OBESITY HAS REACHED an escalating rate since 1980

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1 /07/$15.00/0 Endocrinology 148(12): Printed in U.S.A. Copyright 2007 by The Endocrine Society doi: /en Urocortins and Cholecystokinin-8 Act Synergistically to Increase Satiation in Lean But Not Obese Mice: Involvement of Corticotropin-Releasing Factor Receptor-2 Pathway G. Gourcerol,* L. Wang,* Y. H. Wang, M. Million, and Y. Taché Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women s Health, Department of Medicine, Division of Digestive Diseases, University of California Los Angeles, Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California Interactions between gastrointestinal signals are a part of integrated systems regulating food intake (FI). We investigated whether cholecystokinin (CCK)-8 and urocortin systems potentiate each other to inhibit FI and gastric emptying (GE) in fasted mice. Urocortin 1 and urocortin 2 (1 g/kg) were injected ip alone or with CCK (3 g/kg) in lean, diet-induced obese (DIO) or corticotropin-releasing factor receptor-2 (CRF 2 )-deficient mice. Gastric vagal afferent activity was recorded from a rat stomach-vagus in vitro preparation. When injected separately, urocortin 1, urocortin 2, or CCK did not modify the 4-h cumulative FI in lean mice. However, CCK plus urocortin 1 or CCK plus urocortin 2 decreased significantly the 4-h FI by 39 and 27%, respectively, compared with the vehicle vehicle group in lean mice but not in DIO mice. OBESITY HAS REACHED an escalating rate since 1980 with a current estimate of 33% of the adult population being obese in the United States (1). Several gut peptides are known to influence eating behavior, body weight, and glucose homeostasis (2). Growing evidence supports their possible relevance in the development of eating disorders and obesity (2 4). In particular, recent studies indicate that changes in gastrointestinal peptides and incretin hormones may contribute to the rapid resolution of type 2 diabetes, reduction of food intake, and weight loss after gastric bypass surgery (5 7). Among gut peptides, cholecystokinin (CCK) is expressed in enteroendocrine cells of the intestinal mucosa and secreted predominantly as CCK-58 in response to specific nutrients present in the gut lumen (8 10). CCK-8 interaction with CCK-1 receptors located on endings of vagal sensory fibers represents the major afferent limb of peripheral CCK-8-induced early satiation and inhibition of gastric emptying (11 13). It is now well established that there is a synergistic First Published Online October 11, 2007 * G.G. and L.W. contributed equally to this work. Abbreviations: CCK, Cholecystokinin; CRF, corticotropin-releasing factor; DIO, diet-induced obesity; GD, gastric distension; GVA, gastric vagal afferent; h, human; ia, intragastric artery; m, mouse; Q, response quotient; RF, mechanoreceptive field; Ucn, urocortin. Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. Likewise, CCK-urocortin-1 delayed GE in lean but not DIO mice, whereas either peptide injected alone at the same dose had no effect. CCK-urocortin 2 suppression of FI was observed in wild-type but not CRF 2 -deficient mice. Gastric vagal afferent activity was increased by intragastric artery injection of urocortin 2 after CCK at a subthreshold dose, and the response was reversed by devazepide. These data establish a peripheral synergistic interaction between CCK and urocortin 1 or urocortin 2 to suppress FI and GE through CRF 2 receptor in lean mice that may involve CCK modulation of gastric vagal afferent responsiveness to urocortin 2. Such synergy is lost in DIO mice, suggesting a resistance to the satiety signaling that may contribute to maintain obesity. (Endocrinology 148: , 2007) interaction between CCK-8 and leptin, the long-term signal of energy balance (14 17). We initially reported that the ip coinjection of CCK-8 and leptin results in a sustained inhibition of food intake linked with an increased gastric vagal afferent discharge and Fos expression in brain nuclei regulating food intake in rats and mice (14, 15). CCK satiety action is also modulated by pancreatic/gut hormones, transmitters, and steroids such as insulin, glucagon-like peptide, serotonin, and estradiol in rodents and/or humans (18 21). Brain corticotropin-releasing factor (CRF) signaling pathways play a key role in the stress response, including the alteration of feeding behavior (22, 23). The CRF system in mammals encompasses CRF and three related peptides, urocortin (Ucn) 1, Ucn 2, and Ucn 3 and two receptor subtypes, CRF 1 and CRF 2 (24, 25). CRF ligands interact with CRF receptors with a distinct affinity. CRF binds with higher affinity to CRF 1 than CRF 2 receptors. Ucn 1 displays a high affinity to both CRF 1 and CRF 2 receptors, whereas Ucn 2 and Ucn 3 bind selectively to CRF 2 receptors (24, 25). Several reports established that CRF, Ucn 1, Ucn 2, and Ucn 3 injected into the brain reduce food intake in rodents through CRF 1 and CRF 2 receptor-dependent mechanisms that mediate, respectively, the acute (first hour) and delayed (3 6 h) anorexic responses (26 30). A few studies also demonstrated that the ip injection of Ucn 1, unlike that of CRF, potently reduces food consumption in rodents and marsupials (31 33) suggesting that Ucn 1 may contribute to initiate a satiety signal through peripheral CRF 2 pathway. Prominent expression of 6115

2 6116 Endocrinology, December 2007, 148(12): Gourcerol et al. Synergy between Peripheral Urocortins and CCK Ucn 1, Ucn 2, and CRF 2 receptors have been found in peripheral tissues, including the gastrointestinal tract (34 36), and CRF 2 binding sites have been characterized on rat vagal afferent fibers (37, 38). Taken together, these raise the possibility of interaction between peripheral CCK and CRF 2 ligands to influence food intake. Therefore, we examined the hypothesis of a CCK-CRF signaling synergistic interaction to influence food intake and gastric emptying using consecutive ip injections of CCK-8 and Ucn 1 or the selective CRF 2 agonist, Ucn 2 (39), in wildtype as well as CRF 2 -deficient mice (40). Because CCK-8 inhibitory effects on food intake and gastric emptying involve the activation of capsaicin-sensitive vagal afferent fibers (11, 31), we assessed whether the synergistic interaction between CCK-8-Ucn 1 or CCK-8-Ucn 2 could also influence gastric emptying in mice and gastric vagal afferent activity in a rat stomach-vagus in vitro preparation previously used to establish the peripheral CCK-8-leptin synergistic interaction (15, 41). Lastly, because rats fed with a high-fat diet showed a reduced sensitivity to the satiety effect of low doses of CCK-8 (42), we examined whether CCK-8-Ucn 1 and CCK- 8-Ucn 2 interactions to suppress food intake and gastric emptying are altered in mice fed with a high-fat diet that induces obesity (DIO). Animals Materials and Methods Experiments were conducted in lean and DIO adult male mice (C57BL/6; Harlan, San Diego, CA) and CRF 2 -deficient mice (Oregon Health and Science University, Portland, OR). Mice were maintained under controlled temperature (20 23 C) and lighting ( h) conditions. Lean mice were fed ad libitum with a standard rodent chow (protein 29%, carbohydrate 59%, soybean fat 12%; Prolab RMH 500; PMI Nutrition International, LLC, Brentwood, MO) and tap water. DIO was achieved by feeding adult lean mice with a high-fat chow diet (20% of kilocalories from protein, 20% from carbohydrate, and 60% from fat including 90% lard and 10% soybean; D12492; Research Diets Inc., New Brunswick, NJ) for 7 8 wk before the start of experiments and throughout the study. Basal blood glucose levels in DIO mice were comparable with those of lean mice (130 6 mg/dl, n 10 and mg/dl, n 10, respectively; P 0.05). Male CRF 2 -deficient mice and their littermates were generated at the Oregon Health and Science University (Portland, OR) as previously described (26). Mice were backcrossed onto the C57BL/6J background for eight generations. All experiments were conducted under the Veterans Administration Animal Component of Research Protocol Substances Mouse Ucn 1 and mouse (m) Ucn 2 (Clayton Foundation Laboratories, Salk Institute, La Jolla, CA) in powder form and sulfated CCK-8 (Peninsula Labs, Belmont, CA) aliquoted in distilled water (100 ng/ l) were stored at 80 C. Ucn 1 and Ucn 2 were dissolved in distilled water, and CCK-8 was diluted in saline (sterile 0.9% NaCl; Sigma Chemical Co., St. Louis, MO) immediately before use. The ip injections were performed in 0.1 ml per mouse. For in vitro studies, human (h) Ucn 2 (Clayton Foundation Laboratories, Salk Institute) in powder form was dissolved in saline and devazepide (L-364,718; Merck Sharp and Dohm, Rahway, NJ) in 50 l vehicle (dimethyl sulfoxide, and 50 l Tween 80) to make 1 g/ l stock solution aliquots that were stored at 20 C and further diluted in saline to appropriate concentrations before use. Measurement of food intake Food intake was measured as in our previous studies in mice (31, 43). Mice were fasted overnight and refed ad libitum with either rodent preweighed standard chow (lean mice) or high-fat chow (DIO mice). Food intake was determined by weighing ( 0.01 g) the food and spillage collected on a paper placed at the bottom of each cage. Food intake was assessed as the difference between the food weights before and after each feeding period. Cumulative food intake was calculated by adding values from each consecutive period. Measurement of gastric emptying Gastric emptying of a nonnutrient viscous bolus was determined by the phenol red method as in previous studies in mice (44). The noncaloric bolus consisted of 0.5 ml of a viscous suspension containing 1.5% methylcellulose (4000 centipoises; Sigma) and 0.05% phenol red (Sigma) given by oroesophageal gavage with a stainless steel tube to conscious mice lightly hand restrained. After 20 min, mice were euthanized by a lethal dose of sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL) injected ip followed by bilateral thoracotomy. The stomach was removed, rinsed in 0.9% saline, placed in 15 ml of 0.1 n NaOH, and homogenized (Polytron; Brinkmann Instruments, Westbury, NY). The suspension was allowed to settle for 60 min at room temperature, and then 0.5 ml of 20% trichloroacetic acid (Sigma) was added to 5 ml of supernatant. After centrifugation (3000 rpm at 4 C for 20 min), 3 ml of the supernatant was mixed with 4 ml of 0.5 n NaOH. Samples were read at 560 nm in a spectrophotometer (Shimazu UV-160; Shimadzu Scientific Instruments, Inc., Columbia, MD). The absorbance of the phenol red recovered from similar processing of the stomach from mice euthanized immediately after the gavage of 0.5 ml viscous bolus was used as a standard (0% emptying). The percentage of emptying during the 20-min period was calculated with the following formula: percent emptying (1 absorbance of test sample/absorbance of standard) 100. Measurement of gastric vagal afferent (GVA) activity in vitro Experiments were conducted on the isolated rat stomach-vagus nerve in vitro preparation as we previously described (41). Fasted rats were anesthetized with isoflurane and exsanguinated. The lower-thoracic esophagus, stomach, and proximal duodenum ( 1 cm) were isolated with the attached gastric vagal nerves and left gastric artery. The preparation was then transferred to the main chamber of a Sylgard-coated (Dow Corning, Midland, MI) organ bath (33 1 C) that was perfused continuously with oxygenated Ringer solution at a ml/min flow rate. The left gastric artery was catheterized with a polyethylene tubing (PE-10, outside diameter 0.61 mm; Becton Dickinson & Co., Franklin Lakes, NJ) for intraarterial injections. A latex balloon ( 12 mm diameter) attached to a PE-10 catheter was placed into the center of the stomach through the cut end of the duodenum to perform gastric distension (GD). The ventral gastric branch of the vagal nerve trunk was isolated from the surrounding connective tissue and placed on a miniholder. The distal cut end was positioned on one lead of a bipolar recording electrode (platinum wire, 30 m), and the other lead was connected to a slim connective tissue. The recording electrodes were immersed in paraffin oil to prevent dehydration and short circuiting. The unit action potential of GVA fibers was sent to a preamplifier (DAM-6 100, khz band-pass filter; World Precision Instruments, Sarasota, FL) and further amplified times to give an action potential peak-peak amplitude of 1 5 V. Original data were displayed on a digital storage oscilloscope (model 2211; Tektronix, Allied Electronics, Inc., Fort Worth, TX) and recorded on-line on a digital tape (high-density linear optical digital audio tape deck, DTC-700; Sony, J&R Film/Moviola Digital, Hollywood, CA) or a digital compact disc (Tascam CC-222; Teac Professional Division, West LA Music, Los Angeles, CA). Unit potentials were simultaneously sent to a PC computer equipped with an A/D board (DT2831; Data Translation, Marlboro, MA). Using the acquisition module of WAVEFORM impulse analysis software, units within the upper and lower threshold settings of the preamplifier, were acquired on-line onto the hard drive of a computer. Single units were discriminated from the multiunits recordings based on the amplitude and waveform off-line. The response patterns of different units were obtained by sorting spike waveforms using Spike 2 software. Response magnitudes were normalized by a response quotient (Q) as detailed previously (41) where Q 5-min total spike count postinjec-

3 Gourcerol et al. Synergy between Peripheral Urocortins and CCK Endocrinology, December 2007, 148(12): tion/5-min total spike count preinjection. For GD, 20-sec pulses count before and during distension were compared. Experimental protocols All experiments were conducted in individually housed, 18-h fasted mice that had free access to water. Mice were reused three to five times for food intake experiments with at least a 7-d interval between experiments. Experiments started between 0900 and 0930 h. Food intake was monitored at 30, 60, 120, and 240 min after peptide injection in mice with free access to water and preweighed standard rodent chow (lean mice) or high-fat chow (DIO mice). In all experiments, peptides and their vehicles were given in two consecutive ip injections. To determine the synergistic interaction, lean mice ( g; 2 3 months old) were injected with vehicle or Ucn 1 (1 g/kg) plus vehicle or CCK-8 (1 or 3 g/kg). Peptide doses were selected based on our previous dose-response studies showing that Ucn 1 (1 g/kg) or CCK-8 (3.5 g/kg) injected ip alone were below threshold to influence the 4-h cumulative food intake in lean mice (14, 31). Based on results of the first experiment, CCK-8 at 3 g/kg was selected for all further studies. To assess the CRF receptor/s involved in Ucn 1 action, lean mice ( g; 2 3 months old) were injected ip with vehicle or mucn 2 (1 g/kg) plus vehicle or CCK-8 (3 g/kg). Likewise, CRF 2 -deficient and wild-type littermate mice ( and g, respectively; 8 10 months old) were injected ip with vehicle or CCK-8 (3 g/kg) plus vehicle or mucn 2 (1 g/kg). DIO mice ( g; 5 6 months old) were injected ip with vehicle, Ucn 1 (1 g/kg) or mucn 2 (1 g/kg) plus vehicle or CCK-8 (3 g/kg) and then given free access to preweighed high-fat chow. To investigate peptide interaction on gastric emptying, conscious fasted lean and DIO mice ( and g, respectively) were injected ip with either vehicle or Ucn 1 (1 g/kg) plus vehicle or CCK-8 (3 g/kg). The noncaloric bolus was given by oroesophageal gavage 10 min after the injections, and gastric emptying was determined 20 min thereafter. For electrophysiological recording from GVA fibers in vitro, after a 30-min stabilization period, the following consecutive injections (0.1 ml) into the gastric artery (ia) were performed in three separate experiments: 1) vehicle and hucn 2 (75 ng); 2) vehicle, CCK-8 (1 ng), vehicle, hucn 2 (75 ng), vehicle, and CCK-8 (1 ng); 3) vehicle, devazepide (10 g), CCK-8 (1 ng), vehicle, and hucn 2 (75 ng). The intervals between injections were 10 min for vehicle and 15 min or more for other substances. Doses of CCK-8 and devazepide were based on previous dose-response studies under similar conditions (41) and Ucn 2 from pilot studies. At the end of each experiment ( min after the beginning of the study), GD was performed by injecting air (1 ml, 20 sec) into the latex balloon through the catheter. Thereafter, mechanoreceptive fields (RFs) of the GVA units were located. To identify accurately the RF of activated GVA, the stomach was divided arbitrarily into seven regions: 1) central part of the fundus; 2 4) proximal, central, and distal parts of the funduscorpus borderline, respectively; 5) central part of the corpus; 6) central part of the antrum; and 7) proximal duodenum. At the end of the experiment, toluidine blue (0.1%, 0.1 ml) was injected ia to determine whether the administered solution perfused the target regions and reached the identified receptive field. Statistical analysis All values are presented as mean sem. One-way ANOVA followed by Tukey-Kramer post hoc test was performed to assess differences between groups in food intake and gastric emptying. The additive effect of peptides injected alone were calculated using the Bliss model as reported in other studies (18, 45) using the formula: R AB R A R B (1-R A ) in which A and B are test substances and R is the fractional response of the substances. The synergistic effect between CCK-8 and Ucn 1 or CCK-8 and Ucn 2 was then determined by comparing the response observed with the combination of peptides to their additive effect using the Wilcoxon rank test. Gastric vagal afferent discharge data were compared using Student s paired t test. Differences were considered significant when P Results Ucn 1 and CCK-8 injected ip act synergistically to inhibit food intake in lean mice Ucn1(1 g/kg) vehicle administered in two consecutive ip injections did not significantly modify the feeding response to a fast, compared with the vehicle vehicle control group as monitored at different time intervals or 4-h cumulative period (Fig. 1). CCK-8 (1 and 3 g/kg) vehicle also did not influence the 4-h feeding cumulative response to a fast. However, CCK-8 dose-dependently reduced food intake by 41.2 and 75.7%, respectively, during the first 30-min period (Fig. 1, data not shown). Compared with ip vehicle ip vehicle, Ucn 1 (1 g/kg) CCK-8 (3 g/kg) significantly decreased food consumption for the 0- to 30-min and 30- to 60-min periods as well as the 4-h cumulative food intake (Fig. FIG. 1. Effects of CCK-8 and Ucn 1 injected ip alone or consecutively on food intake in fasted/refed lean mice. Left panel, Food intake for each time period; right panel, 4-h cumulative food intake. Each column represents the mean SEM (eight to 10 animals/group). *, P 0.05 vs. vehicle vehicle;, P 0.05 vs. Ucn 1 CCK-8;, P 0.05 vs. additive effect.

4 6118 Endocrinology, December 2007, 148(12): Gourcerol et al. Synergy between Peripheral Urocortins and CCK 1). The reduction at 4 h was significantly higher (34.6%) than the additive effect of the peptides (Fig. 1). CCK-8 at 1 g/kg Ucn1(1 g/kg) inhibited significantly food intake by 70.2% during the first 30 min, although the 4-h cumulative food intake was not significantly decreased (grams per 4 h: vehicle vehicle, ; vehicle CCK-8, ; Ucn 1 vehicle, ; Ucn 1 CCK, , n 4 10/group, P 0.05, data not shown). Thus, CCK-8 at 3 g/kg and Ucn at 1 g/kg were selected in all other experiments. Ucn 2 and CCK-8 injected ip act synergistically to inhibit food intake in lean mice The ip injections of vehicle CCK-8 (3 g/kg) or Ucn 2 (1 g/kg) vehicle inhibited food intake only during the first 30 min, resulting in a similar 4-h cumulative food intake as in the vehicle vehicle group (Fig. 2). By contrast, Ucn 2 CCK-8 inhibited significantly the feeding response to a fast for 60 min, yielding a significant decrease in 4-h cumulative food intake, compared with the vehicle vehicle group and 8-fold higher inhibitory response than the additive effect of each peptide injected alone (Fig. 2). Ucn 2 and CCK-8 synergistic effect is no longer observed in CRF 2 knockout mice CRF 2 -deficient mice and wild-type littermates injected ip with vehicle vehicle had similar food intake in response to a fast at any time point (Fig. 3). CCK-8 vehicle ip did not significantly reduce food intake in CRF 2 -deficient mice or wild-type littermates for the first hour after injection (Fig. 3). Notably, feeding increased during the 60- to 120-min and 120- to 240-min periods after CCK-8 injected alone in wildtype and CRF 2 -deficient mice, respectively. However, in wild-type mice, the combination of ip CCK-8 (3 g/kg) Ucn2(1 g/kg) significantly inhibited food intake by 62.4 and 46.7% for the first 30 min and the 4 h-cumulative food intake periods, respectively, and masked the rebound effect that was observed in the CCK-8 vehicle group (Fig. 3). By contrast, the suppression of food consumption induced by Ucn 2 CCK-8 was no longer observed in CRF 2 -deficient mice either at 30 min or for the 4-h cumulative period (Fig. 3). Influence of Ucn 1, Ucn 2, and CCK-8 alone or in combination on food intake in DIO mice In DIO mice, CCK-8 (3 g/kg) did not reduce food ingestion of a fat diet during neither the first 30 min nor the 4-h cumulative period, although a significant increase in feeding response was observed during the 60- to 120-min period, compared with vehicle vehicle. Ucn 1 (1 g/kg) vehicle induced a significant 37.7% decrease in the 4-h cumulative food intake of high fat diet (Fig. 4). Ucn 2 vehicle or Ucn 2 CCK-8 did not significantly decrease feeding of a fat diet at any time point in DIO mice, compared with vehicle vehicle (Fig. 4). Contrasting with what was observed in lean mice, ip injections of CCK-8 plus either Ucn 1 or Ucn 2 in DIO mice resulted in a comparable 4-h feeding response as that of Ucn 1 or Ucn 2 alone (Fig. 4). Ucn 1 and CCK-8 inhibit synergistically gastric emptying in lean but not DIO mice Ucn1(1 g/kg) vehicle or CCK-8 (3 g/kg) vehicle did not modify the 20-min rate of gastric emptying of a nonnutrient bolus either in lean mice or DIO mice (Fig. 5). In lean mice, the combination of CCK-8 Ucn 1 decreased significantly gastric emptying, whereas coadministration of both peptides did not reduce significantly gastric emptying in DIO mice (Fig. 5). The inhibitory effect induced by injec- FIG. 2. Effects of CCK-8 and mucn 2 injected ip alone or consecutively on food intake in fasted/refed lean mice. Left panel, Food intake for each time period; right panel, 4-h cumulative food intake. Each column represents the mean SEM (six animals/group). *, P 0.05 vs. vehicle vehicle;, P 0.05 vs. Ucn 2 CCK-8;, P 0.05 vs. additive effect.

5 Gourcerol et al. Synergy between Peripheral Urocortins and CCK Endocrinology, December 2007, 148(12): FIG. 3. Effects of CCK-8 and mucn 2 injected ip alone or consecutively on food intake in fasted/refed lean CRF 2 -deficient (CRF 2 / ) or wild-type littermate (WT) mice. Left panel, Food intake for each time period; right panel, 4-h cumulative food intake. Each column represents the mean SEM (seven to eight animals/group). *, P 0.05 vs. vehicle vehicle;, P 0.05 vs. Ucn 2 CCK-8. tions of both peptides in lean mice was 38.1% higher than the calculated additive effect. Such synergistic effect was not observed in DIO mice. Synergistic action between CCK-8 and hucn 2 to increase gastric vagal afferent activity Gastric afferent fiber recordings were made from the ventral gastric vagus nerve filaments in 19 rat stomach-vagus in vitro preparations. Fifty-five single units had an initial basal activity ranging from 0.01 to 4.18 spikes/sec with an average of spikes/sec. Intraarterial injection of hucn 2 (75 ng) had no significant effect on GVA activity (n 21 U, seven rats, Fig. 6A1), whereas these same units responded to GD applied for 20 sec by a significant increase in GVA impulses per 20 sec from 27 6 before to during the distension (Fig. 6B1). After CCK-8 (1 ng) pretreatment, hucn 2 increased significantly GVA fiber discharge from preinjection Q values of to (P 0.05, n 23 U, seven rats), whereas CCK-8 at such a low dose had no effect on GVA activity either before or after hucn 2 (Fig. 6A2). Gastric distension (1 ml, 20 sec) activated these 23 U as shown by the increase in impulses per 20 sec from 32 9 before to 96 FIG. 4. Effects of CCK-8, Ucn 1, or mucn 2 injected ip alone or consecutively on food intake in fasted/refed high-fat diet in diet-induced obese mice. Left panel, Food intake for each time period; right panel, 4-h cumulative food intake. Each column represents the mean SEM (10 12 animals/group). *, P 0.05 vs. vehicle vehicle;, P 0.05 vs. Ucn 1 CCK-8;, P 0.05 vs. Ucn 2 CCK-8.

6 6120 Endocrinology, December 2007, 148(12): Gourcerol et al. Synergy between Peripheral Urocortins and CCK FIG. 5. Effects of CCK-8 and Ucn 1 injected ip alone or consecutively on gastric emptying of a nonnutrient bolus in fasted lean and DIO mice. The noncaloric bolus was given by oroesphageal gavage 10 min after the ip injection of vehicle or peptides, and gastric emptying was measured 20 min later. Each column represents the mean SEM (six animals/group). *, P 0.05 vs. vehicle group;, P 0.05 vs. additive effect. 26 during GD (P 0.05; Fig. 6B2). The CCK-1 receptor antagonist, devazepide (10 g), inhibited the CCK-8 effect on hucn 2 response (11 U from five rats; Fig. 6A3), whereas the basal activity (impulses per 20 sec) was enhanced from 26 6to89 24 during GD (P 0.05, 11 U from five rats, Fig. 6B3). The corresponding examples of GVA fiber discharge patterns are shown in Fig. 6C. The GVA fibers did not respond to ia injections of vehicle, hucn 2 (75 ng), and CCK-8 (1 ng), whereas hucn 2 injected after CCK-8 pretreatment resulted in GVA fiber discharge, the latter being blocked by devazepide pretreatment. The response to GD at the end of peptide injection had a rapid onset and return to basal, whereas that of hucn 2 after CCK pretreatment showed a slower onset and prolonged duration (Fig. 6C2). The sensitive units to gastric mechanoreceptive field stimulation analyzed in these experiments were distributed mainly in fields 3 and 5, corresponding to the central part of the funduscorpus border and corpus, respectively (Fig. 6C). Discussion We showed that consecutive ip injections of Ucn 1 and CCK-8 resulted in an enhanced inhibition of a feeding response to a fast in lean mice. Vehicle-CCK-8 at 1 and 3 g/kg dose-dependently inhibited food intake by 41 and 76% only for the first 30 min after injection, consistent with the early satiation signal induced by CCK-8 injected ip in doses ranging from 1 to 30 g/kg in mice (14, 46 50). However, when the ip injection of CCK-8 (3 g/kg) was combined with Ucn 1 at the subthreshold dose of 1 g/kg (Ref. 31 and present observation), a significant reduction of food intake was still maintained during the min after injection, leading to a significant decrease of the 4-h cumulative food intake. Overall these findings provide the first evidence of a synergistic interaction between CCK-8 and Ucn 1 injected peripherally to suppress food intake in lean mice as demonstrated in a fast/refeeding paradigm. Ucn 1 is known to interact with an equal high affinity to both CRF 1 and CRF 2 receptors (24). Although both CRF receptor subtypes are expressed in the rat stomach (36), convergent evidence supports that Ucn 1 satiety action is mediated by the activation of CRF 2 receptors. First, we showed that the selective CRF 2 receptor agonist, Ucn 2 (39), injected peripherally with CCK-8 also resulted in a potent synergistic effect as shown by the 8-fold greater inhibition of the 4-h cumulative food intake than the additive effect of peptides during the same period. Second, the Ucn 2 and CCK-8 synergistic inhibitory interaction was reproduced in wild-type mice and no longer observed in CRF 2 -deficient mice. It is unlikely that the unresponsiveness of CRF 2 -deleted mice was confounded by differences in basal feeding behavior. Body weights of CRF 2 -deficient mice and wild-type littermates were indistinguishable, and there was no significant difference in their 4-h feeding response to an overnight fast. Likewise, previous reports showed that CRF 2 -deficient mice displayed similar spontaneous feeding, body weight, metabolic variables, and exploratory behavior as wild-type littermates (26, 40, 51, 52). The 24-h cumulative food intake in response to a 16-h food deprivation in CRF 2 -deficient mice was found identical (40) or reduced by 25% (51), compared with wildtype counterparts. The present studies also provide the first evidence that ip Ucn 2 alone induces a rapid-onset reduction of food intake in lean and wild-type mice that contrasts with the 2- to 6-h delay in the anorexic action of centrally injected Ucn 2 in both food-deprived and freely fed rats (29, 30, 53 55). These findings support a peripherally initiated satiety action of Ucn 2 that warrants further investigations. Pathways through which the synergistic interaction between peripherally administrated Ucn 1 or Ucn 2 and CCK-8 occurred are likely to involve vagal afferent fibers. The vagaldependent, capsaicin-sensitive activation of brain neurocircuitry induced by peripheral CCK-8 injected at low doses has been one of the most thoroughly established mechanism, mediating CCK-related changes in food intake and gastric emptying (11, 12). CCK-1 receptors are present on vagal afferent fibers in which CCK-8 acts to induce a rapid CCK- 1-mediated stimulation of GVA activity (41, 56). The accumulation of CRF binding proximal to vagal ligation and the characterization of CRF 2 receptor in nodose ganglia in rats provide also neuroanatomical support for a CRF 2 receptormediated modulation of vagal afferent (37, 38). Moreover, the present electrophysiological and functional data are consistent with an interaction involving vagal pathways. Using a stomach-vagus in vitro preparation as in our previous studies (41), we showed that intragastric artery injections of CCK-8 followed by Ucn 2 increased gastric vagal afferent activity. The response was observed under conditions in which either peptide, injected at subthreshold doses (41), did not alter basal gastric vagal activity, whereas the same single units were responsive to gastric distension. Chemosensitive units activated by CCK-8 pretreatment plus Ucn 2 are polymodal vagal afferent fibers as shown by their responsiveness to gastric distension and a local mechanical stimulus. Likewise, at higher doses, CCK alone was characterized to recruit rat polymodal gastric vagal afferent (41, 57). The blockade of CCK-8-induced gastric vagal afferent response to Ucn 2 by devazepide (58) established the CCK-1 receptor specificity of CCK action. In addition, we showed that CCK-8-Ucn 1 in-

7 Gourcerol et al. Synergy between Peripheral Urocortins and CCK Endocrinology, December 2007, 148(12): FIG. 6. A, Synergistic interaction between CCK-8 and hucn 2 to increase gastric vagal afferent discharge in a rat gastric stomach in vitro preparation. A, Mean SEM of Q values for gastric vagal afferent discharges in response to consecutive (within min interval) intragastric artery injections of saline and hucn 2 (A1); saline, CCK-8, saline, hucn 2, saline, and CCK-8 (A2); saline, devazepide, CCK-8, saline, and hucn 2 (A3). *, P 0.05, paired t test, compared with respective saline. B, Gastric vagal afferent fibers discharge before (pre GD) and during (during GD) GD (1 ml, 20 sec) performed at the end of experiments 1, 2, and 3 as detailed in A. *, P 0.05 paired t test, compared with pre-gd. C, Representative single unit of gastric vagal afferent fiber response to consecutive injections of saline and hucn 2, GD and RF probing (1); saline, CCK-8, saline, Ucn 2, GD, and RF (2); and saline, devazepide, CCK-8, saline, Ucn 2, GD, and RF (3). Schematic representation of gastricresponsive fields is displayed above the localization of fields 1 7. Injections are indicated by arrows. jected ip at subthreshold doses when given singly significantly suppressed gastric emptying of a nonnutrient meal. It is well established that low doses of CCK-8 inhibit gastric emptying through activation of capsaicin-sensitive vagovagal reflex (11), whereas ip Ucn 1 inhibition of gastric emptying involves activation of peripheral CRF 2 receptors in mice (31, 59). Therefore, CCK-8 and Ucn 1-induced synergistic interaction to reduce both food intake and gastric emptying in lean mice are consistent with an enhanced vagal afferent signaling. The delayed gastric emptying induced by such peptide interaction may also contribute to the extended inhibition of food intake when CCK-8 was administered with Ucn 1 or Ucn 2. Prolonged presence of food in the stomach influences the degree of fullness (60) and conveys satiety signal to reduce food intake (61). Interestingly, whereas a synergic interaction between ip CCK-8 and Ucn 1 or Ucn 2, acting through CRF 2 receptors to suppress food intake and delay gastric emptying, was observed in lean mice, such potentiation did not occur in DIO mice. Although DIO mice tend to have a lower rate of basal gastric emptying than the lean mice, it is unlikely that the lack of synergistic interactions represents a floor effect. In previous dose-response studies, CCK-8 or Ucn 1 injected ip singly in lean or obese mice induced a complete suppression of gastric emptying at maximal effective doses (31, 33). Because the lack of synergy remained unchanged when food intake was assessed with normal chow (data not shown), the composition of the meal is unlikely to be involved in this differential response. The reduced sensitivity of DIO mice to CCK-8 and Ucn 1 or Ucn 2 synergistic effect may be linked to CCK resistance in obese mice. We showed that CCK-8 injected ip at a dose that decreased feeding in lean mice did not reduce meal size in DIO mice. Such a resistance to CCK satiation effect has been reported previously in obese rat models when the peptide was injected at low doses (42, 62). In addition, CCK did not reduce feeding and gastric emptying in DIO rats (63). It remains unclear whether peripheral or central satiety processing is responsible for the reduced sensitivity to CCK. However, ip CCK-induced Fos expression in the nucleus tractus solitarius in rats fed with a low fat diet was nearly abolished in rats fed a high-fat diet for more than 2 wk (64). These data support a role of altered vagal

8 6122 Endocrinology, December 2007, 148(12): Gourcerol et al. Synergy between Peripheral Urocortins and CCK responsiveness to CCK-8 in mice maintained in a high-fat diet, leading to reduced CCK action and synergistic interaction with Ucn 1 or Ucn 2 under these conditions. Chronic exposure to high dietary fat may desensitize synergistic mechanisms that limit food ingestion and have implications in obesity due to the lack of efficient satiety signaling. Although not investigated in the present work, we can speculate that the synergy between the CCK and CRF 2 pathway could impact long-term feeding behavior. First, Ucn 1 or Ucn 2 abolished the pattern of food intake recovery that occurred when CCK-8 was injected alone, suggesting that such interaction may prevent compensatory mechanisms associated with short-term reduction of food intake and take part in reducing the cumulative food intake at 4 h. In addition, there is evidence that CCK-58 is the major detectable endocrine form of CCK in fasted rats (9) and the predominant form in humans released postprandially (8). Other studies showed that CCK-58 injected iv results in a prolonged discharge of intestinal afferent nerve, compared with CCK-8 (65). Therefore, this raises the possibility that the interaction between CCK and Ucn 2 under conditions of CCK-58 release may be longer lasting than observed presently. Lastly, CRF 2 - deficient mice displayed increased high-fat consumption, compared with their littermates (66). Although this could be due to changes in energy expenditure (66), the loss of synergy between CCK and CRF 2 pathways may also contribute to the enhanced consumption of high-fat diet in CRF 2 -mutant mice. The synergy between the CCK and Ucn 1/Ucn 2-CRF 2 system may also be relevant in functional dyspepsia, a condition for which the symptoms (nausea, fullness, inhibition of gastric emptying) are meal related and can be aggravated by stress (67, 68). Certain nutrients, particularly fat, known to release CCK, can generate dyspeptic symptoms in healthy subjects, and injection of CCK-8 induces more symptoms in dyspeptic patients than controls (67). On the other hand, the loss of synergy between CCK-8 and Ucn 1/Ucn 2-CRF 2 system under conditions of a high-fat diet may play a role in the satiety signaling alterations seen in obese patients. For instance, patient suffering from bulimia nervosa have altered satiation thresholds with a reduced sensitivity to gastric distension and a blunted CCK release (69, 70). In summary, our results indicate a synergic interaction between peripheral CCK-8 and Ucn 1 or Ucn 2 leading to the reduction of the 4-h cumulative feeding response to a fast in lean mice. Ucn 1 and Ucn 2 actions are mediated by CRF 2 receptors and likely to involve vagal afferent pathways as shown by the potentiation of GVA response in an isolated stomach-vagus in vitro preparation. Such interaction also encompasses the inhibition of gastric emptying, further supporting the mediation through vagovagal reflex and the possible role of delayed gastric emptying in the reduction of 4-h food intake in response to CCK-8-Ucn 1 or Ucn 2 coinjections. DIO mice no longer showed a synergistic interaction to ip injections of CCK-8-Ucn 1 or Ucn 2, suggesting that chronic exposure to a high-fat diet alters satiety signaling interaction and may play role in maintaining obesity. Acknowledgments We thank Dr. Jean Rivier (Clayton Foundation Laboratories, Salk Institute, La Jolla, CA) for the generous donation of CRF and urocortin peptides and Dr. Mary Stenzel-Poore (Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR) for the supply of CRF 2 / and littermate-type mice. Dr. Jen Yu Wei is acknowledged for his comments on the electrophysiology experiments and Ms. Honghui Liang for technical assistance. Received May 21, Accepted October 2, Address all correspondence and requests for reprints to: Yvette Taché, Ph.D., Center for Neurovisceral Sciences and Women s Health, CURE Building 115, Room 203, VA Greater Los Angeles Healthcare System, Wilshire Boulevard, Los Angeles, California E- mail: ytache@mednet.ucla.edu. 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