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Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
3To whom correspondence should be addressed. E-mail: webbk{at}vt.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • gene expression • chickens • PepT1 • dietary protein
Peptide transporters (PepT)4 are membrane proteins responsible for selective translocation of small peptides across the cell membrane. The cloning and characterization of the peptide transporters, PepT1 and PepT2, provided valuable information on the mechanism of peptide transport in mammalian species (1). PepT1 and PepT2 are distinct in their tissue distributions, substrate kinetics, and specific roles in different tissues. PepT1, which is expressed predominantly in the small intestine, and PepT2, which is expressed in kidney, central nervous system, and several other peripheral tissues, were shown to have nutritional, clinical, and pharmaceutical importance (2–4). In the small intestine, PepT1 is differentially expressed in different sections of the small intestine as well as in different areas along the crypt-villus axis (5).
The expression of PepT1 varies in response to changes in diet and developmental age. Selective amino acids and peptides (6) and growth factors (7) can regulate PepT1 gene expression. Under various conditions of malnourishment, expression of PepT1 is greatly enhanced (8). Shen et al. (9) reported that intestinal expression of PepT1 in rats is induced postpartum. Although PepT1 is located exclusively in the apical brush border of enterocytes in both prenatal and mature rats, it could be detected in the subapical cytoplasm and basolateral membrane of enterocytes immediately after birth (10).
A chicken intestinal peptide transporter (cPepT1) was cloned and characterized in our laboratory (11). Northern blot analysis showed that cPepT1 mRNA was expressed mainly in the small intestine, and at lower levels in the kidney and cecum, but not in the liver, crop, proventriculus, or pectoralis and fibularis longus muscles. The developmental and nutritional changes in PepT1 mRNA abundance have not been investigated in chickens. The objective of the present study was to determine the relative abundance of cPepT1 mRNA from the embryonic d 16 (e 16) to d 35 in broilers fed diets containing different dietary protein levels.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-32P]-dATP was purchased from ICN Pharmaceutical. DNA polymerase I/DNase I was purchased from Invitrogen. Animals and tissue sampling. Cobb-Cobb eggs and day of hatch Cobb-Cobb chicks were obtained from a commercial hatchery. For eggs, the entire small intestine was collected from the embryos. After hatch but before feeding, chicks were sampled and designated d 0. Day of hatch chicks were randomly assigned to heated floor pens with wood shavings. All pens had 24-h lighting and the chicks had free access to water. For a given sampling day, 10–14 chicks from each group were killed by cervical dislocation and sexed by visual inspection of the gonads. Intestinal tissues were collected and separated into duodenum, jejunum, and ileum, washed with ice-cold PBS, frozen in liquid nitrogen, and stored at –80°C. 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 upper and lower portions, corresponding to the jejunum and ileum, respectively. Growth data and tissue samples from male birds only were analyzed for d 1 through 35. All animal procedures were approved by the Institutional Animal Care and Use Committee at Virginia Tech.
Expt. 1. Ten eggs were sampled at e 18. After hatch but before feeding, 10 chicks were sampled (d 0). Other chicks (n = 339) were randomly divided among 3 pens. Chicks in a pen were randomly assigned to diets containing 12, 18, or 24% crude protein (CP). Daily feed intake of the chicks fed diets containing 18 or 24% crude protein was restricted to the intake of chicks fed the 12% CP diet. This was accomplished by feeding the groups of chicks receiving 18 and 24% CP diets an amount of feed equivalent to the feed consumed by the group of chicks fed the 12% CP diet the previous day. The composition of the experimental diets is shown in Table 1. On d 1, 3, 5, 7, 10, 14, 21, 28, and 35, 10–12 chicks from each group were killed by cervical dislocation and intestinal tissues were collected immediately. Total feed consumption was monitored daily and body weight was determined for chickens sampled at each time point.
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Expt. 3. The protocol of this experiment was the same as that of Expt. 1, except for the addition of 1 dietary treatment and a slightly different sampling schedule. In Expt. 3, chicks (n = 388) were randomly divided among 4 pens. The chicks in 3 pens were fed the same diets with the pair-feeding protocol as in Expt. 1. For the 4th treatment, chicks had free access to the 24% CP diet. On d 1, 3, 5, 7, 10, 14, and 35, 11–14 chicks from each group were killed by cervical dislocation and intestinal tissues were collected immediately. Total feed consumption was monitored daily and body weight was determined for chickens sampled at each time point.
Northern blot analysis.
For all experiments, total RNA was extracted using TriReagent. For Northern blot analysis, total RNA (20 µg) was denatured and size-fractionated on 1% agarose gels in 2.2 mol/L formaldehyde, stained with ethidium bromide, and transferred to a nylon membrane. For Expts. 1 and 3, 5 separate gels were run. Each gel comprised 1 replicate including each of the 3 small intestinal sections from 1 of the 5 chickens of each of the treatment groups from each sampling time. After prehybridization for 2 h in a solution containing 50% formamide, 5X Denhardt’s solution, 6X SSPE (1X SSPE = 0.15 mmol/L NaCl, 10 mmol/L NaH2PO4, and 1 mmol/L EDTA), 0.5% SDS and 10 mg/L yeast tRNA at 42°C, RNA blots were hybridized for 16 h under identical conditions with the addition of cPepT1 cDNA as the probe (11). The probe was labeled with [-32P]-dATP by nick translation using DNA polymerase I/DNase I and purified by Sephadex G-50 spin column chromatography. Posthybridization washing was done under high-stringency conditions, which involved washing twice in 5X SSPE, 0.5% SDS at room temperature for 15 min, twice in 1X SSPE, 0.5% SDS at 42°C for 15 min, and twice in 0.1X SSPE, 1% SDS at 65°C for 15 min. Washed filters were exposed with an intensifying screen to Kodak X-ray film. The abundance of cPepT1 mRNA was quantified by densitometry. Expt. 1 was preliminary in nature, and cPepT1 mRNA abundance was not related to the abundance of mRNA from a housekeeping gene. To correct for differences in RNA loading onto gels or in RNA transfer to membranes, membranes were stripped of the cPepT1 probe and rehybridized to a [32P]-labeled 18s rRNA probe in Expts. 2 and 3. The density of the hybridization bands was quantified using the Gelwork 1D digital image analysis system (UVP).
Statistical methods. Data were analyzed with ANOVA using the General Linear Models procedure (SAS/STAT version 7.12 for Windows, SAS Institute). For the variables feed intake and body weight (Expts. 1 and 3), the model included the main effects of diet and time and the diet x time interaction. For hybridization intensity comparisons (Expts. 1 and 3), the model included the main effects of diet, tissue, and time and all two-way interactions. For Expt. 3, the model included the main effects of time and sex and the time x sex interaction. The main effect of time was further tested for linear and quadratic fit using orthogonal contrast statements in the General Linear Model procedure. Significant differences among diets, tissues, or sex was tested using Tukey’s Honestly Significant Difference.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Effects of developmental age and diet on cPepT1 mRNA abundance in the small intestine.
In Expt. 1, PepT1 mRNA abundance was barely detectable at e 18 and increased 
50-fold at d 0 (P < 0.05) in the duodenum, jejunum, and ileum (Fig. 1). After hatch, there was a linear increase (P < 0.0001) in cPepT1 mRNA abundance with time and there was a protein x time interaction (P < 0.0001; Fig. 2). The interaction resulted from cPepT1 mRNA abundance generally decreasing throughout the study in all 3 sections of the small intestine in chicks fed the 12% CP diet (Fig. 2A, upper panel), whereas cPepT1 mRNA abundance increased with time in all 3 sections of the small intestine of chicks fed the 18% CP diet and the 24% CP diet (Fig. 2A, middle and lower panels, respectively). The major change in cPepT1 abundance occurred by d 21 for chicks fed the 12% CP diet (Fig. 2A, upper panel), d 14 for those fed the 18% CP diet (Fig. 2A, middle panel), and d 10 for those fed the 24% CP diet (Fig. 2A, lower panel). Data from the 3 sections were combined to show the main effect of diet (Fig. 2B, upper panel). cPepT1 mRNA level was lowest (P < 0.05) in chickens fed the low-protein diet (12%), highest in chickens fed the 24% CP diet, and intermediate for chicks fed the 18% CP diet (P < 0.05). Data from the chicks fed the 3 diets were combined to show the main effect of tissue (Fig. 2B, lower panel). cPepT1 mRNA abundance was higher in the jejunum (P < 0.05) than in the other 2 tissues.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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To improve our understanding of the mechanisms involved in the nutritional regulation of transporter gene expression, we investigated the effects of dietary protein on the abundance of cPepT1 mRNA to determine whether an increase in dietary protein level was associated with a similar increase in cPepT1 mRNA. In a normal production setting, dietary protein levels would be varied in a stepwise manner beginning at 23% at hatch and decreasing to 20 and 18% at 21 and 42 d of age, respectively (17). Depending on the production system, an even more refined step-wise change may involve 4 or 5 steps. For the current study, we chose not to vary dietary protein percentage with age; rather, we held it constant at 12, 18, or 24% for the duration of the experiment. A number of dietary models could have been chosen for this study, each having its weaknesses and strengths. In the model used, intake was equalized and protein content was varied. Therefore, although protein intake varied, so did the intake of other macronutrients. This point must be considered as the results are evaluated. In Expt. 1, an increase in cPepT1 mRNA abundance in chickens fed 18 and 24% CP diets and a decrease in chickens fed a low 12% CP diet was observed. To investigate the possibility that the increase in cPepT1 mRNA abundance in chickens fed 18 or 24% CP diets may be due to a combination of diet restriction and protein intake rather than dietary protein intake alone, a 4th group with free access to the 24% CP diet in Expt. 3 was added. In chickens with free access to the 24% CP diet, the cPepT1 mRNA abundance declined during the first 14 d and rose from d 14 to 35, but remained lower than that of chickens fed diets containing 18 or 24% CP.
These results are similar to those from studies of PepT1 expression in rats. rPepT1 mRNA abundance dropped suddenly after d 5 to 11% of the level of an adult and rose by d 75 to 25% of the level of an adult (9). Ihara et al. (8) examined the expression of rPepT1 in response to starvation. In those studies, the level of rPepT1 mRNA increased to 179, 164, and 161% of control in rats that were food deprived (no food intake for 4d), partially deprived (50% of control intake), or administered total parenteral nutrition, respectively. rPepT1 protein expression showed similar changes with mRNA. In contrast, there was no change in SGLT1 mRNA level in these treatment groups. Therefore, the higher cPepT1 mRNA abundance in the present study may have been due to the restricted feeding for these 2 groups.
Adaptation to a protein diet was correlated with changes in the activity of enzymes involved in protein digestion (18). A high-protein diet enhanced the levels of intestinal peptidases (19), and a protein-sufficient diet induced lactase synthesis in protein-starved pigs (20). These are consistent with reports on dietary regulation of PepT1 in rats showing that the rPepT1 mRNA abundance was 2-fold higher in rats fed a 50% casein diet than in rats fed a protein-free diet (6). The kinetic analysis from the studies also showed that rPepT1 activity was induced by a 50% casein diet with an increase in Vmax but not Km. The comparable patterns of the induced PepT1 mRNA abundance with increased dietary protein level suggested that dietary protein might have a regulatory effect on cPepT1 expression at the transcriptional level. Furthermore, both PepT1 mRNA abundance and transport rate of Gly-Gln by PepT1 increased when cells were treated with the dipeptide Gly-Sar for 24 h (21), whereas Gly-Gln treatment of the cells also caused an increase in both PepT1 mRNA abundance and Gly-Sar uptake (22). Promoter analysis of rPepT1 showed that the 5' upstream region of rPepT1 contains elements that respond to peptide substrates such as Asp-Lys, Phe-Val, and free amino acids including Phe, Lys, and Arg (6). Therefore, end products of protein digestion, small peptides and amino acids, may participate in pathways that control expression of intestinal transporters like PepT1 and eventually cause increased transporter capacity in the intestine. Mechanisms by which dietary protein regulates cPepT1 gene expression have yet to be determined.
There are reports regarding amino acid transporters showing that gene expression of system A, system L, and system XAG– was upregulated under conditions of amino acid restriction (23,24). It was suggested that gene expression is upregulated through amino acid sensory pathways at times of amino acid starvation (25). Although the regulatory networks have not been fully revealed, a potential amino acid–responsive element was found in the promoter regions of several genes, including mouse PepT1 (25). At present, we do not know whether the upregulatory effect was due to a response to peptide restriction or amino acid restriction derived from dietary protein limitation. Both amino acid–activating and peptide-activating pathways have to be investigated to fully elucidate the regulatory effects of dietary protein limitation. Peptide transporters may be one of the essential proteins induced by a stress response such as starvation for cell survival.
Further studies are required to establish the mechanism of transcriptional control of the cPepT1 gene, particularly through identification of the factors involved in the regulation of the transporters. Amino acids, peptide substrates, insulin, and growth factors were reported as factors regulating peptide transport through PepT1. Information on how cPepT1 is regulated at the molecular level will provide further insight into the basic mechanisms controlling body nitrogen and amino acid homeostasis in chickens.
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FOOTNOTES |
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2 Current address: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL
4 Abbreviations used: CP, crude protein; cPepT1, chicken peptide transporter 1; e, embryonic day; PepT, peptide transporter; SGLT-1, sodium-glucose transporter-1.
Manuscript received 13 July 2004. Initial review completed 10 August 2004. Revision accepted 15 November 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Herrera-Ruiz, D. & Knipp, G. T. (2003) Current perspectives on established and putative mammalian oligopeptide transporters. J. Pharm. Sci. 92:691-714.[Medline]
2. Leibach, F. H. & Ganapathy, V. (1996) Peptide transporters in the intestine and the kidney. Annu. Rev. Nutr. 16:99-119.[Medline]
3. Adibi, S. A. (1997) The oligopeptide transporter (Pept-1) in human intestine: biology and function. Gastroenterology 113:332-340.[Medline]
4. Daniel, H. (2004) Molecular and integrative physiology of intestinal peptide transport. Annu. Rev. Physiol. 66:361-384.[Medline]
5. Freeman, T. C., Bentsen, B. S., Thwaites, D. T. & Simmons, N. L. (1995) H+/di-tripeptide transporter (PepT1) expression in the rabbit intestine. Eur. J. Physiol. 430:394-400.[Medline]
6. Shiraga, T., Miyamoto, K.-I., Tanaka, H., Yamamoto, H., Taketani, Y., Morita, K., Tamai, I., Tsuji, A. & Takeda, E. (1999) Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1. Gastroenterology 116:354-362.[Medline]
7. Nielsen, C. U., Amstrup, J., Steffansen, B., Frokjaer, S. & Brodin, B. (2001) Epidermal growth factor inhibits glycylsarcosine transport and hPepT1 expression in a human intestinal cell line. Am. J. Physiol. 281:G191-G199.
8. Ihara, T., Tsujikawa, T., Fujiyama, Y. & Bamba, T. (2000) Regulation of PepT1 peptide transporter expression in the rat small intestine under malnourished conditions. Digestion 61:59-67.[Medline]
9. Shen, H., Smith, D. E. & Brosius, F. C., III (2001) Developmental expression of Pept1 and PepT2 in rat small intestine, colon, and kidney. Pediatr. Res. 49:789-795.[Medline]
10. Hussain, I., Kellett, G. L., Affleck, J., Shepherd, E. J. & Boyd, C.A.R. (2002) Expression and cellular distribution during development of the peptide transporter (PepT1) in the small intestinal epithelium of the rat. Cell Tissue Res. 307:139-142.[Medline]
11. Chen, H., Pan, Y.-X., Wong, E. A., Bloomquist, J. R. & Webb, K. E., Jr (2002) Molecular cloning and functional expression of a chicken intestinal peptide transporter (cPepT1) in Xenopus oocytes and Chinese Hamster Ovary cells. J. Nutr. 132:387-393.
12. Vieira, S. L. & Moran, E. T., Jr (1999) Effects of egg of origin and chick post-hatch nutrition on broiler live performance and meat yields. World’s Poult. Sci. J. 55:125-142.
13. Noy, Y. & Sklan, D. (1995) Digestion and absorption in the young chick. Poult. Sci. 74:366-373.[Medline]
14. Obst, B. S. & Diamond, J. M. (1992) Ontogenesis of intestinal nutrient transport in domestic chickens (Gallus gallus) and its relation to growth. Auk 109:451-464.
15. Uni, Z., Tako, E., Gal-Garber, O. & Sklan, D. (2003) Morphological, molecular, and functional changes in the chicken small intestine of the late-term embryo. Poult. Sci. 82:1747-1754.
16. Sklan, D., Geyra, A., Tako, E., Gal-Gerber, O. & Uni, Z. (2003) Ontogeny of brush border carbohydrate digestion and uptake in the chick. Br. J. Nutr. 89:747-753.[Medline]
17. National Research Council (1994) Nutrient Requirements of Poultry 9th rev. ed. 1994 National. Academy Press Washington, DC.
18. Moundras, C., Rémésy, C. & Demigné, C. (1993) Dietary protein paradox: decrease of amino acid availability induced by high-protein diets. Am. J. Physiol. 264:G1057-G1065.[Medline]
19. Jean, C., Rome, S., Mathe, V., Huneau, J. F., Aattouri, N., Fromentin, G., Achagiotis, C. L. & Tome, D. (2001) Metabolic evidence for adaptation to a high protein diet in rats. J. Nutr. 131:91-98.
20. Dudley, M. A., Schoknecht, P. A., Dudley, A. W., Jr, Jiang, L., Ferraris, R. P., Rosenberger, J. N., Henry, J. F. & Reeds, P. J. (2001) Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet. Am. J. Physiol. 280:G621-G628.
21. Thamotharan, M., Bawani, S. Z., Zhou, X. & Adibi, S. A. (1998) Mechanism of dipeptide stimulation of its own transport in a human intestinal cell line. Proc. Assoc. Am. Physicians 110:361-368.[Medline]
22. Walker, D., Thwaites, D. T., Simmons, N. L., Gilbert, H. J. & Hirst, B. H. (1998) Substrate upregulation of human small intestinal peptide transporter, hPEPT1. J. Physiol. 507:696-706.
23. Nicholson, B. & McGivan, J. D. (1996) Induction of high affinity glutamate transport activity by amino acid deprivation in renal epithelial cells does not involve an increase in the amount of transporter protein. J. Biol. Chem. 271:12159-12164.
24. Palacin, M., Estevez, R., Bertran, J. & Zorzano, A. (1998) Molecular biology of mammalian plasma membrane amino acid transporters. Physiol. Rev. 78:969-1054.
25. Fafournoux, P., Bruhat, A. & Jousse, C. (2000) Amino acid regulation of gene expression. Biochem. J. 351:1-12.[Medline]
26. Fei, Y.-J., Sugawara, M., Liu, J.-C., Li, H. W., Ganapathy, V., Ganapathy, M. E. & Leibach, F. H. (2000) cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1. Biochim. Biophys. Acta 1492:145-154.[Medline]
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