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Presence and distribution of ghrelin-immunopositive cells in the chicken gastrointestinal tract
ARTICLE IN PRESS Acta histochemica 107 (2005) 3—9
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Presence and distribution of ghrelinimmunopositive cells in the chicken gastrointestinal tract Simona Neglia, Nadia Arcamone, Vincenzo Esposito, Giuliana Gargiulo, Paolo de Girolamo Department of Biological Structures, Functions and Technologies, University of Naples ‘‘Federico II’’, Via Veterinaria 1, I-80137 Naples, Italy Received 16 June 2004; received in revised form 15 October 2004; accepted 17 December 2004
KEYWORDS Ghrelin; Chicken; Gastrointestinal tract; Immunohistochemistry; Western blot
Summary The presence and distribution patterns of ghrelin, a gastric acylated peptide, were studied in the entire gastrointestinal tract of the chicken (Gallus domesticus) using the peroxidase–antiperoxidase immunohistochemical method, western blot analysis and a specific antibody against the C-terminal region of rat ghrelin. Ghrelinimmunopositive cells were observed in the mucosal layer of all segments examined. The largest numbers of ghrelin-positive cells were located at the base of lobuli of the proventriculus gland, along villi of the intestines and in crypts of the duodenum. Lower numbers of ghrelin-immunostained cells were located in crypts of jejunum and ileum and only few ghrelin-immunostained cells were detected at the base of crypts of the large intestine. Closed and open types of cells were observed in all segments. Western blot analysis confirmed the presence of ghrelin-like protein in the entire chicken gastrointestinal tract. The anatomical distribution patterns and the morphological characteristics of chicken ghrelin-positive cells suggest that they are endocrine cells. Furthermore, it is concluded that ghrelin shows a high degree of preservation during evolution from non-mammalian vertebrates to mammals. & 2005 Elsevier GmbH. All rights reserved.
Introduction Growth-hormone secretagogues (GHSs) are small synthetic peptides and non-peptidyl molecules that
stimulate growth hormone (GH) release both in vivo and in vitro from the anterior pitituary gland via a G-protein-coupled receptor (GHS-R; Bowers, 1998). Recently, a novel GHS-R ligand was isolated and
Corresponding author. Tel.: +39 081 2536114; fax: +39 081 2536097.
E-mail address: [email protected] (P. de Girolamo). 0065-1281/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2004.12.001
ARTICLE IN PRESS 4 characterized in rat (Kojima et al., 1999) and human (Date et al., 2000) stomach and was called ghrelin. Ghrelin is a 28-amino-acid peptide in which the third serine residue (Ser3) shows an n-octanoyl modification, essential for its biological activity (Kojima et al., 1999). Accumulating evidence suggests that, besides a potent GH-releasing action, ghrelin regulates food intake, energy balance, body weight, gastric motility and acid secretion, function of the endocrine pancreas and glucose metabolism (Kamegai et al., 2000; Masuda et al., 2000; Wren et al., 2000; Broglio et al., 2001; Date et al., 2001; Kamegai et al., 2001; Nakazato et al., 2001). Ghrelin is mainly produced in the stomach (Kojima et al., 1999; Ariyasu et al., 2001). Besides the gastroenteric tract, ghrelin is also expressed in various peripheral tissues (Mori et al., 2000; Gualillo et al., 2001; Volante et al., 2002; Andreis et al., 2003) and in the arcuate nucleus (ARC) of the hypothalamus, an appetite regulating centre (Lu et al., 2002). In situ hybridization and immunohistochemical studies indicated that ghrelin-producing cells are a distinct type of endocrine cells, known as X/A-like cells, that are localized in the mucosal epithelium of corpus and fundus of the stomach (Date et al., 2000; Dornonville de la Cour et al., 2001; Sakata et al., 2002). Few studies have been carried out on ghrelin expression in non-mammalian vertebrates. Recently, ghrelin was found in the brain and stomach of both frog (Galas et al., 2002) and chicken (Kaiya et al., 2002). Chicken ghrelin consists of 26 amino acids with n-octanoyl modification at Ser3 and has the same sequence in the N-terminal region as in as the human and rat form. In fact, administration of chicken ghrelin increases plasma GH levels in both rats and chicks, and increases plasma corticosterone levels in growing chicks, at a lower dose than mammalian ghrelin (Kaiya et al., 2002). In a previous study, Ahmed and Harvey (2002) showed ghrelin-immunostaining in the chicken hypothalamus with an antibody developed especially for radioimmunoassays and raised in rabbit that recognizes the C-terminal region of rat ghrelin (RAB-031-31; Phoenix Pharmaceuticals, Belmont, CA, USA). Recently, Wada et al. (2003) demonstrated ghrelin-positive cells in the chicken proventriculus using an N-terminal region-recognizing antibody only. Because mammalian ghrelin is also expressed in the intestines (Date et al., 2000), we studied distribution patterns of ghrelin in both the proventriculus and small and large intestines of chicken using immunohistochemistry and western blotting and a specific polyclonal antibody against the C-terminal region of rat ghrelin.
S. Neglia et al.
Material and methods Immunohistochemistry Three-month-old chickens (Gallus domesticus) of both sexes were used in the present study. The gastrointestinal tract was sectioned in small samples of the following segments: proventriculus, ventriculus, duodenum, jejunum, ileum, caecum and colorectum. The tissues were immediately fixed in Bouin’s solution for 24 h, dehydrated in a graded series of alcohol, embedded in paraffin wax and transverse 6–7-mm-thick serial sections were cut. After dewaxing, sections were processed for immunohistochemistry using the peroxidase–antiperoxidase (PAP) method according to Sternberger (1986). Endogenous peroxidase activity was blocked by treating the sections with 0.3% hydrogen peroxide for 20 min at room temperature (RT) and then rinsed for 15 min in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Sections were incubated for 30 min at RT with normal goat serum (dilution, 1:5; Jackson Immunoresearch Laboratories, West Grove, PA, USA) and then with primary antibody (18 h in a moist dark chamber at 4 1C). The primary antibody, a rabbit antibody that recognizes the C-terminal region of rat ghrelin and cross-reacts with chicken ghrelin (H-031-31; Phoenix Pharmaceuticals), was diluted 1:750. After incubation, sections were rinsed in PBS for 15 min and, incubated for 30 min at RT with goat anti-rabbit IgG (dilution, 1:50; Vector Laboratories, Burlingame, CA, USA). Subsequently, sections were rinsed in PBS for 15 min and then incubated for 30 min at RT with PAP complex (dilution, 1:100; Dako, CA, USA). Peroxidase activity was visualized with a solution of 10 mg 3-30 diaminobenzidine tetrahydrocloride (DAB; Sigma, St. Louis, MO, USA) in 15 ml 0.5 M Tris buffer, pH 7.6, containing 0.03% hydrogen peroxide. Specificity of the immunohistochemical staining reaction was tested as follows: (1) substitution of either the ghrelin antibody or the anti-rabbit IgG, or the PAP complex by PBS (negative controls); (2) application of rat stomach tissue known to be positive for ghrelin (positive controls); (3) previous absorption of the primary antibody with homologous antigen (031-31; Phoenix Pharmaceuticals; up to 50 mg/ml antibody in the final dilution). Sections were examined with a Aristoplan light microscope (Leica, Wetzlar, Germany) equipped with a DC300F digital camera (Leica).
Western blot analysis Samples of all parts of the gastrointestinal tract were homogenized (1:2 w/v) with a Potter
ARTICLE IN PRESS Ghrelin in the chicken gastrointestinal tract homogenizer in cold RIPA buffer [50 mM Tris-HCl, pH 8.0, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 150 mM NaCl and 0.5% deoxycholic acid] containing 1 mM phenylmethylsulfonyl fluoride (Sigma) and 100 inhibitor cocktail. Then, suspensions were centrifuged at 14,000g for 15 min at 4 1C and the protein concentrations of the resulting pellets, divided in equal aliquots, were determined by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were separated by electrophoresis on 10% SDS-polyacrylamide gels (NuPAGE Novex 10% bis-Tris gels; Invitrogen, Carlsbad, CA, USA) and, then, gels were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT in PBS-Tween buffer (0.01 M PBS and 0.05% Tween-20) containing 5% non-fat milk and incubated for 1.5 h at RT with the primary antibody, as described above, diluted 1:1500 in PBS-Tween. After washing, membranes were incubated for 1 h at RT with polyclonal goat anti-rabbit IgG conjugated with horseradish peroxidase (dilution, 1:2000; DAKO). Membranes were incubated in chemoluminescence reagent (ECL; Amersham, Little Chalfont, England) to visualize immunocomplexes, and were exposed for 1–30 s to hyperfilm.
Results Immunohistochemistry Ghrelin-immunopositive cells (ghrelin-ic) were observed in mucosal layers of all gastrointestinal tract segments examined: proventriculus, duodenum, jejunum, ileum, caecum and colo-rectum. High numbers of strongly stained ghrelin-ic were found at the base of glandular lobuli of the proventriculus (Fig. 1A and B). In contrast, few ghrelin-ic were observed in the basal zone of proventriculus plicae (Fig. 1C), which showed an intense immunostaining that was restricted to the basal region of the cytoplasm. These cells had a small round shape and were considered to be of the closed cell type (Fig. 1D). Few ghrelin-ic were of the open cell type that showed an elongated shape and apical cytoplasmic processes in contact with the lumen (Fig. 1E). Many ghrelin-ic were found to be scattered in epithelium of crypts (Fig. 2A) and along villi of the duodenum (Fig. 2B). Closed and open cell types (Fig. 2C) were both observed in this intestinal segment as well. Fewer ghrelin-ic were found in epithelia of crypts of the jejunum and ileum and generally they appeared to be of the closed cell
5 type (Fig. 2D). Very few ghrelin-ic, and especially of the closed cell type and triangular in shape, were detected in crypts of the large intestine (Fig. 2E). Negative controls and sections stained with preabsorbed antibodies did not show any specific staining. Specificity of immunostaining was confirmed by the positive controls (data not shown).
Western blot analysis Ghrelin-like protein was detected by western blot analysis of homogenates of all segments of the chicken gastrointestinal tract which was in agreement with our immunohistochemical data. The ghrelin-like protein that was detected had an estimated molecular mass of approximately 44–46 kDa (Fig. 3).
Discussion In the present study, we demonstrated the occurrence and distribution patterns of ghrelin-like protein in the entire chicken gastrointestinal tract using western blot analysis and peroxidase–antiperoxidase immunohistochemistry respectively. Our findings are not in agreement with a recent study of Ahmed and Harvey (2002) who found ghrelin immunostaining to be present in the chicken brain only and not in the gastrointestinal tract. This discrepancy may be due to a difference in antibodies used in the two studies. In fact, both antibodies recognize the C-terminal region of ghrelin, but we used an antibody especially developed for immunohistochemistry, whereas Ahmed and Harvey (2002) used an antibody that was developed especially for radioimmunoassays. The presence of ghrelin-ic in the chicken gastrointestinal tract suggests that this peptide may act as a gastrointestinal hormone besides its function as a neuropeptide (Ahmed and Harvey, 2002). Recently, Wada et al. (2003) demonstrated ghrelin immunostaining in the mucosal layer of the chicken proventriculus. In agreement with these results, we observed the largest number of ghrelin-ic to be present in the chicken proventriculus which corresponds to the gastric fundus in mammals. Somewhat lower numbers of ghrelin-ic were found in jejunum, ileum and large intestine. Our findings agree with distribution patterns reported in human and rat digestive tract. In fact, Kojima et al. (1999) and Date et al. (2000) found ghrelin-ic to be mainly located in the oxyntic mucosa of gastric fundus. These cells were present
ARTICLE IN PRESS 6
S. Neglia et al.
Figure 1. Micrographs of ghrelin-immunostained cells (ghrelin-ic) in the chicken proventriculus. (A and B) Ghrelin-ic (arrows) in the mucosal layer at the base of glandular lobuli. (C) Closed type of ghrelin-ic (arrow) in the basal zone of a plica. (D) Open type of ghrelin-ic (arrow) that is elongated in shape, with an apical cytoplasmic process (arrowhead) in contact with the lumen. (E) Closed type of ghrelin-ic (arrows) without connection with the lumen, round in shape with intense immunostaining located at the basal side of the cytoplasm. Magnifications: A, 80; B, 150; C, 260; D, 450; E, 220.
in larger numbers in the small intestines than in the large intestines. Therefore, it is likely that ghrelin has effects on various gastrointestinal functions such as motility of the gut wall and gastric acid secretion both in rat and chicken. Furthermore, ghrelin probably acts on the pituitary via the systemic circulation, contributing to the regulation of GH secretion and feeding behaviour.
Kojima et al. (1999) and Date et al. (2000) have also characterized ghrelin-producing cells in the digestive tract of human and rat by ultrastructural and immunohistochemical means. These endocrine cells, which are typical of the oxyntic mucosa of both species, correspond to X/A-like cells. Our findings on distribution patterns and morphologic characteristics of chicken ghrelin-ic suggest that
ARTICLE IN PRESS Ghrelin in the chicken gastrointestinal tract
7
Figure 2. Micrographs of ghrelin-ic in chicken small and large intestine. (A) Ghrelin-ic (arrows) in crypts of the duodenum. (B) Ghrelin-ic (arrows) along villi of the duodenum. (C) Closed type of ghrelin-ic (arrows) in crypts of jejunum. (D) Closed type of ghrelin-ic (arrows), triangular in shape, in the crypts of the colo-rectum. (E) Duodenum. Open type of ghrelin-ic cell (arrow), elongated in shape, with an apical cytoplasmatic process (arrowhead) in contact with the lumen. Magnifications: A, 60; B, 130; C, 300; D, 280; E, 450.
they correspond to endocrine cells. However, further investigations are required in order to identify the specific cell types on the basis of the current classification (Solcia et al., 2000), although the types and distribution patterns of avian gut endocrine cells have been described in a detailed manner and have been compared with the situation in mammals (Rawdon and Andrew, 1999). Additionally, we demonstrated that these cells are of the open cell type and closed cell type in their relationship with the lumen. Some studies indicate that the open cell type is functionally affected by the food content and pH in the lumen, whereas the second cell type is affected by
hormones, local factors, neuronal stimulation and/or mechanical stress (Solcia et al., 2000). The occurrence of the open and closed cell type in the chicken gastrointestinal tract suggests that various factors regulate ghrelin synthesis and the cells have specific physiological effect in each region. So far, the presence and structure of ghrelin-like protein have been investigated not only in the chicken but also in others non-mammalian vertebrates, such as amphibians (Kaiya et al., 2001; Galas et al., 2002) and fish (Uniappan et al., 2002; Parhar et al., 2003). The amino acid sequences of ghrelin in the different animal species examined,
ARTICLE IN PRESS 8
Figure 3. Western blot analysis of ghrelin in extracts of chicken gastrointestinal tract. Lane 1, protein markers; lane 2, homogenate of gastrointestinal tract showing a strongly stained band at approximately 44–46 kDa.
show a high preservation of the ‘‘active core’’ (GSSF), essential for its molecular biological activity, in tilapia, eel and chicken (Kaiya et al., 2002; 2003a, b), but not bullfrog (GLTF; Kaiya et al., 2001) and goldfish (GTSF; Uniappan et al., 2002). Interestingly, these first four amino acids are preserved in human and rat as well (Kojima et al., 1999). These findings show that ghrelin is phylogenetically highly preserved from non-mammalian species to mammals not only in its amino acid sequence but also in its distribution patterns.
References Ahmed S, Harvey S. Ghrelin: a hypotalamic GH-releasing factor in domestic fowl (Gallus domesticus). J Endocrinol 2002;172:117–25. Andreis PG, Malendowicz LK, Trejter M, Neri G, Spinazzi R, Rossi GP, Nussdorfer GG. Ghrelin and growth hormone secretagogue receptor are expressed in the rat adrenal cortex:evidence that ghrelin stimulates the growth, but not the secretory activity of adrenal cells. FEBS Lett 2003;536:173–9. Ariyasu H, Takaya K, Tagami T, Ogawa Y, Hosoda K, Akamizu T, Suda M, Koh T, Natsui K, Toyooka S, Shirakami G, Usui T, Shimatsu A, Doi K, Hosoda H, Kojima M, Kangawa K, Nakao K. Stomach is a major source of circulating ghrelin, and feeling state determines plasma ghrelin-like immunoreactivity in humans. J Clin Endocrinol Metab 2001;86:4753–8.
S. Neglia et al. Bowers CY. Growth hormone-releasing peptide (GHRP). Cell Mol Life Sci 1998;54:1316–29. Broglio F, Arvat E, Benso A, Gottero C, Cuccioli G, Papotti M, van der Lely AJ, Deghenghi R, Ghigo E. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001;86:5083–6. Date Y, Kojima M, Hosoda H, Sawaguchi A, Mondal MS, Suganuma T, Matsukura S, Kangawa K, Nakazato M. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and human. Endocrinology 2000;141:4255–66. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 2001;280:904–7. Dornonville de la Cour C, Bjorkqvist M, Sandvik AK, Bakke I, Zhao CM, Chen D, Hakanson R. A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control. Regul Pept 2001;99:141–50. Galas L, Chartrel N, Kojima M, Kangawa K, Vaudry H. Immunohistochemical localization and biochemical characterization of ghrelin in the brain and stomach of the frog Rana esculenta. J Comp Neurol 2002; 450:34–44. Gualillo O, Caminos JE, Blanco M, Garcia-Caballero T, Kojima M, Kangawa K, Dieguez C, Casanueva FF. Ghrelin, a novel placental-derived hormone. Endocrinology 2001;142:788–94. Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I. Central effect of ghrelin, an endogenous growth hormone secretagogue, on hypothalamic peptide gene expression. Endocrinology 2000;141: 4797–800. Kamegai J, Tamura H, Shimizu T, Ishii S, Sugihara H, Wakabayashi I. Chronic central infusion of ghrelin increases hypotalamic neuropeptide Y and agoutirelated protein mRNA levels and body weight in rats. Diabetes 2001;50:2438–43. Kaiya H, Kojima M, Hosoda H, Koda A, Yamamoto K, Kitajima Y, Matsumoto M, Minamitake Y, Kikuyama S, Kangawa K. Bullfrog ghrelin is modified by n-octanoic acid at the third threonine residue. J Biol Chem 2001;276:40,441–8. Kaiya H, Van Der Geyten S, Kojima M, Hosoda H, Kitajima Y, Matsumoto M, Geelissen S, Darras VM, Kangawa K. Chicken ghrelin: purification, cDNA cloning and biological activity. Endocrinology 2002;143:3454–63. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K. Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus. Comp Biochem Physiol B. Biochem Mol Biol 2003a;135:421–9. Kaiya H, Kojima M, Hosoda H, Riley LG, Hirano T, Grau EG, Kangawa K. Amidated fish ghrelin: purification, cDNA cloning in the Japanese eel and its biological activity. J Endocrinol 2003b;176(3):415–23. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing
ARTICLE IN PRESS Ghrelin in the chicken gastrointestinal tract acylated peptide from stomach. Nature 1999;402: 656–60. Lu S, Guan JL, Wang QP, Uehara K, Yamada S, Goto N, Date Y, Nakazato M, Kojima M, Kangawa K, Shioda S. Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus. Neurosci Lett 2002;321:157–60. Masuda Y, Tanaka T, Inomata N, Ohnuma N, Tanaka S, Itoh Z, Hosoda H, Kojima M, Kangawa K. Ghrelin stimulates gastric acid secretion and motility in rats. Biochem Biophys Res Commun 2000;276:905–8. Mori K, Yoshimoto A, Takaya K, Hosoda K, Ariyasu H, Yahata K, Mukoyama M, Sugawara A, Hosoda H, Kojima M, Kangawa K, Nakao K. Kidney produces a novel acylated peptide, ghrelin. FEBS Lett 2000;486:213–6. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001;409:194–8. Parhar IS, Sato H, Sakuma Y. Ghrelin gene fish is modulated by sex and development. Biochem Biophys Res Commun 2003;305:169–75. Rawdon BB, Andrew A. Gut endocrine cells in birds: an overview, with particular reference to the chemistry of gut peptides and the distribution, ontogeny, embryonic origin and differentiation of the endocrine cells. Prog Histochem Cytochem 1999;34:3–82. Sakata I, Nakamura K, Yamakazi M, Matsubara M, Hayashi Y, Kangawa K, Sakai T. Ghrelin-producing cells exist as
9 two types of cells, closed- and opened-type cells, in the rat gastrointestinal tract. Peptides 2002;23:531–6. Solcia E, Rindi G, Buffa R, Fiocca R, Capella C. Gastric endocrine cells: types, function and growth. Regul Pept 2000;93:31–5. Sternberger LA. Immunocytochemistry, 3rd ed. New York, NY, USA: Wiley; 1986. Uniappan S, Lin X, Cervini L, Rivier J, Kaya H, Kangawa K, Peter RE. Goldfish ghrelin: molecular characterization of the complementary deoxyribonucleic acid, partial gene structure and evidence for its stimulatory role in food intake. Endocrinology 2002;143:4143–6. Volante M, Allia E, Gugliotta P, Funaro A, Broglio F, Deghenghi R, Muccioli G, Ghigo E, Papotti M. Expression of ghrelin and of GHS receptor by pancreatic islet cells and related endocrine tumors. J Clin Endocrinol Metab 2002;279:E695–706. Wada R, Sakata I, Kaiya H, Nakamura K, Hayashi Y, Kangawa K, Sakai T. Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adult chicken. Regul Pept 2003;111: 123–8. Wren AM, Small CJ, Ward HL, Murphy KG, Dakin CL, Taheri S, Kennedy AR, Roberts GH, Morgan DCA, Ghatei MA, Bloom SR. The novel hypothalamic peptide ghrelin stimulates food intake and growth hormone secretion. Endocrinology 2000;141:4325–8.
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Presence and distribution of ghrelinimmunopositive cells in the chicken gastrointestinal tract Simona Neglia, Nadia Arcamone, Vincenzo Esposito, Giuliana Gargiulo, Paolo de Girolamo Department of Biological Structures, Functions and Technologies, University of Naples ‘‘Federico II’’, Via Veterinaria 1, I-80137 Naples, Italy Received 16 June 2004; received in revised form 15 October 2004; accepted 17 December 2004
KEYWORDS Ghrelin; Chicken; Gastrointestinal tract; Immunohistochemistry; Western blot
Summary The presence and distribution patterns of ghrelin, a gastric acylated peptide, were studied in the entire gastrointestinal tract of the chicken (Gallus domesticus) using the peroxidase–antiperoxidase immunohistochemical method, western blot analysis and a specific antibody against the C-terminal region of rat ghrelin. Ghrelinimmunopositive cells were observed in the mucosal layer of all segments examined. The largest numbers of ghrelin-positive cells were located at the base of lobuli of the proventriculus gland, along villi of the intestines and in crypts of the duodenum. Lower numbers of ghrelin-immunostained cells were located in crypts of jejunum and ileum and only few ghrelin-immunostained cells were detected at the base of crypts of the large intestine. Closed and open types of cells were observed in all segments. Western blot analysis confirmed the presence of ghrelin-like protein in the entire chicken gastrointestinal tract. The anatomical distribution patterns and the morphological characteristics of chicken ghrelin-positive cells suggest that they are endocrine cells. Furthermore, it is concluded that ghrelin shows a high degree of preservation during evolution from non-mammalian vertebrates to mammals. & 2005 Elsevier GmbH. All rights reserved.
Introduction Growth-hormone secretagogues (GHSs) are small synthetic peptides and non-peptidyl molecules that
stimulate growth hormone (GH) release both in vivo and in vitro from the anterior pitituary gland via a G-protein-coupled receptor (GHS-R; Bowers, 1998). Recently, a novel GHS-R ligand was isolated and
Corresponding author. Tel.: +39 081 2536114; fax: +39 081 2536097.
E-mail address: [email protected] (P. de Girolamo). 0065-1281/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.acthis.2004.12.001
ARTICLE IN PRESS 4 characterized in rat (Kojima et al., 1999) and human (Date et al., 2000) stomach and was called ghrelin. Ghrelin is a 28-amino-acid peptide in which the third serine residue (Ser3) shows an n-octanoyl modification, essential for its biological activity (Kojima et al., 1999). Accumulating evidence suggests that, besides a potent GH-releasing action, ghrelin regulates food intake, energy balance, body weight, gastric motility and acid secretion, function of the endocrine pancreas and glucose metabolism (Kamegai et al., 2000; Masuda et al., 2000; Wren et al., 2000; Broglio et al., 2001; Date et al., 2001; Kamegai et al., 2001; Nakazato et al., 2001). Ghrelin is mainly produced in the stomach (Kojima et al., 1999; Ariyasu et al., 2001). Besides the gastroenteric tract, ghrelin is also expressed in various peripheral tissues (Mori et al., 2000; Gualillo et al., 2001; Volante et al., 2002; Andreis et al., 2003) and in the arcuate nucleus (ARC) of the hypothalamus, an appetite regulating centre (Lu et al., 2002). In situ hybridization and immunohistochemical studies indicated that ghrelin-producing cells are a distinct type of endocrine cells, known as X/A-like cells, that are localized in the mucosal epithelium of corpus and fundus of the stomach (Date et al., 2000; Dornonville de la Cour et al., 2001; Sakata et al., 2002). Few studies have been carried out on ghrelin expression in non-mammalian vertebrates. Recently, ghrelin was found in the brain and stomach of both frog (Galas et al., 2002) and chicken (Kaiya et al., 2002). Chicken ghrelin consists of 26 amino acids with n-octanoyl modification at Ser3 and has the same sequence in the N-terminal region as in as the human and rat form. In fact, administration of chicken ghrelin increases plasma GH levels in both rats and chicks, and increases plasma corticosterone levels in growing chicks, at a lower dose than mammalian ghrelin (Kaiya et al., 2002). In a previous study, Ahmed and Harvey (2002) showed ghrelin-immunostaining in the chicken hypothalamus with an antibody developed especially for radioimmunoassays and raised in rabbit that recognizes the C-terminal region of rat ghrelin (RAB-031-31; Phoenix Pharmaceuticals, Belmont, CA, USA). Recently, Wada et al. (2003) demonstrated ghrelin-positive cells in the chicken proventriculus using an N-terminal region-recognizing antibody only. Because mammalian ghrelin is also expressed in the intestines (Date et al., 2000), we studied distribution patterns of ghrelin in both the proventriculus and small and large intestines of chicken using immunohistochemistry and western blotting and a specific polyclonal antibody against the C-terminal region of rat ghrelin.
S. Neglia et al.
Material and methods Immunohistochemistry Three-month-old chickens (Gallus domesticus) of both sexes were used in the present study. The gastrointestinal tract was sectioned in small samples of the following segments: proventriculus, ventriculus, duodenum, jejunum, ileum, caecum and colorectum. The tissues were immediately fixed in Bouin’s solution for 24 h, dehydrated in a graded series of alcohol, embedded in paraffin wax and transverse 6–7-mm-thick serial sections were cut. After dewaxing, sections were processed for immunohistochemistry using the peroxidase–antiperoxidase (PAP) method according to Sternberger (1986). Endogenous peroxidase activity was blocked by treating the sections with 0.3% hydrogen peroxide for 20 min at room temperature (RT) and then rinsed for 15 min in 0.01 M phosphate-buffered saline (PBS), pH 7.4. Sections were incubated for 30 min at RT with normal goat serum (dilution, 1:5; Jackson Immunoresearch Laboratories, West Grove, PA, USA) and then with primary antibody (18 h in a moist dark chamber at 4 1C). The primary antibody, a rabbit antibody that recognizes the C-terminal region of rat ghrelin and cross-reacts with chicken ghrelin (H-031-31; Phoenix Pharmaceuticals), was diluted 1:750. After incubation, sections were rinsed in PBS for 15 min and, incubated for 30 min at RT with goat anti-rabbit IgG (dilution, 1:50; Vector Laboratories, Burlingame, CA, USA). Subsequently, sections were rinsed in PBS for 15 min and then incubated for 30 min at RT with PAP complex (dilution, 1:100; Dako, CA, USA). Peroxidase activity was visualized with a solution of 10 mg 3-30 diaminobenzidine tetrahydrocloride (DAB; Sigma, St. Louis, MO, USA) in 15 ml 0.5 M Tris buffer, pH 7.6, containing 0.03% hydrogen peroxide. Specificity of the immunohistochemical staining reaction was tested as follows: (1) substitution of either the ghrelin antibody or the anti-rabbit IgG, or the PAP complex by PBS (negative controls); (2) application of rat stomach tissue known to be positive for ghrelin (positive controls); (3) previous absorption of the primary antibody with homologous antigen (031-31; Phoenix Pharmaceuticals; up to 50 mg/ml antibody in the final dilution). Sections were examined with a Aristoplan light microscope (Leica, Wetzlar, Germany) equipped with a DC300F digital camera (Leica).
Western blot analysis Samples of all parts of the gastrointestinal tract were homogenized (1:2 w/v) with a Potter
ARTICLE IN PRESS Ghrelin in the chicken gastrointestinal tract homogenizer in cold RIPA buffer [50 mM Tris-HCl, pH 8.0, 0.1% sodium dodecyl sulfate (SDS), 1% NP-40, 150 mM NaCl and 0.5% deoxycholic acid] containing 1 mM phenylmethylsulfonyl fluoride (Sigma) and 100 inhibitor cocktail. Then, suspensions were centrifuged at 14,000g for 15 min at 4 1C and the protein concentrations of the resulting pellets, divided in equal aliquots, were determined by the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were separated by electrophoresis on 10% SDS-polyacrylamide gels (NuPAGE Novex 10% bis-Tris gels; Invitrogen, Carlsbad, CA, USA) and, then, gels were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). Membranes were blocked for 1 h at RT in PBS-Tween buffer (0.01 M PBS and 0.05% Tween-20) containing 5% non-fat milk and incubated for 1.5 h at RT with the primary antibody, as described above, diluted 1:1500 in PBS-Tween. After washing, membranes were incubated for 1 h at RT with polyclonal goat anti-rabbit IgG conjugated with horseradish peroxidase (dilution, 1:2000; DAKO). Membranes were incubated in chemoluminescence reagent (ECL; Amersham, Little Chalfont, England) to visualize immunocomplexes, and were exposed for 1–30 s to hyperfilm.
Results Immunohistochemistry Ghrelin-immunopositive cells (ghrelin-ic) were observed in mucosal layers of all gastrointestinal tract segments examined: proventriculus, duodenum, jejunum, ileum, caecum and colo-rectum. High numbers of strongly stained ghrelin-ic were found at the base of glandular lobuli of the proventriculus (Fig. 1A and B). In contrast, few ghrelin-ic were observed in the basal zone of proventriculus plicae (Fig. 1C), which showed an intense immunostaining that was restricted to the basal region of the cytoplasm. These cells had a small round shape and were considered to be of the closed cell type (Fig. 1D). Few ghrelin-ic were of the open cell type that showed an elongated shape and apical cytoplasmic processes in contact with the lumen (Fig. 1E). Many ghrelin-ic were found to be scattered in epithelium of crypts (Fig. 2A) and along villi of the duodenum (Fig. 2B). Closed and open cell types (Fig. 2C) were both observed in this intestinal segment as well. Fewer ghrelin-ic were found in epithelia of crypts of the jejunum and ileum and generally they appeared to be of the closed cell
5 type (Fig. 2D). Very few ghrelin-ic, and especially of the closed cell type and triangular in shape, were detected in crypts of the large intestine (Fig. 2E). Negative controls and sections stained with preabsorbed antibodies did not show any specific staining. Specificity of immunostaining was confirmed by the positive controls (data not shown).
Western blot analysis Ghrelin-like protein was detected by western blot analysis of homogenates of all segments of the chicken gastrointestinal tract which was in agreement with our immunohistochemical data. The ghrelin-like protein that was detected had an estimated molecular mass of approximately 44–46 kDa (Fig. 3).
Discussion In the present study, we demonstrated the occurrence and distribution patterns of ghrelin-like protein in the entire chicken gastrointestinal tract using western blot analysis and peroxidase–antiperoxidase immunohistochemistry respectively. Our findings are not in agreement with a recent study of Ahmed and Harvey (2002) who found ghrelin immunostaining to be present in the chicken brain only and not in the gastrointestinal tract. This discrepancy may be due to a difference in antibodies used in the two studies. In fact, both antibodies recognize the C-terminal region of ghrelin, but we used an antibody especially developed for immunohistochemistry, whereas Ahmed and Harvey (2002) used an antibody that was developed especially for radioimmunoassays. The presence of ghrelin-ic in the chicken gastrointestinal tract suggests that this peptide may act as a gastrointestinal hormone besides its function as a neuropeptide (Ahmed and Harvey, 2002). Recently, Wada et al. (2003) demonstrated ghrelin immunostaining in the mucosal layer of the chicken proventriculus. In agreement with these results, we observed the largest number of ghrelin-ic to be present in the chicken proventriculus which corresponds to the gastric fundus in mammals. Somewhat lower numbers of ghrelin-ic were found in jejunum, ileum and large intestine. Our findings agree with distribution patterns reported in human and rat digestive tract. In fact, Kojima et al. (1999) and Date et al. (2000) found ghrelin-ic to be mainly located in the oxyntic mucosa of gastric fundus. These cells were present
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S. Neglia et al.
Figure 1. Micrographs of ghrelin-immunostained cells (ghrelin-ic) in the chicken proventriculus. (A and B) Ghrelin-ic (arrows) in the mucosal layer at the base of glandular lobuli. (C) Closed type of ghrelin-ic (arrow) in the basal zone of a plica. (D) Open type of ghrelin-ic (arrow) that is elongated in shape, with an apical cytoplasmic process (arrowhead) in contact with the lumen. (E) Closed type of ghrelin-ic (arrows) without connection with the lumen, round in shape with intense immunostaining located at the basal side of the cytoplasm. Magnifications: A, 80; B, 150; C, 260; D, 450; E, 220.
in larger numbers in the small intestines than in the large intestines. Therefore, it is likely that ghrelin has effects on various gastrointestinal functions such as motility of the gut wall and gastric acid secretion both in rat and chicken. Furthermore, ghrelin probably acts on the pituitary via the systemic circulation, contributing to the regulation of GH secretion and feeding behaviour.
Kojima et al. (1999) and Date et al. (2000) have also characterized ghrelin-producing cells in the digestive tract of human and rat by ultrastructural and immunohistochemical means. These endocrine cells, which are typical of the oxyntic mucosa of both species, correspond to X/A-like cells. Our findings on distribution patterns and morphologic characteristics of chicken ghrelin-ic suggest that
ARTICLE IN PRESS Ghrelin in the chicken gastrointestinal tract
7
Figure 2. Micrographs of ghrelin-ic in chicken small and large intestine. (A) Ghrelin-ic (arrows) in crypts of the duodenum. (B) Ghrelin-ic (arrows) along villi of the duodenum. (C) Closed type of ghrelin-ic (arrows) in crypts of jejunum. (D) Closed type of ghrelin-ic (arrows), triangular in shape, in the crypts of the colo-rectum. (E) Duodenum. Open type of ghrelin-ic cell (arrow), elongated in shape, with an apical cytoplasmatic process (arrowhead) in contact with the lumen. Magnifications: A, 60; B, 130; C, 300; D, 280; E, 450.
they correspond to endocrine cells. However, further investigations are required in order to identify the specific cell types on the basis of the current classification (Solcia et al., 2000), although the types and distribution patterns of avian gut endocrine cells have been described in a detailed manner and have been compared with the situation in mammals (Rawdon and Andrew, 1999). Additionally, we demonstrated that these cells are of the open cell type and closed cell type in their relationship with the lumen. Some studies indicate that the open cell type is functionally affected by the food content and pH in the lumen, whereas the second cell type is affected by
hormones, local factors, neuronal stimulation and/or mechanical stress (Solcia et al., 2000). The occurrence of the open and closed cell type in the chicken gastrointestinal tract suggests that various factors regulate ghrelin synthesis and the cells have specific physiological effect in each region. So far, the presence and structure of ghrelin-like protein have been investigated not only in the chicken but also in others non-mammalian vertebrates, such as amphibians (Kaiya et al., 2001; Galas et al., 2002) and fish (Uniappan et al., 2002; Parhar et al., 2003). The amino acid sequences of ghrelin in the different animal species examined,
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Figure 3. Western blot analysis of ghrelin in extracts of chicken gastrointestinal tract. Lane 1, protein markers; lane 2, homogenate of gastrointestinal tract showing a strongly stained band at approximately 44–46 kDa.
show a high preservation of the ‘‘active core’’ (GSSF), essential for its molecular biological activity, in tilapia, eel and chicken (Kaiya et al., 2002; 2003a, b), but not bullfrog (GLTF; Kaiya et al., 2001) and goldfish (GTSF; Uniappan et al., 2002). Interestingly, these first four amino acids are preserved in human and rat as well (Kojima et al., 1999). These findings show that ghrelin is phylogenetically highly preserved from non-mammalian species to mammals not only in its amino acid sequence but also in its distribution patterns.
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