Pig Enterocytes Research Articles

Deoxynivalenol affects cell metabolism in vivo and inhibits protein synthesis in IPEC-1 cells

Deoxynivalenol is present in forage crops in concentrations that endanger animal welfare but is also found in cereal-based food. The amphipathic nature of mycotoxins allows them to cross the cell membrane and interacts with different cell organelles such as mitochondria and ribosomes. In our study, we investigated the gene expression of several genes in vivo and in vitro that are related to the metabolism. We observed a significantly higher COX5B and MHCII expression in enterocytes of DON-fed pigs compared to CON-fed pigs and a marked increase in GAPDH and SLC7A11 in DON-fed pigs, but we could not confirm this in vitro in IPEC-1. In vitro, functional metabolic analyses were performed with a seahorse analyzer. A significant increase of non-mitochondrial respiration was observed in all DON-treatment groups (50–2000 ng/mL). The oxygen consumption of cells, which were cultured on membranes, was examined with a fiber-glass electrode. Here, we found significantly lower values for DON 200- and DON 2000-treatment group. The effect on ribosomes was investigated using biorthogonal non-canonical amino acid tagging (BONCAT) to tag newly synthesized proteins. A significantly reduced amount was found in almost all DON-treatment groups. Our findings clearly show that apical and basolateral DON-treatment of epithelial cell layer results in decreasing amounts of newly synthesized proteins. Furthermore, our study shows that DON affects enterocyte metabolism in vivo and in vitro.

The Uptake of Glutamine and Release of Arginine, Citruline and Proline by the Small Intestine of Developing Pigs ,

Arteriovenous (A-V) differences in the plasma concentrations of amino acids across the jejunum were studied in preweaning (14- to 21-d-old) and post-weaning (29- to 58-d-old) pigs in the postabsorptive state. Glutamine was the only amino acid that was extracted by the small intestine in both pre- and post-weaning pigs. The production of citrulline by the jejunum was low in preweaning pigs, but was threefold greater in the post-weaning pigs than in the preweaning pigs. The output of proline by the intestine was observed in the post-weaning pigs but not in the preweaning pigs. Arginine and alanine were the predominant amino acids released by the jejunum of the pre- and post-weaning pigs, respectively. Whereas glutamate was the major amino acid formed from glutamine in pig enterocytes in vitro, the jejunum of the post-weaning pigs released comparable amounts of citrulline, proline and glutamate in vivo. There were no significant A-V differences in the concentrations of urea, ornithine, taurine, tyrosine, serine, glycine and nutritionally essential amino acids in either the preweaning or the post-weaning pigs. Thus, we demonstrated for the first time the uptake in vivo of glutamine and the release of arginine, alanine, citrulline, glutamate and proline by the small intestine of developing pigs. Our findings on the release in vivo of citrulline (the precursor of arginine) and proline by the small intestine of the pre- and post-weaning pigs provide a basis for explaining why arginine and proline are nutritionally essential amino acids for young suckling piglets but not for adult pigs.

Oxidation of amino acids, glucose, and fatty acids as metabolic fuels in enterocytes of developing pigs.

Enterocytes of young pigs are known to use glutamine, glutamate, and glucose as major metabolic fuels. However, little is known about the roles of aspartate, alanine, and fatty acids as energy sources for these cells. Therefore, this study simultaneously determined the oxidation of the amino acids and glucose as well as short- and long-chainfatty acids in enterocytes of developing pigs. Jejunal enterocytes were isolated from 0-, 7-, 14- and 21-day-old piglets, and incubated at 37°C for 30min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5mM D-glucose and one of the following: D-[U-14C]glucose, 0.5-5mM L-[U-14C]glutamate, 0.5-5mM L-[U-14C]glutamine, 0.5-5mM L-[U-14C]aspartate, 0.5-5mM L-[U-14C]alanine, 0.5-2mM L-[U-14C]palmitate, 0.5-5mM [U-14C]propionate, and 0.5-5mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate by enterocytes from all age groups of piglets increased (P < 0.05) gradually with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, and glucose by enterocytes from 0- to 21-day-old pigs and of alanine from newborn pigs were much greater (P < 0.05) than those for the same concentrations of palmitate, propionate, and butyrate. Compared with 0-day-old pigs, the rates of oxidation of glutamate, aspartate, glutamine, alanine, and glucose by enterocytes from 21-day-old pigs decreased (P < 0.05) markedly, without changes in palmitate oxidation. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes of pigs was limited during their postnatal growth. At each postnatal age, the oxidation of glutamate, glutamine, aspartate, and glucose produced much more ATP than alanine, propionate, butyrate and palmitate. The degradation of glutamate was initiated primarily by glutamate-pyruvate and glutamate-oxaloacetatetransaminases. Our results indicated that amino acids (glutamate plus glutamine plus aspartate) are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs.

Cortisol enhances citrulline synthesis from proline in enterocytes of suckling piglets.

There are marked decreases in plasma concentrations of cortisol and arginine (an essential amino acid for neonates) as well as intestinal citrulline synthesis in piglets during the first 14days of life. The objective of this study was to test the hypothesis that increasing plasma cortisol concentrations by cortisol administration may prevent the decline in intestinal citrulline and arginine synthesis from proline, thereby possibly increasing plasma arginine concentration in suckling piglets and their growth. Seven-day-old pigs reared by sows received daily intramuscular injections of hydrocortisone 21-acetate (25mg/kg) or vehicle solution (saline) (n = 10/group). At 14days of age, piglets were used to prepare jejunal enterocytes. Cells were incubated at 37°C for 30min in oxygenated Krebs buffer containing 5mM glucose, 2mM [U-14C]proline, and 2mM glutamine. Cortisol treatment increased plasma cortisol concentration, mitochondrial proline oxidase and N-acetylglutamate synthase activities, cytosolic argininosuccinate lyase activity, and the intracellular concentrations of N-acetylglutamate and carbamoyl phosphate for citrulline and arginine synthesis. However, cortisol treatment induced the expression of intestinal arginase-II for arginine hydrolysis, resulting in no change in plasma arginine concentration. Administration of cortisol had no effect on milk consumption or the whole-body growth rate of piglets, but increased villus height in the jejunum and ileum. Collectively, these results suggest an important role for proline oxidase and N-acetylglutamate in regulating citrulline and arginine synthesis from proline in pig enterocytes. Because proline catabolism plays an important role in modulating protein synthesis, cell proliferation, and arginine production, our findings may have important implications for understanding the role of proline oxidase in the growth and health of the mammalian small intestine.

Effect of Lactobacillus spp. on adhesion, invasion, and translocation of Campylobacter jejuni in chicken and pig small-intestinal epithelial cell lines

BackgroundCampylobacter spp. are a major cause of bacterial food-borne diarrhoeal disease. This mainly arises through contamination of meat products during processing. For infection, Campylobacter spp. must adhere to epithelial cells of the mucus layer, survive conditions of the gastrointestinal tract, and colonise the intestine of the host. Addition of probiotic bacteria might promote competitive adhesion to epithelial cells, consequently reducing Campylobacter jejuni colonisation. Effect of Lactobacillus spp. (PCS20, PCS22, PCS25, LGG, PCK9) on C. jejuni adhesion, invasion and translocation in pig (PSI cl.1) and chicken (B1OXI) small-intestine cell lines, as well as pig enterocytes (CLAB) was investigated.ResultsOverall, in competitive adhesion assays with PSI cl.1 and CLAB cell monolayers, the addition of Lactobacillus spp. reduced C. jejuni adherence to the cell surface, and negatively affected the C. jejuni invasion. Interestingly, Lactobacillus spp. significantly impaired C. jejuni adhesion in three-dimensional functional PSI cl.1 and B1OXI cell models. Also, C. jejuni did not translocate across PSI cl.1 and B1OXI cell monolayers when co-incubated with probiotics. Among selected probiotics, Lactobacillus rhamnosus LGG was the strain that reduced adhesion efficacy of C. jejuni most significantly under co-culture conditions.ConclusionThe addition of Lactobacillus spp. to feed additives in livestock nutrition might be an effective novel strategy that targets Campylobacter adhesion to epithelial cells, and thus prevents colonisation, reduces the transmission, and finally lowers the incidence of human campylobacteriosis.

253 Glutamate and glutamine are the major metabolic fuels in enterocytes of suckling piglets

Abstract Glutamate and glutamine are known to be important energy substrates in pig enterocytes, and aspartate has also been reported to be extensively catabolized by the rat small intestine. However, little is known about the relative role of amino acids, glucose and fatty acids in ATP production by enterocytes. In the present study, enterocytes isolated from 0-, 7-, 14- and 21-day-old piglets were used to determine the rates of oxidation of amino acids, fatty acids and glucose. Enterocytes were incubated at 37oC for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and one of the following: 0.5–5 mM L-[U-14C]glutamate, 0.5–5 mM L-[U-14C]glutamine, 0.5–5 mM L-[U-14C]aspartate, 0.5–5 mM L-[U-14C]alanine, 0.5–2 mM L-[U-14C]palmitate, D-[U-14C]glucose, 0.5–5 mM [U-14C]propionate, or 0.5–5 mM [1-14C]butyrate. At the end of the incubation, 14CO2 produced from each 14C-labeled substrate was collected. Rates of oxidation of each substrate in enterocytes from all age groups of piglets increased (P &amp;lt; 0.01) progressively with increasing its extracellular concentrations. The rates of oxidation of glutamate, glutamine, aspartate, glucose by enterocytes from 0- to 21-day-old pigs were much greater (P &amp;lt; 0.01) than those for the same concentrations of alanine, palmitate, propionate, and butyrate. In cells from all age groups of piglets, rates of oxidation of, and ATP production from 5 mM glutamate or 5 mM glutamine were greater (P &amp;lt; 0.01) than those from 5 mM glucose and aspartate. Oxidation of alanine, propionate, butyrate and palmitate by enterocytes was limited. At each postnatal age, the oxidation of glutamate and glutamine produced more ATP than any other substrates. Our results indicated that glutamate and glutamine are the major metabolic fuels in enterocytes of 0- to 21-day-old pigs. Because of limited uptake of arterial glutamate by enterocytes, dietary glutamate is essential to the integrity and function of the pig small intestine.

Identification of microRNA transcriptome reveals that miR-100 is involved in the renewal of porcine intestinal epithelial cells.

MicroRNAs play important roles in various cellular processes, including differentiation, proliferation and survival. Using a pig model, this study sought to identify the miRNAs responsible for crypt-villus axis renewal of the small intestinal epithelium. Compared to the villus upper cells, there were 15 up-regulated and 41 down-regulated miRNAs in the crypt cells of the jejunum. Notably, we found that miR-100 was expressed more in the villus upper cells than in the crypt cells, suggesting an effect on intestinal epithelium differentiation. Overexpression of miR-100 increased the activity of alkaline phosphatase, confirming that miR-100 promoted IPEC-J2 cell differentiation. MiR-100 can inhibit cell proliferation as evidenced by CCK-8 and cell cycle assay results. We also showed that miR-100 significantly inhibited the migration of IPEC-J2 cells and promoted cell apoptosis through caspase-3-dependent cleavage of Bcl-2. Furthermore, FGFR3 was identified as a potential target of miR-100 by bioinformatics analysis. We confirmed that overexpression of miR-100 suppressed FGFR3 expression in IPEC-J2 cells by directly targeting the FGFR3 3'-UTR. This is the first report of miRNAs acting on the renewal of the intestinal crypt-villus axis. Our results also showed that miR-100 promotes the differentiation and apoptosis, and inhibits the proliferation and migration of enterocytes of pigs.

Gene Editing of Swine Myosin Vb Induces Microvillus Inclusion Disease and Loss of Apical Sodium Transporters with Maintenance of CFTR in Enterocytes

BackgroundThe human genetic disorder Microvillus Inclusion Disease (MVID) results from inactivating mutations in Myosin Vb (MYO5B). MVID is characterized by severe, unremitting diarrhea that typically presents shortly after birth. The necessity of implementing total parenteral nutrition (TPN) and the long‐term administration of TPN in neonates with MVID presents challenges to elucidating the complex pathogenesis of MVID in humans. Thus, a large animal model of MVID that closely recapitulates human physiology is needed. To better understand the pathophysiology of MVID we created a large animal model that closely mimics human MVID.MethodsPigs were cloned using somatic cell nuclear transfer of primary pig fibroblasts edited with TALEN to express a P663L mutation of the MYO5B gene. This swine P663L mutation, referred to as MYO5B(P663L), corresponds to the Navajo P660L mutation seen in MVID patients. Neonatal pigs were euthanized within 1 day of birth and tissue was collected for analysis and generation of intestinal enteroids.ResultsThe MYO5B(P663L) pigs developed diarrhea shortly after birth, similar to patients with MVID. Immunostaining for brush border components F‐actin, ezrin, CD10 and DPPIV showed the presence of subapical intracellular inclusions in enterocytes of MYO5B P663L pigs. Transmission electron microscopy confirmed the presence of inclusions lined with microvilli in the MYO5B(P663L) pigs. Immunostaining for NHE3 and SGLT1, drivers of intestinal sodium and water absorption, showed decreased apical expression and diffuse sub‐apical expression in the MYO5B(P663L) pigs compared to control pigs. In contrast to decreased expression of sodium transporters, CFTR was maintained on the apical membrane of MYO5B(P663L) pigs. Enteroids generated from the proximal small intestine of neonatal control and MYO5B(P663L) pigs grew at similar rates, but showed differences in the brush border. Enteroids derived from MYO5B(P663L) pigs had sub‐apical expression of intestinal alkaline phosphatase and DPPIV and decreased apical expression of NHE3 compared to control enteroids. Administration of forskolin resulted in increased swelling in MYO5B(P663L) enteroids (170.8% ± 15.9) compared to control enteroids (90.4% ± 4.6), indicative of functional Cl− secretion.ConclusionsCollectively, these data suggest that decreased apical expression of NHE3 and SGLT1, which are required for water absorption, are likely responsible for the MVID diarrhea disorder. Moreover, maintenance of functional CFTR on the apical membrane of enterocytes in the MYO5B(P663L) pig may further exacerbate the development of diarrhea by actively secreting Cl−, thus driving further water loss by facilitating fluid secretion. This novel large animal model of MVID could provide an important vehicle for testing novel therapeutic approaches for patients with MVID.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Complete genome characterization of recent and ancient Belgian pig group A rotaviruses and assessment of their evolutionary relationship with human rotaviruses.

Group A rotaviruses (RVAs) are an important cause of diarrhea in young pigs and children. An evolutionary relationship has been suggested to exist between pig and human RVAs. This hypothesis was further investigated by phylogenetic analysis of the complete genomes of six recent (G2P[27], G3P[6], G4P[7], G5P[7], G9P[13], and G9P[23]) and one historic (G1P[7]) Belgian pig RVA strains and of all completely characterized pig RVAs from around the globe. In contrast to the large diversity of genotypes found for the outer capsid proteins VP4 and VP7, a relatively conserved genotype constellation (I5-R1-C1-M1-A8-N1-T7-E1-H1) was found for the other 9 genes in most pig RVA strains. VP1, VP2, VP3, NSP2, NSP4, and NSP5 genes of porcine RVAs belonged to genotype 1, which is shared with human Wa-like RVAs. However, for most of these gene segments, pig strains clustered distantly from human Wa-like RVAs, indicating that viruses from both species have entered different evolutionary paths. However, VP1, VP2, and NSP3 genes of some archival human strains were moderately related to pig strains. Phylogenetic analysis of the VP6, NSP1, and NSP3 genes, as well as amino acid analysis of the antigenic regions of VP7, further confirmed this evolutionary segregation. The present results also indicate that the species barrier is less strict for pig P[6] strains but that chances for successful spread of these strains in the human population are hampered by the better adaptation of pig RVAs to pig enterocytes. However, future surveillance of pig and human RVA strains is warranted. Rotaviruses are an important cause of diarrhea in many species, including pigs and humans. Our understanding of the evolutionary relationship between rotaviruses from both species is limited by the lack of genomic data on pig strains. In this study, recent and ancient Belgian pig rotavirus isolates were sequenced, and their evolutionary relationship with human Wa-like strains was investigated. Our data show that Wa-like human and pig strains have entered different evolutionary paths. Our data indicate that pig P[6] strains form the most considerable risk for interspecies transmission to humans. However, efficient spread of pig strains in the human population is most likely hampered by the adaptation of some crucial viral proteins to the cellular machinery of pig enterocytes. These data allow a better understanding of the risk for direct interspecies transmission events and the emergence of pig rotaviruses or pig-human reassortants in the human population.

Oleate Metabolism in Pig Enterocytes Is Characterized by an Increased Oxidation Rate in the Presence of a High Esterification Rate within Two Days after Birth 2

Oleate (OLE) is the principle fatty acid (FA) in mammalian colostrum, but its role in the energy supply in enterocytes after birth remains unknown. We investigated the metabolic fate of OLE in pig enterocytes at birth (d0) and after 2 d of suckling (d2). Cellular TG and phospholipids (PL) and FA composition were analyzed. Metabolic end-products of [1-14C]OLE were measured in enterocyte incubations. We characterized intestinal carnitine palmitoyltransferase 1 (CPT1), the key enzyme of mitochondrial FA oxidation. The TG content was 6.6-fold higher in enterocytes from pigs on d 2 than in those obtained on d 0, whereas the PL content did not differ. The level of OLE in TG and PL increased from 15 and 11% of total FA, respectively, in enterocytes from newborn piglets to 30 and 17%, respectively, in those from d2 pigs. The capacity for OLE utilization was 2.8-fold greater in d2 than in d0 pig enterocytes. The oxidation and esterification rates were enhanced in enterocytes from piglets on d 2 compared to those obtained on d 0, by 4- and 2.6-fold, respectively. The predominant OLE fate was the esterification pathway, representing >85% of OLE metabolized in both groups. The limited OLE oxidation observed at d 2 may result from the presence of a highly malonyl-CoA–sensitive CPT1A, because the half maximal inhibitory concentration for malonyl-CoA was 162 ± 25 nmol/L. This study highlighted the high esterification capacity for OLE in the newborn pig intestine, which may preserve this major colostrum FA for delivery to other tissues.

Expression of Na+/glucose co-transporter 1 (SGLT1) in the intestine of piglets weaned to different concentrations of dietary carbohydrate

Na+/glucose co-transporter 1 (SGLT1) transports dietary sugars from the lumen of the intestine into enterocytes. Regulation of this protein is essential for the provision of glucose to the body and, thus, is important for maintenance of glucose homeostasis. We have assessed expression of SGLT1 at mRNA, protein and functional levels in the intestinal tissue of 28 d old piglets weaned onto isoenergetic diets with differing concentrations of digestible carbohydrate (CHO). We show that expression of SGLT1 remains constant when piglets are fed up to 40 % CHO-containing diets. However, there is a significant increase in SGLT1 expression when the CHO content of the diet is>50 %. Morphometric analyses indicate that the increased expression is not due to a trophic effect. It has been proposed that in rat intestine, in response to a high-CHO diet, GLUT2 (the classical basolateral membrane monosaccharide transporter) is translocated to the luminal membrane of enterocytes to absorb excess dietary glucose. We show, using immunohistochemistry and Western blotting with antibodies raised to amino acids in different epitopes of GLUT2, that under all dietary conditions, low to high CHO, GLUT2 is expressed on the basolateral membrane of pig enterocytes. Furthermore, functional studies indicate that there is no uptake of 2-deoxy-D-glucopyranoside, a specific substrate of Na+-independent glucose transporters into brush-border membrane vesicles isolated from the intestines of piglets either maintained on low- or high-CHO diets. Thus, SGLT1 is the major route for absorption of dietary sugars across the luminal membrane of swine enterocytes.

Intestinal microbiota differentially affect brush border enzyme activity and gene expression in the neonatal gnotobiotic pig

To study microbial influence on intestinal development pertaining to nutrient digestion, two separate gnotobiotic experiments were performed, each with 16 piglets allocated to four treatment groups: germfree (GF), monoassociation with Escherichia coli, monoassociation with Lactobacillus fermentum or conventionalization with faecal bacteria (CV). Enzyme activity and gene expression of lactase phlorizin hydrolase (LPH) and aminopeptidase N (APN) were measured in isolated enterocytes, harvested on day 14, using specific substrates and quantitative PCR respectively. Enterocytes of CV pigs had reduced APN activity, but had increased gene expression relative to GF, making the specific activity:mRNA (A:G) ratio dramatically lower (p < 0.05). Similarly, LPH A:G ratio was significantly reduced (p < 0.05) in enterocytes of CV pigs as compared with GF. The results of co-incubation of L. fermentum, E. coli and faecal bacteria with APN indicate a direct relationship between enzyme inactivation and specific A:G ratio in enterocytes. We conclude that enterocyte up-regulation of APN expression occurs as either a direct response to microbial colonization or as a feedback mechanism in response to reduced enzyme activity through microbial degradation. This mechanism may play a role in ensuring effective competition of the host with the intestinal microbiota for available nutrients.

Catabolism of nutritionally essential amino acids in developing porcine enterocytes

This study was conducted using the piglet model to test the hypothesis that mucosal cells of the neonatal small intestine can degrade nutritionally essential amino acids (EAA). Enterocytes were isolated from the jejunum of 0-, 7-, 14-, and 21-day-old pigs, and incubated for 45 min in Krebs buffer containing plasma concentrations of amino acids and one of the following L-[1-(14)C]- or L-[U-(14)C]-amino acids plus unlabeled tracees at 0.5, 2, or 5 mM: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In these cells, branched-chain amino acids (BCAA) were extensively transaminated and 15-50% of decarboxylated branched-chain alpha-ketoacids (BCKA) were oxidized to CO(2) depending on the age of piglets. BCAA transamination increased but their decarboxylation decreased between 0 and 14 days of age. Addition of 1 and 2 mM alpha-ketoglutarate to incubation medium dose-dependently stimulated BCAA transamination without affecting their decarboxylation. Western blot analysis revealed that the abundance of mitochondrial BCAA aminotransferase declined but cytosolic BCAA aminotransferase increased between 0 and 14 days of age, with the cytosolic protein being the major isoform in 7- to 21-day-old pigs. BCKA dehydrogenase protein existed primarily as the phosphorylated (inactive) form in enterocytes of newborn pigs and its levels were markedly reduced in older pigs. All measured parameters of BCAA metabolism did not differ between 14- and 21-day-old pigs. In contrast to BCAA, catabolism of methionine and phenylalanine was negligible and that of other EAA was absent in enterocytes from all ages of piglets due to the lack of key enzymes. These results indicate that enterocytes are an important site for substantial degradation of BCAA but not other EAA in the neonatal gut.

L-Glutamine or l-alanyl-l-glutamine prevents oxidant- or endotoxin-induced death of neonatal enterocytes

This study tested the hypothesis that L-glutamine (Gln) or L-alanyl-L-glutamine (Ala-Gln) prevents oxidant- or endotoxin-induced death of neonatal enterocytes. Enterocytes of neonatal pigs rapidly hydrolyzed Ala-Gln and utilized Gln. To determine whether Gln or Ala-Gln has a cytoprotective effect, IPEC-1 cells were cultured for 24 h in Gln-free Dulbecco's modified Eagle's-F12 Ham medium containing 0, 0.5, 2.0 or 5.0 mM Gln or Ala-Gln, and 0, 0.5 mM H(2)O(2) or 30 ng/ml lipopolysaccharide (LPS). Without Gln or Ala-Gln, H(2)O(2)- or LPS-treated cells exhibited almost complete death. Gln or Ala-Gln at 0.5, 2 and 5 mM dose-dependently reduced H(2)O(2)- or LPS-induced cell death by 14, 54 and 95%, respectively, whereas D: -glutamine, alanine, glutamate, ornithine, proline, glucosamine or nucleosides had no effect. To evaluate the effectiveness of Gln or Ala-Gln in vivo, 7-day-old piglets received one-week oral administration of Gln or Ala-Gln (3.42 mmol/kg body weight) twice daily and then a single intraperitoneal injection of LPS (0.1 mg/kg body weight); piglets were euthanized in 24 and 48 h to analyze intestinal apoptotic proteins and morphology. Administration of Gln or Ala-Gln to LPS-challenged piglets increased Gln concentrations in small-intestinal lumen and plasma, reduced intestinal expression of Toll-like receptor-4, active caspase-3 and NFkB, ameliorated intestinal injury, decreased rectal temperature, and enhanced growth performance. These results demonstrate a protective effect of Gln or Ala-Gln against H(2)O(2)- or LPS-induced enterocyte death. The findings support addition of Gln or Ala-Gln to current Gln-free pediatric amino acid solutions to prevent intestinal oxidative injury and inflammatory disease in neonates.

Clostridium difficile Infections in Children

Clostridium difficile Infections in Children

Expression of the Sodium‐neutral amino acid exchanger ASCT2 along the small intestinal crypt‐villus axis in the formula‐fed neonatal pig

The sodium‐neutral amino acid (AA) exchanger ASCT2 gene expressesATB0 protein responsible for the exchange of gut luminal neutral AAacross the apical membrane. The study was to quantify the ASCT2mRNA and protein expression along the jejunal crypt‐villus axis with six Yorkshiregilts at 14–16 d of age fed a milk‐based liquid formula. Threeepithelial cell fractions, representing cells from the uppervillus, the middle villus and the crypt regions, were sequentiallyisolated, with cell viability of 92–95% as assessed bytrypan blue exclusion, along the crypt‐villus axis from theentire jejunum by the distended sac method. Real time RT‐PCR analyses (SmartCycler using SYBRGreen‐I detection kit) revealed a linear decrease (P&lt;0.05)in the ASCT2 mRNA level from the crypt cell to the upper villus cell with β‐actin as thehouse‐keeping control. No significant differences (P&gt;0.05) were observed in ASCT2 protein abundance in cellular homogenate, soluble, and brush border membrane among the cell fractions. These results suggest that neonatal pig enterocytes express ATB0 in the small intestinal enterocyte during the entire process of the cell proliferation and differentiation and the expression of ATB0 is not only regulated at transcriptional level but also at the post transcriptional levels. Supported by NSERC Discovery Program.

Cholera Toxin Entry into Pig Enterocytes Occurs via a Lipid Raft- and Clathrin-Dependent Mechanism

The small intestinal brush border is composed of lipid raft microdomains, but little is known about their role in the mechanism whereby cholera toxin gains entry into the enterocyte. The present work characterized the binding of cholera toxin B subunit (CTB) to the brush border and its internalization. CTB binding and endocytosis were performed in organ-cultured pig mucosal explants and studied by fluorescence microscopy, immunogold electron microscopy, and biochemical fractionation. By fluorescence microscopy CTB, bound to the microvillar membrane at 4 degrees C, was rapidly internalized after the temperature was raised to 37 degrees C. By immunogold electron microscopy CTB was seen within 5 min at 37 degrees C to induce the formation of numerous clathrin-coated pits and vesicles between adjacent microvilli and to appear in an endosomal subapical compartment. A marked shortening of the microvilli accompanied the toxin internalization whereas no formation of caveolae was observed. CTB was strongly associated with the buoyant, detergent-insoluble fraction of microvillar membranes. Neither CTB's raft association nor uptake via clathrin-coated pits was affected by methyl-beta-cyclodextrin, indicating that membrane cholesterol is not required for toxin binding and entry. The ganglioside GM(1) is known as the receptor for CTB, but surprisingly the toxin also bound to sucrase-isomaltase and coclustered with this glycosidase in apical membrane pits. CTB binds to lipid rafts of the brush border and is internalized by a cholesterol-independent but clathrin-dependent endocytosis. In addition to GM(1), sucrase-isomaltase may act as a receptor for CTB.

Enzyme linked immuno-absorbent assay (ELISA) to determine the effectiveness of anti-adhesive factors in blocking the binding of F4(K88)ac E coli to pig intestine

Concern over the use of prophylactic antibiotics and the potential of them being banned has increased interest in other methods of controlling disease. Specifically postweaning diarrhoea in the pig, often associated with E Coli (F4 K88ac) is a candidate for an alternative strategy. If in-feed additives can be demonstrated which effectively block the binding of the F4 (K88)ac pilli to the surface of the pig enterocytes then the severe form of the disease could be controlled without the use of antibiotics. The work reported here provides a screening method to determine whether potential in-feed additives can block the binding of purified F4(K88)ac to isolated brush border membranes taken from neonatal F4(K88)ac positive pigs.

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets.

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.

Lactate inhibits citrulline and arginine synthesis from proline in pig enterocytes.

Hypocitrullinemia and hypoargininemia but hyperprolinemia are associated with elevated plasma concentration of lactate in infants. Because the small intestine may be a major organ for initiating proline catabolism via proline oxidase in the body and is the major source of circulating citrulline and arginine in neonates, we hypothesized that lactate is an inhibitor of intestinal synthesis of citrulline and arginine from proline. To test this hypothesis, jejunum was obtained from 14-day-old suckling pigs for preparation of enterocyte mitochondria and metabolic studies. Mitochondria were used for measuring proline oxidase activity in the presence of 0-10 mM L-lactate. For metabolic studies, enterocytes were incubated at 37 degrees C for 30 min in Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose, 2 mM L-glutamine, 2 mM L-[U-14C]proline, and 0, 1, 5, or 10 mM L-lactate. Kinetics analysis revealed noncompetitive inhibition of intestinal proline oxidase by lactate (decreased maximal velocity and unaltered Michaelis constant). Lactate had no effect on either activities of other enzymes for arginine synthesis from proline or proline uptake by enterocytes but decreased the synthesis of ornithine, citrulline, and arginine from proline in a concentration-dependent manner. These results demonstrate that lactate decreased intestinal synthesis of citrulline and arginine from proline via an inhibition of proline oxidase and provide a biochemical basis for explaining hyperprolinemia, hypocitrullinemia, and hypoargininemia in infants with hyperlactacidemia.