Corticotropin-releasing factor (CRF) or CRF binding-protein ligand inhibitor administration suppresses food intake in mice and elevates body temperature in rats

Brain Research 900 (2001) 177–185 www.elsevier.com / locate / bres

Research report

Corticotropin-releasing factor (CRF) or CRF binding-protein ligand inhibitor administration suppresses food intake in mice and elevates body temperature in rats Stephen C. Heinrichs a ,*,1 , Dominic L. Li b , Smriti Iyengar b b

a Neurocrine Biosciences, Inc., 10555 Science Center Drive, San Diego, CA 92121, USA Lilly Neuroscience, Mail Code 0510, Lilly Research Laboratories, Eli Lilly, Lilly Corporate Center, Indianapolis, IN 46285, USA

Accepted 13 February 2001

Abstract Corticotropin-releasing factor (CRF) receptor agonist and CRF binding-protein (CRF-BP) ligand inhibitor peptides both activate CRF systems but exert very distinct functional profiles in animal models of arousal, energy balance and emotionality. The present studies were designed to extend the dissimilar efficacy profiles of central administration of a CRF agonist, r / h CRF(1–41), versus a CRF-BP ligand inhibitor, r / h CRF(6–33), into mouse and rat models of energy balance in order to further explore in vivo efficacy of these ligands in two separate animal species. In CD-1 mice, food intake was significantly attenuated 3 h after acute administration of CRF(1–41) (0.007–0.2 nmol), but not CRF(6–33). In obese Ob / Ob mice, both CRF(1–41) (0.007–0.2 nmol) and CRF(6–33) (0.02–2.3 nmol) significantly attenuated basal feeding over 3 h following acute peptide administration. In rats, CRF(1–41) (1 nmol) and CRF(6–33) (1.5–7.7 nmol) infusion significantly increased rectal temperature. In studies employing a telemetry apparatus, core temperature was also increased by CRF(1–41) (1 nmol) and CRF(6–33) (1.5 nmol), whereas only CRF(1–41) increased locomotor activity and heart rate. These results suggest that CRF receptor agonist administration is capable of producing a global profile of negative energy balance by reducing food intake in mice and increasing energy expenditure in rats. In contrast, CRF-BP ligand inhibitor administration appears to suppress food intake in a mouse strain selective manner and to elevate rectal and core temperature in rats without accompanying cardiovascular activation.  2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Neuropeptides and behavior Keywords: Corticotropin-releasing factor; Feeding; Rectal temperature; Telemetry; Mouse; Rat

1. Introduction Corticotropin-releasing factor (CRF) is a neurohormone which appears necessary and sufficient for the organism to mount functional, physiological and endocrine responses to stressors [15,35]. It may be possible to test pharmacologically for functional diversity within brain CRF pathways by targeting separate populations of CRF binding sites in brain, namely post-synaptic CRF receptors versus CRF binding-protein (CRF-BP) [35]. Such functional diversity is postulated due to the heterologous anatomical distribu*Corresponding author. Current address: Boston College, Department of Psychology, McGuinn Hall, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA.

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tion of the CRF receptor agonists CRF and urocortin, CRF receptors and CRF-BP in rat and mouse brain [10,27] and due to differential in vivo efficacy of peptide ligands with receptor / binding site selective affinities [3,34]. In particular, a CRF-BP ligand inhibitor, r / h CRF(6–33), binds the CRF-BP, dissociating it from CRF, thereby increasing levels of endogenous CRF in the brain [5,34]. Moreover, CRF receptor agonist and CRF-BP ligand inhibitor peptides both have equivalent actions in animal models of arousal and learning / memory which do not transfer to measures of cardiovascular tone or emotionality [5,19]. Because CRF-BP may alter CRF actions at specific regions in the brain, a CRF-BP inhibitor has the potential to affect physiological and behavioral parameters selectively. This conclusion is supported by the finding that while both

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02286-7

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CRF(1–41) and CRF(6–33) reduce weight gain in obese Zucker rats, only CRF(1–41) increased plasma ACTH and heart rate [18]. Additional evidence favoring a role for CRF-BP in brain is provided by the differential pharmacology of ovine CRF [o CRF(1–41)] versus rat / human CRF [r / h CRF(1–41)]. Given that both peptides are high affinity agonists at CRF 1 and CRF 2 receptors, whereas r / h CRF(1–41), but not o CRF(1–41), binds with equally high affinity at CRF-BP [4], the relatively increased potency and efficacy of ovine CRF in producing electrophysiological [32] and neurochemical [13] excitation provides evidence that CRF-BP may act to limit in vivo actions of rat / human CRF. Conversely, high affinity ligand competition binding by r / h CRF(6–33) [1] would be expected to promote efficacy of endogenous CRF-like peptides. Taken together, these results provide a potential neurobiological mechanism for efficacy of CRF-BP ligand inhibitors. The present studies were designed to extend the dissimilar pharmacological profiles of a CRF agonist, r / h CRF(1– 41), versus a CRF-BP ligand inhibitor, r / h CRF(6–33), into mouse and rat models of energy balance in order to broaden the profile of in vivo efficacy for these CRF system ligands. In particular, well documented anorexic [33] and temperature elevating [31] properties of CRF were studied in the mouse and rat, respectively. In addition, putative increases in arousal and energy expenditure produced by acute infusion of CRF peptides were characterized using a radiotelemetry technique which provides a non-invasive means of obtaining cardiovascular, physiological and behavioral measurements from freely moving, conscious animals. One indispensable feature of telemetry experiments is the ability to confirm the absence of stress artifacts which would otherwise confound studies employing stress-related neuropeptides [8]. The specific a priori hypothesis under test was that global anorexic and temperature-increasing properties of CRF receptor agonist administration would not be reproduced in toto by administration of the CRF-BP ligand inhibitor. In contrast, central infusion of r / h CRF(6–33) was expected to induce negative energy balance properties without accompanying non-specific properties of the receptor agonist [5].

2. Materials and methods

2.1. Animals CD-1 mice (group housed males, 20–25 g) were obtained from Charles River Laboratories (Portage, MI, USA). Ob / Ob mice (group housed males, 50–55 g) were obtained from Harlan Laboratories, UK. Animals were maintained on a 12 h day / night cycle under controlled environmental conditions. Food and water were supplied ad libitum. Mouse testing protocols were approved by the Institutional Animal Care and Use Committee of Eli Lilly and Company.

Rats for the rectal temperature and telemetry experiments were 3 month old male Wistar rats (Charles River, CA, USA) weighing 300–350 g. All animals were group housed in a temperature and light cycle-controlled (lights on 07.00–19.00) vivarium with ad libitum access to water and rodent chow (Picolab Rodent Diet 20). All animals were allowed 1 week to acclimate to the vivarium subsequent to arrival and were tested between 09.00 and 16.00. Rat testing protocols were approved by the Institutional Animal Care and Use Committee of Neurocrine Biosciences, Inc.

2.2. Drug and peptides Rat / human (r / h) CRF(1–41), ovine (o) CRF(6–33), r / h CRF(6–33) and r / h d-Phe CRF(12–41) were synthesized by Nick Ling at Neurocrine Biosciences using solid phase methodology on a peptide synthesizer (Beckman Model 990). Distilled water adjusted to pH 6.7 was used as the peptide diluent. Amphetamine sulfate (Sigma) was diluted in 0.9% physiological saline for intraperitoneal injection in a 1 ml / kg volume.

2.3. Intracerebroventricular ( i.c.v.) cannulations and injections 2.3.1. I.c.v. injection in mice All peptides were administered by i.c.v. injection into the lateral ventricle according to an adaptation of the Laursen and Belknap procedure [23]. A 50 ml Hamilton syringe was fitted with PE-20 tubing so that the tip of the needle was 3.7 mm from the end of the tubing. Bregma was located with the tip of the needle by feeling for a slight depression in the middle of the skull and the needle was moved 2 mm lateral to the bregma for actual injection. Verification of the injection site was made histologically by staining the needle tract with cresyl violet for Nissl substances and also by injecting a tetrazolium dye into the site through the needle. 2.3.2. I.c.v. cannula implantation and injection in rats Animals were anesthetized with isoflurane (2–5% in oxygen) and secured in a stereotaxic instrument (Kopf). A guide cannula (Plastics One, VA, USA) aimed above the lateral ventricle was then implanted and anchored to the skull with one stainless steel screw and dental cement. Stereotaxic coordinates were, with the tooth bar 15.0 mm above interaural zero, 20.6 mm posterior to bregma, 62.0 mm lateral and 23.2 mm below skull surface at the point of entry. Guide cannulae were kept patent until injection by insertion of a dummy stylet. Animals were undisturbed for a 7 day post-surgical recovery period. For injections, the dummy stylet was removed and an injector was inserted through the guide to 1 mm beyond its tip. Five microliters were injected over a 1 min period. Cannula

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placement was checked following each study using cresyl violet dye injection.

2.4. Food intake measurement in mice Mice were individually housed and allowed to acclimate to plastic cages (6312 inches) for approximately 1 h before treatment administration. To allow a comparison of the feeding data with the telemetry studies in the rat, which were conducted over 3 h, the cumulative effects on food consumption were measured over 3 h after peptide administration. Our previous experience with mice [20] has shown that the effects of exogenously applied peptides in CD-1 mice can be measured reliably during daylight hours. Each mouse was weighed and provided with a pre-weighed amount of food (50 g), in the form of food pellets, which allowed minimum spillage. Water was available ad libitum. Food weights were recorded at various times following dosing, using a Sartorius balance (Model LP22005) equipped with automatic printout as described previously [20]. Food consumption was measured by subtracting the weight of food at various times after dosing from the initial weight of food.

2.5. Rectal temperature probe Acute measurement of body temperature in rats was accomplished using a rectal probe thermocouple thermometer (Physitemp, Model BAT-12). Rats were placed supine with the tail averted to the side on a cotton towel and the petroleum jelly covered probe was inserted a standardized distance (3.5 cm) until a stable temperature reading was obtained. Baseline temperature was measured immediately following removal from the home cage prior to administration of experimental treatments. Rats were not previously habituated to rectal probe insertion and in preliminary validation experiments it was determined that a 60 min test–retest delay was necessary for the handling / injection stress-related elevations in temperature to dissipate. Rectal temperature results are expressed as difference scores in which baseline measurements were subtracted from 60 min post-injection measurements for each rat. Ambient room temperature recorded using the probe thermometer was a steady 22.58C. The rectal temperature of vehicle-treated rats used for difference score calculation was 35.58C.

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biopotential leads were inserted subcutaneously and attached to the right shoulder and the left groin area. The device itself was implanted into the abdomen and sutured to the abdominal wall. Once implanted, temperature, activity, heart rate, and blood pressure measurements are collected every 5 min without disturbing the rat by receivers under each cage connected to a computer (Data Sciences). All rats received Benzelline Penicillin (0.25 cc s.c.) and Banamine (2 mg s.c.) following surgery and daily for 2–3 days post-surgery. A 1-week surgical recovery period elapsed prior to baseline measurements. For the telemetry studies, rats were habituated to the i.c.v. injection by a sham acute i.c.v. injection followed the next day by an injection of vehicle. On alternating days thereafter, each rat was administered vehicle, r / h CRF(6– 33), or r / h CRF(1–41), so that each rat received each treatment in random order. Biotelemetry data were collected 1 h prior to injection and for 3 h post-injection during the light phase of the circadian cycle.

2.7. Statistical analysis Amount of food consumed by mice was analyzed with a one-way analysis of variance (ANOVA) followed by posthoc analysis using Dunnett’s test for multiple comparisons as well as the Tukey–Kramer comparison test. Statistical differences were assumed when P , 0.05. Data are presented in Figs. 1 and 2 as mean6S.E.M. Rat temperature data were analyzed by one- or two-way ANOVA as appropriate with treatment and dose as between subject factors. Significant main or interaction effects were followed by specific post-hoc comparisons ´ using the Scheffe test. In order to protect against Type I error, no pairwise comparisons were performed without

2.6. Telemetry implantation and monitoring Rats were anesthetized with isoflurane gas and kept on a heating pad throughout the duration of the telemetry implant. TL11M2-C50-PXT telemetry devices (Data Sciences, St. Paul, MN, USA) were equipped with a pressure catheter and two biopotential leads. The pressure catheter was inserted into the descending aorta, between the renal arteries and bifurcation of the femoral arteries. The

Fig. 1. Anorexic effect of CRF receptor agonist over 3 h in CD-1 mice. Mean6S.E.M. food consumption measured over 3 h following intracerebroventricular administration of r / h CRF(1–41) or r / h CRF(6–33) (n 5 6–20 / group). Data are expressed as percent of baseline intake measured over the first 3 h of the preceding 24 h period. *P , 0.05 relative to baseline.

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controls was approximately 0.15–0.2 g for CD-1 mice and 0.35–0.4 g for Ob / Ob mice. Food intake was significantly (P , 0.05) attenuated 3 h after acute administration of low doses of r / h CRF(1–41) (0.007–0.2 nmol, i.c.v.) in normal CD-1 mice. However, the CRF binding-protein inhibitor, r / h CRF(6–33), had no effect on basal feeding at doses up to 23 nmol i.c.v. (Fig. 1). In contrast, in Ob / Ob mice, both r / h CRF(1–41) (0.007–0.2 nmol, i.c.v.) and r / h CRF(6– 33) (0.02–2.3 nmol, i.c.v.) significantly (P , 0.05) attenuated basal feeding 3 h after acute administration of peptide (Fig. 2). This anorexic effect persisted for up to 6 h post-administration (data not shown).

Fig. 2. Anorexic effects of CRF receptor agonist and CRF-BP ligand inhibitor over 3 h in Ob / Ob mice. Mean6S.E.M. food consumption measured over 3 h following intracerebroventricular administration of r / h CRF(1–41) or r / h CRF(6–33) (n 5 7–16 / group). Data are expressed as percent of baseline intake measured over the first 3 h of the preceding 24 h period. *P , 0.05 relative to baseline.

prior detection of significant omnibus effects by ANOVA. Telemetry data were analyzed using each 5 min data point following treatment in a two-way repeated measures ANOVA with treatment and time as experimental factors.

3. Results

3.1. Effects of r /h CRF(1 – 41) and r /h CRF(6 – 33) on food intake in CD-1 and Ob /Ob mice Baseline food consumption at 3 h in vehicle-treated

3.2. Effects of o CRF(6 – 33), r /h CRF(1 – 41), r /h CRF(6 – 33), d-Phe CRF(12 – 41) and amphetamine on rectal temperature in Wistar rats Validation of the rectal temperature assay (Fig. 3) was performed using a 5 mg / kg intraperitoneal dose of amphetamine which significantly elevated rectal temperature relative to rats administered vehicle [t(20) 5 3.155, P , 0.005]. CRF peptide treatment also produced an overall increase in rectal temperature [F(9,69) 5 3.20, P , 0.05] with the 1 nmol i.c.v. dose of r / h CRF(1–41) and 1.5 and 7.5 nmol i.c.v. doses of r / h CRF(6–33) each producing significant increases relative to rats treated with vehicle (Scheffe´ tests, P , 0.05). Temperature increases induced by threshold 1–1.5 nmol i.c.v. doses of either r / h CRF(1– 41) or r / h CRF(6–33) were significantly [F(1,23) 5 6.33, P , 0.02] attenuated by co-administration (data not shown) of a 2.9 nmol i.c.v. dose of the CRF receptor antagonist, d-Phe CRF(12–41). The inactive control peptide, o CRF(6–33), did not elevate rectal temperature [F(2,26) , 1, n.s.].

Fig. 3. CRF receptor agonist and CRF-BP ligand inhibitor increase rectal temperature in Wistar rats. Mean6S.E.M. change in rectal temperature 60 min following intracerebroventricular administration of vehicle (n 5 17 / group), o CRF(6–33), r / h CRF(1–41) or r / h CRF(6–33) (N 5 5–10 / group) or intraperitoneal administration of 0 or 5 mg / kg doses of amphetamine (N 5 10–12 / group). *P , 0.05 relative to respective vehicle-treated controls.

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Fig. 4. Effects of CRF receptor agonist and CRF-BP ligand inhibitor peptides on telemetered measures of core temperature and activity in Wistar rats. Mean core temperature (top panel) and overall activity (bottom panel) in transponder-implanted rats 1 h prior to and 3 h following intracerebroventricular administration of either r / h CRF(1–41) or r / h CRF(6–33) (n 5 7–8 / group).

3.3. Effects of r /h CRF(1 – 41) and r /h CRF(6 – 33) in telemetry-monitored Wistar rats Central administration of both r / h CRF(1–41) (1 nmol i.c.v.) and r / h CRF(6–33) (1.5 nmol i.c.v.) significantly increased core temperature [F(2,10) 5 7.36, P , 0.01] as compared to vehicle. The temperature increase induced by r / h CRF(1–41) and r / h CRF(6–33), but not vehicle, was noticable 60 min post-injection (Fig. 4) and persisted for 2 h [F(70,350) 5 1.47, P , 0.01]. Activity levels increased only when the animals had been administered r / h CRF(1– 41) [F(2,10) 5 11.75, P , 0.002]. Administration of r / h CRF(1–41) also elevated heart rate significantly [F(2,10) 5 5.26, P , 0.03], although the CRF-induced elevation in blood pressure (Fig. 5) was not significant.

4. Discussion The present results strongly support the a priori hypothesis under test that CRF receptor agonists have a different

pharmacological and functional profile from CRF-BP ligand inhibitors in multiple in vivo models sensitive to activation of brain CRF systems. Consistent with previous reports using rat models [5,18], a clearcut dissociation of in vivo efficacy of r / h CRF(1–41) from r / h CRF(6–33) exists since only the CRF receptor agonist suppressed feeding in CD-1 mice and elevated heart rate in rats. Moreover, r / h CRF(6–33) was slightly less potent than r / h CRF(1–41) as an anorexic treatment in Ob / Ob mice, a finding consistent with the previously reported rightward shift in the CRF-BP ligand inhibitor dose response curve measured using the Morris water maze task [5]. This reduced potency of r / h CRF(6–33) versus r / h CRF(1–41) in suppressing food intake has also been reported recently following peripheral administration of the peptides [2]. In contrast, there was no evidence of differential maximum efficacy of the two peptides in suppressing appetite in Ob / Ob mice or in increasing body temperature in Wistar rats. Taken together, these results suggest that the CRF-BP ligand inhibitor peptide mechanism of action appears distinct from that of the CRF receptor agonist peptide in

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Fig. 5. Effects of CRF receptor agonist and CRF-BP ligand inhibitor peptides on telemetered measures of heart rate and blood pressure in Wistar rats. Mean blood pressure (top panel) and heart rate (bottom panel) in transponder-implanted rats 1 h prior to and 3 h following administration of either r / h CRF(1–41) or r / h CRF(6–33) (n 5 7–8 / group).

that efficacy of r / h CRF(6–33) arises in a strain and testing context-specific manner. Consistent with the present focus on negative energy balance properties of CRF peptides, it has previously been demonstrated that long-term, central administration of o CRF or r / h CRF(6–33) reduces body-weight gain in obese Zucker rats [18]. One recent report replicated these findings and clarified whether the reduction in body weight is attributable to altered feeding and drinking behavior [7]. Obese Zucker rats were fitted with osmotic mini-pumps connected to intracerebroventricular cannulae through which vehicle, o CRF(1–41) (1 nmol / day) or r / h CRF(6– 33) (7.5 nmol / day) was infused for 7 days. In agreement with published results [18], o CRF(1–41) and r / h CRF(6– 33) significantly reduced body-weight gain in the obese Zucker rat [7]. In addition, food intake was reduced, whereas water consumption was unaffected [7]. Together with other reports of in vivo efficacy of r / h CRF(6–33) [28], it appears that CRF-BP ligand inhibition is a viable

strategy for activation of brain CRF circuits in a variety of behavioral and physiological testing contexts. Moreover, the Ob / Ob mouse and Zucker obese rat appear to be two animal models of genetic obesity in which administration of a CRF-BP ligand inhibitor produces a common anorexic effect. Note that appetite suppression following treatment with r / h CRF(6–33) has now been observed both acutely using a 3 h meal in Ob / Ob mice in the present studies as well as chronically using 24 h measurements in Zucker obese rats [7]. Temperature-elevating and anxiogenic effects of central CRF administration are well documented in rat [30,33] to reflect neurotransmitter-like actions of the peptide and to reproduce the profile of physiological and behavioral activation induced in rodents by exposure to naturalistic stressors [9,22]. The body temperature elevating effect of CRF itself is difficult to interpret since the threshold dose for elevating rectal and core temperature is suprathreshold for producing anxiogenic-like suppression of exploratory

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behavior in the plus-maze test [5]. This lack of dose separation raises the possibility that temperature increases were non-specific consequences of general arousal or agitation. Doses of CRF which altered appetite and body temperature in the present studies are also anxiogenic in other animal models of anxiety [26,36]. In contrast, the CRF-BP ligand inhibitor peptide that was equally potent and effective in elevating rectal and core temperature in the present studies is not reported to produce anxiogenic behavior at doses up to 25 times higher [5]. In contrast to the CRF receptor agonist, neither increased overall activity nor elevated heart rate were noted following CRF-BP ligand inhibitor administration. Moreover, the rectal temperature assay appeared to provide specific [31] and CRF receptor selective [25] measures of core temperature in the present studies since o CRF(6–33), which has no affinity for any of the CRF binding sites / receptors [5], was ineffective. In addition, the CRF peptide-induced elevation in rectal temperature was reversible by co-administration of a single dose of a CRF receptor antagonist which was selected based on the CRF agonist / antagonist competition literature [25] to provide a molar excess of antagonist versus agonist. Taken together, these results provide evidence for functional diversity of CRF receptors versus CRF binding protein in rat brain. Neurobiological support for the functional divergence of CRF receptors versus CRF binding protein is provided by a recent study [11] of neuronal activation patterns following in vivo administration of r / h CRF(6–33). Fos expression seen following central administration of a 25 mg dose of CRF-BP ligand inhibitor was most strongly coincident with the distribution of CRF-BP expression [11]. In particular, Fos-immunoreactive neurons in the isocortex, the olfactory system, amygdala and a number of discrete brainstem cell groups were found to co-express CRF-BP mRNA. A subset of these activated, CRF-BP expressing neurons co-expressed CRF 1 receptors [11]. Note that these Fos activation effects of r / h CRF(6–33) were dose dependent over the same dose range used in the present studies. In stark contrast to the widespread Fos activation induced by central administration of CRF itself [6], the discrete and selective profile of CRF-BP ligand inhibitor activation suggests a signalling function for CRF-BP which is dissociable from that of CRF receptors [11]. The present species comparative studies are important in order to address concerns related to the generality of CRF effects in vivo. For example, measures of sympathetic activation, such as oxygen consumption and brown adipose tissue thermogenesis, which are well validated to be sensitive to exogenous CRF administration in several strains of rat [16,30], are insensitive in C57Bl / 6J and Ob / Ob mouse strains administered anorexic doses of CRF [14]. Moreover, a direct comparison of peptide CRF receptor agonist and antagonist actions in multiple paradigms of novelty-induced exploratory behavior in four mouse strains also revealed strain-dependent profiles of

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efficacy [12] which the authors attribute to genetically programmed differences in behavioral stress-reactivity. Finally, an earlier report of CRF-BP ligand inhibitor efficacy described suppression of body-weight gain in Zucker obese, but not Zucker lean, rats [18]. Such factors are addressed in the present studies by inclusion of data from one rat and two mouse strains tested using overlapping dose ranges. Indeed, the present data are consistent with the finding of stain-dependent actions of CRF systems activation since CRF-BP ligand inhibitor administration suppressed appetite in Ob / Ob, but not CD-1, mice. This conclusion is supported by the reported lack of effect of acute CRF-BP ligand inhibitor administration on food intake of Wistar rats [18]. However, strain or paradigm insensitivity to CRF peptide action is not a reasonable explanation for the dichotomous effect of r / h CRF(6–33) on food intake since r / h CRF(1–41) exerted significant effects in all three rodent strains and all three paradigms employed in the present studies. Thus, the possibility that environmental or genetic factors predispose certain organisms to be more or less sensitive to CRF peptide actions must be considered. CRF mutant mice, by virtue of concurrent dysregulation of brain CRF systems and pituitary–adrenal tone, may provide insight into the relative contribution of central versus peripheral mechanisms regulating ingestive behavior and body weight. For example, CRF knockout mice exhibit normal growth, weight and food intake when allowed ad libitum access to food relative to their wildtype littermates [17]. However, restriction of meal size or duration with or without running wheel access resulted in a higher rate of body-weight loss in wildtype mice, suggesting that CRF and / or glucocorticoid secretion plays an important role in the weight loss which occurs during food restriction in normal animals, by a mechanism independent of alteration in food intake. Similarly, the characterization of a CRF-BP overexpressing mutant mouse [24] has revealed that broad expression of CRF-BP in brain, plasma and peripheral organs leads to hypo-reactivity of the pituitary to a stressor and small but significant increases in the rate of body-weight gain relative to wildtype control mice. The physiological relevance of CRF-BP for bodyweight regulation is further supported by the complementary decreased weight gain phenotype of CRF-BP knockout mice [29]. Moreover, male CRF-BP knockout mice exhibited a significant reduction in food intake during the diurnal photoperiod of the circadian cycle [21]. Interestingly, CRF-BP transgenic and wildtype animals exhibit similar plasma corticosterone concentrations under basal and stimulated conditions, suggesting that CRF-BP could participate in an HPA-independent mechanism of bodyweight regulation [24]. Thus, data from CRF mutant mice suggest that brain CRF dysregulation, and deletion or overexpression of CRF-BP in particular, alters energy balance homeostasis. In conclusion, anorexic and body temperature-elevating,

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but not cardiovascular, effects of native CRF peptide administration can be mimicked by central administration of a CRF-BP ligand inhibitor peptide. These results are consistent with earlier findings [5,18,19] in which these same CRF peptides were examined in learning / memory and energy balance contexts. Thus, the anorexic action of CRF-BP ligand inhibitor was strain-specific to Ob / Ob mice and the CRF-BP ligand inhibitor-induced increase in energy expenditure in Wistar rats was achieved without the reactivity and signs of malaise which characterized CRF administration [18]. In contrast, the non-specific behavioral, physiological and cardiovascular activation produced by CRF itself could account in part for the negative energy balance properties of this peptide.

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Acknowledgements We thank Margaret Joppa and Rick Nelson for expert technical assistance. This research was supported, in part, by grant DK 51983 to SCH.

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