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W2005668229 abstract "This study examines the role of glucagon and insulin in the incorporation of 15N derived from 15N-labeled glutamine into aspartate, citrulline and, thereby, [15N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mm NH4Cl and either 2-15N- or 5-15N-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 [15N]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 [2-15N] and [5-15N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of 15N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form U m+1 andU m+2 (urea containing one or two atoms of15N). However, insulin had little effect compared with control. The [5-15N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-15N]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 15NH3 that was generated in the mitochondria by either PDG (from 5-15N) or glutamate dehydrogenase (from 2-15N) 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 glucagon-induced increase in [15N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [15N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis ofN-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 of15N-labeled glutamine into the urea cycle, and the production of urea isotopomers. This study examines the role of glucagon and insulin in the incorporation of 15N derived from 15N-labeled glutamine into aspartate, citrulline and, thereby, [15N]urea isotopomers. Rat livers were perfused, in the nonrecirculating mode, with 0.3 mm NH4Cl and either 2-15N- or 5-15N-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 [15N]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 [2-15N] and [5-15N]glutamine is incorporation into urea. Glucagon significantly stimulated the uptake of 15N-labeled glutamine and its metabolism via phosphate-dependent glutaminase (PDG) to form U m+1 andU m+2 (urea containing one or two atoms of15N). However, insulin had little effect compared with control. The [5-15N]glutamine primarily entered into urea via ammonia incorporation into CP, whereas the [2-15N]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 15NH3 that was generated in the mitochondria by either PDG (from 5-15N) or glutamate dehydrogenase (from 2-15N) 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 glucagon-induced increase in [15N]urea synthesis was associated with a significant elevation in hepatic N-acetylglutamate concentration. Therefore, the hormonal regulation of [15N]urea isotopomer production depends upon the coordinate action of the mitochondrial PDG pathway and the synthesis ofN-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 of15N-labeled glutamine into the urea cycle, and the production of urea isotopomers. phosphate-dependent glutaminase glutamate dehydrogenase N-acetylglutamate carbamyl-phosphate synthetase-I gas chromatography-mass spectrometry atom percent of excess We have previously demonstrated that glutamine is the chief precursor for urea-N (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), following its metabolism via the phosphate-dependent glutaminase (PDG)1 pathway to provide NH3 and glutamate (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). A smaller fraction of ammonia may be derived via the glutamate dehydrogenase (GDH) reaction (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar). However, glutamate rapidly transaminated to aspartate to provide the second nitrogen of urea (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). More recently, we developed a theoretical framework that described the incorporation of 15N from15NH4Cl into urea and that predicted the proportions of U m, U m+1, and U m+2 isotopomers of urea produced (containing no, one, or two atoms of 15N) 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 (4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). We have also examined the incorporation of15N from [5-15N]glutamine into urea in isolated hepatocytes and examined effects of pH and hormones on this process (3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). 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) (5Hall L.M. Metzenberg R.L. Cohen P.P. J. Biol. Chem. 1958; 230: 1013-1021Abstract Full Text PDF PubMed Google Scholar, 6Cohen N.S. Kyan F.S. Kyan S.S. Cheung C.W. Raijman L. Biochem. J. 1986; 229: 205-211Crossref Scopus (58) Google Scholar, 7Fahien L. Schoder J.M. Gehred G.A. Cohen P.P. J. Biol. Chem. 1964; 239: 1935-1942Abstract Full Text PDF PubMed Google Scholar, 8Lund P. Wiggins D. Biochem. J. 1984; 218: 991-994Crossref PubMed Scopus (23) Google Scholar).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 2-15N- or 5-15N-labeled glutamine and GC-MS to address the following questions: (i) What is the relative incorporation of 15NH3, formed from 15N-labeled glutamine via the PDG (from 5-15N) and/or GDH (from 2-15N) pathway, into citrulline or aspartate, and thereby, [15N] urea isotopomers? (ii) Is the hepatic intramitochondrial pool of 15NH3 (formed via either PDG or GDH) in equilibrium with the perfusate NH3pool? (iii) Does production of [15N]urea isotopomers depend on the species of 15N-labeled glutamine,i.e. amino versus amido 15N? The results of these determinations were used to examine the hypothesis that the 5-N of glutamine is directly channeled to carbamyl phosphate synthesis (9Meijer A.J. FEBS Lett. 1985; 191: 249-251Crossref PubMed Scopus (39) Google Scholar).Our methodology employed the stable isotope, 15N, and GC-MS provides an excellent approach to the quantitation and identification of 15N enrichment in metabolic intermediates (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 10Reeds P.J. Berthold H.K. Boza J.J. Burrin D.G. Jahoor F. Jaksic T. Klein P.D. Keshen T. Miller R. Stoll B. Wykes L.J. Eur. J. Pediat. 1997; 156: S50-S58Crossref PubMed Google Scholar, 11Castillo L. Chapman T.E. Sanchez M., Yu, Y.-M. Burke J.F. Ajami A.M. Vogt J. Young V.R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7749-7753Crossref PubMed Scopus (192) Google Scholar). The use of 15N 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 (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar).In a separate series of perfusions, we have examined the role of glucagon or insulin in the regulation of the 15N enrichment of the two nitrogenous precursor pools involved in urea synthesis as well as the production of mass isotopomers of [15N]urea. These hormones are key players in the regulation of hepatic nitrogen and carbohydrate metabolism in normal and disease states (3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Brosnan J.T. Ewart H.S. Squires S.A. Adv. Enzyme Regul. 1995; 35: 131-146Crossref PubMed Scopus (32) Google Scholar, 13Hamberg O. Vilstrup H. J. Hepatol. 1994; 21: 381-387Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 14Magnusson I. Rothman D.L. Gerard D.P. Katz L.D. Shulman G.I. Diabetes. 1995; 44: 185-189Crossref PubMed Google Scholar, 15Blackmore P.F. Assimacopoulos J.F. Chan T.M. Exton J.H. J. Biol. Chem. 1979; 254: 2828-2834Abstract Full Text PDF PubMed Google Scholar, 16Kashiwagura T.K. Erecinska M. Wilson D.F. J. Biol. Chem. 1985; 260: 407-414Abstract Full Text PDF PubMed Google Scholar). The data demonstrate that glucagon stimulated the flux through the PDG pathway and the formation of [15N]urea mass isotopomers from [2-15N]glutamine or [5-15N]glutamine. However, insulin has little effect on the flux through the PDG pathway and the formation of [15N]urea. The increased urea synthesis with glucagon was coupled with an increased hepatic level of N-AG. There was no evidence for a metabolic channeling between glutaminase and carbamyl-phosphate synthetase.DISCUSSIONIn the current study we used a series of liver perfusion experiments to examine the role of glucagon or insulin in the regulation of the 15N enrichment of the two nitrogenous precursor pools involved in urea synthesis as well as the production of mass isotopomers of [15N]urea from either 2-15N- or 5-15N-labeled glutamine. These hormones have a pivotal role in the regulation of hepatic nitrogen and carbohydrate metabolism (3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Brosnan J.T. Ewart H.S. Squires S.A. Adv. Enzyme Regul. 1995; 35: 131-146Crossref PubMed Scopus (32) Google Scholar, 13Hamberg O. Vilstrup H. J. Hepatol. 1994; 21: 381-387Abstract Full Text PDF PubMed Scopus (24) Google Scholar, 14Magnusson I. Rothman D.L. Gerard D.P. Katz L.D. Shulman G.I. Diabetes. 1995; 44: 185-189Crossref PubMed Google Scholar, 15Blackmore P.F. Assimacopoulos J.F. Chan T.M. Exton J.H. J. Biol. Chem. 1979; 254: 2828-2834Abstract Full Text PDF PubMed Google Scholar, 16Kashiwagura T.K. Erecinska M. Wilson D.F. J. Biol. Chem. 1985; 260: 407-414Abstract Full Text PDF PubMed Google Scholar). Elucidation of the hormonal regulation of [15N]urea mass isotopomer production from15N-labeled glutamine is of special importance both in normal and catabolic states such as sepsis or after surgery, when the liver consumes large quantities of glutamine (12Brosnan J.T. Ewart H.S. Squires S.A. Adv. Enzyme Regul. 1995; 35: 131-146Crossref PubMed Scopus (32) Google Scholar, 26Vinnars E. Bergstrom J. Furst P. Ann. Surg. 1975; 182: 665-667Crossref PubMed Scopus (224) Google Scholar, 27Pacitti A.J. Augsten T.R. Souba W.W. J. Surg. Res. 1992; 53: 298-305Abstract Full Text PDF PubMed Scopus (12) Google Scholar).We employ the single-pass isolated perfused rat liver, because this model preserves the normal lobular microcirculation of the liver and avoids problems that may arise because of recycling of substrate (such as products of perivenous hepatocyte metabolism being recycled to periportal hepatocytes) that would occur in isolated hepatocytes or in a recirculating perfusion. The use of [2-15N]glutamine and of [5-15N]glutamine allows us to compare the metabolic fate of these two nitrogen atoms. The amide nitrogen of glutamine was primarily incorporated into urea via carbamyl phosphate, whereas the amino nitrogen was primarily incorporated via aspartate, as is evident from the relative enrichments of aspartate and of citrulline generated from each substrate.Glucagon stimulated flux through the PDG pathway (p < 0.05), as has been previously reported (3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 12Brosnan J.T. Ewart H.S. Squires S.A. Adv. Enzyme Regul. 1995; 35: 131-146Crossref PubMed Scopus (32) Google Scholar, 16Kashiwagura T.K. Erecinska M. Wilson D.F. J. Biol. Chem. 1985; 260: 407-414Abstract Full Text PDF PubMed Google Scholar, 23Haüssinger D. Biochem. J. 1990; 267: 281-290Crossref PubMed Scopus (265) Google Scholar). The activation of glutaminase is confirmed by the decrease in liver glutamine and the increase in glutamate. The fact that glutaminase is irreversible permits reliable estimates of glutaminase flux based on conversion of [5-15N]glutamine to labeled products. However, the enzymes subsequent to glutaminase, glutamate dehydrogenase, and the aminotransferases are reversible so that the incorporation of15N into ammonia and amino acids by these enzymes will reflect isotopic exchange as well as net flux. Our results supply clear evidence for the reversibility of these enzymes. For example, the labeling of glutamate, alanine, and aspartate in the experiments that utilized [5-15N]glutamine depends on the incorporation of ammonia into glutamate via glutamate dehydrogenase acting in the direction of reductive animation. The labeling of ammonia and of citrulline in the experiments that utilize [2-15N]glutamine rely on the deamination of glutamate via glutamate dehydrogenase.These differences of product enrichment in perfusions with the differently labeled glutamines are germane to the question of glutamate dehydrogenase equilibration. It is well established that the equilibrium of the GDH enzyme activity lies far to the glutamate side (28McGivan J.D. Chappell J.B. FEBS Lett. 1975; 52: 1-7Crossref PubMed Scopus (66) Google Scholar), but this holds true only for the purified enzyme in which the concentration of ammonia is in the high millimolar range, well beyond any expected in vivo concentration (28McGivan J.D. Chappell J.B. FEBS Lett. 1975; 52: 1-7Crossref PubMed Scopus (66) Google Scholar, 29Charles A.S. Lieu B.S. Hsu B.Y.L. Alberto B.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Poncz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (610) Google Scholar). Indeed, recent investigation with the hyperammonemia, hyperinsulinemia syndrome, which is caused by a mutant GDH that is no longer subject to tight inhibition by GTP, suggests that the enzyme can function primarily for the purpose of glutamate oxidation, at least in liver and pancreas (29Charles A.S. Lieu B.S. Hsu B.Y.L. Alberto B.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Poncz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (610) Google Scholar). The current data indicate that the glutamate dehydrogenase is reversible; the incorporation of 15N from [5-15N]glutamine into glutamate and from [2-15N]glutamine into ammonia occurs via the amination and deamination reactions, respectively, of glutamate dehydrogenase. However, the flux through the glutamate dehydrogenase is not so great, in comparison with the flux of other relevant pathways (1Nissim I. Cattano C. Nissim I. Yudkoff M. Arch. Biochem. Biophys. 1992; 292: 393-401Crossref PubMed Scopus (33) Google Scholar, 2Nissim I. Cattano C. Zhiping L. Nissim I. J. Am. Soc. Nephrol. 1993; 3: 1416-1427PubMed Google Scholar, 3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), that full 15N equilibration occurs. Glutaminase produces equal quantities of glutamate and ammonia. If the equilibrating effect of glutamate dehydrogenase were absolute, then glutamate and ammonia would become equally labeled within mitochondria regardless of whether [2-15N]glutamine or [5-15N]glutamine were substrates, and there would be no difference between these substrates in the labeling of other metabolites such as alanine, aspartate, or citrulline. There are, however, substantial differences in the labeling of these other metabolites, which is most likely explained by “metabolic competition,” i.e. that aspartate and alanine aminotransferases compete effectively with glutamate dehydrogenase for [15N]glutamate that arises from [2-15N]glutamine metabolism and carbamyl-phosphate synthetase competes effectively with glutamate dehydrogenase for15NH3 that arises from [5-15N]glutamine metabolism. Therefore, it is not necessary to invoke metabolic channeling, merely metabolic competition, to account for the different labeling patterns that are found when these two positional isotopomers of 15N glutamine are employed as substrates.The glutamate dehydrogenase reaction may have also a key role in the regulation of glutamine synthesis. It is possible that a significant portion of the endogenous glutamine production is mediated via de novo synthesis, in which a net flux of carbons into glutamine occurs. In this case α-ketoglutarate formed in the tricarboxylic acid cycle (from pyruvate and lactate added to the perfusate) would be converted to glutamate via the glutamate dehydrogenase reaction and then to glutamine. Our previous investigation with isolated hepatocytes and [3-13C]pyruvate indicated that approximately 15% of glutamate carbons were derived from [3-13C]pyruvate (3Nissim I. Yudkoff M. Brosnan J.T. J. Biol. Chem. 1996; 271: 31234-31242Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). However, further study with 13C-labeled precursor would be required to determine whether a similar portion of glutamate carbon is derived during liver perfusion.Fig. 3 shows that the 15N enrichment of glutamine in the cellular pool is always lower than in the effluent perfusate and that the enrichment of the cellular pool is much higher when [2-15N]glutamine is the substrate. That the enrichment in the cellular pool is lower than in the perfusate means that some of the intracellular glutamine arises from unlabeled sources, i.e.via proteolysis, glutamine recycling, or de novo glutamine synthesis (as indicated above). We previously reported that glutamine production via proteolysis amounted to 7 nmol/min/g (4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar), that is, approximately 3% of the endogenous glutamine production calculated in the current study. Therefore, proteolysis seems unlikely to be the major source for endogenous glutamine production, and thus the dilution of 15N enrichment in the perfusate glutamine must occur following production of glutamine from other unlabeled sources. In the case of [5-15N]glutamine, unlabeled glutamate and15NH3 will be produced via the PDG pathway. In the mitochondria, this 15NH3 can be metabolized to form [15N]glutamate via the GDH pathway. If15N-labeled ammonia and glutamate are used for glutamine synthesis, then glutamine would be labeled at 2-N and 5-N (4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). However, in the current study no doubly labeled glutamine was detected regardless of the experimental conditions, indicating that glutamine was formed from either unlabeled ammonia, unlabeled glutamate, or both. It is possible that the unlabeled mitochondrial glutamate (formed via the PDG pathway) is transported to the cytosol and then recycled to glutamine in the perivenous hepatocytes. Therefore, during perfusion with [5-15N]glutamine, mainly unlabeled glutamine is produced and simultaneously dilutes the [5-15N]glutamine enrichment in the liver (Fig. 3). However, in the case of perfusions with [2-15N]glutamine, [15N]glutamate (between 30–50 APE, depending upon the experimental condition) is formed so that the glutamine produced would be labeled, and thereby, the dilution of the intrahepatic [2-15N]glutamine would be less significant than in the case of perfusions with [5-15N]glutamine. Haüssinger et al. (24Haüssinger D. Gerok W. Sies H. Biochim. Biophys. Acta. 1983; 755: 272-278Crossref PubMed Scopus (84) Google Scholar) have shown that an intrahepatic glutamine cycle exists whereby there is simultaneous catabolism and synthesis of glutamine (in periportal and perivenous hepatocytes, respectively. Haüssinger (23Haüssinger D. Biochem. J. 1990; 267: 281-290Crossref PubMed Scopus (265) Google Scholar) has also shown an effective uptake mechanism for glutamate in the perivenous hepatocytes. We therefore envisage that some glutamate produced in periportal hepatocytes by glutaminase is taken up by the perivenous hepatocytes and converted to glutamine. In this case, the endogenous production of glutamine is mediated primarily via recycling of perfusate glutamine as indicated by Haüssinger et al.(23Haüssinger D. Biochem. J. 1990; 267: 281-290Crossref PubMed Scopus (265) Google Scholar, 24Haüssinger D. Gerok W. Sies H. Biochim. Biophys. Acta. 1983; 755: 272-278Crossref PubMed Scopus (84) Google Scholar).The data in Fig. 7 are of considerable interest in the light of the suggestion that a metabolic channel exists between PDG and CPS-I such that ammonia that arises from 5-15N of glutamine enjoys preferential access to carbamyl-phosphate synthetase (9Meijer A.J. FEBS Lett. 1985; 191: 249-251Crossref PubMed Scopus (39) Google Scholar). Both [5-15N]glutamine and [2-15N]glutamine give rise to ammonia in the same compartment (the mitochondria of glutaminase-containing hepatocytes) but as a result of the action of two different enzymes. 15NH3 will be produced from [5-15N]glutamine by the PDG pathway. It will also be produced from [2-15N]glutamine by the GDH pathway, which acts on [15N]glutamate that arises via PDG. By comparing the metabolic fate of 15NH3 that is produced in the same compartment by these two different enzymes, we can determine whether ammonia that arises via the PDG pathway has preferential access to carbamyl-phosphate synthetase. Ammonia that arises in the mitochondria can have three immediate metabolic fates: (i) to be incorporated into carbamyl phosphate by CPS-I, (ii) to be incorporated into glutamate by glutamate dehydrogenase, and (iii) to leave the mitochondria. 15NH3 that is incorporated into carbamyl phosphate will be reflected in perfusate [15N]citrulline, whereas 15NH3that leaves the mitochondria will be reflected in perfusate NH3 (4Brosnan J.T. Brosnan M.E. Charron R. Nissim I. J. Biol. Chem. 1996; 271: 16199-16207Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Fig. 7 shows the correlation between perfusate15NH3 and perfusate [15N]citrulline at all of the time points in all of the perfusions. An excellent correlation was found between the isotopic enrichment of perfusate ammonia and citrulline, and it is clear that the data from the perfusions with [2-15N]glutamine and [5-15N]glutamine fall on the same line. Thus,15NH3 molecules that arise within the mitochondria by the agency either of glutaminase or of glutamate dehydrogenase enjoy the same partition between incorporation into carbamyl phosphate by means of CPS-I or leaving the mitochondria. Thus, in these experiments, there is no evidence for preferential access for ammonia that arises by the action of glutaminase to carbamyl-phosphate synthetase. To the contrary, we provide strong evidence that such ammonia is metabolized without any such metabolic channel. Raijman and co-workers (31Cheung C.W. Cohen N.S. Raijman L. J. Biol. Chem. 1989; 264: 4038-4044Abstract Full Text PDF PubMed Google Scholar) have shown a channeling of urea cycle intermediates at each of the three cytoplasmic enzymes of the urea cycle. However, there are fundamental differences between the current investigation and that of Raijman and co-workers (31Cheung C.W. Cohen N.S. Raijman L. J. Biol. Chem. 1989; 264: 4038-4044Abstract Full Text PDF PubMed Google Scholar). First, the current observation deals with the first mitochondrial reaction in the urea cycle (the channeling of ammonia derived via the PDG to CPS-I), whereas the observations of Raijman and co-workers deals with the cytoplasmic enzymes of the urea cycle. Second, in the current study we used 15N-labeled glutamine and liver perfusion system, whereas Raijman and co-workers (31Cheung C.W. Cohen N.S. Raijman L. J. Biol. Chem. 1989; 264: 4038-4044Abstract Full Text PDF PubMed Google Scholar) used [14C]HCO3− and isolated hepatocytes.The production of the urea isotopomers requires comment. The time course for the three isotopomers (Fig. 8) shows that the pattern of their production varied throughout the perfusions, and even in the control perfusions, a steady state did not appear to be achieved until about 60–70 min. This is attributable to a slow increase in PDG activity that occurred during the perfusions (Fig. 2 A), except in the case of the glucagon-infused livers, where there was a large activation of glutaminase. But, even there, the activation is progressive, and a steady state is not achieved until about 60 min. Thus the progressive decrease in the proportion ofU m and increase in the proportion ofU m+1 and U m+2 throughout the perfusions are attributable to the increased provision of15N-labeled urea precursors with time. The comparison with experimental and predicted isotopomer production (Table I) showed very good agreement for the proportion of U m andU m+1. However, there was quite a difference between the predicted and experimental values ofU m+2, with the experimental value invariably being greater. We do not know precisely why this is so, but we can make one suggestion. A possible explanation is that the enzymes of the urea cycle and, presumably, ureagenesis occur throughout the liver acinus, except for the last few perivenous cells, which contain glutamine synthetase (24Haüssinger D. Gerok W. Sies H. Biochim. Biophys. Acta. 1983; 755: 272-278Crossref PubMed Scopus (84) Google Scholar). However, the glutaminase location does not exactly parallel that of the urea cycle in that it is expressed only in the early portion of the peri-portal region (24Haüssinger D. Gerok W. Sies H. Biochim. Biophys. Acta. 1983; 755: 272-278Crossref PubMed Scopus (84) Google Scholar). In the experiments reported in this paper, urea synthesis will occur in the early part of the acinus from perfusate NH4Cl and 15N-labeled glutamine and in the later part of the acinus largely from NH4Cl. The urea isotopomers measured in the perfusate will be the sum of those produced in early" @default.
W2005668229 created "2016-06-24" @default.
W2005668229 creator A5003254586 @default.
W2005668229 creator A5037535982 @default.
W2005668229 creator A5046069471 @default.
W2005668229 creator A5066968727 @default.
W2005668229 creator A5079911358 @default.
W2005668229 date "1999-10-01" @default.
W2005668229 modified "2023-09-30" @default.
W2005668229 title "Studies of Hepatic Glutamine Metabolism in the Perfused Rat Liver with 15N-Labeled Glutamine" @default.
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