Des-Acyl Ghrelin Induces Food Intake by a Mechanism Independent of the Growth Hormone Secretagogue Receptor

0013-7227/06/$15.00/0 Printed in U.S.A. Endocrinology 147(5):2306 –2314 Copyright © 2006 by The Endocrine Society doi: 10.1210/en.2005-1357 Des-Acyl Ghrelin Induces Food Intake by a Mechanism Independent of the Growth Hormone Secretagogue Receptor Koji Toshinai, Hideki Yamaguchi, Yuxiang Sun, Roy G. Smith, Akihiro Yamanaka, Takeshi Sakurai, Yukari Date, Muhtashan S. Mondal, Takuya Shimbara, Takashi Kawagoe, Noboru Murakami, Mikiya Miyazato, Kenji Kangawa, and Masamitsu Nakazato Department of Internal Medicine (K.T., H.Y., Y.D., M.S.M., T.SH., T.K., M.N.), Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan; Department of Veterinary Physiology (N.M.), University of Miyazaki, Miyazaki 8892151, Japan; Huffington Center on Aging (Y.S., R.G.S.), Baylor College of Medicine, Texas 77030; Department of Pharmacology (A.Y., T.SA.), University of Tsukuba, Ibaraki 305-8575, Japan; and National Cardiovascular Center Research Institute (M.M., K.K.), Osaka 565-8565, Japan Ghrelin, an acylated peptide produced predominantly in the stomach, stimulates feeding and GH secretion via interactions with the GH secretagogue type 1a receptor (GHS-R1a), the functionally active form of the GHS-R. Ghrelin molecules exist in the stomach and hypothalamus as two major endogenous forms, a form acylated at serine 3 (ghrelin) and a des-acylated form (des-acyl ghrelin). Acylation is indispensable for the binding of ghrelin to the GHS-R1a. Ghrelin enhances feeding via the neuronal pathways of neuropeptide Y and orexin, which act as orexigenic peptides in the hypothalamus. We here studied the effect of des-acyl ghrelin on feeding behavior. Intracerebroventricular (icv) administration of rat des-acyl ghrelin to rats or mice fed ad libitum stimulated feeding during the light phase; neither ip nor icv administration of desacyl ghrelin to fasting mice suppressed feeding. The icv ad- G HRELIN IS A 28-amino-acid peptide isolated from human and rat stomach as an endogenous ligand for the GH secretagogue receptor (GHS-R) (1). The GHS-R, a G protein-coupled seven-transmembrane domain receptor, was initially identified as a receptor for small synthetic molecules termed GH secretagogues (GHSs), such as L-692,429, GHRP-6, and MK-0677, all of which act on the pituitary to stimulate GH secretion (2, 3). Two GHS-R subtypes are generated by alternative splicing of a single gene: the full-length type 1a receptor (GHS-R1a) and a carboxyl-terminally truncated form, the GHS-R type 1b (GHS-R1b), that encodes a protein containing transmembrane domain one to five (2, 3). The GHS-R1a is the functionally active, signal transducing form of the GHS-R, whereas the GHS-R1b is devoid of highaffinity ligand binding and signal transduction activity. Ghrelin molecules, predominantly produced by endocrine cells First Published Online February 16, 2006 Abbreviations: AgRP, Agouti gene-related protein; CRF, corticotropin-releasing factor; 2-DG, 2-deoxy-d-glucose; GHS, GH secretagogue; GHS-R, GH secretagogue receptor; HKRB, Krebs-Ringer bicarbonate buffer; icv, intracerebroventricular(ly); LHA, lateral hypothalamic area; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; PVN, paraventricular nucleus; RP-HPLC, reverse-phase HPLC. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. ministration of des-acyl ghrelin induced the expression of Fos, a marker of neuronal activation, in orexin-expressing neurons of the lateral hypothalamic area, but not neuropeptide Y-expressing neurons of the arcuate nucleus. Peripheral administration of des-acyl ghrelin to rats or mice did not affect feeding. Although icv administration of ghrelin did not induce food intake in GHS-R-deficient mice, it did in orexin-deficient mice. In contrast, icv administration of des-acyl ghrelin stimulated feeding in GHS-R-deficient mice, but not orexin-deficient mice. Des-acyl ghrelin increased the intracellular calcium concentrations in isolated orexin neurons. Central desacyl ghrelin may activate orexin-expressing neurons, perhaps functioning in feeding regulation through interactions with a target protein distinct from the GHS-R. (Endocrinology 147: 2306 –2314, 2006) of the gastric oxyntic glands (4, 5), exist in two major molecular forms, ghrelin and des-n-octanoyl ghrelin (des-acyl ghrelin) (6). These two ghrelin molecules are also produced in the rat hypothalamus, as demonstrated by the combination of reverse-phase HPLC (RP-HPLC) with two separate RIAs recognizing ghrelin and des-acyl ghrelin (7, 8). All ghrelin species identified in fish, amphibians, birds, and many mammals possess a unique structural modification of the hydroxyl group of their third residue, which is either serine or threonine, by n-octanoic acid (9). This acylation is essential for the binding of ghrelin to the GHS-R1a (1, 10, 11); thus, the acylated form has been designated as ghrelin in our original description (1). Administration of ghrelin stimulates food intake in humans and rats (12–16) but does not change feeding behavior in GHS-R-deficient mice (17), suggesting that ghrelin enhances food intake via GHS-R-mediated signaling. Several recent in vitro studies have demonstrated that radiolabeled ghrelin and des-acyl ghrelin bound to the membranes of PC-3 prostate tumor cells, H9c2 cadiomyocytes and isolated adipocytes, none of which expressed the GHS-R (18 –20). This binding could be displaced by ghrelin, des-acyl ghrelin, and synthetic GHSs. Ghrelin and des-acyl ghrelin exhibit similar GHS-R-independent biological activities, including a cytoprotective effect on cultured cardiomyocytes 2306 Toshinai et al. • Des-Acyl Ghrelin Induces Feeding Behavior (18), the inhibition of cell proliferation of breast carcinoma cell lines (19), the reduction of glycerol release from rat epididymal adipocytes (20), an ionotropic effect on guinea pig papillary muscle (21), and the promotion of bone marrow adipogenesis (22). Although the signaling molecules downstream of des-acyl ghrelin remain undefined, des-acyl ghrelin appears to share a subset of biological activities with ghrelin in peripheral tissues through an unidentified receptor or a target protein unique from the GHS-R. The coordination of the regulation of food intake and energy expenditure occurs in the hypothalamus. Glucoprivic states induced by fasting or treatment with 2-deoxy-d-glucose (2-DG), a selective inhibitor of carbohydrate metabolism, increase feeding through the activation of orexigenic peptides, neuropeptide Y (NPY) and agouti gene-related protein (AgRP) in the arcuate nucleus (23). Secretion of desacyl ghrelin from the rat hypothalamus increased in glucoprivic states induced by fasting or treatment with 2-DG (7). The axonal terminals of neurons that produce ghrelin and des-acyl ghrelin make direct synaptic contacts with NPYand orexin-expressing neurons, which participate in hypothalamic feeding (...truncated)