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BEHAVIORAL PHARMACOLOGY
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California (E.P.Z., L.E.R., G.R.V., K.I., G.F.K.); Department of Neuropsychiatry, Osaka City University Medical School, Osaka, Japan (K.I.); and Peptide Biology Laboratory, The Salk Institute for Biological Sciences, La Jolla, California (J.E.R., W.W.V.)
Received for publication
March 18, 2004
Accepted
April 28, 2004.
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Abstract |
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) is hypothesized to mediate behavioral, autonomic, and endocrine responses to stress (Zorrilla and Koob, 2004a). Two genes code for separate families of G-protein-coupled CRF receptors (CRF1 and CRF2) with distinct distributions and pharmacological properties (Zorrilla and Koob, 2004a). Molecular and antagonist studies point to arousing and anxiogenic-like roles for the CRF1 receptor (Zorrilla and Koob, 2004b), but behavioral functions of the CRF2 receptor remain obscure.
The characterization of urocortin 1 (Ucn 1) (Vaughan et al., 1995), a mammalian CRF paralog with greater affinity for the CRF2 receptor than CRF, led to two hypotheses about the behavioral significance of the CRF2 receptor. First, because i.c.v. Ucn 1 had more potent, efficacious, and prolonged anorectic effects than CRF (Spina et al., 1996), it was hypothesized that CRF2 receptor activation suppressed feeding. Studies with a preferential CRF2 receptor antagonist and CRF2 receptor-deficient mice supported this hypothesis (Zorrilla et al., 2003). Second, because i.c.v. Ucn 1 was less effective than CRF in stimulating motor activity in a familiar environment (Spina et al., 1996; Reyes et al., 2001), it was hypothesized that CRF2 receptor activation had motor suppressive effects. Selective agonists for the CRF2 receptor had not been identified, however, precluding determination of whether CRF2 receptor activation was sufficient for these effects.
Genes encoding two selective CRF2 agonists—urocortin 2 (Ucn 2) and urocortin 3 (Ucn 3)—were recently cloned from both mouse and human genomic libraries (Lewis et al., 2001; Reyes et al., 2001). Ucn 2, a putative 38-amino acid CRF-related neuropeptide, is approximately 1000-fold more selective for the CRF2 receptor than is Ucn 1 (Reyes et al., 2001). Conversely, ovine CRF (oCRF) shows almost 200-fold greater affinity for the CRF1 than CRF2 receptor (Behan et al., 1996). Neither peptide has affinity for the human CRF-binding protein (Sutton et al., 1995; Lewis et al., 2001). Thus, as relatively selective, direct receptor agonists, oCRF and Ucn 2 are powerful tools for discriminating the behavioral functions of the CRF1 and CRF2 receptors.
Centrally infused Ucn 2 is anorectic and, like several recognized appetite suppressants, activates interconnected brain regions known to participate in the regulation of autonomic and visceral functions related to energy balance (Inoue et al., 2003). Meal pattern analysis showed that Ucn 2 has satiation-like effects, reducing meal size and eating rate, without altering meal frequency. In the present study, the dose-related anorectic and antidipsogenic effects of Ucn 2, the highly selective CRF2 receptor agonist, and oCRF, the moderately selective CRF1 receptor agonist, were compared in nondeprived rats fed either a standard chow diet or a diet supplemented by highly palatable, energy-dense foods ("cafeteria diet"). The cafeteria diet is a well-validated procedure for inducing hyperphagia (30–100% caloric increase) (Rothwell and Stock, 1979; Rothwell and Stock, 1988). In contrast to models of drug-, lesion-, or deprivation-induced hyperphagia, the cafeteria diet models appetite characteristics that accompany overeating in humans. Using the diet, one also can observe shifts in food selection associated with the composition of or individual preferences for the food items (Rogers and Blundell, 1984; Prats et al., 1989; Esteve et al., 1994).
Potential aversive consequences that could account for ingestive effects also were examined. Although i.c.v. Ucn 2 does not share the anxiogenic-like effects of CRF (Valdez et al., 2002), it can induce a conditioned taste aversion (CTA) at a dose 100-fold higher than its minimal effective anorectic dose (Inoue et al., 2003). Ucn 2's anorectic effects are delayed, and the formation of a CTA depends on temporal proximity between the conditioned stimulus and the internal drug state. Therefore, results from a time-insensitive measure of malaise are needed to evaluate the potential role of malaise in Ucn 2-induced anorexia. Rats increase geophagia (e.g., kaolin clay intake), a form of pica behavior, in response to stimuli that elicit visceral illness (e.g., poisons, toxins, hypergravity, microgravity). Treatments that increase geophagia also can form a CTA, and antiemetics reduce clay intake. Accordingly, geophagia is a proposed rodent analog of emesis (Takeda et al., 1993). Unlike CTA testing, geophagia is an active, unconditioned behavior and is quantitatively dose-dependent. Therefore, we compared the ability of Ucn 2 and oCRF to increase kaolin intake. Motor activity also was monitored. LiCl, an aversive agent that induces gastrointestinal malaise, was used as a positive control.
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Materials and Methods |
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Surgery
Rats that were to receive i.c.v. administration of human Ucn 2 (hUcn 2) or oCRF were implanted with indwelling cannulae directed unilaterally at the lateral ventricle. Anesthetized (halothane, 2–3% in oxygen) subjects were secured in a stereotaxic frame (David Kopf Instruments, Tujunga, CA). Using sterile technique, a straight, stainless steel, 22-gauge guide cannula (Plastics One Inc., Roanoke, VA) was lowered above the lateral ventricle and anchored to the skull with screws and dental cement. With the tooth bar set 5.0 mm above interaural zero, the coordinates were A/P -0.6 mm, M/L ± 2.0 mm relative to bregma, and 3.2 mm ventral from the skull surface (Pellegrino et al., 1979). A dummy stylet (Plastics One) maintained patency. Subjects were allowed 1 week to recover from surgery.
Drugs and Injections
hUcn 2 and oCRF were synthesized manually using solid phase methodology, purified using high-performance liquid chromatography, and fully characterized using capillary zone electrophoresis, high-performance liquid chromatography, and mass spectrometry, as described previously (Reyes et al., 2001). Peptides were dissolved in sterile 0.5x phosphate-buffered saline (pH 7.4) immediately prior to testing and kept on ice. Peptides or vehicle were injected (i.c.v. 5 µl) over 1 min with a Hamilton microsyringe using a 28-gauge stainless steel injector attached to PE 20 tubing. The injector, which projected 1.3 mm past the end of the cannula, was left in place for 1 min after infusion to allow diffusion. Placement was confirmed histologically. For pica assessment, sterile, isotonic (0.15 M) LiCl (Sigma-Aldrich, St. Louis, MO) or NaCl were administered intraperitoneally in a volume of 20 ml/kg.
Behavioral Tests
Chow, Cafeteria Diet, and Water Intake. Rats in the oCRF (n = 18) and hUcn 2 cohorts (n = 14) were randomly assigned to the chow or cafeteria diet conditions (n = 7–9/group). All received ad libitum chow and water for the duration of the study. One week prior to i.c.v. surgery, rats were single-housed. At this time, the following highly palatable, commercially available food items also were provided to cafeteria diet rats: rice cakes (Quaker Oats, Chicago, IL), cheese crackers (Keebler Company, Elmhurst, IL), mini marshmallows (Safeway, Pleasanton, CA), and cookies and cream milk chocolate (Hershey Foods Corporation, Hershey, PA). Items were chosen in pilot studies from an assortment of foods based on a combination of 1) the rats' preferences; 2) the homogeneity, macronutrient composition, unique orosensory characteristics, and energy density of the foods; and 3) the stability of the foods' hydration (weight) over a 6-h period. As shown in Table 1, supplemental foods were uniformly low in protein. Two items, rice cakes and marshmallows, were composed almost exclusively of carbohydrates (CHOs), whereas two items, cheese crackers and chocolate, had mixed-fat/CHO content. After recovery from surgery, rats were reacclimated to the cafeteria diet for 1 week, receiving daily 6-h access to the supplemental items from 11:00 AM to 5:00 PM (i.e., starting 1 h after dark onset). This limited-access schedule induces hyperphagia on both a food weight and caloric basis, but avoids protein malnutrition because rats eat chow when cafeteria diet items are not available (Rothwell and Stock, 1988).
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For testing, chow- and cafeteria-diet fed rats were pretreated at 10:30 AM (i.e., 30 min prior to preweighed food and water access) with hUcn 2 or oCRF (i.c.v. 0, 0.1, 1.0, or 10.0 µg; for reference, 1 µg of hUcn 2 = 241 pmol and 1 µg of oCRF = 214 pmol) in a full Latin square design with 3 to 4 drug-free days between treatments. Peptide cohorts were tested identically on different days. Food, spillage, and water were postweighed after 6 h to determine intake. Each cafeteria diet rat's "preference" for food items was defined as a mean preference ratio (item intake/total intake on a weight basis) averaged from 3 drug-free baseline days.
Kaolin and Food Intake and Motor Activity. To compare the effects of hUcn 2 (n = 27) and oCRF (n = 27) on simultaneous intake of chow and kaolin clay, rats were first acclimated to ad libitum access to hardened kaolin pellets for 1 week in their home cages. To prepare kaolin, 1% acacia gum (w/w) (Sigma-Aldrich), kaolin (aluminum silicate; Sigma-Aldrich), and water (approximately 700 ml/kg kaolin) were heated to form a paste. The paste was extruded through a pastry sleeve to form uniform columns, dried thoroughly under a fume hood for several days, and broken into 1- to 1.5-inch pellets. The testing apparatus consisted of 16 wire mesh cages (20 x 25 x 36 cm), each equipped with two horizontal infrared photocell beams situated along the long axis of the cage, 2 cm above the floor and 16 cm from one another. On the 2 d prior to testing, rats were habituated to the test cages for 3 h with food and kaolin access to reduce the novelty of the environment. On the testing day, rats were acclimated to the test cages for an additional 90 min prior to drug treatment. White noise (70 db) was present during habituation and testing.
For testing, rats were pretreated 30 min prior to access to pre-weighed chow and kaolin with hUcn 2 or oCRF (i.c.v. 0, 0.1, 1.0, or 10.0 µg) in a between-subjects design (n = 6–7/group). Peptide cohorts were tested identically on different days. To mimic the time frame of testing during ad libitum access, access was provided for 6 h starting 1 h after the onset of the dark cycle. Diaper pads were placed below the cages to collect spillage. Photocell counts were recorded automatically by a computer. To rule out that anorectic effects were secondary to antidipsogenic effects of the peptides, water was not provided during pica testing. The effects of isotonic LiCl (n = 8) were compared with those of NaCl (n = 8) as a positive control.
Statistical Analyses
Baseline intake of chow- and cafeteria diet-fed rats was compared by Student's t test. Separate analyses of variance were used to determine drug effects on absolute changes in intake. Proportional changes in intake also were examined to standardize change according to each rat's intake under vehicle conditions, thereby accounting for any baseline differences. Statistics presented in the text reflect absolute caloric analyses, which were substantively identical to those from proportional analyses. For ad libitum intake studies, dose was a within-subject factor, and diet was a between-subject factor. To allow direct comparison of peptide effects on food intake within each diet condition, a two-way mixed analysis of variance also was performed with dose as a within-subject factor and peptide as a between-subject factor. For item analysis, dose and item macronutrient composition (i.e., CHO-rich versus fat/CHO-rich) were within-subject factors. Stability of rats' baseline item preferences was calculated as an intraclass correlation (Shrout and Fleiss, 1979). Pearson correlations were used to examine the relation of baseline preference (percent total food weight) with drug effects on intake (percent caloric change). Differences between correlations of hUcn 2 and oCRF rats were evaluated using the Fisher r-to-z transformation. For kaolin intake analysis, dose was a between-subject factor. Time, as 1-h bins, also was a within-subject factor for motor activity analysis. Following significant omnibus F ratios, linear contrasts were performed to determine the dose dependence of observed effects. Within-subject Dunnett's tests were used to compare drug effects to vehicle levels. Across peptides, Bonferroni-corrected (
= 0.05/3) Student's t tests were used to compare proportionally standardized effects at each dose. The statistical packages used were SPSS 12.0 (SPSS Inc., Chicago, IL) and Instat 3.0 (GraphPad Software Inc., San Diego, CA).
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Water Intake
Both hUcn 2 [F(3,36) = 7.85, p < 0.001) and oCRF [F(3,48) = 4.59, p < 0.01) significantly reduced 6-h water intake during the dark cycle. As shown in Fig. 1, however, lower doses of hUcn 2 than oCRF reduced intake both on an absolute and percent reduction basis [minimum effective dose (MED) of approximately 0.1 µg for hUcn 2 versus 10.0 µg for oCRF by both analyses]. Linear contrasts indicated that effects of hUcn 2 [F(1,12) = 25.47, p < 0.0001] and oCRF [F(1,16) = 11.99, p < 0.005] were dose-dependent. An overall effect of diet revealed that cafeteria diet rats drank less water than chow-fed rats [F(1,28) = 4.90, p < 0.05]. Nonsignificant dose x diet interactions indicated that antidipsogenic effects of hUcn 2 [F(3,36) = 2.02, p > 0.10] and oCRF [F(3,48) = 0.74, p > 0.50] did not differ reliably between diet conditions.
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Food Intake
Total Caloric Intake. The cafeteria diet increased caloric intake during testing in both the hUcn 2 [95% increase in vehicle-treated animals; F(1,12) = 28.95, p < 0.001] and oCRF cohorts [65% increase; F(1,16) = 12.71, p < 0.005]. Both hUcn 2 [F(3,36) = 8.71, p < 0.001] and oCRF [F(3,48) = 13.87, p < 0.001] reduced 6-h total caloric intake of chow- and cafeteria diet-fed rats. Linear contrasts indicated that these effects were dose-dependent [hUcn 2, F(1,12) = 25.03, p < 0.0001; CRF, F(1,16) = 29.96, p < 0.0001]. As shown in Fig. 2, lower doses of hUcn 2 than oCRF reduced chow intake [MED of 0.1 µg for hUcn 2 versus 10.0 µg for oCRF; dose x peptide, F(3,42) = 3.58, p < 0.05]. In contrast, the anorectic effects of oCRF and hUcn 2 on total caloric intake in cafeteria diet rats did not differ significantly from one another [see Fig. 2; dose x peptide, F(3,42) = 0.57, p > 0.60]. This reflected that hUcn 2 less potently reduced total caloric intake in cafeteria diet rats than in chow diet rats.
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Item Analysis by Macronutrient Composition. Results of item analysis according to macronutrient composition are shown in Fig. 3. Item selection did not differ according to macronutrient composition in either cohort (p > 0.20). hUcn 2 reduced intake of both CHO-rich and mixed-fat/CHO items, as indicated by a main effect of dose [F(3,18) = 6.16, p < 0.005]. However, a significant macronutrient x dose interaction [F(3,18) = 4.71, p = 0.01] reflected that hUcn 2 more potently reduced intake of the CHO-rich food items (MED = 0.1 µg) than of the mixed-fat/CHO items (MED = 10 µg) both on an absolute caloric and percent reduction basis. The greater potency of hUcn 2 was observed against both rice cakes and marshmallows (data not shown). In contrast, oCRF comparably reduced CHO-rich and mixed-fat/CHO item intake [MED = 10.0 µg; dose, F(3,24) = 11.96, p < 0.001], as indicated by a nonsignificant macronutrient x dose interaction [F(3,24) = 0.62, p > 0.60].
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Item Analysis by Preference. The relation of baseline preference for food items to subsequent drug-induced changes also differed between hUcn 2 and oCRF. Greater baseline item preference was associated strongly with greater oCRF-induced anorexia (r = -0.57 and -0.74 for CHO-rich and fat/CHO items, respectively, at the 10-µg dose). In contrast, greater baseline preference for fat/CHO items was associated with greater sparing of these items following hUcn 2 treatment (r = 0.36 to 0.60 across the effective dose range; p < 0.05 versus oCRF), and baseline preference for CHO items did not correlate systematically with the degree of hUcn 2-induced anorexia (r = 0.24 to -0.68, not significant).
Pica Testing
Kaolin and Chow Intake. During simultaneous access to food and kaolin, hUcn 2 reduced food intake [MED = 1.0 µg; F(3,23) = 5.22, p < 0.01] (see Fig. 4). hUcn 2 did not significantly increase kaolin clay consumption [F(3,23) = 1.61, p > 0.20], although marginally increased clay intake was observed at the 10-µg dose (p < 0.08). oCRF also significantly reduced food intake [MED = 10.0 µg; F(3,23) = 5.41, p < 0.01) without significantly increasing kaolin intake [F(3,23) = 0.43, p > 0.70]. In contrast, the aversive positive control LiCl elicited an almost 4-fold increase in kaolin intake at a dose similarly anorectic to the highest doses of hUcn 2 and oCRF (see Fig. 4).
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Motor Activity. As shown in Fig. 5, oCRF, hUcn 2, and LiCl differed in their effects on motor activity during access to food and kaolin. Ovine CRF [F(3,23) = 3.14, p < 0.05; linear contrast p < 0.01] but not hUcn 2 [dose, F(3,23) = 1.72, p > 0.15] dose-dependently increased motor activity. In fact, pair-wise comparisons indicated that 0.1 µg of hUcn 2 reduced motor activity during the last2hof testing (see Fig. 5). Neither peptide exhibited a significant dose x time interaction (p > 0.60). LiCl, in contrast to both oCRF and hUcn 2, selectively suppressed motor activity during early portions of the 6-h observation period, as indicated by a significant treatment x time interaction [F(5,70) = 4.36, p < 0.005]. Activity levels varied across the observation period for all compounds, as evidenced by highly significant effects of time (p < 0.001).
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Discussion |
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In the present and previous (Reyes et al., 2001; Inoue et al., 2003) studies, central Ucn 2 infusion had potent, prolonged anorectic effects. Previously, antisauvagine-30, a preferential CRF2 receptor antagonist and antisense knockdown of CRF2 receptor expression, attenuated CRF- and Ucn 1-induced anorexia (Zorrilla et al., 2003). The findings suggest that brain CRF2 receptor activation is sufficient to suppress feeding. Reductions in cumulative 6-h intake ranged from 25 to 60% for both relatively bland (chow) and highly palatable (rice cakes and marshmallows) low-fat/CHO-rich items. In contrast, intake of mixed-fat/CHO items was spared until the 10-µg dose, which tended to increase clay intake and previously promoted a CTA (Inoue et al., 2003), suggesting malaise. Because macronutrient composition is confounded with other food properties (e.g., energy density, texture), it is not clear whether hUcn 2 altered macronutrient preference per se, as opposed to preference for an associated food characteristic (Lawton and Blundell, 1993; Mok et al., 2000). Still, oCRF did not share the macronutrient-related anorexia of hUcn 2 but suppressed intake according to item preference. The different anorexigenic profiles suggest different mechanisms for the peptides' effects on feeding.
Time course and pharmacologic analyses also support the hypothesis that CRF1 and CRF2 receptors mediate distinct forms of anorexia (Zorrilla et al., 2003). Nonselective CRF receptor agonists produced prolonged, short-onset anorexia in wild-type mice; abbreviated, short-onset anorexia in CRF2 receptor null mutant mice; and delayed-onset anorexia in CRF1 receptor knockouts. Similarly, relative to CRF1 receptor agonists, Ucn 2 induced delayed-onset anorexia in rats. Finally, Ucn 1, which activates both CRF receptors, had additive effects on food intake relative to subtype-selective agonists.
Both peptides suppressed drinking, with hypodipsic and anorectic effects corresponding in potency and magnitude. Hypodipsia following i.c.v. CRF/Ucn 1 similarly was reported in mice, rats, chickens, and sheep (Denbow et al., 1999; Weisinger et al., 2000; Inoue et al., 2003). The peptides' anorectic effects were not secondary to hypodipsia since they were retained in pica testing, during which water was not provided. However, the converse relation of hypodipsia to anorexia is less clear. Hypodipsia might reflect secondary decreases in prandial drinking. In rats, most water intake is prandial, and food-associated drinking is motivated by the quantity of food consumed (Fitzsimons and Le Magnen, 1969). This "secondary reduction" hypothesis is supported by pair-feeding studies in sheep, wherein vehicle-treated ewes provided with only the quantity of food consumed by Ucn 1-treated subjects mirrored their hypodipsia. Analogously, many "specific" anorectics, including fenfluramine and leptin, reduce prandial drinking. Alternatively, hypodipsia might reflect a primary effect on drinking, either as independent regulation of body fluid homeostasis (possibly via CRF/urocortin signaling elements in the supraoptic nucleus) (Van Pett et al., 2000; Imaki et al., 2001; Reyes et al., 2001) or as nonspecific, but coordinated, suppression of diverse appetitive behaviors. A better understanding of the functional significance of CRF/Ucn-induced hypodipsia will increase understanding of the significance of CRF receptor-mediated anorexia.
Neither peptide increased kaolin clay intake, an unconditioned behavior reflecting malaise (Takeda et al., 1993). Given that i.c.v. r/hCRF and hUcn 2 induce a strong CTA at doses in the 0.5 to 5.0 (Krahn et al., 1988; Heinrichs et al., 1991; Benoit et al., 2000) and 10-µg range, respectively (Inoue et al., 2003), the lack of effects on clay intake is noteworthy. Kaolin intake is putatively a more specific measure of visceral illness than CTAs, which might be formed in response to any novel state, be it aversive or appetitive (Hunt and Amit, 1987). Supporting the sensitivity of pica testing, LiCl increased clay intake 4-fold. Increased kaolin intake in response to noxious visceral stimuli also has been observed in mice (Santucci et al., 2000), rhesus monkeys (Knezevich, 1998), and humans (Vermeer and Ferrell, Jr., 1985; Grigsby et al., 1999) (c.f., Kaopectate).
Unlike oCRF, which dose-dependently increased motor activity in a familiar environment, hUcn 2 did not increase behavioral arousal. Rather, hUcn 2 nondose-dependently suppressed later motor activity. These findings replicate those of Valdez and colleagues (2002) using the identical photocell testing apparatus but differ slightly from those of Reyes and colleagues (2001), who found no effect of mUcn 2 (i.c.v. 1 µg) on home cage activity using radiotelemetry. Dose, peptide, or procedural differences may account for differences between studies. Clearly, however, i.c.v. Ucn 2 does not increase behavioral arousal, and the present study along with that of Valdez and colleagues (2002) suggests that it can mildly suppress motor activity. Similarly, i.c.v. Ucn 3, another selective CRF2 agonist, acutely suppressed motor activity (Valdez et al., 2003). Sedation does not account for hUcn 2's anorectic effects, however. The dose-response functions for its motor and feeding effects differed, and anorexia was item-specific under choice conditions.
Although oCRF appeared to be a less potent anorectic than hUcn 2, two caveats should be considered when interpreting this finding. First, potency differences may reflect different time courses of the peptides. The 6-h time point of measurement used in the present study, selected based on the time course of i.c.v. Ucn 2 anorexia, biases effects in favor of Ucn 2. At a shorter time point, CRF might have reduced feeding more potently than Ucn 2, as observed previously with 1-h, but not 6-h, chow intake (Reyes et al., 2001). Time course issues reflect not only the delayed onset of Ucn 2 anorexia but also the abbreviated nature of CRF-induced anorexia; previously, doses as high as 5 µg of CRF markedly reduced cumulative food intake 30 to 60 min postinjection but not 3 to 6 h postinjection in nondeprived rats (Arase et al., 1989a,b). The putatively different time courses of the peptide's anorectic activity corresponds to the reviewed time courses of CRF1 and CRF2 receptor-mediated anorexia and suggests different endogenous roles for CRF1 and CRF2 agonists in the regulation of food intake. Because cumulative anorexia in the present study persisted at 10-µg doses of oCRF, it was possible to compare directly the qualitative anorexia produced by hUcn 2 and oCRF. Future studies could examine qualitatively the anorexia resulting from lower doses of CRF1 receptor agonists at briefer posttreatment intervals.
Second, vehicle-treated rats in the oCRF cohorts ate less than those in the hUcn 2 cohorts. Reduced intake was evident on the initial treatment day of Latin square designs as well as in the pica study's between-subject design and was not evident on treatment-free days prior to or following test days (data not shown). Thus, the reduced intake of vehicle-treated rats in CRF cohorts likely resulted from testing factors, perhaps from proximity to "stressed," CRF-treated conspecifics (Hotta et al., 1999), rather than from random sampling error, carryover, or conditioned drug effects. Several findings counter the hypothesis that the reduced vehicle baseline of oCRF cohorts artifactually accounts for the potency differences between hUcn 2 and oCRF. First, differences also were evident as proportional decreases relative to baseline and not only as absolute decreases in intake. Second, potency differences were observed under three separate testing conditions (chow diet, cafeteria diet, and kaolin/chow pica study), suggesting that they did not result from chance distortion of dose-response profiles. Third, under vehicle conditions that achieved similar levels of intake (e.g., oCRF cafeteria diet versus hUcn 2 chow diet), CRF also was less potent than hUcn 2, indicating that potency differences cannot be ascribed solely to baseline intake differences. Still, it remains possible that the relative potency of oCRF may be underestimated because of the consistently reduced baselines. This would especially be true if the reduction of intake in vehicle-treated rats resulted from conspecific stress, rather than from test procedures unrelated to oCRF treatment. Notwithstanding the caveats regarding relative potency, the qualitatively different patterns of anorexia produced by the peptides were consistent across different degrees of anorexia.
In summary, central infusion of a CRF2 receptor agonist suppressed intake of bland and palatable diets without inducing behavioral arousal or malaise—anorexigenic effects that qualitatively differed from those of a CRF1 receptor agonist. Ucn 2 preferentially reduced intake of CHO-rich food items and mildly suppressed motor activity. In contrast, oCRF suppressed preferred food intake and increased behavioral activation. The dissimilar behavioral consequences of hUcn 2 and oCRF administration are consistent with the dissimilar distribution and pharmacology of CRF2 and CRF1 receptors and support the hypothesis of distinct CRF1- and CRF2-mediated forms of anorexia.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: CRF, corticotropin-releasing factor; Ucn 1, urocortin 1; Ucn 2, urocortin 2; Ucn 3, urocortin 3; oCRF, ovine CRF; CTA, conditioned taste aversion; hUcn 2, human Ucn 2; CHO, carbohydrate; MED, minimum effective dose.
Address correspondence to: Dr. Eric P. Zorrilla, Department of Neuropharmacology, CVN-7, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: ezorrilla{at}scripps.edu
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References |
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Arase K, Shargill NS, and Bray GA (1989a) Effects of corticotropin releasing factor on genetically obese (fatty) rats. Physiol Behav 45: 565-570.[CrossRef][Medline]
Arase K, Shargill NS, and Bray GA (1989b) Effects of intraventricular infusion of corticotropin-releasing factor on VMH-lesioned obese rats. Am J Physiol 256: R751-R756.
Behan DP, Grigoriadis DE, Lovenberg T, Chalmers D, Heinrichs S, Liaw C, and De Souza EB (1996) Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders. Mol Psychiatry 1: 265-277.[Medline]
Benoit SC, Thiele TE, Heinrichs SC, Rushing PA, Blake KA, and Steeley RJ (2000) Comparison of central administration of corticotropin-releasing hormone and urocortin on food intake, conditioned taste aversion and c-Fos expression. Peptides 21: 345-351.[CrossRef][Medline]
Denbow DM, Snapir N, and Furuse M (1999) Inhibition of food intake by CRF in chickens. Physiol Behav 66: 645-649.[CrossRef][Medline]
Esteve M, Rafecas I, Fernandez-Lopez JA, Remesar X, and Alemany M (1994) Effect of a cafeteria diet on energy intake and balance in Wistar rats. Physiol Behav 56: 65-71.[CrossRef][Medline]
Fitzsimons TJ and Le Magnen J (1969) Eating as a regulatory control of drinking in the rat. J Comp Physiol Psychol 67: 273-283.[CrossRef][Medline]
Grigsby RK, Thyer BA, Waller RJ, and Johnston GA Jr (1999) Chalk eating in middle Georgia: a culture-bound syndrome of pica? South Med J 92: 190-192.[CrossRef][Medline]
Heinrichs SC, Britton KT, and Koob GF (1991) Both conditioned taste preference and aversion induced by corticotropin-releasing factor. Pharmacol Biochem Behav 40: 717-721.[CrossRef][Medline]
Hotta M, Shibasaki T, Arai K, and Demura H (1999) Corticotropin-releasing factor receptor type 1 mediates emotional stress-induced inhibition of food intake and behavioral changes in rats. Brain Res 823: 221-225.[CrossRef][Medline]
Hunt T and Amit Z (1987) Conditioned taste aversion induced by self-administered drugs: paradox revisited. Neurosci Biobehav Rev 11: 107-130.[CrossRef][Medline]
Imaki T, Katsumata H, Miyata M, Naruse M, Imaki J, and Minami S (2001) Expression of corticotropin releasing factor (CRF), urocortin and CRF type 1 receptors in hypothalamic-hypophyseal systems under osmotic stimulation. J Neuroendocrinol 13: 328-338.[CrossRef][Medline]
Inoue K, Valdez GR, Reyes TM, Reinhardt LE, Tabarin A, Rivier J, Vale WW, Sawchenko PE, Koob GF, and Zorrilla EP (2003) Human urocortin II, a selective agonist for the type 2 corticotropin-releasing factor receptor, decreases feeding and drinking in the rat. J Pharmacol Exp Ther 305: 385-393.
Knezevich M (1998) Geophagy as a therapeutic mediator of endoparasitism in a free-ranging group of rhesus macaques (Macaca mulatta). Am J Primatol 44: 71-82.[CrossRef][Medline]
Krahn DD, Gosnell BA, Levine AS, and Morley JE (1988) Behavioral effects of corticotropin-releasing factor: localization and characterization of central effects. Brain Res 443: 63-69.[CrossRef][Medline]
Lawton CL and Blundell JE (1993) 5-HT manipulation and dietary choice: variable carbohydrate (Polycose) suppression demonstrated only under specific experimental conditions. Psychopharmacology (Berl) 112: 375-382.[CrossRef][Medline]
Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, Reyes TM, Gulyas J, Fischer W, et al. (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 98: 7570-7575.
Mok E, Paquette M, and Thibault L (2000) Effect of quipazine, a selective 5-HT3 agonist, on dietary self-selection of different macronutrient diets in male and female rats. Appetite 34: 313-325.[CrossRef][Medline]
Pellegrino LJ, Pellegrino AS, and Cushman AJ (1979) A Stereotaxic Atlas of the Rat Brain, Plenum, New York.
Prats E, Monfar M, Castella J, Iglesias R, and Alemany M (1989) Energy intake of rats fed a cafeteria diet. Physiol Behav 45: 263-272.[CrossRef][Medline]
Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, Gulyas J, Rivier J, Vale WW, et al. (2001) Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 98: 2843-2848.
Rogers PJ and Blundell JE (1984) Meal patterns and food selection during the development of obesity in rats fed a cafeteria diet. Neurosci Biobehav Rev 8: 441-453.[CrossRef][Medline]
Rothwell NJ and Stock MJ (1979) Regulation of energy balance in two models of reversible obesity in the rat. J Comp Physiol Psychol 93: 1024-1034.[CrossRef][Medline]
Rothwell NJ and Stock MJ (1988) The cafeteria diet as a tool for studies of thermogenesis. J Nutr 118: 925-928.
Santucci D, Corazzi G, Francia N, Antonelli A, Aloe L, and Alleva E (2000) Neurobehavioural effects of hypergravity conditions in the adult mouse. Neuroreport 11: 3353-3356.[Medline]
Shrout PE and Fleiss JL (1979) Intraclass correlations: uses in assessing rater reliability. Psychol Bull 86: 420-428.[CrossRef][Medline]
Spina M, Merlo-Pich E, Chan RK, Basso AM, Rivier J, Vale W, and Koob GF (1996) Appetite-suppressing effects of urocortin, a CRF-related neuropeptide. Science (Wash DC) 273: 1561-1564.[Abstract]
Sutton SW, Behan DP, Lahrichi SL, Kaiser R, Corrigan A, Lowry P, Potter E, Perrin MH, Rivier J, and Vale WW (1995) Ligand requirements of the human corticotropin-releasing factor-binding protein. Endocrinology 136: 1097-1102.[Abstract]
Takeda N, Hasegawa S, Morita M, and Matsunaga T (1993) Pica in rats is analogous to emesis: an animal model in emesis research. Pharmacol Biochem Behav 45: 817-821.[CrossRef][Medline]
Valdez GR, Inoue K, Koob GF, Rivier J, Vale WW, and Zorrilla EP (2002) Human urocortin II: mild locomotor suppressive and delayed anxiolytic-like effects of a novel corticotropin-releasing factor related peptide. Brain Res 943: 142-150.[CrossRef][Medline]
Valdez GR, Zorrilla EP, Rivier J, Vale WW, and Koob GF (2003) Locomotor suppressive and anxiolytic-like effects of urocortin 3, a highly selective type 2 corticotropin-releasing factor agonist. Brain Res 980: 206-212.[CrossRef][Medline]
Vale W, Spiess J, Rivier C, and Rivier J (1981) Characterization of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science (Wash DC) 213: 1394-1397.
Van Pett K, Viau V, Bittencourt JC, Chan RK, Li HY, Arias C, Prins GS, Perrin M, Vale W, and Sawchenko PE (2000) Distribution of mRNAs encoding CRF receptors in brain and pituitary of rat and mouse. J Comp Neurol 428: 191-212.[CrossRef][Medline]
Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, et al. (1995) Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature (Lond) 378: 287-292.[CrossRef][Medline]
Vermeer DE and Ferrell RE Jr (1985) Nigerian geophagical clay: a traditional antidiarrheal pharmaceutical. Science (Wash DC) 227: 634-636.
Weisinger RS, Blair-West JR, Burns P, Denton DA, McKinley MJ, Purcell B, Vale W, Rivier J, and Sunagawa K (2000) The inhibitory effect of hormones associated with stress on Na appetite of sheep. Proc Natl Acad Sci USA 97: 2922-2927.
Zorrilla EP and Koob GF (2004a) The roles of urocortins 1, 2 and 3 in the brain, in Handbook of Stress, Immunology and Behaviour (Steckler T, Kalin N, and Reul H eds) Elsevier, New York, in press.
Zorrilla EP and Koob GF (2004b) The therapeutic potential of CRF1 antagonists for anxiety. Expert Opin Investig Drugs 13: 799-828.[CrossRef][Medline]
Zorrilla EP, Taché Y, and Koob GF (2003) Nibbling at CRF receptor control of feeding and gastrocolonic motility. Trends Pharmacol Sci 24: 421-427.[CrossRef][Medline]
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, p < 0.05 versus oCRF group (Student's t test with Bonferroni correction).
