OBESITY HAS REACHED an escalating rate since 1980

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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. Tache´

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 90073

Interactions between gastrointestinal signals are a part of integrated systems regulating food intake (FI). We investi-gated whether cholecystokinin (CCK)-8 and urocortin sys-tems potentiate each other to inhibit FI and gastric emptying (GE) in fasted mice. Urocortin 1 and urocortin 2 (1g/kg) were injected ip alone or with CCK (3g/kg) in lean, diet-induced obese (DIO) or corticotropin-releasing factor receptor-2 (CRF2)-deficient mice. Gastric vagal afferent activity was

re-corded from a rat stomach-vagusin vitropreparation. 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 vehiclevehicle group in lean mice but not in DIO mice.

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 CRF2-deficient mice. Gastric vagal

affer-ent activity was increased by intragastric artery injection of urocortin 2 after CCK at a subthreshold dose, and the re-sponse was reversed by devazepide. These data establish a peripheral synergistic interaction between CCK and urocor-tin 1 or urocorurocor-tin 2 to suppress FI and GE through CRF2

receptor in lean mice that may involve CCK modulation of gastric vagal afferent responsiveness to urocortin 2. Such syn-ergy is lost in DIO mice, suggesting a resistance to the satiety signaling that may contribute to maintain obesity. ( Endocri-nology148: 6115– 6123, 2007)

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BESITY 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 glu-cose homeostasis (2). Growing evidence supports their pos-sible 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-in-duced early satiation and inhibition of gastric emptying (11– 13). It is now well established that there is a synergistic

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 inhi-bition of food intake linked with an increased gastric vagal afferent discharge and Fos expression in brain nuclei regu-lating 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, seroto-nin, and estradiol in rodents and/or humans (18 –21).

Brain corticotropin-releasing factor (CRF) signaling path-ways 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, uro-cortin (Ucn) 1, Ucn 2, and Ucn 3 and two receptor subtypes, CRF1and CRF2(24, 25). CRF ligands interact with CRF re-ceptors with a distinct affinity. CRF binds with higher affinity to CRF1than CRF2receptors. Ucn 1 displays a high affinity to both CRF1and CRF2receptors, whereas Ucn 2 and Ucn 3 bind selectively to CRF2 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 CRF1and CRF2receptor-dependent mechanisms that mediate, respec-tively, 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) sug-gesting that Ucn 1 may contribute to initiate a satiety signal through peripheral CRF2pathway. Prominent expression of 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.

Endocrinologyis published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

doi: 10.1210/en.2007-0678

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Ucn 1, Ucn 2, and CRF2receptors have been found in pe-ripheral tissues, including the gastrointestinal tract (34 –36), and CRF2binding sites have been characterized on rat vagal afferent fibers (37, 38). Taken together, these raise the pos-sibility of interaction between peripheral CCK and CRF2 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 CRF2agonist, Ucn 2 (39), in wild-type as well as CRF2-deficient mice (40). Because CCK-8 inhibitory effects on food intake and gastric emptying in-volve the activation of capsaicin-sensitive vagal afferent fi-bers (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-vagusin vitropreparation previously used to establish the peripheral CCK-8-leptin synergistic interac-tion (15, 41). Lastly, because rats fed with a high-fat diet showed a reduced sensitivity to the satiety effect of low doses of 8 (42), we examined whether 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).

Materials and Methods

Animals

Experiments were conducted in lean and DIO adult male mice (C57BL/6; Harlan, San Diego, CA) and CRF2-deficient mice (Oregon

Health and Science University, Portland, OR). Mice were maintained under controlled temperature (20 –23 C) and lighting (0600 –1800 h) conditions. Lean mice were fedad libitumwith 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 through-out the study. Basal blood glucose levels in DIO mice were comparable with those of lean mice (130⫾6 mg/dl, n⫽10 and 127⫾5 mg/dl, n⫽ 10, respectively;P⬎0.05). Male CRF2-deficient mice and their

litter-mates 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 03008-05.

Substances

Mouse Ucn 1 and mouse (m) Ucn 2 (Clayton Foundation Laboratories, Salk Institute, La Jolla, CA) in powder form and sulfated CCK-8 (Pen-insula 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. Forin vitrostudies, 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% meth-ylcellulose (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 0.1nNaOH, 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 0.5nNaOH. 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 vitropreparation 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 prep-aration 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 2.0 –2.5 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 (plat-inum 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, 100 –10 kHz band-pass filter; World Precision Instruments, Sarasota, FL) and further amplified 300 – 750 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 Trans-lation, Marlboro, MA).

Using the acquisition module of WAVEFORM impulse analysis soft-ware, units within the upper and lower threshold settings of the pre-amplifier, 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

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postinjec-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 exper-iments. 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 (23.7⫾0.3 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-re-sponse 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 (25.8⫾0.3 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, CRF2-deficient and wild-type littermate

mice (32.5⫾0.7 and 32.7⫾0.5 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 (47.9⫾0.5 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 (32.9⫾0.7 and 48.1⫾0.6 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 fibersin 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 (87–100 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 fundus-corpus borderline, respectively; 5) central part of the fundus-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:

RAB⫽RA⫹RB⫻(1-RA)

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 com-pared using Student’s pairedttest. Differences were considered signif-icant whenPⱕ0.05.

Results

Ucn 1 and CCK-8 injected ip act synergistically to inhibit food intake in lean mice

Ucn 1 (1␮g/kg)⫹vehicle administered in two consecu-tive 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 in-take 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. Eachcolumnrepresents the meanSEM(eight to 10 animals/group). *,P0.05vs.vehicle

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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

⫹ Ucn 1 (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, 0.88⫾0.06; vehicle⫹CCK-8, 0.99⫾0.08; Ucn 1⫹vehicle, 0.81⫾0.08; Ucn 1⫹CCK, 0.75⫾0.05, 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 CRF2knockout mice

CRF2-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 CRF2-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 wild-type and CRF2-deficient mice, respectively. However, in wild-type mice, the combination of ip CCK-8 (3 ␮g/kg)⫹

Ucn 2 (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 CRF2-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 in-gestion 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

Ucn 1 (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. Eachcolumnrepresents the meanSEM(six animals/group). *,P0.05vs.vehicle vehicle; §,P⬍0.05vs.Ucn 2⫹CCK-8; ‡,P⬍0.05vs.additive effect.

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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 ven-tral gastric vagus nerve filaments in 19 rat stomach-vagusin vitropreparations. Fifty-five single units had an initial basal activity ranging from 0.01 to 4.18 spikes/sec with an average of 0.90⫾0.13 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 71⫾24 during the distension (Fig. 6B1). After CCK-8 (1 ng) pretreatment, hUcn 2 increased significantly GVA fiber discharge from preinjection Q values of 1.15⫾0.10 to 1.82⫾0.35 (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 dis-tension (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. Eachcolumnrepresents the meanSEM(10 –12 animals/group). *,P⬍0.05vs.vehicle⫹vehicle; §,P⬍0.05vs.Ucn 1⫹CCK-8; †,P⬍0.05vs.Ucn 2⫹CCK-8.

FIG. 3. Effects of CCK-8 and mUcn 2 injected ip alone or consecutively on food intake in fasted/refed lean CRF2-deficient (CRF2⫺

/⫺) or wild-type littermate (WT) mice.Left panel, Food intake for each time period;right panel, 4-h cumulative food intake. Eachcolumnrepresents the mean

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26 during GD (P⬍0.05; Fig. 6B2). The CCK-1 receptor an-tagonist, 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⫾ 6 to 89⫾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 re-spond 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 sensi-tive units to gastric mechanorecepsensi-tive field stimulation an-alyzed in these experiments were distributed mainly in fields 3 and 5, corresponding to the central part of the fundus-corpus border and fundus-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 re-sponse 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 rang-ing 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 30 – 60 min after injection, leading to a significant decrease of the 4-h cumulative food intake. Overall these findings provide the first evidence of a syn-ergistic interaction between CCK-8 and Ucn 1 injected pe-ripherally to suppress food intake in lean mice as demon-strated in a fast/refeeding paradigm.

Ucn 1 is known to interact with an equal high affinity to both CRF1and CRF2receptors (24). Although both CRF

re-ceptor subtypes are expressed in the rat stomach (36), con-vergent evidence supports that Ucn 1 satiety action is me-diated by the activation of CRF2receptors. First, we showed that the selective CRF2receptor 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 syn-ergistic inhibitory interaction was reproduced in wild-type mice and no longer observed in CRF2-deficient mice. It is unlikely that the unresponsiveness of CRF2-deleted mice was confounded by differences in basal feeding behavior. Body weights of CRF2-deficient mice and wild-type littermates were indistinguishable, and there was no significant differ-ence in their 4-h feeding response to an overnight fast. Like-wise, previous reports showed that CRF2-deficient mice dis-played 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 CRF2-deficient mice was found identical (40) or reduced by 25% (51), compared with wild-type 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 find-ings support a peripherally initiated satiety action of Ucn 2 that warrants further investigations.

Pathways through which the synergistic interaction be-tween peripherally administrated Ucn 1 or Ucn 2 and CCK-8 occurred are likely to involve vagal afferent fibers. The vagal-dependent, capsaicin-sensitive activation of brain neurocir-cuitry 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 8 acts to induce a rapid CCK-1-mediated stimulation of GVA activity (41, 56). The accu-mulation of CRF binding proximal to vagal ligation and the characterization of CRF2receptor in nodose ganglia in rats provide also neuroanatomical support for a CRF2 receptor-mediated modulation of vagal afferent (37, 38). Moreover, the present electrophysiological and functional data are con-sistent with an interaction involving vagal pathways. Using a stomach-vagusin vitropreparation as in our previous stud-ies (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 poly-modal vagal afferent fibers as shown by their responsiveness to gastric distension and a local mechanical stimulus. Like-wise, 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-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. Eachcolumnrepresents the mean⫾SEM(six animals/group). *,P0.05vs.vehicle group; ‡,P0.05vs.additive effect.

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jected ip at subthreshold doses when given singly signifi-cantly 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 vagova-gal reflex (11), whereas ip Ucn 1 inhibition of gastric emp-tying involves activation of peripheral CRF2 receptors in mice (31, 59). Therefore, CCK-8 and Ucn 1-induced syner-gistic interaction to reduce both food intake and gastric emp-tying 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 CRF2receptors to suppress food intake and delay gastric emptying, was ob-served 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 previ-ous 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 com-position of the meal is unlikely to be involved in this dif-ferential 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 emp-tying 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 expres-sion 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

FIG. 6. A, Synergistic interaction between CCK-8 and hUcn 2 to increase gastric vagal afferent discharge in a rat gastric stomachin vitro preparation. A, Mean⫾SEMof Q values for gastric vagal afferent discharges in response to consecutive (within 10 –15 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, pairedttest, 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. *,P0.05 pairedttest, 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 gastric-responsive fields is displayed above the localization of fields 1–7. Injections are indicated byarrows.

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responsiveness to CCK-8 in mice maintained in a high-fat diet, leading to reduced CCK action and synergistic inter-action 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 CRF2 path-way 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 as-sociated with short-term reduction of food intake and take part in reducing the cumulative food intake at 4 h. In addi-tion, 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 dis-charge 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, CRF2 -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 CRF2pathways may also contribute to the enhanced consumption of high-fat diet in CRF2-mutant mice. The synergy between the CCK and Ucn 1/Ucn 2-CRF2 system may also be relevant in functional dyspepsia, a con-dition 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-CRF2 sys-tem under conditions of a high-fat diet may play a role in the satiety signaling alterations seen in obese patients. For in-stance, patient suffering from bulimia nervosa have altered satiation thresholds with a reduced sensitivity to gastric dis-tension 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 CRF2 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 sup-porting the mediation through vagovagal reflex and the pos-sible 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 Micro-biology and Immunology, Oregon Health and Science University, Port-land, OR) for the supply of CRF2⫺/⫺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, 2007. Accepted October 2, 2007.

Address all correspondence and requests for reprints to: Yvette Tache´, Ph.D., Center for Neurovisceral Sciences and Women’s Health, CURE Building 115, Room 203, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Los Angeles, California 90073. E-mail: ytache@mednet.ucla.edu.

This work was supported by National Institutes of Health Grants R01 DK-33061 (to Y.T.), Center Grant DK-41301 (Animal Core; to Y.T.), Veterans Affairs Career Scientist and Merit Awards (to Y.T.), the French Nation Society of Gastroenterology (Socie´te´ Nationale Franc¸aise de Gas-troente´rologie; to G.G.), and the French Foreign Office (to G.G.).

Disclosure Statement: The authors have nothing to disclose.

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