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Endocrinology Vol. 140, No. 1 310-317
Copyright © 1999 by The Endocrine Society

ARTICLES

Glucocorticoid Replacement, but not Corticotropin-Releasing Hormone Deficiency, Prevents Adrenalectomy-Induced Anorexia in Mice1

Lauren Jacobson

Division of Endocrinology, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Lauren Jacobson, Division of Endocrinology, Children’s Hospital, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: jacobson{at}a1.tch.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
There is considerable evidence that CRH can suppress food intake. As hypothalamic CRH, a main site of CRH expression, is also negatively regulated by glucocorticoids, it is unclear whether anorexia and weight loss in adrenal insufficiency are attributable to elevated CRH or to decreased glucocorticoid levels. To distinguish these possibilities, we have measured food intake and body weight in wild-type and CRH-deficient mice after sham adrenalectomy (Sham ADX) or adrenalectomy (ADX) with and without corticosterone (B) replacement. CRH deficiency neither increased basal food intake and body weight nor attenuated decreases in food intake after ADX or Sham ADX. B replacement producing plasma levels above the circadian peak completely blocked ADX-induced decreases in feeding and body weight in all mice and frequently stimulated food intake in CRH-deficient mice. Plasma levels of insulin and leptin, two other hormones involved in appetite regulation, did not differ between genotypes; however, the relationship between food intake and circulating leptin was significantly less negative at B doses that preserved appetite. B replacement levels slightly below circadian peak concentrations did not prevent hypophagia after ADX. We conclude that factors other than or in addition to CRH are more important in mediating appetite responses to adrenalectomy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE is considerable evidence indicating that CRH, the main regulator of pituitary-adrenocortical activity, is an endogenous inhibitor of food intake. Injection of CRH into the brain, specifically into the paraventricular hypothalamus (PVN), a major site of CRH expression, decreases spontaneous or fasting-induced feeding (1, 2). Central pharmacological blockade, immunoneutralization, or immunotoxin targeting of CRH can enhance basal and neuropeptide Y (NPY)-induced feeding (3, 4) or inhibit anorexia evoked by stresses that elevate PVN CRH expression (2, 5, 6). A role for CRH in clinical eating disorders has been suggested by findings of increased cerebrospinal fluid CRH immunoreactivity both in anorexia nervosa and in at least 50% of depressed patients, many of whom may exhibit appetite disturbances (7).

Appetite in humans and animals also parallels changes in adrenal steroid levels. Anorexia and weight loss are hallmarks of adrenal insufficiency in Addison’s disease, whereas increased appetite correlates with glucocorticoid overproduction in Cushing’s syndrome and with exogenous glucocorticoid administration in normal volunteers (8, 9). Adrenalectomy or glucocorticoid treatment, respectively, prevent or restore weight gain in both normal rodents and many models of genetic or experimentally induced obesity, often through effects on food intake (10, 11). As CRH is negatively regulated by glucocorticoids (12), it is not known whether these effects on appetite are directly related to glucocorticoid levels or to reciprocal changes in CRH.

Glucocorticoids may also act independently of CRH to increase appetite. Glucocorticoids increase food intake and weight gain in rats with lesions of the PVN that destroy CRH neurons (13, 14). Glucocorticoids induce the expression of other factors that may enhance food intake, including NPY, the type 1 NPY receptor, and melanin-concentrating hormone (15, 16, 17, 18, 19). To determine whether the effects of glucocorticoid on appetite are mediated by inverse variations in CRH, we measured food intake and weight gain after adrenalectomy with and without glucocorticoid replacement in wild-type (WT) and CRH-deficient [knockout (KO)] mice generated by homologous recombination (20).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
All experiments were approved by the Children’s Hospital animal care and use committee. Male WT and CRH KO mice of a mixed C57/BL6 x 129Sv background were 10–16 weeks old at the time of study. CRH KO mice were generated from matings either 1) between a female heterozygous and a male homozygous for the null allele, or 2) between two homozygous KO parents. Offspring of the former type of cross survive and develop normally without glucocorticoid replacement, whereas 10 µg/ml corticosterone (B) was supplied in the drinking water from embryonic day 12 to weaning to ensure adequate lung development and survival of pups from the latter cross (20). WT mice were generated by interbreeding WT offspring from matings of heterozygous parents. Although different generations of WT and CRH KO mice were used, food intake responses to adrenalectomy and B replacement were similar in CRH KO mice derived by either method and in WT mice studied in different experiments (compare Exp I and IIA).

All mice were housed individually on a 14-h light, 10-h dark cycle (lights on, 0700 h; lights off, 2100 h) and were acclimatized to eating powdered chow from Agway (Syracuse, NY) or from Harlan-Teklad (Madison, WI) from metabolic feeders. Adrenal surgery was performed after food intake had stabilized, usually within 4 days of single housing. Mice were housed either in metabolic cages (Maryland Plastics, Federalsburg, MD) or in standard plastic tubs containing bedding, with metabolic feeders containing food placed inside the cage. Food spillage or contamination under these conditions was negligible. Feeding patterns were similar for mice in metabolic vs. regular cages for a given diet (data not shown).

Adrenal surgery and B replacement
Mice were sham adrenalectomized (Sham ADX) or adrenalectomized (ADX) under 2.5% tribromoethanol anesthesia (21). ADX, B-replaced mice were implanted with a sc pellet at the time of surgery, using previously described techniques (22). In preliminary experiments using WT mice, we determined that a 40-mg pellet containing 10% B and 90% cholesterol provided approximately physiological replacement, in that it prevented increases in circadian nadir ACTH 5 days after adrenalectomy without causing thymic atrophy (data not shown). For Exp I and II, mice were replaced with 40-mg pellets containing 25% B and 75% cholesterol, representing approximately 2.5 times physiological replacement (ADX+25% B). In Exp III, mice were replaced with pellets containing 10% B and 90% cholesterol (ADX+10% B). All ADX mice were given 0.9% saline to drink after surgery, whereas Sham ADX mice received tap water.

Experiments
Exp I. Food intake was measured daily for 7 days after ADX, Sham ADX, or ADX+25% B replacement. Body weight was measured on the morning of adrenal surgery and every 2 days thereafter. Mice were given standard powdered rodent chow to eat (Agway, Syracuse, NY). This diet supplied 3.46 Cal/g and was composed of 22.5% protein, 52% carbohydrate (primarily starch), and 6% fat by weight (remainder as moisture, fiber, vitamins, and minerals). Mice were killed within 2 h of lights on, 7 days after adrenal surgery. CRH KO mice for this experiment were bred from heterozygous females and homozygous null males.

Exp II. Food intake was measured for 2 days after ADX, Sham ADX, or ADX+25% B pellet replacement. Body weight was measured on the morning of adrenal surgery. Mice were killed within 2 h of lights on of the second day for measurement of plasma B, insulin, and leptin. This experiment was performed twice, once (Exp IIA) replicating the conditions of Exp I and once (Exp IIB) using a refined diet from Harlan-Teklad because the powdered standard chow was not available. The refined diet provided 3.76 Cal/g and contained 20.4% protein (casein), 61% carbohydrate (43% sucrose and 18% corn starch), and 5.5% fat (corn oil) by weight (remainder as moisture, fiber, vitamins, and minerals). CRH KO mice in both repetitions of this experiment were offspring of homozygous CRH-/- parents and had been supplemented with B in the drinking water until weaning as described above.

Exp III. Food intake was measured for 2 days after ADX or ADX+10% B pellet replacement. Mice were otherwise treated as described in Exp II, but were given conventional chow (Agway). CRH KO mice in this experiment were offspring of homozygous CRH-/- parents and had been treated with B until weaning, as described above.

Data analysis
Food intake and body and thymus weight measurements. Food and body weights were measured to the nearest 0.01 and 0.05 g, respectively, using a Mettler balance (Toledo, OH). Food intake was calculated as the change in weight of the metabolic feeder plus food. Measurements of 24-h food intake were performed within 2 h of the same time each day and were normalized to initial body weight for each mouse. Thymus glands (Exp I) were collected as previously described (22). Thymus wet weight was measured to the nearest milligram and normalized to body weight measured on the afternoon before death.

Assays. Plasma B was measured by a commercial RIA kit (ICN, Costa Mesa, CA), using previously described modifications (23). Plasma insulin and leptin were measured using RIA kits from Linco (St. Louis, MO) for rat insulin and mouse leptin, respectively, with all reagents and samples at half-volume. This modification did not affect the performance of either assay, with interassay coefficients of variation being less than 10% for both assays.

Statistics. Hormone and organ data were analyzed by two-way ANOVA (Sigmastat, Jandel Corp., San Rafael, CA). Data that were not normally distributed were log transformed before analysis. Newman-Keuls multiple range post-hoc tests were performed when main effects or their interaction were significant. Food intake and body weight data were analyzed by three-way ANOVA (StatView, SAS Institute, Inc., Cary, NC), with post-hoc testing by t test with Bonferroni correction for multiple comparisons (24). Plasma hormone levels for the two repetitions of Exp II were analyzed by three-way ANOVA for diet, genotype, and treatment effects to determine whether data from Exp IIA and IIB could be pooled. As discussed below, there was a significant main effect of diet, so data from these two experiments are reported separately. Plasma insulin levels in mice from Exp IIA were analyzed by the nonparametric Mann-Whitney rank sum test with Bonferroni’s correction for multiple comparisons, because most values in ADX mice were below the limit of detection. Regression of food intake on plasma leptin was analyzed as a two-way factorial analysis of covariance (25) using SPSS for Windows (SPSS, Inc., Chicago, IL), with genotype and adrenal group defined as the two main factors, and plasma leptin as the continuous covariate. Regression slopes were compared by an interaction F test between the adrenal group factor and the covariate, plasma leptin, as previously described (26). Significance was defined as P < 0.05. Except where indicated, data are presented throughout as the mean ± SEM; where no error bars are evident in graphs, the SEM was smaller than the scale of the symbol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of adrenalectomy and glucocorticoid replacement on food intake and body weight over 7 days in WT and CRH KO mice
As previously observed (20), body weight did not differ significantly between male WT and CRH KO mice before surgery [WT, 28.80 ± 0.88 g (n = 12); KO, 30.00 ± 1.02 g (n = 15)]. Therefore, food intake data were normalized to initial body weight to control for individual differences in weight. Similar results were obtained when food intake data were analyzed without normalization. WT and KO mice exhibited similar 24-h food intake before adrenal surgery (Fig. 1Go, A–C). Anesthesia and surgery tended to decrease food intake, as indicated by the slight, but nonsignificant, decrease in Sham ADX mice on day 1; however, the magnitude of the decrease was similar in both genotypes (Fig. 1AGo). After adrenalectomy without B replacement (ADX), a greater and more prolonged decrease in food intake occurred that was comparable and even more prolonged in KO vs. WT mice (Fig. 1BGo). In contrast, food intake on day 1 was at least as high as presurgery levels in mice replaced with B at the time of adrenalectomy (ADX+25% B; Fig. 1CGo). In WT mice, B replacement prevented the decrease in feeding after adrenalectomy, and the ADX+25% B group ate significantly more on day 1 than did ADX or Sham ADX mice. In KO mice, B replacement not only maintained food intake at significantly higher levels than those in ADX (days 1–3) or Sham ADX (days 1–2) mice, but also significantly increased food intake on days 1–3 over presurgery levels. Food consumption on day 2 was also significantly greater in KO vs. WT ADX+25% B mice (Fig. 1CGo).



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  		Figure 1. Changes in food intake (A–C) and body weight (D–F) in male WT (closed symbols) and CRH KO mice (open symbols) from Exp I. Mice were Sham ADX (A and D, triangles), ADX (B and E, squares), or ADX+25% B (C and F, crosses), as described in Materials and Methods, and were studied for 7 days after surgery on day 0. Food intake and body weight are normalized to presurgery body weight, which did not differ between genotypes (see text). Basal levels of food intake (day 0) were measured over the 24 h before adrenal surgery. n = 3–7/group. Significant main effects on food intake by three-way ANOVA: adrenal group, time. Significant interactions: genotype x adrenal group; time x adrenal group. Significant main effect in change in body weight by three-way ANOVA: adrenal group. Significant interactions: genotype x adrenal group; time x adrenal group. Significant differences by post-hoc testing: #, P < 0.05 vs. time zero levels in the same genotype and adrenal group; *, P < 0.05 vs. ADX mice in the same genotype; {dagger}, P < 0.05 vs. ADX and Sham ADX mice in the same genotype; §, P < 0.05 vs. WT mice in the same adrenal group.

 
Changes in body weight paralleled those in food intake after adrenal surgery. Body weight did not change after sham adrenalectomy (Fig. 1DGo), but the more sustained decreases in food intake after adrenalectomy were associated with a decrease in body weight in both genotypes that was more prolonged in KO mice (Fig. 1EGo). This decrease in body weight did not occur when adrenalectomy was combined with 25% B pellet replacement, although WT ADX+25% B mice tended to lose weight by day 6 (Fig. 1FGo). Consistent with the more marked and prolonged changes in food intake in response to adrenalectomy with or without 25% B replacement, ADX+25% B KO mice exhibited significantly higher body weight relative to ADX KO mice on day 2 and relative to both ADX and Sham ADX KO mice on day 4 (Fig. 1FGo).

Morning plasma B 7 days after adrenal surgery did not differ statistically between genotypes in a given adrenal group (Table 1Go). B replacement in the ADX+25% B group was associated with significant thymic atrophy in both genotypes (Table 1Go).


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  		Table 1. Plasma corticosterone and normalized thymus weight in male WT and CRH KO mice from Exp I

 
Plasma hormones associated with changes in feeding 2 days after adrenalectomy and glucocorticoid replacement
To determine whether genotype-associated differences in peripheral hormones might have accounted for changes in feeding after adrenal surgery, mice were monitored for food intake and were killed for measurement of plasma B, insulin, and leptin 2 days after adrenalectomy, sham adrenalectomy, or adrenalectomy with 25% B pellet replacement. This time was estimated from Exp I to be the point of maximal differences in food intake between adrenal groups. As in Exp I, presurgery body weight did not differ between WT and CRH KO mice [WT, 28.52 ± 0.91 g (n = 14); KO, 27.87± 0.67 g (n = 15)]. In mice given standard rodent chow (Exp IIA), food intake closely matched the profile seen in Exp I. Presurgery food intake was not significantly different between genotypes (Fig. 2Go). As in Exp I, changes in food intake associated with ADX or Sham ADX were comparable between genotypes, although KO mice exhibited significant decreases in food intake 1 day after Sham ADX that were not evident in WT mice. However, by day 2, food intake was significantly lower in ADX vs. Sham ADX mice of both genotypes. As in Exp I, 25% B pellet replacement not only completely prevented decreases in food intake associated with ADX or Sham ADX in both WT and KO mice, but KO ADX+25% B mice given standard rodent chow consumed significantly more food by day 2 than did their WT counterparts (Fig. 2Go).



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  		Figure 2. Normalized food intake in male WT (left) and CRH KO (right) mice from Exp IIA. Mice given free access to conventional chow were studied for 2 days after sham adrenalectomy (triangles), adrenalectomy (squares), or adrenalectomy with 25% B pellet replacement (crosses; n = 4–6/group). Significant main effects by three-way ANOVA: adrenal group, time. Significant interactions: genotype x adrenal group; time x adrenal group. Significant differences by post-hoc testing: #, P < 0.05 vs. time zero levels in the same genotype and adrenal group; *, P < 0.05 vs. ADX mice in the same genotype; {dagger}, P < 0.05 vs. ADX and Sham ADX mice in the same genotype; §, P < 0.05 vs. WT mice in the same adrenal group.

 
Plasma hormones were similar between genotypes for each adrenal group in Exp IIA and are presented at the top of Table 2Go. Morning plasma B decreased with adrenalectomy and increased to about 27 µg/dl in the ADX+25% B group, but did not differ significantly between genotypes for a given adrenal group. Plasma B in ADX+25% B mice was higher at 2 days than that in Exp I (Table 1Go), reflecting the spike in B occurring soon after pellet implantation (27). Plasma insulin tended, although not significantly, to be lower in Sham ADX KO vs. WT mice and lower in ADX vs. Sham ADX mice of both genotypes. B replacement significantly increased insulin levels relative to those in ADX in both genotypes and relative to those in Sham ADX in KO mice. Leptin levels were similar between genotypes in each adrenal group, although in this experiment only, ADX and Sham ADX KO mice had significantly different plasma leptin levels (Table 2Go, top).


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  		Table 2. Plasma corticosterone, insulin, and leptin levels in mice from Exp II

 
In an experiment that replicated all adrenal surgery groups but used a refined rather than a standard rodent diet (Exp IIB), overall feeding patterns were comparable to those in previous experiments. Although KO mice tended to have lower initial food intake, this difference was not significant. CRH deficiency also did not significantly affect either initial body weight [WT, 30.39 ± 0.96 g (n = 15); KO, 31.20 ± 0.48 g (n = 15)] or the decreases in food intake after ADX or Sham ADX (Fig. 3Go). In this experiment, both genotypes displayed significant decreases in food intake for 2 days after Sham ADX; however, food intake was still significantly greater in Sham ADX than in ADX mice, and feeding responses were comparable between genotypes. Replacing ADX mice with a 25% B pellet still maintained food intake at presurgery levels in both genotypes. However, food consumption in ADX+25% B KO mice did not significantly exceed that in WT mice when the refined diet was used (Fig. 3Go). Although there were no significant differences between repetitions of Exp II in plasma insulin and leptin levels for Sham ADX or ADX mice or in plasma B levels for ADX+25% B mice, plasma insulin and leptin were significantly higher in ADX+25% B mice fed the refined vs. the standard diet. Nevertheless, there were no differences in either plasma insulin or plasma leptin between WT and KO mice in a given adrenal group from Exp IIB (Table 2Go, bottom).



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  		Figure 3. Normalized food intake in male WT (left) and CRH KO (right) mice from Exp IIB. Mice were treated as described in Fig. 2Go, but were given a refined diet (see Materials and Methods; n = 4–6/group). Significant main effects by three-way ANOVA: genotype, adrenal group, time. Significant interaction: adrenal group x time. Significant differences by post-hoc testing: #, P < 0.05 vs. time zero levels in the same genotype and adrenal group; *, P < 0.05 vs. ADX mice in the same genotype; {dagger}, P < 0.05 vs. ADX and Sham ADX mice in the same genotype.

 
To determine whether the effects of B on feeding could have been influenced by circulating leptin or insulin, we regressed cumulative postsurgical food intake on plasma hormone levels for individual ADX and ADX+25% B mice. There were no significant correlations between food intake and either plasma insulin or plasma B concentrations (data not shown). However, there was a significant negative linear correlation between cumulative postsurgery food intake and plasma leptin 2 days after adrenalectomy in mice fed standard chow from Exp IIA (Fig. 4Go). Furthermore, the negative relation between food intake and plasma leptin was significantly steeper for ADX than for ADX+25% B mice. Although there was a tendency for KO regression lines to be right-shifted relative to those of WT mice, this trend was not significant (Fig. 4Go). Qualitatively similar results were obtained when cumulative intake was analyzed against plasma leptin for mice fed the refined diet in Exp IIB and when 24 h food intake for day 2 alone was regressed on leptin in either repetition of Exp II (data not shown).



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  		Figure 4. Linear regression of postsurgery normalized food intake vs. plasma leptin in ADX (squares) and ADX+25% B (crosses) WT and KO mice from Exp IIA. Data are compiled from plasma leptin values on day 2 (Table 2Go) and cumulative normalized food intake after surgery for the individual mice in Fig. 2Go. Equations and coefficients of correlation (r) and determination (r2) are given below. There was a significant difference between regression slopes of ADX and ADX+25% B mice, as described in Materials and Methods and Results. WT mice: ADX, y = -5.052x + 31.691; r = 0.97; r2 = 0.941; ADX+25% B, y = -1.003x + 36.114; r = 0.785; r2 = 0.617; KO mice: ADX, y = -2.496x + 34.608; r = 0.639; r2 = 0.409; ADX+25% B, y = -0.723x + 40.911; r = 0.656; r2 = 0.430.

 
Effect of lower glucocorticoid replacement on adrenalectomy-induced decreases in food intake
To test whether lower B concentrations were sufficient to maintain food intake after adrenalectomy, we compared consumption of standard rodent chow between WT and KO mice that had or had not been replaced with a 10% B pellet at the time of adrenalectomy (ADX+10% B). At 2 days after adrenalectomy and 10% B pellet implantation, ADX+10% B mice had plasma B levels that were similar between genotypes and approximately 40% of those in the previous experiment (WT, 11.6 ± 2.2 µg/dl; KO, 12.3 ± 1.6 µg/dl; n = 3–4/group). WT ADX+10% B mice exhibited decreases in food intake on day 1 after surgery that were statistically indistinguishable from those in ADX mice (Fig. 5Go). Although food consumption on day 1 tended to be slightly higher in ADX+10% B vs. ADX KO mice, this trend was not significant relative to levels in either KO ADX or WT ADX+10% B mice (Fig. 5Go). By day 2, ADX, but not ADX+10% B, mice exhibited significant depressions in food consumption relative to presurgery levels (Fig. 5Go).



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  		Figure 5. Food intake, normalized to initial body weight, in male WT (filled symbols) and CRH KO (open symbols) mice from Exp III. Consumption of conventional chow was measured before and 2 days after adrenalectomy with (circles) or without (squares) 10% B pellet replacement. n = 3–4/group. Significant main effects by three-way ANOVA: genotype, adrenal group, time. Significant interaction: adrenal group x time. Significant differences by post-hoc testing: #, P < 0.05 vs. time zero levels in the same genotype and adrenal group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that genetic CRH deficiency neither augments basal food intake nor attenuates decreases in feeding after adrenal surgery. Furthermore, we have found that restoration of food intake in ADX mice by glucocorticoids does not depend on inhibition of CRH. These results suggest that at least in mice, factors other than or in addition to CRH are more important for controlling basal and glucocorticoid-associated effects on food intake.

CRH-deficient mice did not exhibit increased body weight or food intake under basal conditions, nor did they display smaller decreases in feeding after adrenalectomy and sham adrenalectomy. These data seemingly disagree with the large body of clinical and animal data linking CRH with anorexia (2, 3, 4, 5, 6, 7, 8). We cannot exclude the possibility that peripheral or central factors have compensated for developmental CRH deficiency to control feeding in the KO mouse. Nevertheless, the close similarity of the graded decreases in food intake between WT and KO after both sham surgery and adrenalectomy indicates that such compensation, if occurring, is quite precise.

Adrenalectomy-induced decreases in appetite could be mediated by CRH-related peptides, whose effects might be mimicked by central administration of CRH and blocked by CRH antagonists or antisera. For example, urocortin displays 45% amino acid identity with CRH, has similar or higher affinity for known CRH-binding sites, and can suppress food intake after intracerebroventricular administration (28, 29). However, there is currently no strong evidence that hypothalamic urocortin changes with adrenalectomy (30), and it remains to be determined whether endogenous urocortin is actually a physiological mediator of anorexia.

Unlike humans and rats, in which chronic adrenal insufficiency is associated with sustained decreases in appetite (8, 10), food consumption in mice returned to presurgery levels within 6 days of adrenalectomy. Hypophagia after adrenalectomy was not solely an effect of surgery, as decreases in food consumption after sham adrenalectomy tended to be smaller and shorter in both genotypes. Restoration of food consumption was not due to incomplete adrenalectomy, as plasma B levels in both genotypes were low and unresponsive to increases in ACTH at 7 days postadrenalectomy (Jacobson, L., unpublished observations). The transience of the adrenalectomy-induced depression in feeding may relate to the species or strain used; other researchers also found no sustained effects of adrenalectomy on food intake or body weight in the C57/BL6 mouse strain, one of the two strains in the genetic background of the mice used in this study (31).

Although adrenalectomy removes multiple hormonal and neural factors, replacement of only B was sufficient to preserve food intake after adrenalectomy. Regardless of variations in feeding responses of KO ADX+25% B mice, this level of B replacement consistently prevented adrenalectomy-induced hypophagia in both genotypes. Thus, some appetite-stimulating effects of glucocorticoids do not require inhibition of CRH. These results are consistent with the ability of glucocorticoids to enhance food intake and weight gain in rats with PVN lesions (13, 14).

In the present study, glucocorticoid levels required to maintain food intake after adrenalectomy were relatively high, as indicated by thymic atrophy in the ADX+25% B group at 7 days. Catabolic effects of high glucocorticoid levels were also suggested by weight loss in WT ADX+25% B mice by day 6, an effect that was less evident in KO mice, possibly due to their greater food intake. Replacement with lower B levels, producing plasma concentrations 2 days after surgery slightly below those observed at the normal circadian peak (23), did not prevent adrenalectomy-induced decreases in food intake. Although plasma B levels 2 days after 10% B pellet implantation are elevated relative to the constant levels of 5–6 µg/dl reached by 5 days (our unpublished observations), our findings agree with the observed requirement for B levels in the 30 µg/dl range to stimulate food intake in lean mice (31) and are consistent with the dependence of peak feeding in rats on occupancy of type 2 (glucocorticoid) receptors by maximal circadian levels of B (32).

Interestingly, despite their low circulating B levels and peripheral evidence of adrenal insufficiency (20, 23), CRH KO mice exhibit normal basal food intake and decrease food intake as much as WT mice after adrenalectomy. These observations suggest that as a consequence of chronic glucocorticoid deficiency, KO mice are more sensitive to lower B levels. Such increased sensitivity could account for the transient stimulation of food intake frequently observed in KO mice replaced with 25% B pellets, and for the trend toward attenuated hypophagia in ADX+10% B KO mice. In agreement with this interpretation, long term adrenalectomized wild-type mice increase food intake after acute B treatment (33).

It is also possible, in accord with evidence for type I B receptor-mediated stimulation of feeding (10, 32), that low glucocorticoid levels contribute to maintaining food intake under normal conditions, and that decreased food intake after adrenalectomy in WT and KO mice partly reflects the loss of these low levels. The inability of the 10% pellet replacement to prevent this decrease fully may be due to the combined effects of glucocorticoid removal and surgery stress, the latter being suggested by the slight, but often significant, decreases in feeding after sham adrenalectomy. Nevertheless, although food intake did not differ statistically between ADX and ADX+10% B mice at any time, the 10% B pellet replacement did appear to shorten the duration of adrenalectomy-induced anorexia, as by 2 days after surgery, food intake was similar to presurgery levels in ADX+10% B mice but was still significantly reduced in ADX mice. Removing and replacing B without concurrent surgery should reveal whether lower B levels influence food intake.

The selective augmentation of feeding in KO mice after B replacement is unlikely to be due solely to CRH deficiency, as KO mice did not increase intake of the refined diet after ADX+25% B replacement. Further stimulation of feeding in this group may have been prevented by the higher circulating levels of both insulin and leptin (Table 2Go), either of which can inhibit food intake (34). Although the current data do not exclude the possibility that CRH influences glucocorticoid-induced feeding, these results suggest that dietary, metabolic, or other hormonal factors are more important than CRH in regulating appetite.

Correlation of food intake with plasma leptin revealed that the negative relationship between food intake and circulating leptin was significantly steeper in ADX mice of either genotype. The difference in regression slopes between ADX and ADX+25% B mice is probably not due solely to glucocorticoid-dependent differences in the induction of leptin by postsurgery food intake, because the latter relationship should have a positive slope. These results are highly suggestive that glucocorticoids reduce sensitivity to leptin and may resolve the conundrum of how glucocorticoids stimulate appetite and leptin simultaneously in human subjects (35).

Although the slope of the regression of food intake on leptin tended to be less negative in ADX KO vs. ADX WT mice, suggesting a lower sensitivity to leptin in the absence of CRH, this trend was not significant. Although it is possible that this trend would reach significance if more mice were analyzed, the effect of glucocorticoid replacement on the relationship of feeding to leptin levels was robust even with relatively few animals, suggesting that glucocorticoid levels are a stronger factor regulating food intake under these circumstances. In addition, although leptin has been reported to stimulate CRH (36, 37), the significant negative relationship between food intake and plasma leptin in adrenalectomized KO mice implies that at least some of the inhibitory effects of leptin on appetite can occur independently of CRH.

If glucocorticoids regulate leptin signaling, they are likely to do so at the level of the brain. Although it is conceivable that glucocorticoids could regulate the proportion of free, presumably active, leptin (38, 39) in the periphery, there is evidence that these steroids affect central actions of leptin. Peripherally administered glucocorticoids have been shown to prevent the inhibition of food intake due to intracerebroventricular leptin administration (40). This interference with leptin signaling probably involves factors downstream or separate from leptin binding, as glucocorticoids can restore food intake and obesity in rodents genetically deficient in leptin or its receptor (11). NPY, which is induced by glucocorticoids and partially accounts for the hyperphagia and obesity of leptin-deficient (ob/ob) mice (15, 16, 41), could potentially mediate the effects of glucocorticoid on appetite. Other hypothetical mediators include central melanocortin-4 receptor pathways, defects in which can induce obesity independently of leptin, and the recently described orexin and CART systems (18, 42, 43, 44).

In summary, our results demonstrate that glucocorticoids can influence food intake by CRH-independent mechanisms, possibly in part by altering sensitivity to leptin. There are inherent caveats about the potential effects of species, strain, and developmental compensation in conventional gene KO models. However, our findings in the CRH KO mouse suggest that other factors are more important than CRH in mediating anorexia after the surgical stress of sham adrenalectomy and the composite stimulus of adrenalectomy, as well as in regulating the orexigenic effects of glucocorticoids. Elucidation of the neural mechanisms by which glucocorticoids maintain food intake should provide important tools for the clinical management of appetite disorders and obesity.


   Acknowledgments
 
The author is indebted to Drs. Joseph Majzoub and Louis Muglia for their generous input into this manuscript, including the provision of CRH WT and KO mice, and to Dr. David Zurakowski for gracious assistance with statistical analysis. The technical assistance of Jennifer Lee and Jassy J. Upender is gratefully acknowledged.


   Footnotes
 
1 Portions of this work were presented at the 10th International Congress of Endocrinology, San Francisco, California, June 12–13, 1996. This work was supported in part by grants to the author from the NIH (DK-49333) and the National Alliance for Research on Schizophrenia and Depression. Back

Received April 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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