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Poult Sci 2007. 86:1739-1753
© 2007 Poultry Science Association
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MOLECULAR, CELLULAR, AND DEVELOPMENTAL BIOLOGY

Developmental Regulation of Nutrient Transporter and Enzyme mRNA Abundance in the Small Intestine of Broilers1

E. R. Gilbert*, H. Li*, D. A. Emmerson{dagger}, K. E. Webb, Jr.* and E. A. Wong*,2

* Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061; and {dagger} Aviagen, untsville, AL 35805

2 Corresponding author: ewong{at}vt.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this study was to investigate intestinal nutrient transporter and enzyme mRNA in broilers selected on corn- and soybean-based (line A) or wheat-based (line B) diets. We investigated the peptide transporter PepT1, 10 amino acid transporters (rBAT, bo,+AT, ATBo,+, CAT1, CAT2, LAT1, y+LAT1, y+LAT2, BoAT, and EAAT3), 4 sugar transporters (SGLT1, SGLT5, GLUT5, and GLUT2), and a digestive enzyme (aminopeptidase N). Intestine was collected at embryo d 18 and 20, day of hatch, and d 1, 3, 7, and 14 posthatch. The mRNA abundance of each gene was assayed using real-time PCR and the absolute quantification method. For PepT1, line B had greater quantities of mRNA compared with line A (P = 0.001), suggesting a greater capacity for absorption of amino acids as peptides. Levels of PepT1 mRNA were greatest in the duodenum (P < 0.05), whereas the abundances of SGLT1, GLUT5, and GLUT2 mRNA were greatest in the jejunum (P < 0.05). Abundances of EAAT3, bo,+AT, rBAT, BoAT, LAT1, CAT2, SGLT5, and aminopeptidase N mRNA were greatest in the ileum (P < 0.05). Quantities of PepT1, EAAT3, BoAT, SGLT1, GLUT5, and GLUT2 mRNA increased linearly (P < 0.01), whereas CAT1, CAT2, y+LAT1, and LAT1 mRNA decreased linearly (P < 0.05) with age. Abundance of y+LAT2 mRNA changed cubically (P = 0.002) with peaks of expression at day of hatch and d 7, and aminopeptidase N and SGLT5 mRNA changed quadratically (P = 0.005) with age. These results provide a comprehensive profile of the temporal and spatial expression of nutrient transporter mRNA in the small intestine of chicks.

Key Words: amino acid transporter • broiler • monosaccharide transporter • PepT1


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the small intestine, the final digestion and absorption of dietary nutrients occurs through digestive enzymes and transporter proteins expressed in the apical membrane of enterocytes. Amino acids are transported into the enterocyte as di- or tripeptides by the H+-dependent peptide transporter 1, PepT1 (Leibach and Ganapathy, 1996; Chen et al., 2002), or as free amino acids by a variety of different amino acid transporters that vary in substrate specificity (Kanai and Hediger, 2004; Palacin and Kanai, 2004; Verrey et al., 2004). Reports from our laboratory suggest that, from a nutritional perspective, peptide transport through PepT1 is important in chickens, cows, pigs, and sheep (Matthews et al., 1996; Pan et al., 1997; Chen et al., 1999). Less is known about free amino acid transport in livestock and poultry. Because optimal growth of broilers depends on the amino acid supply, studies of genes involved in the process of protein digestion and absorption are important (Galgarber and Uni, 2000).

The process of dietary carbohydrate absorption in the small intestine occurs through the action of facilitated and Na+-dependent transport proteins expressed in the enterocyte (Thorens, 1996). Glucose and galactose are transported into the cell by the Na+-dependent transporter SGLT1 (Thorens, 1996; Ferraris, 2001; Wright and Turk, 2004), whereas fructose enters through GLUT5 by facilitated diffusion (Ferraris, 2001; Uldry and Thorens, 2004; Wright and Turk, 2004). The exit of monosaccharides from the cell is mediated by GLUT2 through facilitated diffusion (Ferraris, 2001; Uldry and Thorens, 2004; Wright and Turk, 2004). Absorption of carbohydrates from the intestinal lumen is critical for maintaining energy supplies in animals, and is influenced by luminal digestion, apical membrane digestion, and transport into the enterocyte by SGLT1 (Sklan et al., 2003). Using White Leghorn chickens, Barfull et al. (2002) found that SGLT1 mRNA and protein declined with age after hatch, matching the described decline in sugar transport activity, whereas Sklan et al. (2003) observed low mRNA levels of SGLT1 at hatch followed by a slight increase to d 7. Others demonstrated that uptake of glucose was low right after hatch and increased slowly with age, suggesting that transport may be a limiting factor in glucose assimilation during the early posthatch period (Sulistiyanto et al., 1999), possibly because of the presence of hydrophobic yolk in the lumen at hatch and low concentrations of luminal sodium (Noy and Sklan, 1999). Understanding sugar transporter gene regulation is important for the elucidation of mechanisms that facilitate changes in rates of nutrient uptake (Ferraris, 2001).

Genetics can also influence nutrient requirements in chickens, including Arg requirements in White Leghorn chicks (Nesheim and Hutt, 1962) and protein requirements for egg production in layers (Harms and Waldroup, 1962). It follows that genetic variation in nutrient requirements may be attributed to digestive and absorptive capacities at the intestinal level and postabsorptive utilization of nutrients (NRC, 1975), and these differences may be most significant during the first few days of life. Uni et al. (1995) observed differences in starch digestion between 2 broiler strains between d 4 and 14 posthatch. Starch digestion in the heavy (Arbor Acres) chicks was 90 to 95% from d 4 to 14, respectively, whereas in the light (Lohman) chicks, it increased from 80% on d 4 to 93% on d 14. Nutritional conditions in the early posthatch period may have the greatest impact on the overall lifetime performance of chickens (Lilja, 1983; Geyra et al., 2001) and nutrient deficiencies during the first few days after hatch will depress mucosal development (Uni et al., 1998b) and compromise immune function (Casteel et al., 1994). Although intestinal absorption of nutrients may be of minimal significance in ovo, digestive and absorptive functions are established before the onset of exogenous feeding to prepare the chick (Sklan, 2001). In chicks from embryo d 16 to day of hatch (DOH), there was a 14- to 50-fold increase in mRNA levels of PepT1 (Chen et al., 2005). Similarly, Uni et al. (2003) observed that intestinal mRNA expression of aminopeptidase, adenosine triphosphatase, maltase, and SGLT1 increased 9- to 25-fold from d 15 of incubation to d 19 in chick embryos. Thus, a greater understanding of the influence of both genetics and development on expression of nutrient transporters may facilitate improvements in growth performance through dietary manipulation to take advantage of differential gene expression. Additionally, to reduce the cost of providing protein in the diet and to reduce excess nitrogen excretion into the environment, a greater understanding of amino acid absorption in chickens is needed. The objective of this study was to investigate the developmental regulation of intestinal peptide, amino acid, and monosaccharide transporter mRNA in 2 genetically selected lines of broiler chicks. This study is the first to report analysis of mRNA abundance of these genes by absolute quantification, and is the most comprehensive study of chicken nutrient transporter mRNA abundance to date.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Collection
The chickens in the present experiment represented 2 commercial broiler lines, which were genetically selected under different nutritional environments. Line A was developed in a nutritional environment in which corn- and soy-based diets and lower relative amino acid concentrations were fed. Line B was fed wheat-based diets and amino acid concentrations that were 15 to 20% higher than for line A. When fed a corn- and soy-based diet with approximately 20% CP, line A had a higher BW than line B at 40 d. In contrast, line B attained a higher 40-d BW compared with line A on a 20% CP wheat-based diet (Figure 1Go). Both lines have been subjected to balanced selection for improved growth, feed conversion, meat yields, reproduction, and general fitness. These lines were chosen for this study to determine whether the difference in protein responsiveness was related to expression of nutrient transporters.


Figure 1
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  		Figure 1. Body weights at 40 d for mixed-sex birds from lines A and B fed either a corn-based or a wheat-based diet.

 
Eggs from each genetic line were obtained from Aviagen (Huntsville, AL). Day-of-hatch chicks from each genetic line were randomly assigned to heated floor pens with wood shavings. Day of hatch was defined as the birds having cleared the shell on d 21 of incubation. All pens had 24-h lighting and chicks had free access to water. After hatch, birds were given ad libitum access to a corn-and soy-based diet that was formulated to contain 3,064 kcal/kg of ME, 20.0% CP, 1.17% total Lys, 0.50% Met, 0.85% TSAA, 0.76% Thr, 1.33% Arg, 0.83% Ile, 0.95% Val, 0.23% Trp, 0.94% calcium, 0.45% phosphorus, and 0.21% sodium. Birds were killed by cervical dislocation at the following time points: embryo d 18 (E18), embryo d 20 (E20), DOH (after hatch but before feeding), 1 d posthatch (D1), 3 d posthatch (D3), 7 d posthatch (D7), and 14 d posthatch (D14). Birds were processed in a manner similar to the procedure described by Chen et al. (2005). Body weights of all birds were recorded. For all time points except E18, the intestine was separated into the duodenum, jejunum, and ileum. The proximal enlarged loop of the small intestine was taken as the duodenum. The rest of the small intestine was divided by Meckel’s diverticulum into proximal and distal portions, corresponding to the jejunum and ileum, respectively. For E18, the entire small intestine was collected because of the difficulty in handling the tissue. Intestinal segments were rinsed with ice-cold PBS, minced with a razor blade, frozen as aliquots in liquid nitrogen, and stored at –80°C. Sex of the birds was determined by PCR. Deoxyribonucleic acid was isolated from liver samples using the DNeasy Tissue kit (Qiagen, Valencia, CA). Polymerase chain reaction was performed with liver DNA and primers for tyrosinase (positive control; forward primer: 5'-TCGAGAGGCATAATAATGCATCCA-3', reverse primer: 5'-AGAGCTT GCTGAGGAAGGAGTG-3'), and primers for the W chromosome (female specific; forward primer: 5'-CTGTGAT AGAGACCGCTGTGC-3', reverse primer: 5'-CAACGCT GACACTTCCGATGT-3'), and reactions were electrophoresed on a 1.5% agarose gel and stained with ethidium bromide. All reaction products contained the 400-bp tyrosinase-specific band. Female birds were identified by the presence of the 1,200-bp band corresponding to the W chromosome, whereas male birds lacked this band. Four male birds were selected from each line at each time point to use for real-time PCR. All animal procedures were approved by the Institutional Animal Care and Use Committee at Virginia Tech.

Real-Time PCR
Total RNA was isolated from each tissue sample using the RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. Total RNA was quantified spectrophotometrically at 260/280 nm and stored at –80°C. Real-time PCR was used to determine the number of molecules of mRNA present for each gene of interest per nanogram of total RNA starting template. An RNA standard curve for each gene was generated based on modification of the protocol of Fronhoffs et al. (2002). Briefly, chicken-specific cDNA were amplified and subcloned into a vector. Total RNA from D7 jejunum and gene-specific primers (Table 1Go) were used to perform reverse transcription PCR (RT-PCR). Primers were designed using the Primer3 program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Two-step RT-PCR was performed using Promega reagents (Madison, WI) in a PTC-200 Peltier DNA Engine (MJ Research, Reno, NV), following the manufacturer’s protocols. The following PCR conditions were used: 95°C for 5 min and 36 cycles of 94°C for 1 min, 54°C for 1 min, 72°C for 1 min, and a final step of 73°C for 10 min. The PCR products were electrophoresed on a 2% agarose gel, excised for purification using the QIAquick-Gel Extraction kit (Qiagen), and ligated into the pGEM-T Easy Vector (Promega). Escherichia coli competent cells were transformed using a BTX-Harvard Apparatus ECM Electro Cell Manipulation System (Holliston, MA), and plated out overnight in the presence of 100 µg/mL of ampicillin, isopropyl thiogalactoside, and X-Gal. The vector DNA was purified from cells containing the vector with the PCR insert using the QIAprep Spin Miniprep kit (Qiagen). Purified plasmid samples were then sequenced at the Virginia Bioinformatics Institute at Virginia Tech. The HiSpeed Plasmid Midi kit (Qiagen) was used to purify and obtain high yields of the plasmid samples following the manufacturer’s protocol. Nested primers were designed (Table 1Go) within cloned chicken cDNA sequences with the Primer Express software, optimized for use with Applied Biosystems Real-Time PCR Systems (Foster City, CA).


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  		Table 1. Primers used for cloning chicken cDNA1 and real-time PCR2
 
To generate a standard curve, plasmids containing amplified chicken cDNA were linearized opposite a T7 or SP6 promoter, depending on the orientation of the insert sequence. The Pst I and Sac II restriction enzymes (New England Biolabs, Ipswich, MA) were used to linearize the plasmid for synthesis of RNA from the T7 and SP6 promoters, respectively. Digested DNA were purified using a High Pure PCR Product Purification kit (Roche, Pleasanton, CA), and quantified spectrophotometrically. In vitro transcription was performed on linearized plasmids with the MEGAscript T7 or SP6 in vitro transcription kit (Ambion, Austin, TX). Samples were treated with DNase I to remove the DNA template and cRNA was precipitated with lithium chloride and quantified using the RiboGreen assay (Molecular Probes, Eugene, OR) and a FLUOstar Optima microplate reader (BMG Labtech, Offenburg, Germany).

The number of molecules per microliter (N) was calculated using the following equation, with a molecular mass constant derived from Avogadro’s constant:


Formula

A dilution series of 1011 to 104 molecules per microliter was performed in the presence of yeast tRNA at 10 µg/mL. The dilution series for each standard curve was reverse-transcribed in parallel with chick intestinal total RNA samples using the High-Capacity cDNA Archive kit (Applied Biosystems). Each reverse transcription reaction contained 2,000 ng of RNA at a concentration of 100 ng/µL, and an equal volume of each standard curve dilution cRNA was added to its respective reaction. The cDNA was then diluted 1:30 before addition to PCR reactions.

Real-time PCR was performed on an Applied Biosystems 7300 instrument with ABI plates using the absolute quantification method. For each 25-µL PCR reaction, 2 µL of the cDNA diluted 1:30, 12.5 µL of SYBR Green Master Mix (Applied Biosystems), 9.5 µL of water, and 0.5 µL of the forward and reverse primer at a 5-µM stock concentration were added. Polymerase chain reaction was performed under the following conditions: 50°C for 10 min and 40 cycles of 95°C for 1 min and 60°C for 1 min. A dissociation step, consisting of 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s, was performed at the end of each PCR reaction to ensure amplification of a single product.

Statistical Analysis
All data were analyzed using SAS PROC MIXED (SAS Institute, Cary, NC). The model included the main effects of genetic line, intestinal segment, age, and all appropriate 2-way interactions. The 3-way interactions were removed from the model as they were determined to be nonsignificant (P < 0.05). Differences among segments were evaluated by the Tukey test for multiple comparisons. The E18 time point was not included in the statistical model. The main effects of age were further tested for linear, quadratic, and cubic responses using orthogonal contrasts in PROC MIXED. The contrast coefficient matrix for unequal spacing was generated using PROC IML.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
After hatch, birds were given ad libitum access to a 20% CP corn- and soy-based diet, because this has been shown to accentuate differences in BW between the 2 lines over 42 d. Body weights were not significantly different between genetic lines by D14 (P = 0.5; Figure 2Go), although weights of line A and B birds were starting to diverge, with line A weights being greater than line B weights.


Figure 2
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  		Figure 2. Bird BW from day of hatch to d 14 posthatch. Means represent BW (g) ± SEM from 35 mixed-sex birds. Four male birds were randomly selected for the experiment from both genetic lines at each time point. DOH = day of hatch (after hatch but before feeding); D1 = d 1 after hatch; D3 = d 3 after hatch; D7 = d 7 after hatch; D14 = d 14 after hatch.

 
The mRNA abundance for a number of amino acid, peptide, and monosaccharide transporters was determined using real-time PCR and the absolute quantification method. Thus, the results presented herein represent mRNA, and it is unknown at this time whether these data parallel expression and activity of the functional proteins. The functions of all genes evaluated in this study are listed in Table 1Go. The mRNA expression data are summarized in Tables 2Go and 3Go, with separate rows for main effects of developmental age, intestinal segment, and genetic line, interactions, and orthogonal contrasts across developmental age. If no significant differences were observed between genetic lines, data for lines A and B were combined and displayed as a single graph.


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  		Table 2. Developmental regulation of peptide and amino acid transporter mRNA in the small intestine of broiler chicks1
 

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  		Table 3. Developmental regulation of amino acid and sugar transporter and enzyme mRNA in the small intestine of broiler chicks1
 
At the brush border membrane, the uptake of amino acids is mediated by the action of the peptide transporter PepT1 and a number of different amino acid transporters, which transport neutral, cationic, and anionic amino acids. The number of molecules of peptide transporter PepT1 mRNA per nanogram of total RNA ranged from 700 to 12,000 (Figure 3Go, panels A and B). Abundance of PepT1 mRNA was greater in line B than in line A (P = 0.0007). In line B, PepT1 mRNA rose continuously with age, whereas in line A, mRNA leveled off after d 3. There was an approximate 2-fold difference between lines A and B in concentration of PepT1 mRNA per nanogram of total RNA (3,600 vs. 6,300, respectively). Quantities of PepT1 mRNA were greatest in the duodenum compared with the ileum (P = 0.04), whereas jejunal mRNA was intermediate. Abundance of PepT1 mRNA increased linearly (P = 0.0001) from E20 to D14.


Figure 3
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  		Figure 3. The number of PepT1 mRNA molecules per nanogram of total RNA in line A (A) and line B (B) chicks was determined by real-time PCR. Quantities represent the means of 4 male birds ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a main effect of genetic line (P = 0.0007), intestinal segment (P = 0.031), and linear effect (P = 0.0001) with age.

 
The rBAT and bo,+AT proteins heterodimerize and serve as Na+-independent amino acid exchangers on the apical membrane, with a high extracellular affinity for cationic amino acids and a high intracellular affinity for neutral amino acids (Palacin and Kanai, 2004). Molecules of bo,+AT mRNA per nanogram of total RNA ranged from 35,000 to 170,000 (Figure 4Go, panels A and B). Neither genetic line nor age of bird influenced abundance of bo,+AT mRNA. The quantities of bo,+AT mRNA were greater in the ileum compared with the duodenum and jejunum (P = 0.0001). There was a line x intestinal segment interaction (P = 0.04), where line A mRNA levels were greatest in the ileum, lowest in the jejunum, and intermediate in the duodenum, whereas in line B, mRNA levels were also greatest in the ileum but lowest in the duodenum and intermediate in the jejunum. There was also an intestinal segment x age interaction (P = 0.01). Quantities of mRNA in the ileum increased to D1, whereas mRNA levels decreased to D1 in the duodenum and jejunum, and quantities of mRNA in the ileum increased most dramatically after D3 compared with the duodenum and jejunum. The rBAT mRNA quantities ranged from 5,900 to 57,000 molecules per nanogram of total RNA (Figure 5Go, panel A). Differences observed between genetic lines and among ages were not significant. Quantities of rBAT mRNA were greater in the ileum compared with the duodenum and jejunum (P < 0.01).


Figure 4
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  		Figure 4. The number of bo,+AT mRNA molecules per nanogram of total RNA in line A (A) and line B (B) chicks was determined by real-time PCR. Quantities represent the means of 4 male birds ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a main effect of intestinal segment (P = 0.0001), a genetic line x intestinal segment interaction (P = 0.041), and an intestinal segment x age interaction (P = 0.011).

 

Figure 5
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  		Figure 5. Real-time PCR analysis of brush border membrane amino acid transporter and enzyme mRNA in the small intestine of 2 genetically selected lines of broiler chicks. The number of rBAT (A), EAAT3 (B), BoAT (C), and APN (D) mRNA molecules per nanogram of total RNA. Quantities represent the means of 4 male line A and line B birds (n = 8) ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a main effect of intestinal segment (P < 0.001) on rBAT, EAAT3, BoAT, and APN mRNA and a linear increase (P < 0.01) with age on EAAT3, BoAT, and APN mRNA. There was an intestinal segment x age interaction (P < 0.05) for EAAT3 and BoAT mRNA.

 
The EAAT3 brush border membrane-bound protein is a Na+-dependent transporter with a high affinity for anionic amino acids, including aspartate and glutamate (Kanai and Hediger, 2004). Quantities of EAAT3 mRNA molecules per nanogram of total RNA ranged from 1,000 to 12,500 (Figure 5Go, panel B). There was no effect of genetic line on EAAT3 mRNA quantities. Quantities of EAAT3 mRNA were greatest in the ileum, lowest in the duodenum, and intermediate in the jejunum (P = 0.0001). Quantities of EAAT3 mRNA increased linearly (P = 0.001) from E20 to D14. There was an intestinal segment x age interaction (P = 0.0001), where EAAT3 mRNA increased most dramatically after D3 in the ileum compared with the duodenum and jejunum. Molecules of BoAT mRNA per nanogram of total RNA ranged from approximately 300 to 11,000 (Figure 5Go, panel C). Quantities of mRNA were not influenced by genetic line. Molecules of BoAT mRNA were greater in the ileum compared with the duodenum and jejunum (P < 0.001). Quantities of BoAT mRNA increased linearly (P = 0.0001) from E20 to D14. There was an intestinal segment x age interaction (P = 0.002), where BoAT mRNA levels increased most dramatically with age in the ileum as compared with the duodenum and jejunum, where mRNA remained fairly constant with age.

Amino acid and peptide supply to the enterocyte is dependent on terminal digestion at the brush border membrane by aminopeptidases, such as aminopeptidase N (APN), which cleaves neutral and basic amino acids from the N-terminal end of peptides (Sanderink et al., 1988). The amount of APN mRNA per nanogram of total RNA ranged from 47,000 to 300,000 molecules (Figure 5Go, panel D). The quantities of APN mRNA were not influenced by genetic line. Quantities of APN mRNA were greatest in the ileum, lowest in the duodenum, and intermediate in the jejunum (P = 0.0001). Quantities of APN mRNA increased quadratically (P = 0.02) from E20 to D14, with a decline from DOH to D1, and subsequent increase in mRNA to D14.

At the basolateral membrane, amino acids are similarly transported out of the enterocyte and into the blood via amino acid transporters with specificity for neutral, anionic, and cationic amino acids. The y+LAT1 and y+LAT2 proteins mediate the Na+-dependent transport of neutral amino acids with high affinity, in exchange for intracellular cationic amino acids, whereas LAT1 exhibits a high affinity for branched-chain and aromatic amino acids (Verrey et al., 2004). The CAT1 and CAT2 proteins both transport cationic amino acids with high affinity. The number of molecules of LAT1 mRNA per nanogram of total RNA ranged from 40 to 530 (Figure 6Go, panel A). Abundance of LAT1 mRNA was not influenced by genetic line. Abundance of LAT1 was greater in the ileum compared with the duodenum (P = 0.007), and levels were intermediate in the jejunum. Quantities of LAT1 mRNA decreased linearly (P = 0.05) from E20 to D14. There was an intestinal segment x age interaction (P = 0.04), where LAT1 mRNA increased from E20 to DOH in the ileum, in contrast to the duodenum and jejunum, where mRNA levels did not change, and duodenal mRNA increased after D3 as compared with the jejunum and ileum, where mRNA decreased.


Figure 6
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  		Figure 6. Real-time PCR analysis of basolateral amino acid transporter mRNA in the small intestine of genetically selected lines of broiler chicks. The number of LAT1 (A), y+LAT1 (B), y+LAT2 (C), CAT1 (D), and CAT2 (E) mRNA molecules per nanogram of total RNA. Quantities represent the means of 4 male line A and line B birds (n = 8) ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a linear decline (P < 0.05) with age of y+LAT1, CAT1, CAT2, and LAT1 mRNA. There was a cubic effect (P = 0.002) with age on y+LAT2 mRNA. There was a main effect of intestinal segment (P < 0.01) on CAT2 and LAT1 mRNA. There was an interaction of age x segment (P = 0.04) on LAT1 mRNA.

 
The number of molecules of y+LAT1 mRNA per nanogram of total RNA ranged from 120 to 550 (Figure 6Go, panel B). No significant main effects were observed. The quantities of y+LAT1 mRNA decreased linearly (P = 0.003) from E20 to D14. Abundance of y+LAT2 mRNA per nanogram of total RNA ranged from 2,700 to 11,000 molecules (Figure 6Go, panel C). There was no effect of genetic line or intestinal segment on y+LAT2 mRNA abundance. Quantities of y+LAT2 mRNA changed cubically (P = 0.002) with age, with the greatest mRNA quantities observed at DOH and D7.

The number of molecules of CAT1 mRNA per nanogram of total RNA ranged from 180 to 4,400 molecules (Figure 6Go, panel D). The abundance of CAT1 mRNA was not influenced by genetic line or intestinal segment. The quantities of CAT1 mRNA decreased linearly (P = 0.0001) from E20 to D14. Quantities of CAT2 mRNA per nanogram of total RNA ranged from 30 to 500 (Figure 6Go, panel E). Quantities of CAT2 mRNA were not influenced by genetic line. Abundance of CAT2 mRNA was greatest in the ileum, lowest in the duodenum, and intermediate in the jejunum (P < 0.05). Quantities of CAT2 mRNA decreased linearly from E20 to D14 (P = 0.0001). The Na+-dependent and Cl–-dependent cationic and neutral amino acid transporter ATBo,+ was also evaluated in this study; however, there was no amplification of the PCR product during real-time PCR. Thus, we concluded that it is most likely not expressed in the chicken small intestine.

In a manner similar to amino acid assimilation, oligosaccharides are broken down into monosaccharides, which are then taken up by sugar transporters located on the brush border membrane. The SGLT1 protein mediates the Na+-dependent uptake of glucose and galactose across the brush border membrane (Thorens, 1996) and is considered to be the primary mediator of glucose assimilation in the small intestine (Wright, 1993; Hediger and Rhoads, 1994). A search of the chicken genome revealed 2 predicted SGLT genes, with 1 located on chromosome 8 (Gen-Bank accession number XM_422459) and the other on chromosome 15 (GenBank accession number XM_415247). A multiple-sequence alignment (http://workbench.sdsc.edu) of amino acids from the 2 predicted SGLT genes and all 11 members of the human SLC5 gene family predicted that SGLT on chromosome 15 was the most similar to human SGLT1, whereas SGLT on chromosome 8 was the most similar to SGLT5, a kidney glucose transporter. Thus, in this paper, the SGLT gene on chromosome 15 will be referred to as SGLT1 and the SGLT gene on chromosome 8 will be referred to as SGLT5.

Quantities of SGLT1 mRNA were greatest in comparison with the other monosaccharide transporters examined in this study, and ranged from 1,200 to 63,300 molecules per nanogram of total RNA (Figure 7Go, panel A). Abundance of SGLT1 mRNA was not influenced by genetic line. Levels of SGLT1 mRNA increased linearly (P = 0.0001) from E20 to D14. The quantities of SGLT1 mRNA were greater in the jejunum compared with the duodenum and ileum (P = 0.0001). There was an intestinal segment x age interaction (P = 0.007), in which SGLT1 mRNA increased most dramatically with age in the jejunum as compared with the duodenum and ileum.


Figure 7
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  		Figure 7. Real-time PCR analysis of monosaccharide transporter mRNA in the small intestine of 2 genetically selected lines of broiler chicks. The number of SGLT1 (A), SGLT5 (B), GLUT5 (C), and GLUT2 (D) mRNA molecules per nanogram of total RNA. Quantities represent the means of 4 male line A and line B birds (n = 8) ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a main effect of intestinal segment (P = 0.0001) on SGLT1, SGLT5, GLUT5, and GLUT2 mRNA, a linear effect (P = 0.0001) with age on SGLT1, GLUT5, and GLUT2, and a quadratic effect (P = 0.005) with age on SGLT5. There was an intestinal segment x age interaction (P < 0.01) on SGLT1, GLUT5, and GLUT2.

 
Abundance of SGLT5 mRNA per nanogram of total RNA ranged from 1,200 to 7,300 molecules (Figure 7Go, panel B). Quantities of SGLT5 mRNA changed quadratically (P = 0.005) with time, with mRNA quantities declining to D1 and increasing thereafter. The mRNA was greatest in the ileum, lowest in the duodenum, and intermediate in the jejunum (P < 0.01).

Transport of fructose across the brush border membrane is facilitated by GLUT5 (Uldry and Thorens, 2004). Quantities of GLUT5 mRNA ranged from 300 to 4,300 molecules per nanogram of total RNA (Figure 7Go, panel C). Quantities of GLUT5 were greater in the jejunum and ileum compared with the duodenum (P = 0.0001). Abundance of GLUT5 mRNA increased linearly (P = 0.0001) from E20 to D14. An intestinal segment x age interaction (P = 0.0006) was observed, in that abundance of GLUT5 mRNA increased more dramatically after DOH in the jejunum and ileum as compared with the duodenum.

The GLUT2 transporter mediates the facilitated transport of glucose, galactose, and fructose across the basolateral membrane (Uldry and Thorens, 2004). The number of molecules of GLUT2 mRNA per nanogram of total RNA ranged from 60 to 2,300 (Figure 7Go, panel D). The mRNA levels of GLUT2 were greatest in the jejunum, lowest in the ileum, and intermediate in the duodenum (P = 0.0001). Abundance of GLUT2 mRNA increased linearly (P = 0.0001) from E20 to D14. There was an intestinal segment x age interaction (P = 0.0002), in which GLUT2 mRNA increased more dramatically in the jejunum after D3 compared with the duodenum and ileum.

In addition to nutrient transporters and a digestive enzyme, mRNA for a housekeeping gene, GAPDH, was evaluated in this study. The housekeeping gene GAPDH is assumed to be uniformly expressed across cell types and is commonly used as an internal standard in relative quantification studies to correct for differences in RNA loading amounts (Jain et al., 2006). Our results demonstrated that there was a linear decrease (P = 0.0001) in GAPDH mRNA quantities from E20 to D14. The quantity of GAPDH mRNA per nanogram of total RNA ranged from 31,700 to 190,000 (Figure 8Go). Quantities of GAPDH mRNA were greater in the duodenum than in the ileum (P = 0.002) and levels were intermediate in the jejunum. Abundance of GAPDH mRNA was not influenced by genetic line.


Figure 8
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  		Figure 8. The number of GAPDH mRNA molecules per nanogram of total RNA was determined by real-time PCR. Quantities represent the means of 4 male line A and line B birds (n = 8) ± SEM at embryo d 18 (E18) and embryo d 20 (E20), day of hatch (DOH; after hatch but before feeding), and d 1 (D1), d 3 (D3), d 7 (D7), and d 14 (D14) after hatch. The statistical model did not include the E18 time point. There was a main effect of intestinal segment (P = 0.002) and a linear effect (P = 0.0001) with age.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chicken lifetime performance is greatly influenced by diet quality and nutrient availability during the early posthatch period (Lilja, 1983; Geyra et al., 2001). The growth of chicks is dependent on the ability of the intestine to assimilate nutrients efficiently as the bird shifts from the lipid-rich yolk as the sole source of energy to a carbohydrate- and protein-based diet (Sklan, 2001). Because the developmental regulation of nutrient transporter mRNA has not been thoroughly evaluated in chickens, in this study we compared developmental changes in mRNA abundance in 2 genetically selected lines of broilers. A greater understanding of the capacity of the intestine to assimilate amino acids and glucose is critical to formulating diets that best accommodate the profile of digestive enzymes and nutrient transporters expressed in the small intestine at any point in time. Amino acids are required for many life processes including for protein synthesis and energy, yet little is known about the process of amino acid transport in the chicken intestine. Broilers are raised on diets consisting of approximately 60% carbohydrates (Galgarber et al., 2000); thus, an understanding of intestinal sugar transport is also critical to improving efficiency of growth.

At the brush border membrane, a number of proteases and peptidases break down proteins into peptides and free amino acids for uptake into cells by peptide and amino acid transporters. Peptide transport is one of the major routes of amino acid assimilation by the enterocyte and is H+-dependent (Chen et al., 2002). Interestingly, in our study, PepT1 was the only gene for which mRNA abundance was influenced by genetic line. Line B expressed approximately 2-fold higher levels of mRNA than line A. If protein expression data parallel these findings, it is possible that line B may have a greater capacity to assimilate amino acids in the form of di- and tripeptides. If this is the case, a diet that optimizes availability of amino acids as small peptides may improve growth performance and feed efficiency in line B broilers. Daniel (2004) suggested, based on evidence from numerous studies, that peptides are absorbed faster and more efficiently than free amino acids in the small intestine. Chen et al. (2005) found that chicken PepT1 mRNA levels varied with both dietary protein level and developmental stage. In this study, PepT1 mRNA increased linearly with time to D14. Similarly, Chen et al. (2005) observed a linear increase in cPepT1 with age, suggesting an importance in peptide transport in the posthatch chick. We found that mRNA for PepT1 was expressed at the greatest levels in the duodenum, in agreement with previous studies (Chen et al., 2002), whereas the putative brush border membrane-expressed amino acid transporters were expressed at the greatest levels in the ileum. It is likely that the spatial expression pattern of these genes corresponds to their transport kinetics, with PepT1 exhibiting a greater Vmax and capacity to absorb large amounts of substrate entering the proximal intestine (Daniel, 2004), whereas the free amino acid transporters, with higher affinities and lower transport capacities (Kanai and Hediger, 2004; Palacin and Kanai, 2004; Verrey et al., 2004), extract the remaining unabsorbed amino acids from the lumen of the distal intestine before they are excreted.

Aminopeptidase N is a digestive enzyme that cleaves amino acids from the N-terminal end of peptides, and is fairly specific for peptides with an N-terminal neutral or basic amino acid (Sanderink et al., 1988), which then generates substrate for the amino acid transport systems. Abundance of APN mRNA was greater than all transporter genes examined. Maintaining high expression levels of APN mRNA throughout the small intestine would provide a constant supply of substrates for the amino acid transporters.

The BoAT is a Na+-dependent neutral amino acid transporter expressed on the apical membrane. This transporter was recently cloned in mammals (Broer et al., 2004), and in this study, a search of the chicken genome using a mammalian consensus sequence revealed a similarity to a predicted sequence given the gene name "X transporter 2" (GenBank accession number: XM_419056). Quantities of BoAT mRNA were of a similar magnitude to the quantities of mRNA observed for EAAT3 and PepT1. Quantities of EAAT3 mRNA increased with age and were greatest in the ileum of the small intestine. With the rapid rate of intestinal growth as a proportion of total body mass (Sklan, 2001) and the use of glutamate as an oxidative fuel source in the intestine (Wu, 1998; Nissim, 1999), increasing amounts of energy are needed to meet the needs of the rapidly growing chick. Thus, it is not surprising that mRNA expression of the glutamate transporter EAAT3 increased more than 3-fold from E20 to D14 in our study. Rome et al. (2002) found that levels of EAAT3 mRNA increased from rat postnatal d 4 to 21 all along the small intestine. The reported cellular distribution of EAAT3 also supports its role in providing energy to intestinal cells and stimulating cellular proliferation, with protein expression localized to the crypts and lower villi and decreased abundance toward the villus tip (Rome et al., 2002; Iwanaga et al., 2005). Both groups also reported an increased gradient of expression from proximal to distal intestine, similar to our findings, suggesting increased capacity for glutamate absorption in the distal small intestine. For 3 of the amino acid transporters, bo,+AT, EAAT3, and BoAT, there was an interaction of age x segment with ileal mRNA increasing most dramatically with age in comparison with the duodenum and jejunum, suggesting that the ileum is an important site for free amino acid assimilation in the growing chick.

Expression of the amino acid transporters on the basolateral membrane differed from the amino acid transporters on the brush border membrane. CAT1, CAT2, y+LAT1, and LAT1 are basolaterally expressed in mammalian epithelial cells (Kizhatil and Albritton, 2003; Dave et al., 2004; Verrey et al., 2004), and in this study mRNA quantities were highest at E18 and decreased with age, suggesting an importance for these transporters in the chick embryo. While the chick relies on yolk for nourishment during embryological development, nutrients are obtained in the intestine through the basolateral surface from the bloodstream until exogenous feeding at hatch. In contrast, mRNA quantities of y+LAT2 were lowest at E18 and increased with age, suggesting an important role in the posthatch chick. The Na+-dependent and Cl–-dependent cationic and neutral amino acid transporter ATBo,+ was also evaluated in this study; however, we were unable to detect ATBo,+ mRNA and concluded that it is most likely not expressed in the chicken small intestine.

Expression of monosaccharide transporters also differed temporally and spatially in the chick small intestine. The mRNA for SGLT1 was expressed from 6- to 29-fold greater than the other 3 monosaccharide transporters, indicating that SGLT1 is the major pathway for glucose assimilation in the small intestine. Obst and Diamond (1992) observed a dramatic increase in glucose uptake 14 d posthatch, and in our study, we observed an approximate 9-fold increase in SGLT1 mRNA from E18 to DOH, followed by a 2-fold increase to D14, suggesting that the capacity for glucose uptake increases with age in chickens. Uni et al. (1998b) observed that broiler mucosal sucrase and maltase activities were lower in the duodenum than in the jejunum and ileum, with activities increasing to adult levels from DOH to d 2 posthatch in all segments of the small intestine. After d 2, sucrase and maltase activities declined to d 4, after which enzyme activities gradually rose to d 11 in the jejunum. Activities plateaued after d 4 in the duodenum and ileum. We observed that SGLT1 mRNA was greater in the jejunum and ileum as compared with the duodenum, matching the gradient of disaccharidase activity in broiler small intestine, but we also observed a continual increase in mRNA expression of SGLT1 from DOH to d 14 posthatch. It may be that digestion at the intestinal level is the limiting factor to assimilation of glucose, and not expression of the transporter per se.

For GLUT2, GLUT5, and SGLT1, there was an interaction of age x intestinal segment, with mRNA levels increasing most dramatically with age in the jejunum, suggesting that the jejunum is the primary site of sugar assimilation in the chicken intestine. Garriga et al. (2002) found that apical Na+-dependent D-glucose transport was reduced in the ileum compared with the duodenum and jejunum, also paralleling protein expression of SGLT1. This is in agreement with our results, in which we observed greater mRNA levels of SGLT1 in the jejunum and duodenum. In our study, SGLT5 mRNA was greatest in the ileum, in contrast to the highest expression in the jejunum observed for the other sugar transporters. It is possible that SGLT5 may have a higher affinity for substrate, and thus serves to transport glucose at the distal intestine, whereas SGLT1 transports the majority of dietary glucose in the proximal intestine. In addition, whereas expression of SGLT1, GLUT2, and GLUT5 mRNA increased linearly from E20 to D14, SGLT5 mRNA changed quadratically, with the lowest expression at D1 and a subsequent increase to D14. To date, SGLT1 is the only intestinal transporter reported in chickens that is known to be responsible for the uptake of glucose (Wright and Turk, 2004). That mRNA for SGLT5, a transporter not yet reported to be present in the intestine, was present and exhibited a regional pattern of expression different from SGLT1 suggests a unique role for this transporter in the uptake of glucose. Zhao et al. (2005) reported that bovine SGLT5 mRNA was found predominantly in bovine kidney and was undetectable in bovine mammary gland, liver, lung, and small intestine.

Quantities of GLUT5 mRNA increased with age. The diet offered to chicks lacked fructose; therefore, it is surprising that GLUT5 mRNA was up-regulated with age despite the lack of luminal substrate. In rabbits, fructose uptake increases during the last week of gestation (Phillips et al., 1990), and after birth, fructose transport decreases and then increases again after weaning with the introduction of fructose-containing diets, demonstrating substrate-dependent induction (Buddington and Diamond, 1990). Quantities of GLUT2 mRNA increased with age but represented the lowest quantities compared with the other sugar transporters.

Expression of nutrient transporters and aminopeptidase showed different developmental patterns. In general, brush border membrane transporter mRNA increased with age, whereas basolateral membrane transporter mRNA decreased with age. Transporter expression changed dramatically between E18 and DOH. The expression of nutrient transporters and digestive enzymes is induced before hatch to prepare the chick for exogenous feeding (Sklan, 2001). We observed an approximate 3-to 5-fold increase in mRNA for APN (56,000 to 147,500 molecules), y+LAT2 (3,000 to 8,500 molecules), bo,+AT (43,000 to 106,300), EAAT3 (1,600 to 4,300 molecules), rBAT (7,200 to 36,200 molecules), and PepT1 (750 to 3,600 molecules) and a 7- to 9-fold increase in mRNA for GLUT2 (70 to 490 molecules), BoAT (500 to 4,000 molecules), and SGLT1 (1,500 to 14,400 molecules) from E18 to DOH. Birds sampled at DOH had not yet received exogenous feed, suggesting that the rise in mRNA levels was genetically programmed to prepare the chick for a carbohydrate- and protein-based diet at hatch, and was not induced by dietary substrate. Moreno et al. (1996) observed uptake of {alpha}-methyl-D-glucoside, a nonmetabolizable glucose analogue in chick small intestine, 2 d before hatch and an increase in Vmax to 1 d posthatch, and suggested that genotypic programs of development influence changes in the absorptive capabilities of the intestine before hatch. Uni et al. (2003) observed a 9- to 25-fold increase in aminopeptidase, adenosine triphosphatase, maltase, and SGLT1 mRNA abundance from d 15 of incubation to d 19 in chick embryos, and a subsequent decline to hatch. Chen et al. (2005) found that PepT1 mRNA increased 14- to 50-fold from embryo d 16 to DOH in mixed-sex Cobb chicks.

Induction of gene expression of intestinal digestive enzymes and nutrient transporters prenatally also occurs in mammals. In a review of the regulation of sugar transport, Ferraris (2001) described the appearance of intestinal GLUT5, GLUT2, and SGLT1 mRNA in the developing rat. At early gestation, GLUT2 and SGLT1 mRNA are detected, whereas GLUT5 is not detected in significant amounts until weaning. Phillips et al. (1990) described a 3-fold increase in the active transport of glucose and galactose during the final week of gestation of rabbits. Thus, it is clear that in both avian and mammalian species, mechanisms exist to prepare the developing animal for nutrient uptake posthatch or postnatally. Buddington and Diamond (1990) suggested that in the newborn rabbit, intestinal uptake rates of glucose, galactose, Pro, Leu, Lys, and Met are at levels 2 to 2.5 times higher than maximum adult values, and that levels declined until weaning. The authors hypothesized that this may be partially explained by the occurrence of transport along the entire cryptvillus axis at birth. The suckling of milk induces mucosal hypertrophy and establishment of a well-defined crypt-villus gradient of expression, resulting in a dilution of mature enterocytes with immature cells, with localization restricted to the mid to upper villus. In the chicken small intestine, in contrast to mammals, cell proliferation is not restricted to the crypts, but occurs along the entire length of the villus during the first few days posthatch (Uni et al., 1998a). Future studies should attempt to correlate changes in cell proliferation and development of the mucosal layer with changes in nutrient transporter mRNA and protein quantities, site-specific localization, and transport activity in the chicken.

In summary, we used the method of absolute quantification by real-time PCR to quantify mRNA abundance for 10 amino acid transporters, a peptide transporter, 4 monosaccharide transporters, a digestive enzyme, and a housekeeping gene. Our study represents a comprehensive profile of the mRNA abundance of nutrient transporters and an aminopeptidase in the small intestine of broilers. Transporter mRNA levels ranged from as low as 100 molecules per nanogram of total RNA for CAT1 to as high as 100,000 molecules for bo,+AT. Of the genes examined in this study, the mRNA levels for the digestive enzyme APN were the highest at 200,000 molecules per nanogram of total RNA. We evaluated the temporal and spatial distribution of these genes in the small intestine of 2 genetically selected lines of broiler chicks. Developmentally, mRNA for PepT1, the brush border membrane-associated amino acid transporters, and the sugar transporters increased with age from E18 to D14, whereas the basolateral amino acid transporters decreased with age. This may be associated with the consumption of feed, reducing the need for basolateral uptake of nutrients from the bloodstream. In terms of spatial distribution, PepT1 was expressed at the greatest levels in the duodenum, the sugar transporters were expressed at the greatest levels in the jejunum, and the amino acid transporters were expressed at the greatest levels in the ileum. This suggests differences in absorptive capacity along the length of the small intestine. Future studies will involve manipulation of the diet to exploit differences in transporter gene expression to better meet the nutritional requirements of the growing chick.


   FOOTNOTES
 
1 This project was supported by National Research Initiative Competitive Grant no. 2005-35206-15271 from the USDA Cooperative State Research, Education, and Extension Service. This research was also funded in part by the John Lee Pratt Animal Nutrition Program at Virginia Tech. Back

Received for publication January 24, 2007. Accepted for publication April 20, 2007.


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