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Faculty of Nutrition and Department of Animal Science, Texas A&M University, College Station, TX 77843-2471
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
FOOTNOTES
LITERATURE CITED
ABSTRACT 
Weaning is associated with increased intestinal metabolism of glutamine and arginine as well as elevated plasma concentrations of cortisol (the major circulating glucocorticoid) in pigs. The objective of this study was to determine if cortisol plays an important role in mediating the enhanced amino acid metabolism in enterocytes of weaned pigs by administering RU486 (a glucocorticoid receptor antagonist). Eighteen 21-d-old pigs were randomly assigned to three groups of six. Two of these groups received intramuscular injections of 0 or 10 mg RU486 per kg body weight 5 min before and 24 and 72 h after weaning to a corn-soybean meal-based diet. The third group was allowed to suckle freely from sows. When the pigs were 29 d old, jugular venous blood was obtained and pigs were killed for preparation of jejunal enterocytes. The activities of arginase, argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, cells were incubated for 0 or 30 min at 37°C in 2 mL of Krebs-bicarbonate buffer (pH 7.4) containing 0 or 2 mmol/L L-[U-14C]arginine or 2 mmol/L L-[U-14C]glutamine. In comparison with suckling pigs, weaning resulted in increases in the following: 1) the activities of arginase, ASS, ASL and P5C synthase, 2) the metabolism of arginine to CO2, proline and ornithine, and 3) the conversion of glutamine to ornithine, citrulline and CO2. The effects of the administration of RU486 were as follows: 1) attenuation of the increase in arginase activity and the production of ornithine from arginine, 2) abolition of the induction of ASL and P5C synthase, and 3) prevention of the increase in glutamine metabolism and the production of proline and CO2 from arginine in enterocytes of weaned pigs. These data suggest that glucocorticoids play an essential role in mediating the enhanced intestinal degradation of arginine and glutamine during weaning.
KEY WORDS: cortisol · RU486 · arginine · glutamine · weaning · pigsINTRODUCTION
Weaning is a critical stage of development in the life of an animal and is characterized by a shift from a high fat to a high carbohydrate diet in mammals, including pigs (Henning 1981
, Pluske et al. 1995). There are extraordinary hormonal and metabolic alterations as well as adaptations of many organs in weanling animals (Deschodt-Lanckiman et al. 1974, Henning 1981, Warshaw et al. 1980). Although much effort has been directed towards the studies of developmental changes of intestinal digestive enzymes (Chappel et al. 1989, Henning 1981, Lund and Smith 1987), there have been only a few studies of intracellular amino acid metabolism in intestinal epithelial cells of weanling mammals. We recently demonstrated increases in the metabolism of arginine and glutamine (two conditionally essential amino acids) in enterocytes of 29-d-old pigs weaned at 21 d of age, compared with suckling pigs (Wu 1995, Wu et al. 1994b). Our studies further showed that the induction of citrulline synthesis from glutamine and the metabolism of arginine to ornithine and proline in enterocytes of postweaning pigs are primarily independent of age or change in diet (Dugan et al. 1995, Wu 1995), suggesting an important role for nondietary factors in mediating enhanced intestinal amino acid metabolism during weaning.
Weaning in pigs is associated with increased plasma concentrations of cortisol (hydrocortisone) , the major circulating glucocorticoid in pigs (Worsae and Schmidt 1980) and humans (Ganong 1991). This steroid hormone is a potent inducer of hepatic urea cycle enzymes including arginase (Morris 1992). Cortisol administration increases intestinal activities of digestive enzymes [lactase, maltase and aminopeptidases in prenatal pigs (Sangild et al. 1995), amylase and maltase in 26-d-old suckling pigs (Chappel et al. 1989)], and of arginase in suckling and postweaning rats (Herzfeld and Raper 1979). We recently demonstrated that the administration of hydrocortisone-21-acetate (HYD)4 to 21-d-old suckling pigs increased the activities of arginase and pyrroline-5-carboxylate (P5C) synthase and the metabolism of arginine and glutamine in enterocytes (Flynn and Wu 1997). These findings imply, but do not provide direct evidence for, an essential role of glucocorticoids in the enhanced metabolism of glutamine and arginine in enterocytes during weaning. We hypothesized that if cortisol mediates the increased intestinal metabolism of amino acids in postweaning pigs, this effect may be attenuated or prevented by a glucocorticoid receptor antagonist. This hypothesis was tested in the present study with the use of RU486 (mifepristone), a potent antagonist of glucocorticoid receptors (Baulieu 1989). Our results demonstrate that the administration of RU486 attenuated the increase in arginase activity and the production of ornithine from arginine, abolished the induction of argininosuccinate lyase (ASL) and P5C synthase, and prevented increases in the metabolism of glutamine and the production of proline and CO2 from arginine in enterocytes of postweaning pigs.
MATERIALS AND METHODS
, Klobasa et al. 1987, Noblet and Etienne 1989, Wu and Knabe 1994). At 21 d of age, 18 pigs (6.07 ± 0.16 kg, means ± SEM) were randomly assigned to one of three groups of six pigs. One group was allowed to continue to nurse freely from the sow, while the other two groups received intramuscular injections of vehicle solvent (sesame oil) or RU486 (10 mg/kg body wt) 5 min before weaning and 24 and 72 h later. The dose of RU486 used was based on previous in vivo studies with a number of species, including rats, humans, guinea pigs (Baulieu 1989, Goascogne et al. 1991, Speiser et al. 1993) and piglets (Flynn and Wu 1997). The weaning diet was based on corn and soybean meal and was formulated to meet NRC requirements for nutrients (Wu et al. 1994a). Dry matter, energy content and amino acid composition in the weaning diet and in sow's milk on d 22 of lactation are given in Table 1. Porcine milk composition is similar between d 22 and 29 of lactation (Klobasa et al. 1987, Wu and Knabe 1994). Milk consumption by suckling pigs was estimated on the basis of weight gain and the ratio (4.5:1) of milk consumption to weight gain (Noblet and Etienne 1989), instead of by the weigh-suckle-weigh technique (Noblet and Etienne 1989) that would inflict stress on piglets and increase plasma cortisol levels. When pigs were 29 d of age, blood (3 mL) was withdrawn from the jugular vein into heparinized tubes between 1000 and 1100 h, after which pigs were killed for preparation of enterocytes (Wu 1995).
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Table 1. Dry matter, energy content and amino acid composition of sow's milk and weanling diet |
). Plasma used for the cortisol assay was not deproteinized and was stored at 
20°C until analysis. Total plasma concentrations of cortisol were determined using the Coat-A-Count® cortisol kit (Diagnostic Products, Los Angeles, CA). This kit and the procedure used have been validated for the determination of plasma concentrations of cortisol in young pigs (Klemcke et al. 1989 and 1993). Heparin, unlike EDTA, has no effect on the accuracy or precision of the assay (Klemcke et al. 1993).
Preparation and incubation of enterocytes.
Enterocytes were prepared from the jejunum of 29-d-old pigs with the use of oxygenated (95% O2-5% CO2) Ca2+-free Krebs-Henseleit bicarbonate (KHB) buffer (pH 7.4) containing 5 mmol/L glucose and 5 mmol/L EDTA, as previously described (Watford et al. 1979, Wu 1995). Cells (4-5 g protein/L) were incubated for 0 or 30 min at 37°C in 2 mL of oxygenated (95% O2-5% CO2) KHB buffer containing 0 or 2 mmol/L [U-14C]arginine or [U-14C]glutamine (4.2 kBq/nmol). Incubations were terminated by addition of 0.2 mL of 1.5 mol/L HClO4, and 14CO2 was collected in 0.2 mL NCS (Wu et al. 1995). NCS is a surface-active organic base tissue solubilizer (Amersham, Arlington Heights, IL). The acidified incubation medium was neutralized with 0.1 mL of 2 mol/L K2CO3 and used for analysis of amino acids by HPLC methods involving precolumn derivatization with o-phthaldialdehyde (Wu et al. 1994b). Rates of production of arginine and glutamine metabolites were calculated by subtraction of basal (0 min incubation) concentrations of amino acids in cell extracts; rates were linear during the 30-min incubation period. Incubated enterocytes were viable for at least 30 min on the basis of linear O2 consumption, as determined by Clark-type polarographic oxygen probes (Wu et al. 1994b).
Determination of enzyme activities.
The activities of arginase, argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were determined as previously described (Wu 1995). Briefly, enterocytes (20 g protein/L) were suspended in KHB buffer and lysed by three cycles of freezing and thawing. Cell lysates were centrifuged for 1 min in a microcentrifuge (13,000 × g) and the supernatant was used for enzyme assays. For arginase assay, a solution (100 µL) consisting of 50 µL lysate supernatant (0.5 mg protein) and 50 µL of 50 mmol/L MnCl2 was incubated for 5 min at 55°C. After cooling to room temperature, the mixture was incubated at 37°C for 0 or 20 min with 50 µL of 30 mmol/L L-arginine (in 150 mmol/L potassium phosphate buffer, pH 7.5). The incubation was terminated by addition of 50 µL of 1.5 mol/L HClO4, and the acidified incubation mixture was neutralized with 25 µL of 2 mol/L K2CO3. The neutralized extract was used for analysis of ornithine by HPLC. For ASS assay, a solution (200 µL), which consisted of 75 mmol/L potassium phosphate buffer (pH 7.5), 10 mmol/L citrulline, 10 mmol/L aspartate, 5 mmol/L ATP, 5 mmol/L MgSO4 and cell lysates (0.5 mg protein), was incubated at 37°C for 0 or 20 min. The incubation was terminated by addition of 50 µL of 1.5 mol/L HClO4, and neutralized extracts were used for analysis of argininosuccinate and arginine by HPLC. For ASL assay, a solution (40 µL) containing 129 mmol/L sodium phosphate buffer (pH 7.0), 20 mmol/L argininosuccinate and 65 mmol/L EDTA was incubated at 37°C for 0 or 15 min. The incubation was terminated by addition of 20 µL of 1.5 mol/L HClO4, and neutralized extracts were used for determination of arginine by HPLC.
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Table 2. Glutamine metabolism in enterocytes from suckling pigs and from postweaning pigs that were or were not treated with RU4861 |
). Briefly, the assay mixture (1.0 mL) consisted of 0.1 mol/L HEPES (pH 7.4), 20 mmol/L MgCl2, 1 mmol/L gabaculine [an inhibitor of ornithine aminotransferase (OAT)], 1 mmol/L [U-14C]glutamate (16.8 kBq/nmol), 3 mmol/L ATP, 0.2 mmol/L NADPH, 15 mmol/L phosphocreatine, 10 U of creatine kinase, 0.25% Nonidet P-40 and mitochondria (1 mg protein). At the end of 0 or 30 min incubation periods at 23°C, the assay mixture was acidified with 100 µL of 1.5 mol/L HClO4, and the acidified assay mixture was neutralized with 40 µL of 2 mol/L K2CO3. [14C]P5C was separated from [14C]glutamate with the use of a Dowex AG 1-X8 resin (acetate form, 200-400 mesh, BioRad, Richmond, CA). The recovery of P5C from the resin column was >99% as determined with a purified P5C standard (Wu and Knabe 1995).
). DNA in enterocytes was measured by a fluorometric method involving Hoescht H-33258 with calf thymus DNA as a standard, as previously described (Wu and Knabe 1995).
Statistical analysis.
Data were analyzed by one-way ANOVA with Student-Newman-Keuls test for identifying differences among means (Steel and Torrie 1980). P < 0.05 was taken to indicate statistical significance. Values in the text are means ± SEM, n = 6.
RESULTS
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Table 3. Arginine metabolism in enterocytes from suckling pigs and from postweaning pigs that were or were not treated with RU4861 |
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Table 4. Activities of argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), arginase and pyrroline-5-carboxylate (P5C) synthase in enterocytes from suckling pigs and from postweaning pigs that were or were not treated with RU4861 |
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Table 5. Plasma concentrations of ammonia, urea, and cortisol in suckling pigs and in postweaning pigs that were or were not treated with RU4861 |
DISCUSSION
) and rats (Windmueller 1982, Windmueller and Spaeth 1976). About 20-25% of circulating glutamine is taken up by the small intestine of suckling and postweaning pigs (Wu et al. 1994a). Similarly, 25-30% of circulating glutamine is taken up by the small intestine of adult rats (Windmueller 1982). The small intestine of weaned pigs (Wu 1995) and rats (Wakabayashi 1995) has high activities of arginase, OAT, ornithine carbamoyltransferase (OCT) and P5C reductase and thus extensively metabolizes arginine to ornithine, urea and proline. About 32-37% of metabolized arginine carbons are recovered in ornithine in enterocytes of postweaning pigs (Wu et al. 1996a) and in the small intestine of adult rats (Windmueller and Spaeth 1976). Thus, the small intestine plays a quantitatively important role in utilizing dietary and circulating glutamine and arginine and therefore in regulating the homeostasis of these two amino acids. Interestingly, we have recently found that enterocytes of postweaning pigs have all of the enzymes necessary for ureagenesis and are capable of synthesizing urea from ammonia (Wu 1995). Synthesis of urea from glutamine occurs in enterocytes of postweaning pigs as a result of the induction of arginase but is absent in cells from suckling pigs because of negligible activity of arginase (Wu 1995).
, Visek 1984, Wakabayashi et al. 1994). For example, glutamine is required for the synthesis of purine and pyrimidine nucleotides, NAD+ and aminosugars, and is a major energy substrate for rapidly proliferating cells (Lacey and Wilmore 1990). Arginine is the precursor of ornithine, creatine, agmatine and nitric oxide and is a potent endocrine secretagogue (Barbul 1995, Galea et al. 1996). In nutritional practice, dietary supplementation of glutamine prevents jejunal atrophy in early weaned pigs (Wu et al. 1996b), probably by providing energy and purine and pyrimidine nucleotides for enterocytes (Wu et al. 1995) and by stimulating enterocyte ornithine decarboxylase (Kandil et al. 1995). Also, dietary supplementation of arginine to mice improves survival in gut-derived sepsis and peritonitis by modulating bacterial clearance via NO synthesis (Gianotti et al. 1993). Thus, characterization of the regulation of intestinal metabolism of glutamine and arginine is of nutritional and physiological importance.
) and provide direct evidence for an essential role of glucocorticoids in mediating the induction of P5C synthase and the enhanced metabolism of glutamine to ornithine, citrulline, proline and CO2 in enterocytes of weaned pigs. RU486 treatment decreased arginase activity by 73% (Table 4) and the production of ornithine, proline, and CO2 from arginine by 52, 100 and 84% (Table 3), respectively, in enterocytes of postweaning pigs. Because there are two isozymes of arginase in pig enterocytes (M
Rabet-Touil et al. 1996), it is not clear at present which form(s) of arginase may be induced by cortisol and repressed by RU486. On the basis of the recovery of metabolized arginine carbons in ornithine, citrulline, proline and CO2 in enterocytes from pigs treated or not with RU486 [40 vs. 169 nmol carbon/(mg protein·30 min), calculated from Table 3], it can be estimated that RU486 treatment decreased arginine metabolism via the arginase pathway by 78%. These results suggest that glucocorticoids play an important role in mediating the induction of arginase and the enhanced metabolism of arginine to ornithine, proline and CO2 in enterocytes of postweaning pigs. It is noteworthy that weaning is also associated with increased plasma concentrations of thyroid hormones (Angel and Back 1985), which has been reported to increase hepatic arginase activity and mRNA levels (Callery and Elinson 1996, Iwase et al. 1995). This may explain why RU486 completely blocked the induction of intestinal arginase in cortisol-treated suckling pigs (Flynn and Wu 1997) but not in postweaning pigs (Table 4). The finding that RU486 failed to attenuate or prevent the increase in ASS activity in enterocytes of postweaning pigs (Table 4) suggests that factors other than glucocorticoids (e.g., thyroid hormones) may play a key role in the induction of intestinal ASS.
), probably because piglets used in the present study, unlike the pigs used previously, were weighed and separated from litter mates and thus were stressed at 21 d of age. Second, a 10-fold increase in intestinal arginase activity in postweaning pigs resulted in eight- and fivefold increases, respectively, in the production of ornithine and CO2 from 2 mmol/L arginine, compared with age-matched suckling pigs. The induction of arginase resulted in the synthesis of large amounts of proline from arginine in enterocytes of postweaning pigs, and an inhibition of arginase activity by RU486 resulted in no production of proline from arginine. Third, RU486 treatment prevented weaning's induction of P5C synthase and consequently abolished the increase in the synthesis of ornithine, citrulline and proline from glutamine in postweaning pig enterocytes, further suggesting that P5C synthase is a key regulatory enzyme in the synthesis of citrulline from glutamine (Wakabayashi 1995, Wu et al. 1994b). Fourth, in enterocytes from RU486-treated pigs, a 73% decrease in arginase activity compared with untreated weaned pigs was associated with a similar (78%) decrease in the overall metabolism of arginine to ornithine, proline and CO2, further substantiating a major role of arginase in the degradation of arginine in pig enterocytes (Wu et al. 1996a).
). This explains our observation that there was no difference in plasma cortisol concentrations between suckling pigs and untreated 29-d-old weaned pigs (d 8 postweaning) (Table 5). It appears that an early increase in plasma cortisol concentrations on d 2 postweaning was sufficient to induce arginase, ASL and P5C synthase in pig enterocytes, which was then sustained. Interestingly, RU486 administration increased the plasma concentration of cortisol by 356% compared with untreated weaned pigs. This finding was similar to that reported for humans administered RU486 (Bertagna et al. 1986), which has been suggested to result from the hypersecretion of cortisol by the adrenal cortex (Bertagna et al. 1986).
) and weaning (this study) is of nutritional relevance. First, inductions of arginase and ASL by cortisol and weaning make it possible for the small intestine to detoxify as urea locally produced ammonia due to enhanced metabolism of glutamine [a major fuel (Wu et al. 1995)] by enterocytes of postweaning pigs (Table 2). Second, the formation of large amounts of ornithine from arginine as a result of the induction of arginase resulted in the synthesis of large amounts of proline via arginase, OAT and P5C reductase in postweaning pig enterocytes. Third, the induction of P5C synthase by cortisol and weaning enhanced the synthesis of citrulline and proline from glutamine and thus increased the availability of citrulline for renal synthesis of arginine. Our findings may help to explain why arginine and proline are nutritionally indispensable amino acids for young pigs but not for adult pigs (Ball et al. 1986, Mertz et al. 1952), and may have relevance to infant nutrition because the pig is a widely used animal model for studying intestinal amino acid metabolism in humans (Kandil et al. 1995, Reeds et al. 1996, Wu et al. 1996a).
ACKNOWLEDGMENTS
FOOTNOTES
-hydroxy-11-(4-dimethylaminophenyl)17
-(prop-1-ynyl)estra-4,9-dien-3-one.
Manuscript received 18 September 1996. Initial reviews completed 30 October 1996. Revision accepted 13 January 1997.
LITERATURE CITED
s syndrome.
J. Clin. Endocrinol. Metab.
1986;
63:639-643
[Abstract]
Rabet-Touil H.,
Lerminiaux H.,
Duee P. H.,
Blachier F.
Evidence for increased anionic arginase activity in pig enterocytes during development.
Biochem. Mol. Biol. Int.
1996;
38:197-204
[Medline]
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