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Glutamine Synthesis in the Developing Porcine Placenta¹

Departments of Animal Science³ Veterinary Anatomy and Public Health,⁴ Texas A&M University, College Station, Texas 77843-2471

	ABSTRACT

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Glutamine plays a vital role in fetal carbon and nitrogen metabolism and exhibits the highest fetal:maternal plasma ratio among all amino acids in pigs. Such disparate glutamine levels between mother and fetus suggest that glutamine may be actively synthesized and released into the fetal circulation by the porcine placenta. We hypothesized that branched-chain amino acid (BCAA) metabolism in the placenta plays an important role in placental glutamine synthesis. This hypothesis was tested by studying conceptuses from gilts on Days 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day). Placental tissue was analyzed for amino acid concentrations, BCAA transport, BCAA degradation, and glutamine synthesis as well as the activities of related enzymes (including BCAA transaminase, branched-chain -ketoacid dehydrogenase, glutamine synthetase, glutamate-pyruvate transaminase, and glutaminase). On all days of gestation, rates of BCAA transamination were much greater than rates of branched-chain -ketoacid decarboxylation. The glutamate generated from BCAA transamination was primarily directed to glutamine synthesis and, to a much lesser extent, alanine production. Placental BCAA transport, BCAA transamination, glutamine synthesis, and activities of related enzymes increased markedly between Days 20 and 40 of gestation, as did glutamine in fetal allantoic fluid. Accordingly, placental BCAA levels decreased after Day 20 of gestation in association with a marked increase in BCAA catabolism and concentrations of glutamine. There was no detectable catabolism of glutamine in pig placenta throughout pregnancy, which would ensure maximum output of glutamine by this tissue. These novel results demonstrate glutamine synthesis from BCAAs in pig placentae, aid in explaining the abundance of glutamine in the fetus, and provide valuable insight into the dynamic role of the placenta in fetal metabolism and nutrition.

placenta

	INTRODUCTION

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Amino acids play a central role in cell functions, acting as precursors for the synthesis of peptides, neurotransmitters, nitric oxide, polyamines, carnitine, cell proteins, hormones, nucleotides, creatine, and porphyrins [1]. Additionally, these building blocks of protein provide a vital source of energy for fetal growth [2]. Amino acids are also signaling molecules, thereby regulating embryonic development [3, 4]. Of particular interest is glutamine, which plays an important role in fetal and placental nutrition [2, 5]. For example, glutamine is essential for the synthesis of nucleotides and amino sugars that are necessary for cell proliferation and differentiation. Furthermore, animal studies have established that glutamine is crucial for interorgan carbon and nitrogen metabolism in the conceptus (embryos/ fetus and associated placental membranes) [6, 7]. Consistent with its versatile functions, glutamine exhibits high fetal:maternal ratio in plasma among all amino acids in mammals [8, 9]. For example, in pigs, fetal:maternal plasma ratios for glutamine are 3.87 and 2.88, respectively, at Days 45 and 110 of gestation [8]. Such disparate glutamine levels between mother and fetus suggest that glutamine is actively synthesized and released by the placenta.

Previous studies with skeletal muscle indicate that de novo glutamine synthesis involves the transamination of branched-chain amino acids (BCAAs; isoleucine, leucine, and valine) [10]. Interestingly, the placentae of humans [11] and sheep [12] exhibit high BCAA transaminase activity. This would result in the transamination of BCAAs with - ketoglutarate to form branched-chain -ketoacids (BCKA) and glutamate, which is a precursor of glutamine (Fig. 1). Such a pathway for placental glutamine synthesis is possible in view of the previous finding that the rat placenta contains glutamine synthetase activity [13]. However, metabolic studies of BCAA catabolism and glutamine synthesis in placental tissues have not been reported.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Branched-chain amino acid transamination coupled with glutamine and alanine synthesis in pig placentae. Enzymes that catalyze the indicated reactions are: 1) BCAA transaminase; 2) BCKAD; 3) glutamine synthetase; 4) glutamate-pyruvate transaminase; 5) glucose metabolism via glycolysis and the degradation of glucogenic amino acids [10]; and 6) glucose metabolism via glycolysis and the Krebs cycle as well as the degradation of amino acids via multiple pathways (including the Krebs cycle) [10]. The corresponding -ketoacids of leucine, isoleucine, and valine are -ketoisocaproate, -keto-ß-methylvalerate, and -ketoisovalerate, respectively. Placentae transport BCAAs and release glutamine through specific transporters on the plasma membrane.

In contrast to glutamine, fetal:maternal plasma ratios of BCAAs are relatively low in mammals throughout pregnancy [8, 9]. For example, plasma fetal:maternal ratio of leucine in pigs is less than 0.70 between Days 45 and 110 of gestation [8], suggesting extensive catabolism of BCAAs in the porcine placenta. On the basis of this observation, we hypothesized that BCAA metabolism plays an important role in placental glutamine synthesis, which contributes to an increase in fetal glutamine availability. This hypothesis was tested using placentae from pigs between Days 20 and 110 of gestation (term, 114 days). Elucidating a link between BCAA metabolism and glutamine production in placental tissue would provide valuable insight into the dynamic role of the placenta in fetal amino acid metabolism and nutrition.

	MATERIALS AND METHODS

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Chemicals

Methanol and HPLC-grade water were procured from Fisher Scientific (Fair Lawn, NJ). Radioactive reagents were obtained from American Radiolabeled Chemicals (St. Louis, MO). Immediately before use, [¹⁴C]BCAAs were purified using AG 1-X8 (acetate form, 200–400 mesh) as resin bed (0.6 x 6 cm) and deionized water (2 ml) as the eluting solvent [14]. Lactate dehydrogenase and protein phosphatase were from Roche (Basel, Switzerland). AG 1-X resin was purchased from Bio-Rad Laboratories (Hercules, CA), and Soluene 350 and UltimaGold were obtained from Perkin Elmer (Boston, MA). NCS-II came from Amersham Biosciences (Piscataway, NJ). All other chemicals utilized in this study were obtained from Sigma (St. Louis, MO).

Experimental Animals

Sexually mature crossbred gilts (Yorkshire x Landrace dams and Duroc x Hampshire sires) were observed daily for estrous behavior. Gilts exhibiting at least two estrous cycles of normal duration (18–21 days) were bred to crossbred boars (Yorkshire x Landrace dams and Duroc x Hampshire sires) and hysterectomized on Day 20, 30, 35, 40, 45, 50, 60, 90, or 110 of gestation (n = 6 per day) [15]. Briefly, gilts received an i.m. injection of Telazol (1 mg/kg body weight) to induce anesthesia, followed by administration of isofluorane (1% to 5%) via a face mask during surgery. The uterus was removed by midventral laparotomy. Placentae (the chorioallantois) were exposed and isolated by dissection. A portion of placental tissue was used immediately for metabolic studies (BCAA transport, BCAA degradation as well as glutamine and alanine metabolism). Remaining placental tissue was stored at –80°C for determination of amino acid content and enzyme assays within 1 wk. The pig has a true epitheliochorial placenta [16]. We collected the entire chorioallantois from each fetal-placental unit and took great care not to include the amnion or necrotic tips. The chorioallantois portion of the placenta is rather homogeneous when compared with the synepitheliochorial placentae of ruminants or the hemochorial placentae of humans and laboratory rodents [16]. Given the relative homogeneity of the porcine placenta, we would not expect different parts of the placenta to exhibit differences in BCAA transport and degradation. This is supported by our preliminary findings that there were no significant differences (P > 0.05) in BCAA transport or BCAA degradation in different parts of placentae at Day 40 or Day 110 of gestation. This study was approved by the Texas A&M University Institutional Agricultural Animal Care and Use Committee.

Amino Acid Concentrations in Pig Placenta

Amino acids were determined using HPLC methods involving precolumn derivatization with ophthaldialdehyde [17]. Briefly, 100 mg of placental tissue were homogenized in 2 ml 1.5 M HClO₄ and then neutralized with 1 ml K₂CO₃. Samples were centrifuged at 2000 x g for 10 min, and the supernatant fluids were used for amino acid analysis. The HPLC system (Waters Inc., Milford, MA) consisted of: 1) a model 600E Powerline multisolvent delivery system with 100-µl heads, a model 712 WISP Autosampler, and a Millennium-32 workstation; 2) a model 474 scanning fluorescence detector (excitation 340 nm, emission 450 nm, gain 100); and 3) a Supelco C₁₈ reversed-phase guard column (4.6 mm x 5 cm, 20–40 µm) coupled to a Supelco C₁₈ reversed-phase column (4.6 mm x 15 cm, 3 µm). Amino acids in samples were quantified on the basis of known amounts of standards.

Branched-Chain Amino Acid Transport in Placenta

BCAA transport in pig placentae was determined using ¹⁴C-labeled leucine, isoleucine, or valine, as described for other tissues [18]. Briefly, samples of placentae (200 mg) were washed three times in oxygenated (95% O₂:5% CO₂; v/v) Krebs-Henseleit bicarbonate (KHB) buffer containing 20 mM Hepes (pH 7.4) and 5 mM glucose. Samples were then incubated at 37°C for 5 min in 1 ml of oxygenated KHB buffer consisting of 20 mM Hepes, 5 mM glucose, 0.5 or 2 mM each of BCAAs, 0.05 µCi each of [U-¹⁴C]BCAAs, and 0.05 µCi [³H] inulin (an extracellular marker). After the 5-min incubation, the tissues were rinsed thoroughly with fresh KHB buffer, and then solubilized in 0.5 ml of Soluene. The solution was measured for ¹⁴C and ³H radioactivities using a dual-channel counting program in a Packard 1900 liquid scintillation counter (Meriden, CT). The specific activity of [¹⁴C]BCAAs in the medium was used to calculate BCAA uptake by placentae. Results from preliminary experiments established that BCAA uptake was linear over a 5-min period.

Branched-Chain Amino Acid Degradation in Placentae

BCAA degradation was quantified using ¹⁴C-labeled leucine, isoleucine, or valine, as previously described for skeletal muscle [19]. Samples of placentae (200 mg) were preincubated at 37°C for 30 min in 2 ml of oxygenated (95% O₂:5% CO₂, v/v) KHB buffer and then incubated at 37°C for 2 h in 2 ml of oxygenated KHB containing 5 mM glucose, 20 mM Hepes (pH 7.4), 0.5 mM NH₄Cl, 0.5 or 2 mM each of BCAAs, and 0.5 µCi each of [U-¹⁴C]BCAAs. After a 2-h incubation at 37°C, 0.2 ml Soluene 350 was injected through the rubber cap into suspended center wells and 0.2 ml of 1.5 M HClO₄ acid was injected into the incubation medium to liberate ¹⁴CO₂ from corresponding BCKAs. After a 60-min incubation, suspended wells were transferred to scintillation vials containing 15 ml of cocktail for measurement of ¹⁴CO₂ produced from enzymatic decarboxylation of BCKA by placental tissue. To measure net production of [¹⁴C]BCKA by placental tissue, replacement center wells each received 0.2 ml Soluene, and 0.7 ml of 30% H₂O₂ was introduced into the acidified incubation medium to chemically decarboxylate BCKA. The center wells were collected following a 1-h incubation at 37°C. Scintillation vials were mixed with cocktail for measurement of ¹⁴CO₂ radioactivity using a Packard liquid scintillation counter [20].

Production of Glutamine and Alanine in Placentae Incubated with BCAAs

Production of glutamine and alanine in porcine placental tissue was determined using established methods [19]. Briefly, placental samples (200 mg) were preincubated at 37°C for 30 min in 2 ml of oxygenated (95% O₂:5% CO₂, v/v) KHB buffer and then incubated at 37°C for 2 h in 2 ml of oxygenated KHB buffer containing 5 mM D-glucose; 20 mM Hepes (pH 7.4); and 0, 0.5, or 2 mM each of BCAAs. Following the 2-h incubation, 0.5 ml of 2% (w/v) trichloroacetic acid was added to the incubation medium. The whole mixture (medium plus tissue) was homogenized, and the extract was analyzed for amino acids using HPLC [17]. Rates of glutamine and alanine production were calculated on the basis of concentrations in cell extracts in the presence or absence of added BCAA substrates.

Glutamine and Alanine Degradation in Placentae

To determine whether glutamine and alanine could be catabolized in porcine placenta, tissues (200 mg) were preincubated at 37°C for 30 min in 2 ml of oxygenated (95% O₂:5% CO₂, v/v) KHB buffer and then incubated at 37°C for 2 h in 2 ml of oxygenated KHB buffer containing 5 mM D-glucose, 20 mM Hepes (pH 7.4), and 0 or 2 mM glutamine or alanine. Following the 2-h incubation, the whole mixture (medium plus tissue) was analyzed for amino acids as described previously [17].

BCAA Transaminase and BCKA Dehydrogenase Activities in Placentae

Activities of both BCAA transaminase (EC 2.6.1.42) and BCKA dehydrogenase (EC 1.2.4.4) in pig placentae were determined as described for ovine tissues [12]. Placental samples (200 mg) were homogenized in 1 ml freshly prepared buffer consisting of 50 mM Hepes (pH 7.5), 3 mM EDTA, 5 mM dithiothreitol (DTT), 2% (v/v) Triton X-100, and 0.1% (w/v) protease inhibitor (aprotinin, chymostatin, pepstatin A, and PMSF). Homogenates were centrifuged at 600 x g for 10 min and the supernatant fluids subjected to three cycles of freezing (in liquid nitrogen) and thawing (in a 30°C waterbath). The BCAA transaminase assay mixture consisted of 0.5 ml of 50 mM Tris/HCl buffer (pH 8.6), 0.1 ml of 1.6 mM pyridoxal phosphate, 0.2 ml of 50 mM -ketoglutarate, 1 ml of 10 mM leucine plus 0.1 µCi purified [1-¹⁴C]leucine. After warming in a 30°C water bath, sample tubes received 0.2 ml tissue extract, and blanks received 0.2 ml homogenization buffer. Following a 20-min incubation, 0.2 ml Soluene was injected, through the rubber stopper, into each suspended center well, and 1 ml of 1.5 M HClO₄/30% (w/w) H₂O₂ (0.3:0.7) was injected, through the same stopper, into the assay solution. After a 60-min incubation, center wells were transferred to scintillation vials containing 15 ml cocktail for measurement of ¹⁴C radioactivity in a Packard liquid scintillation counter.

The BCKA dehydrogenase (BCKAD) assay mixture consisted of 0.1 ml of 20 mM MgCl₂, 0.1 ml of 10 mM DTT, 0.1 ml of 4 mM thiamine pyrophosphate plus 4 mM coenzyme A plus 10 mM NAD⁺, 0.4 ml of 50 mM potassium phosphate buffer (pH 7.5), 0.1 ml of tissue extract, and 0.1 ml protein phosphatase (1 U/ml) or 0.1 ml of 1 mM potassium fluoride. Protein phosphatase, which converts BCKAD from an inactive, phosphorylated form into an active, dephosphorylated form, was used to measure total BCKAD activity. Potassium fluoride, an inhibitor of protein kinase, was used to assess BCKAD activity in its active state. Blanks were prepared similarly, using 0.1 ml homogenization buffer instead of tissue extract. All samples and blanks were preincubated for 10 min in a 30°C water bath, after which 0.1 ml of 10 mM -ketoisocaproic acid plus 0.1 µCi [1-¹⁴C]-ketoisocaproic acid was added into all tubes. Tubes were then returned to the water bath for a 15-min incubation at 30°C. The collection of ¹⁴CO₂ was performed as described previously.

Glutamine Synthetase and Glutamate-Pyruvate Transaminase Activities in Placentae

Glutamine synthetase (EC 6.3.1.2) activity was measured as previously described for skeletal muscle [21]. Briefly, 200 mg of placental sample was homogenized in 1 ml buffer consisting of 50 mM Tris (pH 7.9), 2 mM EDTA, and 2 mM DTT. Homogenates were centrifuged at 10 000 x g and 4°C for 10 min. Then 100 µl of the supernatant were mixed with 100 µl of 100 mM MgCl₂ plus 75 mM ATP plus 50 mM phosphocreatine, 100 µl of 100 mM NH₄Cl, 100 µl creatine kinase (100 U/ml), and 100 µl of 100 mM glutamate plus 0.05 µCi [1-¹⁴C]glutamate. This mixture was incubated at 37°C for 30 min, and the reaction was then terminated by addition of 200 µl of 1.5 M HClO₄. After 2 min, 100 µl of 2 M K₂CO₃ was added into each tube. An aliquot (500 µl) of the neutralized extract was loaded into an ion-exchange column (AG 1-X8, acetate form, 200– 400 mesh; 0.6 x 6 cm resin bed). The column was eluted with 2 ml H₂O, and this effluent was collected in a scintillation vial containing 15 ml UltimaGold scintillation cocktail (Packard) for measurement of ¹⁴C radioactivity.

For determination of glutamate-pyruvate transaminase (EC 2.6.1.2) activity, placental tissue (200 mg) was homogenized in 1 ml of 250 mM sucrose plus 1 mM EDTA plus 50 mM potassium phosphate buffer (pH 7.5) containing 0.5% (v/v) Triton X-100, 2.5 mM DTT, and protease inhibitors (5 µg/ml PMSF, 5 µg/ml aprotinin, 5 µg/ml chymostatin, 5 µg/ ml pepstatin A). Homogenates were centrifuged at 10 000 x g and 4°C for 10 min, and the supernatant was used for enzyme assays. The activity of glutamate-pyruvate transaminase was determined at 25°C using a UV/ VIS spectrophotometer as previously described [22]. Briefly, the assay mixture (3 ml) consisted of 80 mM alanine, 7 mM -ketoglutarate, 0.16 mM NADH, 25 µg L-lactate dehydrogenase, cell extracts (0.2–0.5 mg protein), and 80 mM sodium phosphate buffer (pH 7.6). The decrease in NADH was used to determine enzyme activity.

Glutaminase Activity in Placentae

The activity of phosphate-dependent glutaminase (EC 3.5.1.2) was determined as previously described for pig enterocytes [20]. Briefly, placental tissue (200 mg) was homogenized in 2 ml of buffer containing 300 mM sucrose, 1 mM EDTA, and 5 mM Hepes (pH 7.4). The homogenates were subjected to three cycles of freezing (in liquid nitrogen) and thawing (37°C water bath) to release glutaminase from mitochondria. The assay mixture (1 ml), which consisted of 20 mM glutamine, 150 mM potassium phosphate (pH 8.2), and tissue extract (0.2–1 mg protein), was incubated at 37°C for 0 or 20 min. At the end of the incubation, glutamate in the assay mixture was determined using HPLC [17].

Statistical Analysis

Data were analyzed by one-way or two-way ANOVA and the Student- Newman-Keul multiple range test [23], using the Statistical Analysis System software (SAS Institute, Cary, NC). Statistical significance was set at P = 0.05. Data are presented as means with pooled SEM because it is the preferred estimate of experimental error in ANOVA when numbers of observations in treatment groups are equal [23].

	RESULTS

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Concentrations of Amino Acids in Pig Placentae

Data for concentrations of seven glutamine-family amino acids in placentae between Days 20 and 110 of gestation are summarized in Table 1. The most salient observation relates to the abundance of glutamine in the placentae. Although Day 20 measurements showed similar concentrations of glutamate and glutamine (P > 0.05), at all other stages glutamine levels were higher (P < 0.01). Placental concentrations of glutamine increased (P < 0.01) progressively between Days 20 and 40 of gestation. Glutamine levels were highest between Days 40 and 60 of gestation at approximately 200% of Day 20 values (P < 0.01) and declined thereafter (P < 0.01). Concentrations of glutamine in placentae were similar (P > 0.05) between Days 90 and 110 of gestation. Concentrations of aspartate in placentae increased (P < 0.01) progressively between Days 20 and 35 of gestation, were similar (P > 0.05) between Days 35 and 50 of gestation (P > 0.05), and declined thereafter (P < 0.01). In contrast, concentrations of glutamate and alanine in placentae were relatively constant throughout gestation (P > 0.05). Concentrations of all BCAAs in placentae were similar (P > 0.05) between Days 20 and 30 of gestation, declined (P < 0.01) on Day 35 of gestation, and were similar (P > 0.05) between Days 35 and 110 of gestation.

Transport of BCAAs in Pig Placentae

Placental transport of BCAAs increased (P < 0.01) with increasing extracellular concentrations from 0.5 to 2 mM (Table 2). For all days of gestation, rates of placental transport were in the following order: leucine > isoleucine > valine (P < 0.01). Between Days 20 and 40 of gestation, placental transport of BCAAs increased (P < 0.01) progressively and then declined (P < 0.01) between Days 40 and 60 of gestation. Rates of placental transport of leucine, isoleucine, and valine were similar (P > 0.05) between Days 60 and 110 of gestation.

Degradation of BCAAs in Pig Placentae

Data on placental degradation of leucine, isoleucine, and valine at an extracellular concentration of 2 mM are summarized in Table 3. Rates of oxidative decarboxylation of all BCKAs generated from BCAA transamination were remarkably low (P < 0.01) when compared with the net production of BCKAs. Approximately 83% to 90% of the BCAA-derived BCKAs were released from the placental tissue into the incubation medium. For all BCAAs, rates of transamination, BCKA production, and BCKA decarboxylation increased (P < 0.01) progressively between Days 20 and 40 of gestation and then declined (P < 0.01) between Days 40 and 60 of gestation. The highest values represented 4.0-, 3.9-, and 4.4-fold increases (P < 0.01) in degradation for leucine, isoleucine, and valine, respectively, compared with Day 20 of gestation. Rates of BCAA degradation were similar (P > 0.05) between Days 60 and 110 of gestation. At all days of gestation, rates of net transamination, net BCKA production, and BCKA oxidative decarboxylation in pig placentae incubated with 0.5 mM leucine, 0.5 mM isoleucine, and 0.5 mM valine were about 28% to 35% of those obtained in the presence of 2 mM BCAAs (data not shown).

Production of Glutamine and Alanine in Pig Placentae Incubated with BCAAs

Relatively large amounts of glutamine and, to a lesser extent, glutamate and alanine were produced from BCAA degradation in pig placenta at all days of gestation (Table 4). There was no detectable net synthesis of other amino acids from BCAAs in pig placentae (data not shown). Extracellular concentrations of BCAAs, from 0.5 to 2 mM, increased (P < 0.01) placental production of glutamine, glutamate, and alanine (Table 4). In a pattern similar to that of BCAA degradation, production of glutamine, glutamate, and alanine increased (P < 0.01) progressively between Days 20 and 40 of gestation and then declined (P < 0.01) between Days 40 and 60 of gestation.

Lack of Degradation of Glutamine and Alanine in Pig Placentae

There was no detectable disappearance (P > 0.05) of glutamine or alanine from the placenta-containing medium after a 2-h incubation period. There was no detectable production of glutamate, aspartate, or alanine from glutamine by pig placentae between Days 20 and 110 of gestation. Likewise, there was no detectable formation of amino acids (including glutamate, glutamine, and aspartate) from alanine by pig placentae throughout pregnancy. These results indicate the lack of glutamine and alanine degradation via glutaminase and transaminase pathways, respectively, in pig placentae.

Activities of BCAA Transaminase, BCKA Dehydrogenase, Glutamine Synthetase, Glutamate-Pyruvate Transaminase, and Glutaminase in Pig Placentae

The activities of BCAA-metabolic enzymes and glutamine- and alanine-synthetic enzymes were in the following order: BCAA transaminase > glutamate-pyruvate transaminase > glutamine synthetase > BCKAD between Days 20 and 110 of gestation (Table 5). Interestingly, BCAA transaminase activity was about 10- to 15-fold higher (P < 0.01) than that of BCKAD at various days of gestation. BCKAD activity was low in pig placentae regardless of the presence or absence of phosphatase. Activities of BCAA transaminase, glutamine synthetase, and glutamate-pyruvate transaminase increased markedly (P < 0.01) between Days 20 and 40 of gestation, decreased (P < 0.01) between Days 40 and 60 of gestation, and remained at reduced levels during late gestation. Placental BCKAD activity increased (P < 0.01) between Days 20 and 35 of gestation, remained at high levels through Day 40 of gestation, and declined thereafter (P < 0.01) to Day 30 values. The activities of all of these enzymes were highest on Day 40 of gestation. There was no detectable activity of phosphate-dependent glutaminase in pig placentae between Days 20 and 110 of gestation.

	DISCUSSION

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This is the first report of a link between BCAA metabolism and glutamine production in porcine placental tissue. The present study provided the following significant findings: 1) placental BCAA degradation, glutamine synthesis, and activities of related enzymes in the placenta are highest at Day 40 of gestation in association with the highest concentration of glutamine in allantoic fluid; 2) placental concentrations of BCAAs decrease after Day 20 in tandem with a marked increase in glutamine synthesis and concentrations; 3) throughout gestation, rates of BCAA transamination are much greater than rates of BCKA decarboxylation; and 4) the glutamate generated from BCAA transamination was directed primarily to the synthesis of glutamine and, to a much lesser extent, alanine.

Despite reports of the high fetal:maternal plasma ratio for glutamine in mammals [8, 9], little is known about the role of the placenta regarding this phenomenon. In postnatal mammals, skeletal muscle is the major source of glutamine, which derives an -amino group from BCAAs via glutamate formation [10]. The presence of BCAA transaminase in ovine and human placentae [11, 12] and glutamine synthetase in rat placenta [13] suggests a capacity for de novo glutamine synthesis in placentae. However, direct evidence for placental glutamine synthesis is lacking in the literature. Ovine [24] and porcine placentae actively transport BCAAs (Table 2), but plasma fetal:maternal ratios for these amino acids are relatively low, compared with glutamine in mammals, including pigs and sheep [8, 9]. These results imply extensive catabolism of BCAAs by both porcine and ovine placentae. Consistent with this suggestion, uteroplacental tissues accounted for 42% of the total BCAAs utilized by ovine fetus uteroplacenta at Day 130 of gestation [25].

BCAA transaminase converts BCAAs and -ketoglutarate to corresponding BCKAs and glutamate, respectively [26]. Glutamate is then utilized for glutamine synthesis and alanine production by glutamine synthetase and glutamate- pyruvate transaminase, respectively (Fig. 1). Similar to reports for rat placenta [13], these two enzymes exhibit high activities in pig placenta (Table 5). Consistent with the enzymological data, pig placentae produced both glutamine and alanine from BCAAs in a concentration-dependent manner at all days of gestation (Table 4). The ammonia used for placental glutamine synthesis is likely derived from maternal and fetal plasma and is not expected to be limiting. Although catabolism of some amino acids (e.g., valine, isoleucine, serine, and aspartate) may be a source of -ketoglutarate and pyruvate for BCAA transaminase and glutamate-pyruvate transaminase, respectively, glucose metabolism probably plays a major role in providing carbon skeletons for glutamine and alanine synthesis in pig placenta (Fig. 1). This view is substantiated by our findings that rates of glutamine and alanine formation were exceedingly low when pig placentae were incubated in the absence of glucose (G. Wu, unpublished observations). In further support of the role of BCAA metabolism in glutamine synthesis, rates of BCAA transamination closely matched rates of the synthesis of glutamine plus alanine in the placentae (Table 4). For example, at Day 40 of gestation, rates of net transamination of leucine plus isoleucine plus valine in placentae incubated with 2 mM BCAAs averaged 4.45 nmol/ mg tissue per 2 h (Table 3), whereas rates of the synthesis of glutamine plus alanine were 4.23 nmol/mg tissue per 2 h, respectively (Table 4). Similar findings have been reported for skeletal muscle [10, 14, 18, 19].

Results of the present study indicate that, under optimal assay conditions, glutamate-pyruvate transaminase activity was 2- to 3-fold higher than glutamine synthetase activity in pig placentae between Days 20 and 110 of gestation (Table 5). Similar results have been reported for rat placenta [13]. Paradoxically, glutamine, rather than alanine, was the major nitrogenous product of placental glutamate metabolism (Table 4). For example, the rate of glutamine synthesis was 260% greater than the rate of alanine synthesis in Day 40 placentae. This result suggests that BCAA-derived glutamate is a preferential substrate for glutamine synthetase rather than for glutamate-pyruvate transaminase in pig placentae, possibly because of intracellular compartmentalization of BCAAs, -ketoglutarate, and pyruvate metabolism. Thus, as placental glutamine synthesis increased, placental concentrations of glutamine rose markedly between Days 20 and 110 of gestation (Table 1). These results indicate that placental synthesis of glutamine from BCAAs is an important source of glutamine in fetal plasma, thereby contributing to its high fetal:maternal plasma ratio (e.g., > 2.5 in pigs) [8].

Previous in vivo studies detected a low activity of BCKAD [12] and a low rate of leucine oxidation [25] in the ovine placenta. Similarly in pig placentae, BCKAD activity (either phosphorylated or dephosphorylated form of the enzyme) represented <10% of BCAA transaminase activity (Table 5), and BCKA decarboxylation was limited (Table 3). Accordingly, the release of BCKAs into the incubation medium accounted for over 80% of BCAAs transaminated in pig placentae at all days of gestation (Table 3). Thus, the major function of placental BCAA catabolism is to transfer an amino group to -ketoglutarate for glutamate formation (Fig. 1), rather than oxidizing BCKA carbon skeletons for placental ATP production. BCKAs produced by placenta may be oxidized by extraplacental tissues (including liver and kidneys), utilized by the fetal liver, or both to synthesize glucose and ketone bodies (acetoacetate and ß-hydroxybutyrate), which are important fuels for fetal extrahepatic tissues, including the brain, small intestine, kidneys, and skeletal muscle.

Intracellular glutamine levels are determined by glutamine synthesis and catabolism. As reported for rat placenta [13], pig placenta lacks phosphate-dependent glutaminase (the major enzyme for initiating glutamine degradation in animal cells) and, therefore, has little ability to oxidize glutamine or convert glutamine to glutamate. Phosphate-dependent glutaminase activity is widespread in animal cells [27], but the placenta is perhaps one of the very few mammalian tissues that does not degrade glutamine via the glutaminase pathway. The lack of glutamine hydrolysis may maximize the output of glutamine by pig placentae, thereby maintaining high levels of glutamine in fetal plasma. Thus, glutamine synthesis from BCAAs plays a major role in regulating glutamine concentration in, and its release by, pig placentae.

Placental transport of BCAAs (Table 2) as well as BCAA transaminase and BCKA dehydrogenase activities (Table 5) increased markedly between Days 20 and 40 of gestation. During this period, placental BCAA catabolism (primarily via transamination) increased sharply (Table 3). Similarly, glutamine synthase activity (Table 5) and synthesis of glutamine from BCAA-derived glutamate (Table 4) were highest at Day 40 of gestation and remained high at Day 45 of gestation. Accordingly, concentrations of BCAAs in pig placentae decreased, whereas those of glutamine increased, between Days 20 and 40 of gestation. Importantly, the greatest rate of glutamine synthesis in the pig placenta was associated with a marked increase in concentrations of glutamine in allantoic fluid from 0.69 mM at Day 30 to the highest value of 3.44 mM at Day 40 of gestation [15]. Because of technical difficulty, we could not obtain Day 40 fetal umbilical plasma for glutamine analysis. However, we have shown that the plasma fetal:maternal plasma ratio for glutamine is highest at Day 45 of gestation (the earliest Day for sampling of fetal umbilical blood) in pigs [8]. The Day 45 value is 3.87, in comparison with values for Days 60–110 of gestation (2.44–2.88) [8], indicating that glutamine is more abundant in the fetus than in the dam. These results illustrate metabolic coordination among several integrated pathways that support the high rate of glutamine synthesis in pig placenta during early gestation, when placental development is maximum and fetal growth increases rapidly [28].

There is active uptake of amino acids by the porcine uterus during pregnancy [29]. Although glutamine is synthesized de novo from BCAAs in the placenta, other fetal tissues may also produce glutamine. For example, fetal skeletal muscle contains both BCAA transaminase [12, 13] and glutamine synthetase (G. Wu, unpublished observation) and would be expected to synthesize glutamine. During early gestation when the fetal mass is relatively low [30], compared with the placenta [28], placental synthesis of glutamine is likely the major source of glutamine in fetal blood. Thus, promoting placental growth and BCAA transport will have an important implication for increasing glutamine availability and metabolic function in the conceptus. Further studies are necessary to determine glutamine synthesis by fetal tissues other than placenta. Such knowledge will enhance our understanding of the regulation of glutamine homeostasis in the growing and developing fetus.

Our findings have broad implications for embryonic and fetal development. Glutamine is an essential precursor for the synthesis of not only protein but also other molecules of enormous biological importance. These substances include aminosugars (UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine, a precursor for the formation of all glycoproteins), purine and pyrimidine nucleotides, as well as amino acids (ornithine, citrulline, and arginine) [3]. During early pregnancy, porcine, bovine, and ovine embryos synthesize and release relatively large amounts of glycoproteins [31, 32], which are essential for embryo maturation and implantation [33]. Accordingly, glutamine has been shown to enhance the embryonic development of mammals, including the pig [34]. Moreover, porcine placental growth is most rapid between Days 30 and 60 of gestation [28] and, therefore, requires high levels of glutamine for both DNA and protein syntheses to support cell proliferation. These roles of glutamine are consistent with our findings that both placental glutamine synthesis (Table 4) and concentrations of glutamine in allantoic fluid [15] increased markedly between Days 30 and 45 of gestation (peak values on Day 40 of gestation), which is a critical period for porcine fetal development [35]. In the second half of pregnancy, the absolute growth of fetal pigs increases most rapidly [28], which must be supported by an increase in glutamine provision. Importantly, the uptake of glutamine by porcine [30] and ovine [36] fetuses is the highest among amino acids during late gestation, and some of this glutamine is derived from placental synthesis. Finally, glutamine is a major substrate for the fetal synthesis of arginine (an essential amino acid for the fetus) [37]. This metabolic pathway is expected to play an important role in maintaining arginine homeostasis in the porcine fetus because uterine uptake of arginine is not sufficient to support the requirement for fetal growth during late pregnancy [30].

In summary, results of this study provide the first evidence for a connection between metabolism of BCAAs and glutamine production in pig placentae. Additionally, there is clear evidence for metabolic coordination to maximize placental glutamine synthesis at Day 40 of gestation when glutamine is most abundant in fetal allantoic fluid and placental growth is most rapid. These findings highlight the physiological significance of the placenta in fetal glutamine production and also stimulate further interest in the role of amino acids in fetal growth and development.

	ACKNOWLEDGMENTS

 
We would like to express our appreciation to all of the research personnel in our laboratories for their dedicated work, to Mr. Kenton Lillie for his invaluable assistance with animal husbandry, and to Dr. Susan M. Hutson for her expert advice on BCAA transaminase and BCKAD assays. The excellent office support of Frances Mutscher is also gratefully appreciated.

	FOOTNOTES

 
¹ This research was supported, in part, by U.S. Department of Agriculture/ National Research Initiative Grant 2001-35203-11247.

² Correspondence: Dr. Guoyao Wu, Department of Animal Science, 212 Kleberg Center, 2471 TAMUS, Texas A&M University, College Station, TX 77843-2471. FAX: 979 845-6057; g-wu{at}tamu.edu

Received: 13 November 2003.

First decision: 6 December 2003.

Accepted: 9 January 2004.

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