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Endocrinology Vol. 141, No. 8 2715-2724
Copyright © 2000 by The Endocrine Society

ARTICLES

Modulation of Urocortin-Induced Hypophagia and Weight Loss by Corticotropin-Releasing Factor Receptor 1 Deficiency in Mice1

Margaret J. Bradbury, Mary I. McBurnie, Derek A. Denton, Kuo-Fen Lee and Wylie W. Vale2

The Clayton Foundation Laboratories for Peptide Biology (M.J.B., K.-F.L., W.W.V.), The Salk Institute, La Jolla, California 92037; and The Howard Florey Institute of Experimental Physiology and Medicine (M.I.M., D.A.D.), University of Melbourne, Parkville, Victoria, 3052, Australia

Address all correspondence and requests for reprints to: Wylie Vale, Ph.D., Clayton Foundation Laboratories for Peptide Biology, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intracerebroventricular injection of CRF or urocortin (Ucn) reduces appetite and body weight. CRFR1 and CRFR2, the receptors for CRF and Ucn, are expressed in neurons associated with appetite-control and metabolism, but their relative contributions in mediating CRF- or Ucn-induced hypophagia and weight loss are not known. We used homozygous mice lacking CRFR1 (CRFR1-/-) and wild-type littermates to determine the role of CRFR1 in mediating the changes in food intake and body weight following intracerebroventricular administration of Ucn. CRFR1-/- mice, which are glucocorticoid deficient, were given corticosterone in their drinking water to induce diurnal variations in circulating corticosterone. A 7-day intracerebroventricular infusion of Ucn transiently suppressed ad libitum food intake equally in CRFR1-/- and wild-type mice. Body weight reduction during Ucn infusion paralleled food intake in wild-type mice, but persisted throughout the infusion in CRFR1-/- mice. After food-deprivation, acute intracerebroventricular injection of Ucn suppressed food intake for 1.5 h in wild-type mice. By contrast, CRFR1-/- mice did not respond to Ucn 1.5 h after injection. At later time points, Ucn suppressed food intake equally in both genotypes. The distinct time courses of CRF-receptor-induced hypophagia suggest that separate pathways act cooperatively to adjust food intake during challenges to homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REDUCED food intake (1), increased sodium appetite (2), and weight loss (3) follow exposure to acute and chronic stresses in laboratory animals. CRF family peptides in the brain and pituitary are thought to contribute to stress-related changes in nutrient intake and energy expenditure following exposure to stressors. Well-established stress-responses such as activation of the hypothalamic-pituitary-adrenal axis, increases in sympathetic nervous system activity and reductions in exploratory behavior are replicated by the administration of CRF into brain ventricles (4, 5). Central administration of CRF also mimics hypophagia and weight loss measured during and after exposures to stressors in rats (6) and mice (7, McBurnie and Denton, personal communication), whereas administration of CRF receptor antagonists blocks stress-induced anorexia (8). Thus, the decrease in food intake and body weight following CRF administration coupled with the known role of CRF in stress responses suggests that CRF family ligands and receptors are involved in coordinating appropriate changes in energy intake with information about challenges to homeostasis.

It is not known which endogenous CRF ligands and CRF receptors modulate food intake and energy expenditure. There are two CRF family ligands identified in mammals, CRF and urocortin (Ucn) (9). Currently, two G protein-coupled CRF-ligand receptors, CRFR1 (10) and CRFR2 (11, 12, 13, 14), have been isolated from mammalian tissue. CRF-immunoreactive fibers are more often colocalized with CRFR1 than they are with CRFR2 in the brain (9). In addition, the affinity for CRF at CRFR2 is approximately 15-fold less than that at CRFR1 (9), suggesting that CRF acts as an endogenous ligand for CRFR1. By contrast, Ucn has a higher affinity for both receptors than does CRF (9). There is, however, little Ucn-like immunoreactivity in brain areas containing CRFR1 (15). Instead, fibers positive for Ucn-like peptides are often, but not exclusively, colocalized with CRFR2 (9, 15). Thus, it appears that Ucn is not an endogenous ligand for CRFR1 but is well able to stimulate both receptors when administered during experimental conditions.

CRF or Ucn administration in rodents suppresses appetite and causes weight loss, with Ucn exhibiting greater potency. It is not known which CRF receptors are responsible for these effects and which ligand may naturally fulfill these functions. Both ligands are expressed in central nervous system areas implicated in the regulation of appetite and metabolism. For example, CRFR1 is expressed in the dorsomedial nuclei and medial preoptic areas of the hypothalamus (16) and the central nucleus of the amygdala (16), all areas documented to modulate energy expenditure (17, 18). Localization of CRFR2 messenger RNA (mRNA) in the ventromedial nucleus (16, 19) suggests that this receptor may modulate food intake and energy expenditure, as proposed from numerous lesion studies (i.e. Ref. 20). Identification of magnocellular neurons within the paraventricular and supraoptic nuclei of the hypothalamus positive for CRFR2 mRNA also raises the possibility that stimulation of CRFR2 may regulate water and sodium intake and excretion (21). It is difficult to determine the singular effects of CRFR1 and CRFR2 in food intake, fluid balance, and weight regulation because the affinities for many agonists and peptide antagonists for each receptor overlap. Although small molecule CRFR1 antagonists have been developed (22, 23), there is currently no small molecule CRFR2 antagonist available. In addition, central or peripheral injection of ligands with high affinity for CRFR1 leads to increased production of glucocorticoids. Corticosteroids enhance food intake (17) and suppress sympathetic nervous system activity (24), countering the effects of central CRF on appetite and body weight. Thus, the inexact receptor pharmacology and the glucocorticoid-mediated counterregulatory effects of CRFR1 stimulation hamper the determination of the relative contributions of CRFR1 and CRFR2 in appetite and metabolism.

One approach to distinguish the roles of related receptors is to use genetic tools to manipulate the expression of receptor proteins. Smith et al. (25) and Timpl et al. (26) each engineered a mouse in which the CRFR1 was functionally removed. These mice have little endogenous hypothalamic-pituitary-adrenal axis activity under basal conditions as well as after exposure to stressors compared with wild-type mice (25, 26). We used mice homozygous for the CRFR1 deletion (CRFR1-/-) (25) and their wild-type littermate controls to assess the role of CRFR1 on food intake under ad libitum (ad lib) conditions and during Ucn-induced hypophagia and weight loss. We chose Ucn as a CRF receptor ligand to ensure stimulation of both CRFR1 and CRFR2. We administered Ucn through indwelling cannulae in an attempt to minimize handling-induced stress responses on food intake and body weight. The lack of endogenous hypothalamic-pituitary-adrenal axis activity in CRFR1 -/- mice allowed us to control their peripheral corticosterone concentrations through the use of corticosterone administration in the drinking fluids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The Salk Institute for Biological Research Animal Care and Use committee approved all experiments. Male mice in which exons 5–8 of the CRFR1 were replaced with a PGK-neomycin-resistant cassette (CRFR1-/-) (25) and their wild-type littermates used in these experiments were bred from a colony at the Salk Institute for Biological Research. The genetic background of the mice in this study was a mixture of 129sV and C57BL/6. Mice began participation in the experiments below when they were between 8 and 16 weeks of age. The mouse colony lighting was 12-h light, 12-h dark. Mice received water and Teklad 8002 or 8604 diet (standard chow) ad lib until the diet modifications described below commenced.

Diet modifications and data collection
Some wild-type mice and some CRFR1-/-mice were provided with 0.2% EtOH (VEH) or 10 mg/liter corticosterone (CORT) (Sigma, St. Louis, MO) in VEH. Some mice received a low sodium diet (ICN Biochemicals, Inc., Cleveland, OH; Na+ 4–5 mmol/kg, K+ 230–235 mmol/kg) in place of the standard chow. Mice on the low sodium diet received additional drinking tubes containing NaCl diluted in VEH (wild-type) or 0.3 M NaCl in CORT (CRFR1-/-). Daily food intake and body weights were measured to the nearest 0.01 g. Fluid intakes were measured to the nearest 0.1 ml.

Food intake and body weight in wild-type and CRFR1-/- mice
Thirteen wild-type and twelve CRFR1-/- mice were adapted to CORT or VEH and provided with standard mouse chow for 3 weeks, after which food intake, fluid intakes, and body weights were measured 2 h after lights on and 2 h before lights off for 3 days. These mice were also food-deprived 4 h before lights off. The next morning, 1 h after lights on, preweighed standard mouse chow was returned to the cage, and food intake was measured periodically. After a 7-day recovery period, mice were food-deprived again. One hour after lights on, 300 µl of blood were collected through the retroorbital sinus into serum clot activator tubes within 1 min of touching the home cage. After 1.5–2.0 h of feeding ad lib, the mice were decapitated. Blood was collected into serum clot activator tubes. Serum was stored at -70 C.

Intracerebroventricular surgery
A set of mice separate from that above was anesthetized with 41 mg of ketamine and 0.2 mg of xylazine. Cannulae made from 30-g needles were inserted 3.2 mm below the surface of the brain approximately 0.1 mm posterior to the intersection of the bregmoid and saggital skull sutures and secured to the skull with cranioplastic cement and dental screws as previously described (27). Flexible tubing attached the cannulae to osmotic minipumps (Model 2002, Alza Corp., Palo Alto, CA) that delivered 0.5 µl/h of artificial cerebrospinal fluid (aCSF), pH 7.2 (27). The tubing and minipump were tunneled sc and the skin was sutured over the tubing. Mice were injected sc with 2 ml of lactated ringer’s solution. Mice recovered their eating and drinking patterns approximately 5–6 days after surgery.

Effects of ICV administration of Ucn on food intake
Seven wild-type and nine CRFR1-/- (age >7 weeks) male mice were adapted to VEH or CORT. To assess sodium appetite, mice were adapted to low-sodium diet and 0.3 M NaCl drinking tubes for several weeks. Mice received third ventricular cannulae as above. Fourteen days after surgery, mice were anesthetized with isoflurane 4 h before lights off. The aCSF-containing minipumps were removed and replaced with osmotic minipumps (Model 2001, flow rate of 1.0 µl/h) containing 0.1 µg/ul Ucn, pH 7.4–7.8, delivering 2.4 µg (0.59nmol) Ucn/day. This procedure was completed in 5–10 min and the mice were active approximately 10 min later. We estimate that the aCSF in the tygon tubing was cleared and replaced with Ucn in approximately 12 h. Seven days later, the Ucn-containing minipumps were exchanged for aCSF-containing minipumps (Model 2001). Seven days later, the aCSF minipumps were removed from the mice and 1 cm pieces of flexible tubing prefilled with either aCSF or 0.5 ng Ucn/µl aCSF were attached to the cannulae. Mice were deprived of food but allowed fluids ad lib. Seventeen hours later, 1 h after lights on, mice were restrained and 0.5 µl of aCSF or 0.25 µg (0.061 nmol) Ucn in 0.5 µl aCSF was injected through the cannulae over 1 min. Subsequent food intake, fluid intakes and body weights were measured periodically after injection. Seven days later, mice were anesthetized and food-deprived again. Mice who had received aCSF injections received Ucn injections. The remaining mice were either injected with aCSF or restrained for 1 min. Subsequent intake data from aCSF-injected and restrained mice were not different; together these mice comprised the control (CON) treatment group. Four days later, mice were weighed and exposed to CO2 for 3 min. One ul of Evans Blue dye was injected through the tubing into the cannulae. Mice were decapitated after a 1-min exposure to CO2. Trunk blood was collected into serum separation tubes (Microtainer, Becton Dickinson and Co., Franklin Lakes, NJ). Serum was stored at -70 C. Thymus glands were collected for wet weight. Mice used for data analysis (6 wild-type and 6 CRFR1-/- mice) had either dye in brain ventricles or a cannula tract ending in the third ventricle.

Effects of ACTH injection on post food-deprivation food intake
Seven wild-type male mice were food-deprived 4 h before lights on. Seventeen hours later, mice were injected sc with either saline (100 µl, n = 3) or with 0.16 U ACTH gel in saline (Rh|$$|Axone-Poulenc Rorer, Inc., Collegeville, PA, n = 4). Mice were given preweighed food pellets. 1.5 h later, mice were decapitated and trunk blood was collected into serum separation tubes. Food intake was measured.

Serum hormone assays
Serum corticosterone concentrations were assayed with a RIA kit from ICN Biochemicals (Costa Mesa, CA). Serum and reagent volumes were halved. Serum was diluted 1:50. Serum leptin concentrations were assayed with a RIA kit from Linco Research, Inc. (St. Louis, MO). Serum and reagent volumes were halved. Serum glucose concentrations in 5 µl of serum were determined using a glucose oxidase reaction (Trinder’s reagent, Sigma, St. Louis, MO).

Data analysis
Food intake, fluid intakes and body weight data in this study were analyzed with two-way repeated measures (RM) ANOVA, two-way ANOVA, or Student’s tests using SigmaStat version 2.03 (SSPS, Inc., San Rafael, CA). Data that were not normally distributed were rank transformed before analysis. Sources of significant differences contributing to significant main effects after ANOVA were identified with the Newman-Keul’s post hoc test. Significance was defined as P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of CRFR1 -/- mutation and corticosterone-containing drinking fluid on food intake, fluid intake and body weight
Body weight and nutrient intakes were averaged during a 3-day period of feeding ad lib starting 3 weeks after introduction of CORT-containing or VEH-containing drinking fluids to adult CRFR1-/- and wild-type mice. Two-way ANOVA demonstrated that CRFR1-/- mice weighed less than wild-type mice and that this dose of corticosterone provision did not alter body weight (Table 1Go). To assess the effects of corticosterone replacement on food and fluid intake, measurements were normalized to the most recently measured body weight. There were no main effects of genotype or corticosterone replacement on normalized food or fluid intake. The gross diurnal pattern of food intake was not altered by genotype or fluid drunk; 85% of the 24-h food intake occurred from 2 h before to 2 h after lights off in all mice (data not shown).


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  		Table 1. Body weight, food intake, and fluid intake in wild-type and CRFR1-/- mice drinking vehicle or corticosterone (10 mg/liter) during ad libitum conditions

 
The effects of CRFR1 deletion and corticosterone provision on body weight, food intake, and fluid intake were tested in this cohort of mice after a 17-h period of food deprivation ending 1-h after lights on. By two-way ANOVA, genotype and corticosterone availability did not affect body weight loss during the overnight fast, an average of 15% in all mice (data not shown). Body weights returned to prefasting levels 24 h after refeeding, with no effect of genotype or corticosterone availability (data not shown). As previously noted (28), voluntary fluid intake was severely reduced during the period of food deprivation to only 10% of ad lib values, with no significant effect of genotype or corticosterone replacement on fluid consumption (data not shown).

Normalized food and fluid intakes after food deprivation are shown in Fig. 1Go, A and B, respectively. Data were analyzed with two-way ANOVA at each interval, with genotype and presence of corticosterone in the drinking fluid as the dependent variables. During the 0–1.5 h interval, there was a significant stimulatory effect of corticosterone replacement on normalized food intake with no effect of genotype. Neither genotype nor corticosterone replacement had an effect on normalized food intake during any other interval, or on the cumulative 24-h normalized food intake. There was a significant main effect of corticosterone replacement on fluid intake between 9 and 24 h and on cumulative fluid intake, with no main effect of genotype. Fluid intake during other intervals was not affected by genotype or corticosterone provision.



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  		Figure 1. Effects of corticosterone or VEH on normalized food intake (A) and normalized water intake (B) in previously food-deprived wild-type and CRFR1-/- mice. Wild-type and CRFR1-/- mice were provided with standard chow and water with either 10 mg/liter corticosterone (CORT) or VEH for 3 weeks. Mice were then deprived of food for 17 h. Fluid was available ad lib. One hour after lights on, mice were given preweighed food. Food intake, fluid intake, and body weight were measured periodically thereafter. By two-way ANOVA, CORT increased food consumption during the 0–1.5 h interval and fluid consumption during the 9–24 h and 0–24 h intervals after food was returned to cages (P < 0.01). There were no main effects of genotype. n = 6 or 7/group.

 
To assess hormonal consequences of CRFR1 gene deletion and glucocorticoid replacement during food deprivation in the presence or absence of CRFR1, the mice were food-deprived again after 4 days of feeding ad lib. Immediately before food restoration and 1.5–2.0 h after feeding, blood was collected from the retroorbital sinus within 1 min of cage handling. Serum concentrations of corticosterone, glucose, leptin, and retroperitoneal fat mass are shown in Table 2Go. As expected with food deprivation, serum corticosterone concentrations in wild-type mice, 61.7 ± 12.6 ng/ml, were elevated beyond previously reported early morning values measured in ad lib fed male mice, (i.e. 11 ± 2.25 ng/ml in Ref. 25). By two-way ANOVA, we found a main effect of genotype on serum corticosterone levels, but not one of corticosterone provision. Before food repletion, serum glucose was approximately 65 mg/dl in all mice, with no effect of genotype or corticosterone treatment. Serum glucose rose after food repletion to a similar degree in all groups. Two-way ANOVA revealed that food-deprived CRFR1-/- mice had significantly lower leptin levels before and after refeeding with no main effect of fluid type (Table 2Go). At the end of the refeeding period, retroperitoneal fat mass was less in CRFR1-/- mice, an effect not reversed by corticosterone replacement.


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  		Table 2. Serum hormone concentrations and fat measurements in wild-type and CRFR1-/- mice drinking vehicle or corticosterone (10 mg/liter) immediately following a 17-h period or food deprivation and after 1.5 h of food availability

 
Effects of 7-day intracerebroventricular Ucn infusion on ad lib food intake, fluid intake, and body weight
Results from our initial experiments indicated that food intake as a proportion of body mass was not different between wild-type and CRFR1-/- mice drinking VEH. Thus, we felt that these mice provided an acceptable model for testing the effects of ICV Ucn administration on ad lib food intake. We wanted to provide these mice with a circadian pattern of corticosterone to promote surgical recovery and to minimize the confound of glucocorticoid deficiency on Ucn-induced effects on food intake. As the experiments above demonstrated that this dose of corticosterone did not alter food or fluid intake during ad lib feeding, we adapted the CRFR1-/-mice to 10 mg/liter corticosterone (CORT, as above) in the drinking fluid to replace this diurnal rhythm and to provide beneficial steroids during cranial surgery. Wild-type mice received VEH. We also adapted mice to low sodium food and provided additional drinking tubes containing 0.3 M NaCl in VEH (wild-type) or CORT (CRFR1-/-) to assess sodium appetite.

After adaptation to CORT or VEH and low sodium diet, mice received chronic ICV implants. All mice first received 14 day infusions of aCSF. Measurements from the last 7 days of these infusions were averaged. These values were considered as the baseline control. Mice subsequently received 7-day infusions of 0.1 µg/µl/h Ucn. At the end of Ucn infusion, mice received 7-day infusions of aCSF. By two-way RM ANOVA, there was a significant interaction between genotype and day after baseline on normalized food and water intake. Post hoc analysis revealed that within both wild-type and CRFR1-/- mice, normalized food intake (Fig. 2AGo) and water intake (Fig. 2BGo) decreased significantly compared with baseline intake on the second day after placement of the Ucn pump. The magnitudes of these decreases, to approximately 30% of baseline intake values, were similar in wild-type and CRFR1-/- mice. It is not clear from these data whether the time course of Ucn-induced hypophagia in CRFR1-/- and wild-type mice differed. We estimate that the Ucn began to flow into the brain ventricles at approximately 0200 h on day 1. Intake and body weight measurements on day 1 were taken at 0800 h. It is unlikely that mice consumed a significant quantity of food during this 6-h interval. We also predict that corticosterone consumption in CRFR1-/- was reduced only on the second day of the Ucn infusion.



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  		Figure 2. Effects of central Ucn infusion and subsequent aCSF infusion on normalized low sodium food intake (A) and water intake (B) in wild-type and CRFR1-/- mice. Water contained either 10 mg/liter corticosterone (CRFR1-/-) or 0.2% EtOH (wild-type) VEH. Osmotic minipumps delivered aCSF into the third ventricles through indwelling cannulae. Food and fluid intakes and body weights were taken at 0800 h, 2 h after lights on. Data from the last 7 days of the 14-day baseline aCSF infusion were averaged and considered the baseline day. Intakes were normalized to individual body weights. Minipumps delivered 0.1 µg/µl/h Ucn beginning on approximately 0200 h on day 1 and recovery aCSF beginning at approximately 0200 h on day 8. Significant interactions between genotype and experimental day were found on normalized food and water intakes by two-way RM ANOVA. Significant differences by post hoc tests (all P < 0.007): *, intake different from baseline day within genotype. +, intake different between genotypes within experimental day. n = 6/group.

 
In CRFR1-/- mice, normalized food intake increased above baseline levels on the first and second days after the Ucn-containing minipump was replaced with one containing aCSF. In wild-type mice, normalized food intake decreased on the second day after exposure to this second aCSF infusion compared with baseline. This second, transient decrease in food intake was less than that measured after infusion of Ucn. Normalized food intake differed between genotypes on the third day of exposure to Ucn and on the first 2 days of the second aCSF infusion. Water consumption, and presumably corticosterone consumption, in the CRFR1-/- mice did not differ from baseline throughout this aCSF infusion. Consumption of 0.3 M NaCl did not change during the experiment and did not differ between genotypes. Daily, mice drank approximately 0.3 ml, the equivalent of 36 µmol of NaCl (data not shown).

Body weights during the baseline aCSF, Ucn, and second aCSF infusions are shown in Fig. 3AGo. Two-way RM ANOVA demonstrated a significant interaction between genotype and day on body weight. Post hoc analysis revealed that within wild-type mice, body weight dropped below the baseline value only on the second day of Ucn exposure. Conversely, in CRFR1-/- mice, body weight dropped below baseline values on the second through sixth days of exposure to Ucn. To facilitate comparison of body weights between genotypes, averaged body weights from each mouse during the baseline period were considered 100%. The subsequent body weights were normalized to this control value (Fig. 3BGo) and analyzed as above. Two-way RM ANOVA demonstrated a significant interaction between genotype and day on normalized body weight. Normalized body weight in wild-type mice dropped below baseline values on the second day of Ucn exposure to 93% of the baseline body weight. Normalized body weight in CRFR1-/- mice was less than baseline on the second through sixth days of Ucn infusion, with a sustained loss of approximately 7%. Genotype differences in normalized body weight were found on the fourth through seventh days of Ucn infusion.



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  		Figure 3. Effects of central Ucn infusion and restoration of aCSF infusion on raw (A) and normalized (B) body weight in wild-type and CRFR1-/- mice. Data are from mice shown in Fig. 2Go. Body weights during the baseline period were averaged for the baseline day data point. This value was considered 100% for normalization in (B). Significant interactions between genotype and experimental day were found in raw body weight and in normalized body weight data by two-way RM ANOVA. Significant differences by post hoc tests (all P < 0.025): *, intake different from baseline day within genotype. +, intake different between genotypes within experimental day. n = 6/group.

 
Effects of acute intracerebroventricular Ucn injection on food intake, fluid intake, and body weight after food deprivation
To better discriminate the time course of Ucn-induced hypophagia in wild-type and CRFR1-/- mice, the mice above were injected ICV with Ucn or CON-treated (aCSF injection or restraint) 1 h after lights-on, after 17 h of food deprivation. One week later, mice were food-deprived again and received the alternate treatment in a cross-over design (see Materials and Methods). Seventeen hours of food deprivation caused similar weight loss in wild-type and CRFR1-/- mice, to 93 ± 0.5% and 92 ± 0.6% of pre-food-deprivation weights, respectively. Weight gain after refeeding was similar in all mice (data not shown).

Two-way ANOVAs were applied to food and fluid intake data from each time interval after refeeding, with genotype and treatment as the independent variables. Data are shown in Fig. 4Go, A and B, respectively. During the 0–1.5 h interval, there was a significant interaction between genotype and treatment; normalized food intake in wild-type, but not CRFR1-/- mice, was reduced after Ucn injection. Food consumption during the 0–1.5 h interval in all CRFR1-/- mice and in CON-treated wild-type mice was similar to that of unhandled mice in Fig 1Go. Normalized food intake at 1.5–3 h was not affected by genotype or treatment. Between 3 and 11 h after food intake, there was a significant main effect of treatment, with Ucn-treated mice eating less. There was no effect of genotype on food intake during this interval. Normalized food intake between 11 and 24 h after treatment was not affected by treatment in either wild-type or CRFR1-/- mice. Cumulative food intake during the 24-h period was reduced in Ucn-treated mice with a trend for a main effect of genotype (P = 0.07), but no significant interaction. Normalized fluid intakes were measured 3, 11, and 24 h after treatment (Fig. 4BGo). Between 0 and 3 h, Ucn reduced fluid intake in wild-type, but not CRFR1-/- mice. Fluid intake was not affected by genotype or treatment at any other time point. NaCl intake during the experiment was less than 0.1 ml/measurement/mouse with no effect of treatment or genotype (data not shown).



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  		Figure 4. Effects of acute central Ucn injection on normalized food intake (A) and normalized water intake (B) in previously food-deprived wild-type and CRFR1-/- mice. Mice from Fig. 2Go were deprived of food for 17 h. Fluids were available ad lib. One hour after lights on, mice were injected with Ucn or control-treated and given preweighed food. Food and fluid intake and body weights were measured periodically thereafter. One week later, mice were food-deprived again and given the alternate treatment in a cross-over design. *, Intake after Ucn-treatment less than that after CON treatment within genotype by two-way ANOVA (all P < 0.008): six wild-type and 5 CRFR1-/- mice completed the cross-over design.

 
After an additional 4 days of ad lib food, VEH (wild-type) and CORT (CRFR1-/-) availability, mice were killed after a 3-min exposure to CO2. At the end of this stressor, serum corticosterone concentrations in CRFR1-/- mice were less than those in wild-type mice (wild-type: 100.0 ± 13.5, CRFR1 -/-: 36.7 ± 14.0 ng corticosterone/ml serum, Student’s t test: P = 0.03). Thymus weights in wild-type and CRFR1-/- mice were not different (wild-type: 114.9 ± 7.7, CRFR1 -/-: 117.0 ± 5.8 mg thymus/100 g body weight). This suggests that corticosterone replacement in CRFR1-/- mice was within a physiological range. We also examined serum leptin in these mice. As in our previous experiments with food-deprived mice, serum leptin concentrations were lower in CRFR1-/- mice (wild-type: 7.5 ± 0.5, CRFR1 -/- with corticosterone: 4.2 ± 0.8 ng leptin/ml serum, Student’s t test: P = 0.005).

Effect of ACTH injection on food intake
Compared with injection of saline, injection of ACTH in a long-lasting gel form increased serum corticosterone concentrations from 12.2 ± 4.1 to 463.8 ± 61.7 ng/ml 1.5 h after injection in wild-type mice. Serum ACTH increased from 399.8 ± 90 to 4597.3 ± 1577.4 pg/ml. Despite these increases in peripheral hormone levels, food intake was unaffected by ACTH administration (saline-injected: 27.6 ± 4.3 mg/g BW, ACTH-injected: 26.5 ± 5.1 mg/g BW).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we found that CRFR1-/- mice had normal food intake and fluid intake during ad lib and postdeprivation conditions, despite the lack of CRFR1 and attendant hypocorticoidism. Restoration of the glucocorticoid circadian rhythm through the provision of corticosterone in the drinking water of CRFR1-/- mice had small effects on these parameters. Chronic third ventricular Ucn infusion through indwelling cannulae transiently reduced ad lib food intake in wild-type and corticosterone-replaced CRFR1-/- mice to a similar degree. After food deprivation, acute injection of Ucn through indwelling third ventriclar cannulae reduced initial (0–1.5 h) food intake in wild-type, but not CRFR1-/- mice. Thereafter, Ucn effects on food intake were not dependent on genotype. These results suggest that CRF receptors each alter food intake with a distinct time course.

Ad lib and postdeprivation food intake and physiological responses to food deprivation in wild-type and CRFR1-/- mice
Consistent with previous reports, CRFR1-/- mice had profoundly reduced circulating corticosterone concentrations compared with wild-type mice. The hypocorticoidism in CRFR1-/- mice did not recapitulate the reduced food intake shown previously in adrenalectomized rats (17) and mice (29). Our results are consistent with the observation of normal food intake measured in mice lacking the gene for CRF (29). It is possible that developmental compensations in both genetic models for hypothalamic-pituitary-adrenal axis deficiency correct for the lack of adrenocortical activity. Alternatively, residual adrenocortical function or secretion of mineralocorticoids in CRF-/- and CRFR1-/-mice may be sufficient to maintain food intake through the stimulation of mineralocorticoid or glucocorticoid receptors in the brain. Finally, the effects of adrenalectomy on food intake may relate to either surgery or removal of peripheral epinephrine stores. In accordance with the notion that basal food intake is not altered by CRF receptor signaling, administration of the nonselective CRF receptor antagonist {alpha}-helical-CRF (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) (8) does not alter food intake under ad lib conditions.

CRFR1-/- mice had smaller retroperitoneal fat pads after an overnight period of food deprivation followed by 1.5 h of food availability. This effect was not ameliorated by replacement with corticosterone in the drinking fluid. These results are in agreement with the lower total carcass triglycerides measured in CRF-deficient mice (30). It is not known whether the reduction in circulating glucocorticoids is entirely responsible for the reduction in fat levels in CRF and CRFR1 deficient mice. Corticosterone-replaced CRFR1-/- mice also had lower serum leptin concentrations after fasting and after refeeding than wild-type mice. The adipocyte-derived hormone leptin suppresses appetite when delivered centrally into rats (31) and mice (32). Leptin is hypothesized to inform appetite and metabolic centers in the brain about the adipose stores of the organism (33). According to the adipocyte model of appetite regulation (33), the low levels of leptin measured in CRFR1-/- mice would be expected to stimulate appetite. The lack of appetite stimulation by the low leptin levels in CRFR1-/- mice may have been due to increased leptin receptor sensitivity. Adrenalectomized mice were more sensitive to both endogenous (29) and exogenous (34) leptin, an effect reversed with tonic corticosterone replacement (29). Thus, the appetite-stimulating effects of reduced leptin levels in CRFR1-/- mice may have been counterbalanced by increased leptin receptor sensitivity.

The dose of corticosterone used throughout this study in drinking fluids, 10 mg/liter (CORT), was previously shown to restore the lights-on plasma corticosterone concentrations in CRFR1-/- mice to those measured in wild-type mice (25). In preliminary studies, we have found that the amount of corticosterone measured in 24 h urine samples in wild-type mice drinking VEH and CRFR1-/- drinking CORT is not different (Bradbury and Vale, unpublished results), suggesting that CORT replacement in CRFR1-/- mice was within a physiological range for mice feeding ad lib. Certainly this dose was not in excess; CORT water did not reduce thymus weights in CRFR1-/- mice beyond those measured in wild-type mice drinking VEH. It is of interest to note that after an overnight fast, both CORT-treated wild-type and CORT-treated CRFR1-/- mice ate more than VEH-treated mice during the first 1.5 h after food availability (Fig. 1Go). Corticosterone at low doses is known to stimulate the appetite (17), an effect that was observed in this study only after a period of food deprivation. The similarity of appetite stimulation in both genotypes of mice suggests that after 4 weeks of CORT treatment, corticosterone receptors in CRFR1-/- mice function without impairment and without supersensitivity. In accordance with (29), this observation also suggests a role for corticosterone on appetite that is independent from negative feedback inhibition of CRF expression and attendant suppression of CRF’s anorectic influences.

Effects of central Ucn administration on food intake and weight loss
The effect of CRFR1 deficiency on Ucn-induced hypophagia observed in the present study depended upon the interval after introduction of Ucn into the brain ventricles. During continuous central infusions, food intake was measured approximately 6 h after Ucn administered through osmotic minipumps began to enter the brain ventricles. This interval occurred during a circadian time with little food intake. Food intake measured 24 h later demonstrated a strong hypophagic effect of Ucn that was equivalent in wild-type and CRFR1-/- mice, suggesting a lack of CRFR1 involvement in food intake during these times. In CRFR1-/- mice, this time point was accompanied by a reduction in CORT consumption. The lack of effect of CORT on ad lib food intake (Table 1Go) makes it unlikely that reduced corticosterone consumption was responsible for the transient decrease in food intake in Fig. 2AGo. Nonetheless, the possibility remains that variable corticosterone consumption in CRFR1-/- mice modulated food intake.

To better measure the onset of Ucn-induced hypophagia, we also monitored food intake after acute injection of Ucn through the third ventricular cannulae of food-deprived wild-type and CRFR1-/- mice. In wild-type mice, Ucn injection reduced food intake during the 0–1.5 and the 3–11 h intervals. By contrast, CRFR1-/- mice did not respond to Ucn 0–1.5 h after injection. CRFR1-/- and wild-type mice were equally responsive to Ucn during the 3–11 h interval. The stress of the acute injection procedure itself did not appear to have a large effect on food intake; food consumption in both Ucn-injected and CON-treated CRFR1-/- mice (Fig. 4Go) and all unhandled mice (Fig. 1Go) were similar. The interval during which CRFR1-/- mice began to respond to Ucn is not clear. All mice, including Ucn-treated CRFR1-/- mice, ate little during the 1.5–3 h interval. Control-treated mice of both genotypes and Ucn-treated CRFR1-/- mice may have been in a refractory phase during this interval, having consumed approximately 25% of previously observed daily food intake during the 0–1.5 h interval. Conversely, Ucn injection may have suppressed food intake at this time in CRFR1-/- mice. These data are reciprocal to those of Coste et al. (35), in which mice lacking CRFR2 had reduced food intake early after central Ucn injection, but were unresponsive to the hypophagic effects of Ucn several hours later. The early responsiveness to Ucn in wild-type mice is not likely due to changes in circulating corticosterone concentrations; a 40-fold increase in corticosterone after injection of ACTH in wild-type mice did not alter post food-deprivation food intake.

The rapid, apparently CRFR1-dependent, phase of hypophagia may relate to previously established CRFR1-mediated responses to stressful stimuli. Removal of the CRFR1 or administration of CRF receptor antagonists were shown previously to decrease behavioral and endocrine responses to stressors such as restraint and exposure novel environment (25, 36). Smagin et al. (8) have recently demonstrated that anorexia following restraint stress in rats was attenuated by central injection of the CRF receptor antagonist {alpha}-helical-CRF (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41). Although there is some preference of {alpha}-helical-CRF (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41) for CRFR2, the doses used in (8) were not sufficient to allow for discrimination of the receptor type required. Our results suggest that stress-induced anorexia may involve stimulation of CRFR1 during the first hours of the response.

It is currently not understood how CRFR1-dependent processes communicate with neural areas influencing food intake. One possible neural pathway involved in coordinating food intake may be between the amygdala and the hypothalamus. The amygdala coordinates behavioral and hormonal responses to stress (37). Within the amygdala, the central nucleus of the amygdala contains both CRF fibers (37) and CRFR1 mRNA (38). Injection of CRF receptor antagonists into the central nucleus of the amygdala or into the neighboring CRFR1-rich medial amygdala attenuates hormonal, cathecholaminergic and behavioral responses to stress (37). The central and medial nuclear divisions of the amygdala each project to the ventromedial and paraventricular nuclei of the hypothalamus (39, 40), areas involved in the regulation of appetite and energy metabolism (17, 18). Whether these regions of the amygdala contribute to stress-induced anorexia remains to be examined.

The persistence of Ucn-induced hypophagia in CRFR1-/- mice 3–11 h after acute injection and during the continuous 7-day infusion indicates that other CRF receptors mediate this appetite suppression. CRFR2 is well situated to influence food intake during exposures to stressors as well as to changes in food availability or changes in energy expenditure. The VMH is one of the densest neural areas containing CRFR2 mRNA (16). Neurons in the VMH are directly responsive to administration of glucose, insulin, and FFA (17). Results from lesion studies support the hypothesis that the VMH links the nutritive status of the organism to appropriate neural, hormonal and food intake responses (17). Although there is no direct evidence demonstrating a role of CRFR2 in these responses, CRFR2 mRNA expression is linked to food drive, increasing after leptin administration (41) and decreasing during food deprivation (42, 43). Furthermore, the expression of CRFR2 in the VMH is altered by restraint stress (44), indicating that this area and these receptors may help coordinate food intake behavior with adaptive responses to perceived homeostatic threats. Thus, stimulation of CRFR2 may be part of either the afferent sensing mechanism or an efferent response system that coordinates food intake through the VMH.

In addition to effects at the VMH, CRFR2 may modulate food intake through serotonergic pathways. Drugs that reduce serotonin reuptake (45) or that activate serotonin 2A/C receptors (46) suppress appetite in humans and rodents. Neurons in the dorsal and medial raphe, nuclei which produce serotonin, contain CRFR2 and are in close apposition with fibers demonstrating Ucn-like immunoreactivity (9, 15). Thus, it is possible that Ucn injection into the brain will also reduce appetite through the release of 5HT and subsequent activation of 5HT 2A/C receptors. Alternatively, Ucn administration may activate a subtype of CRF receptors involved in food intake that have not been identified.

Ucn administration in this study may have also reduced food intake as a consequence of delayed gastric emptying. Central injection of Ucn and CRF into rats reduces the rate at which food exits the stomach, presumably through actions on vagal and sacral parasympathetic nerve activity (47). Subsequent delays in gastric emptying may reduce food intake through increased stimulation of gastric stretch receptors (47). Although the pharmacology for these effects is not fully understood, in vivo pharmacological studies indicate that central CRFR2 is capable of delaying gastric emptying.

VEH and CORT consumption in these studies were reduced in parallel with food intake during both food restriction and during Ucn administration. This passive, voluntary hypodipsia during reduced food intake has been previously observed in mice (28). Ucn had been proposed to alter sodium appetite because the supraoptic nucleus, a nucleus involved in osmotic regulation, contains both Ucn-like immunoreactivity (15) and CRFR2 mRNA (16). Ucn administration in mice was predicted to mimic the increase sodium ingestion induced by CRF administration measured in rabbits (48). In this study, and in wild-type Balb/C mice (McBurnie and Denton, personal communication), however Ucn had little effect on sodium appetite early in the 7-day infusion. The lack of sodium appetite stimulation after Ucn administration in mice may indicate a species-specific neural circuitry in areas of the brain regulating fluid and sodium intake.

Weight loss was sustained in CRFR1-/- mice but not wild-type mice during the 7-day Ucn infusion, despite equivalent food intake in both genotypes. As opposed to food intake, which did not vary with predicted circulating corticosterone concentrations, maintenance of body weight during the Ucn infusion may have been modulated by the hypocorticoidism observed in CRFR1-/- mice. Central infusion of CRF receptor agonists increases several indices of sympathetic nervous system activity (4). In wild-type mice, this effect is likely moderated by an increase in adrenocortical activity. For example, sympathetic activation of BAT is stimulated by central CRF receptor ligands and inhibited by endogenous glucocorticoids (24). We have found that the weight of the thymus, an organ whose weight correlates negatively with circulating corticosterone concentrations (49), was reduced by 20% after a 6-day infusion of 0.1 µg Ucn/h into wild-type mice (Bradbury, unpublished results). By contrast, it is unlikely that CRFR1-/- mice in the present study were able to mount additional hypothalamic-pituitary-adrenal axis responses during Ucn infusion. Thus, in CRFR1-/- mice, Ucn-induced stimulation of the sympathetic nervous system likely proceeded without the usual abatement brought about by increased circulating concentrations of corticosterone.

In summary, we have found that ad lib and postdeprivation food intake was not altered by CRFR1 deficiency. Ucn-induced hypophagia was dependent upon CRFR1 in the first hours after food deprivation. Thereafter, and during ad lib conditions, other CRF receptors mediated Ucn effects on food intake. The altered time course of Ucn-induced hypophagia in food-deprived CRFR1 -/- mice complements the recently reported effects of resistance of CRFR2-deficient mice to the late effects of Ucn on appetite (35). Ucn infusion into the brain reduced weight in CRFR1 -/- mice, an effect that may have been masked in wild-type mice due to enhanced adrenocortical secretions. Together, these results support the notion CRFR1 is not involved in basal food intake. Stressors that increase CRF or Ucn release in the central nervous system may exact rapid decreases in food intake through CRFR1.


   Acknowledgments
 
We thank Dr. Jean Rivier and Ron Kaiser for generously providing us with urocortin and Dr. Chien Li for valiant assistance in the animal facility.


   Footnotes
 
1 This work was supported by the Adler Foundation (to M.J.B), the Kleberg Foundation (to K.-F.L., W.W.V), NIH, DK-26741 (to M.J.B, K.-F.L., W.W.V.), the Harold G. and Leila Y. Malher Charitable Fund (D.A.D.), and the Foundation for Research (to M.J.B, K.-F.L., W.W.V.). Back

2 FFR Senior Investigator. Back

Received December 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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