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J Biol Chem, Vol. 274, Issue 41, 28958-28965, October 8, 1999


Studies of Hepatic Glutamine Metabolism in the Perfused Rat Liver with 15N-Labeled Glutamine*

Itzhak NissimDagger §, Margaret E. Brosnan, Marc YudkoffDagger , Ilana NissimDagger , and John T. Brosnan

From the Dagger  Division of Child Development and Rehabilitation, Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelpia, Pennsylvania 19104 and the  Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland A1B 3X9, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 Um+1 and Um+2 (urea containing one or two atoms of 15N). 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 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 15N-labeled glutamine into the urea cycle, and the production of urea isotopomers.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that glutamine is the chief precursor for urea-N (1-3), following its metabolism via the phosphate-dependent glutaminase (PDG)1 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 15N from 15NH4Cl into urea and that predicted the proportions of Um, Um+1, and Um+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 (4). We have also examined the incorporation of 15N from [5-15N]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 2-15N- 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 NH3 pool? (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 (9).

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 (1- 4, 10, 11). 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 (1-4).

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 (3, 12-16). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Animals-- Chemicals were of analytical grade and obtained from Sigma-Aldrich. Enzymes and cofactors for the enzymatic analyses were obtained from Roche Molecular Biochemicals. The 2-15N- and 5-15N-labeled glutamine were from Cambridge Isotopes Laboratories, Inc. (Andover, MA). Harlan Sprague-Dawley rats were from the Memorial University colony and were fed on Agway Prolab rat chow.

Liver Perfusion-- Livers from fed, male Harlan Sprague-Dawley rats (weighing about 200-270 g) were perfused in the nonrecirculating mode as described by Sies (17). The perfusion medium was a Krebs' saline continuously gassed with 95% O2, 5% CO2 and containing lactate (2.1 mM) and pyruvate (0.3 mM) as metabolic fuels. Perfusion flow rate, pH, pCO2, and pO2 (in influent and effluent media) were monitored throughout and O2 consumption was determined. After 10 min of perfusion, glutamine (1 mM), either [5-15N]glutamine or [2-15N]glutamine, 99 atom percent of excess (APE), and NH4Cl (0.3 mM) were added to the medium. Perfusion continued for another 60 min with hormones (10-7 M glucagon or 10-7 M insulin) infused between 40 and 70 min. Control perfusions had saline infusion from 40 to 70 min.

Samples were taken at the indicated times from influent and effluent media for chemical and GC-MS analysis. At the end of the perfusion (70 min), livers were freeze-clamped with aluminum tongs precooled in liquid N2. The frozen livers were ground to a fine powder and extracted into perchloric acid, and the neutralized extracts were used for analysis of adenine nucleotides by enzymatic techniques (18) and amino acids by high pressure liquid chromatography, using precolumn derivatization with o-phthalaldehyde (19). Urea and ammonia were determined by standard methods (20, 21). The level of N-AG in freeze-clamped livers was determined using GC-MS and a modification of the conventional isotope dilution technique as we have previously described (3).

GC-MS Determination of 15N-Labeled Metabolites-- GC-MS measurements of 15N-isotopic enrichment were performed on a Hewlett Packard 5970 HP-MS coupled with a 5890 HP-GC, as described previously (3, 4). Briefly, 15NH3 enrichment was measured after conversion of ammonia to glutamate (22). Isotopic enrichment in glutamate, aspartate, or N-acetylglutamate was determined following separation of these amino acids from glutamine and asparagine (3). 15N enrichment in citrulline, alanine, urea, and glutamine was measured following removal of any arginine that might be present as this can interfere with the GC-MS analysis of citrulline. A 500-µl aliquot of the medium or liver perchloric acid extract was added to an equal volume of NaHCO3 buffer (pH 7.1), and this was applied to an AG-50 (Na+; 20-50 mesh, 0.5 × 2.5 cm) column. Arginine remained bound to the resin, whereas citrulline, urea, and other amino acids were eluted with 3 ml of water. The effluent was collected and then applied to an AG-50 (H+; 100-200 mesh; 0.5 × 2.5 cm) column that was washed with 4 ml of water. Urea and amino acids were eluted with 3 ml of 4 N NH4OH (3, 4).

For measurement of 15N enrichment, urea and amino acids were converted into t-butyldimethylsilyl derivatives. The m/z 231, 232, 233, and 234 of the urea t-butyldimethylsilyl derivative was monitored for singly labeled and doubly labeled urea determination (3, 4). Isotopic enrichment in citrulline, glutamine, glutamate, aspartate, and alanine was monitored using ratios of ions at m/z of 443/442, 432/431, 433/432, 419/418, and 261/260, respectively (3, 4).

Data Presentation and Analysis-- The formation of 15N-labeled metabolites was determined by the product of their isotopic enrichment (APE/100) times concentration (nmol/g wet wt) and is expressed as nmol 15N-metabolite/ wet wt. Flux through the PDG pathway during the course of the perfusion was calculated from the sum of 15N-labeled urea, ammonia, alanine, and glutamate formation from [5-15N]glutamine (3).

The endogenous production of glutamine (Pr) during the course of perfusion was calculated according to the equation: Pr = I × [(E1/E2) - 1], where I is the rate of 15N-labeled glutamine infusion (nmol·min-1·g-1), E1 is the isotopic enrichment of the influent glutamine, and E2 is the 15N enrichment of effluent glutamine at the steady state (in most cases between 50 and 70 min).

The distribution of [15N]urea mass isotopomers was calculated using the mathematical model we have previously described (4). Briefly, when 15N-labeled glutamine is provided as substrate the urea formed may have a mass of 60, 61, or 62 molecular weight depending on whether zero, one, or two 15N atoms of urea are labeled. This in turn depends on the enrichment of 15N in the two relevant nitrogen pools, i.e. the mitochondrial ammonia pool and the cytoplasmic aspartate pool. Let the fractional abundance of 15N in the mitochondrial ammonia pool be x; then the fractional abundance of 14N in the same pool is 1 - x. Similarly, let the fractional abundance of 15N in the cytoplasmic aspartate pool be y; then the fractional abundance of 14N in the same pool is 1 - y. Then the fractional abundance of the urea isotopomers will be: Um = (1 - x)(1 - y), where Um is the fraction of urea containing no atom of 15N; Um+1 = 1 - [xy + (1 - x)(1 - y)], where Um+1 is the fraction of urea containing one atom of 15N; Um+2 = xy, where Um+2 is the fraction of urea containing two atoms of 15N. Therefore, Um, Um+1, and Um+2 sum to unity. This relationship permits one to predict the fractional abundance of Um, Um+1, and Um+2 at any given abundance of 15N in the mitochondrial ammonia and cytoplasmic aspartate pools, i.e. at any values of x and y.

Statistical analyses were carried out by the use of Student's t test or analysis of variance test, as appropriate. A p value less than 0.05 was taken as indicating a statistically significant difference. Regression analysis was carried out using the Sigma Plot Program.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Characterization of the Perfused Livers-- A total of eighteen perfusions were carried out: nine with [5-15N] glutamine, of which three each were infused with saline, glucagon, or insulin from 40 to 70 min, and nine with [2-15N] glutamine, of which three each were infused with saline, glucagon, or insulin from 40 to 70 min. Because all perfusions were presented with the same concentrations of substrates (1 mM glutamine, 0.3 mM NH4Cl, 2.1 mM lactate, and 0.3 mM pyruvate), it was possible to combine those with [2-15N] and those with [5-15N] glutamine when basic perfusion characteristics were assessed. As illustrated in Fig. 1, in the control perfusions (saline infused) O2 consumption was stable, at about 2.5 µmol, min-1·g-1 from 25 to 70 min. Likewise the outputs of alanine, glutamate, and urea were stable as well as the uptake of ammonia and glutamine (the apparent increase in urea output between 50 and 70 min was not significant). At 70 min the measured uptake of nitrogen (from glutamine and ammonia) was about 100 nmol of nitrogen min-1·g-1, whereas the output of nitrogen (in urea, alanine, and glutamate) was about 150 nmol of nitrogen min-1·g-1, indicating that endogenous sources contributed about a third of the nitrogen measured in the perfusion effluent (Fig. 1A).


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  		Fig. 1.   Total nitrogen metabolism and balance and O2 consumption in the perfused liver. Livers were perfused with 1 mM 15N-labeled glutamine and 0.3 mM NH4Cl. The data are the means ± S.D for six livers. diamond , O2; triangle , aspartate; black-diamond , alanine; black-square, urea; black-triangle, glutamine; black-down-triangle , ammonia; , glutamate.

When glucagon was infused from 40 to 70 min (Fig. 1B), there was a significant increase in O2 consumption as well as a significantly increased uptake of glutamine and output of urea and glutamate. At 70 min the measured uptake of nitrogen was about 143 nmol·min-1·g-1, and output was about 214 nmol·min-1·g-1. Fig. 1C shows that when insulin was infused there was stable O2 consumption of 2.5 µmol min-1·g-1). In addition, nitrogen uptake was about 103 nmol of nitrogen min-1·g-1, and output was about 161 nmol of nitrogen min-1·g-1.

In control, the liver content of adenine nucleotides after 70 min perfusion (ATP, 2.59 ± 0.29 µmol/g; ADP, 1.34 ± 0.18 µmol/g; and AMP, 0.29 ± 0.09 µmol/g) were quite similar to levels found in vivo (18). There is no significant difference following glucagon or insulin infusion. Similarly, there is no difference in the levels of aspartate and citrulline. The only significant (p < 0.05) differences found were increased levels of glutamate (1.95 ± 0.25 versus 3.26 ± 0.71 µmol/g in the control and glucagon-infused livers, respectively), decreased levels of glutamine (3.38 ± 0.44 versus 2.45 ± 0.4 µmol/g in control and glucagon-infused livers, respectively), decreased ornithine level (0.17 ± 0.03 versus 0.09 ± 0.02 µmol/g in control and glucagon-infused livers, respectively), and increased levels of alanine from 1.35 ± 0.3 in control to 2.22 ± 0.64 µmol/g during insulin perfusions.

15N-Labeled Glutamine Uptake and Metabolism-- Fig. 2 depicts the uptake of 15N-labeled glutamine during the course of perfusion. The curves of 15N glutamine uptake (Fig. 2A) are derived from experiments with [2-15N]glutamine and [5-15N]glutamine. As indicated above, this is possible because the experimental conditions are the same. Insulin had little effect on glutamine uptake (p > 0.05, compared with saline perfusion), whereas glucagon significantly (p < 0.05) stimulated glutamine uptake.


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  		Fig. 2.   15N-Labeled glutamine uptake (A) and its metabolism via the flux through the PDG pathway (B) during the course of liver perfusion for saline-infused livers (open circle ) glucagon-infused livers (black-triangle), and insulin-infused livers (triangle ). Data for glutamine uptake are combined from [2-15N]glutamine and [5-15N]glutamine, and flux through the PDG was estimated from experiments with [5-15N]glutamine as detailed under "Experimental Procedures." Bars are the means ± S.D for three livers. *, p < 0.05 compared with saline perfusions.

If we take the sum of 15N-labeled products from [5-15N]glutamine as an index of glutamine metabolism via the flux through the PDG pathway, the data indicate a good agreement between glutamine uptake (Fig. 2A) and glutamine metabolism via the PDG pathway (Fig. 2B). It has been well established that glutamine metabolism and ammonia formation from glutamine are mediated essentially via flux through the PDG pathway (1, 3, 12, 23). In the current study we estimated the flux through PDG as the sum of 15N-labeled ammonia, urea, alanine and glutamate production during the course of perfusion. We have found that the formation of other amino acids from [5-15N]glutamine accounted for less than 5% of [5-15N]glutamine consumption. The calculation shows that the rates of the flux through PDG are 354 ± 64, 756 ± 77, and 412 ± 86 nmol·min-1·g-1 (means ± S.D. after 70 min) in the control, glucagon, and insulin perfusions, respectively. Such calculations assume that the 15N enrichment of intracellular glutamine is the same as that in the influent perfusate, but this is not so. Fig. 3 illustrates that the average enrichment of the effluent [5-15N]glutamine at the end of the perfusion (70 min) is about 89 APE regardless of the hormonal treatment. At the same time, in the freeze-clamped livers it averaged 67, 71, and 73 APE in the control, glucagon-infused and insulin-infused livers, respectively. These enrichments were not significantly different from each other, but such intracellular dilution of the perfusate glutamine indicates that the estimates of flux through glutaminase, calculated above, should each be increased by about 25%. The rates of the PDG pathway then will be 417, 892, and 486 nmol·min-1·g-1 in control, glucagon, and insulin perfusion, respectively. The current rates of flux via the PDG are in agreement with previous studies using [14C]glutamine (12, 23). Therefore, the current data demonstrate that glucagon significantly (p < 0.05) stimulated the flux through the PDG pathway, whereas insulin had little effect on glutamine metabolism via the PDG pathway compared with control perfusion (Fig. 2B).


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  		Fig. 3.   Isotopic enrichment of the precursor glutamines in the perfusate (hatched bars) and liver extract (solid bars) at the end of 70 min of perfusion. Bars are the means ±S.D for three livers. *, p < 0.05 compared with perfusate enrichment.

When we examine the hormonal regulation of glutamine metabolism, we must take into account the rate of hepatic glutamine synthesis (recycling) as indicated previously (2, 23). The initial 15N enrichment of effluent glutamine (at 15 min) is 93.5 ± 1.3 APE (mean ± S.D, n = 18), regardless of the experimental condition. However, the 15N enrichment of glutamine in the effluent at 70 min is decreased to 89.3 ± 3.1 APE (p < 0.001). There are two possibilities to explain this result. One possibility is that because of slow uptake of perfusate glutamine, there is little opportunity for equilibration, but after 70 min of perfusion this seems unlikely. The second possibility is that intracellular isotopic enrichment is diluted by the endogenous production of unlabeled glutamine. From the isotopic enrichment of 15N-labeled glutamine at the steady state (50-70 min), we were able to calculate the rate of the endogenous glutamine production. In perfusions with [5-15N]glutamine, these rates are 191 ± 109, 204 ± 108, and 274 ± 22 nmol·min-1·g-1 (mean ± S.D, n = 3) in the control, glucagon-infused, and insulin-infused livers, respectively. These rates are not significantly different from each other. The estimated rates of the endogenous glutamine synthesis are about 30% higher in the livers perfused with [5-15N]glutamine compared with those perfused with [2-15N]glutamine. We consider the data obtained with [5-15N]glutamine to be more reliable because the APE of effluent glutamate in these perfusions is less than 10, whereas in livers perfused with [2-15N]glutamine it ranged between 25-50 APE. Thus, if uptake of glutamate by the perivenous hepatocytes were a significant source of glutamate for glutamine synthesis in these cells, there would be an appreciable underestimation of the rate of glutamine recycling if this glutamate were already appreciably labeled.

Production of 15N-Labeled Products-- Fig. 4 shows the production of labeled products in livers perfused with [5-15N]glutamine and infused with saline (Fig. 4A), glucagon (Fig. 4B), or insulin (Fig. 4C). As indicated above, the PDG pathway will convert [5-15N]glutamine to 15NH3, which may be released as 15NH3 or incorporated into glutamate by glutamate dehydrogenase or into carbamyl phosphate via CPS-I (and then, into urea). 15N in glutamate may be transaminated to other amino acids that are released by the liver (e.g. alanine) or transaminated to aspartate, which can be incorporated into urea. As indicated in Fig. 4, the principal nitrogenous product released was urea. After 40 min of perfusion, [15N]urea production amounted to about 100-150 nmol·min-1·g-1 in each of the perfusions and continued to increase to 240-300 nmol·min-1·g-1 at 70 min in the perfusions with saline or insulin infusions. In the case of the glucagon perfusions, however, [15N]urea production increased to about 650 nmol·min-1·g-1. The [15N]ammonia production profiles showed a similar response to glucagon: 15NH3 production plateaued at about 80 nmol·min-1·g-1 in the control and insulin-perfused livers but increased to 130 nmol·min-1·g-1 in the glucagon perfusions. Production of alanine and of glutamate were minor (5-20 nmol·min-1·g-1).


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  		Fig. 4.   Production of 15N-labeled metabolites from [5-15N]glutamine during the course of liver perfusion. Data are the products of 15N enrichment (APE/100) times concentration (nmol·min-1·g-1). Bars are the means ± S.D for three livers. black-square, urea; triangle , ammonia; black-down-triangle , alanine; open circle , glutamate.

Fig. 5 shows the production of labeled products in livers perfused with [2-15N] glutamine and infused with saline (Fig. 5A), glucagon (Fig. 5B), and insulin (Fig. 5C). Glutaminase will convert [2-15N]glutamine to [15N]glutamate within hepatic mitochondria, and this glutamate can then be transaminated to form [15N]aspartate or alanine or be deaminated via glutamate dehydrogenase to yield 15NH3. 15N from [2-15N]glutamine may be incorporated into urea either through aspartate or through carbamyl-phosphate synthetase. As in the experiments with [5-15N]glutamine, experiments with [2-15N]glutamine demonstrate that in the control perfusions (Fig. 5A) the principal labeled product was urea that was produced at a rate of about 120-200 nmol·min-1·g-1 at 40 min, and this increased to 320, 760, and 277 nmol·min-1·g-1 at 70 min in the control, glucagon-infused, and insulin-infused perfusions, respectively. The other 15N-labeled products were relatively minor, but there were important differences between them. At 70 min in the control perfusions, the production of 15N-labeled ammonia, glutamate, and alanine were 27, 19, and 19 nmol·min-1·g-1. In the glucagon-infused livers, these rates were 66, 81, and 12 nmol·min-1·g-1 and in the insulin-perfused livers rates of 40, 16, and 29 nmol·min-1·g-1 were found. The increased production of 15N-labeled ammonia and glutamate in the glucagon-infused livers were significantly different from controls, as was the increased production of [15N]alanine in the insulin-infused perfusions.


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  		Fig. 5.   Production of 15N-labeled metabolites from [2-15N]glutamine during the course of liver perfusion. Data are the products of 15N enrichment (APE/100) times concentration (nmol·min-1·g-1). Bars are the means ± S.D for three livers. black-square, urea; triangle , ammonia; black-down-triangle , alanine; open circle , glutamate.

Comparison of 15N Enrichment in Perfusate with That in the Liver-- An important aim of the current study is to address the following question: Is the 15N labeling of intracellular metabolites in equilibrium with the effluent metabolites? To this end, we determined the 15N enrichment in liver metabolites after freeze-clamping at 70 min and in samples taken simultaneously in the perfusate outflow. These data are shown in Fig. 6, demonstrating a highly significant relationship (p < 0.0001) between 15N enrichment in perfusate and liver for citrulline, aspartate, and glutamate.


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  		Fig. 6.   15N enrichment (APE) in perfusate at the end of 70 min of perfusion versus freeze-clamped liver. black-square, [5-15N]glutamine-control; black-diamond , [5-15N]glutamine-glucagon; , [5-15N]glutamine-insulin; , [2-15N]glutamine-control; diamond , [2-15N]glutamine-glucagon; open circle , [2-15N]glutamine-insulin.

Fig. 6 also shows the differing degrees to which these amino acids were labeled in the different experimental situations. It is apparent that aspartate and glutamate were more heavily labeled by [2-15N]glutamine than by [5-15N]glutamine. This is to be expected as glutaminase will produce [15N]glutamate, which will be readily transaminated to aspartate. Such a product-precursor relationship is borne out by the slightly higher 15N enrichment in the precursor (glutamate) than in the products (aspartate). Of course the 15N enrichment in glutamate is considerably lower than in its precursor, glutamine. Citrulline was more heavily labeled by [5-15N]glutamine. This is consistent with the 15NH3 produced through the PDG pathway being efficiently removed by carbamyl-phosphate synthetase.

Fig. 7 shows a plot of the 15N enrichment of perfusate ammonia with that of perfusate citrulline. Data points from the perfusions with [2-15N]glutamine and [5-15N]glutamine are denoted with separate symbols. It is evident that there was a highly significant relationship (p < 0.0001) between 15N enrichment in perfusate ammonia and citrulline for either substrate, and furthermore, it is clear that the points obtained with [2-15N]glutamine fall on the same line as those obtained with [5-15N] glutamine. This will be discussed in more detail under "Discussion."


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  		Fig. 7.   15N enrichment (APE) in perfusate ammonia as a function of perfusate citrulline. open circle , perfusions with [2-15N]glutamine; , perfusions with [5-15N]glutamine.

Production of Urea Mass Isotopomers-- Fig. 8 illustrates the actual distribution of urea mass isotopomers when [2-15N]glutamine or [5-15N]glutamine was used as labeled precursor. The data indicate that glucagon rapidly and remarkably stimulated Um+1 and Um+2 production regardless of the position of 15N in glutamine. However, insulin had little effect on the formation of [15N]urea mass isotopomers compared with control perfusions. It should be emphasized that a steady state level of isotopomer production was not achieved immediately, and indeed, the proportions of Um+1 and Um+2 continued to increase, and the proportion of Um continued to decrease until about 60 min. We therefore used the 15N enrichment in the effluent citrulline and aspartate to calculate [15N]urea isotopomer distribution at 70 min using the mathematical model we have previously described (4).


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  		Fig. 8.   Production of [15N]urea mass isotopomers during the course of perfusion. Livers were perfused with 1 mM [5-15N]glutamine or [2-15N]glutamine and other nutrients as indicated under "Experimental Procedures." Um, Um+1, and Um+2 are urea isotopomers containing no, one, or two 15N, respectively, for saline-infused livers (open circle ), glucagon-infused livers (black-triangle), and insulin-infused livers (triangle ). Each data point represents the mean ± S.D for three livers. *, p < 0.05 compared with saline perfusions.

In our previous work we showed that during perfusions with 15NH4Cl, the 15N enrichment in perfusate citrulline and aspartate were reliable proxies for the enrichment, respectively, of mitochondrial ammonia and cytoplasmic aspartate (4). It is important, therefore, to determine whether these relationships would also hold when 15N-labeled glutamine was the precursor. These data are shown in Table I. In this table we provide data at 40 min (when n = 9 for each group) and at 70 min (n = 3 for each group). It is quite apparent that we were able to predict, very accurately, the proportion of unlabeled urea produced (Um) and also the proportion of singly labeled produced (Um+1); however, in the case of doubly labeled urea (Um+2), the agreement was not good, because there was always a larger proportion of this isotopomer found than predicted.

                              
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Table I
Comparison of experimental and predicted isotopomer percentages
Data are given as means ± S.D. Experimental details are given under "Experimental Procedures." Isotopomer prediction was from the 15N enrichment of perfusate aspartate and citrulline as described in the text.

Hormonal Regulation of N-Acetylglutamate Levels-- In a study of nitrogen metabolism in isolated hepatocytes, we observed that glucagon increased and insulin decreased N-acetylglutamate levels (3). In the present study we measured N-AG in livers freeze-clamped at the end of the 70-min perfusions. The current measurements substantiate our previous observation and demonstrate that glucagon caused a significant increase in N-AG, 130 ± 29 nmol·min-1·g-1 compared with 61 ± 8 nmol·min-1·g-1 for the control (means ± SD, n = 6). A decrease of approximately 20% was observed in the insulin perfusions (48 ± 12 nmol·min-1·g-1), but this did not quite reach statistical significance (p = 0.06) from the control values. Previous investigations have indicated a short term regulation of urea synthesis through rapid changes in N-AG levels, because the mitochondrial carbamyl-phosphate synthetase-I is inactive when N-AG is absent (6, 7, 25). The current study indicates a linear correlation between hepatic N-AG levels and the production of [15N]urea mass isotopomers, suggesting that the hormonal regulation of N-AG synthesis may play a key role in ureagenesis from glutamine.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In 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 (3, 12-16). Elucidation of the hormonal regulation of [15N]urea mass isotopomer production from 15N-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 (12, 26, 27).

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 (3, 12, 16, 23). 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 of 15N 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 (28), 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 (28, 29). 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 (29). 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 (1-3), 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 for 15NH3 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 alpha -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 (3). 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 (4), 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 and 15NH3 will be produced via the PDG pathway. In the mitochondria, this 15NH3 can be metabolized to form [15N]glutamate via the GDH pathway. If 15N-labeled ammonia and glutamate are used for glutamine synthesis, then glutamine would be labeled at 2-N and 5-N (4). 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. (24) have shown that an intrahepatic glutamine cycle exists whereby there is simultaneous catabolism and synthesis of glutamine (in periportal and perivenous hepatocytes, respectively. Haüssinger (23) 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. (23, 24).

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 (9). 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 15NH3 that leaves the mitochondria will be reflected in perfusate NH3 (4). Fig. 7 shows the correlation between perfusate 15NH3 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 (31) 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 (31). 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 (31) 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. 2A), 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 of Um and increase in the proportion of Um+1 and Um+2 throughout the perfusions are attributable to the increased provision of 15N-labeled urea precursors with time. The comparison with experimental and predicted isotopomer production (Table I) showed very good agreement for the proportion of Um and Um+1. However, there was quite a difference between the predicted and experimental values of Um+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 (24). 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 (24). 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 and later portions of the acinus. Support for this explanation is provided in our previous study demonstrating a significant correlation between observed and predicted Um+1 and Um+2 when various 15NH4Cl enrichments were used (4). Brunengraber et al. (30) have emphasized the errors that can occur in measuring biosyntheses by means of mass isotopomer distribution when there are variations in the enrichment of the labeled precursor because of compartmentation. A similar explanation may underlie the differences we find between the predicted and experimentally determined values for the Um+2 isotopomer of urea.

In conclusion, the current investigation indicates that there is no channeling of [5-15N]glutamine toward the synthesis of mitochondrial carbamyl phosphate. Ammonia formed from glutamine, whether from [5-N] via PDG or from [2-N] via GDH, enjoys the same partition between incorporation into carbamyl phosphate or leaving the mitochondria. This observation is of importance in terms of understanding perturbations of hepatic glutamine metabolism and ureagenesis in vivo, as we have previously demonstrated (32).

    ACKNOWLEDGEMENT

We acknowledge the excellent technical assistance provided by B. Hall and Zhiping Lin.

    FOOTNOTES

* 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. The 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.

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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