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*
U.S. Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030 and the
Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, TX 77843
3To whom correspondence should be addressed. E-mail: tdavis{at}bcm.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • somatotropin • urea cycle • protein metabolism • amino acids • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). Recently, studies in our laboratory have demonstrated that ST treatment in growing swine enhances metabolic efficiency of amino acid utilization by minimizing protein loss during food deprivation and by maximizing protein gain during meal absorption (3
,4
). This is accomplished by attenuating the reduction in protein synthesis that occurs with fasting and by enhancing the reduction in proteolysis that occurs with feeding. This conservation of amino acids results in an improvement in protein balance and likely contributes to the reduction in circulating amino acid concentrations that we have observed in fully fed ST-treated pigs.
Another characteristic effect of ST administration is a decrease in blood urea nitrogen (BUN) concentrations (3
,5
–7
) and whole-body leucine oxidation (3
), suggesting a reduction in amino acid catabolism. Two possible mechanisms could explain the effect of ST on the rate of amino acid catabolism. First, because this rate is a direct function of the circulating amino acid concentrations, the reduction in plasma amino acid concentrations in ST-treated growing swine could be responsible for the reduced rate of amino acid catabolism (3
). Second, lower rates of amino acid oxidation and urea production may reflect a direct effect of ST on the pathways of amino acid catabolism. Previous studies indicate that ST treatment decreases urea synthesis in swine and rats via the urea cycle (3
,8
,9
), the primary pathway for disposal of ammonia produced from protein and amino acid degradation in mammals. The reduced ureagenesis in ST-treated adult rats is associated with a reduction in the gene expression of some urea cycle enzymes in liver (8
,10
).
Extensive catabolism of essential and nonessential amino acids, including glutamine, glutamate, aspartate, proline, lysine, and branched-chain amino acids (BCAA) (11
–13
), occurs within the small intestine. It has been shown that urea synthesis from ammonia occurs via the urea cycle enzymes not only in hepatocytes within the liver, but also in enterocytes within the small intestine (14
). However, the kinetics of urea cycle enzymes differ markedly between the liver and small intestine (15
). In addition, studies in rats have shown that urea cycle enzymes in liver and intestine differ in their response to physiological and nutritional perturbations, such as glucagon treatment (16
–18
) and high-protein diets (19
,20
). Whether ST treatment affects porcine urea cycle enzymes differentially in hepatocytes and enterocytes is unknown.
Therefore, the first objective of this study was to determine the effect of 7 d of exogenous ST treatment in growing swine on the activities of urea cycle enzymes and closely related enzymes in both hepatocytes and enterocytes. A second objective was to determine the relationship between urea cycle enzyme activity and the availability of substrate for ureagenesis in ST-treated and control pigs. A third objective was to determine whether the ST-induced reduction in BUN concentration was associated with the decrease in amino acid catabolism, as indicated by oxidation of phenylalanine. Studies were performed in rapidly growing pigs (
25 kg) in which protein intake and ST treatment were rigorously controlled over a 7-d treatment period and during a 6-h isotope infusion study in the fed state.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The protocol was approved by the Animal Care and Use Committee of Baylor College of Medicine and was conducted in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Housing and care of the animals conformed to U. S. Department of Agriculture guidelines. Twenty-six crossbred (Landrace x Yorkshire x Hampshire x Duroc) female pigs were purchased from the Agriculture Headquarters at the Texas Department of Criminal Justice (Huntsville, TX). The pigs were received at the Baylor College of Medicine Animal Facility at 8–10 wk of age, weighing 10 kg. During the 2-wk acclimation and treatment period, pigs were fed a high-protein dry matter diet (Baby Pig Meal; Producers Cooperative Association, Bryan, TX) at a rate of 6% of body weight per day.
The pigs received 1750 kJ · kg-1 · d-1 metabolizable energy in the dry matter diet. The dry matter diet consisted of protein (264 g · kg-1), carbohydrate (471 g · kg-1), and fat (70 g · kg-1; Table 1
). To ensure that pigs were in the fully fed state throughout the infusion period, control and ST-treated pigs were infused intraduodenally with an elemental nutrient solution (Table 2
), similar to the diet by Stoll et al. (21
). The elemental nutrient solution consisted of glucose (104 g · L-1), lipid (21 g · L-1; Intralipid; Baxter Healthcare, Deerfield, IL), a complete amino acid mixture (55 g · L-1; Ajinomoto, Toyko, Japan), electrolytes, and trace minerals sufficient to meet or exceed the requirements for young growing pigs (22
). The pigs received 900 kJ · kg-1 · d-1, 13 g amino acid · kg-1 · d-1, and a fluid intake of 240 mL · kg-1 · d-1.
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Twenty female pigs were housed in individual cages. During the initial 2-wk acclimation and treatment period, pigs were fed a high-protein dry matter diet (Table 1)
. Pigs were weighed every other day to calculate feed intake at 6% body weight, thus ensuring that 90% of ad libitum intake for pigs of this age was consumed. Pigs were offered food in two equal amounts (one-half the total amount of feed, twice daily) each day at 0800 and 1500 h. Pigs generally consumed all the food presented to them, and unconsumed food was accounted for when estimating daily food intake and feed efficiency. Water was continuously available.
Following the 2-wk acclimation period, pigs were deprived of food overnight and the carotid artery, jugular vein, portal vein, and duodenum were catheterized using sterile techniques under general anesthesia, as previously described (23
), to measure phenylalanine oxidation, and hormone and substrate concentrations as reported in this manuscript. The caudal vena cava was also catheterized and flow probes were placed around the portal vein and caudal aorta to measure amino acid kinetics in the hindquarter and portal-drained viscera using a dual, stable isotopic tracer/mass transorgan balance technique, which will be presented elsewhere. The catheters were externalized, flushed with 100 kU heparin · L-1 saline, tied off to prevent discharge, and housed within a pocket of a swine jacket (Domestic Swine Jacket; Harvard Apparatus, Holliston, MA). Pigs received nutrition (nutrient solution; Table 2
) intravenously during the 2- to 3-d recovery period before returning to their normal dietary regimen (dry matter diet; Table 1
). Antibiotics were administered daily to prevent infection.
Pigs were weight-matched and randomly assigned to one of two treatment groups: ST treatment (n = 10) or control group (n = 10). The treatment group received daily injections of recombinant porcine ST at a rate of 150 µg · kg-1 · d-1 for 7 d. The control group received equal volume injections of sterile saline for 7 d. The injections were divided into two equal daily doses and administered into the hindlimb musculature concurrent with the feeding sessions. To minimize the confounding effect of differences in feed intake, control pigs were pair fed to the level of their respective weight-matched ST-treated counterpart during the 7-d treatment period.
Infusions.
Pigs were deprived of food overnight, given their final injection of ST (150 µg · kg-1 · d-1) or saline, and secured in a swine hammock (Walter Terry Distributor, Houston, TX). The hammock supported the animal’s body weight and allowed the legs to hang comfortably and without encumbrance. To ensure that pigs were in the fully fed state throughout the infusion period, control and ST-treated pigs were infused intraduodenally for 7 h with the nutrient solution (Table 2
; 11 mL · kg-1 · h-1) beginning 1 h before the onset of the tracer infusion. To estimate CO2 production rate, the jugular vein was infused with a primed (7.5 µmol · kg-1), continuous (10 µmol · kg-1 · h-1) infusion of NaH 13CO3 ([1-13C]bicarbonate; Cambridge Isotope Laboratories, Andover, MA) from 0 to 120 min. Arterial blood samples for estimation of CO2 production were obtained at baseline and every 15 min throughout the 2-h NaH 13CO3 infusion. To quantify phenylalanine oxidation rate, the duodenum was infused with a primed (20 µmol · kg-1), continuous (20 µmol · kg-1 · h-1) infusion of [1-13C]phenylalanine (Cambridge Isotope Laboratories) from 120 to 360 min. Arterial blood samples for analysis of isotopic enrichment of [1-13C]phenylalanine and 13CO2 were obtained at baseline and every 30 min throughout the 4-h [1-13C]phenylalanine infusion. Arterial and portal venous blood was taken at baseline and every 30 min throughout the infusion for determination of amino acid concentration. Arterial blood was also taken at 0, 240 and 360 min for determination of BUN concentration. Breath samples were obtained at baseline and every 30 min throughout the infusion for isotopic enrichment of 13CO2. Pigs were killed by exsanguination under anesthesia (pentobarbital sodium), upon which tissue samples from liver and jejunum were rapidly removed, immediately frozen in liquid nitrogen, and stored at -80°C for 2–3 d for subsequent measurement of urea cycle enzyme activities.
Blood urea nitrogen, ammonia and bicarbonate concentrations.
Heparinized blood (1.0 mL) samples were obtained, centrifuged at 2500 x g for 15 min at 4°C, and the plasma was stored at -80°C until analyzed for BUN and ammonia. BUN was analyzed via an endpoint colorimetric assay (Vitros Chemistry Products; Johnson & Johnson Clinical Diagnostics, Rochester, NY). Briefly, the urease reaction with plasma generates ammonia which passes through a semipermeable membrane before reacting with bromophenol blue. Plasma ammonia concentrations were analyzed via endpoint colorimetric assay (Vitros Chemistry Products; Johnson & Johnson Clinical Diagnostics). Briefly, ammonium ions are converted to gaseous ammonia and pass through a semipermeable membrane layer before reacting with bromophenol blue. Whole blood samples (0.2 mL) were obtained and immediately analyzed on a pH/blood gas analyzer (Ciba Corning Diagnostics, Medfield, MA) for determination of bicarbonate (HCO3-) concentration.
Plasma amino acid concentrations.
Heparinized whole blood samples (0.5 mL) were centrifuged at 2500 x g for 15 min at 4°C, and the plasma was stored at -80°C until analyzed for amino acid concentrations. Plasma amino acid concentrations were determined using reverse-phase HPLC, as previously described (24
). Plasma spiked with methionine sulfone (internal standard) was filtered through a 10,000 molecular weight filter. Amino acids were precolumn derivatized with phenyl isothiocyanate, separated on a PICO-TAG reverse-phase column (Waters, Milford, MA), and detected on-line by spectrophotometry. Concentrations were calculated with the use of an amino acid standard (Pierce Chemical, Rockford, IL).
Analysis of tracer enrichment.
Blood and breath samples for 13CO2 production were analyzed using gas chromatography isotope ratio mass spectrometry. Briefly, a 1.0-mL aliquot of whole blood was placed in a 10-mL vacutainer (Becton Dickinson, Franklin Lakes, NJ) with 1.0 mL of perchloric acid (10 g/100 g), gently mixed, and placed on ice for 60 s. Room air was filtered via a soda lime filter (Sodasorb; Grace Container Products, Lexington, MA) to obtain air devoid of CO2. The air (8 mL) was injected into the 10-mL vacutainer containing 1:1 whole blood:perchloric acid and 8–10 mL of air was withdrawn again. This newly extracted gas sample was transferred to a second vacutainer for subsequent analysis of isotopic enrichment of 13CO2 via continuous flow gas chromatography isotope ratio mass spectrometry (ANCA RoboPrep-G; Europa Instruments, Crewe, UK).
Mass spectrometric analysis of plasma [1-13C]phenylalanine was conducted via heptaflurobutyric anhydride derivative (25
). Phenylalanine was isolated via cation exchange chromatography (AG-50W resin; BioRad, Hercules, CA). The isotopic enrichment of derivatized [1-13C]phenylalanine was determined by negative chemical ionization gas chromatography mass spectrometry (Hewlett-Packard 5890 Series II GC equipped with a Europa Orchid 20/20 stable isotope analyzer; Hewlett-Packard, Palo Alto, CA) by monitoring the m/z ratio of ions at 383/384.
Determination of the urea cycle enzyme activities in liver and jejunum.
The urea cycle enzymatic pathway leading to urea production in the liver and gut is depicted in Figure 1
. Sample tissues of liver and jejunum (0.5 g) were homogenized in a 4-mL buffer solution containing 300 mmol · L-1 sucrose, 1 mmol · L-1 EDTA, 5 mmol · L-1 hepes, 3 mmol · L-1 dithiothreitol, 0.5% Triton X-100, 5 mg · L-1 phenylmethylsulfonylfluoride, 5 mg · L-1 chymostatin, and 5 mg · L-1 aprotinin at pH 7.4. The homogenates were centrifuged at 600 x g and 4°C for 10 min. The supernatant was used directly for assays of argininosuccinate synthase (ASS; EC 6.3.4.5) (4
) and argininosuccinate lyase (ASL; EC 4.3.2.1) (4
), and was treated by three cycles of freezing (in liquid nitrogen) and thawing (at 37°C) before use for assays of carbamoylphosphate synthase-I (CPS-I; EC 6.3.4.16) (3), ornithine carbamoyltransferase (OCT; EC 2.1.3.3) (3), arginase (EC 3.5.3.1), ornithine aminotransferase (OAT; EC 2.6.1.13) (4), glutaminase (EC 3.5.1.2), and glutamate dehydrogenase (GDH; EC 1.4.1.2) (4). The activities of urea cycle enzymes (CPS-I, OCT, ASS, ASL and arginase), OAT and glutaminase were determined at 37°C and two protein levels for 0, 5, 10 and 15 min as previously described (15
,26
). Briefly, the CPS-I assay mixture (0.5 mL) contained 150 mmol · L-1 potassium phosphate buffer (pH 7.5), 25 mmol · L-1 ATP, 25 mmol · L-1 MgCl2, 5 mmol · L-1 N-acetylglutamate, 20 mmol · L-1 NH4Cl, 5 mmol · L-1 ornithine, 100 mmol · L-1 NaHCO3, 10 U of added OCT (from streptococcus faecalis), and tissue extracts (0.25 and 0.5 mg protein). The OCT assay mixture (2 mL) contained 100 mmol · L-1 potassium phosphate buffer (pH 7.5), 15 mmol · L-1 ornithine, 40 mmol · L-1 carbamoyl phosphate, and tissue extracts (20 and 40 µg protein). The ASS assay mixture (0.2 mL) contained 75 mmol · L-1 potassium phosphate buffer (pH 7.5), 10 mmol · L-1 citrulline, 5 mmol · L-1 aspartate, 5 mmol · L-1 ATP, 5 mmol · L-1 MgSO4, and tissue extracts (0.25 and 0.5 mg protein). The ASL assay mixture (0.2 mL) contained 129 mmol · L-1 sodium phosphate buffer (pH 7.0), 10 mmol · L-1 argininosuccinate, 65 mmol · L-1 EDTA, and tissue extracts (0.25 or 0.5 mg protein). The arginase assay mixture (0.3 mL) contained 20 mmol · L-1 arginine, 3.3 mmol · L-1 MnCl2, 50 mmol · L-1 Tris-HCl buffer (pH 7.5), and tissue extracts (0.25 and 0.5 mg protein for jejunum; 5 and 10 µg protein for liver). The OAT assay mixture (2 mL) contained 75 mmol · L-1 potassium phosphate (pH 7.5), 20 mmol · L-1 ornithine, 0.45 mmol · L-1 pyridoxal phosphate (PLP), 5 mmol · L-1 O-aminobenzaldehyde, 0 or 3.75 mmol · L-1 
-ketoglutarate, and tissue extracts (0.25 or 0.5 mg protein for jejunum; 1 or 2 mg protein for liver). The assay mixture for jejunal glutaminase (0.5 mL) contained 150 mmol · L-1 potassium phosphate buffer (pH 8.2), 20 mmol · L-1 glutamine, and tissue extracts (50 and 100 µg protein). Liver glutaminase activity was measured at 37°C as described by Watford and Smith (27
) in which the assay mixture (0.6 mL) contained 50 mmol · L-1 potassium phosphate (pH 8.0), 20 mmol · L-1 Tris-HCl buffer (pH 8.0), 100 mmol · L-1 glutamine, 2 mmol · L-1 NH4Cl, and tissue extracts (50 or 100 µg protein). GDH activity was determined as described by Wu et al. (28
) in which the assay mixture (3 mL) contained 110 mmol · L-1 NH4Cl, 7 mmol · L-1 -ketoglutarate, 0.16 mmol · L-1 NADH, 25 µg L-lactate dehydrogenase, 80 mmol · L-1 sodium phosphate buffer (pH 7.6), and tissue extracts (0.25 and 0.5 mg protein for jejunum; 10 or 20 µg protein for liver). Only the supernatant fluid of 600 x g centrifugation of the tissue homogenate was used for the enzyme assays. Protein concentration in the supernatant was used to calculate enzyme activities. Protein concentrations in the liver and jejunum, expressed on a tissue weight basis, did not differ significantly (P > 0.01) between control and ST-treated pigs. An average of the values obtained at the three time-points (5, 10 and 15 min) of the assay, after subtraction of the blank value (0 min incubation) represented the enzyme activity. Enzyme activities were expressed on the basis of tissue protein used in assay mixtures. All enzyme assays except for jejunal glutaminase were performed under Vmax conditions and in the linear portion of the V vs. enzyme curve (15
). Jejunal glutaminase activity (a kidney type isoenzyme) was measured under established optimal conditions to ensure linearity of the assay (26
).
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Determination of N-acetylglutamate content in liver and jejunum.
N-acetylglutamate content in the liver and jejunum was determined by HPLC as described by Alonso and Rubio (29
). Liver or jejunum tissue (0.6 g) was homogenized with 1 mL of 1.5 mol · L-1 HClO4, and the acidified solution was neutralized with 0.5 mL 2 mol · L-1 K2CO3. The extract was adjusted to pH 2.0 with 70 µL of 1 mol · L-1 HCl, and applied to Dowex 50W-X8 resin (H+ form, 200–400 mesh; 0.6 x 5 cm resin bed). The column was washed with 2 mL H2O, and the effluent was adjusted to pH 6–8 with 52 µL of 1 mol · L-1 KOH and brought to pH 7.5 with 0.3 mL of 250 mmol · L-1 potassium phosphate buffer (pH 7.5). An aliquot (1 mL) of the solution was incubated with 0.5 mg aminoacylase (Sigma, St. Louis, MO) at 37°C for 0 or 90 min, and this aminoacylase reaction was terminated by addition of 0.2 mL of 1.5 mol · L-1 HClO4. The acidified solution was used for glutamate analysis by HPLC (26
). Determination of N-acetylglutamate in the liver and jejunum included correction for endogenous amounts of tissue glutamate.
Determination of carbamoylphosphate content in liver and jejunum.
Tissues (0.5 g) were homogenized in 2 mL of 1.5 mol · L-1 HClO4, and then neutralized with 1 mL of 2 mol · L-1 K2CO3. The extracts were used for determination of carbamoylphosphate concentration through its conversion to citrulline in the presence of OCT, as previously described (26
). Determination of carbamoylphosphate in the liver and jejunum included correction for endogenous amounts of tissue citrulline.
Calculations.
Phenylalanine oxidation rate was determined via the following standard steady-state equations. CO2 production rate was determined by the following equation:
 |
where Ei is the infusate NaH 13CO3 enrichment, Eb1 is the enrichment of expired 13CO2 at plateau during NaH 13CO3 infusion, and IR is the infusion rate of NaH 13CO3.
The effect of ST on whole-body phenylalanine oxidation rates was determined by the following equation, which incorporates the production of CO2 and the isotopic enrichments of both CO2 and [1-13C]phenylalanine:
 |
where PR is the CO2 production rate, Eb2 is the 13CO2 enrichment in the breath at plateau during phenylalanine infusion, and Ep is the enrichment of plasma [1-13C]phenylalanine during the last 2 h of phenylalanine infusion. Figure 2
shows the isotopic enrichment (atom percent excess) of 13CO2 in the expired breath at steady-state conditions during NaH13CO3 infusion (75–120 min) and [1-13C]phenylalanine infusion (270–360 min).
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Individual t tests were performed to detect significant differences between treatment groups for urea cycle enzyme activity as well as N-acetylglutamate and carbamoylphosphate content. ANOVA with repeated measures was used to detect changes with treatment during sampling over the 7-h infusion for plasma amino acids and BUN concentrations and isotopic enrichments. Correlation analysis of regulatory liver urea cycle enzymes (CPS-I and ASS) and key circulatory amino acids that are substrates or intermediates of the urea cycle and of urea cycle enzymes and analyzed blood variables were performed to determine whether a significant relationship existed between these variables. Three control and one ST-treated pigs died during the treatment period, and, therefore, the resulting sample size was seven for the control group and nine for the ST-treated group. Results are presented as means ± SD. Probability values of P 
0.01 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The initial starting weight of the pigs was 10.1 ± 0.8 kg. At the end of the 2-wk acclimation period and catheter implantation, pigs were weight-matched and randomly assigned to either the control or ST treatment group (15.6 ± 1.7 vs. 17.1 ± 1.6 kg, respectively). Control pigs were pair fed to the level of ST-treated pigs during the 7-d treatment period to limit any variations due to the effect of feeding. At the end of the 7-d treatment period, the body weight of the ST-treated pigs was significantly (P < 0.05) greater than that of the control pigs (22.1 ± 2.2 vs. 19.5 ± 2.3 kg, respectively). The weight-scaled average daily gain tended to be higher (P = 0.1) in the ST-treated vs. control pigs during the 7-d treatment period (33.4 ± 6.2 vs. 27.2 ± 11.5 g · kg-1 · d-1, respectively). Feed efficiency, as reflected by weight gain-to-feed ratio, during the 7-d treatment period also tended (P = 0.2) to be higher in ST-treated vs. control pigs (0.56 ± 0.10 vs. 0.48 ± 0.18 g gain · g intake-1, respectively). These data indicate that the efficiency with which dietary protein was utilized for growth was increased by 7 d of ST treatment in growing, fed pigs, consistent with previous studies in ST-treated pigs in the fed state (1
–3
).
Amino acid concentrations.
Consistent with previous studies (3
,4
), there was a significant decrease (P < 0.05) in systemic total amino acid (TAA) concentrations (-32%) in the ST-treated pigs versus controls (Fig. 3
A). There was also a significant decrease (P < 0.05) in essential amino acid (EAA; -34%), nonessential amino acid (NEAA; -53%), and BCAA (-37%) concentrations in the systemic circulation. ST treatment also decreased (P < 0.05) in plasma amino acid concentrations (i.e., TAA, -23%; EAA, -23%; NEAA, -43%; and BCAA, -28%) in the portal-drained viscera circulation that supplies the liver (Fig. 3
B).
|
. Consistent with the decrease in systemic circulating concentrations of TAA, EAA, NEAA, BCAA, systemic concentrations (Fig. 4
A) of citrulline (-49%), arginine (-37%), and ornithine (-32%), and portal venous concentrations (Fig. 4
B) of aspartate (-30%), citrulline (-47%), arginine (-32%), proline (-27%), and ornithine (-42%) were significantly reduced (P < 0.05) in ST-treated pigs.
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There was a significant (P < 0.05) decrease (-30%) in whole-body phenylalanine oxidation with ST treatment vs. control (178 ± 14 vs. 137 ± 18 µmol phenylalanine · kg-1 · h-1, respectively) indicative of a reduction in the loss of carbon from the amino acid pool. This decrease in whole-body phenylalanine oxidation is consistent with the decreased oxidation rate of leucine observed in a similar animal model (3
). The reduction in whole-body phenylalanine oxidation indicates a reduction in amino acid catabolism and, thus, a conservation of amino acids, due to ST treatment in growing pigs.
Blood urea nitrogen concentration.
As an indication of the effectiveness of our animal model, we measured BUN concentrations. Consistent with our previously published data in a similar animal model (3
,4
), there was a significant (P < 0.001) decrease (-46%) in BUN concentrations following 7 d of ST treatment compared with those in controls (3.5 ± 0.8 vs. 6.5 ± 0.9 mmol · L-1, respectively). These data indicate a decrease in ureagenesis and an improvement in nitrogen retention due to ST treatment in growing pigs in the fed state.
Plasma ammonia and bicarbonate concentration.
Plasma ammonia and bicarbonate concentrations were determined because the majority of ammonia and bicarbonate that is produced from amino acid catabolism is converted to urea through the urea cycle (30
). Systemic plasma ammonia concentrations were significantly (P < 0.04) lower (-45%) in ST-treated pigs compared with controls (61.2 ± 5.5 vs. 110.6 ± 25.4 µmol · L-1, respectively). The bicarbonate concentration in neither the systemic (control vs. ST, 24.7 ± 1.3 vs. 23.3 ± 0.8 mmol · L-1, respectively) nor the portal venous (control vs. ST, 26.2 ± 3.3 vs. 25.3 ± 2.0 mmol · L-1, respectively) circulation was significantly affected (P > 0.05) by ST treatment in growing pigs. The decrease in ammonia concentration indicates a reduction in overall amino acid catabolism in accordance with the decrease in BUN concentration and whole-body phenylalanine oxidation.
Activities of urea cycle enzymes in liver and jejunum.
The activities of CPS-I, OCT, ASS, ASL, arginase, OAT, GDH and glutaminase in pig liver and jejunum are presented in Table 3
. After 7 d of ST treatment in growing pigs in the fed state, the activities of CPS-I (-45%), ASS (-38%), ASL (-23%), arginase (-27%), and glutaminase (-18%) were significantly decreased (P < 0.05) in ST-treated pig liver vs. control pig liver. There were trends for the activities of OCT (-6%; P = 0.1), OAT (-6%; P = 0.2), and GDH (-4%; P = 0.3) to be reduced in ST-treated pig liver.
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), OAT activity was much greater in pig jejunum than in pig liver (Table 3)
. N-acetylglutamate and carbamoylphosphate concentrations in liver and jejunum.
Consistent with the reduction in the urea cycle enzyme activities with ST treatment in this study, there was a significant decrease (P < 0.05) in the concentrations of carbamoylphosphate (-20%) and N-acetylglutamate (-26%) in the liver of ST-treated vs. control pigs. Interestingly, ST treatment increased (P < 0.05) carbamoylphosphate (+28%) and N-acetylglutamate (+32%) concentrations in the jejunum (Table 4
). The levels of carbamoylphosphate (P 0.001) and N-acetylglutamate (P 0.001) were significantly greater in liver than in jejunum of control pigs.
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R2 correlation values are outlined in Tables 5
and 6. There were significant correlations (P < 0.05) between the two key regulatory hepatic urea cycle enzymes (CPS-I and ASS) and BUN concentration, plasma ammonia concentration, and liver carbamoylphosphate and N-acetylglutamate content (Table 5)
. As CPS-I and ASS enzyme activity decreased, BUN, plasma ammonia, whole-body phenylalanine oxidation, and liver carbamoylphosphate and N-acetylglutamate content decreased. There was also a significant relationship (P < 0.05) between liver CPS-I and ASS activity and substrate availability for the liver urea cycle (Table 6)
. CPS-I and ASS activity decreased in conjunction with decreased concentrations of systemic arginine, proline and ornithine, and of portal citrulline, arginine, proline and ornithine. There was not a significant relationship (P > 0.05) between intestinal urea cycle enzyme activity and the aforementioned study variables (data not shown). These correlations indicate a significant relationship between the reduction in liver urea cycle enzyme activity and substrate availability for urea production in the liver.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Effects of ST on urea cycle enzyme activity.
The urea cycle consists of CPS-I, OCT, ASS, ASL and arginase, and it is present primarily in the liver (30
). Metabolic and enzymatic studies have established that CPS-I and ASS are rate-controlling enzymes for hepatic ureagenesis (17
). Importantly, recent studies have shown that urea is also synthesized from ammonia via the urea cycle enzymes in enterocytes of the small intestine (14
), where large amounts of ammonia are produced from extensive catabolism of many nutritionally EAA and NEAA, including glutamine, glutamate, aspartate, proline and BCAA (11
,12
). CPS-I and OCT are mitochondrial enzymes, whereas ASS and ASL are located in the cytosol. There are two distinct arginase isoforms (arginase I and arginase II), which are encoded by different genes and differ in molecular and immunological properties, tissue distribution, subcellular location, and regulation of expression (31
). Arginase I (a cytosolic enzyme) is highly expressed in the liver and to a much lesser extent, in a few other tissues including the small intestine, whereas arginase II (a mitochondrial enzyme) is widespread (32
). Arginase II is the principal arginase isoform in the small intestine of postweaning pigs (33
). Urea cycle enzymes differ between the liver and small intestine in enzyme kinetics (15
) and in response to physiological and nutritional alterations (16
–20
). In view of urea cycle enzyme activities as well as urea synthesis by enterocytes and hepatocytes (15
,30
), we have previously suggested that the liver is the primary organ for ureagenesis compared with the small intestine in the pig (14
,15
).
The results of this study demonstrate that the activities of CPS-I, ASS, ASL and arginase in liver are reduced by ST treatment in growing pigs (Table 3)
. These results are consistent with previous studies that showed that ST treatment decreased the mRNA levels and activities of CPS-I, ASS, ASL and arginase in rat liver (8
,10
,34
), as well as arginase activity in pig liver (9
). Another important finding of this study is that porcine urea cycle enzymes differed between the liver and small intestine in their response to ST treatment, in that the activities of nearly all intestinal urea cycle enzymes were unaltered in ST-treated swine (Table 3)
. Interestingly, the activities of all urea cycle enzymes except OCT were substantially lower in the small intestine than in the liver, further supporting the view that the liver is the principal site of ureagenesis in mammals, including pigs (14
,15
). Our findings suggest that there is tissue-specific regulation of ureagenesis and expression of urea cycle enzymes during exogenous ST administration.
Regulators of urea cycle enzyme activity in ST-treated pigs.
To identify the factors involved in the regulation of urea cycle enzyme activity in ST-treated pigs, we measured the concentration of N-acetylglutamate, an essential allosteric activator of CPS-I (30
). Hepatic concentrations of N-acetylglutamate were reduced in ST-treated pigs (Table 4)
, as previously reported for rats (35
), and the reduction in N-acetylglutamate was correlated with the reduction in both CPS-I and ASS activities (key regulatory enzymes of hepatic urea synthesis). In contrast, intestinal concentrations of N-acetylglutamate were greater in ST-treated pigs (Table 4)
. This difference may be related to the major metabolic functions of these two organs. For example, the decrease in N-acetylglutamate availability in the liver would reduce the conversion of ammonia into urea, whereas the increase in N-acetylglutamate concentration in the small intestine would enhance the synthesis of carbamoylphosphate from ammonia, ATP and bicarbonate for the generation of citrulline and arginine, an EAA for young piglets (32
,36
).
ST treatment reduced glutaminase activity but had no effect on the activity of GDH or OAT in porcine liver (Table 3)
. Thus, ST regulates the expression of only some but not all enzymes involved in hepatic amino acid metabolism. The available evidence suggests that the ST-induced reduction in some of the mitochondrial urea cycle enzymes indicates a potential species-specific response of hepatic urea cycle enzymes to ST treatment. Studies in rats showed that ST treatment reduced the activities of mitochondrial hepatic urea cycle enzymes (37
), and the activity of hepatic lysine -ketoglutarate reductase (38
). A decrease in intramitochondrial ammonia supply may be responsible in part for reduced ureagenesis in ST-treated pigs.
Substrate availability for urea production.
We are unaware of any studies examining whether ST exerts a direct or indirect effect on the expression of hepatic urea cycle enzymes. ST does, however, induce a conservation of amino acids and blunts the loss of nitrogen and carbon from the amino acid pool, as indicated by reductions in amino acid oxidation rates and reduced BUN concentrations. In this study, the reduction in hepatic urea cycle enzyme activity in ST-treated pigs was associated with a reduction in TAA concentrations in the systemic and portal venous circulation (Fig. 2)
. Citrulline, arginine, ornithine, aspartate and proline serve as precursors for urea production in the liver and small intestine (Fig. 1)
. Systemic concentrations of citrulline, arginine and ornithine were lower after ST treatment. Amino acid concentrations in the portal venous circulation that flows directly into the liver represent major effluent contributions from the spleen, stomach, gut and pancreas. Portal venous concentrations of citrulline, arginine, ornithine, aspartate and proline were lower in the ST-treated pigs vs. the controls, indicating a possible reduction in the availability of substrates for urea production in the liver. These reductions in amino acid concentrations were correlated with the reductions in hepatic CPS-I and ASS activities. Cohen et al. (39
) showed that when external ornithine concentrations are in the range of 0.03–0.2 mmol/L, ornithine transport into mitochondria is a rate-controlling step in hepatic citrulline and urea synthesis. This finding supports our view that the changes in substrate availability in ST-treated pigs may play a role in regulating hepatic ureagenesis.
In the current study, both control and ST-treated pigs were continuously infused (7 h) with a nutrient solution consisting of EAA, NEAA, glucose and fat. Thus, two sources of amino acids were available to the intestine for utilization: arterial and dietary. Intestinal influx of systemic and efflux of portal venous concentrations of TAA, NEAA, EAA and BCAA were reduced in the ST-treated pigs. Although the systemic substrate availability of amino acids for urea production was reduced, the influx of amino acids from duodenal luminal sources into the enterocyte was similar in the control and ST-treated pigs. This duodenal source may have presented the intestine with an adequate source of substrate for urea production, resulting in no detectable change in urea cycle enzyme activity in enterocytes of ST-treated pigs. Previous studies in pigs in the fed state have shown that a significantly greater percentage of phenylalanine (59%) used for mucosal protein synthesis was derived from dietary sources than from arterial sources (40
). Moreover, the reduction in portal-drained viscera efflux of amino acid in ST-treated pigs and the increase in gut weight per unit length (data not shown) suggest an increased utilization of amino acids for protein synthesis by the gut in ST-treated pigs. Furthermore, our current study demonstrated that ST treatment of growing pigs reduced ammonia production, indicating a reduction in amino acid catabolism and a conservation of amino acids in the body. Ammonia concentrations were correlated with urea cycle enzyme activity, and, thus, the decrease in hepatic urea cycle enzyme activity was consistent with the decreases in both the availability of amino acid substrates and ammonia supply.
The results of the current study showed that ST treatment reduced whole-body phenylalanine oxidation, consistent with previous studies in growing pigs in the fed state using [1-13C]leucine as the isotopic tracer (3
). Oxidation of phenylalanine represents an irreversible loss of that amino acid from the body. A lower rate of phenylalanine oxidation would be indicative of a conservation of that amino acid and a decrease in the level of nitrogen availability for urea production. The reduction in phenylalanine oxidation may be the result of a general reduction in systemic and portal venous amino acid precursor concentrations, secondary to the enhanced protein synthesis and reduced proteolysis of ST treatment. Together, these changes result in a more efficient use of amino acids for growth in ST-treated pigs.
In conclusion, ST treatment of growing swine reduced hepatic activities of urea cycle enzymes (CPS-I, ASS, ASL and arginase) and glutaminase, as well as N-acetylglutamate and carbamoylphosphate concentrations. There was also a decrease in available substrates for urea production (i.e., amino acids and ammonia) with ST treatment. These results provide a biochemical basis for explaining the decreased ureagenesis in ST-treated pigs. Our findings also demonstrate that splanchnic tissues responded differentially to ST treatment, raising an important question of tissue-specific regulation of urea cycle enzymes and related enzymes in mammals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the U. S. Department of Agriculture National Research Initiative Grants 96-35206-3657, 00-35206-9405, and 97-35206-5096, the U. S. Department of Agriculture, Agriculture Research Service under Cooperative Agreement 58-6250-6-001, National Institute of Child Health and Human Development Training Grant T32-HD-07445, and Hatch Project 8200. This work is a publication of the U. S. Department of Agriculture/Agriculture Research Service Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, and Texas Children’s Hospital, Houston, TX. The contents of this publication do not necessarily reflect the views or policies of the U. S. Department of Agriculture, and mention of trade names, commercial products or organizations does not imply endorsement by the U. S. Government.
4 Abbreviations used: ASL, argininosuccinate lyase; ASS, argininosuccinate synthase; BCAA, branched-chain amino acid; BUN, blood urea nitrogen; CPS-I, carbamoylphosphate synthase-I; EAA, essential amino acid; GDH, glutamate dehydrogenase; NEAA, nonessential amino acid; OAT, ornithine aminotransferase; OCT, ornithine carbamoyltransferase; ST, somatotropin; TAA, total amino acid.
Manuscript received 12 July 2001. Initial review completed 19 August 2001. Revision accepted 9 October 2001.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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