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Endocrinology Vol. 140, No. 8 3543-3551
Copyright © 1999 by The Endocrine Society

ARTICLES

Lower Weight Loss and Food Intake in Protein-Deprived, Corticotropin Releasing Hormone-Deficient Mice Correlate with Glucocorticoid Insufficiency1

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, Department of Pharmacology and Neuroscience, Mail Code 136, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To determine if CRH and glucocorticoids are respectively required for hypophagia and catabolism in malnutrition, we have subjected wild-type (WT) and CRH knockout (KO) mice to dietary protein deprivation. Compared with WT mice, CRH KO mice exhibited greater decreases in food intake and negligible change in plasma corticosterone after 7 days of protein-free diet. Restricting consumption of normal or protein-free diet for 9 days to the lower intake in protein-deprived CRH KO mice increased evening plasma corticosterone in WT but not KO mice. Restricted intake of protein-free diet increased morning corticosterone more in both genotypes than restricted intake of normal diet, although corticosterone levels were much lower in CRH KO mice. CRH deficiency attenuated body and thymus weight loss induced by restricted diets. Lower weight loss in CRH KO mice was associated with lower fractional loss of body water and protein. The remaining catabolic response in CRH KO mice did not correlate with morning plasma catecholamines or insulin. Corticosterone, but not the progestational appetite stimulant megestrol acetate, prevented hypophagia in CRH KO mice given protein-free diet. We conclude that differences in feeding and metabolic responses to protein deprivation between WT and CRH KO mice are primarily attributable to glucocorticoid insufficiency.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MALNUTRITION continues to be a prevalent and powerful predictor of morbidity and mortality in chronic disease (1). Understanding the physiological effects of malnutrition may aid in minimizing its adverse consequences. Increased plasma glucocorticoids are frequently observed in malnutrition, and we have shown that protein deficiency, in the absence of disease, specifically increases drive for ACTH as well as glucocorticoid production in rats (2). Because elevated glucocorticoids can cause catabolism (3), it is of interest to determine whether malnutrition-induced increases in glucocorticoids are necessary for the mobilization of body nutrient stores elicited by this nutritional stress.

We have also found that induction of protein malnutrition by providing protein-deficient food is associated with reduced food intake in rodents (2). In light of the drive for ACTH secretion induced by dietary protein deprivation, this hypophagia might be mediated by CRH. CRH is not only a major regulator of hypothalamic-pituitary-adrenocortical axis activity, but it has been suggested to function as an endogenous inhibitor of food intake (4). In support of this possibility, intracerebroventricular administration of the {alpha}-helical9–41 CRH antagonist increases food intake in rats given protein-free diet (5). CRH might therefore provide a link between malnutrition and anorexia in chronic disease states such as AIDS or cancer cachexia, in which malnutrition and impaired appetite are frequently associated (1).

To address the hypothesis that CRH is necessary for both the hypophagia and glucocorticoid-induced catabolism observed in malnutrition, we have compared effects of protein and calorie restriction in WT and CRH-deficient (knockout; CRH KO) mice. The CRH KO mouse, generated at the Division of Endocrinology, Children’s Hospital, exhibits normal viability and longevity but has severely impaired adrenocortical responses to circadian and stressful stimuli (6, 7). Although adrenomedullary epinephrine production may also be reduced secondary to glucocorticoid insufficiency, this deficit is less severe than that of glucocorticoid secretion, and mineralocorticoid levels appear normal (6, 8). In minimizing deficiencies in other major adrenal or pituitary hormones, the CRH KO model affords advantages over adrenalectomy or hypophysectomy for evaluating physiological effects of glucocorticoid secretion. We have therefore compared food intake, plasma hormones, body composition, and thymus weight in WT and CRH KO mice to assess the impact of CRH and glucocorticoid deficiency on metabolic and behavioral responses to protein deprivation.


   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 and female wild-type (WT) and CRH-deficient (knockout, KO) mice of a mixed C57BL/6 x 129 Sv background were generated in our colony as previously described (9). Mice were 3–8 months old at the time of use but were matched between genotypes to within 2 months of age and 10 g of body weight for each experiment. Diet group assignments were also designed to minimize variations in weight between groups. All mice were housed individually in conventional Plexiglas tub cages on a 12-h light, 12-h dark cycle (lights on at 0700 h). All mice were given several days to adapt to eating a nutritionally complete, refined diet (Harlan-Teklad, Madison, WI). The diet supplied 21% of calories as protein, 67% as carbohydrate, and 13% as fat, and has been previously described (2). The diet was either provided in powdered form in a metabolic feeder placed inside the cage, or in a pelleted form placed in the food holder of the cage. We have consistently found that spillage is minimal and that accurate food intake measurements can be obtained with either approach (2).

Experiments
Experiments 1–3 were performed in separate sets of mice. Following adaptation to the normal form of the experimental diet, mice were either allowed to continue eating this diet (21% protein), switched to an equicaloric, protein-free form of the diet supplying carbohydrate in place of protein (0% protein), or given a restricted amount of normal diet (pair-fed) matched to the intake of a paired mouse in the 0% protein group. Mice were weighed to the nearest 0.05 g before assignment to diet groups and on the day before the mice were killed. Food intake was weighed to the nearest 0.01 g and provided within 2 h of lights-off to avoid disturbing the circadian rhythms of pair-fed mice (10). Food intake measurements were normalized to initial, pre-diet body weight for each mouse; similar results were obtained from analysis of raw data (not shown). Blood samples were collected into heparinized capillary tubes by retro-orbital puncture within 30 sec of cage opening. All mice were killed by decapitation in the morning within 4 h of lights-on.

Exp 1
To evaluate feeding, hormone, and metabolic responses to dietary protein deprivation, male or female WT and CRH KO mice were given free access to normal (21%) protein or protein-free diets for 7 days. In the female mouse experiment, additional mice of each genotype were concurrently pair-fed according to intake by a matched mouse in the 0% protein group.

Exp 2
Exp 2 was performed to control for lower consumption of the protein-free diet by CRH KO mice, and to study the effects of protein deprivation for a slightly longer period of time (9 days). For the first day of experimental dietary regimens, mice in the 0% protein group were allowed to feed freely, and pair-fed mice were given a restricted amount of food estimated from Exp 1 to match this consumption. The voluntary intake measured in CRH KO mice on day 1 was used to determine the rations of mice in the 0% protein (restricted, 0% protein) and pair fed groups (restricted, 21% protein) for the rest of the experiment. This level was verified to be the minimum restriction necessary to equalize caloric intake between WT and KO mice by allowing a subset of WT and CRH KO mice in the 0% protein group to feed freely again on day 5. Rations of all restricted mice were adjusted to equalize total intake across both restricted diet groups. Blood samples were collected for circadian peak and trough corticosterone measurements within 1 h of lights-off on day 7 and when mice were killed on the morning of day 9. Thymus glands were collected on saline-moistened filter paper and weighed; decapitated carcasses were frozen for body composition analysis.

Exp 3
Food intake was measured in WT and CRH KO mice for 2 days before and 5 days after changing to a protein-free diet. To evaluate the ability of specific steroids to reverse diet-induced hypophagia, mice were injected 1 h before lights-out with either dimethylsulfoxide (DMSO) vehicle, 50 mg/kg corticosterone, or megestrol acetate, sc, in a volume of 2 µl/g. These doses were given for 4 days, beginning 1 day after introduction of the protein-free diet.

Assays and statistics
Plasma corticosterone (ICN Pharmaceuticals, Inc., Costa Mesa, CA) and insulin (Linco Research, Inc., St. Louis, MO) were assayed using commercial kits as previously described, using all reagents at half-volume (9). Body composition analysis was performed as previously described (11) after drying carcasses to constant weight at 65 C. Body water was calculated as the percent difference between wet and dry carcass weight. After drying, carcasses were subjected to alcoholic potassium hydroxide hydrolysis and saponification at 60 C. Protein content was assayed using Biuret reagent with bovine BSA as a standard. Triglyceride content was assayed by enzymatic conversion to glycerol using a commercial kit (GPO-Trinder, Sigma Chemical Co.). Catecholamines were measured in alumina-extracted plasma by liquid chromatography with electrochemical detection as previously described (12).

Data were analyzed by two-way ANOVA (SuperANOVA, Abacus Concepts, Berkeley, CA), with posthoc testing by t test with Bonferroni correction where main effects were significant. Typically, three comparisons were planned pre-hoc: (1, 2) between one diet group and the other diet groups in the same genotype and (3) between WT and CRH KO mice in the same diet group. Therefore, P values were multiplied by 3. Significance was defined at P < 0.05 after correction by multiplication by the number of posthoc comparisons. Data are shown throughout as mean ± SEM. Where no error bars are visible in figures, the scale of the error was smaller than that of the symbol.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1. Food intake and hormone responses to dietary protein deprivation in WT and CRH KO mice allowed to feed ad lib
In initial experiments, we assessed changes in food intake and plasma hormones of male WT and CRH KO mice over 7 days of normal vs. protein-free diet, to determine if mice displayed similar responses to those we had observed in rats (2). Similar to rats, male WT mice exhibited hypophagia in response to the protein-deficient diet (Fig. 1AGo). CRH KO mice also decreased food intake on the protein-free diet, although in contrast to all previous observations, in which basal food intake was similar between WT and CRH KO mice (9), this set of CRH KO mice ate significantly less of the normal diet compared with WT mice (Fig. 1AGo). Correlating with differences in normal diet consumption, WT mice given the 21% protein chow gained weight, whereas KO mice only maintained weight. However, although total caloric intake was even lower in protein-deprived CRH KO mice, this group did not lose more weight than WT mice in the same diet group (Fig. 1BGo). Plasma corticosterone was significantly increased in WT mice given free access to protein-free diet (Fig. 1CGo). Even though caloric intake was even lower in CRH KO mice, no significant increase in morning plasma corticosterone occurred in protein-deprived CRH KO mice (Fig. 1CGo). Consistent with previous observations (7), plasma insulin tended, although not significantly, to be higher in WT vs. CRH KO mice fed ad libitum (WT, 3.5 ±.1.5 ng/ml; KO, 1.0 ± 0.1 ng/ml; n = 4–5/group). Plasma insulin levels decreased significantly in WT mice subjected to dietary protein deprivation, although no further reduction occurred in the already low levels in CRH KO mice (WT, 0.2 ± 0.1 ng/ml; KO, 0.5 ± 0.4 ng/ml; n = 4–5/group).



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  		Figure 1. Cumulative food intake (A), change in body weight (B), and plasma corticosterone (C) in male wild-type (WT) and CRH KO (KO) mice that had free access to either normal (21% protein) or protein-free (0% protein) diet in Exp 1. Cumulative food intake and change in body weight were normalized to body weight for each mouse at the time of assignment to diet groups. n = 4–5 per group. *, P < 0.05 vs. 21% protein, same genotype. {dagger}, P < 0.05 vs. WT, same diet group.

 
To ensure that the lack of a significant glucocorticoid response to protein deprivation in CRH KO mice was not solely due to adrenal hyporesponsiveness, we performed similar experiments in female CRH KO mice, which exhibit less adrenocortical impairment (6). To control for reduced caloric intake in mice given the protein-free diet, additional mice were fed a reduced amount of normal diet equivalent to that eaten by a paired mouse of the same genotype in the 0% protein diet group. Basal food intake did not differ between female WT and CRH KO mice given the 21% protein diet (Fig. 2AGo). However, only CRH KO female mice significantly decreased their food intake when given protein-free diet. Despite this lower caloric intake, CRH KO mice tended, although not significantly, to lose less weight than WT mice in the same diet group (Fig. 2BGo). While there were no significant differences in daily (not shown) or cumulative food intake between diet groups in WT mice, protein-deprived and pair-fed mice exhibited significant elevations in morning plasma corticosterone relative to their freely feeding counterparts (Fig. 2CGo). In contrast to their ability to achieve plasma corticosterone levels of 12 µg/dl after some acute stimuli (6), female CRH KO mice did not exhibit detectable increases in corticosterone by 7 days of protein deprivation or food restriction, despite their significantly lower overall caloric intake (Fig. 2CGo).



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  		Figure 2. Cumulative food intake (A), change in body weight (B), and plasma corticosterone (C) in female WT and CRH KO mice from Exp 1 that had free access to either normal (21% protein) or protein-free (0% protein) diet, or were pair-fed to a mouse of the same genotype in the 0% protein group. Symbols and abbreviations are as in Fig. 1Go. n = 4–6 per group.

 
Exp 2. Hormone and body composition changes induced by dietary protein deprivation in WT and CRH KO mice restricted to equivalent caloric intake
To control for lower consumption of protein-free diet by CRH KO mice, and to investigate the effects of dietary manipulations over a slightly longer period, we restricted male WT and KO mice for 9 days to the caloric intake observed in CRH KO mice given protein-free food. In this experiment, ad libitum consumption of the normal, 21% protein diet was similar between WT and CRH KO mice. Both WT and CRH KO mice exhibited decreased food intake over the first 24 h of access to the 0% protein diet, although this decrease was only significant relative to the 21% diet group in CRH KO mice (day 1; Fig. 3Go, left). The average caloric intake measured in CRH KO mice was used to determine the rations of mice in the 0% protein (restricted, 0% protein) and pair-fed groups (restricted, 21% protein) for the rest of the experiment, and amounted to about 55% of normal diet intake before the diet change in all groups. To confirm that consumption of protein-free diet measured on day 1 was representative, we allowed one set of WT and CRH KO mice to feed ad libitum on protein-free chow after 4 days of restricted access to the this diet (day 5; Fig. 3Go, right). Previously restricted WT mice increased their consumption of the protein-free food to levels equivalent to those in controls eating the normal 21% protein diet. However, CRH KO mice did not exhibit similar compensatory increases, and consumption in this group remained significantly below that in both their respective normal diet controls and in protein-deprived WT mice (Fig. 3Go, right).



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  		Figure 3. Food intake, normalized to pre-diet body weight, in a subset of WT and CRH KO mice on days 1 (left) and 5 (right) of Exp 2. The left-hand panel depicts ad libitum food intake on the first day of 0% protein diet (open bars), with intake of mice given unrestricted access to normal diet shown for comparison (21% protein, filled bars). WT and KO mice in the 0% Protein group were then restricted for 4 days to the intake measured in CRH KO mice on day 1. The right-hand panel depicts food intake on day 5, when mice in the 0% protein group were again allowed free access to the protein-free diet. Symbols are as in Fig. 1Go. n = 4–5 per group. *, P < 0.05 vs. 21% Protein, ad libitum, same genotype. {dagger}, P < 0.05 vs. WT, same diet group.

 
Circadian peak and nadir plasma corticosterone levels were determined at 7 and 9 days, respectively, of experimental diets and are shown in Fig. 4Go. Plasma corticosterone showed a strong diurnal rhythm in all WT mice and was significantly elevated in calorically restricted WT mice at both times of day, regardless of diet composition. However, at the same relative degree of caloric restriction, plasma corticosterone was even higher in WT mice given the protein-free diet. This increase was significant relative to mice with restricted intake of the 21% protein diet in the morning, and tended, although not significantly, to be greater in the evening as well (Fig. 4Go). While plasma corticosterone levels were significantly lower relative to WT mice at all times, CRH KO mice in the 0% protein group did display a significant increase in morning plasma corticosterone compared with both their counterparts given either free or restricted access to 21% protein diet (Fig. 4Go, left).



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  		Figure 4. Morning (AM, left) and evening (PM, right) plasma corticosterone levels measured in mice from Exp 2. WT and CRH KO mice were either allowed free access to normal diet (21% protein, ad libitum) or were given a restricted amount of either normal (21% protein, restricted) or protein-free diet (0% protein, restricted) based on the consumption of protein-free diet by CRH KO mice on day 1 of experimental diets. n = 6–9 per group. *, P < 0.05 vs. 21% protein, ad libitum, same genotype. {dagger}, P < 0.05 vs. WT, same diet group. §, P < 0.05 vs. 21% protein, restricted, same genotype

 
Epinephrine and norepinephrine were also measured in plasma obtained at the time mice were killed to evaluate potential differences between WT and CRH KO mice in other counter-regulatory hormone responses to calorie and protein restriction. Plasma epinephrine was significantly elevated in WT mice after 9 days of restricted access to normal diet, relative to levels in both WT ad libitum fed controls and CRH KO mice in the restricted, 21% protein diet group. Plasma norepinephrine did not vary significantly between genotypes or diet groups (Table 1Go).


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  		Table 1. Morning plasma catecholamines after 9 days of experimental diets in mice from Exp 2

 
To assess the metabolic effects of the differential adrenal responses to protein and calorie restriction, we compared body weight, thymus weight, and carcass composition in WT and CRH KO mice from Exp 2. WT and CRH KO mice had similar body weights before dietary manipulation (WT, 27.64 ± 0.67 g; CRH KO, 26.72 ± 0.58 g, n = 22/group). Despite similar levels of food intake in this experiment, CRH KO mice given free access to the normal, 21% protein diet gained significantly less weight than did WT mice. However, CRH KO mice also lost significantly less weight than WT when subjected to the same degree of protein and/or calorie restriction (Fig. 5AGo). This difference was most marked in mice given reduced amounts of the 21% protein diet but was significant even in the 0% protein group (Fig. 5AGo). Thymus weight was significantly reduced relative to body weight in WT mice subjected to caloric restriction with complete or partial protein deprivation (Fig. 5BGo). As previously reported, thymus weight tended to be higher relative to body weight in CRH KO mice (6). Normalized thymus weight did not change in this genotype with dietary manipulations and was significantly higher in CRH KO vs. WT mice in the 0% or 21% protein restricted diet groups (Fig. 5BGo).



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  		Figure 5. Change in (A) body weight, and (B) the ratio of thymus weight to body weight, in male WT and CRH KO mice from Exp 2. Change in body weight is expressed as a percent of initial weight at the beginning of experimental diets, and thymus weight is expressed as fraction of final body weight, measured the day before mice were killed. Symbols and groups are as in Fig. 4Go.

 
To determine whether the lower diet-induced weight loss in CRH KO mice was associated with defense of specific body compartments, we performed carcass composition analysis on mice from Exp 2. Carcass dry weight was significantly lower in CRH KO vs. WT mice fed the 21% protein diet ad lib, correlating with the significantly lower triglyceride content in this genotype (Table 2Go). Carcass protein also tended, although not significantly, to be lower in CRH KO mice (Table 2Go), whereas percent body water tended, also not significantly, to be higher (not shown). To evaluate diet-induced changes while accounting for genotype-associated differences in basal body composition, we normalized data to the mean of the normal diet controls (21% protein, ad libitum) for each genotype. When compared with their respective normally nourished controls, WT mice on either restricted diet lost a significant amount of body water, whereas CRH KO mice showed little change in this variable. CRH KO mice also lost a significantly lower fraction of body water than did WT mice in either the 0% or 21% protein restricted diet groups (Fig. 6AGo). WT mice lost protein to a significant extent on both restricted diets (Fig. 6BGo). However, relative carcass protein depletion was significantly less in CRH KO vs. WT mice in each of these diet groups. Furthermore, unlike WT mice, protein loss in CRH KO mice on restricted intake of the 21% protein diet could not be statistically discriminated from that in genotype controls fed ad libitum (Fig. 6BGo). In contrast to their attenuated diet-induced changes in protein content, and in spite of their lower basal body triglyceride levels, CRH KO mice exhibited similar proportional, significant decreases relative to WT mice in carcass triglyceride content (Fig. 6CGo).


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  		Table 2. Carcass water, dry weight, protein, and triglyceride content in ad lib fed male WT and CRH KO mice from Exp 2

 


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  		Figure 6. Carcass water (A), protein (B), and triglyceride content (C) in male WT and CRH KO mice from Exp 2. Data are expressed as a percentage of the mean of the 21% protein, ad libitum group in the same genotype. Symbols and group sizes are as in Fig. 4Go.

 
Exp 3. Effect of glucocorticoid replacement on hypophagia induced by protein-free diet in WT and CRH KO mice
To determine if glucocorticoid deficiency might be implicated in the more pronounced hypophagic response to protein-free food in CRH KO mice, consumption of the 0% protein diet was measured in WT and CRH KO mice injected sc with either 50 mg/kg corticosterone or vehicle (DMSO), once per day, 1 h before lights-off. To compare the efficacy of glucocorticoid vs. progestational appetite stimulants in this model, additional mice were treated with 50 mg/kg megestrol acetate in DMSO. Both genotypes ate similar amounts of normal, 21% protein diet before being switched to the protein-free diet. Both genotypes also exhibited significant decreases in food intake on day 1 of the protein-deficient diet, before steroid treatment began (Fig. 7Go). In WT mice, consumption of the protein-free diet did not remain suppressed, but returned within 2 days to levels observed before the diet change. Neither steroid treatment significantly modified food intake in WT mice (Fig. 7Go, left). In CRH KO mice, consistent with their lack of compensatory increases in food intake after restricted access to protein-free diet, consumption of the 0% protein diet remained suppressed in vehicle-injected mice (Fig. 7Go, right). Corticosterone reversed this hypophagia within the first day of treatment, and maintained food consumption at levels equivalent both to those in WT mice and to intake of normal diet in KO mice before diet change. In contrast to corticosterone, a comparable dose of megestrol acetate was ineffective in increasing food intake in protein-deprived CRH KO mice (Fig. 7Go, right). Administering a single, higher dose of steroids (100 mg/kg) did not increase consumption of 0% protein diet in either genotype beyond that observed at the end of the initial treatment period (data not shown).



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  		Figure 7. Daily consumption of protein-free diet in male WT and CRH KO mice treated with DMSO vehicle, corticosterone (Cort), or megestrol acetate (MegAc). Mice eating normal diet were switched to equicaloric, protein-free diet on day 0 (arrow). Both genotypes exhibited significant decreases in consumption of protein-free food on d1 relative to their prior intake of normal diet, indicated by asterisks on d1. Steroid or vehicle treatment (shaded box) was given by sc injection once per day, 1 h before lights-out, beginning on day 1. Mice were treated with either 50 mg/kg corticosterone, 50 mg/kg megestrol acetate, or an equal volume of DMSO vehicle. Food intake measurements are normalized to d0 body weight. n = 3–4 per group. *, P < 0.05 vs. intake of 21% protein diet on day -1, same genotype. {dagger}, P < 0.05, CRH KO vs. WT in same treatment group. §, P < 0.05, vs. vehicle-treated mice in same genotype.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although protein-calorie malnutrition has long been known to increase plasma glucocorticoids (13, 14), the neuroendocrine mechanisms, as well as the metabolic consequences, of this increase remain unclear. Our current data support a requirement for CRH to induce glucocorticoid responses to the metabolic stress of reduced protein or caloric intake but also indicate that, of the two endocrine mediators, glucocorticoids are more important in facilitating catabolic and compensatory feeding responses to protein deprivation.

Exp 1. Effects of dietary protein deprivation in WT and CRH KO mice fed ad lib: the role of CRH in mediating decreased food intake
In WT and CRH KO mice allowed to feed freely, CRH deficiency prevented the adrenocortical activity but not the hypophagia associated with dietary protein deprivation. Comparable to previous observations during fasting (6), only WT mice increased plasma corticosterone in response to protein or food restriction. The role of CRH in mediating pituitary-adrenal responses to food restriction is unclear because CRH expression is typically decreased by food deprivation (15). Nevertheless, the negligible change in corticosterone evoked by dietary restriction in female CRH KO mice contrasts both with the marked adrenocortical responses in WT mice and with the 10 µg/dl increments in corticosterone in female CRH KO mice following other stimuli (6). The lack of comparable responses in female CRH KO mice in the present study argues for a active role of CRH in maintaining the elevated glucocorticoid levels during protein or food restriction.

The more severe hypophagia induced in CRH KO mice by protein-free diet was unexpected in light of evidence implicating CRH as a mediator of anorexia (16). CRH has been specifically implicated in the hypophagia of protein-deprived rodents by the ability of the {alpha}-helical9–41 CRH antagonist to increase intake of protein-free diet in rats (5). Although this pharmacological evidence and our previous studies (2) suggest that CRH could mediate both the hypophagia and the pituitary-adrenal activation induced by protein deprivation, our present data indicate the CRH dependence of food intake and endocrine responses to this stress differ dramatically. We have recently shown similar CRH independence for the hypophagia induced by adrenalectomy, a known stimulus to neuroendocrine CRH expression (9).

Exp 2. Effects of protein and calorie deprivation in WT and CRH KO mice restricted to equivalent intake: the role of CRH-induced glucocorticoid secretion in mediating catabolism
Comparison of endocrine and metabolic responses of WT and CRH KO mice limited to equivalent intake revealed several significant, although incomplete, deficiencies in the ability of CRH-deficient mice to respond to protein or calorie restriction. As expected from Exp 1, WT mice exhibited sustained increases in glucocorticoid production. Morning plasma corticosterone levels in the restricted, 21% protein WT group approximated the circadian maximum levels in their counterparts fed ad libitum The further increase in circadian nadir corticosterone levels in the restricted, 0% protein WT group corroborated the differential increases induced by protein deprivation vs. calorie restriction on glucocorticoid levels in rats (2). Lack of similar differences between protein-deprived and pair fed female mice in Exp 1 may have been due to gender differences or to the relative severity of caloric restriction. Despite the marked increase in morning corticosterone, which in normal rodents would render the adrenocortical axis refractory to most stimuli (17, 18), circadian peak corticosterone levels were instead further increased in undernourished WT mice, indicating an overall, prolonged increase in glucocorticoid production.

Consistent with the selective increase in morning plasma corticosterone induced by protein deprivation in WT mice and rats, CRH KO mice also exhibited slight but significant increases in morning but not evening plasma corticosterone after restricted intake of protein-free diet. This increase could be due to decreases in clearance associated with protein malnutrition (14) or to other factors such as TRH178–199, a TRH fragment with ACTH-inhibiting activity that would decrease in parallel with malnutrition-induced inhibition of its precursor gene (19, 20, 21). It is also conceivable that the CRH-related molecule, urocortin, accounts for adrenocortical activation in protein deprivation, although expression of this peptide in neurons affecting adrenocortical activity remains controversial (22, 23).

Sustained elevations in glucocorticoids in protein- and food-restricted WT mice were associated with more severe symptoms of immune suppression and catabolism. Lymphoid atrophy and immune suppression occur in protein-calorie malnutrition and are linked to increased glucocorticoid levels (14). Our findings that thymus weight, a classic indicator of glucocorticoid exposure (24), decreased with diet-induced changes in corticosterone levels only in WT mice corroborate a role for glucocorticoids in this deficit.

Elevated glucocorticoid production in diet-restricted WT mice was also associated with greater mobilization of body water and protein. Underscoring the caveats of using body weight as an index of catabolism, but supporting the requirement for glucocorticoids to facilitate free water clearance (25), differential weight loss between genotypes was largely attributable to water retention in CRH KO mice. The mechanisms for this effect are poorly understood but may involve inhibition of vasopressin or direct effects of glucocorticoids (25). Consistent with the catabolic effects of high glucocorticoid levels (3), WT mice exhibited greater proportional loss of body protein after complete or partial protein deprivation than did CRH KO mice.

Nevertheless, our data also indicate that considerable protein loss occurred in CRH KO mice without sustained increases in glucocorticoids. These results agree with our previous findings that carcass protein depletion induced by central administration of interleukin-1 is not ameliorated by keeping glucocorticoid levels low (26). While increased adrenomedullary activity can also contribute to catabolism (3, 14), and CRH KO mice, consistent with their adrenocortical insufficiency (8), had lower plasma epinephrine levels, plasma epinephrine did not correlate directly with catabolism in either WT or CRH KO mice. Protein loss in CRH KO mice might have been mediated by reductions in insulin, which we have consistently found to be low in protein-deprived or food-restricted rodents (Results, paragraph one, and data not shown), or to decreased activity of the GH-insulin-like growth factor axis (27).

In contrast to the changes in body protein, relative loss of body fat was similar in WT and CRH KO mice on restricted diets. Body fat content was significantly lower in male CRH KO mice, in agreement with their lower levels of insulin and glucocorticoids (7, 9). However, CRH KO mice were able to decrease their already low levels of body fat after protein or food restriction by 80%, a fractional reduction similar to that in WT mice.

It is still possible that the very low glucocorticoid production in CRH KO mice was sufficient to mediate the observed catabolic responses. We attempted to address this issue by including adrenalectomized mice of both genotypes in Exp 2. Although body composition of surviving adrenalectomized mice in the restricted diet groups closely resembled that in intact CRH KO mice, too few adrenalectomized mice survived at the restricted intake of protein-deprived, CRH KO mice to permit experimental and statistical comparison to the other diet groups (not shown). We did not feel that it was either feasible or humane to repeat this experiment. The survival of CRH KO but not ADX mice on restricted diets also reiterates, consistent with the normal longevity of CRH KO mice, that the impairment of adrenal function in this genotype is not physiologically equivalent to adrenalectomy.

Exp 3. Effect of glucocorticoid replacement on consumption of protein-free diet by WT and CRH KO mice: role of glucocorticoids in stimulating food intake
Consistent with genotype-associated differences in glucocorticoid-sensitive metabolic endpoints during dietary restriction, the decreased intake of protein-free diet in CRH KO mice could also be attributed to glucocorticoid insufficiency. Glucocorticoids rapidly normalized of caloric intake in CRH KO mice eating 0% protein diet, suggesting, as we have shown for adrenalectomy-induced hypophagia (9), that glucocorticoids are a more important determinant than CRH of food intake. We did not observe any effect of corticosterone treatment on consumption of the 0% protein diet in WT mice, possibly because the hypophagia was transient, and steroid treatment began after food intake had already normalized.

Although megestrol acetate and related synthetic progestins have been used clinically to stimulate appetite (28), megestrol acetate had no effect on intake of the protein-free diet in either CRH KO or WT mice. The dose of megestrol acetate was pharmacologically comparable to that of corticosterone and to therapeutic doses in humans (28), as well as three times the dose that increases feeding in rats (Jacobson, L., unpublished observations). Although the mechanisms underlying hypophagic responses of mice to protein-free diet may differ from those for human anorexia, the specific reversal of the former by glucocorticoids, along with evidence for glucocorticoid activity of megestrol acetate (29), argue that appetite effects of this progestin may be mediated via glucocorticoid receptors.

In summary, we have shown that the differential changes in food intake, thymus weight, and body composition exhibited by CRH-deficient mice during protein and/or calorie restriction are most consistently attributable to their tertiary glucocorticoid deficiency. Our findings underscore the need for CRH to mediate diet-induced increases in glucocorticoid secretion, but emphasize that glucocorticoids are more important in determining the feeding as well as metabolic responses to these nutritional stresses.


   Acknowledgments
 
We are indebted to Dr. Karel Pacak for performing plasma catecholamine measurements, and to Drs. Joseph Majzoub and Louis Muglia for providing WT and CRH knockout mice. The expert assistance of Jennifer Lee, Chris Lage, Allison Carrigan, and Joel Solano is gratefully acknowledged.


   Footnotes
 
1 Portions of this work were presented in abstract form at the 79th Annual Meeting of The Endocrine Society, June 11–14, 1997, Minneapolis, Minnesota. This work was supported by grants to LJ from NIH (DK-49333) and the National Association for Research on Schizophrenia and Depression. Back

Received November 25, 1998.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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