaction (1). However, glutamate rapidly transaminated to aspartate to provide the second nitrogen of urea (1–3). More recently, we developed a theoretical framework that described the incorporation of N from NH4Cl into urea and that predicted the proportions of Um, Um11, and Um12 isotopomers of urea produced (containing no, one, or two atoms of N) as a function of the isotopic enrichment of the two nitrogenous precursor pools for urea. We experimentally validated this model in the isolated perfused rat liver (4). We have also examined the incorporation of N from [5-N]glutamine into urea in isolated hepatocytes and examined effects of pH and hormones on this process (3). These latter studies showed that our theoretical framework for prediction of the labeling patterns of urea was also valid in the hepatocyte model and that alkalosis and glucagon were powerful stimuli for increased flux through hepatic glutaminase and the urea cycle. They also showed important effects of hormones and of pH on the hepatocyte concentration of N-acetylglutamate (N-AG), the obligatory activator of carbamyl-phosphate synthetase-I (CPS-I) (5–8). In this study we used this framework to explore the role of insulin or glucagon in the production of mass isotopomers of urea. We used 2-Nor 5-N-labeled glutamine and GC-MS to address the following questions: (i) What is the relative incorporation of NH3, formed from N-labeled glutamine via the PDG (from 5-N) and/or GDH (from 2-N) pathway, into citrulline or aspartate, and thereby, [N] urea isotopomers? (ii) Is the hepatic intramitochondrial pool of NH3 (formed via either PDG or GDH) in equilibrium with the perfusate NH3 pool? (iii) Does production of [N]urea isotopomers depend on the species of N-labeled glutamine, i.e. amino versus amido N? The results of these determinations were used to examine the hypothesis that the 5-N of glutamine is directly channeled to carbamyl phosphate synthesis (9). Our methodology employed the stable isotope, N, and GC-MS provides an excellent approach to the quantitation and identification of N enrichment in metabolic intermediates (1– 4, 10, 11). The use of N as a metabolic tracer is pivotal to the precise definition of precursor-product relationships and quantitation of N-flux from either the 2-N or 5-N of glutamine to NH3, carbamyl phosphate, aspartate, citrulline, and, thereby, urea (1–4). In a separate series of perfusions, we have examined the role * This work was supported by National Institutes of Health Grants DK-53761, HD-34900, and NS-37915 and by the Medical Research Council (Canada). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: Div. of Child Development, Abramson Pediatric Research Ctr., Rm. 510C, 34th St. and Civic Center Blvd., Philadelphia, PA 19104-4318. Fax: 215-590-5199. 1 The abbreviations used are: PDG, phosphate-dependent glutaminase; GDH, glutamate dehydrogenase; N-AG, N-acetylglutamate; CPS-I, carbamyl-phosphate synthetase-I; GC-MS, gas chromatography-mass spectrometry; APE, atom percent of excess. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 274, No. 41, Issue of October 8, pp. 28958–28965, 1999 © 1999 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.