Rabbit SLC15A1, SLC7A1 and SLC1A1 genes are affected by site of digestion,stage of development and dietary protein content

Peptide transporter 1 (SLC15A1, PepT1), excitatory amino acid transporter 3 (SLC1A1, EAAT3) and cationic amino acid transporter 1 (SLC7A1, CAT1) were identified as genes responsible for the transport of small peptides and amino acids. The tissue expression pattern of rabbit (SLC15A1, SLC7A1 and SLC1A1) across the digestive tract remains unclear. The present study investigated SLC15A1, SLC7A1 and SLC1A1 gene expression patterns across the digestive tract at different stages of development and in response to dietary protein levels. Real time-PCR results indicated that SLC15A1, SLC7A1 and SLC1A1 genes throughout the rabbits’ entire development and were expressed in all tested rabbit digestive sites, including the stomach, duodenum, jejunum, ileum, colon and cecum. Furthermore, SLC7A1 and SLC1A1 mRNA expression occurred in a tissue-specific and time-associated manner, suggesting the distinct transport ability of amino acids in different tissues and at different developmental stages. The most highly expressed levels of all three genes were in the duodenum, ileum and jejunum in all developmental stages. All increased after lactation. With increased dietary protein levels, SLC7A1 mRNA levels in small intestine and SLC1A1 mRNA levels in duodenum and ileum exhibited a significant decreasing trend. Moreover, rabbits fed a normal level of protein had the highest levels of SLC15A1 mRNA in the duodenum and jejunum (P<0.05). In conclusion, gene mRNA differed across sites and with development suggesting time and sites related differences in peptide and amino acid absorption in rabbits. The effects of dietary protein on expression of the three genes were also site specific.     

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.     

Peptide Transporter in the Rat Small Intestine: Ultrastructural Localization and the Effect of Starvation and Administration of Amino Acids

Peptide transporter-1 is a H+/peptide cotransporter responsible for the uptake of small peptides and peptide-like drugs, and is present in the absorptive epithelial cells of the villi in the small intestine (duodenum, jejunum, and ileum). It has been localized to the apical microvillous plasma membrane of the absorptive epithelial cells of the rat small intestine using the immunogold electron microscopic technique. Digital image analysis of the jejunum revealed that the transporter protein was abundant at the tip of the villus and that the amount decreased from the tip of the villus to its base. The effect of dietary administration of amino acids and starvation on the expression of PepT1 in the jejunum was examined by immunoblotting and image analysis of immunofluorescence. Starvation markedly increased the amount of peptide transporter present, whereas dietary administration of amino acids reduced it. The gradient of the transporter protein along the crypt-villus axis was maintained under either condition. These observations show that it is specific to the microvillous plasma membrane and that its expression is regulated by the nutritional condition.     

Sequence,tissue distribution and developmental changes in rat intestinal oligopeptide transporter

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day-old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.     

Characterization and Regulation of the Amino Acid Transporter SNAT2 in the Small Intestine of Piglets

The sodium-dependent neutral amino acid transporter 2 (SNAT2), which has dual transport/receptor functions, is well documented in eukaryotes and some mammalian systems, but has not yet been verified in piglets. The objective of this study was to investigate the characteristics and regulation of SNAT2 in the small intestine of piglets. The 1,521-bp porcine full cDNA sequence of SNAT2 (KC769999) from the small intestine of piglets was cloned. The open reading frame of cDNA encodes 506 deduced amino acid residues with a calculated molecular mass of 56.08 kDa and an isoelectric point (pI) of 7.16. Sequence alignment and phylogenetic analysis revealed that SNAT2 is highly evolutionarily conserved in mammals. SNAT2 mRNA can be detected in the duodenum, jejunum and ileum by real-time quantitative PCR. During the suckling period from days 1 to 21, the duodenum had the highest abundance of SNAT2 mRNA among the three segments of the small intestine. There was a significant decrease in the expression of SNAT2 mRNA in the duodenal and jejunal mucosa and in the expression of SNAT2 protein in the jejunal and ileal mucosa on day 1 after weaning (P < 0.05). Studies with enterocytes in vitro showed that amino acid starvation and supplementation with glutamate, arginine or leucine enhanced, while supplementation with glutamine reduced, SNAT2 mRNA expression (P < 0.05). These results regarding the characteristics and regulation of SNAT2 should help to provide some information to further clarify its roles in the absorption of amino acids and signal transduction in the porcine small intestine.     

Effects of glucocorticoids on the gene expression of nutrient transporters in different rabbit intestinal segments

Glucocorticoids (GCs) are counterregulatory hormones with broad effects on the digestion and absorption of dietary carbohydrates, lipids and proteins, but the underlying molecular mechanisms of these effects remain unclear. The present experiment was conducted to investigate the main expression sites of nutrient transporters and the effects of GCs on the gene expression of these transporters in the rabbit small intestine. The results showed that peptide transporter 1 (PepT1), facultative amino acid transporter (rBAT), neutral amino acid transporter (B⁰AT), excitatory amino acid transporter 3 (EAAT3), sodium-glucose transporter 1 (SGLT1) and glucose transporter 5 (GLUT5) were mainly expressed in the distal segment, glucose transporter 2 (GLUT2) and fatty-acid-binding protein 4 (FATP4) were mainly expressed in the proximal segment and cationic amino acid transporter 1 (CAT1) was mainly expressed in the middle segment of the rabbit small intestine. In addition, we analysed the effects of 3 h (short-term) or 7 days (long-term) dexamethasone (DEX) treatment on the gene expression of most nutrient transporters. The results showed that short-term DEX treatment significantly decreased PepT1, B⁰AT, EAAT3, rBAT and SGLT1 expressions in all small intestinal segments, while it significantly decreased GLUT2 in the duodenum and FATP4 in the duodenum and ileum (P < 0.05). Long-term DEX treatment also significantly decreased PepT1, CAT1, B⁰AT, EAAT3, rBAT and SGLT1 in all small intestinal segments and significantly decreased GLUT2 in the jejunum and FATP4 in the ileum (P < 0.05). In conclusion, DEX could decrease the gene expression of most nutrient transporters (except GLUT5) and affect the transport of intestinal amino acids, monosaccharides and fatty acids.     

Enrofloxacin and Probiotic Lactobacilli Influence PepT1 and LEAP-2 mRNA Expression in Poultry

Expression of peptide transporter 1 (PepT1) and liver-expressed antimicrobial peptide 2 (LEAP-2) in chickens can be influenced by food deprivation, pathological conditions and drug administration. Effect of three putative probiotic Lactobacillus strains and enrofloxacin on the expression of PepT1 and LEAP-2 mRNA was investigated in Ross 308 chickens. One-day-old chicks (n = 24) were allocated to following groups: control (without treatment); group treated with probiotics via feed; group treated with a combination of probiotics and enrofloxacin; and a group given enrofloxacin only. The drug was administered at a dose of 10 mg kg^?1, via drinking water for 5 days. Samples from liver, duodenum and jejunum were collected 126 h after the start of the treatment. Expression levels of PepT1 and LEAP-2 were determined by real-time polymerase chain reaction and were statistically evaluated by Mann–Whitney test. Enrofloxacin administered alone or in combination with probiotics provoked a statistically significant up-regulation of PepT1 mRNA levels in the measured organ sites. These changes can be attributed to a tendency of improvement in utilization of dietary peptide and in body weight gain. LEAP-2 mRNA expression levels did not change significantly in enrofloxacin-treated chickens in comparison with control group.     

Cloning and expression analysis of an ovine PAP-like protein cDNA, a gene differentially expressed in scrapie

In a previous study, the mRNA level of a pancreatitis-associated protein (PAP)-like protein was found to be elevated in the ileal Peyer's patch of lambs during the early phase of scrapie infection. Here, we report the isolation of the ovine PAP-like protein cDNA which encodes a putative 178 amino acid protein with a signal peptide and a C-lectin binding domain. Comparisons of REG/PAP proteins between various species showed that the deduced amino acid sequences were conserved. The overall amino acid identity between the ovine PAP-like protein and bovine, human and rat REG/PAP proteins varied from 23% to 85%. In Northern blot analysis the expression of the ovine PAP-like protein mRNA was restricted to the ileal and jejunal Peyer's patches. The cellular expression of the PAP-like protein mRNA in the ovine intestine was further characterized by in situ hybridization. PAP-like protein mRNA was detected in cells of the epithelial lining in most crypts and in some intestinal villi in the ileum and jejunum while in the colon and rectum, the PAP-like protein mRNA expression was only detected in the deep portion of a few crypts. The data provided will offer the possibility to search for a link between this PAP-like protein and early events in the development of scrapie.     

Small intestinal morphology and sugar transporters expression when consuming diets of different energy levels: comparison between Tibetan and small-tailed Han sheep

Some non-structural carbohydrates, especially starch, escape ruminal fermentation, are converted into glucose, and are absorbed from the small intestine. This glucose provides an important source of energy, and its usage is more efficient than glucose from carbohydrates which are fermented as short chain fatty acids in the rumen and, subsequently, undergo hepatic gluconeogenesis. Tibetan sheep graze on the harsh Qinghai-Tibetan Plateau (QTP) all year round and their carbohydrate and energy intakes fluctuate greatly with seasonal forage availability. Consequently, a high capacity to absorb glucose from the small intestine would be particularly beneficial for Tibetan sheep to allow them to cope with the inconsistent dietary intakes. This study examined how the small intestinal morphology and sugar transporters’ expression of Tibetan and Small-tailed Han (Han) sheep respond to fluctuating energy intakes under the harsh conditions of the QTP. Han sheep graze on the QTP only in summer and are generally raised in feedlots. Twenty-four Tibetan sheep and 24 Han sheep, all wethers, were assigned randomly to four groups (n = 6 per breed/group), with each group offered a diet differing in digestible energy content: 8.21, 9.33, 10.45 and 11.57 MJ/kg DM. After 49 d, all sheep were slaughtered, tissues of the small intestine were collected, and measurements were made of the morphology and glucose transporters and the related regulation gene expressions. At intakes of low energy levels, Tibetan sheep had a greater villus surface area in the duodenum, jejunum and ileum and higher mRNA expression of sodium-dependent glucose transporter 1 in the duodenum and ileum (P < 0.05) than Han sheep. In the glucose transporter 2 (GLUT2) mediated glucose absorption pathway, Tibetan sheep had higher GLUT2 and taste receptor family 1 member 2 and 3 mRNA expressions than Han sheep in the duodenum, jejunum and ileum (P < 0.05). We concluded that the differences between breeds indicated a greater glucose absorption capacity in the small intestine of Tibetan than Han sheep, which would confer an advantage to Tibetan over Han sheep to an inconsistent energy intake on the harsh QTP. These findings suggested that ruminants raised under harsh environmental conditions with highly fluctuating dietary intakes, as is often the case in grazing ruminants worldwide, are able to absorb glucose from the small intestine to a greater extent than ruminants raised under more moderate conditions.     

鳜小肽转运载体PepT1基因分子特征及其表达研究

小肽转运载体（PepT1）是低亲和力、高容量的肽转运载体,在小肽的吸收过程中发挥着重要的作用。研究采用同源克隆和RACE技术克隆了鳜鱼（Siniperca chuatsi） PepT1基因全长cDNA序列,其cDNA序列全长为2480 bp,包含43 bp的5'UTR序列,232 bp的3'UTR序列,以及2205 bp开放阅读框,编码735个氨基酸。 氨基酸序列同源性分析结果显示,鳜鱼与石斑鱼（Epinephelus aeneus）、鲈鱼（Dicentrarchus labrax）PepT1间同源性均为89%,与其他非鱼类物种的同源性则在46%56%。经预测,鳜鱼PepT1编码蛋白的分子量为64.8 kD,等电点为8.97,该蛋白具有与哺乳动物同源蛋白相似的12 个螺旋跨膜结构,并且在跨膜区9和10之间有一个大的外环;跨膜区氨基酸高度保守,并存在有5个膜外N-糖基化位点和3个膜内含蛋白激酶C基序的相同区域。实时荧光定量表达分析表明,鳜鱼PepT1基因在前肠和中肠中表达量显著高于后肠（P0.05）,这说明前、中肠是鳜鱼肠道吸收小肽的主要部位;在胚后不同发育阶段鳜鱼前肠均能检测到PepT1基因的表达,并且在10 g个体中表达量最高,之后随着体重的增加其表达量维持在一个稳定水平。本研究结果首次报道了鳜鱼PepT1基因全序列及其分子表达特征,为鱼类营养及生理学的研究提供有价值的参考资料。       

Diurnal rhythm in mRNA expression of genes encoding amino acid transporter and circadian gene cry in intestinal mucosa of piglets

Long chain PUFA contents in plasma and liver both exhibited diurnal rhythms in pigs. However, whether mRNA expression of amino acid transporter and circadian gene Cry in intestinal mucosa is also rhythmic is yet to be known. The purpose of this study aims to investigate the diurnal rhythm in mRNA expression of genes encoding amino acid transporter and whether their rhythm was related to the expression of circadian gene Cry in intestinal mucosa of piglets. Thirty-six piglets (Duroc?×?Landrace?×?Large Yorkshire) at the age of 35 days were selected and fed for three weeks, and then samples were collected at 3:00 am (Clo3), 7:00 am (Clo7), 11:00 am (Clo11), 3:00 pm (Clo15), 7:00 pm (Clo19), and 11:00 pm (Clo23) at the age of 56 days. At each time point, small intestinal mucosa samples were collected from duodenum, jejunum, and ileum for detection of mRNA expression of the amino acid transporters and circadian gene Cry. The results showed that mRNA expression of most amino acid transporters in intestinal mucosa was higher at night and lower during the daytime. Expression of SLC1A2, SLC6A20, SLC7A1, and SLC6A14 in duodenal mucosa reached the peak at Clo3 and Clo7; the diurnal rhythm of expression of SLC1A2, SLC6A20, and SLC7A1 was similar to Cry1, while the diurnal rhythm of expression of SLC6A14 had a similar trend to Cry2. Expression of SLC16A10, SLC1A2, and SLC7A1 in jejunal mucosa reached the peak at Clo7, while SLC6A14 reached the peak at Clo3; the diurnal rhythm of expression of SLC1A2 showed a similarity with Cry1, while the diurnal rhythm of expression of SLC16A10, SLC7A1, and SLC6A14 was similar to Cry2. Expression of SLC6A14, SLC6A20, and SLC7A1 in ileal mucosa reached the peak at Clo3; the diurnal rhythm of expression of SLC6A20 has a similarity with Cry1, while the diurnal rhythm of expression of SLC7A1 and SLC6A14 was similar to Cry2. The results suggested that the mRNA expression of most genes encoding amino acid transporters exhibited diurnal rhythms in the intestinal mucosa of piglets, and SLC7A1, SLC6A14, and SLC1A2 have a similar rhythm with circadian clock genes Cry1 and 2, and they reached the peak at Clo3 and Clo7.     

Molecular cloning, tissue distribution and ontogenetic expression of sodium proton exchanger isoform 2 ( NHE-2) mRNA in the small intestine of pigs

Molecular cloning, tissue distribution and ontogenetic regulation of sodium/proton exchanger isoform 2 (NHE-2) mRNA expression were evaluated in the pig small intestine during postnatal development. The 2872-bp porcine full cDNA sequence of the NHE-2 (EF672046) cloned in this study showed 80% and 70% homology with known human and mouse gene sequence, respectively. Hydrophobic prediction suggests 13 putative membrane-spanning domains within porcine NHE-2. The porcine NHE-2 mRNA was detected in the brain, liver, kidney, heart, lung, small intestine and muscle. The small intestine had the highest NHE-2 mRNA abundance and the brain, lung and liver had the lowest NHE-2 mRNA abundance (P < 0.05). Along the longitudinal axis, the duodenum had the highest NHE-2 mRNA abundance and the ileum and colon had the lowest NHE-2 mRNA abundance (P < 0.05). The NHE-2 mRNA level was increased from day 1 to day 26 in the duodenum (P < 0.05) and dropped dramatically on day 30 (P < 0.05). There is no difference between day 1 and day 7 (P > 0.05). After day 30, the NHE-2 mRNA level remained the same except on day 90 (P > 0.05). The mRNA expression of NHE-2 was not only differentially regulated by age but also differentially distributed along the small intestine of piglets at early stages and growing stages of life, which may contribute to changes in NHE activity.     

Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes

Fatty acid translocase (FAT)/CD36 is one of several putative plasma membrane long-chain fatty acid (LCFA) transport proteins; however, its role in intestinal absorption of LCFA is unknown. We hypothesized that FAT/CD36 would be differentially expressed along the longitudinal axis of the gut and during intestinal development, suggesting specificity of function. We found that intestinal mucosal FAT/CD36 mRNA levels varied by anatomic location along the longitudinal gut axis: stomach 45 +/- 7, duodenum 173 +/- 29, jejunum 238 +/- 17, ileum 117 +/- 14, and colon 9 +/- 1% (means +/- SE with 18S mRNA as control). FAT/CD36 protein levels were also higher in proximal compared with distal intestinal mucosa. Mucosal FAT/CD36 mRNA was also regulated during intestinal maturation, with a fourfold increase from neonatal to adult animals. In addition, FAT/CD36 mRNA levels and enterocyte LCFA uptake were rapidly downregulated by intraduodenal oleate infusion. These findings suggest that FAT/CD36 plays a role in the uptake of LCFA by small intestinal enterocytes. This may have important implications in understanding fatty acid absorption in human physiological and pathophysiological conditions.     

Site-specific effects of dietary salt intake on guanylin and uroguanylin mRNA expression in rat intestine

Guanylin and uroguanylin are newly discovered intestinal peptides that have been shown to affect NaCl transport in both the intestine and kidney. The present study tests the hypothesis that guanylin and uroguanylin mRNA expression in each major region of the intestine is regulated by NaCl intake. Semiquantitative multiplex RT-PCR analysis was used to determine the molecular expression of guanylin and uroguanylin in the duodenum, jejunum, ileum, and colon in rats maintained on low (LS), normal (NS), or high (HS) NaCl intake for 4 days. LS intake reduced the expression of uroguanylin, and to a lesser degree, guanylin mRNA in all intestinal segments compared to NS intake. The duodenum was the site of the greatest decrease for both. In contrast, HS intake significantly increased the expression of guanylin mRNA only in the duodenum and jejunum and had minimal effect on uroguanylin mRNA. The minimum time required for altered gene expression was determined by delivering an oral NaCl challenge directly to the gastrointestinal tract by oro-gastric administration to LS or NS animals. In LS rats, NaCl oro-gastric administration significantly increased mRNA expression of both peptides in all intestinal segments. Furthermore, the increases in guanylin and uroguanylin mRNA were detected within 4 h and plateaued by 8 h. Conversely, acute oro-gastric administration of the same NaCl solution to NS rats caused elevations of guanylin mRNA only in the duodenum and jejunum, and of uroguanylin mRNA only in the ileum and colon. In conclusion, the data demonstrate that variations in NaCl intake lead to intestinal segment-specific changes in guanylin and uroguanylin mRNA expression.     

Growth and morphological changes in the small and the large intestine in piglets during the first three days after birth.

Growth and morphological changes in the small and the large intestine of piglets were examined during the first three days after birth. There was a 72% increase in small intestinal weight, virtually all of which occurred during the first day and was due primarily to a 115% increase in the weight of the mucosa. Associated with the tissue weight gain there was a 24% increase in small intestinal length, a 15% increase in small intestinal diameter, a 33-90% increase in villus height and a 14-51% increase in villus diameter, during the first day. The cellular population in the small intestinal mucosa, as indicated by its DNA content, increased progressively with age, and at three days had increased by 84-154%. The percentage increase in mucosal DNA content was highest in the duodenum, intermediate in the jejunum and lowest in the ileum. Histological features and tissue protein contents revealed a transient epithelial cellular swelling related to intracellular accumulation of protein on the first day. Protein accumulation was evident in the jejunum and ileum but not in the duodenum. The positions of the nuclei in the epithelial cells suggested that on the first day protein absorption was at a more advanced stage in the jejunum and the proximal ileum than in the distal ileum. Large intestinal weight increased by 33% during the first day and had doubled by the third day, and this weight gain was due to both mucosal and non-mucosal tissue growth. Villus-like structures were observed in the caecum and the proximal colon in piglets at birth and one day after birth but not in piglets three days after birth. It is speculated that such villus-like structures may have a functional significance during the transition to complete dependence on oral nutrition in newborns.     

藏羚羊低氧诱导因子1α基因的克隆与组织表达

为探讨低氧诱导因子1α (hypoxia inducible factor l alpha,HIF-1α)在藏羚羊(Pantholops hodgsonii)高原低氧适应机制中的作用,采用RT-PCR和RACE技术克隆藏羚羊HIF-1α基因的cDNA序列,同时采用real-time PCR和Western blot方法...