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Changes of activity and mRNA expression of urea cycle enzymes in the liver of developing Holstein calves¹

M. Takagi^*, T. Yonezawa^*, S. Haga, H. Shingu, Y. Kobayashi, T. Takahashi^*, Y. Ohtani^*, Y. Obara^* and K. Katoh^*,2

^* Department of Animal Physiology, Graduate School of Agricultural Science, Tohoku University, 1-1 Amamiyamachi, Sendai, Miyagi, 981-8555, Japan; and and National Institute of Livestock and Grassland Science, Ikenodai, Tsukuba, Ibaraki, 305-0901, Japan

	Abstract

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Urea is an important reutilizable nitrogen source for the ruminant and is mainly synthesized through the urea cycle in the liver. The cycle is undertaken by 5 enzymes: carbamoyl phosphate synthetase (CPS), ornithine transcarbamoylase (OTC), arginino-succinate synthetase (AS), argininosuccinate lyase (AL), and arginase. The purpose of this study was to investigate changes in the activity of the enzymes and mRNA expression, given that previous observations have indicated an increase in plasma urea concentrations with age in Holstein calves. First, plasma concentrations of metabolites and hormones were determined in calves at 1, 3, 8, 13, and 19 wk of age (n = 4, weaned at 6 wk of age). The plasma concentration of urea drastically increased after weaning (P < 0.001). The plasma concentration of glucose was lowest at 8 wk. The plasma concentration of IGF-I gradually increased with age, although those of NEFA, glucagon, and cortisol decreased (P < 0.001). Concentrations of triglyceride, -amino nitrogen, growth hormone, and insulin did not change significantly with age of the calf. Next, using the liver tissues taken from calves at 2, 13, and 19 wk of age (n = 4 to 6 at each time point, weaned at 6 wk of age), we measured the activity and mRNA expression of the enzymes by biochemical methods and quantitative reverse transcription-PCR, respectively. The activities of CPS (P < 0.001), OTC (P = 0.001), and AS (P = 0.015) increased with age, whereas AL (P = 0.003) decreased. Although mRNA expression was decreased with age for AL (P = 0.002) and arginase (P = 0.007), no significant change was observed for CPS, OTC, or AS mRNA expression. We conclude that the increased urea production in the liver may be explained not only by an increase in the activities of the urea cycle enzymes, but also by increased ammonia production by rumen fermentation and gluconeogenesis from amino acids around weaning time.

Key Words: calf • development • liver • mRNA • urea cycle enzyme • weaning

	INTRODUCTION

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The digestive and metabolic mechanisms of the ruminant change drastically around the time of weaning. In postweaning ruminants, solid diets are mainly fermented to VFA as the main source of energy and to ammonia, methane, and carbon dioxide in the rumen by bacterial activity. A portion of the ammonia produced is utilized in the foregut for the synthesis of bacterial proteins, which are digested and absorbed as peptides or amino acids in the small intestine, and are finally utilized as body proteins, nucleic acids, and substrates of gluconeogenesis (Lapierre and Lobley, 2001).

Excess ammonia is converted into urea in the liver and is excreted into the urine. In the ruminant, however, urea is partially transferred back into the digestive tract by either secretion into saliva or transport across the rumen wall, and is recycled for the synthesis of bacterial proteins (Obara and Shimbayahsi, 1980; Lapierre and Lobley, 2001). Thus, urea is an important reutilizable nitrogen source for the ruminant.

The urea cycle, which is involved in the conversion of toxic ammonia into the less toxic urea, consists of 5 key enzymes: carbamoyl phosphate synthetase (CPS, EC 6.3.4.16), ornithine transcarbamoylase (OTC, EC 2.1.3.3), argininosuccinate synthetase (AS, EC 6.3.5.4), argininosuccinate lyase (AL, EC 4.3.2.1), and arginase (ARG, EC 3.5.3.1). These enzymes are regulated by nutritional and hormonal factors in rats (Takiguchi and Mori, 1995).

Recently, some reports have suggested that the plasma concentration of urea is increased after weaning in calves (Quigley et al., 1994; Cowles et al., 2006; Quigley et al., 2006; Khan et al., 2007a,b). However, there have been no reports on changes in the urea cycle enzyme activities around the weaning time of the ruminant. In this study, we therefore investigated changes in the activity of the urea cycle enzymes and mRNA expression from the point of view of an increase in plasma urea concentrations around weaning time in Holstein calves.

	MATERIALS AND METHODS

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This study was conducted according to the "Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences" (The Physiological Society of Japan, Tokyo), and was approved by The Animal Care Committee of Tohoku University.

Animals
Holstein male calves were fed colostrum from their dams once after birth on each farm. They were then gathered at the Mito Research Farm (Meiji Feed Co., Tokyo, Japan) and fed artificial colostrum (Head Start, The Saskatoon Colostrum Co. Ltd., Saskatoon, Canada) for 3 d. Beginning on d 4 after birth, the calves were fed milk replacer (Meiji Feed Co.), which was diluted 1:7 (vol/vol) in water, and calf starter (Snow Brand Seed Co., Sapporo, Japan), as shown in Table 1 and as described in our previous paper (Kitade et al., 2002), until 19 wk of age. The calves were weaned at 6 wk of age. The artificial colostrum contained 57.4% CP (15.5% IgG) and 4.2% crude fat on a DM basis. The milk replacer contained 24.5% CP and 21.9% crude fat on a DM basis. The calf starter contained 20% CP and 75% TDN on a DM basis. Timothy hay, water, and mineral salts were fed ad libitum. The calves were fed according to the Japanese Feeding Standard (AFFRC, 1990).

Blood samples were taken by jugular venipuncture feeding at 0900 h. Samples (5 mL) were immediately transferred into heparinized (VP-H100K, Termo, Tokyo, Japan) polyethylene tubes cooled in ice water. Samples (2 mL) for the glucagon assay were immediately transferred into heparinized (VP-100K, Termo) polyethylene tubes containing aprotinin (500 kIU/mL of blood (Wako Pure Chemical Industries, Osaka, Japan). The samples were centrifuged at 8,000 x g for 15 min at 4°C to obtain plasma. The plasma samples were divided into portions, which were immediately frozen at –30°C until assayed for metabolites and hormones.

Approximately 50-g liver tissue samples were taken after slaughter by exsanguination after anesthesia was induced by sodium thiopental (1 g/head; Ravonal, Tanabe, Osaka, Japan); samples were immediately snap-frozen in liquid nitrogen and stored at –80°C until they were assayed for the activity and expression of hepatic urea cycle enzymes.

Plasma Concentrations of Metabolites and Hormones
Plasma concentrations of glucose, NEFA, and triglyceride were determined by using commercially available kits (Wako Pure Chemical Industries Ltd., Osaka, Japan). Plasma concentrations of urea and cortisol were determined by using the urea-ammonia F-kit (J. K. International Inc., Tokyo, Japan) and cortisol ELISA kit (Oxford Biomedical Research Inc., Oxford, MI), respectively, according to the manufacturer’s instructions. The plasma concentrations of -amino nitrogen were determined by using the method described by Lee and Takahashi (1966). Plasma concentrations of GH, insulin, IGF-I, and glucagon were measured by RIA, as described by Kuhara et al. (1991). The intraassay CV for the GH, insulin, IGF-I, and glucagon assays were 8.1, 6.7, 16.9, and 12.2%, respectively. The minimum detectable concentration for GH, insulin, IGF-I, and glucagon was 0.2 ng/mL, 0.1 µU/mL, 50 pg/mL, and 1 pg/mL, respectively. All samples were analyzed in the same assay to eliminate interassay variation.

Activities of Liver Urea Cycle Enzymes
Extraction of Homogenate from the Liver Tissue.
The liver tissue samples were homogenized and extracted according to a method slightly modified from that described by Kharbuli et al. (2006). A 10% (wt/vol) homogenate of the tissues was prepared in a homogenizing buffer containing 100 mM Tris-HCl buffer (pH 7.5), 50 mM KCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 500 kIU/mL of aprotinin, using a Multi-beads shocker cell disruptor (MB400U, Yasui Kikai, Osaka, Japan). The homogenate was subjected to mild sonication for proper breakage of mitochondria only for the assay of CPS and OTC. Sonication was conducted on ice 5 times for 2 s, with a 2-s interval between each sonication, using a Branson sonifier (Model S 450, micro-tip, 4 A output, Branson Ultrasonics Corp., Danbury, CT). After the homogenate was centrifuged at 10,000 x g for 10 min, the supernatant was used for the assay. All steps were carried out at 4°C.

Determination of Urea Cycle Enzyme Activities.
Activities of the liver urea cycle enzymes were determined according to a method modified from those described by Saha et al. (1995) and Mohamed et al. (2005). The assay procedure was basically similar for all the enzymes. The consumption of citrulline or urea was measured for the assay of CPS, OTC, and AS, and AL or ARG, respectively.

Determination of CPS Activity.
The assay mixture contained 50 µmol of potassium phosphate buffer (pH 7.5), 50 µmol of ammonium chloride, 50 µmol of sodium bicarbonate, 10 µmol of ATP, 10 µmol of L-ornithine, 15 µmol of MgSO₄, 5 µmol of N-acetyl-L-glutamate, and 0.2 mL of tissue extract, all in a final volume of 1 mL. The reaction time was 60 min. The enzyme reaction was carried out at 30°C and was stopped by adding 0.5 mL of 10% perchloric acid per 1 mL of reaction mixture, followed by centrifugation to remove precipitated protein. Citrulline in the supernatant was measured by the method of Boyde and Rahmatullah (1980). One unit of enzyme activity was defined as the amount of enzyme catalyzing 1 µmol of product formed or substrate used per hour at 30°C (µmol·h^–1). Enzyme activity was normalized according to the protein content, which was measured by using a protein quantification kit (Dojindo Laboratories, Kumamoto, Japan).

Determination of OTC Activity.
The assay mixture contained 90 µmol of glycylglycine buffer (pH 8.3), 20 µmol of L-ornithine, 20 µmol of carbamoylphosphate, and 0.1 mL of tissue extract in a final volume of 2 mL. The reaction time was 15 min.

Determination of AS Activity.
The assay mixture contained 50 µmol of potassium phosphate buffer (pH 7.0), 3 µmol of L-citrulline, 5 µmol of L-aspartate, 10 µmol of ATP, 10 µmol of MgSO₄, 20 units of urease, and 0.2 mL of tissue extract in a final volume of 1 mL. The reaction time was 60 min.

Determination for AL Activity.
The assay mixture contained 50 µmol of potassium phosphate buffer (pH 7.3), 4 µmol of argininosuccinate, and 0.2 mL of tissue extract in a final volume of 1 mL. The reaction time was 60 min. Urea in the supernatant was measured by using a urea-ammonia F-kit (J. K. International Inc.).

Determination of ARG Activity.
The assay mixture contained 50 µmol of glycine buffer (pH 9.5), 50 µmol of L-arginine, 0.5 µmol of MnCl₂, and 0.1 mL of tissue extract in a final volume of 2 mL. The reaction time was 40 min.

mRNA Expression of Liver Urea Cycle Enzymes
Reverse Transcription.
Total RNA was extracted by using NucleoSpin RNA II (Nippon Genetics Co. Ltd., Tokyo, Japan) according to the manufacturer’s instructions. A 5-µg quantity of total RNA was then reverse transcribed using SuperScript III (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions.

Quantitative Reverse Transcription-PCR.
Quantitative reverse transcription-PCR was performed by using primer sets and the SYBR Green qPCR kit (Finnzymes, Espoo, Finland) in a DNA engine Opticon 2 Continuous Fluorescence Detector (MJ Research Inc., Watertown, MA) according to the method described by Yonezawa et al. (2004), but with modifications. The sequences, expected amplicon sizes, and GenBank accession numbers of each primer set are shown in Table 2. The reactions were performed in 10 µL of final volume containing 1 x SYBR Green Master Mix, 0.5 µM primer mixtures, and 0.5 µL from the reverse transcription product. To reduce variability between replicates, PCR premixes, which contained all reagents except for the template, were prepared and aliquoted into 0.2-mL thin-well plates (MJ Research Inc.). The PCR conditions were 40 cycles of the following protocol: 10 s of denaturation at 95°C, 20 s of annealing at 60°C, followed by 20 s of extension at 72°C. The post-PCR melting curves confirmed the specificity of single-target amplification. All products were sequenced to confirm the identity of each gene. The expression of each gene relative to glyceraldehyde-3-phosphate dehydrogenase was determined. A significant difference in glyceraldehyde-3-phosphate dehydrogenase was not seen among different ages (P = 0.98; data not shown).

	RESULTS

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All the amounts of milk replacer given to the calves were consumed within 15 min. The plasma concentrations of the metabolites and hormones are shown in Table 3. Although the plasma concentration of urea gradually increased by 3 wk of age, it drastically increased after weaning at 6 wk of age (P < 0.001). The plasma concentration of glucose was lowest at 8 wk of age (P < 0.001), but gradually recovered to the preweaning level by 19 wk of age (P < 0.001). The plasma concentration of NEFA was decreased with age (P < 0.01).

The activities and mRNA expression of the urea cycle enzymes are shown in Table 4. The enzyme activity of CPS was the lowest of the 5 enzyme activities, and was greater at 13 and 19 wk of age than at 2 wk of age (P < 0.001). Although the activities of OTC (P = 0.001) and AS (P = 0.015) also gradually increased with age, that of AL at 13 and 19 wk of age was smaller than that at 2 wk of age (P = 0.003). The activity of ARG was not changed. Although mRNA expression was not changed in CPS, OTC, or AS over this period of time, those of AL (P = 0.002) and ARG (P = 0.007) at 13 and 19 wk of age were smaller than that at 2 wk of age.

	DISCUSSION

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Ruminants synthesize glucose, preferentially from propionate and amino acids, by gluconeogenesis in the liver, particularly after weaning (Hayashi et al., 2006). A reciprocal association between glucose and urea metabolism in ruminants has been reported. Intravenous glucose infusion improved nitrogen retention and lowered plasma urea nitrogen concentration in lambs (Eskeland et al., 1974), and also decreased urinary nitrogen and increased the nitrogen balance (Matras and Preston, 1989). Supplementation of adult ruminants with dietary sucrose altered the metabolism of urea and glucose in plasma via the rate of ammonia and propionate production in the rumen (Sutoh et al., 1993, 1996).

Hayashi et al. (2006) reported that plasma urea nitrogen as well as the rates of irreversible urea loss and recycling gradually increased with age as well as with the development of the reticulorumen in calves. Ammonia is produced from dietary nitrogen and endogenous urea by the protease, peptidase, and urease activities of rumen microbes. After ammonia is produced, it is absorbed through the rumen epithelium into the portal bloodstream and is converted into urea by urea cycle enzymes in the liver. Urea, which is recycled to the rumen, is an important nitrogen source for the synthesis of microbial protein in ruminants. Recycling of urea is thought to increase with development of the rumen. Therefore, urea cycle enzymes may change as rumen function develops in calves. The urea nitrogen pool and irreversible loss rate have been shown to increase with increasing serum urea concentrations as well as with increasing nitrogen intake in adult ruminants (Obara and Shimbayashi, 1980; Obara and Dellow, 1993).

Results on the activities of hepatic urea cycle enzymes in other animal species have been reported previously. Generally, absolute values of enzyme activities for OTC and ARG are approximately 100 times greater than those for CPS, AS, and AL, as was also demonstrated in the present study. Although AS showed the least activity, and is considered a rate-limiting enzyme in rats and goats (Charbonneau et al., 1967; Ide and Shimbayashi, 1968), in the present study CPS showed the least activity of the 5 enzymes, and is considered a rate-limiting enzyme in calves. The regulation mechanism of the urea cycle in calves may differ from that of other animal species.

Boling and Nuzum (1975) reported that aging did not affect the activities of the urea cycle enzymes in calves at a range in age of 3 to 12 yr. Therefore, this finding suggests that urea cycle enzymes may be affected by aging only around weaning time. Variation of the urea cycle enzymes with aging in other animal species has been reported. The activities of hepatic urea cycle enzymes, except for AL, were increased with aging in rats (Charbonneau et al., 1967). There was a slight decrease in CPS mRNA with aging in mice (Dhahbi et al., 1999). These reports indicate that activities of the urea cycle enzymes may be increased without the induction of mRNA, as suggested by the present study. However, because the metabolism of weaned ruminants is fundamentally different from that of rodents, it is not reasonable to compare these activities among different animal species.

Generally, mammalian fetuses transfer ammonia to their mothers. Neonatal infants, however, must remove ammonia in some way after birth. This acute change in environment is thought to lead to an increase in plasma concentration of cortisol to induce urea cycle enzymes (Takiguchi and Mori, 1995). Plasma concentration of cortisol in neonatal calves is also high (Knowles et al., 2000). Additionally, induction of mRNA expression of the hepatic urea cycle enzymes by gluco-corticoids, such as dexamethasone, was reported for rats (Kimura et al., 2001). The high plasma concentration of cortisol at 1 wk of age and increased mRNA expression of urea cycle enzymes at 2 wk of age in our results are consistent with these reports.

Gene expression is regulated by posttranscriptional and posttranslational modifications as well as by transcription and translation. In the former, the stability of mRNA and translation initiation is regulated by specific nucleotide sequences and many binding proteins that bind these sequences (Day and Tuite, 1998). In the latter, proteins change their activity state, turnover, and interactions with other proteins by the addition of a modifying group to one or more amino residues (Mann and Jensen, 2003). The long-term control of metabolic pathways can also be affected by the rates of synthesis and degradation of enzymes. Additionally, N-acetylglutamate is absolutely required for activation of CPS as an allosteric activator (Meijer et al., 1990). Thus, because the regulation of enzyme activity is complex, the cause of the mismatch during developmental changes between the activity and the level of a particular mRNA is not clear. Possible causes for the mismatch are the increasing stability of mRNA, the decreasing turnover rate of enzyme proteins, modification of the enzymes, and an increase in allosteric activator with development.

In conclusion, we demonstrated that activities of the hepatic urea cycle enzymes CPS, OTC, and AS increased in developing Holstein calves, which seemed to contribute to increased urea synthesis and recycling. Increased activities of urea cycle enzymes may also be secondarily caused by the demand on urea synthesis after weaning, such as by an increased production of ammonia by ruminal microbes or gluconeogenesis from amino acids.

	Footnotes

 
¹ We express our appreciation to S. Hayashi (Laboratory of Functional Morphology, Tohoku University), T. Takimoto (Laboratory of Animal Nutrition, Tohoku University), and Y. Hoshino (Laboratory of Animal Reproduction, Tohoku University) for their helpful advice on this study, and to A. F. Parlow for the antibodies to ovine GH and IGF-I provided by NIDDK (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD).

² Corresponding author: kato{at}bios.tohoku.ac.jp

Received for publication December 12, 2007. Accepted for publication March 3, 2008.

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