Chapter III Ontogenesis of peptide transport and morphological changes in the ovine gastrointestinal tract
Nutrient absorption is important in all stages of life. As the diet of an animal changes from birth on, morphological and biochemical adaptation can be anticipated in order to accommodate changing demands. The main focus of the present study was to examine the relationship between age and diet on the potential for peptide transport via PepT1 in the gastrointestinal tract of lambs and to relate changes of peptide transport capability to morphological changes. A 2×4 factorial arrangement of treatments was used with 32 crossbred lambs. Four blocks were created based upon gender, birth type (single or twin), birth weight, and birth date. Lambs were randomly allotted at birth to receive or not to receive a creep diet. All lambs were allowed to nurse. Sampling times of 2, 4, 6, or 8 wk were randomly allotted to lambs. Samples for RNA extraction and histological evaluation were taken from the dorsal rumen, ventral rumen, omasum, duodenum, jejunum, and ileum. Villi were about 7% shorter (P < 0.09) in lambs receiving creep feed. Papillary height and width increased linearly (P < 0.001 and P < 0.0001, respectively) with age. Total and keratinized epithelial cells in the stomach decreased (P 03 and P < 0.004, respectively) with age and were fewer (P < 0.0002 and P < 0.0001, respectively) in lambs receiving creep feed. Creep feeding appears to have slightly altered the mucosal structure of the small intestine and it was advantageous in that it stimulated papillary growth and thus predisposed the rumen for the introduction of feed into the diet. A 2.8 kb oPepT1 mRNA was present in all tissues studied by 2 wk and age did not significantly influence the abundance of oPepT1 mRNA in the small intestine or stomach. In the small intestine, abundance of oPepT1 mRNA was greatest (P < 0.0007) in the jejunum. In the stomach, abundance of oPepT1 mRNA was greatest (P < 0.01) in the dorsal rumen. In the stomach, particularly in the rumen, a greater abundance of oPepT1 mRNA was observed in lambs not receiving the creep diet. It seems likely that a stimulus for development is coming from the non-luminal direction, possibly blood-borne, and may be involved in the ontogenesis of oPepT1. Peptide transport appears to be a physiologically important process in the young lamb and the rumen appears to be involved in the transport of peptides, particularly in lambs that only nurse
The PepT1 transports di- and tripeptides as well as some peptidomemetics such as β-lactam antibiotics and angiotensin converting enzyme (ACE) inhibitors (Daniel, 1996). The PepT1 has 12 transmembrane domains with a large extracellular loop between transmembrane domains nine and ten (Fei et al., 1994). Data from our laboratory indicated the presence of peptide transport protein(s) in the forestomach of sheep (Matthews et al., 1996; Pan et al., 1997). Peptide transport protein one (PepT1) was observed to be present in the rumen, omasum, and small intestine of sheep (Chen et al., 1999).
Data on the ontogenesis of peptide transport indicate that the young may have a greater ability to transport peptides than older animals. Influxes of radiolabelled glycyl-L-proline and glycyl-L-phenylalanine were reported to be greater in suckling rabbits than in adults (Rubino and Guandalino, 1977). Using isolated, everted intestinal segments Himukai et al. (1980) reported glycylglycine and glycyl-L-leucine influxes were greatest in sucklings, intermediate in weanlings, and least in adult guinea pigs. A decrease in maximal transport capacity between the suckling and adult guinea pigs (Vmax; 50 and 20 nmoles·cm-2·min-1, respectively) was attributed to the age-dependent difference observed.
Molecular data on the ontogenesis of PepT1 in laboratory animal species indicate greater mRNA abundance and protein expression during the early postnatal period (Miyamoto, 1996; Shen et al., 2001). Dietary regulation has been implicated as an important factor in the intestinal transport of peptides (Ferraris et al., 1988; Erickson et al., 1995; Shiraga et al., 1999). Therefore, the focus of the present study was to examine the relationship between age and diet on the potential for peptide transport via PepT1 in the gastrointestinal tract of lambs. It was also of interest to relate changes of peptide transport capability to morphological changes
Materials and Methods
Materials. All chemicals used were of enzyme, molecular biology, or histological grade. Diethyl pyrocarbonate (DEPC), EDTA, sodium acetate, formaldehyde, chloroform, and isopropanol were purchased from Sigma (St. Louis, MO). Sodium chloride and MOPS were purchased from Fisher Scientific (Fair Lawn, NJ). RNASIN Ribonuclease Inhibitor was purchased from Promega (Madison, WI). Restriction enzymes were purchased from New England Biolabs (Beverly, MA). Nylon membranes used were from MSI (Westboro, MA). The [∝-32P]dATP was purchased from ICN Pharmaceuticals (Costa Mesa, CA). DNA polymerase I/DnaseI and the RNA ladder (0.24 – 9.5 Kb) were from Gibco Life Technologies (Gaithersburg, MD). TriReagent was purchased from MRC (Cincinnati, OH).
Experimental Design. A 2 x 4 factorial arrangement of treatments was used with 32 crossbred lambs. Four blocks were created based upon gender, birth type (single or twin), birth weight, and birth date. Lambs were randomly allotted at birth to receive or not to receive a creep diet and all lambs were allowed to nurse. Sampling times of either 2, 4, 6, or 8 wk were randomly allotted to lambs at this time. Ewes and their lamb(s) were placed in their assigned pens on the third day following the birth of the lamb(s). There were 10 sets of twins and 12 singles. Twin lambs were assigned to receive the same diet, but were assigned to different sampling times. Numbers of twins and singles were equalized among treatments.
Animal Management and Diets. Ewes and their lambs were housed in four pens on raised wire mesh flooring. The pens were divided by a barrier that allowed only the lambs access to a portion of the pen. Creep feed was provided in this portion of the pen for the lambs receiving the creep diet. This area was also gated, thus allowing the separation of ewes and lambs while the ewes were being fed.
Diets were formulated to meet NRC requirements (NRC, 1985). Ingredient and chemical composition are presented in Tables 1 and 2, respectively. Diets were analyzed for dry matter (AOAC, 1990), N (Kjeldahl; AOAC, 1990), NDF (VanSoest and Wine, 1967; Goering and VanSoest, 1970), and ADF (VanSoest, 1963; Goering and VanSoest, 1970).
Ewes were fed, based upon average weight (62 kg), to meet requirements for lactating ewes during the first 6 to 8 wk of lactation with suckling twins (NRC, 1985). Ewes were fed at 0630 and 1530 and were allowed 3-h to eat at each feeding. Lambs were fed at 1530 and allowed 24-h access to the creep feed. Refused feed was removed from the bunk, weighed, and recorded after each feeding period.
Tissue Sampling. Final weights of the lambs were obtained and recorded prior to sampling. Lambs were stunned by a captive bolt pistol and killed by exsanguination. The abdomen was opened immediately and the gastrointestinal tract was removed from the caudal end of the esophagus to the ileo-cecal junction. Samples for RNA extraction and histological evaluation were taken from the dorsal rumen, ventral rumen, omasum, duodenum, mid-jejunum, and terminal ileum. Intestinal segments were cut open longitudinally and all tissues were washed in ice-cold 0.9% saline. For RNA extraction, intestinal epithelium was collected by scraping the mucosal surface with a glass slide.
Samples were then wrapped in aluminum foil and frozen in liquid N2. Whole tissue sections of the dorsal and ventral rumen and omasal plies were collected, wrapped in aluminum foil, and frozen in liquid N2. All samples were stored at –80°C until further analysis. For histological evaluation, approximately 1 cm2 sections of tissue were cut and mounted serosal side down on cardboard. In the case of omasal plies, one of the epithelial surfaces was mounted against the cardboard. Samples were fixed in 10% phosphate buffered formalin. Each sample was trimmed, placed in a tissue cassette, processed, and embedded in paraffin wax by routine methodology (Bancroft and Stevens, 1990). Sections (4 µm) were cut in duplicate, mounted on glass slides, and stained with haematoxylin and eosin (Bancroft and Stevens, 1990) for analysis by light microscopy.
Morphometric Measurements. Digital images were obtained at 10X with a Polaroid Digital Microscope Camera, DMCIe (Polaroid, Cambridge, MA). Measurements were made using Sigmascan Pro 5.0 (SPSS, Inc., Chicago, IL). For the duodenum and jejunum, ten intact, well oriented, and transversely cut villi and crypts were measured per animal. Measurements of villus height, villus width, and crypt depth were made as previously described (Kik et al., 1990). Villus width was defined as the distance from the outside epithelial edge to the outside of the opposite epithelial edge determined at mid-height of the villus. Ratios were calculated for villus height:crypt depth and villus height:villus width.
For ruminal tissues, ten intact, well oriented, and transversely cut papillae were measured per animal from both the dorsal and ventral rumen. Papillary height was defined as the distance from the tip of the papillae to the base of the papillae. Papillary width was defined as the distance from the outside epithelial edge to the opposite outside epithelial edge determined at mid-height of the papillae. The ratio of papillary height:papillary width was calculated. Total epithelial cells, keratinized epithelial cells, and nonkeratinized epithelial cells were determined at 400X. Keratin stains red in haematoxylin and eosin stain, thus allowing for the differentiation of keratinized and nonkeratinized epithelial cells (Bancroft and Stevens, 1990). The ratio of keratinized epithelial cells to nonkeratinized epithelial cells was calculated.
For omasal tissues, total epithelial cells, keratinized epithelial cells, and nonkeratinized epithelial cells were determined at 400X for ten papillae per animal. Keratinized cells were differentiated in the same manner as was performed in the rumen. The ratio of keratinized to nonkeratinized epithelial cell thickness was also determined.
Preparation of Total RNA. Total RNA was extracted from the duodenum, jejunum, and ileum, as well as the dorsal and ventral rumen using TriReagent. Total RNA was recovered per the manufacturer’s protocol. Briefly, tissue samples of approximately 0.25 g were homogenized in 3 mL of TriReagent. Next, 200 µL chloroform were added to each homogenate and mixed well. Mixtures were centrifuged at 12,000 x g for 15 min at 4°C and the aqueous layer was transferred to a new tube. This was followed by the addition of 10U of RNASIN Ribonuclease Inhibitor and an equal volume of 100% isopropanol to each tube. All samples were then placed at -80°C for 2-Tubes were then centrifuged at 12,000 x g for 15 min at 4° Pellets were washed once in DEPC treated 70% ethanol and centrifuged at 12,000 x g for 15 min at 4°C. Total RNA was then recovered by dissolving pellets in 10 mM Tris-HCl (pH 7.6), 1mM EDTA (DEPC-treated). RNA content and purity was determined by measuring absorption at 260 and 280 nm. Samples were stored at -80°C.
Abundance of oPepT1 mRNA. Twenty micrograms of total RNA were electrophoresed through a 1% agarose gel containing 2.2 M formaldehyde and gels were stained with 0.05 mg/mL ethidium bromide. The RNA was transferred to a nylon membrane by downward capillary transfer with 10 mM NaOH, 5X SSC (3 M NaCl, 0.3 M sodium-citrate pH 7.0) and crosslinked with UV light for 30 s. Membranes were probed with oPepT1 cDNA (Pan et al., 2001). One hundred nanograms of probe were labeled with [∝-32P]dATP by nick translation and purified using Sephadex G-50 spin column chromatography. Prehybridization was conducted for 2-to 2.5-h at 42°C in a solution containing 50% deionized formamide, 5X Denhardt’s solution, 6X saline-sodium phosphate-EDTA (SSPE), 0.2% sodium lauryl sulfate (SDS), and 10 µg/mL tRNA. Hybridization was conducted at 42°C overnight. Following hybridization, membranes were washed: twice at room temperature for 30 min in 5X SSPE, 0.5% SDS; twice at 42°C for 30 min in 1X SSPE, 0.5% SDS; and twice at 65°C for 30 min in 0.1X SSPE and 1% SDS. Membranes were exposed to Kodak XAR-5 film at -80°C. Next, membranes were stripped in a solution containing 50% deionized formamide, and 6X SSPE for 30 min at 65°C and washed twice in 2X SSPE. Membranes were then probed with 18S rRNA as an internal control using the same hybridization conditions as were used for oPepT1
Chapter I. Introduction
Chapter II. Literature Review
Cloning of Mammalian Peptide Transporters
Tissue Distribution of PepT1
Ruminal Histology and Papillary Development
Omasal Histology and Laminae Development
Small Intestinal Histology and Mucosal Development
Ontogenesis of Nutrient Transport .
Chapter III. Ontogenesis of Peptide Transport and Morphological Changes. in the Ovine Gastrointestinal Tract
Materials and Methods
Animal Management and Diets.
Preparation of Total RNA.
Abundance of oPepT1 mRNA
Chapter IV. Epilogue.
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