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ANIMAL NUTRITION |
Department of Animal and Food Sciences, University of Kentucky, Lexington 40546-0215
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: amino acid transport • bovine • cationic amino acid transporter-1 • regulated gene expression • SLC7A1 • small intestine
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Merchen and Titgemeyer, 1992) and the second-limiting AA when microbes serve as the only protein supply (Richardson and Hatfield, 1978). Although bypass proteins (which have a relatively high L-Lys content) are typically fed as a source of supplemental L-Lys, very little is known about the transport proteins expressed by the small intestinal epithelia that are responsible for absorbing free CAA from luminal digesta (Moe et al., 1987; Matthews, 2000).
In mammals, 4 CAA transport system activities have been identified (y+, y+L, b0,+, B0,+). Of these, system y+ activity (Na+-independent uptake of L-Lys, L-Arg, L-ornithine, and, to a lesser degree, L-His) is reported to be expressed along the entire small intestine on both the apical and basolateral membranes of absorptive epithelial cells (Devés and Boyd, 1998; Palacín et al., 1998; Chairoungdua et al., 1999; Krehbiel and Matthews, 2003). The small intestinal epithelia of Holstein bulls and steers possess large amounts of Na+-independent Lys uptake (Moe et al., 1987; Wilson and Webb, 1990). Four proteins [cationic AA transporter-1 (CAT-1), CAT-2a, CAT-2b, and CAT-3] are known to possess system y+ activity. Of these, CAT-1 is the only one 1) abundantly expressed by the small intestinal epithelium, 2) possessing a high affinity (Km in the µM range) for L-Lys and L-Arg, and 3) that is sensitive to trans-stimulation (Devés and Boyd, 1998; Hatzoglou et al., 2004). In cell culture models, the relative steady-state expression levels of CAT-1 mRNA are considered proportional to CAT-1 activity (Hyatt et al., 1997; Fernandez et al., 2001), indicating that the content of CAT-1 mRNA likely represents relative CAT-1 activity. Understanding the expression pattern of CAT-1 mRNA by the small intestinal epithelium as steers develop from the suckling through the finishing production stage will facilitate the matching of dietary supplementation of CAA with small intestinal absorption capacity.
The first goal of this research was to determine whether bovine steers express a CAT-1 mRNA ortholog. The second goal was to test the hypotheses that expression of CAT-1 mRNA by duodenal, jejunal, and ileal epithelia differs among suckling, weanling, growing, and finishing steers, and within a particular stage, among the epithelia.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Care of Animals. Research protocols were approved by the University of Kentucky Institutional Animal Care and Use Committee.
Twenty-four purebred Angus cow-bull calf pairs were obtained from the University of Kentucky Agricultural Research Center Beef Unit. Calves were weighed and castrated from 12 to 24 h after birth, and vitamins A and D (1.0 mL; Sparhawk Labor Inc., Shawnee Mission, KS) and E with Se (0.5 mL; MU-SE, Schering-Plough Animal Health, Union, NJ) were administered. After all calves were born, steers were assigned randomly to 1 of 4 treatment groups, which were the suckling, weanling, growing, and finishing production stages (n = 6). All cow-calf pairs were maintained on a mixed pasture consisting of approximately 50% orchardgrass, 25% endophyte-free fescue, and 25% clover. All steers had ad libitum access to water and a mineral mix throughout the trial. Calves had unrestricted access to their dams, and the cow-calf pairs were observed daily for any signs of sickness. At approximately 84 d of age, the calves were vaccinated with Clostri Shield 7 Way and Vira Shield 5 plus Somnus (Grand Laboratories, Larchwood, IA), except for those in the suckling treatment group.
The compositions of the diets for the 4 production stages are presented in Table 1. Suckling (suckling milk) and weanling (grazing grass and suckling milk) treatment calves remained with their dams until slaughter at 32 ± 1 and 184 ± 1 d of age, respectively, whereas growing (fed a corn silage diet) and finishing (fed a high-concentrate corn diet) treatment calves were weaned as a group in a pen at 180 ± 5 d of age. The weaned calves were fed a high-stress adaptation diet consisting of (as-fed basis) 34.3% cracked corn, 15% cottonseed hulls, 15% whole oats, 16% soybean meal, 5% cane molasses, 4% dicalcium phosphate, 6% limestone, 3% trace mineralized salt (94% salt, 5.50 g/kg of Zn, 9.28 g/kg of Fe, 4.79 g/kg of Mn, 1.84 g/kg of Cu, 1.15 mg/kg of I, 0.18 mg/kg of Se, 0.65 mg/kg of Co; Burkman Feeds, Danville, KY), 0.3% vitamin A (8,811 IU/g), D (1,762 IU/g), and E (11 IU/g) premix (ADM Alliance Nutrition, Quincy, IL), 1.2% decoquinate pre-mix (6% decoquinate; Deccox, Alpharma Inc., Bridgewater, NJ), and 0.2% chlortetracycline premix (110 g of chlortetracycline/kg, Auromycin 50, Alpharma Inc.). After 7 d, these steers were moved to individual 3 x 3.7 m pens in an environmentally controlled room (21°C) with 16-h light:8-h dark cycles. For the first 30 d in the room, the steers were fed enough of a fescue hay (76%) plus soybean hull (23%) diet to maintain BW and were allowed to adapt to the individual pens. After the adaptation period, steers of both groups were fed enough growing diet (Table 1) to gain BW at 1 kg/d (NRC, 2000). After 31 to 33 d of feeding, growing treatment steers were slaughtered (at 248 ± 2 d of age), whereas finishing treatment steers were moved and housed as 2 groups of 3 steers in 2 open-air, 8 x 30 m pens. Steers were fed enough of a high-concentrate, corn-based diet (finishing diet; Table 1) to gain BW at 1 kg/d for 175 ± 5 d. Fourteen days before slaughter at 423 ± 3 d of age, finishing calves were housed individually to facilitate determination of individual feed intake.
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Slaughter and Tissue Collection. Suckling treatment steers were anesthetized by intrajugular administration of pentobarbital (80 mg/kg of BW) and then exsanguinated during tissue removal. Steers of all other treatment groups were stunned with a captive-bolt pistol and exsanguinated. Serially, the liver, kidney, small intestine, and pancreas were removed. The total length of the small intestine (pyloric valve to ileal-cecal junction) was determined by looping the intestine across a board that was fitted with metal pegs at 2-m increments. Looping of the small intestine was performed without tension to minimize stretching, and the board was kept wet to minimize tissue shrinkage.
The sites and protocol of tissue sampling for RNA extraction were described previously by Howell et al. (2001). More specifically, 1-m sections of the duodenum (0.5 to 1.5 m caudal to the pyloric junction), jejunum (middle of the first half of the nonduodenal small intestine), and ileum (middle of the second half of the nonduodenal small intestine) were removed. Each section was bisected, inverted, and rinsed with cold (4°C) physiological saline (0.9% NaCl). One half of each intestinal section was scraped to collect the epithelia (Matthews et al., 1996) and the other half was frozen in liquid N2.
Total RNA Extraction and Reverse Transcription-PCR
Total RNA Extraction. Total RNA was extracted from approximately 1 g of scraped epithelia of duodenal, jejunal, and ileal samples from each steer. The RNA extraction was performed by using TRIzol reagent, following the instructions of the manufacturer (Invitrogen, Carlsbad, CA). To obtain RNA for reverse transcription (RT) reactions, 1 g of epithelial tissue was homogenized in 5 mL of TRIzol. After total RNA was recovered, the RNA pellet was washed gently with 75% ethanol (0.5 mL) and centrifuged at 7,500 x g at 4°C for 5 min. The cleaned RNA was then suspended in RNase-free distilled H2O and stored at –80°C. The quality of the total RNA samples was determined by electrophoresis of RNA solutions on agarose gels and visualization of the integrity of the 18S and 28S RNA bands. The concentration, purity, and amount of total RNA were determined by UV spectrophotometry. With these standard procedures, nearly all RNA samples used for the assay were of high quality and purity (260/280 absorbance >1.7).
RT-PCR.
For partial-length cloning of bovine CAT-1, the RT-PCR was performed by using a 2-step protocol designed in accordance with the manufacturer’s guidelines (Invitrogen). For the first step, 5 µg of total RNA was reverse-transcribed to complementary DNA (cDNA) by using a SuperScript III RT, Ologo(dT)20 primer, Random Hexamer, and deoxy nucleotide 5'-tri-phosphate (dNTP) mix. For the second step, all of the PCR reactions were performed in a Hybaid MultiBlock PCR System (Thermo Electron Corporation, Waltham, MA). Each 50-µL reaction mixture contained 4.0 µL of RT product (>200 ng) as DNA template, 0.8 µM each forward and reverse primer (Table 2), 1.5 U of Platinum Taq DNA Polymerase, 4.0 mM MgCl2, and 0.2 mM of each dNTP (all from Invitrogen). The thermal cycle program for the PCR consisted of 1 cycle at 94°C (5 min), 38 cycles at 94°C (0.5 min), 62.5°C (0.5 min), and 72°C (1.0 min), 1 cycle at 72°C (10 min), and 1 cycle at 4°C. The forward and reverse primers were designed based on a pig CAT-1 cDNA sequence (GenBank accession no. NM_001012613; Table 2 and Figure 1).
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Cloning.
The putative CAT-1 PCR products were purified from the PCR reaction mixture by using a Pure-Link Quick Gel Extraction Kit (Invitrogen) and then amplified by using the TA Cloning Kit (Invitrogen) in accordance with the manufacturer’s instructions. Briefly, the PCR product was inserted into pCR II plasmid vector by ligation with T4 DNA Ligase overnight at 14°C; the resulting construct was then transfected into One Shot INV
F' Chemically Competent Escherichia coli cells. The transfected E. coli cells were grown on Luria-Bertani agar plates containing ampicillin (at 100 µg/mL) and X-Gal (40 µL of a 40 mg/mL solution). After incubation of the plates at 37°C overnight, 10 to 20 white colonies were selected for further culture in 3 to 4 mL of Luria-Bertani-ampicillin (100 µg/mL) broth overnight at 37°C with vigorous shaking (
250 to 300 rpm/min). The plasmids were then isolated from the E. coli cells by using the Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI) according to the manufacturer’s instructions. Recovered plasmids were subjected to restriction analysis by using endonucleases EcoRI and NcoI. The cDNA yielding the appropriate restriction patterns were sent to the DNA Sequencing Core Laboratory at the University of Florida (Gainesville) for sequencing.
Bioinformatic Analysis of RT-PCR Products. The sequences of the molecular cloning products were compared with the template sequences for validation purposes by using the National Center for Biotechnology Information (NCBI) BLASTN computer program (version 2.2.12; www.ncbi.nlm.nih.gov/BLAST; last accessed Nov. 12, 2007). Multiple alignment of bovine CAT-1 cDNA sequences with those of other animal species reported in the literature was performed by using the ClustalW computer program (version 1.83) at the European Molecular Biology Laboratory–European Bioinformatics Institute (EMBL-EBI) Web site (http://www.ebi.ac.uk/clustalw/index.html; last accessed Nov. 12, 2007). During the alignment work, the default parameters with the program were used.
Real-Time RT-PCR Quantification of Bovine CAT-1 mRNA
RT.
For relative quantification of CAT-1 gene expression, a real-time RT-PCR analysis was performed by using a 2-step protocol designed according to the manufacturer’s guidelines (Applied Biosystems, 2004). Total RNA was treated with DNase I (amplification grade, Invitrogen) as described by the manufacturer. Briefly, 1 µg of total RNA was combined with 1 µL of 10x reaction buffer, 7 µL of diethylpyrocarbonate-treated H2O, and 1 µL of DNase I and incubated at room temperature for 15 min. One microliter of 25 mM EDTA was then added to the DNase-treated total RNA and incubated at 65°C for 10 min to stop the reaction. The entire volume of DNAse-treated total RNA (approximately 11 µL) was then used in the RT reaction, as suggested by the manufacturer (Invitrogen). Briefly, a solution of Hexamers (50 ng/µL) and Oligo(dT)20 (50 µM) primers (1 µL each) was added to each reaction. The reaction mixture was incubated at 70°C for 10 min and then chilled on ice for 1 min. Afterward, a solution containing 2 µL of RT buffer (10x), 2 µL pf dithiothreitol (0.1 M), 4 µL of dNTP (10 mM each), 4 µL of MgCl2 (25 mM), and 1 µL of RNAse Out (Invitrogen) was added to the reaction. After incubation at 37°C for 2 min, the reaction was incubated with SuperScript II reverse transcriptase (1 µL) at room temperature for 10 min, and then incubated at 37°C for 50 min. To stop the reaction, the reaction mixture was incubated at 70°C for 10 min and then chilled on ice. The resulting reaction products, which were cDNA, were then stored at –20°C until use in the real-time RT-PCR analysis of CAT-1 mRNA content.
Real-Time RT-PCR.
The real-time PCR of RT products was performed by using an ABI Prism 7000 Sequence Detection System and a Custom TaqMan Primer and Probe set (Applied Biosystems, Foster City, CA), consisting of 2 unlabeled PCR primers and 1 Taq-Man Minor Groove Binding probe with FAM, a reporter dye labeled at the 5' end (Table 2
). Components of a 25-µL PCR reaction were the Assays-by-Design Primer and Probe set (1.25 µL, Applied Biosystems), DNAse/RNAse-free H2O (10.25 µL), cDNA template (1.0 µL), and TaqMan Universal PCR Master Mix-No AmpErase UNG (12.5 µL, Applied Biosystems). The PCR conditions used for the amplification and quantification were an initial denaturing stage (95°C for 10 min), followed by 40 cycles of 2 amplification stages of denaturing (95°C for 15 s) and annealing-extension (60°C for 1 min), with a melting curve program (60 to 95°C), a heating rate of 0.15°C/s, and continuous fluorescence measurements. Each tissue cDNA sample of each steer was performed in triplicate.
Relative quantification of CAT-1 mRNA expression was conducted by using a relative standard curve method and a serial dilution of RT cDNA. The standard curves for CAT-1 and 18S were constructed with a common basis cDNA sample, which was an aliquot pool of all the cDNA samples generated for quantification (Applied Biosystems, 2004; Bustin, 2004; Larionov et al., 2005). The quantified levels of 18S ribosomal RNA were used as an endogenous control to normalize variations in mRNA inputs and RT reaction efficiencies (Bustin et al., 2005). Specifically, bovine small intestinal cDNA samples were serially diluted by 2.5-, 5-, 25-, 125-, 625-, 3,125-, 15,625-, and 78,125-fold, and the linear range for target quantification was established to ascertain an appropriate amount of cDNA to be used for the standard curve determination. The minimal threshold (CT) values detected by using these dilutions of cDNA were 34 to 38 for the target and control genes. Accordingly, the dilutions of the RT cDNA stocks for CAT-1 and 18S quantification were defined as being 5-and 15,625-fold, respectively. The CT values for 18S mRNA across samples of this study were statistically analyzed to determine whether there was any treatment or tissue effect on 18S expression. For the control of system error during sample preparation, the relative CAT-1 RNA expression was normalized to that of 18S by calculating the ratios of CAT-1:18S relative mRNA quantities. These 18S-normalized ratios were then used for statistical analysis of CAT-1 mRNA expression.
Sequence Validation of Real-Time RT-PCR Products
To prepare the real-time RT-PCR products for sequencing, a PureLink Quick Gel Extraction Kit (Invitrogen) was used to isolate the generated real-time RT-PCR products from the reaction mixtures, which contained unincorporated primers, unincorporated nucleotides, DNA polymerase, and FAM dye that was bound to the probe by the manufacturer (Applied Biosystems). Briefly, 40 to 160 µL of the pooled reaction mixture was electrophoresed on a 1.2% agarose slab gel. A single cDNA band at the desired size was then identified under a UV light, cut out of the gel, placed into a sterile 1.5-mL polypropylene centrifuge tube, dissolved with gel solubilization buffer, and passed through an extraction column. The column-bound cDNA was washed with the washing buffer and eluted with 35 to 50 µL of DNAse/RNAse-free H2O in accordance with the manufacturer’s instructions (Invitrogen).
The purified real-time PCR products were then sequenced by the University of Florida DNA Sequencing Core Laboratory by using the appropriate forward and reverse primers (Table 2). The resulted sequences were aligned to the expected sequences to validate the real-time RT-PCR methodology used in this study.
Statistical Analysis
Data were analyzed as a completely randomized design by ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC). Animal performance data were analyzed in an 1-way ANOVA model using production stages (suckling, weanling, growing, and finishing) as a treatment factor, and Fisher’s protected LSD procedure was used to separate the means (P 
0.10). The relative quantities of 18S and CAT-1 RNA expression were analyzed by a split-plot design ANOVA using the GLM procedures of SAS. The production stage effect was tested in the main plot with individual steers as experimental units, and the tissue distribution effect was tested in the subplot with intestinal regions as experimental units. Sums of squares were partitioned to production stage, intestinal region, the production stage x intestinal region interaction, and the residual error. The production stage x intestinal region interaction was used as the error term for the main plot, and the residual error was used as the error term for the subplot. In the absence of interactions (P > 0.10), main effect means were separated by using Fisher’s LSD procedure. When the interactions were significant (P 0.10), the effect of production stage on RNA expression was further analyzed by the 1-way ANOVA model for each individual intestinal region (duodenum, jejunum, or ileum) to more precisely delineate the production stage effect. Analogously, the effect of intestinal region on RNA expression was also further analyzed by the 1-way ANOVA model for each individual production stage. Fisher’s LSD procedure was used to separate the means as preplanned. Unequal experimental observations (n = 4, 5, or 6) resulted from loss of samples or sample integrity during sample collection, processing, or storage.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Average daily gain did not differ among treatment groups. As planned and expected, the birth BW for suckling, weanling, growing, and finishing treatment groups did not differ (P = 0.83), whereas the final BW at slaughter did (P < 0.001; Table 3). The final BW increased (P < 0.001) significantly from the suckling to weanling, weanling to growing, and growing to finishing stages. However, growth rates, measured either as overall ADG (calculated for the whole experiment period from birth to slaughter) or as final period ADG (calculated for the last 14 d before slaughter for suckling and the last 21 d before slaughter for the other treatment groups), did not differ (P = 0.37 and 0.21, respectively), with the values ranging from 1.0 to 1.2 kg/d.
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). The total absolute length of the small intestine increased (P < 0.05) from suckling to weanling steers and then stabilized, with the weanling, growing, and finishing steers having a small intestinal length that was 45, 48, and 52% longer than suckling steers, respectively. In contrast, the relative small intestinal length (ratio to BW) was greatest (P < 0.05) for suckling steers, being 133, 154, and 367% longer than weanling, finishing, and growing steers. Thus, the relative length of the small intestine decreased with steer development. Generation of a Partial-Length Bovine CAT-1 cDNA
At the initiation of this project, no ruminant-specific CAT-1 cDNA sequences had been reported. Accordingly, the full-length pig CAT-1 cDNA sequence (NM_001012613; Cui et al., 2005) was used as a template for designing the PCR primers (Table 2) for a putative bovine CAT-1 mRNA. The pig CAT-1 cDNA sequence was chosen because it shares the highest identities (93%, 108/115) with a reported expressed sequence tag (GenBank accession no. BE758285) from a bovine cDNA library (Smith et al., 2001), although there was only a fragment of 115 bp that shared these identities in a NCBI nucleotide-nucleotide BLAST search. The CAT-1 sequences of humans, mice, and rats (Gen-Bank accession nos. NM_003045, NM_007513, and NM_013111, respectively) shared only 86 to 88% identities with this reported bovine expressed sequence tag, and also only the same 115-bp fragment shared these identities in the BLAST search.
Two single-gel-band RT-PCR products (approximately 700 bp) were generated from 2 jejunal cDNA samples of 1 suckling and 1 growing Angus steer. These 2 products were then purified and cloned, and several clones were generated from each of 2 PCR products and then were preliminarily verified by an EcoRI and NcoI endonuclease digestion check (data not shown). At least 2 digestion-verified clones for each PCR product were chosen randomly and sequenced. The sequencing results showed that each of these digestion-verified clones had a PCR product insert that was 695 bp in length. Alignment of these 4 sequences with the NCBI BLAST engine (Tatiana and Thomas, 1999) showed 100% (695/695) identities among them. This putative jejunal CAT-1 sequence then was aligned (Figure 1) with the corresponding regions of the pig, human, mouse, rat, and a newly predicted bovine CAT-1 cDNA sequence (Gen-Bank accession no. NM_001012613, NM_003045, NM_007513, NM_013111, and XM_587329, respectively), revealing shared identities of 89, 87, 84, 85, and 99%, respectively. Consequently, this partial-length bovine CAT-1 cDNA sequence now resides in GenBank with accession number DQ399522.
From this partial-length bovine CAT-1 cDNA sequence, the corresponding AA sequence was deduced, and then aligned to these AA sequences deduced from the aforementioned pig, human, mouse, and rat sequences and the predicted bovine CAT-1 cDNA sequence. This analysis revealed that the partial-length bovine CAT-1 AA sequence shared 97, 92, 91, 92, and 100% homology with the pig, human, mouse, and rat sequences and the predicted bovine CAT-1 AA sequence, respectively (data not shown).
Real-Time RT-PCR Methodology Validation
Validation of Real-Time RT-PCR Products.
As shown in Figure 2, the sequence of the CAT-1 real-time PCR product had 100% (74/74 bp) identity with the corresponding region of the end-point PCR product obtained in this study (GenBank accession no. DQ399522), which was used as the template for real-time PCR primer and probe design (Table 2). The sequence of the 18S real-time PCR product had 96% (96/100 bp) identity with the corresponding region of the reported bovine sequence (GenBank accession no. DQ222453), which was used as the template for real-time PCR primer and probe design (Table 2 and Figure 2). This 18S product, however, had 100% (100/100 bp) identity with the corresponding region of another reported bovine 18S gene sequence (GenBank accession no. AF176811). The DQ222453 sequence was reported by a research group in China, whereas AF176811 was reported by a research group in Canada. Differences between cattle breeds or geographic locations may contribute to the difference between North American (DQ399522, AF176811) and Asian (DQ222453) bovine 18S sequences. Overall, these sequence results validated the real-time PCR reactions used in this study, for either the target gene or the endogenous control.
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). Although numerous publications have revealed that 18S rRNA is the most stable reference gene that can be used as an expression control without checking for a treatment effect, potential treatment effects on the expression levels of 18S rRNA were evaluated by comparing the CT values for the 18S rRNA isolated from all small intestinal samples (Bustin et al., 2005). Split-plot ANOVA analysis revealed that there were no production stage effects (P = 0.89), no small intestinal region effects (P = 0.35), and no stage x region interactions (P = 0.72; Table 4) identified. The 18S expression data were further analyzed by 1-way ANOVA to check whether there was any treatment effect at each individual treatment level, because CAT-1 mRNA expression data warranted a further analysis at each individual treatment level (as follows) and 18S data were to be used as normalizers. One-way ANOVA results (Table 4) revealed that 18S rRNA expression did not differ among the 4 production stages for every intestinal region (P = 0.45 to 0.82) and also did not differ among the 3 intestinal regions for every production stage (P = 0.14 to 0.52).
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The transport of CAA and the expression of CAT-1 are known to be modulated by a variety of stimuli, including cell proliferation, growth factors, hormones, cytokines, and levels of substrates (Devés and Boyd, 1998; Hatzoglou et al., 2004). However, how physiological development or production stages affect the expression of CAT-1 in the small intestine of mammals, including ruminants, is not known (Pácha, 2000). To test the effect of 4 commercially relevant production stages on CAT-1 expression in Angus steers, the relative content of CAT-1 mRNA expressed by duodenal, jejunal, and ileal epithelia of suckling, weanling, growing, and finishing steers was analyzed. Cationic AA transporter-1 mRNA was expressed by duodenal, jejunal, and ileal epithelia at all production stages (Table 5). Two-way ANOVA analysis revealed that there was a significant interaction (P = 0.03) between production stage and small intestinal region. Consequently, 1-way ANOVA was further conducted to delineate the expression responses to production stage within each small intestinal region. No differences (0.20 < P 0.47) in CAT-1 mRNA expression were observed among the production stages in either duodenal or ileal epithelial tissues (Table 5). In contrast, production stages affected (P = 0.005) jejunal expression of CAT-1, with that by growing steers being approximately 120, 130, and 60% greater than those by suckling, weanling, and finishing steers, respectively. Given that CAT-1 mRNA encodes for CAT-1 protein, which possesses Na+-independent uptake capacity of CAA activity, our detection of CAT-1 mRNA in jejunal and ileal epithelia of the developing Angus steers is consistent with the observation of Na+-independent uptake capacity of L-Lys measured in brush border membrane vesicles isolated from jejunal and ileal epithelia of growing Holstein steers (Wilson and Webb, 1990).
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) found that expression of ovine transporters was affected by differential rates of growth. Therefore, in the current study, we purposefully formulated the growing and finishing diets to maintain a common ADG (i.e., approximately 1 kg/d) to match that achieved by the suckling and weanling treatment groups. The use of these diets to maintain a constant ADG was successful (Table 3). Accordingly, the increase in jejunal CAT-1 mRNA expression by growing steers found in this study was likely the result of steer developmental stage, not the difference in growth rate. Effect of Small Intestinal Epithelia on CAT-1 mRNA Expression
Cationic AA transporter-1 expression varies considerably across tissues and cell types (Hatzoglou et al., 2004). To understand the influence of small intestinal region on the expression of CAT-1 transporter, the content of CAT-1 mRNA relative to 18S mRNA was compared among duodenal, jejunal, and ileal epithelia within a production stage. Although CAT-1 mRNA was expressed in all 3 regions at each of 4 production stages, no difference (0.27 P 0.37) in terms of CAT-1 expression among 3 regions was observed for suckling, weanling, or finishing steers (Table 5). However, in growing steers the expression of CAT-1 by jejunal epithelia was approximately 130% greater (P = 0.002) than by duodenal or ileal epithelia. Why CAT-1 mRNA expression did not change in duodenal and ileal epithelia was probably because in the duodenum there was not enough free CAA available to stimulate CAT-1 mRNA expression because of incomplete feedstuff digestion, whereas in the ileum there was no need for increasing absorption capacity because a large proportion of free CAA had already been absorbed by jejunal epithelia. In terms of the potential for luminal AA appearance in different regions of the small intestine to affect AA transporter expression, Tagari and Bergman (1978) reported in mature sheep that the amount of total and amino nitrogen decreased on passage into the duodenum, but increased in the jejunum and then again decreased steadily toward the terminal ileum. If CAA levels are proportional to the appearance of total and amino nitrogen, or if CAT-1 mRNA expression is sensitive to total or amino nitrogen load, then this finding in mature sheep may be in agreement with our observation with growing steers.
In general, the absorption mechanisms in ruminants for the end-products of protein digestion appear to be similar to those described in nonruminants (Matthews, 2000; Krehbiel and Matthews, 2003). Likewise, the duodenum, jejunum, and ileum of small intestinal epithelium appear to have different capacities to absorb AA, with essential AA being preferentially absorbed over nonessential AA (Bergen, 1978). In contrast to nonruminants, which appear to possess the greatest absorption capacity in the jejunum, the ileal region of sheep and cattle has been identified as possessing the greatest potential for free AA absorption (Johns and Bergen, 1973; Phillips et al., 1979; Guerino and Baumrucker, 1987). In growing steers, however, this may not be the case for CAA because the Na+-independent uptake capacity for Lys by jejunal and ileal brush border membrane vesicles of growing steers does not appear to differ (Wilson and Webb, 1990). Furthermore, if CAT-1 mRNA is proportional to CAT-1-mediated system y+ uptake capacity (Hyatt et al., 1997; Fernandez et al., 2001), then the findings of this study (Table 5) indicate that the jejunum expresses the greatest capacity for Na+-independent CAA uptake in growing animals (as appears to be the case for nonruminants), whereas no difference exists along the small intestinal epithelia in suckling, weanling, and finishing steers. However, it is still unknown whether the cattle small intestinal epithelium also expresses another AA transporter, BAT1, that also possesses Na+-independent CAA uptake activity (Chairoungdua et al., 1999).
In summary, understanding how developmental stages and dietary substrates regulate system y+ transport of CAA in the ruminant small intestine should facilitate the rational design of dietary CAA supplements for different stages of beef production by matching dietary load to absorptive capacity. To this end, we generated a partial-length cDNA for a bovine CAT-1 mRNA ortholog and then used this sequence information to generate primers for real-time PCR analysis of bovine CAT-1 mRNA expression by small intestinal epithelia. Our data identify, for the first time, that the mRNA for CAT-1, a major mammalian intestinal CAA transporter, is expressed by the duodenal, jejunal, and ileal epithelia of Angus steers during 4 commercially relevant production stages (suckling, weanling, growing, and finishing). This finding of CAT-1 mRNA in cattle small intestinal epithelia is consistent with previously reported Na+-independent uptake of Lys by jejunal and ileal epithelia. We also found that more CAT-1 mRNA was expressed by the jejunal epithelium of growing steers than the jejunum of other steers, and that, within growing steers, the jejunum expressed more CAT-1 mRNA than did the duodenal or ileal epithelia.
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Footnotes |
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2 Corresponding author: e-mail: jmatthew{at}uky.edu
Received for publication November 3, 2006. Accepted for publication November 1, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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