Development of active jejunal glucose absorption in broiler chickens.

Growth in chickens, especially meat-type chickens (broilers), is extremely rapid, but studies on the regulatory mechanism of intestinal glucose absorption with growth are few, contradictory, and unclear. Here, we investigated the regulation of intestinal glucose absorption with growth in broiler chickens using oral glucose gavage, intestinal Evans blue transit, intestinal glucose absorption, scanning electron microscopy, and glucose absorption- and cell junction-related gene expression analyses. Peak blood glucose levels after oral glucose gavage occurred at 10 and 50 min in chickens at 1 wk (C1W) and 5 wk (C5W) of age, respectively. The area under the curve for glucose levels was greater for the C5W than the C1W (P = 0.035). The stain ratio in the small intestine in the C5W was lower than that in the C1W (P = 0.01), but there were no differences in the tissue regions stained with Evans blue and the migration distance of Evans blue from Meckel's diverticulum. In everted sac and Ussing chamber experiments, we observed reduced intestinal glucose uptake and electrogenic glucose absorption in the jejunum of the C5W. Phloridzin, an inhibitor of sodium/glucose cotransporter 1 (SGLT1), suppressed the glucose-induced short-circuit current in the C1W (P = 0.016) but not the C5W. Although the addition of NaCl solution stimulated the glucose-induced short-circuit current in the C1W, no differences between the treatments were observed (P = 0.056), which was also the case in the C5W. Additionally, tissue conductance was diminished in the C5W compared with that in the C1W. Moreover, in the C5W, the intestinal tract was more developed and the jejunal villi were enlarged. In conclusion, glucose absorption throughout the intestine could be greater in C5W than in C1W; however, reduced SGLT1 sensitivity, decreased ion permeability, and intestinal overdevelopment lead to decreased local glucose absorption in the jejunum with growth in broiler chickens. These data provide a detailed analysis of intestinal glucose absorption in growing broiler chickens, and can contribute to the development of novel feeds.

Secondary bile acid lithocholic acid attenuates neurally evoked ion transport in the rat distal colon.

The inhibitory action of the secondary bile acid lithocholic acid (LCA) on neurally evoked Cl-/HCO3- secretion was investigated using the Ussing-chambered mucosal-submucosal preparation from the rat distal colon. Electrical field stimulation (EFS) evoked cholinergic and noncholinergic secretory responses in the rat distal colon. The responses were almost completely blocked by TTX (10-6 M) but not atropine (10-5 M) or hexamethonium (10-4 M). The selective antagonist for VIP receptor 1 (VPAC1) greatly reduced the EFS-evoked response. Thus, the rat distal colon may be predominantly innervated by noncholinergic VIP secretomotor neurons. Basolateral addition of 6 × 10-5 M LCA inhibited the EFS-evoked response. The inhibitory action of LCA was partly rescued by the Y2R antagonist BIIE0246. The bile acid receptor TGR5 agonist INT-777 mimicked the LCA-induced inhibitory action. Immunohistochemical staining showed the colocalization of TGR5 and PYY on L cells. TGR5 immunoreactivity was also found in VIP-immunoreactive submucosal neurons which also expressed the PYY receptor, Y2R. These results suggest that LCA inhibits neurally evoked Cl-/HCO3- secretion through the activation of TGR5 on L cells and cholinergic- and VIP-secretomotor neurons in the submucosal plexus. Furthermore, the inhibitory mechanism may involve TGR5-stimulated PYY release from L cells and Y2R activation in VIP-secretomotor neurons.

Minimum biological domain of xenin-25 required to induce anion secretion in the rat ileum.

Xenin-25 has a variety of physiological functions in the gastrointestinal tract, including ion transport and motility. Xenin-25 and neurotensin show sequence homology, especially near their C-terminal regions. The sequence similarity between xenin-25 and neurotensin indicates that the effects of xenin-25 is mediated by the neurotensin receptor but some biological actions of xenin-25 are independent. We have previously reported that xenin-25 modulates intestinal ion transport and colonic smooth muscle activity. However, minimal biological domain of xenin-25 to induce ion transport was not clear. To improve the mechanistic understanding of xenin-25 and to gain additional insights into the functions of xenin-25, the present study was designed to determine the minimal biological domain of xenin-25 required for ion transport in the rat ileum using various truncated xenin fragments and analogues in an Ussing chamber system. The present results demonstrate that the minimum biological domain of xenin-25 to induce Cl-/HCO3- secretion in the ileum contains the C-terminal pentapeptide. Furthermore, Arg at position 21 is important to retain the biological activity of xenin-25 and induces Cl-/HCO3- secretion in the rat ileum.

Age-related Regulation of Active Amino Acid Transport in the Ileum of Broiler Chickens.

Broiler chickens grow rapidly within a short period; in this regard, our group had previously reported a decrease in the active transport of glucose in the intestines of broiler chickens with their growth. Therefore, in this study, we compared the active transport process of amino acids in the intestines between 1- and 5-week-old broilers using everted sac, Ussing chamber techniques, and real-time quantitative polymerase chain reaction (RT-PCR). The everted sac experiment showed that amino acids were absorbed from all segments of the small intestine in both age groups. There were no significant differences in the serosal to mucosal ratio between 1- and 5-week-old broilers. The Ussing chamber experiment showed that amino acid-induced short-circuit current (ΔIsc) in the ileal epithelium was significantly greater in the 5-week-old chickens than in the 1-week-old chicks (P=0.035). Membrane conductance, an indicator of ion permeability, showed no significant difference between the two groups. Moreover, the mRNA expression levels of amino acid transporters (ASCT1, EAAT3, B0AT1, and y+LAT1) were significantly elevated in the distal ileum of the 5-week-old broilers compared to those in the 1-week-old broilers (P<0.05), while no significant differences were observed in the mRNA levels of ATB0'+, B0/+AT, rBAT, CAT1, and CAT2 in both groups. Our study provides clear evidence that age-dependent increase in the active transport of amino acid across the ileal epithelium is caused by the high expression of Na+-dependent amino acid transporters in broiler chickens.

Leptin and ghrelin expressions in the gastrointestinal tracts of calves and cows.

This study aims to investigate and compare the expressions of leptin and ghrelin in the gastrointestinal tracts of calves and cows. The mRNA expression of leptin in the rumen, abomasum, and jejunum of calves was significantly higher than that in cows. In both calves and cows, abomasum ghrelin mRNA expression was significantly higher than that in other gastrointestinal tracts. In calves, leptin protein expression in the abomasum was the highest. In addition, leptin protein expression in the abomasum and jejunum of calves was significantly higher than that in cows. Results indicated that leptin in the abomasum and jejunum plays an important role during the suckling period in a ruminant.

Effect of feeding behavior on circadian regulation of endothelin expression in mouse colon.

AIMS: The function, regulation and gene expression of the endothelin (ET) system in the intestine is not well understood. We investigated the dependence on feeding schedule and biological clock of the regulation of ET-1 gene expression in mouse colon. MAIN METHODS: Mice were fed freely, fasted for 48 h and re-fed after fasting. KEY FINDINGS: Where indicated ET-1 gene expression was highest in the colon compared with other tissues examined in fasted mice. Fasting increased the level, while maintaining the rhythmicity, of ET-1 gene expression in epithelial colonic tissue. Re-feeding, however, decreased ET-1 gene expression and suppressed rhythmic oscillation, and the rhythmicity also changed for gene expression for circadian clocks, period-1 and period-2 (Per1 and Per2). Furthermore, the decrease in ET-1 gene expression induced by re-feeding was blocked by pre-treatment with hexamethonium and atropine. The daily change in ET-1 gene expression in colon, which depends on feeding schedule via the autonomic nervous system, is synchronized with peripheral circadian oscillators under conditions of free feeding and fasting but not re-feeding. The decrease in ET-1 gene expression in the proximal colon induced by re-feeding occurs via the nervous system. SIGNIFICANCE: ET-1 plays an important physiological role, which is dependent on feeding behavior.

Fatty acid-binding protein expression in the gastrointestinal tract of calves and cows.

Fatty acid-binding protein (FABP) has high affinity for long-chain fatty acids and appears to participate in the metabolism and intracellular transport of lipids. Liver- and intestinal-type FABP (L-FABP and I-FABP, respectively) are expressed in the small intestine. However, in the gastrointestinal tract of ruminants, expression and localization of FABPs are unknown. In this study, we investigated the expression of I-FABP and L-FABP in the gastrointestinal tract of cattle. I- and L-FABP had higher messenger RNA (mRNA) and protein expression levels in the duodenum and jejunum relatively to other gastrointestinal regions in both calves and cows. Furthermore, L-FABP mRNA and protein expression were high in the colon. Both these protein types were confirmed to be in the cytosol of jejunal epithelial cells, where they were found in the villi rather than in the crypts. We concluded that duodenal and jejunal FABPs might be involved in the metabolism of fatty acids mainly in epithelial cells in cattle.

APC and Smad7 link TGFß type I receptors to the microtubule system to promote cell migration.

Cell migration occurs by activation of complex regulatory pathways that are spatially and temporally integrated in response to extracellular cues. Binding of adenomatous polyposis coli (APC) to the microtubule plus ends in polarized cells is regulated by glycogen synthase kinase 3ß (GSK-3ß). This event is crucial for establishment of cell polarity during directional migration. However, the role of APC for cellular extension in response to extracellular signals is less clear. Smad7 is a direct target gene for transforming growth factor-ß (TGFß) and is known to inhibit various TGFß-induced responses. Here we report a new function for Smad7. We show that Smad7 and p38 mitogen-activated protein kinase together regulate the expression of APC and cell migration in prostate cancer cells in response to TGFß stimulation. In addition, Smad7 forms a complex with APC and acts as an adaptor protein for p38 and GSK-3ß kinases to facilitate local TGFß/p38-dependent inactivation of GSK-3ß, accumulation of ß-catenin, and recruitment of APC to the microtubule plus end in the leading edge of migrating prostate cancer cells. Moreover, the Smad7-APC complex links the TGFß type I receptor to the microtubule system to regulate directed cellular extension and migratory responses evoked by TGFß.

Regulation of endothelin-1 expression and function by nutrient stress in mouse colon epithelia.

OBJECTIVE: The endothelin (ET) system is influenced by a variety of stress conditions in many tissues. However, the effects of nutrient stress conditions on ET expression and its function are not well understood in the intestinal tract, while ET-1 gene expression and peptide were found in the intestinal tract. The aim of this study was to investigate the effect of feeding and fasting on the expression of ET-1 and short-circuit current (Isc) induced by ET-1 in mouse colon. MATERIAL AND METHODS: Mice were fed freely, fasted for 48 h, and re-fed after fasting, respectively. ET-1 mRNA levels and peptide concentrations were analyzed using real-time polymerase chain reaction (PCR) and sandwich ELISA, respectively. Isc of epithelial tissue was measured under short-circuit conditions using a Ussing chamber. RESULTS: ET-1 mRNA expression and peptide concentrations in epithelial colonic tissue were significantly increased 48 h after fasting, and decreased within 2 h of re-feeding after a 48-h fast. Furthermore, the addition of ET-1 to the serosal but not the mucosal side increased Isc in colonic epithelia. An increase in Isc was caused by chloride ion (Cl(-)) secretion because Isc induced by ET-1 was blocked by bumetanide and Cl(- -) free conditions. In addition, an increase in Isc induced by ET-1 in colon excised from fasted mice was much lower than that obtained from free-fed mice. CONCLUSIONS: Gene expression, peptide concentration, and the function of ET-1 in mouse colonic epithelia are regulated by nutrient stress.

Possible involvement of prolactin in the synthesis of lactoferrin in bovine mammary epithelial cells.

Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.

Establishment of cell lines that exhibit pluripotency from miniature swine periodontal ligaments.

OBJECTIVE: The periodontal ligament (PDL) is a fibrous connective tissue composed of heterogeneous cell types, including PDL fibroblasts. It is not clear whether cells within the PDL fibroblast population retain the potency to differentiate into other cell types. DESIGN: In the present study, clonal cell lines, derived from Clawn miniature swine PDLs, were established by gene transfection for a human telomerase reverse transcriptase, and characterized. RESULTS: These cell lines, denoted TesPDL1-4, had PDL fibroblasts that showed fibroblastic morphology and expressed procollagen alpha1(I), osteopontin, periostin and alkaline phosphatase mRNA. Under the specific culture conditions, TesPDL3 cells also have the ability to express CD31, vascular endothelial cadherin, von Willebrand factor, osteocalcin, and to form extracellular mineralized nodules. CONCLUSIONS: Our data indicate that TesPDL3 cells have unique properties of expressing several phenotype of fibroblasts, vascular endothelial cells and osteoblasts in cultures.

Rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction.

We have developed a simple method for rapid detection of mycoplasma contamination in cell cultures using SYBR Green-based real-time polymerase chain reaction (PCR). To detect eight common contaminant mollicutes, including Mycoplasma (M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum) and Acholeplasma laidlawii, four primers were prepared based on the 23S rRNA regions. Using these primers and a minimum of 100 fg of mycoplasma genomic DNA, the 23S rRNA regions of these eight mycoplasma species were consistently amplified by real-time PCR. In contrast, no specific amplification product was observed using DNA templates prepared from various mammalian cell lines. Frozen and cultured samples of several cell lines were tested for mycoplasma contamination to evaluate the utility of this method. Of 25 samples that tested positive for mycoplasma by Hoechst staining, which requires two passages of cell cultures started from frozen samples, mycoplasma was detected by real-time PCR in 24 samples of cell extracts prepared directly from frozen samples. When cultured samples were used for this assay, the accuracy of the diagnoses was further improved. Thus, this technique, which is simple, rapid, and sensitive enough for practical application, is suitable for handling many samples and for routine screening for mycoplasma contamination of cell cultures.

Differential expression of endothelin-2 along the mouse intestinal tract.

Endothelin (ET)-2, an ET family peptide, is highly expressed in intestine. However, the specific distribution and function of ET-2 remain unknown. We elucidated the expression profile and localization of ET-2 in mouse gastrointestinal tract. Real-time PCR analysis revealed that ET-2 gene expression in the gastrointestinal tract of healthy animals was relatively high in the colon. Immunohistochemical analysis revealed ET-2-like immunoreactivity mainly in epithelial cells of the mucosa throughout the intestinal tract of healthy animals. Intracellularly, ET-2 was concentrated close to the basement membrane of intestinal epithelial cells. A weak ET-2-like immunoreactivity was also localized to some neurofibers and the myenteric plexus of the muscle layer, coexpressing with vasoactive intestinal peptide. ET-2-like immunoreactivity was also detected at Brunner's glands of the duodenum and follicle-associated epithelium of Peyer's patch. In contrast, ET-1-like immunoreactivity was uniformly distributed in epithelial cells. In dextran sulfate sodium (DSS)-induced colitis, colonic ET-2 was upregulated during the late stage of DSS treatment. These results suggest that in intestinal epithelial cells ET-2 could be secreted into the lamina propria and the dome region in Peyer's patch, and that it might modulate immune cells in these sites for mucosal defense.

Luteolin inhibits endothelin-1 secretion in cultured endothelial cells.

We discovered that luteolin, a typical flavonoid contained in various kinds of plants, inhibits the secretion and gene expression of endothelin-1 (ET-1), a potent vasoconstrictor regulating blood pressure, in porcine aortic endothelial cells. Its ED50 was about 10 microM. In addition, the inhibition of ET-1 by a glycoside compound of luteolin (luteolin-6-C-glucoside) was weak.

Immunolocalization of endothelin-B receptor in mouse intestinal tract.

The endothelin-B (ETB) receptor is a G-protein-coupled receptor that binds endothelin ligands and is essential for the development of epidermal melanocytes and enteric neurons. Recent reports indicate that ETB is localized to nuclei in cardiac ventricular myocytes, although it has been thought that ETB is localized mainly on the plasma membrane. It remains unknown, however, whether this unique distribution of ETB occurs in other tissues. To elucidate the subcellular distribution of ETB in the intestine, we performed immunofluorescence of ETB in mouse intestine using a specific antibody. ETB-like immunoreactivity was detected in both the mucosal and muscle layers. In the mucosal layer, villous epithelial cells, stromal cells of the lamina propria, and cryptic cells were immunostained. Subcellularly, ETB is localized mainly to the nuclei of villous epithelial cells. In the muscle layer, immunoreactivity of ETB was localized to the myenteric plexus. These findings suggest that ETB may function as an "intracrine" receptor for intracellular endothelin ligands in villous epithelial cells and may regulate intestinal function.

Vasoactive intestinal contractor/endothelin-2 gene expression in the murine central nervous system.

Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) family. We have investigated the regional distribution of VIC/ET-2 and of ET-1 gene expression in the adult murine brain and pituitary gland. We used real-time quantitative reverse transcription-linked polymerase chain reaction. VIC/ET-2 gene expression was observed at high levels in the pituitary gland and medulla oblongata in both the mouse and rat. Moderate to low levels of expression were observed in other brain regions. On the contrary, ET-1 gene expression was quite low in the pituitary gland in comparison with the levels observed in the cerebral cortex, striatum, and midbrain. Cold injury to the mouse cerebral cortex caused a significant decrease in VIC/ET-2 gene expression in this structure, whilst expression of the ET-1 gene was increased. These results suggest that VIC/ET-2 may have certain physiological roles that differ from those of ET-1 in the brain and pituitary gland.

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice.

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.

Dietary magnesium increases calcium absorption of ovine small intestine in vivo and in vitro.

The purpose of this study was to investigate whether or not an increase in dietary Mg intake increases Ca absorption in the ovine gastrointestinal tract. In an in vivo experiment, an increase in the infused MgCl2 level (0.0, 25.0 and 75.0 mg Mg x kg BW(-1) x day(-1) with 75.0 mg Ca x kg BW(-1) x day(-1) as CaCl2) into the rumen for ten days significantly decreased fecal excretion but increased urinary excretion (P < 0.05) of Ca in five castrated male sheep. Apparent Ca absorption tended to increase (P = 0.067) whilst the retention and plasma concentration of Ca were not changed. In an in vitro experiment with isolated segments from the rumen, upper jejunum, cecum and upper colon under the presence of an electrochemical gradient, the mucosal to serosal Ca flux rate was significantly greater in the presence of 60.0 mM as compared with 1.2 mM MgCl2 (P < 0.05). From these results, we conclude that the mucosal Mg has the ability to increase the Ca absorption in the gastrointestinal tract in sheep when the dietary Mg level is raised.

Expression of endothelin-1 and vasoactive intestinal contractor genes in mouse organs during the perinatal period.

In an attempt to understand the significance of endothelin-1 (ET-1) and vasoactive intestinal contractor (VIC)/ET-2 peptides in organs during perinatal development, we performed quantitative analysis of ET-1 and VIC gene expression in mouse organs obtained from embryos at days 14 and 17 (E-14 and E-17) of pregnancy, neonates at days 0, 1, 3 and 7 after birth (N-0, -1, -3 and -7), and adult mice (10 weeks old). In intestine, VIC gene expression progressively increased between E-14 and N-1 (approximately 10-fold) and then remained constant into adulthood. ET-1 gene expression exhibited a one-step increase between E-17 and N-0, subsequently remaining constant. In lung, a sharp increase in ET-1 mRNA level (approximately 10-fold) was noticed between E-14 and N-0. The gene expression pattern of VIC, with a peak at N-0, was similar to that of ET-1 although the expression level of VIC was two to three orders of magnitudes lower than that of ET-1. Gene expression patterns of ET-1 and VIC remained nearly constant in brain, heart, liver and kidney throughout the period examined. Considering that the intestinal and pulmonary gene expression levels of both genes reached almost the same level as observed in adult soon after birth, we suggest that these peptides may be involved in the emergence and maintenance of intestinal and pulmonary functions vital after birth.

Effect of aging on gene expression rates of endothelin-1 and endothelin-2/vasoactive intestinal contractor in ethanol-induced gastric mucosal injury of the mouse.

To elucidate the physiological roles of endothelin-1 (ET-1) and endothelin-2 (ET-2)/vasoactive intestinal contractor (VIC) in gastric injury of mice, we measured the gene expression rates of ET-1 and ET-2/VIC in gastric injury induced by ethanol in young (8 weeks) and old (>33 weeks) mice. Mice that were fasted for 24 h were injected with absolute ethanol intragastrically and killed after 1 or 4 h of ethanol exposure. The size of the gastric lesions increased gradually after ethanol exposure and was at its greatest after 4 h, in both young and old mice. The gene expression of ET-1 tended to increase after 1 h and to decrease by 4 h of ethanol exposure in both young and old mice. However, the gene expression of ET-2/VIC in young mice increased significantly after 1 and 4 h of ethanol exposure, whereas the gene expression of ET-2/VIC in the old mice did not change after ethanol exposure. Based on these results, we conclude that aging influences the gene expression of ET-2/VIC but not lesion size or gene expression of ET-1 in ethanol-induced gastric injury in the mouse. We therefore suggest that regulation of gene expression of these two genes differs during the course of aging.