117 Ghrelin: a hypothalamic GH-releasing factor in domestic fowl (Gallus domesticus) S Ahmed and S Harvey Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada (Requests for offprints should be addressed to S Harvey; Email: [email protected]) Abstract Ghrelin, a recently discovered peptide in the mammalian ghrelin. Ghrelin immunoreactivity was also present in hypothalamus and gastrointestinal tract is thought to be the clusters of large ovoid magnocellular cells in the nucleus endogenous ligand for the GH secretagogue (GHS) recep- magnocellularis preopticus pars medialis, nucleus magno- tor and it stimulates GH release in rats and humans. The cellularis preopticus supraopticus and in the chiasma- possibility that ghrelin is present in birds was therefore opticus. Immunoreactivity for ghrelin was restricted to the assessed, since a GHS receptor is present in the chicken cytoplasm of the perikarya and their axonal sprouts. pituitary gland. Immunoreactivity for ghrelin was not seen in any other Although immunoreactive ghrelin is readily detectable hypothalamic nuclei. in the rat stomach and ileum, ghrelin immunoreactivity In a preliminary experiment, circulating GH concen- could not be detected in the chicken proventriculus, trations in conscious immature chicks were promptly stomach, ileum or colon, whereas somatostatin immuno- increased following bolus i.v. administration of human reactivity, in contrast and as expected, was readily ghrelin. The increase in GH concentration (approximately detectable in the chicken gastrointestinal tract. Ghrelin three times that in the controls) was comparable with that immunoreactivity was, however, present in the chicken induced by the same dose (10 µg/kg) of human GH- hypothalamus, although not in the arcuate (infundibular) releasing hormone, although less than that (approximately nucleus, as in rats. Discrete parvocellular cells and neuronal sixfold) induced by thyrotropin-releasing hormone. fibers with ghrelin immunoreactivity were present in the These results demonstrate the presence of a ghrelin-like anterior medial hypothalamus. This immunoreactivity protein in the chicken hypothalamus and suggest that it was specific and completely abolished following the pre- participates in the regulation of GH secretion in birds. absorption of the antibody with an excess of human Journal of Endocrinology (2002) 172, 117–125 Introduction In birds, the control of GH secretion differs from that in mammals, since chicken GHRH has minimal GH- Growth hormone (GH) secretion is regulated by a myriad releasing activity in chickens (Harvey 1999) and because of interacting, multihierarchical factors that dynamically thyrotropin-releasing hormone (TRH) is a potent GH- modify somatotroph function at neural and humeral inter- releasing factor (Harvey 1990). The possibility that ghrelin faces in neurocrine, neuroendocrine, endocrine and para- may participate in GH release in birds was therefore crine ways (Harvey 1995). The hypothalamus is, however, assessed, especially as ghrelin is thought to be the endog- thought to primarily regulate GH release in mammals enous ligand for a GH secretagogue (GHS) receptor through antagonistic interactions between stimulatory identified in chicken pituitary glands (Toogood et al. 1999, (GH-releasing hormone; GHRH) and inhibitory (soma- Gaylinn et al. 2000). tostatin; SRIF) hypophysiotropic-releasing hormones. A third hypothalamic factor, ghrelin, may also be involved (Dieguez & Casanueva 2000, Kojima et al. 2001), since it Materials and Methods is present in the arcuate nucleus of the mouse, rat and human hypothalamus (Kojima et al. 1999, Hosoda et al. Immunocytochemistry 2000a, Korbonits et al. 2001) and is three to five times more potent than GHRH in stimulating GH release in White Leghorns at 4 weeks of age were killed by cervical these species (Kojima et al. 1999, Arvat et al. 2000, Seoane dislocation (with approval from the University of Alberta et al. 2000). It is, however, not known if ghrelin is Health Sciences Animal Welfare Committee) and hypo- involved in GH regulation in other species. thalami were rapidly dissected from the heads, washed in Journal of Endocrinology (2002) 172, 117–125 Online version via http://www.endocrinology.org 0022–0795/02/0172–117 2002 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 09:50:06AM via free access 118 S AHMED and S HARVEY · GHRH: ghrelin in birds ice-cold phosphate-buffered saline (PBS; pH 7·4) and White Leghorn chicks, in comparison with human collected into freshly prepared paraformaldehyde (4% GHRH1–44 and TRH (Bachem, Torrance, CA, USA). w/v) (Sigma, Mississauga, Ontario, Canada). The hypo- Groups of birds (n=8–16) were given a bolus i.v. injection thalami were removed in one piece, with two parallel cuts of peptide or the 0·9% (w/v) NaCl vehicle (1 ml/kg body 2·0–2·5 mm lateral to the midline, a transverse cut in front weight). Each peptide was administered at a dose of of the preoptic area and the roots of the oculomotor nerves 10 µg/kg, since this is the maximally effective dose of and a cut 4–5 mm deep parallel to the base of the brain TRH and GHRH in chickens (Harvey & Scanes 1984) (MacNamee & Sharp 1989). After overnight fixation, at and is comparable to doses of ghrelin that stimulate GH 4 C, the tissues were dehydrated in a graded series of release in rats (4–20 µg/kg in rats, Hosoda et al. 2000b) alcohol (50% v/v, 15–30 min; 70%, 30–60 min; 95%, and humans (5 µg/kg, Takaya et al. 2000; 33–66 µg/kg, 30–60 min; 100%, 30–60 min) and cleared with hemo-de Peino et al. 2000). Venous blood samples were collected (Fisher Scientific, Edmonton, Alberta, Canada) for from each bird 10 min after injection, at the time of the 30 min. Tissues were then infiltrated with paraffin wax for maximal GH response to TRH and GHRH (Harvey & 24hat60C, under normal atmospheric pressure. Serial Scanes 1984). transverse sections (4–8 µm) were cut with a microtome Following centrifugation and separation, the plasma was and mounted onto treated slides (Fisher Scientific). The stored at20 C prior to GH radioimmunoassay, using sections were cut anterior-dorsally, using co-ordinates NIADD reagents (Bethesda, MD, USA, kindly provided A10·0toA5·6, according to the histological atlas of by Dr A F Parlow). Statistical differences in the results Kuenzel & Masson (1988). Magnocellular nuclei were were determined by analysis of variance. identified according to Yasuda (1980) and Mikami (1985). Immunocytochemical staining was performed with commercial reagents (Vector Laboratories, Burlington, Results CA, USA and Sigma), using an avidin–biotin–peroxidase (ABC) (Hsu et al. 1981) method. Sections were incubated Immunohistochemistry with a specific commercial antibody raised in rabbits against rat ghrelin (RAB-031–31; Phoenix Pharmaceuti- As expected (Kojima et al. 1999, Date et al. 2000a), ghrelin cals, Belmont, CA, USA), with which human ghrelin has immunoreactivity was abundantly present within the 100% cross-reactivity. The primary antibody was diluted X/A-like endocrine cells of the rat stomach (Fig. 1A and 1:500 in 1–5% normal goat serum, overnight, at 4 C. After C). Staining was completely abolished following the pre- incubation, the slides were washed three times for 15 min absorption of the antiserum with human ghrelin (Fig. 1B). in PBS. Sections were then incubated for 1 h at room In contrast, sections of the chicken stomach (and the temperature in biotinylated goat anti-rabbit immuno- proventriculus, ileum and colon) had no ghrelin immu- globulin G (Sigma; 1:200). The slides were washed in PBS noreactivity (data not shown), but were stained for SRIF and incubated in ABC reagent for 1 h at room temperature immunoreactivity (Fig. 1D and F). In the hypothalamus, and washed in PBS. Staining was visualized using the ghrelin immunoreactivity was present in discrete cells in chromogenic substrate diaminobenzidine tetrahydrochlo- the nucleus anterior medialis hypothalami (AM) (Fig. ride (Sigma), which resulted in a brown coloration. The 2A–C and Fig. 4), and was also present in clusters of large specificity of staining was determined by preabsorbing the ovoid cells in the nucleus magnocellularis preopticus pars primary antibody with synthetic human ghrelin (Phoenix medialis (PRM) (Fig. 2A, E and F and Fig. 4), nucleus Pharmaceuticals; 1 mg/ml) for 1 h prior to section incu- magnocellularis preopticus pars supraopticus (PRS) (Fig. bation. Non-specific staining was determined by replacing 3A, E and F and Fig. 4) and in the chiasma opticus (CO) the primary antibody with pre-immune rabbit serum or (Fig. 3A–C and Fig. 4). Immunoreactivity for ghrelin was with PBS or by omission of the secondary antibody. As a restricted to the cytoplasm of the perikarya and their positive control (Kojima et al. 1999), sections of the rat axonal sprouts. Ghrelin staining was lost following the stomach were similarly stained for ghrelin immunoreactiv- preabsorption of the antibody with human ghrelin (Fig. ity. Sections of the chick stomach and gastrointestinal tract 2D and Fig. 3D) and not seen when the ghrelin antibody were similarly stained for SRIF immunoreactivity, using was replaced by normal rabbit serum or PBS (data not shown). Immunoreactivity for ghrelin was not present in an antibody raised in rabbits against SRIF14 (Di Scala- Guenot et al. 1984) at a dilution of 1:200, since SRIF any other hypothalamic nuclei. immunoreactivity has been found throughout the chicken gastrointestinal tract (Denbow 2000). GH-releasing activity Circulating GH concentrations in conscious, immature GH-releasing activity chicks were promptly increased (P<0·001) following the The GH-releasing activity of human ghrelin (Phoenix i.v. administration of human ghrelin (Fig. 5). The increase Pharmaceuticals) was assessed in conscious 4-week-old in GH concentration (approximately three times that in Journal of Endocrinology (2002) 172, 117–125 www.endocrinology.org Downloaded from Bioscientifica.com at 09/29/2021 09:50:06AM via free access GHRH: ghrelin in birds · S AHMED and S HARVEY 119 Figure 1 (A) Ghrelin immunoreactivity in the rat stomach (100).