QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rome, S. Articles by Tomé, D. Search for Related Content PubMed PubMed Citation Articles by Rome, S. Articles by Tomé, D.

---

Nutrition and Aging
Research Communication

The Regionalization of PepT1, NBAT and EAAC1 Transporters in the Small Intestine of Rats Are Unchanged from Birth to Adulthood

Sophie Rome¹, Laurence Barbot^,12, Eugénie Windsor^*, Nathalie Kapel^*,, Viviane Tricottet^**,, Jean-François Huneau, Michel Reynes, Jean-Gérard Gobert^*, and Daniel Tomé

UMR 914, Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, Paris, France; ^* Laboratoire de Biologie Animale et Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques René Descartes, Paris, France; Service de Coprologie Fonctionnelle, GH Pitié-Salpêtrière, Paris, France; ^** Service Commun d’Imagerie Cellulaire et Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques René Descartes, Paris, France; Service d’Anatomie Pathologique, Hôpital Paul Brousse, Villejuif, France

²To whom correspondence should be addressed. E-mail: laurence.barbot{at}psl.ap-hop-paris.fr.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The ontogenetic development of PepT1, NBAT and EAAC1 along the vertical and horizontal axes of the rat small intestine was evaluated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The proximodistal profiles of mRNA levels showed that PepT1 was evenly distributed, whereas NBAT had greater expression in the proximal part, and EAAC1 in the distal part. These regionalizations were the same from postnatal days 4 to 50. PepT1 and NBAT proteins were detected in the microvilli of enterocytes along the length of the villi. NBAT was also found in the cytoplasm. Surprisingly, EAAC1 was located exclusively in the microvilli of enterocytes in the crypt and the bases of the villi. These protein expression patterns were similar in all parts of the small intestine (proximal, median and distal), at all ages. We conclude that the expression of PepT1, NBAT or EAAC1 are differently regulated according to both the horizontal and vertical axes.

KEY WORDS: • ontogenesis • amino acid transporters • small intestine • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The small intestine of animals from altricial species such as rat, mouse or rabbit is not mature at the time of birth. In rats, morphogenesis is not complete until wk 3 after birth, when profound changes occur, including increases in intestinal weight, villous and crypt height, cell migration rate, RNA and DNA content (1 ), and the adaptation of intestinal enzymatic activity (2 ). Although the consequences of postnatal development of the small intestine on nutrient absorption are now recognized (3 ), little is known about the ontogenesis of amino acid and peptide transport and transporters. The aim of this study was to describe the postnatal maturation patterns of three amino acid and peptide transporters in rat small intestine, namely PepT1, NBAT and EAAC1. EAAC1 is responsible for the uptake of glutamate, a major metabolic fuel of the enterocyte through the Na⁺-dependent transport system X_A,G- (4 ). NBAT represents the heavy chain of a heterodimeric transporter involved in the heteroexchange of cationic and neutral amino acids corresponding to the b^0,+ transport system (5 ). PepT1 was identified as the protein-mediating H⁺-peptide cotransport in the brush border membrane of intestinal epithelial cells (6 ). The expression of mRNA coding for these transporters was measured, and the distribution of these proteins along the crypt-villus axis was studied from birth to adulthood. Because the intestinal absorption of nutrients is known to vary along the proximodistal axis, we considered the evolution of these transporters in the proximal, median and distal parts of the small intestine.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Maintenance and dissection of animals.

Outbred female Sprague-Dawley rats with litters of 11 pups were used throughout this study (Iffa Credo, L’Arbresle, France) according to the guidelines of the French National Animal Care Committee. The litter was kept with the dam until postnatal day 50 (d 50) and began to nibble solid food at d 21 (maintenance rat diet A04, U.A.R, France: 12.1 kJ/g; 83.9% cereal and cereal products, 4.1% vitamin and mineral mixture, 4% animal proteins and 8% vegetable proteins). Rats were killed by chloroform asphyxiation in random order without regard to sex on d 4, 8, 10, 12, 21, 30 and 50. The small intestine was rapidly removed and divided into three segments of equal length, i.e., proximal, median and distal parts. At the distal part of each segment, a portion of 0.5 cm was removed for immunohistochemistry.

RNA analysis.

Because it was not possible to scrape the small intestine, the entire mucosa was used for all experiments. Intestinal segments were finely crushed in liquid nitrogen and resuspended in a denaturing solution (4 mol/L guanidinium thiocyanate, 25 mmol/L sodium citrate, pH 7, 5 g/L sarcosyl) for mRNA extraction according to Chomczynski et al. (7 ). After RNA integrity had been confirmed by ethidium bromide staining, first strand cDNAs were synthesized from 2 µg of total RNA using oligo(dT)_12–18 as primers in the presence of MML-V reverse transcriptase (RT)³ (Invitrogen, Cergy Pontoise, France), for 1 h at 37°C; 5 µL of each RT product was used for polymerase chain reaction (PCR) (50 µL final reaction volume) with primers chosen from the conserved part of the coding regions for EAAC1 [forward primer (P1): 5'-GGGAAGATCATAGAAGTTGAA-3', reverse primer (P2): 5'-TGAACCGGTCCAGGAGCCAGT-3'], NBAT (P1: 5'-CCTCGGGAGATCCTCTTCTGG-3', P2: 5'-TGCCAGCTGGAGTTTCCATACAC-3') and PepT1 (P1: 5'-GGACTGGGCTAAAGAGAAATACG-3', P2: 5'-GAATCTGATTCCGTTCTCCC-3'). Amplification was performed using a MJ Research PTC-200 Thermocycler (Merck-Eurolab, Fontenay sous Bois, France) for 35 cycles, which consisted of denaturation (95°C, 45 s), annealing (56°C, 45 s) and extension (72°C, 1 min) with Taq DNA Polymerase (Invitrogen, Cergy Pontoise, France). To enable semiquantitative analysis, the housekeeping gene ß-actin was also amplified (P1: 5'-TGGAATCCTGTGGCATCCATGAAA-3', P2: 5'-TAAAACGCAGCTCAGTAACAGTCCG-3') (8 ). ß-Actin (473 bp) and EAAC1 (539 bp), NBAT (542 bp) or PepT1 (640 bp) PCR products were separated by electrophoresis through a 2% agarose gel, stained with ethidium bromide and quantified using a digital imaging system (Alpha Innotech Corporation, San Leandro, CA). All PCR products were verified by sequencing (Genset, Paris, France).

Immunohistochemistry.

Fragments collected at the distal part of each proximal, median and distal segment of freshly dissected small intestine were fixed in alcohol formalin acetic acid fixative (ethyl alcohol/formol/acetic acid (water, 75/2/5/8, v/v/v/v) for 4 h at room temperature. Paraffin sections (4 µm) were first autoclaved at 120°C for 40 min in target retrieval buffer (10 mmol/L citrate buffer, pH 6) for demasking, then rinsed with 150 mmol/L, pH 7.2 PBS. Affinity-purified rabbit anti-rat PepT1 and anti-rat NBAT antibodies were produced against two synthetic peptides, VGKENPYSSLEPVSQTNM for PepT1 (9 ) and NSDYHTVNVDVQK for NBAT (10 ) (Agro Bio, La Ferté St Aubin, France). After endogenous peroxidases were blocked, the sections were incubated overnight either at 4°C with rabbit anti-rat EAAC1 antibodies (10 mg/L; EAAC11A, Alpha Diagnostic International, San Antonio, TX) (11 ) or at room temperature with anti-rat PepT1 antibodies (5 mg/L). For NBAT detection, sections were incubated for 24 h at room temperature with anti-rat NBAT antibodies (5 mg/L). All sections were then incubated with a goat anti-rabbit immunoglobulin G coupled with a dextran-peroxidase complex (EnVision Kit, DAKO, Trappes, France). Detection was performed using 3,3'-diaminobenzidine tetrahydrochloride. Rabbit preimmune serum was used instead of the specific antibodies as a negative control.

Statistical analyses.

Data are given as means ± SD. Comparisons between the different days and intestinal segments were performed using two-way ANOVA and Fisher’s test (StatView). Differences with P < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Quantification of PepT1, NBAT and EAAC1 mRNA along the rat small intestine from postnatal d 4 to 50.

PepT1, NBAT and EAAC1 mRNA were detected all along the small intestine, from d 4 to 50 (Fig. 1 ). Each of these transporters showed a specific pattern of distribution. PepT1 mRNA was evenly distributed from the proximal to the distal small intestine. The level of NBAT mRNA showed an oral to aboral gradient with a higher expression in the proximal than in the median small intestine on d 4 and 10 (P < 0.05). The NBAT mRNA level tended to be higher (P < 0.06) in the proximal than in the distal small intestine on d 4, 10 and 21. In comparison, EAAC1 mRNA exhibited an inverse gradient, with higher expression in the distal small intestine compared with the proximal on d 10 (P < 0.05) and with the median on d 12 and 30 (P < 0.05). The absence of significant gradient at the other ages may have resulted from the high standard deviations related to the outbred characteristic of the rat strain. PepT1, NBAT and EAAC1 distribution patterns remained the same from birth to adulthood.

View larger version (45K): [in this window] [in a new window]   Figure 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of PepT1, NBAT and EAAC1 expression in rat small intestine from d 4 to 50. Results are the means ± SD of 3 rats from different litters. L, DNA ladder. *P < 0.05; P < 0.05 compared with d 21; P < 0.05 compared with d 30.

Immunohistochemical localization of PEPT1, NBAT and EAAC1 proteins along the crypt-villus axis of rat small intestine.

Staining for PEPT1 was detected from d 4 to 50 on the brush border of epithelial cells in the villi (Fig. 2 ) with a gradient from the crypt-villus junction toward the tips of villi. No labeling was observed in crypt cells. NBAT immunoreactivity was observed in villi from base to tip from d 4 to 50 (Fig. 2) . Staining was observed mainly in the brush border membrane of epithelial cells, but a light staining was also detected in their cytoplasm. No NBAT immunoreactivity was seen in crypt cells. EAAC1 immunoreactivity was restricted to the brush border membrane of epithelial cells located at the intervillous space at d 4 and of those lining the crypt on d 21 and 50 (Fig. 2) . No labeling was observed in the upper two thirds of the villi between d 4 and 50. No difference in the distribution of PEPT1, NBAT and EAAC1 immunoreactivities along the crypt-villus axis were detected in the proximal, median and distal parts of the small intestine.

View larger version (142K): [in this window] [in a new window]   Figure 2. Immunolocalization of PEPT1, NBAT and EAAC1 in the rat distal small intestine at d 4, 21 and 50. Arrows indicate specific staining. Slight residual peroxidase activity could be detected in the lamina propria (L) of some sections, but did not interfere with the observation of specific staining. This figure is representative of three independent experiments. Similar staining was observed in the proximal and median parts of the small intestine and on d 8, 10 and 12 (not shown). C, crypt; IV, intervillous space; E, enterocytes. Original magnifications: X64 (except for PEPT1 at d 50: X33, and EAAC1 at d 4 and 21: X132).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Although mRNA coding for PepT1 was found equally in the three parts of the intestine, levels of NBAT mRNA were higher in the proximal part than in the median and distal parts. Cationic amino acids have a high digestibility (12 ) and the distribution of NBAT may thus parallel those of its substrates. However, it should be noted that the amino acid system b^0,+ consists of two proteins (NBAT and b^0,+ AT) encoded by two different genes; thus, the expression of NBAT described in this study may not necessarily correlate with that of the functional b^0,+ system. In contrast to PepT1 and NBAT, EAAC1 mRNA exhibited a strong gradient from the proximal to the distal part. Glutamate, glutamine and glucose are the preferred energy sources for rapidly dividing enterocytes in the small intestine. There is substantial evidence that glutamine and glucose transport and oxidation are not constant along the length of the intestinal tract (13 ,14 ). Glutamate may therefore be the principal fuel for enterocytes in the distal small intestine, compensating for the weak absorption and use of glutamine and glucose in this area.

PEPT1, NBAT and EAAC1 exhibited the same patterns from postnatal d 4 to 50. Although crypts were not yet formed on d 4, the unambiguous localization of each transporter suggests that a clear gradient in epithelial cell function already exists from the bottom of the villi to the top. A marked crypt-villus gradient was observed for PEPT1 immunoreactivity, which is in accordance with previous reports (15 ), but also for NBAT immunoreactivity. Unexpectedly, the crypt-villus distribution of EAAC1 differed completely from those of the two other transporters. Using affinity-purified antibodies described by Rosthein et al. (11 ), EAAC1 immunoreactivity was observed in the microvilli of enterocytes confined to the lower third of the villi and to the crypts. To our knowledge, this is the first report of an amino acid transporter with decreased expression during epithelial cell differentiation in vivo. Our results suggest that the principal function of EAAC1 may be to meet the anabolic requirements of rapidly proliferating epithelial tissue rather than to provide the body with dietary glutamate.

In conclusion, our results provide the first demonstration that EAAC1 and NBAT expression is strong during the first days after birth. The regionalization of PepT1, NBAT and EAAC1 mRNAs along the entire small intestine and the localization of corresponding proteins along the crypt-villus axis are the same in newborn and adult rats. Conversely, PepT1 and EAAC1 mRNA levels vary as a function of age.

	FOOTNOTES

 
¹ Sophie Rome and Laurence Barbot contributed equally to this work.

³ Abbreviations used: P1, forward primer; P2, reverse primer; PCR, polymerase chain reaction; RT, reverse transcriptase.

Manuscript received 21 November 2001. Initial review completed 4 January 2002. Revision accepted 21 February 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Buts, J. P. & Meyer, R. (1981) Postnatal proximodistal development of the small bowel mucosal mass in growing rats. Biol. Neonate 40:62-69.[Medline]

2. Henning, S. J. (1981) Postnatal development: coordination of feeding, digestion, and metabolism, Am. J. Physiol. 241:G199-G214.

3. Pacha, J. (2000) Development of intestinal transport function in mammals. Physiol. Rev. 80:1633-1667.[Abstract/Free Full Text]

4. Kanai, Y. & Hediger, M. A. (1992) Primary structure and functional characterization of a high-affinity glutamate transporter. Nature (Lond.) 360:467-471.[Medline]

5. Tate, S. S., Yan, N. & Udenfriend, S. (1992) Expression cloning of a Na⁺-independent neutral amino acid transporter from rat kidney. Proc. Natl. Acad. Sci. U.S.A. 89:1-5.[Abstract/Free Full Text]

6. Fei, Y. J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F. H., Romero, M. F., Singh, K. & Borons, W. F. (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature (Lond.) 368:563-366.[Medline]

7. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159.[Medline]

8. Jean, C., Rome, S., Mathé, V., Huneau, J. F., Aattouri, N., Fromentin, G., Larue-Achagiotis, C. & Tomé, D. (2001) Metabolic evidence for adaptations to a high protein diet in rats. J. Nutr. 131:91-98.[Abstract/Free Full Text]

9. Tanaka, H., Miyamoto, K. I., Morita, K., Haga, H., Segawa, H., Shiraga, T., Fujioka, A., Kouda, T., Taketani, Y., Hisano, S., Fukui, Y., Kitagawa, K. & Takeda, E. (1998) Regulation of the PepT1 peptide transporter in the rat small intestine in response to 5-fluorouracil-induced injury. Gastroenterology 114:714-723.[Medline]

10. Mosckovitz, R., Udenfriend, S., Felix, A., Heimer, E. & Tate, S. S. (1994) Membrane topology of the rat kidney neutral and basic amino acid transporter. FASEB J 8:1069-1074.[Abstract]

11. Rothstein, J. D., Martin, L., Levey, A. I., Dykes-Hoberg, M., Jin, L., Wu, D., Nash, N. & Kuncl, R. W. (1994) Localization of neuronal and glial glutamate transporters. Neuron 13:713-725.[Medline]

12. Baglieri, A., Mahé, S., Benamouzig, R., Savoie, L. & Tomé, D. (1995) Digestion patterns of endogenous and different exogenous proteins affect the composition of intestinal effluents in humans. J. Nutr. 125:1894-1903.

13. Fleming, S. E. & Kight, C. E. (1994) The TCA cycle as an oxidative and synthetic pathway is suppressed with aging in jejunal epithelial cells. Can. J. Physiol. Pharmacol. 72:266-274.[Medline]

14. Yoshida, A., Takata, K., Kasahara, T., Aoyagi, T., Saito, S. & Hirano, H. (1995) Immunohistochemical localization of Na⁺-dependent glucose transporter in the rat digestive tract. Histochem. J. 27:420-426.[Medline]

15. Ogihara, H., Suzuki, T., Nagamachi, Y., Inui, K. I. & Takata, K. (1999) Peptide transporter in the rat small intestine: ultrastructural localization and the effect of starvation and administration of amino acids. Histochem. J. 31:169-174.[Medline]

This article has been cited by other articles:

				E. R. Gilbert, H. Li, D. A. Emmerson, K. E. Webb Jr., and E. A. Wong Developmental Regulation of Nutrient Transporter and Enzyme mRNA Abundance in the Small Intestine of Broilers Poult. Sci., August 1, 2007; 86(8): 1739 - 1753. [Abstract] [Full Text] [PDF]

				D. T. Thwaites and C. M. H. Anderson H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine Exp Physiol, July 1, 2007; 92(4): 603 - 619. [Abstract] [Full Text] [PDF]

				P. Marquet, L. Barbot, A. Plante, J. F. Huneau, J. G. Gobert, and N. Kapel Cryptosporidiosis Induces a Transient Upregulation of the Oligopeptides Transporter (PepT1) Activity in Neonatal Rats Experimental Biology and Medicine, March 1, 2007; 232(3): 454 - 460. [Abstract] [Full Text] [PDF]

				E. Fernandez, D. Torrents, A. Zorzano, M. Palacin, and J. Chillaron Identification and Functional Characterization of a Novel Low Affinity Aromatic-preferring Amino Acid Transporter (arpAT): ONE OF THE FEW PROTEINS SILENCED DURING PRIMATE EVOLUTION J. Biol. Chem., May 13, 2005; 280(19): 19364 - 19372. [Abstract] [Full Text] [PDF]

				A. Howard, R. A. Goodlad, J. R. F. Walters, D. Ford, and B. H. Hirst Increased Expression of Specific Intestinal Amino Acid and Peptide Transporter mRNA in Rats Fed by TPN Is Reversed by GLP-2 J. Nutr., November 1, 2004; 134(11): 2957 - 2964. [Abstract] [Full Text] [PDF]

				M. Z. Fan, J. C. Matthews, N. M. P. Etienne, B. Stoll, D. Lackeyram, and D. G. Burrin Expression of apical membrane L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G385 - G398. [Abstract] [Full Text] [PDF]

				S. A. Adibi Regulation of expression of the intestinal oligopeptide transporter (Pept-1) in health and disease Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G779 - G788. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rome, S. Articles by Tomé, D. Search for Related Content PubMed PubMed Citation Articles by Rome, S. Articles by Tomé, D.