Acute Central Ghrelin and GH Secretagogues Induce Feeding and Activate Brain Appetite Centers

0013-7227/02/$03.00/0 Printed in U.S.A. Endocrinology 143(1):155–162 Copyright © 2002 by The Endocrine Society Acute Central Ghrelin and GH Secretagogues Induce Feeding and Activate Brain Appetite Centers CATHERINE B. LAWRENCE, AMELIE C. SNAPE, FLORENCE M.-H. BAUDOIN, AND SIMON M. LUCKMAN University of Manchester School of Biological Sciences, Manchester, United Kingdom M13 9PT Ghrelin was recently identified as the endogenous ligand for the GH secretagogue (GHS) receptor. Like the synthetic GHSs [e.g. GH-releasing peptide-6 (GHRP-6)], ghrelin stimulates feeding and increases body weight in rats. The aim of this study was to identify brain regions that are activated by GHSs and determine whether the responses observed were secondary to food intake. In addition, possible mediators of GHS actions were examined. Intracerebroventricular (icv) injection of ghrelin or GHRP-6 into rats significantly stimulated food intake and transiently reduced core body temperature. The effect of both ghrelin and GHRP-6 on food intake was blocked by preadministration of a Y1 NPY receptor antagonist (BIBO3304). Using c-Fos immunohistochemistry, we demon- strated that icv ghrelin or GHRP-6 activated several hypothalamic brain regions, including the arcuate nucleus, paraventricular nucleus, dorsomedial nucleus, lateral hypothalamus, and two regions of the brainstem, the nucleus of the tractus solitarius and the area postrema. The cell activation induced by GHRP-6 was independent of food intake, as the same pattern and extent of c-Fos expression were observed in animals that were denied access to food following treatment. Finally, double immunohistochemistry indicated that orexin-containing, but not melanin-concentrating hormone-containing, neurons in the lateral hypothalamus were activated significantly by central administration of GHRP-6. (Endocrinology 143: 155–162, 2002) G H SECRETAGOGUES (GHS) are small synthetic molecules that stimulate the release of GH from the pituitary (1, 2). The endogenous ligand for the GHS receptor (GHS-R) (3), ghrelin, has recently been isolated from the rat stomach (4), and ghrelin immunoreactivity also has been located in the hypothalamic arcuate nucleus (4). In addition to being expressed in the anterior pituitary, the GHS-R is found in various hypothalamic and thalamic nuclei, the dentate gyrus, substantia nigra, ventral tegmentum, and facial nucleus of the brainstem (5– 8), suggesting a central role for ghrelin. Indeed, central administration of ghrelin causes GH release in rats (9, 10), and GHSs have been implicated in the regulation of energy balance. Single central injections of GHSs, including ghrelin, stimulate feeding in rodents (10 – 18). Likewise, daily injections or infusions of GHSs increase food intake and body weight (17, 19 –21). Ghrelin’s effect on body weight in rodents is due in part to altered metabolism and energy expenditure (16, 21). The precise mechanism of the anabolic actions of GHSs has yet to be fully clarified, although they do not appear to act indirectly via the secretion of GH (12, 15, 17, 18, 21). Previous functional mapping studies have shown that systemic or central administration of GHSs in rodents induce the immediate-early gene c-fos only in the hypothalamic arcuate nucleus (22–24) despite the relatively wide distribution of the receptor within the brain (5– 8). The highest proportion of arcuate neurons activated by systemic GHS contain NPY (25), and many more neurons are activated if the animal is first fasted, a manipulation known to remove inhibitory influences from NPY neurons (26). Moreover, greater than 90% of arcuate NPY neurons possess GHS-R mRNA, suggesting that they are an important target (27). In support, it has been reported that blocking the action of endogenous NPY inhibits GHS-induced feeding in rodents (15–18). Recent studies have demonstrated that additional brain regions can express c-Fos protein after systemic synthetic GHS (28, 29) or central administration of ghrelin (17, 30). However, no systematic quantification of cell number or identification of neuronal phenotype activated by ghrelin in these additional areas has been reported. Furthermore, it has yet to be determined whether the neuronal activation observed after GHSs is secondary to food intake, because the consumption of a large meal may itself lead to c-Fos expression in the brain. For example, a difference in the c-Fos expression pattern is observed between groups of animals allowed access to food or not after central NPY injection (31, 32). Thus, in this study we compared firstly the effects of central administration of ghrelin and the synthetic GHS, GH-releasing peptide-6 (GHRP-6), on feeding and core body temperature. Secondly, we examined whether the feeding response to ghrelin or GHRP-6 was affected by preadministration of a Y1 NPY receptor-selective antagonist (BIBO3304). Thirdly, we quantified the induction of c-Fos (as a marker for neuronal activation) in the forebrain and brainstem and determined whether any of the neuronal activity observed was secondary to food intake rather than a consequence of the initial stimulus. Finally, as arcuate NPYcontaining neurons are implicated in the feeding response of GHSs, and these neurons project to orexin- and melaninconcentrating hormone (MCH)-containing neurons in the lateral hypothalamus (33, 34), we sought to determine whether these latter cell types are activated by GHS treatment. Abbreviations: GHRP-6, GH-releasing peptide-6; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; icv, intracerebroventricular; MCH, melanin-concentrating hormone. 155 156 Endocrinology, January 2002, 143(1):155–162 Materials and Methods Animals and surgery Male Sprague Dawley rats (Charles River Laboratories, Inc., Sandwich, UK), weighing 250 –300 g (8 –10 wk old), were used in all studies and were housed at a constant ambient temperature of 21 ⫾ 2 C on a 12-h light, 12-h dark cycle (lights on at 0800 h). Rat chow (Beekay International, Hull, UK) and tap water were provided ad libitum, except in Exp 4. All procedures conformed to the requirements of the United Kingdom Animals (Scientific Procedures) Act, 1986. To allow for intracerebroventricular (icv) injections, rats were anesthetized with 2.5% halothane (AstraZeneca, Macclesfield, UK) and stereotaxically implanted with guide cannulas into the lateral ventricle [posterior, 0.8 mm from bregma; lateral, 1.5 mm from bregma; according to the atlas of Paxinos and Watson (35)]. The tip of the guide cannula was positioned 1 mm above the injection site (ventral, 3.5 mm from the surface of the skull). In some experiments core body temperature was monitored, remotely in undisturbed animals, by radiotransmitters (TA10TA-F40, Data Sciences, Minneapolis, MN) that were implanted into the peritoneum at the same time as cannulation. All animals were allowed to recover from surgery for a minimum of 5–7 d and then were housed individually. Icv injections were c (...truncated)