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Endocrinology Vol. 145, No. 7 3174-3181
Copyright © 2004 by The Endocrine Society

Impaired Leptin Expression and Abnormal Response to Fasting in Corticotropin-Releasing Hormone-Deficient Mice

Kyeong-Hoon Jeong, Satoru Sakihara, Eric P. Widmaier and Joseph A. Majzoub

Division of Endocrinology (K.-H.J., S.S., J.A.M.), Children’s Hospital, and Division of Endocrinology, Diabetes, and Hypertension (K.-H.J.), Brigham and Women’s Hospital, Harvard Medical School; and Department of Biology (E.P.W.), Boston University, Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Joseph A. Majzoub, Division of Endocrinology, Children’s Hospital, Harvard Medical School, Enders 416, 300 Longwood Avenue, Boston, Massachusetts 02115. E-mail: joseph.majzoub{at}tch.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leptin has been postulated to comprise part of an adipostat, whereby during states of excessive energy storage, elevated levels of the hormone prevent further weight gain by inhibiting appetite. A physiological role for leptin in this regard remains unclear because the presence of excessive food, and therefore the need to restrain overeating under natural conditions, is doubtful. We have previously shown that CRH-deficient (Crh–/–) mice have glucocorticoid insufficiency and lack the fasting-induced increase in glucocorticoid, a hormone important in stimulating leptin synthesis and secretion. We hypothesized that these mice might have low circulating leptin. Indeed, Crh–/– mice exhibited no diurnal variation of leptin, whereas normal littermates showed a clear rhythm, and their leptin levels were lower than their counterparts. A continuous peripheral CRH infusion to Crh–/– mice not only restored corticosterone levels, but it also increased leptin expression to normal. Surprisingly, 36 h of fasting elevated leptin levels in Crh–/– mice, rather than falling as in normal mice. This abnormal leptin change during fasting in Crh–/– mice was corrected by corticosterone replacement. Furthermore, Crh–/– mice lost less body weight during 24 h of fasting and ate less food during refeeding than normal littermates. Taken together, we conclude that glucocorticoid insufficiency in Crh–/– mice results in impaired leptin production as well as an abnormal increase in leptin during fasting, and propose that the fast-induced physiological reduction in leptin may play an important role to stimulate food intake during the recovery from fasting.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN, THE ADIPOCYTE product of Lep gene, is postulated to be part of an adipostat that signals satiety to the brain when energy (fat) stores are high (1, 2), thus decreasing food intake and increasing metabolism (3). Leptin has been shown to inhibit appetite: leptin administration in normal lean and in leptin-deficient Lepob/ob mice (4, 5) and leptin overexpression in transgenic skinny mice (6) reduce food intake.

It is questionable, however, how a mechanism to restrict food intake might have evolved in mammals because, under most natural conditions, the presence of either excessive fat stores or an overabundance of food is unlikely. Supporting this idea, captive baboons have higher body weight, fat mass, and leptin levels than those in the wild, where food is not as abundant (7), suggesting that the elevated leptin levels in the captive animals do not effectively control energy balance in the setting of food abundance. Similarly, as many as 50% of adult humans living in the United States are overweight, and half of these are clinically obese (8), despite their having elevated leptin levels (9), indicating that leptin is not an effective adipostat in many, if not most, humans presented with food abundance. The concept of leptin resistance attempts to reconcile these reports with a role for leptin as an adipostat (10). Chronic exposure of rodents to high-energy diets results in hyperphagia and obesity despite an elevation in serum leptin levels. This may be due in part to decreased signaling between the leptin receptor and nuclear targets, mediated by the suppressor-of-cytokine-signaling-3 (11). Nonetheless, these data suggest that hyperleptinemia caused by chronic excessive food intake does not effectively inhibit appetite or regulate body weight, and that low leptin status may have more physiological impacts in regulating appetite and food intake. Indeed, as a monitor of energy stores, leptin might be better suited to signal the more common state of energy deprivation, where in fact, reduced leptin levels function to inhibit reproduction and growth (12).

The activity of the hypothalamic-pituitary-adrenal (HPA) axis is linked to leptin regulation. The diurnal rhythms of plasma corticosterone and leptin are inversely related in mice (12) and in humans (13), implying a tight reciprocal relationship between these two hormones and a possible role of leptin in regulating the diurnal pattern of corticosterone (12). It is unclear, however, whether this relationship is the result of leptin affecting the HPA axis activity, or it is due to the HPA axis affecting leptin expression and secretion from adipocytes.

Several hormones have been shown to regulate leptin secretion and Lep gene expression. Glucocorticoid treatment increases Lep gene expression and leptin secretion in rodents (14, 15, 16, 17, 18) and in humans (18, 19, 20), whereas adrenergic input inhibits leptin production via ß3-adrenoceptors (15, 21, 22, 23, 24, 25, 26), demonstrating an opposite regulation of glucocorticoid and catecholamines for leptin.

CRH-deficient (Crh–/–) mice exhibit glucocorticoid insufficiency with minimum diurnal change and lack the fasting-induced increase in glucocorticoid (27, 28, 29), thus allowing a unique approach to investigate the regulation and role of leptin during starvation in a glucocorticoid-deficient state. We hypothesized that these mice may have low circulating leptin and impaired leptin responses to fasting. Here we show that leptin does not fall, but rather increases during fasting in Crh–/– mice, and that this is a consequence of glucocorticoid deficiency. Furthermore, the relative leptin excess in Crh–/– mice during fasting is associated with less food intake and weight gain upon refeeding, revealing a potentially important role for physiological leptin reduction in stimulation of appetite during the recovery from fasting.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal husbandry
Wild-type (WT) and Crh–/– littermates (27) were maintained on a 12-h light, 12-h dark cycle with lights on at 0700 h. The mice that were used for all studies were of a C57B6/129 genetic background maintained by breeding within the population. All mice were singly housed for 3–5 d before beginning experiments and fed ad libitum except during fasting. Drinking water was available at all times. All experiments were approved by the Children’s Hospital Animal Care and Use Committee.

CRH infusion and corticosterone implantation
Singly housed adult WT and Crh–/– male mice were subjected either to chronic CRH (human/rat CRH; Bachem, Torrance, CA) infusion using a previously reported method (30), or to corticosterone (Aldrich, Milwaukee, WI) implantation. Briefly, CRH was reconstituted in 1% acetic acid containing 1% BSA, and 0.1% L-ascorbic acid (vehicle), and loaded in osmotic minipumps (Alzet 1002, volume 100 µl, 14-d lifetime; Durect, Cupertino, CA), calculated to deliver at a rate of 1 µg/d. This procedure has been shown to restore the plasma corticosterone rhythm in Crh–/– female mice (30). Control mice were infused with vehicle. Corticosterone pellets were made as 50% (wt/wt) in cholesterol under mild heat. CRH-loaded osmotic minipumps or corticosterone pellets were sc implanted between the scapulae of the mice under anesthesia. Blood was withdrawn 5 d after CRH pump implantation in the evening (1700 h). Mice were subjected to blood collection one more time 9 d after implantation in the morning (0500 h), and epididymal fat pads were collected immediately after second sampling for RNA extraction (see below).

Food withdrawal
Singly housed adult WT and Crh–/– male mice were subjected to fasting for 36 h. All mice were placed in new cages without food at the beginning of the fasting period (1900 h) until blood sample collection (0700 h). In a second set of experiments, WT and Crh–/– male mice were initially subjected to fasting for 36 h, and blood samples were collected (0700 h). After 10 d of a recovery period, they were subjected to corticosterone pellet implantation. Food was withdrawn again 3 d after corticosterone implantation, and blood samples were collected after 36 h (0700 h). In a separate experiment, mice were fasted for 24 h (0700–0700 h), and food intake and body weight change were measured during refeeding.

Sample collection
Blood sampling was performed by retro-orbital sinus phlebotomy into heparinized capillary tubes without anesthesia. Phlebotomy during the lights-off period was performed under a red light to minimize any disturbance of circadian rhythmicity. When animals were subjected to repeated phlebotomy, blood was withdrawn from each animal at a frequency of 4–5 d. Otherwise, only one blood sample was collected from each mouse. Blood was collected on ice, and plasma was immediately separated from cells by centrifugation at 4 C, and stored at –80 C in aliquots until analyses. Decapitation was followed in vehicle- or CRH-infused mice immediately after phlebotomy to collect epididymal fat pads for Lep mRNA analysis. Epididymal fat pads were snap-frozen in liquid N2 and stored at –80 C until homogenization and RNA purification for Northern blot analysis.

Northern blot analysis
Amplification of Lep cDNA from epididymal fat pad total RNA was performed using RT-PCR. Synthesized first strand poly(A) cDNA products were amplified using murine Lep cDNA-specific primers (14). Amplified Lep cDNA fragment was subcloned into pBluescriptIISK vector (Stratagene, La Jolla, CA) and was used as a template for cRNA probe for Northern blot hybridization. Ten micrograms of total RNA from each sample was size-fractionated in a denaturing agarose gel, transferred to a nylon membrane (GeneScreen; NEN Life Science Products, Boston, MA), and hybridized with [32P]-labeled cRNA probe. The hybridized blot was exposed onto the PhosphorScreen (Molecular Dynamics, Sunnyvale, CA), scanned with the PhosphorImager (Molecular Dynamics), and quantitated as density using the ImageQuant program. All signals were normalized to the levels of the ß-actin mRNA signal in the same lane.

Hormone and glucose analyses
Immunoreactive leptin, insulin (Linco, St. Charles, MO), and corticosterone (ICN Biomedicals, Orangeburg, NY) were measured by commercial RIA kits according to the manufacturers’ instructions. Glucose was measured using an automatic glucose analyzer (Apec, West Peabody, MA). Epinephrine and norepinephrine levels were kindly determined by Karel Pacák (National Institutes of Health, Bethesda, MD) using a previously reported method (31).

Data analysis
Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s protected least significant difference (PLSD) test. When appropriate, the unpaired t test was performed for the comparison of mean differences between two different groups. P < 0.05 was considered statistically significant. All data are presented as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CRH-deficient Crh–/– mice have low plasma leptin levels
To test the hypothesis that Crh–/– mice might have low circulating leptin concentrations due to glucocorticoid insufficiency, we first determined plasma leptin levels every 3 h in WT and Crh–/– littermate mice between 0800 h and 2000 h (Fig. 1Go). WT mice showed a clear diurnal variation of plasma leptin with the highest level at 0800 h (3.8 ± 0.6 ng/ml), whereas Crh–/– mice had significantly lower leptin and no variation over the course of the sampling period (Fig. 1AGo). The plasma leptin rhythm in WT mice was reciprocal to that of corticosterone (Fig. 1BGo), consistent with a prior work (12). As expected, plasma corticosterone in Crh–/– mice was blunted at all times with no diurnal variation as previously reported (30). In a separate measurement at 0400 h, WT mice showed a further increase in leptin level, but did not Crh–/– mice (5.9 ± 2.1 ng/ml vs. 1.4 ± 0.2 ng/ml, respectively; P < 0.05, unpaired t test). These data suggest that the defective glucocorticoid rhythm in Crh–/– mice coexists with the defective leptin rhythm.



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  		FIG. 1. CRH-deficient mice have low plasma leptin levels. Plasma of WT and Crh–/– mice was collected at different day times, and leptin (A) and corticosterone (B) levels were determined. n = 5/group. Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s PLSD test. *, Significant difference vs. WT 0800 h; #, significant difference between genotypes at same time point; KO, Crh–/– mice.

 
CRH infusion normalizes leptin in Crh–/– mice
To determine whether the lower leptin expression in Crh–/– mice is a direct consequence of CRH deficiency, we infused CRH into Crh–/– mice sc using osmotic minipumps, and measured plasma corticosterone and leptin (Fig. 2Go). As expected (30), plasma corticosterone concentration and diurnal rhythmicity in Crh–/– mice were increased by CRH infusion (Fig. 2AGo) up to WT levels (see Fig. 1BGo). This was also accompanied by a rise in plasma leptin to WT levels (Fig. 2BGo). Lep mRNA content in epididymal fat pad was also increased significantly by CRH infusion up to the vehicle-infused WT level (Fig. 2Go, C and D). These results indicate that recovery of glucocorticoid levels in Crh–/– mice by CRH infusion correlates with recovery of leptin secretion as well as with recovery of Lep gene expression.



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  		FIG. 2. CRH infusion normalizes leptin in Crh–/– mice. Crh–/– mice were subjected to chronic CRH infusion. A and B, Plasma was collected in late afternoon (1700 h) on d 5 and in the morning (0500 h) on d 9 after the onset of infusion, and corticosterone (A) and leptin (B) levels were determined. n = 4–5/group. Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s PLSD test. *, Significant difference between afternoon vehicle and afternoon CRH infusion. C and D, Epididymal fat pads were collected on d 9 after the onset of CRH infusion, and the Lep mRNA levels were detected by Northern blot hybridization (C) and quantitated by ImageQuant analysis (D). n = 4–7/group. Results were analyzed by unpaired t test. *, Significant difference between WT vehicle and Crh–/– vehicle; #, significant difference between Crh–/– vehicle and CRH infusion; –, vehicle; +, CRH; KO, Crh–/– mice.

 
Leptin is increased during fasting in Crh–/– mice
Circulating leptin levels normally fall during fasting, presumably to minimize nonessential metabolic functions (12). To characterize the leptin response to fasting in Crh–/– mice, we analyzed plasma leptin levels immediately after 36 h of fasting (Fig. 3AGo). As expected, plasma leptin in WT mice significantly dropped after fasting. Paradoxically, plasma leptin in fasted Crh–/– mice, rather than falling, increased significantly. Insulin levels fell in both WT and Crh–/– mice with fasting (Fig. 3BGo), whereas plasma glucose was maintained in WT mice and fell markedly in Crh–/– mice (Fig. 3CGo).



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  		FIG. 3. Leptin is increased during fasting in Crh–/– mice. WT and Crh–/– mice were subjected to fasting for 36 h. Plasma was collected immediately after fasting, and leptin (A), insulin (B), and glucose (C) levels were determined. n = 4–16/group. Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s PLSD test. *, Significant difference vs. WT control; #, significant difference between Crh–/– control and Crh–/– fasting; **, significant difference between WT fasting and Crh–/– fasting; Control, food ad libitum; KO, Crh–/– mice.

 
The differences in the counterregulatory response to fasting in WT and Crh–/– mice were mirrored by changes in corticosterone (Fig. 4AGo), epinephrine (Fig. 4CGo), and norepinephrine (Fig. 4DGo), which increased in WT, but not in Crh–/–, mice. We had previously shown that CRH deficiency, acting via glucocorticoid insufficiency, causes impaired epinephrine secretion due to decreased phenylethanolamine N-methyltransferase expression and activity in the adrenal medulla (31, 32).



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  		FIG. 4. Glucocorticoid restores leptin response to normal in fasted Crh–/– mice. WT and Crh–/– mice were subjected to fasting for 36 h with or without corticosterone implantation, and their plasma was analyzed for corticosterone (A), leptin (B), epinephrine (C), and norepinephrine (D) levels. n = 4–8/group. Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s PLSD test. *, Significant difference vs. control in a genotype; **, significant difference between genotypes in same treatment; #, significant difference between corticosterone implantation/control and corticosterone implantation/fasting in a genotype; Control, food ad libitum; Cort/Control, corticosterone implantation/control; Cort/Fasting, corticosterone implantation/fasting; KO, Crh–/– mice. Note that in panel A, plasma corticosterone levels of WT control (0.3 ± 0.04 µg/dl), Crh–/– control (0.2 ± 0.03 µg/dl), and Crh–/– fasting (0.2 ± 0.04 µg/dl) mice are too low to be visible in the graph.

 
Glucocorticoid restores leptin response to normal in fasted Crh–/– mice
We reasoned that raising glucocorticoid levels in fasted Crh–/– mice to levels found in fasted normal mice would restore the leptin response to fasting to normal. Subcutaneous corticosterone pellet implantation in fasted Crh–/– mice significantly raised corticosterone levels into the normal fasting range (Fig. 4AGo) and prevented the fasting-induced fall in epinephrine (Fig. 4CGo). Corticosterone supplementation of Crh–/– mice also restored the normal fall in leptin after fasting (Fig. 4BGo). In both genotypes, fasting-induced changes in leptin were inversely related to those of both corticosterone and catecholamines. Thus, the normal fasting-induced reduction in leptin depends upon the rise in glucocorticoid and catecholamines as part of the counterregulatory response to starvation.

Crh–/– mice eat less food and gain less weight after fasting
To examine the physiological significance of the paradoxical rise in leptin during fasting in Crh–/– mice, we measured food intake and body weight during the recovery from 24 h of fasting (Fig. 5Go). Crh–/– mice ate less food than WT mice during the recovery period (7.7 ± 0.5 g in Crh–/– mice, 9.5 ± 0.3 g in WT mice, Fig. 5AGo). Crh–/– mice also lost less body weight during fasting and recovered it more slowly than WT mice during the refeeding period (Fig. 5BGo). Because leptin is a potent anorectic hormone, the lack of reduction in leptin during fasting in Crh–/– mice may prevent an appropriate increase in food intake and body weight after the termination of the fast.



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  		FIG. 5. Crh–/– mice eat less food and gain less weight after fasting. WT and Crh–/– mice were subjected to fasting for 24 h. Food was given back after fasting, and the cumulative amount of food intake (A) and change of body weight (B) were measured. n = 5/group. Results were analyzed by two-way ANOVA followed by post hoc comparisons with Fisher’s PLSD test. *, Significant difference between genotypes at same time point; KO, Crh–/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have characterized impaired circulating leptin and epididymal Lep gene expression levels in Crh–/– mice and show that CRH replacement restores leptin mRNA and protein levels to normal. We have also described a paradoxical fasting-induced increase in leptin levels in Crh–/– mice, which coincides with a decrease in catecholamines and is normalized by corticosterone replacement.

Furthermore, we have demonstrated a relationship between the normal fasting-induced fall in leptin and the subsequent increase in food intake at the end of fasting. Crh–/– mice lose less weight than WT mice during fasting, suggesting that Crh–/– mice are not efficient in energy expenditure under fasting conditions, probably due to their insufficiencies of glucocorticoid and catecholamines, which are required for normal fasting-induced lipolysis and proteolysis. Consistent with this, glucocorticoid treatment of fasted Crh–/– mice restores their weight loss to normal (33). After refeeding, Crh–/– mice eat significantly less than WT mice over the ensuing 36 h, and regain their prefasting weight more slowly. The glucocorticoid insufficiency of Crh–/– mice is unlikely to be the direct cause of their decreased food intake at the end of fasting. Under ad libitum feeding conditions, Crh–/– mice have normal food intake and body weight gain despite glucocorticoid insufficiency (27), and treatment of them with glucocorticoid to achieve the levels found in fasted normal mice does not cause an increase in food intake or body weight (34). Likewise, leptin insensitivity or resistance, perhaps due to CRH deficiency, is unlikely to be the explanation for the decreased food intake of Crh–/– mice at the end of fasting because these mice display a normal anorectic response after leptin administration (35). Rather, the relative elevation in leptin and/or less weight loss during fasting in Crh–/– mice are likely to cause their reduced food intake and slower recovery of body weight at the termination of the fast. Therefore, we propose that the normal fall in leptin secretion may play a key role in stimulating appetite after a fast. In support of this, fasted rats infused with a constant amount of leptin that achieves elevated fasting levels (5–7 ng/ml) similar to those in fasted Crh–/– mice show attenuated food intake and weight gain in the period after the fast (36). The normal fall in leptin stimulates hypothalamic neuropeptide Y and agouti-related peptide, and inhibits hypothalamic proopiomelanocortin and {alpha}-MSH, all of which accompany the fasting state and increase appetite (3, 37). By this mechanism, the amount of food ingested in the post-fasting meal would be enhanced. This might be most important in animals which must survive for long intervals between meals.

Recently, a mechanism for the regulation of leptin expression by hexosamine acting as an intracellular energy sensor has been proposed (38, 39). In general, factors that increase cellular glucose uptake and utilization tend to increase leptin synthesis and secretion, whereas factors that promote energy efflux decrease them (39). Thus, food intake (40), insulin (41), and glucocorticoid (15) all promote leptin expression, whereas catecholamines (22, 24, 42) and glucocorticoid during fasting (12) are associated with a fall in leptin. Normally, intracellular hexosamine falls during fasting due to the decrease in insulin coupled with the increase in glucocorticoid and catecholamines, which inhibit glucose uptake (43, 44, 45, 46), enhance breakdown of triglycerides into glycerol and free fatty acid (FFA) by stimulating hormone-sensitive lipase (47), and increase FFA release (48, 49), thus limiting glucose and FFA availability for oxidation. Hexosamine biosynthesis directly regulates leptin biosynthesis and secretion in mouse (38, 50) and human (51) adipocytes. Thus, as hexosamine production decreases during fasting due to limited intracellular glucose and FFA availability, leptin expression and secretion decrease.

Over the long term, leptin levels are proportional to fat mass, whereas in the short-term, leptin levels are nutritionally regulated and function as a rapid mechanism for nutrient sensing (39). Decreased fat stores (29) are the likely basis for the low basal leptin levels in Crh–/– mice. The absence of the fasting-induced fall in leptin is due, at least in part, to the glucocorticoid insufficiency of Crh–/– mice because the administration of CRH or corticosterone restores to normal both basal leptin as well as the fasting-induced fall in the hormone. Catecholamines, epinephrine and norepinephrine, are known to suppress leptin release (22, 24), and their levels are also low after fasting in Crh–/– mice. Both glucocorticoid and catecholamines are required for activation of hormone-sensitive lipase (52), and their deficiencies would result in decreased glycerol and FFA release from adipocytes of Crh–/– mice. We suggest that the resulting increase in intracellular fuel availability would prevent depletion of hexosamine, and thus promote leptin production and secretion during fasting. In support of this hypothesis, both the abnormal epinephrine and leptin responses to fasting in Crh–/– mice are reversed by corticosterone replacement.

Despite normal food intake (27), Crh–/– mice have a blunted diurnal rhythm in plasma leptin. This coincides with the blunted plasma corticosterone rhythm in Crh–/– mice and suggests that the circadian rhythm in corticosterone, along with other factors such as food intake and insulin, may regulate the diurnal leptin rhythm. The most likely mechanism is that increases in glucocorticoid, food intake, and insulin result in energy accumulation in the adipocyte and other cells, thus stimulating leptin secretion. Our data are more consistent with this model than with the possibility that the leptin rhythm drives the diurnal glucocorticoid rhythm (12, 13). Ahima et al. (36) have found that constant infusion of high physiological amounts of leptin in rats causes a dampening, but not disappearance, of the nocturnal rise in glucocorticoid, which is likely related to the decreased food intake caused by the treatment, rather than to a direct effect on circadian rhythmicity. Nevertheless, there is about a 12-h difference between the peaks of normal corticosterone and leptin in WT mice. Thus, any effect of corticosterone on leptin secretion may operate via a mechanism that includes a significant phase delay.

Leptin has been postulated to have a primary role to limit overeating (1, 4, 5, 6). How a mechanism to restrict food intake might have evolved in mammals is puzzling because, under most natural conditions, the presence of either excessive fat stores or an overabundance of food is unlikely. Our present study suggests that the physiological reduction in leptin may play an important role to stimulate food intake during the recovery from fasting. This is consistent with the physiologic role that the fall in leptin has during fasting to inhibit several anabolic hormone systems (12). Consistent with this idea, only diseases caused by insufficient leptin production or action, leading to hyperphagia, obesity, and decreased metabolic function of multiple organ systems, have been described in humans (53, 54) and animals (55, 56, 57), with no examples described of diseases caused by increased leptin production or action. Our findings suggest that the fasting-induced fall in leptin facilitates recovery when food becomes available by stimulating food intake and weight gain more than would occur if leptin did not fall, to more quickly restore homeostasis after fasting. This, rather than an appetite-suppressing role to prevent overeating, provides a rationale for the evolution of leptin as an appetite-regulating hormone.


   Acknowledgments
 
We thank Kolbeinn Gudmundsson and Frederick D. Grant for helpful discussions, Lauren Jacobson for the corticosterone pellet protocol, and Karel Pacák for catecholamine measurements.


   Footnotes
 
Present address for S.S.: Third Department of Medicine, School of Medicine, Hirosaki University, 5 Zaifu-Cho, Hirosaki, Aomori 036-8562, Japan.

This work was supported in part by National Institutes of Health Grant DK50511 (to J.A.M.) and National Science Foundation Grant IBN9513926 (to E.P.W.).

Abbreviations: FFA, Free fatty acid; HPA, hypothalamic-pituitary-adrenal; KO, Crh–/–; PLSD, protected least significant difference; WT, wild-type.

Received November 18, 2003.

Accepted for publication March 9, 2004.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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