Changes in expression of neuropeptides and their receptors in the hypothalamus and gastrointestinal tract of calorie restricted hens.

The list of orexigenic and anorexigenic peptides, those are known to alter feed intake, is continuously growing. However, most of them are studied in mammalian species. We aimed to investigate plasma level and mRNA expression of the pituitary adenylate cyclase-activating polypeptide (PACAP), gene expression of its receptor (PAC1), furthermore the gene expression of galanin (GAL), neuromedin U (NMU), and its two receptors (NMUR1 and NMUR2) in the hypothalamus, proventriculus, and jejunum of hens exposed to 40% calorie restriction. Feed restriction resulted in a 88% increase in mRNA and a 27% increase in peptide level of PACAP in proventriculus measured with qPCR and RIA, respectively. Increases were found in the gene expression of PAC1 (49%) and NMUR1 (63%) in the hypothalamus. Higher expressions of peptide encoding genes (76% for PACAP, 41% for NMU, 301% for NMUR1 and 308% for GAL, P < 0.05) were recorded in the jejunum of hens exposed to restricted nutrition. The results indicate that PACAP level responds to calorie restriction in the proventriculus and jejunum, but not in the hypothalamus and plasma.

The differences between the localizations of MUC1, MUC5AC, MUC6 and osteopontin in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts.

Mucins are high molecular weight glycoproteins which constitute the major component of the mucus layer and are produce by many epithelial tissues in vertebrates. Osteopontin (OPN) is an adhesive phosphorylated glycoprotein that is expressed by a broad range of tissues and cells. Although gastric mucins MUC1, MUC5AC, MUC6 and OPN have been widely used in histological studies and in diagnostic pathology in order to diagnose gastric carcinomas, their localizations in the stomach of quail have not yet been studied. In this study, the localizations of MUC1, MUC5AC, MUC6 and OPN in the proventriculus and gizzard of Japanese quail during the post-hatching period were compared at light microscope levels by applying immunohistochemical methods. In all ages studied, the immunoreactivity of MUC5AC was present in the lining epithelium of both folds and superficial proventricular glands in the proventriculus, whereas MUC1, MUC6 and OPN reactivity was found in the oxynticopeptic cells of profound proventricular glands. In addition, some cells in the fold epithelium of the proventriculus showed a positive reaction to OPN. The immunoreactivity of MUC1 in gizzard was different from that of MUC5AC. Although MUC5AC was expressed in the cells of both the surface epithelium and profound glands of the gizzard, MUC1 was only localized in the profound glands of the gizzard. However, MUC6 and OPN immunoreactivity was absent in the gizzard. The results indicated that the differences between the localizations of MUC1, MUC5AC, MUC6 and OPN in quail proventriculus and gizzard may be a reflection of functional differences of stomach parts. Although the biological significances of the expressions of MUC1, MUC5AC, MUC6 and OPN in the quail stomach remains unknown, these notable glycoproteins may be associated with barrier function, host defence, and/or secretion.

Ghrelin ligand-receptor mRNA expression in hypothalamus, proventriculus and liver of chicken (Gallus gallus domesticus): studies on ontogeny and feeding condition.

We report here the ontogenic changes in mRNA expression of chicken ghrelin (cGhrelin) and its receptor (cGHS-R1a) and the effects of fasting and refeeding on cGhrelin and cGHS-R1a mRNAs expression in 30-day-old broiler chickens. The level of cGhrelin mRNA in the proventriculus was low from embryo--day 15 (E15) to E19, but dramatically increased at post-hatching-day 2 (P2), then remained constant until P30 and followed by a significant decrease at P44 when there was a diet transition at P31 and thereafter. The decreased level was reversed at P58. Hypothalamic cGhrelin mRNA and proventriculus and hepatic cGHS-R1a mRNA were significantly increased at P30. The cGhrelin mRNA level in the proventriculus significantly increased in response to either 12-h or 36-h fasting but did not decrease after subsequent 12-h refeeding. The level of cGHS-R1a mRNA in the proventriculus was significantly upregulated in response to a 12-h fast but not to a 36-h fast and returned to the control level upon 12-h refeeding. Interestingly, it was apparent that the mRNA levels of both cGhrelin and cGHS-R1a in the liver were upregulated in response to fasting in a time-dependent manner and returned to the control level with subsequent refeeding. These results suggest that the expression pattern of ghrelin and its receptor mRNAs distinctly change in tissues depending on ontogenic stages and feeding states in poultry.

Changes in ghrelin levels of plasma and proventriculus and ghrelin mRNA of proventriculus in fasted and refed layer chicks.

This is a test-report of ghrelin levels in plasma and proventriculus, the glandular portion of the avian stomach, by using a specific radioimmunoassay for acylated ghrelin, as well as the expression of the ghrelin gene in the proventriculus after a 12-h fasting period followed by a 6-h feeding period with 6-day-old layer chicks. After fasting, the plasma ghrelin levels increased from 21.3+/-4.5 to 32.9+/-5.0 fmol/ml, but once refed it returned to the control value. After fasting, the ghrelin mRNA and the peptide levels in the proventriculus increased, and ghrelin mRNA levels remained high but once refed the ghrelin content returned to the control level. Furthermore, in order to examine the effect of increased circulating ghrelin on food intake, a bolus intravenous injection of 500 pmol of chicken ghrelin was given to 8-day-old chicks. The ghrelin injection did not cause any significant changes in food intake. These results indicate that the levels of ghrelin and its mRNA with layer chicks are altered according to the feeding state and this in a similar manner as has been observed in mammals. Unlike in mammals, an increase in circulating ghrelin does not cause the promotion of food intake in chicks.

An 8bp indel in exon 1 of Ghrelin gene associated with chicken growth.

Ghrelin, acts as the endogenous ligand for growth hormone secretagogues receptor (GHS-R), is a novel growth hormone (GH) releasing peptide with reported effects on food intake in chickens. In this study, an 8 bp indel polymorphism in exon 1 of the chicken Ghrelin (cGHRL) gene was genotyped in a F(2) designed full-sib population to analyze its associations with chicken growth and carcass traits. Later, mRNA level in the proventriculus was determined by real-time PCR to reveal the expression feature of cGHRL gene. Result showed that this 8 bp indel was significantly associated with body weight at the age of 28 days (BW28) and 56 days (BW56), eviscerated weight (EW) and leg muscle weight (LMW) (P<0.05), highly significantly associated with hatch weight (HW), BW14, 21, 35, 42, 49, 90 and body length (BL), dressed weight (DW), eviscerated weight with giblet (EWG), wing weight (WW), breast muscle weight (BMW) and head and neck weight (HNW) (P<0.01). Meanwhile, A allele (with 'CTAACCTG') was positive for chicken growth as individuals with AA genotype had the highest value of all traits. Analysis on cGhrelin mRNA level revealed that it differed significantly among individuals with three genotypes (P<0.05). Individuals with AB genotype had the highest mRNA level, whereas that of AA had the lowest one. It was concluded that this 8 bp indel of cGHRL gene was significantly associated with most body weight and body composition traits, and negative effect of endogenous Ghrelin on chicken growth were indicated by this study.

Expression and function of Wnt5a in the development of the glandular stomach in the chicken embryo.

The epithelium of the chicken embryonic glandular stomach (proventriculus) differentiates into both a glandular and a luminal epithelium, the cells of which express specific marker genes. The subsequent formation and differentiation of the glands then proceed under the influence of the mesenchyme. To search for possible candidates for the mesenchymal factors involved, we have now investigated the expression and function of Wnt5a in this process. Our current results show that Wnt5a is expressed in the mesenchyme during active gland formation and that overexpression of this gene in ovo results in the increased and ectopic expression of some of the marker genes of the luminal and glandular epithelia. In particular, the overexpression of Wnt5a markedly enhances the expression of the embryonic chicken pepsinogen gene, a marker of the glandular epithelium, indicating its role as a mesenchymal factor that regulates the differentiation of the proventricular epithelium.

Characterization of turkey and chicken ghrelin genes, and regulation of ghrelin and ghrelin receptor mRNA levels in broiler chickens.

Ghrelin, a peptide hormone produced by the stomach in mammals, stimulates growth hormone release and food intake. Recently, ghrelin was identified and characterized in chicken proventriculus and shown to stimulate growth hormone release but inhibit feed intake. The purpose of this work was to identify and further characterize the ghrelin gene in chickens and in turkeys. Using molecular cloning techniques we have sequenced cDNAs corresponding to chicken (White Leghorn) and turkey ghrelin mRNAs. A total of 844 (chicken) or 869 (turkey) bases including the complete coding regions (CDS), and the 5'- and 3'-untranslated regions (UTRs) were determined. Nucleotide sequence (CDS) predicted a 116 amino acid precursor protein (preproghrelin) for both the chicken and the turkey that demonstrated complete conservation of an N-terminal 'active core' (GSSF) including a serine (position 3 of the mature hormone) known to be a modification (acylation) site important for ghrelin bioactivity. Additional nucleotide sequence was found in the 5'-UTRs of both Leghorn and turkey cDNAs that was not present in broilers or the red jungle fowl. The turkey ghrelin gene, sequenced from genomic DNA templates, contained five exons and four introns, a structure similar to mammalian and chicken ghrelin genes. Ghrelin was highly expressed in proventriculus with much lower levels of expression in other tissues such as pancreas, brain, and intestine. RT-PCR was used to quantify ghrelin mRNA levels relative to 18S rRNA in 3-week-old male broiler chickens. The level of ghrelin mRNA increased in proventriculus in response to fasting but did not decline with subsequent refeeding. Plasma ghrelin levels did not change significantly in response to fasting or refeeding and did not appear to reflect changes in proventriculus ghrelin mRNA levels. Ghrelin mRNA levels declined in broiler pancreas after a 48 h fast and increased upon refeeding. Expression of the gene encoding the receptor for ghrelin (growth hormone secretagogue receptor, GHS-R) and a variant form was detected in a variety of tissues collected from 3-week-old male broiler chickens possibly suggesting autocrine/paracrine effects. These results offer new information about the avian ghrelin and ghrelin receptor genes and the potential role that this system might play in regulating feed intake and energy balance in poultry.

Correlation between Musashi-1 and c-hairy-1 expression and cell proliferation activity in the developing intestine and stomach of both chicken and mouse.

Musashi-1 (Msi-1) is an RNA-binding protein that plays key roles in the maintenance of neural stem cell states and in their differentiation into neural cells. Msi-1 has also been proposed as a candidate marker gene of mammalian intestinal stem cells and their immediate lineages. In this study, we examined Msi-1 expression in the small intestine and the stomach of both chicken and mouse during embryonic, fetal and postnatal development. In addition, we analyzed the expression of c-hairy-1, a chicken homologue of mouse Hes1, and assessed the proliferative activity of the cells expressing both of these factors. Significantly, during the development of these digestive organs in both species Msi-1 expression showed dynamic changes, suggesting that it is important for digestive organ development, particularly for epithelial differentiation. Based on our observations of the expression patterns of Msi-1 and c-hairy-1 in the adult small intestine, we speculate that Msi-1 is also a stem cell marker of the chicken small intestinal epithelium.

Exogenous administration of octanoic acid accelerates octanoylated ghrelin production in the proventriculus of neonatal chicks.

Ghrelin is modified by fatty acid at the third serine residue. In this study, derivation of fatty acid for acylation of ghrelin was investigated using a hatchling chicken model. We first studied ghrelin gene expression and production in the neonatal chick proventriculus and then investigated the effect of exogenous octanoic acid (OA) administration on acylated ghrelin production. In a free-feeding condition on day 2.5 after hatching, the density of ghrelin mRNA-expressing (ghrelin-ex) cells was greater than that of ghrelin-immunopositive (ghrelin-ip) cells, but no difference was found between those densities in adult chickens. Intraperitoneal or oral administration of OA for a few days significantly increased the density of ghrelin-ip cells without any changes in ghrelin-ex cells and elevated only octanoylated ghrelin levels in the proventriculus. The results indicate that fatty acid absorbed from food is directly utilized in acylated ghrelin production in the chicken.

Topography and neurochemistry of the enteric ganglia in the proventriculus of the duck (Anas platyrhynchos).

The topographical distribution of the enteric ganglia has been investigated in the proventriculus of the duck using protein gene product 9.5 (PGP 9.5) immunohistochemistry. Myenteric ganglia were usually located between the outer longitudinal and the inner circular muscle layer. Submucous ganglia were sparsely distributed and seemed to be substituted by ganglia located in the tunica mucosa. The neurochemical profile of proventricular ganglion cells was also investigated using nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-histochemistry and pituitary adenylate cyclase activating peptide (PACAP)/galanin (Gal) double-labelling immunohistochemistry. The majority of mucosal ganglion cells were shown to contain the NADPH-d enzyme and both the investigated peptides. These findings provide evidence for the presence of a mucosal ganglionated plexus in the glandular stomach of birds. Moreover, the neurochemical characteristics of this plexus suggest that it plays an important role in regulating several mucosal functions and, in particular, the production and the composition of the gastric juice.

Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken.

Ghrelin was isolated from the rat stomach as an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and has been found in the gastrointestinal tract of many vertebrates. Although the sequence and structure of chicken ghrelin has recently been determined, morphological characteristics of ghrelin cells in the chicken gastrointestinal tract are still obscure. In this study, we investigated ghrelin expression and distribution of ghrelin-producing cells in the hatching and adult chicken gastrointestinal tract by RT-PCR, immunohistochemistry and in situ hybridization. Ghrelin mRNA expression was observed mainly in the proventriculus in the hatching chicken and in the proventriculus, pylorus and duodenum of the adult chicken by RT-PCR. Ghrelin-immunopositive (ghrelin-ip) cells in the proventriculus were located at the mucosal layer but not in the myenteric plexus or smooth muscle layer. The number of ghrelin-ip cells in the adult chicken was greater than that in the hatching chicken. Interestingly, in the adult chicken, the number of ghrelin-ip cells were almost the same as that of ghrelin mRNA-expressing (ghrelin-ex) cells; however, in the hatching chicken, the number of ghrelin-ex cells was greater than that of ghrelin-ip cells. These results clearly demonstrate that ghrelin-producing cells exist in the chicken gastrointestinal tract, especially in the proventriculus, from hatching to adult stages of development, as well as in mammals.

Ontogeny of PACAP immunoreactivity in extrinsic and intrinsic innervation of chicken gut.

An immunohistochemical study was conducted on the ontogeny of pituitary adenylate cyclase-activating polypeptide-27 (PACAP) immunoreactive elements within the extrinsic and intrinsic nerve supply of the chicken embryo gut. The first PACAP-immunoreactivity was detected in the extrinsic nerve supply at E 4 within the pharyngeal region and the primary sympathetic chain. At E 5.5 it appeared in the vagus nerve, the spinal cord, the secondary sympathetic chain, some perivascular plexuses and the Remak ganglion. In the intrinsic nerve supply, the first PACAP-immunoreactive elements were shown at E 4.5-E 5 in the mesenchymal bud of the proventriculus/gizzard. Then they gradually appeared also cranially and caudally both in myenteric and submucous plexuses.

Histochemical characterization of the mucins of the epithelial cells in the chick embryo proventriculus.

The histochemical characteristics of the epithelial mucins, which are produced by the lining epithelium, the superficial glands and the compound glands of the proventriculus were investigated in the chick embryo from the 7th day of incubation to hatching. Results showed that the time of appearance of the carboxylated, sulphated and neutral mucosubstances differed at the three investigated sites. From the 13th to the 19th day of incubation, intense production of the various mucins was detected at the three sites. However, the modality of intracytoplasmatic storage and the production of mucins in both the cells of the lining epithelium and the superficial glands appeared to differ from those observed in the cells of the compound glands. At the 20th and 21st day, the mucins were primarily produced by the lining epithelium and the superficial glands, whereas production diminished in the compound glands. Furthermore, from the 7th to the 17th day of incubation, the cells of the principal ducts of the compound glands showed the same histochemical characteristics and modality of production as those of the glandular cells of the compound glands. On the other hand, from the 18th day to hatching, these cells assumed the same characteristics as those of the lining epithelium and of the superficial glands.

Development of the diffuse endocrine system in the chicken proventriculus.

The development of endocrine cells in the chicken proventriculus has been investigated using light- and electron-microscopy in conjunction with silver and immunocytochemical techniques. The first morphologically detectable endocrine cells were found in 5-day-old embryos by electron microscopy. From the 9th to the 13th day, endocrine cells in contact with the lumen of the organ could be detected both by electron and light (silver impregnation) microscopy. The number of open-type endocrine cells progressively decreased and the number of closed-type increased after this stage. Until the 16th day, endocrine cells were located exclusively in the luminal epithelium, but afterwards they appeared in progressively greater numbers in the compound glands. After hatching, long cytoplasmic processes could be seen in the endocrine cells. Immunoreactivities to regulatory substances appeared in the following order: serotonin (day-14), avian pancreatic polypeptide, glucagon and somatostatin (day-16), bombesin and neurotensin (day-18), and finally, met-enkephalin (day-21).

Immunocytochemical and ultrastructural characterization of endocrine cells in chicken proventriculus.

The endocrine cells of the chicken proventriculus were investigated immunocytochemically, using the peroxidase-antiperoxidase technique on paraffin and semithin sections for light microscopy, and immunogold staining in osmium-fixed material for electron microscopy. The fixation procedure also allowed a detailed ultrastructural investigation. Twenty-three antisera were tested and 7 immunoreactive cell-types were identified: D-cells containing somatostatin-like peptide; EG-cells immunoreactive to anti-glucagon, anti-GLP1 and anti-neurotensin; NT-cells labelled only with anti-neurotensin; BN-cells containing bombesin-like material; ENK-cells showing met-enkephalin immunoreactivity; EC-cells reactive to anti-serotonin; and APP-cells positive to anti-avian pancreatic polypeptide. In addition, enterochromaffin-like (ECL) cells, were also detected by electron microscopy. The presence of ENK-cells and the ultrastructure of these and NT-cells are described for the first time in chicken proventriculus, and glucagon. GLP1 and neurotensin are shown to be colocalized in the EG-cells.

Isolation, sequence and biosynthetic significance of a novel fragment of gastrin-releasing peptide from chicken proventriculus.

The isolation of bombesin-related peptides in chicken proventriculus was monitored by radioimmunoassay using a C-terminal specific bombesin antibody. Two peptides were identified, one corresponded to the 27-residue, chicken gastrin-releasing peptide (GRP-27) previously identified; the other corresponded to its C-terminal hexapeptide. Chicken GRP-27 stimulated pancreatic and gastric acid secretion in anaesthetized turkeys, but the hexapeptide was inactive. No evidence could be found to suggest that the hexapeptide was an artifact of degradation generated during extraction or isolation. It is proposed that the hexapeptide is produced either by chymotryptic-like cleavage of GRP-27 or by trypsin-like cleavage followed by two cycles of dipeptidylaminopeptidase cleavage. This type of biosynthetic processing may be more common than formerly supposed.

Gizzerosine raises the intracellular cyclic adenosine-3',5'-monophosphate level in isolated chicken proventriculus.

Gizzerosine, which was originally found in fish meal, is a compound that causes gizzard erosion and ulceration in chicks. The action of gizzerosine on the isolated cells of chicken proventriculus was studied in the present investigations. Gizzerosine increased intracellular cyclic adenosine-3',5'-monophosphate (cAMP) levels, reaching a plateau within 30 min. Similar maxima of cAMP level were observed in the presence of histamine or gizzerosine. However, the potency of gizzerosine was approximately 1,000-fold higher than that of histamine. The action of gizzerosine was depressed by cimetidine, a histamine H2-receptor antagonist, but not by pyrilamine, a histamine H1-receptor antagonist, indicating that gizzerosine is a very strong histamine H2-receptor agonist. The mucosal cells isolated from the proventriculus by the present procedure had both histamine H1 and H2-receptors. Gizzerosine showed a higher affinity to the cell surface histamine receptor than histamine. These results partly explain the potent activity of gizzerosine in inducing gastric acid secretion and causing gizzard erosion and ulceration in chickens.

Effect of gizzerosine on acid secretion by isolated mucosal cells of chicken proventriculus.

Mucosal cells of the chicken proventriculus were isolated by a collagenase perfusion method and O2 uptake by the isolated cells was measured as an index of the activity of gastric acid secretion. Oxygen consumption was enhanced by histamine; this effect was augmented by the coexistence of isobutylmethylxanthine, an inhibitor of cyclic adenosine 5.-monophosphate (AMP) phosphodiesterase, and suppressed by imidazole, an activator of the enzyme. The action of histamine was inhibited by cimetidine, an antagonist of the histamine H2 receptor. These results indicate that the isolated cells retained the capacity to take up O2, responding to histamine via the H2 receptor and probably the cyclic AMP level. Gizzerosine (2-amino-9-(4-imidazolyl)-7-azanonanoic acid) also stimulated O2 consumption by the isolated cells. The effect of gizzerosine was cancelled by cimetidine, suggesting that the mechanism by which gizzerosine acts on the mucosal cells is similar to that of histamine action. These observations are consistent with our previous presumption that gizzerosine causes gizzard erosion by enhancing gastric acid secretion in chickens.

The relative frequency and topographical distribution of somatostatin-, GRP-, APP-, glucagon-, 5-HT-, and neurotensin-immunoreactive cells in the proventriculus of seven species of birds.

The relative frequency and topographical distribution of proventricular endocrine cells were examined immunohistochemically in seven species of birds: common finch, pigeon, quail, chicken, duck, gull and kite. Gastrin releasing peptide (GRP), somatostatin-, avian pancreatic polypeptide (APP)-, glucagon-, 5-hydroxytryptamine (5-HT)- and neurotensin-immunoreactive cells were observed in this study. GRP- and somatostatin-immunoreactive cells were found in all species examined. All six kinds of immunoreactive cells were found with varying frequency in the pigeon, quail and gull, but not all immunoreactives were found in the other species examined. Species differences with regard to the relative frequency and topographical distribution of proventricular endocrine cells were observed.

Ontogenesis of bombesin-like immunoreactive cells in the chicken proventriculus.

The distribution and morphological behaviour of bombesin like immunoreactive cells in the proventriculus of chick embryos, newborns and adults were investigated by indirect immunoperoxidase procedure. The first immunoreactive cells appear around the 11th day of incubation. Initially few, they increase progressively to reach a peak around the 18th day. Throughout the embryonic period almost all immunoreactive cells are column-shaped, display a typical "open" behaviour, and are situated among other superficial lining epithelial cells. Around hatching time, these "open" cells suddenly decrease in number while another immunoreactive "closed" cell type becomes more evident in a deeper location. In newborns and adults, the majority of immunoreactive cells are oval shaped and are characteristically situated near the neck of lobular glands; they seem to display a "closed" behaviour. The results demonstrate two different kinds of peptide-containing cells which probably exert different functions. During the embryonic period, in fact, they resemble a chemioreceptor, but in postnatal life they appear as a paracrine/endocrine elements.