Peripheral Interaction of Ghrelin with Cholecystokinin on Feeding Regulation
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内分泌学杂志 2005年第8期
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
Address all correspondence and requests for reprints to: Yukari Date, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan. E-mail: dateyuka@med.miyazaki-u.ac.jp.
Abstract
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 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.
Introduction
IN 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 primarily in endocrine cells of the stomach, is released into circulation (5, 6, 7). Whereas multiple gastrointestinal hormones have been implicated in feeding regulation (8, 9, 10, 11, 12), ghrelin stimulates appetite, food intake, and GH secretion when administered to humans and rodents (5, 13, 14, 15, 16, 17). In humans, the circulating ghrelin levels increase before and decrease after every meal (18, 19, 20, 21, 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, 24, 25, 26, 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, 31, 32). This peptide, released from the proximal small intestine, functions as a postprandial satiety signal (33, 34, 35, 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 starvation 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, 39, 40, 41, 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, Long-Evans 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, 43, 44, 45, 46, 47, 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, 403.2 ± 6.0 g; LETO, 392.8 ± 2.6 g; P > 0.1; n = 20), obtained from Otsuka Pharmaceutical (Tokushima, Japan), were used in the experiments for feeding. Male Wistar rats (body weight: 361.6 ± 1.3 g; n = 20) (Charles River Japan, Inc., Shiga, Japan) were used for immunohistochemistry, Fos expression, and electrophysiological studies. Rats were housed individually in plastic cages at constant room temperature in a 12-h light (0800–2000)/12-h dark cycle. Animals were given standard laboratory chow and water ad libitum. Intravenous cannulas were implanted into the right jugular vein under anesthesia after an ip injection of sodium pentobarbital (80 mg/kg body weight) (Abbott Laboratories, Chicago, IL). Rats were sham-injected before the study and weighed and handled daily. We also injected heparin daily (1 U/100 μl 0.9% saline) into the cannulas of the animals to prevent coagulation. Only animals exhibiting progressive weight gain after surgery were used in subsequent experiments. All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.
Preparation of anti-GHS-R serum
The [Cys0]-rat GHS-R [342–364] peptide was synthesized using the Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA), then purified by reverse phase-HPLC. The synthesized peptide (10 mg) was conjugated to maleimide-activated mariculture keyhole limpet hemocyanin (6 mg) (mcKLH; Pierce, Rockford, IL) in conjugation buffer (Pierce). The conjugate was emulsified with an equal volume of Freund’s complete adjuvant and was used to immunize New Zealand white rabbits by intracutaneous and sc injection. Animals were boosted every 2 wk and bled 7 d after each injection. The specificity of the antisera was confirmed by immunoreactivity of GHS-R-expressing (CHO-GHSR62 cells), but not of control cells.
Feeding experiments
Experiments were performed 1 wk after iv cannulation. First, CCK (Peptide Institute, Inc., Osaka, Japan) was dissolved in 0.9% saline, and this solution (10 pmol-5 nmol/100 μl) was administered iv at 1000 h to LETO rats after fasting for an 8-h period to determine the lowest effective dose of CCK on feeding (n = 10 per group). Second, a solution of rat ghrelin (Peptide Institute) dissolved in 0.9% saline (1.5 nmol/100 μl or 3 nmol/100 μl) was administered iv at 1000 h to OLETF and LETO rats fed ad libitum (n = 10 per group). After injection, rats were immediately returned to their cages, then 2-h food intake was measured. Third, a solution of CCK dissolved in 0.9% saline (1 nmol/100 μl) was administered iv at 1000 h to OLETF and LETO rats after fasting for an 8-h period (n = 10 per group). After returning rats to their cages immediately after injection, 2-h total food intake was measured. Fourth, after fasting for an 8-h period, OLETF and LETO rats were treated with CCK (1 nmol/100 μl). They were not given any food until the next injections. Animals were subsequently given ghrelin (3 nmol/100 μl) or saline (100 μl) 30 min after CCK injection (n = 10 per group). After ghrelin or saline injection, 2-h food intake was measured. Fifth, after an 8-h fasting period, LETO rats were first treated with ghrelin (3 nmol/100 μl), then subsequently given CCK (1 nmol/100 μl) or saline (100 μl) 30 min after ghrelin injection (n = 10 per group). The rats were fasted between ghrelin and CCK or saline injections. After this second injection, 2-h food intake was measured. These feeding studies were performed in a crossover design. Rats were allowed at least 4 d without injections between experimental days.
Immunohistochemical double-staining
Three Wistar rats, weighing 300–350 g, were perfused transcardially with 0.1 M phosphate buffer (pH 7.4), then with 4% paraformaldehyde in a 0.1 M phosphate buffer. The nodose ganglia were sectioned into 12 μm-thick slices at –20 C using a cryostat. Sections were stored at –80 C. Primary neurons were also obtained from the nodose ganglia of five Wistar rats, ranging from 5–6 wk of age. These neurons were submitted to collagenase dispersion as described (49, 50), then seeded and cultured for 4 d in polyethylenimine-coated Lab-Tek chamber slides (Electron Microscopy Sciences, Hatfield, PA) in complete DMEM (25 mM glucose) containing 5% newborn calf serum, 5% horse serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 30 ng/ml nerve growth factor 2.5S (Sigma Chemical Co., St. Louis, MO), and 2 mM L-glutamine at 37 C in 5% CO2. The medium was replaced every 2 d. Slides were washed in 0.01 M PBS (pH 7.4), then fixed in 10% formaldehyde. The slides of both the primary culture and sectioned nodose ganglia were incubated overnight at 4 C in rabbit anti-GHS-R antiserum (dilution 1/1000). Antibody staining was detected using Alexa Fluor 594-conjugated chicken antirabbit IgG (Molecular Probes, Inc., Eugene, OR). Samples were subsequently incubated with anti-CCK-AR antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution 1/100), then with Alexa Fluor 488-conjugated donkey antigoat IgG (Molecular Probes, Inc.). Slides were observed by fluorescence microscopy (BH2-RFC; Olympus, Tokyo, Japan). The number of neurons expressing GHS-R or CCK-AR immunoreactivity in the nodose ganglion was quantified by counting two randomly selected visual fields in two sections from each of the three rats.
Fos expression and image analysis
The lowest effective dose of ghrelin or CCK on feeding was used for Fos expression studies. CCK (1 nmol/100 μl) or saline was injected iv into three male Wistar rats weighing 341.6 ± 1.3 g. Ghrelin (1.5 nmol/100 μl) was then injected into these rats 30 min after CCK or saline injection. Ninety minutes after ghrelin or saline injection, rats were perfused transcardially with fixative containing 4% paraformaldehyde. The brain was sectioned into 40-μm-thick samples. Immunohistochemistry of Fos was performed as described (51). Quantitation of Fos-immunoreactive cells in the nucleus of the solitary tract (NTS), parabranchial nucleus (PBN), and hypothalamic arcuate nucleus (bregma: –11.30 to –14.60 for NTS, –9.16 to –10.04 for PBN, –2.30 to –3.30 for the arcuate nucleus from Paxinos and Watson’s rats brain atlas) was performed bilaterally. Fos-expressing cells of the arcuate nucleus for a 0.7-mm right triangle (0.245 mm2) were counted in every fifth section (200 μm frequency) (five tissue sections per rats) using a cell counting program written for NIH Image (version 1.62; National Institutes of Health, Bethesda, MD).
Electrophysiological study
Multiunit neural discharge in gastric vagal afferent fibers was recorded extracellularly. Male Wistar rats, fasted for an 8-h period, were anesthetized by an ip injection of urethan (1 g/kg) (Sigma). The electrophysiological study was performed under anesthetization throughout. The rat trachea was intubated, and the electrocardiogram was recorded. Body temperature was maintained at 37 C. Standard methods of extracellular recording from vagal nerve filaments were used, as described in detail elsewhere (52). After laparotomy, a small catheter (Intramedic PE-10; Clay Adams, Parsippany, NJ) was inserted into the inferior vena cava. After gastric branches of the vagus nerve were visualized, we placed filaments isolated from the peripheral cut end of the ventral branch for recording of afferent nerve activity on a pair of silver wire electrodes. Silver wire electrodes, connected through an alternating current-coupled differential amplifier (DAP-10E; Dia Medical Systems, Co., Tokyo, Japan) to an oscilloscope and magnetic tape recorder, were used for display and storage of the neural activity. A window discriminator (DSE-325A) converted spikes to constant amplitude pulses for analysis of spike frequency with a rate meter that reset at 5-sec intervals. Output from the rate meter was recorded on a chart paper (8K20 recorder; NEC-SAN E1 Co., Tokyo, Japan). Ghrelin (0.03 pmol–1.5 nmol/100 μl) or CCK (0.01 pmol–1 nmol/100 μl) was administered iv to rats through a catheter inserted into the inferior vena cava (n = 10 per group). After administration, nerve discharges from the multiunit afferents were recorded for 60 min and analyzed. In addition, CCK (1 nmol/100 μl) or ghrelin (1.5 nmol/100 μl), considered as a standard dose for feeding (38), was administered iv to rats through a catheter inserted into the inferior vena cava (n = 5 per group) before recording the multiunit afferent nerve discharges for 30 min. After the subsequent iv administration of either ghrelin (1.5 nmol/100 μl) or CCK (1 nmol/100 μl) to these rats, multiunit afferent nerve discharges were recorded for 30 min and analyzed.
Statistical analysis
Groups of data (mean ± SEM) were compared using ANOVA and post hoc Fisher’s test. P < 0.05 was considered to be significant.
Results
Effects of ghrelin and CCK on feeding in OLETF or LETO rats
We first tested various doses of CCK ranging from 10 pmol to 5 nmol in a food intake experiment using LETO rats fasted for an 8-h period (Fig. 1A). The lowest effective dose of CCK administered iv was 1 nmol, which also applied to feeding examination using Wistar rats (data not shown). Therefore, we used 1 nmol as a standard dose of CCK in a subsequent series of experiments. The lowest effective dose of ghrelin on feeding in Wistar rats was 1.5 nmol (38); however, a single iv administration of 1.5 nmol ghrelin to OLETF and LETO rats did not induce feeding (LETO: saline, 0.35 ± 0.19 g; 1.5 nmol ghrelin, 0.28 ± 0.14 g; P > 0.7, n = 10; OLETF: saline, 0.36 ± 0.19 g; 1.5 nmol ghrelin, 0.33 ± 0.21 g; P > 0.9, n = 10). Because a single iv administration of 3.0 nmol ghrelin significantly increased food intake in both OLETF and LETO rats (Fig. 1B), we used this dose as a standard dose of ghrelin in feeding experiments. Although a single iv administration of CCK significantly decreased food intake in LETO rats, it did not affect food intake in CCK-AR-deficient OLETF rats (Fig. 1C). When CCK was administered iv to LETO rats 30 min before ghrelin administration, we could not observe a ghrelin-induced increase in food intake (Fig. 2A). Administration of CCK iv to OLETF rats 30 min before ghrelin treatment, however, induced similar increases in food intake as those seen in rats without pretreatment (Fig. 2A). Conversely, when ghrelin was administered iv to LETO rats 30 min before CCK treatment, the CCK-induced feeding reduction could not be observed (Fig. 2B).
FIG. 1. The effect of iv administration of ghrelin or CCK on food intake in LETO and OLETF rats. A, Two-hour food intake (mean ± SEM) of 8-h fasting rats after a single iv administration of CCK (0.01–5 nmol). *, P < 0.0005 vs. control. B, After a single iv administration of ghrelin (3 nmol) or saline to LETO and OLETF rats, 2-h food intake from 1000–1200 h was measured. *, P < 0.0001 vs. control. C, After a single iv administration of CCK (1 nmol) or saline to LETO and OLETF rats after an 8-h fasting period, 2-h food intake from 1000–1200 h was measured. Control rats were administered saline iv. *, P < 0.001 vs. control.
FIG. 2. The interaction between ghrelin and CCK on food intake in LETO and OLETF rats. A, After a single iv administration of ghrelin or saline after CCK treatment of LETO and OLETF rats after an 8-h fasting period, 2-h food intake from 1000–1200 h was measured. *, P < 0.01 vs. OLETF rats administered saline after CCK treatment. B, After a single iv administration of CCK or saline after ghrelin or saline treatment of LETO rats after an 8-h fasting period, 2-h food intake was measured from 1000–1200 h. Control rats were administered saline iv. **, P < 0.001 vs. control.
Immunohistochemistry
GHS-R- and CCK-AR-immunoreactive neurons were found throughout the nodose ganglion (Fig. 3A). Approximately 70% of GHS-R-immunoreactive neurons in the nodose ganglion also expressed CCK-AR (Fig. 3, B–D). Double-staining studies also demonstrated the colocalization of GHS-R with CCK-AR in cultured nodose ganglion neurons (Fig. 3, E–G). GHS-R immunoreactivity was observed in GHS-R-expressing CHO cells (CHO-GHSR62 cells), but not in control CHO cells (data not shown). No GHS-R-specific immunoreactivity could be detected in the nodose ganglion using either normal rabbit serum or antiserum absorbed with excess synthetic GHS-R [342–364] (Fig. 3H).
FIG. 3. Colocalization of GHS-R with CCK-AR in neurons of the nodose ganglion. A, GHS-R-immunoreactive neurons (arrows) are distributed throughout the nodose ganglion. Antisera for GHS-R [342–364] (A, B, D, E, and G) and CCK-AR (C, D, F, and G) were used to assess the (D) immunofluorescence double staining of GHS-R and CCK-AR in the nodose ganglion or (G) immunofluorescence double staining of GHS-R and CCK-AR in primary cultured neurons of the nodose ganglion. H, No GHS-R-immunoreactivity was observed with antiserum absorbed with excessive synthetic GHS-R [342–364]. R, Rostral; C, caudal; D, dorsal; V, ventral. Scale bar, 200 μm (A), 100 μm (B–H)
Fos expression
A single iv administration of ghrelin induced Fos protein in the arcuate nucleus of the hypothalamus of rats (Fig. 4A). Fos-positive neurons were mainly distributed from the anterior to the middle region of the arcuate nucleus. Ghrelin-induced Fos expression in the NTS and PBN was not found (data not shown). When CCK was administered to rats iv 30 min before ghrelin treatment, the number of Fos-expressing neurons was significantly decreased compared with that induced by ghrelin alone (Fig. 4, B and E) and was not different from that of control rats (Fig. 4, C and E).
FIG. 4. Fos expression in the arcuate nucleus induced by ghrelin administration. A, Fos expression in response to iv ghrelin administration to rats after saline treatment. B, Fos expression in response to iv ghrelin administration after CCK treatment. C, Fos expression in response to iv saline to rats. D, A schematic drawing of an area in which Fos-positive neurons were counted. Fos-expressing neurons in a 0.7-mm right triangle (0.245 mm2) were evaluated. Arc, arcuate nucleus; DMD, dorsomedial nucleus, dorsal; F, fornix; MTu, medial tuberal nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle. Scale bar, 50 μm. C, The number of cells per section (bilateral). Data are expressed as mean ± SEM (n = 3). *, P < 0.0001 vs. rats administered ghrelin after saline treatment.
Electrophysiological study
Intravenous administration of ghrelin to rats significantly suppressed gastric vagal afferent activity (Fig. 5A), whereas iv administration of CCK significantly enhanced the afferent activity (Fig. 5B). The lowest effective doses of ghrelin and CCK were 0.3 pmol and 0.1 pmol, respectively (Fig. 5, A and B). To investigate the interaction between ghrelin and CCK in electrophysiological studies, we used 1.5 nmol ghrelin and 1 nmol CCK, considered as their respective standard doses for feeding. When ghrelin was administered to rats after CCK treatment, gastric vagal afferent activity was not suppressed (Fig. 5C). Conversely, when CCK was administered to rats after ghrelin treatment, the CCK-mediated enhancement of afferent activity could not be observed (Fig. 5D).
FIG. 5. The electrophysiological effect of ghrelin and CCK on gastric vagal afferent activity. A, Alterations of gastric vagal afferent discharge after a single iv administration of ghrelin (0.03 pmol–1.5 nmol). *, P < 0.05 vs. value at 0 min of 1.5 nmol ghrelin. #, P < 0.05 vs. value at 0 min of 0.3 pmol ghrelin. B, Alterations of gastric vagal afferent discharge after a single iv administration of CCK (0.01 pmol–1 nmol). *, P < 0.05 vs. value at 0 min of 1 nmol CCK. #, P < 0.05 vs. value at 0 min of 0.1 pmol CCK. C, Ghrelin (1.5 nmol) administration after CCK treatment (1 nmol) does not attenuate gastric vagal afferent activity. *, P < 0.05 vs. value at 0 min. D, CCK (1 nmol) administration after ghrelin (1.5 nmol) treatment does not activate gastric vagal afferent activity. *, P < 0.05 vs. value at 0 min. Representative data of gastric vagal afferent discharge rates are shown in the upper panels. Vertical bar, 100 impulses/5 sec; horizontal bar, 30 min.
Discussion
Signals produced within gastrointestinal tract affect feeding patterns (53). Distension of the stomach inhibits feeding, while nutrients in the small intestine induce the release of several gastrointestinal hormones, including CCK, an intestinal anorectic peptide (8, 9). The vagus nerve plays an important role in regulating feeding behavior by transmitting chemosensory and mechanosensory information from the viscera (54). Both neural and humoral signals for satiety and starvation generated in the gastrointestinal tract can be conveyed to the brain via the vagal afferent nerve and the blood circulation.
In the present study, we investigated peripheral interaction of ghrelin, an orexigenic gut peptide, with CCK on feeding regulation. Ghrelin, discovered in the stomach, stimulates food intake and GH secretion after iv administration (5, 16, 38, 55). Because the existence of an orexigenic peptide-based system in the periphery has yet to be discovered, ghrelin is thought to be the first peptide acting in the periphery as a starvation signal. Plasma ghrelin levels are up-regulated under conditions of negative energy balance including starvation, whereas they are down-regulated under conditions of positive energy balance (19, 56, 57, 58, 59). Glucose load and food intake lead to a rapid fall in plasma ghrelin concentration, indicating that endogenous ghrelin serves as an indicator of short-term energy balance (19). Very recently, Cummings et al. (22) showed the preprandial increase of ghrelin levels among humans without time- or food-related cues and the overlap between these levels and hunger scores. These findings indicate ghrelin would be a candidate for peripheral meal initiator. In contrast, CCK, the most well-studied gastrointestinal peptide functioning in feeding, transmits a satiety signal to the NTS via vagal afferents (41, 43, 60). CCK decreases food intake when peripherally administered to rats (30). In humans, postprandial CCK levels are increased about five times higher than fasting levels (35, 61, 62). Thus, CCK has been thought to be a meal terminator.
Ghrelin is produced not only in the stomach but also in the hypothalamus (5, 63). Centrally administered ghrelin also stimulates both food intake and GH secretion (5, 13, 15, 64, 65, 66), and the ghrelin receptor is expressed in neuropeptide Y (NPY)- and GHRH-producing neurons in the hypothalamic arcuate nucleus, where it is incompletely isolated from the general circulation by the blood-brain barrier (67). These findings indicate that central ghrelin, peripheral ghrelin, or both may increase food intake and GH secretion via NPY and GHRH directly. However, we recently demonstrated that blockade of the gastric vagal afferent abolished ghrelin-induced feeding, GH secretion, and activation of NPY and GHRH neurons (38). These data suggest a possibility that ghrelin’s signals for starvation and GH secretion are conveyed to the brain via the gastric vagal afferent system. Therefore, ghrelin and CCK, both produced within the gastrointestinal tract, exert opposite effects on feeding behavior through the vagal afferent, thereby regulating food intake on a short-term basis as a meal initiator and terminator, respectively.
In this study, we examined the interaction of ghrelin with CCK in the regulation of feeding behavior using CCK-AR-deficient OLETF rats. Ghrelin increased food intake in both OLETF and their lean littermates, LETO rats. In contrast, CCK decreased food intake in LETO rats fasted for 8-h period, but did not affect food intake in OLETF rats. These findings indicate that CCK-AR is required for CCK, but not ghrelin, regulation of feeding and that exogenous CCK reduces food intake of rats whose endogenous ghrelin levels are increased. Preadministration of CCK to LETO, but not to OLETF, rats blocked the food intake induced by peripheral administration of ghrelin. Conversely, the preadministration of ghrelin to LETO rats blocked the feeding reduction induced by peripheral CCK administration. These findings suggest that the effect of CCK or ghrelin administered after ghrelin or CCK, respectively, on feeding, might not be displayed, while some information to determine feeding behavior induced by exogenously preadministered ghrelin or CCK is transmitting via the vagal afferent system to the brain. When ghrelin or CCK was administered to rats, each plasma level transiently increases over their physiological ranges, which may also have cause complete blockade of the effect of serially administered ghrelin or CCK on feeding.
We also investigated the colocalization of GHS-R with CCK-AR in the rat nodose ganglion. Receptors of the vagal afferent are generated by nodose ganglion neurons and are transported to the nerve terminal through axonal transport (68, 69). Although we failed to demonstrate the colocalization of GHS-R and CCK-AR in the nerve terminal, immunohistochemical double staining of the nodose ganglion demonstrated that the majority of the GHS-R-immunoreactive neurons express CCK-AR. These findings suggest that the vagus nerve plays a major role in determining the peripheral parameters of energy balance.
Signals mediated by ghrelin secretion by the stomach are thought to be transmitted to the hypothalamus of the brain via the NTS, as iv administration of ghrelin induces Fos expression in the arcuate nucleus of the hypothalamus (38). Ghrelin suppresses gastric vagal afferent discharges when administered iv (38), whereas CCK enhances these discharges (43, 44, 45, 46, 47, 48). Preadministration of CCK reduced the number of Fos-immunoreactive neurons induced by ghrelin. Very recently, Kobelt et al. (70) showed that peripherally administered CCK simultaneously with ghrelin inhibited ghrelin-induced feeding and ghrelin-induced Fos expression in the hypothalamic arcuate nucleus. These results are consistent with our data presented here. In addition, treatment with ghrelin after CCK administration did not affect the vagal afferent discharges induced by CCK. The effect of some peptides on vagal afferent discharge is known to be rapid (71, 72). However, in our experimental system, the changes in firing rate of the vagal afferent fibers induced by several substances continued over 60 min (38, 42, 73, 74, 75, 76, 77, 78, 79). These findings suggest that alteration of the firing rate counted by the interval and/or number of firing fibers may be caused by several messengers after peptides bound to their receptors. For example, a single somatostatin administration to rats actually increased the vagal afferent discharge for over 60 min. The afferent discharge stimulated by somatostatin was canceled by an injection of a monoclonal antibody for somatostatin before, but is ineffective after, the somatostatin injection (79). These results suggest the involvement of a unique postreceptor mechanism in the chemoreception as responsible for this long-acting effect of somatostatin on the afferent discharge. Such a postreceptor mechanism may apply to the time course of the ghrelin-induced decrease or CCK-induced increase of the vagal afferent discharge, although the precise mechanism remains to be elucidated. Recently, Królczyk et al. (80) performed electrophysiological recordings in both fasted and fed rats and demonstrated that the firing rate of the vagal afferent discharge in fasted rats was lower than that in fed rats. In that study, the increase in the firing rate after food administration to the fasted rats lasted for 15 min. Considering that ghrelin concentration increases in the fasting state and CCK concentration increases after feeding, exogenous administrations of ghrelin and CCK may induce in part starvation and satiety conditions on the basis of circulating hormones, respectively. The actual linkage of these peripheral signals with the vagal afferent pathway is likely to be more complicated given the remarkable number of neurotransmitters, neuropeptides, and neuromodulators. Feeding is a complicated interaction of many factors such as orexigenic or anorectic signals, emotion, learning, memory, etc. We believe that the alternation of the firing rate of the vagal afferent induced by ghrelin, CCK, or the combination of ghrelin with CCK is only a part of feeding regulation. Although it is difficult to clearly explain the reasons why the afferent activity lasts for such a long period after the single administration of ghrelin or CCK, we suggest that the long-acting effect on the afferent discharge may provide sufficient time for the brain to receive feeding-related conditions originating throughout the body. In addition, there may be a limitation on connecting the electrophysiological findings of rats under anesthetization with the feeding behavior of free-moving rats.
In summary, this study demonstrates that ghrelin administration after CCK treatment does not induce feeding; CCK administration after ghrelin treatment does not reduce it. We assume some mechanism whereby the intracellular signaling pathway induced by preadministered ghrelin or CCK interferes with signal transmission of serially administered CCK or ghrelin. In addition, the efficiency of ghrelin and CCK signal transport may depend on the balance in the plasma concentrations of these factors. In normal subjects, plasma ghrelin levels rise before the onset of meals and decline 30 min after feeding. In obese subjects, however, these declinations in plasma ghrelin levels are absent (81). The lack of suppression of ghrelin secretion after a meal may be a critical factor in the pathophysiology of obesity and eating disorders. CCK is released postprandially, eliciting satiety signals (82, 83, 84, 85). Plasma CCK concentrations in lean subjects fed a solid meal peak around 60 min after eating (86, 87). CCK also interacts synergistically in rats with other hormones released postprandially, including insulin, leptin, and glucagon (88, 89, 90). Abnormalities in the release of or sensitivity to ghrelin and/or CCK may be involved in alterations of food intake. Further investigation of the mechanisms controlling ghrelin and CCK release will help our understanding of the multifactorial regulation of feeding behavior, potentially leading to new treatments for obesity and eating disorders.
Acknowledgments
We thank Rie Matsuura for assistance.
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Address all correspondence and requests for reprints to: Yukari Date, M.D., Ph.D., Third Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan. E-mail: dateyuka@med.miyazaki-u.ac.jp.
Abstract
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 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.
Introduction
IN 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 primarily in endocrine cells of the stomach, is released into circulation (5, 6, 7). Whereas multiple gastrointestinal hormones have been implicated in feeding regulation (8, 9, 10, 11, 12), ghrelin stimulates appetite, food intake, and GH secretion when administered to humans and rodents (5, 13, 14, 15, 16, 17). In humans, the circulating ghrelin levels increase before and decrease after every meal (18, 19, 20, 21, 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, 24, 25, 26, 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, 31, 32). This peptide, released from the proximal small intestine, functions as a postprandial satiety signal (33, 34, 35, 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 starvation 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, 39, 40, 41, 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, Long-Evans 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, 43, 44, 45, 46, 47, 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, 403.2 ± 6.0 g; LETO, 392.8 ± 2.6 g; P > 0.1; n = 20), obtained from Otsuka Pharmaceutical (Tokushima, Japan), were used in the experiments for feeding. Male Wistar rats (body weight: 361.6 ± 1.3 g; n = 20) (Charles River Japan, Inc., Shiga, Japan) were used for immunohistochemistry, Fos expression, and electrophysiological studies. Rats were housed individually in plastic cages at constant room temperature in a 12-h light (0800–2000)/12-h dark cycle. Animals were given standard laboratory chow and water ad libitum. Intravenous cannulas were implanted into the right jugular vein under anesthesia after an ip injection of sodium pentobarbital (80 mg/kg body weight) (Abbott Laboratories, Chicago, IL). Rats were sham-injected before the study and weighed and handled daily. We also injected heparin daily (1 U/100 μl 0.9% saline) into the cannulas of the animals to prevent coagulation. Only animals exhibiting progressive weight gain after surgery were used in subsequent experiments. All procedures were performed in accordance with the Japanese Physiological Society’s guidelines for animal care.
Preparation of anti-GHS-R serum
The [Cys0]-rat GHS-R [342–364] peptide was synthesized using the Fmoc solid-phase method on a peptide synthesizer (433A; Applied Biosystems, Foster City, CA), then purified by reverse phase-HPLC. The synthesized peptide (10 mg) was conjugated to maleimide-activated mariculture keyhole limpet hemocyanin (6 mg) (mcKLH; Pierce, Rockford, IL) in conjugation buffer (Pierce). The conjugate was emulsified with an equal volume of Freund’s complete adjuvant and was used to immunize New Zealand white rabbits by intracutaneous and sc injection. Animals were boosted every 2 wk and bled 7 d after each injection. The specificity of the antisera was confirmed by immunoreactivity of GHS-R-expressing (CHO-GHSR62 cells), but not of control cells.
Feeding experiments
Experiments were performed 1 wk after iv cannulation. First, CCK (Peptide Institute, Inc., Osaka, Japan) was dissolved in 0.9% saline, and this solution (10 pmol-5 nmol/100 μl) was administered iv at 1000 h to LETO rats after fasting for an 8-h period to determine the lowest effective dose of CCK on feeding (n = 10 per group). Second, a solution of rat ghrelin (Peptide Institute) dissolved in 0.9% saline (1.5 nmol/100 μl or 3 nmol/100 μl) was administered iv at 1000 h to OLETF and LETO rats fed ad libitum (n = 10 per group). After injection, rats were immediately returned to their cages, then 2-h food intake was measured. Third, a solution of CCK dissolved in 0.9% saline (1 nmol/100 μl) was administered iv at 1000 h to OLETF and LETO rats after fasting for an 8-h period (n = 10 per group). After returning rats to their cages immediately after injection, 2-h total food intake was measured. Fourth, after fasting for an 8-h period, OLETF and LETO rats were treated with CCK (1 nmol/100 μl). They were not given any food until the next injections. Animals were subsequently given ghrelin (3 nmol/100 μl) or saline (100 μl) 30 min after CCK injection (n = 10 per group). After ghrelin or saline injection, 2-h food intake was measured. Fifth, after an 8-h fasting period, LETO rats were first treated with ghrelin (3 nmol/100 μl), then subsequently given CCK (1 nmol/100 μl) or saline (100 μl) 30 min after ghrelin injection (n = 10 per group). The rats were fasted between ghrelin and CCK or saline injections. After this second injection, 2-h food intake was measured. These feeding studies were performed in a crossover design. Rats were allowed at least 4 d without injections between experimental days.
Immunohistochemical double-staining
Three Wistar rats, weighing 300–350 g, were perfused transcardially with 0.1 M phosphate buffer (pH 7.4), then with 4% paraformaldehyde in a 0.1 M phosphate buffer. The nodose ganglia were sectioned into 12 μm-thick slices at –20 C using a cryostat. Sections were stored at –80 C. Primary neurons were also obtained from the nodose ganglia of five Wistar rats, ranging from 5–6 wk of age. These neurons were submitted to collagenase dispersion as described (49, 50), then seeded and cultured for 4 d in polyethylenimine-coated Lab-Tek chamber slides (Electron Microscopy Sciences, Hatfield, PA) in complete DMEM (25 mM glucose) containing 5% newborn calf serum, 5% horse serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 30 ng/ml nerve growth factor 2.5S (Sigma Chemical Co., St. Louis, MO), and 2 mM L-glutamine at 37 C in 5% CO2. The medium was replaced every 2 d. Slides were washed in 0.01 M PBS (pH 7.4), then fixed in 10% formaldehyde. The slides of both the primary culture and sectioned nodose ganglia were incubated overnight at 4 C in rabbit anti-GHS-R antiserum (dilution 1/1000). Antibody staining was detected using Alexa Fluor 594-conjugated chicken antirabbit IgG (Molecular Probes, Inc., Eugene, OR). Samples were subsequently incubated with anti-CCK-AR antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution 1/100), then with Alexa Fluor 488-conjugated donkey antigoat IgG (Molecular Probes, Inc.). Slides were observed by fluorescence microscopy (BH2-RFC; Olympus, Tokyo, Japan). The number of neurons expressing GHS-R or CCK-AR immunoreactivity in the nodose ganglion was quantified by counting two randomly selected visual fields in two sections from each of the three rats.
Fos expression and image analysis
The lowest effective dose of ghrelin or CCK on feeding was used for Fos expression studies. CCK (1 nmol/100 μl) or saline was injected iv into three male Wistar rats weighing 341.6 ± 1.3 g. Ghrelin (1.5 nmol/100 μl) was then injected into these rats 30 min after CCK or saline injection. Ninety minutes after ghrelin or saline injection, rats were perfused transcardially with fixative containing 4% paraformaldehyde. The brain was sectioned into 40-μm-thick samples. Immunohistochemistry of Fos was performed as described (51). Quantitation of Fos-immunoreactive cells in the nucleus of the solitary tract (NTS), parabranchial nucleus (PBN), and hypothalamic arcuate nucleus (bregma: –11.30 to –14.60 for NTS, –9.16 to –10.04 for PBN, –2.30 to –3.30 for the arcuate nucleus from Paxinos and Watson’s rats brain atlas) was performed bilaterally. Fos-expressing cells of the arcuate nucleus for a 0.7-mm right triangle (0.245 mm2) were counted in every fifth section (200 μm frequency) (five tissue sections per rats) using a cell counting program written for NIH Image (version 1.62; National Institutes of Health, Bethesda, MD).
Electrophysiological study
Multiunit neural discharge in gastric vagal afferent fibers was recorded extracellularly. Male Wistar rats, fasted for an 8-h period, were anesthetized by an ip injection of urethan (1 g/kg) (Sigma). The electrophysiological study was performed under anesthetization throughout. The rat trachea was intubated, and the electrocardiogram was recorded. Body temperature was maintained at 37 C. Standard methods of extracellular recording from vagal nerve filaments were used, as described in detail elsewhere (52). After laparotomy, a small catheter (Intramedic PE-10; Clay Adams, Parsippany, NJ) was inserted into the inferior vena cava. After gastric branches of the vagus nerve were visualized, we placed filaments isolated from the peripheral cut end of the ventral branch for recording of afferent nerve activity on a pair of silver wire electrodes. Silver wire electrodes, connected through an alternating current-coupled differential amplifier (DAP-10E; Dia Medical Systems, Co., Tokyo, Japan) to an oscilloscope and magnetic tape recorder, were used for display and storage of the neural activity. A window discriminator (DSE-325A) converted spikes to constant amplitude pulses for analysis of spike frequency with a rate meter that reset at 5-sec intervals. Output from the rate meter was recorded on a chart paper (8K20 recorder; NEC-SAN E1 Co., Tokyo, Japan). Ghrelin (0.03 pmol–1.5 nmol/100 μl) or CCK (0.01 pmol–1 nmol/100 μl) was administered iv to rats through a catheter inserted into the inferior vena cava (n = 10 per group). After administration, nerve discharges from the multiunit afferents were recorded for 60 min and analyzed. In addition, CCK (1 nmol/100 μl) or ghrelin (1.5 nmol/100 μl), considered as a standard dose for feeding (38), was administered iv to rats through a catheter inserted into the inferior vena cava (n = 5 per group) before recording the multiunit afferent nerve discharges for 30 min. After the subsequent iv administration of either ghrelin (1.5 nmol/100 μl) or CCK (1 nmol/100 μl) to these rats, multiunit afferent nerve discharges were recorded for 30 min and analyzed.
Statistical analysis
Groups of data (mean ± SEM) were compared using ANOVA and post hoc Fisher’s test. P < 0.05 was considered to be significant.
Results
Effects of ghrelin and CCK on feeding in OLETF or LETO rats
We first tested various doses of CCK ranging from 10 pmol to 5 nmol in a food intake experiment using LETO rats fasted for an 8-h period (Fig. 1A). The lowest effective dose of CCK administered iv was 1 nmol, which also applied to feeding examination using Wistar rats (data not shown). Therefore, we used 1 nmol as a standard dose of CCK in a subsequent series of experiments. The lowest effective dose of ghrelin on feeding in Wistar rats was 1.5 nmol (38); however, a single iv administration of 1.5 nmol ghrelin to OLETF and LETO rats did not induce feeding (LETO: saline, 0.35 ± 0.19 g; 1.5 nmol ghrelin, 0.28 ± 0.14 g; P > 0.7, n = 10; OLETF: saline, 0.36 ± 0.19 g; 1.5 nmol ghrelin, 0.33 ± 0.21 g; P > 0.9, n = 10). Because a single iv administration of 3.0 nmol ghrelin significantly increased food intake in both OLETF and LETO rats (Fig. 1B), we used this dose as a standard dose of ghrelin in feeding experiments. Although a single iv administration of CCK significantly decreased food intake in LETO rats, it did not affect food intake in CCK-AR-deficient OLETF rats (Fig. 1C). When CCK was administered iv to LETO rats 30 min before ghrelin administration, we could not observe a ghrelin-induced increase in food intake (Fig. 2A). Administration of CCK iv to OLETF rats 30 min before ghrelin treatment, however, induced similar increases in food intake as those seen in rats without pretreatment (Fig. 2A). Conversely, when ghrelin was administered iv to LETO rats 30 min before CCK treatment, the CCK-induced feeding reduction could not be observed (Fig. 2B).
FIG. 1. The effect of iv administration of ghrelin or CCK on food intake in LETO and OLETF rats. A, Two-hour food intake (mean ± SEM) of 8-h fasting rats after a single iv administration of CCK (0.01–5 nmol). *, P < 0.0005 vs. control. B, After a single iv administration of ghrelin (3 nmol) or saline to LETO and OLETF rats, 2-h food intake from 1000–1200 h was measured. *, P < 0.0001 vs. control. C, After a single iv administration of CCK (1 nmol) or saline to LETO and OLETF rats after an 8-h fasting period, 2-h food intake from 1000–1200 h was measured. Control rats were administered saline iv. *, P < 0.001 vs. control.
FIG. 2. The interaction between ghrelin and CCK on food intake in LETO and OLETF rats. A, After a single iv administration of ghrelin or saline after CCK treatment of LETO and OLETF rats after an 8-h fasting period, 2-h food intake from 1000–1200 h was measured. *, P < 0.01 vs. OLETF rats administered saline after CCK treatment. B, After a single iv administration of CCK or saline after ghrelin or saline treatment of LETO rats after an 8-h fasting period, 2-h food intake was measured from 1000–1200 h. Control rats were administered saline iv. **, P < 0.001 vs. control.
Immunohistochemistry
GHS-R- and CCK-AR-immunoreactive neurons were found throughout the nodose ganglion (Fig. 3A). Approximately 70% of GHS-R-immunoreactive neurons in the nodose ganglion also expressed CCK-AR (Fig. 3, B–D). Double-staining studies also demonstrated the colocalization of GHS-R with CCK-AR in cultured nodose ganglion neurons (Fig. 3, E–G). GHS-R immunoreactivity was observed in GHS-R-expressing CHO cells (CHO-GHSR62 cells), but not in control CHO cells (data not shown). No GHS-R-specific immunoreactivity could be detected in the nodose ganglion using either normal rabbit serum or antiserum absorbed with excess synthetic GHS-R [342–364] (Fig. 3H).
FIG. 3. Colocalization of GHS-R with CCK-AR in neurons of the nodose ganglion. A, GHS-R-immunoreactive neurons (arrows) are distributed throughout the nodose ganglion. Antisera for GHS-R [342–364] (A, B, D, E, and G) and CCK-AR (C, D, F, and G) were used to assess the (D) immunofluorescence double staining of GHS-R and CCK-AR in the nodose ganglion or (G) immunofluorescence double staining of GHS-R and CCK-AR in primary cultured neurons of the nodose ganglion. H, No GHS-R-immunoreactivity was observed with antiserum absorbed with excessive synthetic GHS-R [342–364]. R, Rostral; C, caudal; D, dorsal; V, ventral. Scale bar, 200 μm (A), 100 μm (B–H)
Fos expression
A single iv administration of ghrelin induced Fos protein in the arcuate nucleus of the hypothalamus of rats (Fig. 4A). Fos-positive neurons were mainly distributed from the anterior to the middle region of the arcuate nucleus. Ghrelin-induced Fos expression in the NTS and PBN was not found (data not shown). When CCK was administered to rats iv 30 min before ghrelin treatment, the number of Fos-expressing neurons was significantly decreased compared with that induced by ghrelin alone (Fig. 4, B and E) and was not different from that of control rats (Fig. 4, C and E).
FIG. 4. Fos expression in the arcuate nucleus induced by ghrelin administration. A, Fos expression in response to iv ghrelin administration to rats after saline treatment. B, Fos expression in response to iv ghrelin administration after CCK treatment. C, Fos expression in response to iv saline to rats. D, A schematic drawing of an area in which Fos-positive neurons were counted. Fos-expressing neurons in a 0.7-mm right triangle (0.245 mm2) were evaluated. Arc, arcuate nucleus; DMD, dorsomedial nucleus, dorsal; F, fornix; MTu, medial tuberal nucleus; VMH, ventromedial hypothalamic nucleus; 3V, third ventricle. Scale bar, 50 μm. C, The number of cells per section (bilateral). Data are expressed as mean ± SEM (n = 3). *, P < 0.0001 vs. rats administered ghrelin after saline treatment.
Electrophysiological study
Intravenous administration of ghrelin to rats significantly suppressed gastric vagal afferent activity (Fig. 5A), whereas iv administration of CCK significantly enhanced the afferent activity (Fig. 5B). The lowest effective doses of ghrelin and CCK were 0.3 pmol and 0.1 pmol, respectively (Fig. 5, A and B). To investigate the interaction between ghrelin and CCK in electrophysiological studies, we used 1.5 nmol ghrelin and 1 nmol CCK, considered as their respective standard doses for feeding. When ghrelin was administered to rats after CCK treatment, gastric vagal afferent activity was not suppressed (Fig. 5C). Conversely, when CCK was administered to rats after ghrelin treatment, the CCK-mediated enhancement of afferent activity could not be observed (Fig. 5D).
FIG. 5. The electrophysiological effect of ghrelin and CCK on gastric vagal afferent activity. A, Alterations of gastric vagal afferent discharge after a single iv administration of ghrelin (0.03 pmol–1.5 nmol). *, P < 0.05 vs. value at 0 min of 1.5 nmol ghrelin. #, P < 0.05 vs. value at 0 min of 0.3 pmol ghrelin. B, Alterations of gastric vagal afferent discharge after a single iv administration of CCK (0.01 pmol–1 nmol). *, P < 0.05 vs. value at 0 min of 1 nmol CCK. #, P < 0.05 vs. value at 0 min of 0.1 pmol CCK. C, Ghrelin (1.5 nmol) administration after CCK treatment (1 nmol) does not attenuate gastric vagal afferent activity. *, P < 0.05 vs. value at 0 min. D, CCK (1 nmol) administration after ghrelin (1.5 nmol) treatment does not activate gastric vagal afferent activity. *, P < 0.05 vs. value at 0 min. Representative data of gastric vagal afferent discharge rates are shown in the upper panels. Vertical bar, 100 impulses/5 sec; horizontal bar, 30 min.
Discussion
Signals produced within gastrointestinal tract affect feeding patterns (53). Distension of the stomach inhibits feeding, while nutrients in the small intestine induce the release of several gastrointestinal hormones, including CCK, an intestinal anorectic peptide (8, 9). The vagus nerve plays an important role in regulating feeding behavior by transmitting chemosensory and mechanosensory information from the viscera (54). Both neural and humoral signals for satiety and starvation generated in the gastrointestinal tract can be conveyed to the brain via the vagal afferent nerve and the blood circulation.
In the present study, we investigated peripheral interaction of ghrelin, an orexigenic gut peptide, with CCK on feeding regulation. Ghrelin, discovered in the stomach, stimulates food intake and GH secretion after iv administration (5, 16, 38, 55). Because the existence of an orexigenic peptide-based system in the periphery has yet to be discovered, ghrelin is thought to be the first peptide acting in the periphery as a starvation signal. Plasma ghrelin levels are up-regulated under conditions of negative energy balance including starvation, whereas they are down-regulated under conditions of positive energy balance (19, 56, 57, 58, 59). Glucose load and food intake lead to a rapid fall in plasma ghrelin concentration, indicating that endogenous ghrelin serves as an indicator of short-term energy balance (19). Very recently, Cummings et al. (22) showed the preprandial increase of ghrelin levels among humans without time- or food-related cues and the overlap between these levels and hunger scores. These findings indicate ghrelin would be a candidate for peripheral meal initiator. In contrast, CCK, the most well-studied gastrointestinal peptide functioning in feeding, transmits a satiety signal to the NTS via vagal afferents (41, 43, 60). CCK decreases food intake when peripherally administered to rats (30). In humans, postprandial CCK levels are increased about five times higher than fasting levels (35, 61, 62). Thus, CCK has been thought to be a meal terminator.
Ghrelin is produced not only in the stomach but also in the hypothalamus (5, 63). Centrally administered ghrelin also stimulates both food intake and GH secretion (5, 13, 15, 64, 65, 66), and the ghrelin receptor is expressed in neuropeptide Y (NPY)- and GHRH-producing neurons in the hypothalamic arcuate nucleus, where it is incompletely isolated from the general circulation by the blood-brain barrier (67). These findings indicate that central ghrelin, peripheral ghrelin, or both may increase food intake and GH secretion via NPY and GHRH directly. However, we recently demonstrated that blockade of the gastric vagal afferent abolished ghrelin-induced feeding, GH secretion, and activation of NPY and GHRH neurons (38). These data suggest a possibility that ghrelin’s signals for starvation and GH secretion are conveyed to the brain via the gastric vagal afferent system. Therefore, ghrelin and CCK, both produced within the gastrointestinal tract, exert opposite effects on feeding behavior through the vagal afferent, thereby regulating food intake on a short-term basis as a meal initiator and terminator, respectively.
In this study, we examined the interaction of ghrelin with CCK in the regulation of feeding behavior using CCK-AR-deficient OLETF rats. Ghrelin increased food intake in both OLETF and their lean littermates, LETO rats. In contrast, CCK decreased food intake in LETO rats fasted for 8-h period, but did not affect food intake in OLETF rats. These findings indicate that CCK-AR is required for CCK, but not ghrelin, regulation of feeding and that exogenous CCK reduces food intake of rats whose endogenous ghrelin levels are increased. Preadministration of CCK to LETO, but not to OLETF, rats blocked the food intake induced by peripheral administration of ghrelin. Conversely, the preadministration of ghrelin to LETO rats blocked the feeding reduction induced by peripheral CCK administration. These findings suggest that the effect of CCK or ghrelin administered after ghrelin or CCK, respectively, on feeding, might not be displayed, while some information to determine feeding behavior induced by exogenously preadministered ghrelin or CCK is transmitting via the vagal afferent system to the brain. When ghrelin or CCK was administered to rats, each plasma level transiently increases over their physiological ranges, which may also have cause complete blockade of the effect of serially administered ghrelin or CCK on feeding.
We also investigated the colocalization of GHS-R with CCK-AR in the rat nodose ganglion. Receptors of the vagal afferent are generated by nodose ganglion neurons and are transported to the nerve terminal through axonal transport (68, 69). Although we failed to demonstrate the colocalization of GHS-R and CCK-AR in the nerve terminal, immunohistochemical double staining of the nodose ganglion demonstrated that the majority of the GHS-R-immunoreactive neurons express CCK-AR. These findings suggest that the vagus nerve plays a major role in determining the peripheral parameters of energy balance.
Signals mediated by ghrelin secretion by the stomach are thought to be transmitted to the hypothalamus of the brain via the NTS, as iv administration of ghrelin induces Fos expression in the arcuate nucleus of the hypothalamus (38). Ghrelin suppresses gastric vagal afferent discharges when administered iv (38), whereas CCK enhances these discharges (43, 44, 45, 46, 47, 48). Preadministration of CCK reduced the number of Fos-immunoreactive neurons induced by ghrelin. Very recently, Kobelt et al. (70) showed that peripherally administered CCK simultaneously with ghrelin inhibited ghrelin-induced feeding and ghrelin-induced Fos expression in the hypothalamic arcuate nucleus. These results are consistent with our data presented here. In addition, treatment with ghrelin after CCK administration did not affect the vagal afferent discharges induced by CCK. The effect of some peptides on vagal afferent discharge is known to be rapid (71, 72). However, in our experimental system, the changes in firing rate of the vagal afferent fibers induced by several substances continued over 60 min (38, 42, 73, 74, 75, 76, 77, 78, 79). These findings suggest that alteration of the firing rate counted by the interval and/or number of firing fibers may be caused by several messengers after peptides bound to their receptors. For example, a single somatostatin administration to rats actually increased the vagal afferent discharge for over 60 min. The afferent discharge stimulated by somatostatin was canceled by an injection of a monoclonal antibody for somatostatin before, but is ineffective after, the somatostatin injection (79). These results suggest the involvement of a unique postreceptor mechanism in the chemoreception as responsible for this long-acting effect of somatostatin on the afferent discharge. Such a postreceptor mechanism may apply to the time course of the ghrelin-induced decrease or CCK-induced increase of the vagal afferent discharge, although the precise mechanism remains to be elucidated. Recently, Królczyk et al. (80) performed electrophysiological recordings in both fasted and fed rats and demonstrated that the firing rate of the vagal afferent discharge in fasted rats was lower than that in fed rats. In that study, the increase in the firing rate after food administration to the fasted rats lasted for 15 min. Considering that ghrelin concentration increases in the fasting state and CCK concentration increases after feeding, exogenous administrations of ghrelin and CCK may induce in part starvation and satiety conditions on the basis of circulating hormones, respectively. The actual linkage of these peripheral signals with the vagal afferent pathway is likely to be more complicated given the remarkable number of neurotransmitters, neuropeptides, and neuromodulators. Feeding is a complicated interaction of many factors such as orexigenic or anorectic signals, emotion, learning, memory, etc. We believe that the alternation of the firing rate of the vagal afferent induced by ghrelin, CCK, or the combination of ghrelin with CCK is only a part of feeding regulation. Although it is difficult to clearly explain the reasons why the afferent activity lasts for such a long period after the single administration of ghrelin or CCK, we suggest that the long-acting effect on the afferent discharge may provide sufficient time for the brain to receive feeding-related conditions originating throughout the body. In addition, there may be a limitation on connecting the electrophysiological findings of rats under anesthetization with the feeding behavior of free-moving rats.
In summary, this study demonstrates that ghrelin administration after CCK treatment does not induce feeding; CCK administration after ghrelin treatment does not reduce it. We assume some mechanism whereby the intracellular signaling pathway induced by preadministered ghrelin or CCK interferes with signal transmission of serially administered CCK or ghrelin. In addition, the efficiency of ghrelin and CCK signal transport may depend on the balance in the plasma concentrations of these factors. In normal subjects, plasma ghrelin levels rise before the onset of meals and decline 30 min after feeding. In obese subjects, however, these declinations in plasma ghrelin levels are absent (81). The lack of suppression of ghrelin secretion after a meal may be a critical factor in the pathophysiology of obesity and eating disorders. CCK is released postprandially, eliciting satiety signals (82, 83, 84, 85). Plasma CCK concentrations in lean subjects fed a solid meal peak around 60 min after eating (86, 87). CCK also interacts synergistically in rats with other hormones released postprandially, including insulin, leptin, and glucagon (88, 89, 90). Abnormalities in the release of or sensitivity to ghrelin and/or CCK may be involved in alterations of food intake. Further investigation of the mechanisms controlling ghrelin and CCK release will help our understanding of the multifactorial regulation of feeding behavior, potentially leading to new treatments for obesity and eating disorders.
Acknowledgments
We thank Rie Matsuura for assistance.
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