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© 2008 American Society for Nutrition J. Nutr. 138:262-271, February 2008

Biochemical, Molecular, and Genetic Mechanisms

Dietary Protein Quality and Feed Restriction Influence Abundance of Nutrient Transporter mRNA in the Small Intestine of Broiler Chicks1,2

Elizabeth R. Gilbert3, Huifeng Li3, Derek A. Emmerson4, Kenneth E. Webb, Jr3 and Eric A. Wong3,*

3 Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 and 4 Aviagen, Huntsville, AL 35805

* To whom correspondence should be addressed. E-mail: ewong{at}vt.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The objective of this study was to evaluate the effect of dietary protein quality on intestinal peptide transporter (PepT1), amino acid transporter [Na+-independent cationic and zwiterionic amino acid transporter (bo,+AT), excitatory amino acid transporter 3 (EAAT3), Na+-independent cationic and Na+-dependent neutral amino acid transporter (y+LAT2), and Na+-independent cationic amino acid transporter 2 (CAT2)], glucose transporter [Na+-dependent glucose and galactose transporter 1 (SGLT1) and Na+-independent glucose, galactose, and fructose transporter 2 (GLUT2)], and digestive enzyme [aminopeptidase N (APN)] mRNA abundance in 2 lines of broilers (A and B). At day of hatch (doh), chicks from both lines were randomly assigned to corn-based diets containing 24% crude protein with either soybean meal (SBM) or corn gluten meal (CGM) as the supplemental protein source. Chicks were given unlimited access to feed and water. Groups of chicks from both lines were also assigned to the SBM diet at a quantity restricted to that consumed by the CGM group (SBM-RT). Intestinal transporter and enzyme mRNA abundance was assayed by real-time PCR using the absolute quantification method. Abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B (P < 0.03), whereas APN and SGLT1 were greater in Line A (P < 0.04). When feed intake was equal (CGM vs. restricted SBM), a greater abundance of PepT1 and bo,+AT mRNA was associated with the higher quality SBM (P < 0.04), whereas a greater abundance of EAAT3 and GLUT2 mRNA was associated with the lower quality CGM (P < 0.01). When feed intake was restricted (SBM vs. SBM-RT), a greater abundance of PepT1 mRNA was associated with the restricted intake (P < 0.04). These data demonstrate that both dietary protein quality and feed restriction influence expression of nutrient transporter mRNA in the small intestine of broiler chicks.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Intestinal nutrient absorption is mediated by transporter proteins expressed on enterocytes. Amino acids are transported into the cell in their free form by a variety of transporters that vary in specificity (1–3) or as di- and tripeptides by the peptide transporter, PepT15 (4). In addition to humans and laboratory species, peptide transport through PepT1 is nutritionally important in agricultural species such as chickens, cows, pigs, and sheep (5–7); however, the free amino acid transporters have not been as well studied in these species. Amino acid transporters have varying substrate specificities. For example, transporters were characterized that are specific for basic amino acids [rBAT and Na+-independent cationic and zwiterionic amino acid transporter (bo,+AT), Na+-independent cationic amino acid transporter 1 (CAT1) and CAT2, Na+-independent cationic and Na+-dependent neutral amino acid transporter 1 (y+LAT1) and y+LAT2], neutral amino acids (BoAT, LAT1), and anionic amino acids [excitatory amino acid transporter 3 (EAAT3)], and most of these transporters exhibit some substrate overlap and various ion dependencies and mechanisms for movement of amino acids across the cell membrane (8–13). The absorption of glucose is mediated by the Na+-dependent glucose and galactose transporter 1 (SGLT1) at the brushborder membrane (14) and exit of glucose across the basolateral membrane is mediated by the facilitated transporter Na+-independent glucose, galactose, and fructose transporter 2 (GLUT2) (15).

The dietary regulation of nutrient transporters has been studied extensively, especially with regard to the influence of fasting and starvation. Ferraris and Diamond (16) described the regulation of nutrient transporters as a way to match uptake capacity to requirements without wasting energy on unnecessary transporters. Thus, the study of regulation of transporters leads to the elucidation of mechanisms that can change transport rates (17). The objective of this experiment was to determine the influence of dietary protein quality and feed restriction on peptide, amino acid, and monosaccharide transporter and aminopeptidase mRNA abundance in the small intestine of 2 genetically selected lines of broilers. Chicks were fed diets containing either a higher quality protein source [soybean meal (SBM)] or lower quality protein source [corn gluten meal (CGM)] as the sole supplemental protein during the first 2 wk.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Animals and tissue collection. The chickens in this experiment represent 2 commercial broiler lines that were genetically selected under different nutritional environments. Line A was developed on a corn- and soy-based diet, whereas Line B was developed on a wheat-based diet with amino acid concentrations that were 15–20% higher than for line A. The lines share a common origin but have been separated for at least 30 generations and have been selected in the different nutritional environments for >10 generations. Both lines have been subjected to balanced selection for improved growth, feed conversion, meat yields, reproduction, and general fitness. Additional information on these lines has been previously described (18). These lines were chosen for this study because they respond differently to dietary amino acid concentrations.

Eggs from both genetic lines were obtained from Aviagen. A total of 40 d of hatch (doh) chicks from both genetic lines (total 80) were sampled for intestinal tissue before consumption of feed. The remaining chicks (n = 810; 405/genetic line) were randomly assigned to 1 of 6 heated floor pens with wood shavings. All pens had 24-h lighting and chicks had free access to water. Birds in a pen were randomly assigned to diets containing either SBM or CGM as the supplemental protein source (Table 1). A comparison of essential amino acid compositions of the 2 experimental diets to NRC requirements (19) and a comparison of nonessential amino acid compositions are shown (Table 2). Additionally, to separate the effect of feed intake from protein quality, chicks from both lines were also randomly assigned to the diet containing SBM at a quantity restricted to that consumed by chicks fed the CGM diet the previous 8 h (SBM-RT). Feed intake data were collected at 3 time intervals each day (0700, 1500, and 2300). Birds were killed by cervical dislocation at the following time points: doh (after hatch but before feeding), 1 d (d1), d3, d7, and d14 posthatch. At d1, d3, and d7, 40, 29, and 29 chicks were removed from each pen for sampling, respectively. At d14, the remaining birds were sampled in each pen. Birds and intestinal samples were processed in a manner similar to the procedure described by Chen et al. (20). The intestine was separated into duodenum, jejunum, and ileum. Digesta was squeezed out of the intestine and segments were rinsed 3 times in PBS (NaH2PO4, 1.47 mmol/L; Na2HPO4, 8.09 mmol/L; and NaCl, 145 mmol/L). Whole segments were minced using a razor blade, mixed thoroughly, and frozen as aliquots at –80°C. Sex of the birds was determined by PCR as previously described (18). A total of 5 males were randomly selected from each group for RNA isolation. Remaining bird samples were used for other laboratory analyses. All animal procedures were approved by the Institutional Animal Care and Use Committee at Virginia Tech.


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  		TABLE 1 Ingredient and chemical composition of the experimental diets

 

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  		TABLE 2 Amino acid composition of experimental diets in comparison to NRC requirements for broilers1

 
    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 and the absolute quantification method was used to determine the number of molecules of mRNA present for each gene of interest per nanogram of total RNA starting template (18). An RNA standard curve for each gene was generated based on modification of the protocol of Fronhoffs et al. (21). Total RNA from d7 jejunum and gene-specific primers (see 18 for list of primers) were used to perform RT-PCR. Two-step RT-PCR followed by subcloning into the pGEM-T Easy Vector (Promega) was performed as previously described (18). Purified plasmid samples were sequenced at the Virginia Bioinformatics Institute at Virginia Tech. Nested primers were designed (see 18 for list) within cloned chicken cDNA sequences using the Primer Express software, optimized for use with Applied Biosystems Real-Time PCR Systems.

β-Actin primers were designed in a similar manner for cloning (forward primer, 5'-GTCCACCTTCCAGCAGATGT-3'; reverse primer, 5'-AGTCAAGCGCCAAAAGAAAA-3') and real-time PCR (forward, 5'-GTCCACCGCAAATGCTTCTAA-3'; reverse, 5'-TGCGCATTTATGGGTTTTGTT-3').

Standard curves were made as previously described (18). Briefly, plasmids containing amplified chicken cDNA were linearized opposite a T7 or SP6 promoter depending on the orientation of the insert sequence. In vitro transcription was performed on linearized plasmids using the MEGAscript T7 or SP6 in vitro transcription kit (Ambion) and cRNA was precipitated with lithium chloride and quantified using the ribogreen assay (Molecular Probes) and a FLUOstar OPTIMA microplate reader (BMG LABTECH). A dilution series of 1011–104 molecules/microliter was performed in the presence of yeast tRNA at 10 mg/L.

Each RT reaction contained 2000 ng of RNA at a concentration of 100 ng/µL or an equal volume of a dilution series of cRNA (High-Capacity cDNA Archive kit; Applied Biosystems). The cDNA was diluted 1:30 before addition to PCR that contained primers and SYBR green master mix (Applied Biosystems). PCR 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 using an Applied Biosystems real-time PCR 7300 system. 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 to verify amplification of a single product.

    Statistical analysis. All data were analyzed using the Proc MIXED procedure of SAS (SAS Institute). The model included the main effects of genetic line, intestinal segment, age, diet, and all appropriate 2-way interactions. Differences among segments and diets were evaluated by Tukey's test for multiple comparisons. Differences were considered significant at P < 0.05. The doh and d1 time points were not included in this model, because the feeding restriction was not imposed until after intestinal sampling on d1.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Feed intakes and body weights. Feed intake rose with age in Line A and Line B chicks with free access to feed containing SBM as the supplemental protein, with a consumption of 31 g · bird–1 · d–1; ~32 g/d for Line A chicks and 30 g/d for Line B chicks (Fig. 1). In contrast, in all chicks with free access to the CGM diet and in chicks fed the SBM-RT diet, there was a lower intake throughout the course of the feeding trial, with an intake of 7.8 g · bird–1 · d–1); 8.7 g/d for Line A chicks and 6.9 g/d for Line B chicks. This represented an ~3-fold greater feed intake in birds consuming SBM compared with those consuming CGM. Reflective of the differences in intake were differences in body and intestinal weights among the diet groups. There were interactions of age x diet for body weight (BW), total intestinal weight, relative intestinal weight, and segmental weight (Table 3; Fig. 2; P = 0.0001). Intestinal weights increased with age more rapidly in chicks that ate SBM ad libitum compared with SBM-RT chicks and increased faster in these chicks than in those fed equal amounts of CGM (Table 3; Fig. 2). Chicks consuming SBM ad libitum were heaviest in terms of BW, total intestinal weight, and individual segmental weights; chicks consuming CGM were lightest; and SBM-RT chicks had intermediate intestinal weight, segmental weight, and BW (P = 0.0001; Table 3). Although Line A chicks had greater total BW and intestinal, jejunal, and ileal weight than Line B chicks (P = 0.0001), when total intestine was expressed relative to BW there was no significant difference. Intestinal mass expressed relative to BW was greater in chicks consuming SBM ad libitum than in SBM-RT chicks (P = 0.0001) and was greater in SBM-RT chicks compared with chicks that consumed CGM (P = 0.0001).


Figure 1
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  		FIGURE 1  Pen feed intakes from doh to d13 for 2 genetically selected lines of broiler chicks fed diets that differed in the source of protein. Lines represent feed intake per bird (calculated based on total pen feed intake divided by the number of chicks) for mixed-sex chicks from both lines and each dietary group from doh through d13.

 

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  		TABLE 3 Intestinal weight, BW, and relative intestinal weight in 2 lines of broiler chicks subjected to feeding restriction and diets differing in protein quality from doh to d14

 

Figure 2
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  		FIGURE 2  Intestinal and BW of male chicks (n = 10; 5 from Lines A and B) from 2 genetic lines at d3, d7, and d14 in chicks fed SBM ad libitum, SBM-RT, and CGM. Data for BW (A) and weights of total intestine (B), relative intestine (C), duodenum (D), jejunum (E), and ileum (F) represent means of 10 male chicks (5 from both lines) ± SEM. Relative intestine weights represent total intestinal weight expressed as a percentage of BW. There was an interaction of diet x age for duodenum, jejunum, ileum, intestine, body, and relative intestinal weight, P = 0.0001.

 
    Effects of genetic line and diet on mRNA abundance. The mRNA abundance for PepT1, 4 amino acid transporters (bo,+AT, EAAT3, y+LAT2, and CAT2), 2 glucose transporters (SGLT1 and GLUT2), a digestive enzyme [aminopeptidase N (APN)], and 2 housekeeping genes [β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] 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 if these data parallel expression and activity of the functional proteins. The mRNA expression data are summarized in Table 4, with separate rows for main effects of genetic line, diet, intestinal segment, developmental age, and 2-way interactions. Only significant interactions are expressed graphically. Effects of genetic line, age, diet, and intestinal segment were observed for most of the genes; however, for brevity, only effects involving genetic line, diet, and age will be discussed. To facilitate a more meaningful discussion, only 2 dietary comparisons, the effect of feed restriction (SBM vs. SBM-RT) or protein quality (SBM-RT vs. CGM), will be discussed.


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  		TABLE 4 Dietary regulation of nutrient transporter, enzyme, and housekeeping gene mRNA in the small intestine of broiler chicks1

 
Multiple main effects of genetic line were observed in this study (Table 4). The abundance of PepT1, EAAT3, and GLUT2 mRNA was greater in Line B compared with Line A chicks (P < 0.03). Abundance of APN and SGLT1 mRNA was greater in Line A chicks (P < 0.04). The dietary response of bo,+AT, EAAT3, y+LAT2, APN, and SGLT1 mRNA was differentially regulated by the 2 genetic lines. Line A chicks varied little in expression of bo,+AT (Fig. 3A), y+LAT2 (Fig. 3B), EAAT3 (Fig. 3C), and APN (Fig. 3D) when fed SBM ad libitum or SBM-RT; however, Line B chicks showed greater expression of these genes in feed-restricted chicks compared with chicks consuming SBM ad libitum (P = 0.0003). For the glucose transporter, SGLT1, mRNA abundance was greater in ad-libitum fed chicks than in feed-restricted chicks within Line A, but did not differ in Line B chicks between the 2 groups consuming SBM (P = 0.0001; Fig. 3E).


Figure 3
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  		FIGURE 3  Effect of genetic line and diet on transporter and enzyme mRNA abundance in 2 genetically selected lines of broilers fed diets that differed in the source of protein from doh to d14. The number of bo,+AT (A), y+LAT2 (B), EAAT3 (C), APN (D), and SGLT1 (E) mRNA molecules per nanogram of total RNA is shown on the y-axis. Data represent the means of male chickens (n = 5) from Line A or Line B ± SEM in SBM, SBM-RT, and CGM birds at d3, d7, and d14. There was an interaction of genetic line x diet for all 5 genes, P < 0.05.

 
There was reduced expression of bo,+AT (P = 0.0003; Fig. 3A) and y+LAT2 (P = 0.0001; Fig. 3B) in Line A chicks consuming CGM compared with SBM-RT chicks, whereas dietary groups did not differ in Line B chicks. Abundance of EAAT3 mRNA was greater in chicks consuming CGM compared with chicks consuming equal amounts of SBM, in both lines; however, the difference was more accentuated in Line B chicks (P = 0.005; Fig. 3C). In Line A chicks, mRNA abundance of APN was lower in chicks consuming the CGM diet, in contrast to Line B chicks where mRNA was greater in chicks consuming the CGM diet compared with chicks that consumed equal quantities of the SBM diet (P = 0.0001; Fig. 3D). In Line A chicks, there was also a lesser quantity of SGLT1 mRNA in response to the low-quality protein CGM, whereas in Line B chicks, there was a dramatic increase in SGLT1 mRNA in chicks consuming CGM compared with chicks consuming equal amounts of the SBM diet (P = 0.0001; Fig. 3E).

PepT1 was the only gene influenced by feed restriction with greater expression in the feed-restricted chicks compared with chicks given free access to the diet (P < 0.03; Table 4). Abundance of PepT1 and bo,+AT mRNA were both influenced by dietary protein quality, with greater expression in the chicks that consumed the diet containing the higher quality protein, SBM, compared with chicks that consumed CGM (P < 0.04). The abundance of EAAT3 and GLUT2 mRNA was greater in chicks that consumed CGM compared with chicks consuming an equal amount of SBM (P < 0.01).

Feed restriction and protein quality influenced the age response of PepT1, bo,+AT, EAAT3, y+LAT2, CAT2, APN, and SGLT1 (P < 0.01). In the small intestine of SBM-RT chicks, expression of PepT1 (Fig. 4A), bo,+AT (Fig. 4B), EAAT3 (Fig. 4C), y+LAT2 (Fig. 4D), and CAT2 (Fig. 4E) mRNA increased with age of the chicken, from d3 to d14, whereas in chicks consuming feed ad libitum, mRNA levels increased less dramatically (CAT2), decreased (PepT1), or remained relatively unchanged (bo,+AT, EAAT3, y+LAT2, and APN). Quantities of APN mRNA remained unchanged in response to restricted feeding (Fig. 4F). Protein quality influenced the age response of transporters and APN in a similar manner. In contrast to the general upregulation in gene expression observed in the SBM-RT chicks from d3 to d14, the chicks consuming CGM showed decreased expression of PepT1, bo,+AT, EAAT3, y+LAT2, CAT2, APN, and SGLT1 from d3 to d7, followed by an increase to d14 (P < 0.01; Fig. 4). Quantities of SGLT1 mRNA remained unchanged with age in response to both ad libitum and restricted consumption of the SBM diet but changed in response to the CGM diet (Fig. 4G).


Figure 4
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  		FIGURE 4  Effect of age and diet on transporter and enzyme mRNA abundance in 2 genetically selected lines of broilers fed diets that differ in the source of protein from doh to d14. The number of PepT1 (A), bo,+AT (B), EAAT3 (C), y+LAT2 (D), CAT2 (E), APN (F), and SGLT1 (G) mRNA molecules per nanogram of total RNA is shown on the y-axis. Data represent the means of male birds (n = 10; 5 from both genetic lines) ± SEM in SBM, SBM-RT, and CGM birds at d3, d7, and d14. There was an interaction of age x diet for all 7 genes, P < 0.01.

 
Expression of 2 housekeeping genes, GAPDH and β-actin, was evaluated in this study and both were influenced by genetic line, age, and dietary treatment. β-actin was expressed in greater quantities in Line A chicks than Line B chicks (Table 4; P = 0.0021). Expression between lines of both housekeeping genes differed according to dietary treatment (P < 0.03). In Line A chicks, GAPDH mRNA abundance decreased in response to feed restriction, whereas in Line B chicks, the 2 groups did not differ (Fig. 5A). In response to protein quality, there was a more accentuated difference in GAPDH expression between the Line B SBM-RT and CGM groups (Fig. 5A). β-actin also displayed a differential pattern of expression across dietary groups in the 2 genetic lines, with Line B chicks expressing greater quantities of mRNA when fed the CGM diet than chicks fed an equal amount of SBM (Fig. 5B). Expression of GAPDH was greater in the chicks consuming CGM than in the chicks consuming equal amounts of SBM (P = 0.002). There was an interaction of age x diet on both GAPDH (P = 0.0001) and β-actin (P = 0.005) mRNA quantities. The decrease in mRNA abundance of GAPDH with age was less pronounced in feed-restricted chicks than in ad-libitum fed chicks (Fig. 5C), and for β-actin, there was a less pronounced decrease in feed-restricted chicks from d3 to d7 and a more pronounced increase in expression from d7 to d14 compared with ad-libitum fed chicks (Fig. 5D). In response to dietary protein quality, both GAPDH and β-actin decreased from d3 to d7 and increased from d7 to d14 in chicks that consumed the lower-quality protein, CGM. Thus, β-actin and GAPDH mRNA levels did not remain constant and were deemed unsuitable as internal controls for relative quantification.


Figure 5
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  		FIGURE 5  Effect of genetic line, age, and diet on housekeeping gene mRNA abundance in 2 genetically selected lines of broilers fed diets that differ in the source of protein from doh to d14. The number of GAPDH (A) and β-actin (B) mRNA molecules per nanogram of total RNA in Line A and B chicks is shown in the 2 upper graphs. Data represent the means of male birds (n = 5) from Line A or Line B ± SEM in SBM, SBM-RT, and CGM. There was an interaction of line x diet for both genes, P < 0.03. Quantities of GAPDH (C) and β-actin (D) mRNA in birds at d3, d7, and d14 are shown in the 2 lower graphs. Quantities represent the means of male chicks (n = 10; 5 from both genetic lines) ± SEM in SBM, SBM-RT, and CGM birds at d3, d7, and d14. There was an interaction of age x diet for both genes, P < 0.01.

 

   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Protein quality is reflected by the balance of amino acids required for maximal growth and protein synthesis and the limiting amino acids present (22). Additionally, physical properties of a protein (i.e. other chemical constituents) may also contribute to the availability of amino acids to the animal. In our study, these physical differences in the 2 protein sources may contribute to differences in digestion and absorption at the intestinal level. Both SBM and CGM are deficient in essential amino acids and contain differences in concentrations of specific amino acids, amino acid ratios, and other physical properties. However, these 2 protein sources represent a good model for evaluating the effect of a single protein source on nutrient transporter gene expression in the small intestine. Consumption of a diet with protein that contains an imbalance of essential amino acids and amino acid levels lower than the nutritional requirements leads to metabolic disturbances and reduced feed intake (23). Lower intake observed in birds that consumed the CGM diet (25% of SBM) may be related to a deficiency in essential amino acids and to the metabolic cost associated with removal of amino acids that are in excess of the most limiting for protein synthesis. Differences in body and intestinal weight were reflective of differences in intake and protein quality, with greatest weights and rates of growth in chicks that consumed SBM ad libitum and the least growth in chicks that consumed CGM. Greater BW and intestinal weight of SBM-RT chicks compared with chicks that consumed CGM illustrates that SBM was a better dietary protein source than CGM. As expected, there was a difference between genetic lines, with chicks from Line A weighing more than Line B chicks. Total intestinal weight and the individual weights of the jejunum and ileum were greater in Line A chicks, but when intestinal weight was expressed relative to BW, there was no difference. This indicates that per unit body mass, intestines were developing at the same rate in both lines of broilers.

    Effect of genetic line on transporter expression. The rationale for comparing the 2 genetically selected lines of broilers was to evaluate the idea that differential growth responses to varying amino acid concentrations in the diet is correlated with differences in the profile of nutrient transporters expressed in the small intestine. We previously reported that of the 15 genes evaluated (1 peptide transporter, 9 amino acid transporters, 1 digestive enzyme, and 4 sugar transporters), only 1 was influenced by genetic line, with Line B expressing more PepT1 mRNA than Line A (18). In that experiment, a standard commercial starter diet [20% crude protein (CP); corn-soy] was fed, with no dietary treatment. In this experiment, it is clear that there is a differential response to dietary perturbations, with multiple effects of genetic line and interactions of line and diet. We find that genetic selection has changed expression of intestinal nutrient transporters but that these differences are masked when birds are fed a balanced diet and accentuated when fed deficient diets. We found, in our experiment, an effect of genetic line for 5 transporter or digestive enzyme genes. We observed greater expression of EAAT3, GLUT2, and, similar to the previous study, PepT1 mRNA, in Line B chicks. Expression of APN and SGLT1 mRNA was greater in Line A chicks. Although the implications of these differences are unknown, they clearly suggest that genetic selection based on nutrition has robust effects on gene expression of nutrient transporters. Future experiments will involve exploitations of these differences through the diet in attempts to drive changes in growth performance and intestinal development.

    Effect of feed restriction on transporter expression. Of the genes evaluated in this study, PepT1 was the only 1 for which there was greater expression in the feed-restricted birds than in birds with ad libitum consumption of SBM. As a low-affinity, high-capacity transporter that is considered to be a faster and more energetically efficient means of assimilating amino acids (24–27), increased expression of PepT1 in response to feed restriction may serve as a way to conserve energy while maximizing amino acid uptake despite protein and energy deficiency.

There was an interaction of age x diet for 7 genes associated with digestion or transport, including PepT1, bo,+AT, EAAT3, y+LAT2, CAT2, APN, and SGLT1. There was a similar response to feed restriction with age, in which there was upregulation in expression, which occurred as early as d7 for some genes (PepT1, EAAT3, y+LAT2, and CAT2) and at d14 for others (bo,+AT). Regardless of whether expression increased with age in chicks fed SBM ad libitum (CAT2 and EAAT3), the response to restriction with age was of a greater magnitude.

We observed little response of APN, GLUT2, and SGLT1 mRNA to feed restriction. Susbilla et al. (28) observed increased activities of amino- and dipeptidases in the small intestines of broiler chicks that were feed restricted to 40% of the control intake from 5 to 11 d of age. In feed-restricted leghorn chicks (54% of control intake) from 12 to 17 d, duodenal APN activity was greater than in control or refed birds (29). Food restriction in rats (8–10 g food/d; 30) and protein-energy restriction in postnatal rabbits (31) increased glucose transport. In 6-wk-old male broiler chickens, SGLT1 mRNA was enhanced in birds that were totally deprived of feed for 4 d compared with birds with ad libitum intake (32).

Feed restriction, fasting, or other sources of malnutrition result in reduced intestinal absorptive surface area, which may explain why expression of nutrient transporters increased with age in response to feed restriction. Delayed access to feed for 36 h posthatch resulted in depressed villus height and crypt depth, and depressed growth in all intestinal segments (33). Silva et al. (34) subjected male broiler chicks to feed restriction (30% of ad libitum) and found that feed restriction decreased the surface area of the tip of the enterocytes in the small intestine at 14 d. Thus, the mechanism leading to a fasting- or malnutrition-related increase in nutrient transport may be a combination of increased gene expression and ratio of transporting to nontransporting cells (35).

    Effect of dietary protein quality on transporter expression. In this study we found 4 transporter genes that were expressed differentially due to the effect of protein quality. PepT1 and bo,+AT were expressed at greater quantities in SBM-RT chicks than in chicks that consumed CGM. Quantities of EAAT3 and GLUT2 were greater in chicks that consumed CGM. Dietary protein level and feeding restriction influenced PepT1 expression in a previous study. Chen et al. (20) reported that, by feeding diets to broiler chicks containing 12, 18, or 24% CP at intakes restricted to that consumed by birds fed the 12% CP diet, greater levels of CP in the diet were associated with greater expression of PepT1 during the first 35 d posthatch. Chen et al. (20) found that in the group of birds fed the 24% CP diet ad libitum (40% greater intake), mRNA abundance was lower than that observed for the feed-restricted group, demonstrating that feed restriction upregulated PepT1 expression, similar to what we found in this study.

Karasov et al. (36) described the relationship between dietary amino acid levels and intestinal amino acid uptake. Amino acids can be used as sources of energy, some amino acids are more essential to a cell than others, or more toxic, and to further complicate matters, enterocytes express transporters with overlapping substrate specificity and transporters that mediate the movement of both essential and nonessential amino acids. Thus, it becomes difficult to predict whether a transporter should be upregulated in response to certain amino acid deficiencies or imbalances.

Kilberg et al. (37) described regulation of gene expression in response to reduced amino acid availability. The amino acid response pathway detects and acts in response to an amino acid deficiency, increasing translation of activating transcription factor 4. This pathway is thought to be cell-type specific, with an insufficient amount of an amino acid essential to a particular cell, triggering pathway activation, and subsequent changes in RNA splicing, chromatin remodeling, nuclear RNA export, mRNA stabilization, and translational control. Stein et al. (38) observed that amino acids given as dietary supplements to mice were potent inducers of transporters, differing in their ability to regulate the same amino acid transporter, with a discrepancy between amino acids that were inducers and those that were transporter substrates. Shiraga et al. (39) found elements in the 5' upstream region of rat PepT1 responsive to peptides and free amino acids. Hence, nutrient transporter genes are likely to be responsive to changes in dietary protein.

The age-related response in gene expression to dietary protein quality showed an interesting pattern. For PepT1, bo,+AT, EAAT3, y+LAT2, CAT2, APN, SGLT1, β-actin, and GAPDH, there was an identical age-related pattern of expression in chicks consuming the CGM diet, in which mRNA abundance decreased from d3 to d7 and then rose sharply from d7 to d14. This pattern may be related to depletion of yolk sac reserves, which occurs between wk 1 and wk 2 (40). Because of the severe imbalance of amino acids in the diet, the bird experiences metabolic disturbances (41–43). In general it is hypothesized that depressed intake is related to a conservation of energy, as there is a metabolic cost associated with disposing of excess amino acids. Similarly, the cost associated with upregulating the nutrient transporters may outweigh the benefits to be derived from assimilating those amino acids, especially if the overlap in substrate specificity and potential coordinate regulation prevents them from better controlling the balance of amino acids entering the cell.

Expression of mRNA for GAPDH and β-actin, 2 commonly used housekeeping genes, showed significant interactions of age x diet, line x diet, and diet x segment, similar to the digestive enzyme and nutrient transporters. The age response for chicks consuming CGM was similar for all genes, suggesting that this is a generalized effect. Because GAPDH and β-actin are proteins involved in vital cellular processes including glucose metabolism and cytoskeleton structure and movement, respectively, it is not surprising that they are also influenced by diet. These data demonstrate that correcting gene expression to a housekeeping gene is in some cases inappropriate and should be exercised with caution.

In conclusion, our data demonstrate that dietary protein quality and feed restriction do influence mRNA abundance of peptide, amino acid, and sugar transporters, and a digestive enzyme in the small intestine of 2 genetically selected lines of broilers. The influence of protein quality and feed restriction is dependent on age. In response to feed restriction, expression of peptide and amino acid transporters increased at d7, whereas in response to ad libitum intake of a diet containing the low-quality protein, CGM, expression of transporters decreased at d7 and then increased at d14. In general, the response to feed restriction and to the lower quality protein was greater in Line B chicks, although for those genes, Line A chicks expressed greater overall quantities. A greater understanding of how dietary protein and genetic selection regulates nutrient transporter gene expression will allow for the discovery of ways to potentially manipulate regulatory processes and exploit differences to enhance nutrient uptake and improve nutrient utilization in chickens.


   ACKNOWLEDGMENTS
 
We express our gratitude to Dr. Junmei Zhao for statistical consultations and to Dr. Kristi Thompson, Consulting Nutritionist (Purina TestDiet) for her assistance with the dietary formulations.


   FOOTNOTES
 
1 Supported by the 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 (to E.R.G.). Back

2 Author disclosures: E. R. Gilbert, H. Li, D. A. Emmerson, K. E. Webb Jr, and E. A. Wong, no conflicts of interest. Back

5 Abbreviations used: APN, aminopeptidase N; bo,+AT, Na+-independent cationic and zwiterionic amino acid transporter; BW, body weight; CAT2, Na+-independent cationic amino acid transporter 2; CGM, corn gluten meal; CP, crude protein; doh, day of hatch; d1, d 1 posthatch; EAAT3, excitatory amino acid transporter 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT2, Na+-independent glucose, galactose, and fructose transporter; PepT1, peptide transporter 1; SBM, soybean meal; SBM-RT, soybean meal restricted; SGLT1, Na+-dependent glucose and galactose transporter 1; y+LAT2, Na+-independent cationic and Na+-dependent neutral amino acid transporter. Back

Manuscript received 30 August 2007. Initial review completed 26 September 2007. Revision accepted 21 November 2007.


   LITERATURE CITED
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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