Peripheral Interaction of Ghrelin with Cholecystokinin on Feeding Regulation

0013-7227/05/$15.00/0 Printed in U.S.A. Endocrinology 146(8):3518 –3525 Copyright © 2005 by The Endocrine Society doi: 10.1210/en.2004-1240 Peripheral Interaction of Ghrelin with Cholecystokinin on Feeding Regulation Yukari Date, Koji Toshinai, Shuichi Koda, Mikiya Miyazato, Takuya Shimbara, Tomoko Tsuruta, Akira Niijima, Kenji Kangawa, and Masamitsu Nakazato Third Department of Internal Medicine (Y.D., K.T., S.K., T.S., T.T., M.N.), Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692; Daiichi Suntory Biomedical Research Co., Ltd. (S.K.), Osaka 681-8513; National Cardiovascular Center Research Institute (M.M., K.K.), Osaka 565-8565; and Department of Physiology (A.N.), Niigata University School of Medicine, Niigata 951-8510, Japan Ghrelin and cholecystokinin (CCK) are gastrointestinal hormones regulating feeding. Both transmitted via the vagal afferent, ghrelin elicits starvation signals, whereas CCK induces satiety signals. We investigated the interaction between ghrelin and CCK functioning in short-term regulation of feeding in Otsuka Long-Evans Tokushima fatty (OLETF) rats, which have a disrupted CCK type A receptor (CCK-AR), and their lean littermates, Long-Evans Tokushima Otsuka (LETO) rats. Intravenous administration of ghrelin increased 2-h food intake in both OLETF and LETO rats. Because OLETF rats are CCK insensitive, iv-administered CCK decreased 2-h food intake in LETO, but not in OLETF, rats. Although preadministration of CCK to LETO rats blocked food intake induced by ghrelin, CCK preadministration to OLETF rats did not affect ghrelin-induced food intake. Conversely, preadministration of ghrelin to LETO rats blocked feeding reductions induced by I N ADULT ANIMALS and humans, body weight usually remains within a relatively narrow range, despite large day-to-day changes in the amount of food consumed. Even when the restriction of food intake or excessive overfeeding induces changes in body adiposity, both body weight and adiposity in humans and animals return to baseline levels after the resumption of regular feeding (1, 2, 3). Multiple peripheral signals (e.g. nutrients, nutrient metabolites, or hormones) regulate short-term and long-term food intake and energy balance through diverse but interacting pathways (4). Signals affecting short-term food uptake have significantly different mechanisms than the long-term regulators of energy homeostasis activated in proportion to both body adipose stores and the food consumed over prolonged periods. Using an intracellular calcium assay of stable cell lines expressing rat GH secretagogue receptor (GHS-R), we recently discovered in rat stomach a novel endogenous ligand for the GHS-R (5) named ghrelin. Ghrelin, produced primar- First Published Online May 12, 2005 Abbreviations: CCK, Cholecystokinin; CCK-AR, CCK type A receptor; GHS-R, GH secretagogue receptor; LETO, Long-Evans Tokushima Otsuka; NPY, neuropeptide Y; NTS, the nucleus of the solitary tract; OLETF, Otsuka Long-Evans Tokushima fatty; PBN, parabranchial nucleus. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community. CCK. In electrophysiological studies, once gastric vagal afferent discharges were altered by ghrelin or CCK administration, they could not be additionally affected by serial administrations of either CCK or ghrelin, respectively. The induction of Fos expression in the hypothalamic arcuate nucleus by ghrelin was also attenuated by CCK preadministration. Using immunohistochemistry, we also demonstrated the colocalization of GH secretagogue receptor (GHS-R), the cellular receptor for ghrelin, with CCK-AR in vagal afferent neurons. These results indicate that the vagus nerve plays a crucial role in determining peripheral energy balance. The efficiency of ghrelin and CCK signal transduction may depend on the balance of their respective plasma concentration and/or on interactions between GHS-R and CCK-AR. (Endocrinology 146: 3518 –3525, 2005) ily in endocrine cells of the stomach, is released into circulation (5–7). Whereas multiple gastrointestinal hormones have been implicated in feeding regulation (8 –12), ghrelin stimulates appetite, food intake, and GH secretion when administered to humans and rodents (5, 13–17). In humans, the circulating ghrelin levels increase before and decrease after every meal (18 –22), suggesting that ghrelin functions as a meal initiator. The effect of ghrelin on feeding is rapid and short-lived, implying that ghrelin functions in short-term regulation of feeding. The inverse correlation between ghrelin levels and body mass index, as well as ghrelin-mediated promotion of adipogenesis, suggests that ghrelin may also participate in long-term regulation of body weight (14, 19, 23–27). Most gastrointestinal hormones regulating feeding, with the exception of ghrelin, inhibit food intake (28, 29). Cholecystokinin (CCK) decreases meal size in rats and humans when administered peripherally (30 –32). This peptide, released from the proximal small intestine, functions as a postprandial satiety signal (33–36). The anorectic effect of CCK is also rapid and short-lived; long-term peripheral administration of CCK does not reduce overall food intake or induce maintained weight loss (37). These results suggest that CCK plays an essential role in the short-term regulation of feeding. Although ghrelin has an opposite effect on feeding as CCK, this peptide exhibits characteristics similar to CCK on the short-term regulation of feeding. Both ghrelin and CCK, after release from the gastrointestinal tract, transmit starva- 3518 Date et al. • Interaction of Ghrelin with Cholecystokinin tion and satiety signals to the brain through receptors, GHS-R and CCK type A receptor (CCK-AR), respectively, located in the vagal capsaicin-sensitive afferents (38 – 42). Thus, vagal afferent fibers represent a major target of these peripheral feeding regulators, ghrelin and CCK. In this study, we examined the functional relationship between ghrelin and CCK in the short-term regulation of food intake using CCK-AR-deficient Otsuka Long-Evans Tokushima fatty (OLETF) rats and their lean littermates, LongEvans Tokushima Otsuka (LETO) rats. We also investigated the colocalization of GHS-R with CCK-AR in rat vagal afferents. Because iv administration of ghrelin induces Fos expression in the hypothalamic arcuate nucleus of rats through gastric vagal afferents, we examined the induction of Fos expression in the arcuate nucleus by iv administration of ghrelin after CCK treatment. The electrical discharge of gastric vagal afferents is attenuated by ghrelin and stimulated by CCK (38, 42– 48). In this study, we evaluated changes in vagal afferent firing induced by iv treatment of ghrelin and CCK after CCK and ghrelin administration, respectively. Materials and Methods Animals Ten-week-old OLETF and lean littermate LETO rats (body weight: OLETF (...truncated)