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Endocrinology Vol. 144, No. 8 3547-3554
Copyright © 2003 by The Endocrine Society

Corticotropin-Releasing Hormone-Mediated Pathway of Leptin to Regulate Feeding, Adiposity, and Uncoupling Protein Expression in Mice

Takayuki Masaki, Go Yoshimichi, Seiichi Chiba, Tohru Yasuda, Hitoshi Noguchi, Tetsuya Kakuma, Toshiie Sakata and Hironobu Yoshimatsu

Department of Internal Medicine, School of Medicine, Oita Medical University, Oita 879-5593, Japan

Address all correspondence and requests for reprints to: Takayuki Masaki, Department of Internal Medicine, School of Medicine, Oita Medical University, Hasama, Oita 879-5593, Japan. E-mail: masaki{at}oita-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To examine the functional role of CRH in the regulation of energy homeostasis by leptin, we measured the effects of the CRH antagonist, {alpha}-helical CRH 8–41 ({alpha}CRH) on a number of factors affected by leptin activity. These included food intake, body weight, hypothalamic c-fos-like immunoreactivity (c-FLI), weight and histological characterization of white adipose tissue, and mRNA expressions of uncoupling protein (UCP) in brown adipose tissue (BAT) in C57Bl/6 mice. Central infusion of leptin into the lateral cerebroventricle (icv) caused significant induction of c-FLI in the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus, and arcuate nucleus. In all these nuclei, the effect of leptin on expression of cFLI in the PVN and VMH was decreased by treatment with {alpha}CRH. Administration of leptin markedly decreased cumulative food intake and body weight with this effect being attenuated by pretreatment with {alpha}CRH. In peripheral tissue, leptin up-regulated BAT UCP1 mRNA expression and reduced fat depositions in this tissue. Those changes in BAT were also decreased by treatment with {alpha}CRH. As a consequence of the effects on food intake or energy expenditure, treatment with {alpha}CRH attenuated the leptin-induced reduction of body adiposity, fat cell size, triglyceride contents, and ob mRNA expression in white adipose tissue. Taken together, these results indicate that CRH neurons in the PVN and VMH may be an important mediator for leptin that contribute to regulation of feeding, adiposity, and UCP expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ADIPOCYTOKINE LEPTIN plays an important role in the regulation of energy intake and expenditure (1, 2, 3, 4). The effects of leptin are mediated by receptors in several hypothalamic regions including the ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), arcuate nucleus (ARC), and paraventricular nucleus (PVN) (5, 6, 7, 8). In these nuclei, leptin affects the activity of a variety of neurons containing orexigenic or anorexigenic neuropeptides (9, 10, 11). These peptides include neuropeptide Y (12), agouti-related protein (13), proopiomelanocortin-derived peptide (14, 15), and CRH (16, 17, 18, 19). These peptides constitute the major neuronal network regulating the neuroendocrine system and energy metabolism. The cell body of CRH neurons in the PVN project into several other hypothalamic regions including the VMH to regulate neuroendocrine system and energy homeostasis (20, 21, 22, 23, 24). These neurons and receptors are involved in food intake and/or stress responses (25, 26, 27, 28, 29, 30). Central administration of CRH has been shown to increase sympathetic nerve activity in brown adipose tissue (BAT) involved in energy expenditure and thermogenesis (31, 32, 33). On the basis of the data that CRH neurons may regulate both food intake and peripheral energy expenditure, we carried out a series of studies to investigate the role of CRH neurons in the control of leptin signaling.

Leptin has been demonstrated to increase the release, concentration, and mRNA expression of CRH in the PVN (19). The concentration or turnover rate of hypothalamic CRH has been shown to be lower in leptin receptor-mutated Zucker obese rats (34, 35). Furthermore, leptin-induced feeding suppression is attenuated by pretreatment with the CRH antagonist, {alpha}-helical CRH ({alpha}CRH) or anti-CRH antibody (18, 36, 37). These findings suggest that signal transduction from leptin to CRH neurons may be involved in the central regulation of feeding behavior and energy expenditure.

Despite the rapidly advancing understanding on the functional role of CRH neurons as mediators of leptin, there have been only a small number of studies on the central pathways and the effect of their peripheral action on adiposity and energy expenditure. To address this issue, we investigated the effects of {alpha}CRH on leptin-induced changes in the expression of c-fos-like immunoreactivity (c-FLI) in the hypothalamus, mRNA expressions of uncoupling protein (UCP) in BAT and adiposity of white adipose tissue (WAT). The main objective of these experiments was to clarify how CRH neurons transmit leptin information to the peripheral adipose tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mature male C57Bl/6 mice (Seac Yoshitomi Ltd., Fukuoka, Japan), 12–14 wk of age, were housed in a room illuminated daily from 0700 to 1900 h (a 12-h light, 12-h dark cycle) at a temperature of 21 ± 1 C and humidity 55% ± 5%. The mice were allowed access to standard mouse food CE-2 (CLEA Japan Ltd., Tokyo, Japan) and tap water ad libitum. Selected mice were monitored at least 7 d before each experiment, with food consumption and body weight being measured during three of these days. Total body weight and fat weight were measured using an analytical balance (Mettler, Toledo, Osaka, Japan). Segments of WAT and BAT were obtained by dissection, weighed, and immediately frozen in liquid nitrogen and stored at -80 C. All studies were conducted in accordance with the Oita Medical University Guidelines based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Implantation of a cannula into the lateral ventricle
The mice were anesthetized with an ip injection of nembutal (1 mg/kg) and placed in a stereotactic device (Narishige, Tokyo, Japan) to implant a 29-gauge stainless steel cannula chronically into the left lateral cerebroventricle (icv) (0.5 mm posterior, 1.0 mm lateral, and 2.0 mm ventral to the bregma) (38). Following the procedure, a stainless wire stylet was inserted into the cannula to prevent coagulation. The mice had 1 wk of postoperative recovery. After the completion of the experiments, the animals were decapitated, and the cannula location was verified histologically.

Reagents
Recombinant murine leptin (Amgen, Inc. Biologicals, Thousand Oaks, CA) was dissolved in PBS to concentrations of 0.5 µg/µl. the {alpha} CRH 8–41 (Sigma, St. Louis, MO) was also dissolved in PBS to concentrations of 0.5 µg/µl, respectively. Each solution was freshly prepared on the day of its administration. The pH of each solution was adjusted to 6.4–7.2.

Behavioral analysis of C57Bl/6 mice in leptin and {alpha}CRH infusion study
Body weight and food intake were measured during the adaptation period to assign them evenly into four groups: {alpha}CRH/leptin, PBS/leptin, {alpha}CRH/PBS, and PBS/PBS control. Mice were infused with 0.5 µg/mice leptin and/or 0.5 µg/mice {alpha}CRH through icv cannula for 10 min at an infusion rate of 0.1 µl/min, respectively. These doses and the infusion speed were selected on the basis of earlier results (26, 39, 40) and our preliminary study, including a dose-response relation between dose of leptin, {alpha}CRH, and food intake. Pretreatment with {alpha}CRH or PBS was performed 2 h before administration of leptin or PBS infusion at 1700 once a day for 3 consecutive days. Cumulative food intake, body weight change, histological changes of BAT and WAT, WAT ob mRNA, and BAT UCP1 mRNA expression were measured in each group 15–16 h after the last injection. To rule out the difference in food intake as a factor to influence other parameters, we also created a pair-fed group for each experimental group.

Triglyceride content in adipose tissues
One hundred-milligram sections of adipose tissue from the epididymal were homogenized for 1 min in 2 ml of a solution containing 150 mmol/liter sodium chloride, 0.1% Triton X-100, and 10 mmol/liter Tris (pH 8.0) using a polytron homogenizer (NS-310E; Micro Tech Nichion, Chiba, Japan) The triglyceride content of the homogenized solution was determined using a Determiner triglyceride kit (Eiken Med, Tokyo, Japan).

Histological analysis
Small pieces of epididymal WAT were dissected, washed in saline, fixed with 10% formalin, and embedded in paraffin. Tissue sections were cut at a thickness of 20 µm and stained with hematoxylin and eosin. To examine the size of the white and brown adipocytes, the number of adipocytes was counted in five appropriate limited areas (WAT, 25 x 10-3 mm2; BAT, 1 x 10-3 mm2) of each stained specimen. Multilocular adipocytes in the sections were not counted.

RNA extraction and Northern blot analysis
Total cellular RNA was prepared from various mouse tissues using TRIzol (Lifetech, Tokyo, Japan) according to the manufacturer’s protocol. Total RNA (20 µg) was electrophoresed on 1.2% formaldehyde-agarose gels, and the separated bands transferred by capillary blotting onto a Biodyne B membrane (Pall Canada Co. Ltd., Ontario, Canada) in 20x saline sodium citrate and immobilized by exposure to UV light (0.80 J). Prehybridization and hybridization were carried out according to the method described by the manufacturer’s protocol. Membranes were washed under highly stringent conditions. After washing, the hybridization signals were analyzed with a BIO-image analyzer BAS 2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The membranes were then stripped by exposure to boiling 0.1% SDS and normalized with ribosomal RNA to quantify the amounts of RNA species on the blots.

Preparation of cDNA probe
Primers for polymerase chain reactions were designed for the coding region of the UCP1 gene (GenBank accession no. U63419; upstream primer, 5'-TACACGGGGACCTACAATGCT-3': reverse primer, 5'-TCGCACAGCTTGGT-ACGCTT-3'). Reverse transcription of 10 µg total RNA from C57Bl/6 mice was performed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD) and PCR carried out with Taq DNA polymerase (Amersham International, Buckinghamshire, UK) and 20 pmol of the primers. The reaction profile was denaturation at 94 C for 1 min, annealing at 50 C for 1 min, and extension at 72 C for 1 min, for 30 cycles. The PCR fragment was subcloned into pCRTM2.1 vector (TA cloning kit, Invitrogen, San Diego, CA) and the nucleotide sequence of amplified cDNA confirmed by sequencing. The nucleotide sequences were determined by the dideoxynucleotide chain termination method, using synthetic oligonucleotide primers, which were complementary to the vector sequence and ABI373A automated DNA sequencing system (Perkin-Elmer Co., Norwalk, CT). The ob (GenBank accession no. U18812; upstream primer, 5'-AAGATCCCAGGGAGGAAA-3': reverse primer, 5'-CTGGTGGCCTTTGAAACT-3') probes were generated in an analogous fashion.

c-Fos-like immunohistochemistry
To prevent stress-induced c-FLI on the test day, mice were regularly handled during their recovery from surgery. Mice were assigned to one of four groups: {alpha}CRH/leptin, PBS/leptin, {alpha}CRH/PBS, and PBS/PBS control. Mice were infused with 0.5 µg/mice leptin and/or 0.5 µg/mice {alpha}CRH through icv cannula for 10 min at an infusion rate of 0.1 µl/min, respectively. Pretreatment with {alpha}CRH or PBS was performed 2 h before administration of leptin or PBS infusion at 1700 h. Mice were anesthetized with nembutal (3.3 ml/kg ip) and perfused transcardially with isotonic PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer 1.5 h after the leptin or PBS injection. Brains were removed and postfixed for 24 h before processing for c-FLI. Forty-micrometer slices were cut from the brain with a vibrotome. Forebrain slices were made in the coronal plane to allow visualization of the different nuclei of the hypothalamus [i.e. PVN, VMH, lateral hypothalamic area (LHA), ARC, and DMH]. Tissues were washed three times in PBS, incubated for 1 h in 0.3% H2O2 to quench endogenous peroxidase. Slices were then transferred without rinsing to the primary antibody solution, consisting of 0.005 g/ml polyclonal rabbit antiserum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), with specificity to residues 3–16 of the c-Fos protein. After 24 h of incubation on ice, the slices were washed three times in PBS and processed with the ABC method (Vector Laboratories, Burlingame, CA). Slices were transferred to biotinylated antirabbit antibody for 1 h, washed, transferred to avidin-biotinylated peroxidase for 1 h, washed, and developed with diaminobenzidine substrate for 6 min. Slices were then washed, mounted on slides, and coverslipped with Permount.

Cell counting for c-FLI
An experimenter naive to the treatment prepared camera lucida projections of c-Fos-positive brain structures schedules of the treatment groups. Care was taken to score the structures in approximately the same plane, and scoring was undertaken by blinded individuals who recorded the number and location of c-Fos-positive nuclei (Olympus Corp., Tokyo, Japan). Sections examined for c-FLI included the PVN, DMH, LHA, ARC and VMH area based on Paxinos and Flanklins mice atlas (41). We analyzed an average number of Fos-containing cells over a number of sections in the coordinate ranges. Data represented are the mean number of Fos-positive cells/section determined unilaterally from each mouse. Data were analyzed by ANOVA methods to specifically examine differences between treatment groups (PBS vs. {alpha}-CRH vs. leptin vs. leptin-{alpha}CRH injected at each brain site).

Statistical analyses
All the data were expressed as the mean ± SEM. Differences between treatment groups were assessed by ANOVA or the unpaired t test for multiple comparisons, where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of c-FLI in the hypothalamus
The numbers of c-FLI-positive cells in discrete hypothalamic nuclei are shown in Fig. 1Go and representative photomicrographs of these hypothalamic regions in the PVN and VMH are presented in Fig. 1Go, B and D. In the hypothalamus, administration of leptin caused significant induction (P < 0.01) of c-FLI in the PVN (CONT vs. LEP 43 ± 6 vs. 319 ± 51, P < 0.01), VMH (CONT vs. LEP 11 ± 2 vs. 38 ± 4, P < 0.01), ARC (CONT vs. LEP 12 ± 3 vs. 54 ± 7, P < 0.01), and DMH (CONT vs. LEP 10 ± 2 vs. 22 ± 4, P < 0.01) but not the LHA (CONT vs. LEP 7 ± 1 vs. 5 ± 2, P > 0.1) (Fig. 1Go, A and C). Leptin-induced induction of c-FLI in the PVN and VMH was reduced by pretreatment with {alpha}CRH (PVN: LEP vs. LEP-{alpha}CRH 319 ± 51 vs. 132 ± 28; VMH: LEP vs. LEP-{alpha}CRH 38 ± 4 vs. 25 ± 2, P < 0.01 for each) (Fig. 1Go, A and C). There was no significant change in leptin-induced c-FLI expression after treatment with {alpha}CRH in the DMH (LEP vs. LEP-{alpha}CRH 22 ± 4 vs. 25 ± 6, P > 0.1), ARC (LEP vs. LEP-{alpha}CRH 54 ± 7 vs. 50 ± 5, P > 0.1) and LHA (LEP vs. LEP-{alpha}CRH 5 ± 2 vs. 7 ± 3, P > 0.1).



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  		FIG. 1. Effects of {alpha}CRH on leptin-induced c-FLI in PVN (A) and VMH (B) of C57Bl/6 mice. CONT, C57Bl/6 mice treated with PBS. {alpha}CRH, C57Bl/6 mice treated with {alpha}CRH. LEP, C57Bl/6 mice treated with leptin. LEP-{alpha}CRH, leptin-treated C57Bl/6 mice pretreated with {alpha}CRH. Same abbreviations were used in all figures. Each value and vertical bar represents the mean ± SEM. Statistical significance: **, P < 0.01 vs. the corresponding PBS controls. ++, P < 0.01 vs. the corresponding LEP controls. Representative photomicrographs c-FLI in PVN (C) (x100) and VMH (D) (x400). All sections were cut to a thickness of 40 µm.

 
Effects of {alpha}CRH on leptin-induced changes in food intake, body weight, and BAT UCP1 mRNA expression
Administration of leptin decreased cumulative food intake by 39% and body weight by 12%, compared with PBS-treated controls (P < 0.01 for each) (Fig. 2Go). Pretreatment with {alpha}CRH attenuated the regulatory effects of leptin on food intake and body weight (P < 0.05 for each). No difference was found in food intake and body weight between animals treated with either {alpha}CRH alone or PBS (P > 0.1) (Fig 2Go). As shown in Fig. 2Go, C and D, administration of leptin increased UCP1 mRNA expression in BAT by 86%, compared with appropriate pair-fed control mice (P < 0.01). The regulatory effect of leptin on UCP1 mRNA expression in BAT was attenuated by pretreatment with {alpha}CRH (P < 0.05). Representative photomicrographs of UCP1 mRNA expression in the BAT are presented in Fig. 2DGo. There was no significant difference in BAT UCP1 mRNA expression between {alpha}CRH treatment alone and PBS treatment (P > 0.1) (Fig. 2CGo).



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  		FIG. 2. Effects of{alpha}CRH on leptin-induced changes of food intake (A), body weight (B), and BAT UCP1 mRNA expression (C) in C57Bl/6 mice. Representative photomicrographs of UCP1 mRNA expression in BAT (D). Statistical significance: *, P < 0.05; **, P < 0.01 vs. the corresponding appropriate PBS controls. +, P < 0.05 vs. the corresponding appropriate LEP controls.

 
Histological analysis of WAT and BAT
Leptin treatment reduced the cell size of adipocytes in the epididymal WAT (Fig. 3Go). This change was reflected by an increase in the cell density of adipocytes in restricted areas of WAT in the leptin-treated animals, compared with controls (cell count: leptin 209 ± 16 vs. PBS 112 ± 10, P < 0.01) (Fig. 3Go). These morphological changes in WAT adipocytes were reduced by pretreatment with {alpha}CRH (cell count: leptin 209 ± 16 vs. leptin-{alpha}CRH 156 ± 10, P < 0.01) (Fig. 3Go). Leptin also increased adipocyte deposition in the BAT. Cell density of adipocytes in the restricted area of BAT was higher in the leptin-treated group, compared with controls (cell count: PBS 120 ± 20 vs. leptin 438 ± 41, P < 0.01) (Fig. 3Go). These effects of leptin on BAT adipocytes were reduced by pretreatment with {alpha}CRH (cell count: leptin 438 ± 41 vs. leptin-{alpha}CRH 284 ± 26, P < 0.01) (Fig. 3Go). No significant changes were shown in the histology of WAT and BAT between animals treated with either {alpha}CRH alone or PBS.



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  		FIG. 3. Histological analysis of WAT (A) and BAT (C) in C57Bl/6 mice. All sections were cut to a thickness of 20 µm. Scale bar, 100 µm. Cell count analysis of WAT (B) and BAT (D) in C57Bl/6 mice. Statistical significance: **, P < 0.01 vs. the corresponding PBS controls. ++, P < 0.01 vs. the corresponding LEP controls.

 
Effects of {alpha}CRH on leptin-induced changes in body fat accumulation, triglyceride content, and ob mRNA expression in WAT
Administration of leptin decreased epididymal, mesenteric, and retroperitoneal WAT weight by 38%, 47%, and 41%, respectively, compared with PBS-treated controls (P < 0.01 for each) (Fig. 4AGo). Similarly, the triglyceride content in epididymal WAT was decreased by leptin treatment (P < 0.01) (Fig. 4BGo). Pretreatment with {alpha}CRH significantly attenuated both effects (P < 0.05 for each) (Fig. 4Go, A and B), but there was no significant change in WAT weight or triglyceride content between {alpha}CRH treatment alone and PBS treatment (P > 0.1) (Fig. 4Go, A and B). As shown in Fig. 4CGo, leptin treatment decreased ob mRNA expression in epididymal WAT by 38% (P < 0.01), with this inhibitory effect being reduced by incubation with {alpha}CRH (P < 0.05). Representative photomicrographs of ob mRNA expression in the WAT are presented in Fig. 4DGo.



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  		FIG. 4. Effects of by {alpha}CRH on leptin-induced changes in fat accumulation in the epididymal (Epi), mesenteric (Mes), and retroperitoneal (Ret) regions (A); triglyceride contents in 100 mg epididymal fat (B); and ob mRNA expression in epididymal fat (C). Representative photomicrographs of ob mRNA expression in WAT (D). Statistical significance: *, P < 0.05; **, P < 0.01 vs. the corresponding PBS controls. +, P < 0.05 vs. the corresponding LEP controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To investigate how leptin is mediated by CRH neurons, we measured the expression of c-FLI in the hypothalamus in the above study. We found that leptin induced expression of c-FLI in the PVN, VMH, DMH, and ARC nuclei of the hypothalamus, all of which contain large numbers of leptin receptors (5, 6, 7, 8). This suggests that leptin directly activates the nuclei in these neurons. However, treatment with{alpha}CRH partially attenuated the c-FLI response in the PVN and VMH, indicating that CRH neurons mediated leptin information in these nuclei. Taken together, these findings indicate that neurons in the PVN and VMH may receive leptin stimuli not only directly by the leptin receptor but also indirectly through CRH neurons.

In the hypothalamus, the cell body of CRH neurons is localized mainly in the PVN (20, 21, 22, 23). CRH neurons project diffusely from this nucleus to other hypothalamic nuclei including the VMH (21, 22, 23), but CRH type 1 and/or type 2 receptors are distributed throughout the PVN and VMH (42, 43, 44). From this anatomical background, the reduction of leptin-induced c-FLI expression in the VMH by {alpha}CRH treatment indicates that CRH neurons are linking the PVN to the VMH. On the other hand, CRH neurons in the PVN have been shown to be autoregulated by an ultrashort, loop-positive feedback mechanism acting through the type 1 CRH receptor (43). This suggests that CRH neurons may be activated by CRH itself. Thus, the PVN and VMH may be leptin pathways mediated by CRH neurons, whereas other hypothalamic regions such as the DMH and ARC may be upstream or independent of leptin-CRH signaling. Evidence for this possibility is that these latter regions are activated by leptin but not modulated by treatment with {alpha}CRH. In the present study, we showed the increase in c-FLI in the PVN and VMH with leptin treatment, which were partially attenuated with {alpha}CRH. The reason the decrease was partial may be due to the low dose of {alpha}CRH and/or the involvement of other neurons in the PVN and VMH. Further studies are needed to clarify the role of other neuropeptides beside CRH.

Our study also assessed the functional role of the leptin-CRH pathway by analyzing the effect of {alpha}CRH on leptin-induced physiological responses. There is considerable evidence that the PVN and VMH, which we have identified as the major targets for leptin-CRH pathway, are involved in regulating feeding behavior. Therefore, we demonstrated that blockade of the CRH signaling message attenuated the effect of leptin on food intake and body weight, a result consistent with the finding of earlier studies (18, 36, 37). Central administration of CRH has been shown to suppress food intake (25, 26). In addition, administration of leptin increases mRNA expression of CRH type 2 receptor in the VMH (44). These findings, considered together, imply that suppression of feeding by leptin may be mediated by CRH neurons from the PVN to the VMH in combination with their type 2 receptors in the VMH. It is possible that the localized circuit of CRH neurons in the PVN is also involved in leptin-induced feeding suppression because injection of CRH into the PVN or lesion of this nucleus has been shown to produce hypophagia and hyperphagia, respectively (26). CRH signaling is also important for stress responses (28, 29) that are known to affect both feeding behavior and the expression of hypothalamic c-FLI. Thus, it is conceivable that the change of stress responses by a CRH antagonist could attenuate the expression of c-FLI in the PVN and VMH.

Another important function of leptin-CRH signaling in the PVN and VMH is the regulation of peripheral energy metabolism. Several studies have demonstrated the involvement of these hypothalamic nuclei in the regulation of peripheral autonomic function (31, 32, 33). In our study, leptin treatment increased mRNA expression of BAT UCP1, a marker of energy expenditure, and reduced fat deposition in this tissue independent of its inhibitory action on food intake. Blockade of CRH decreased these leptin-induced changes in BAT function and morphology. Within the BAT, UCP1 is regulated by the sympathetic nerves that occur in abundance in this tissue (45). Administration of leptin has been reported to affect BAT UCP1 by activation of sympathetic nerves (46). Central administration of CRH has been demonstrated to increase sympathetic nerve activity in the BAT (31, 32, 33), whereas other studies have reported chemical stimulation of the PVN and VMH produces similar effects on BAT sympathetic nerves (47). However, a neuroanatomical study using a transsynaptic retrograde tracer showed that many neurons in the PVN, but few neurons in the VMH, were the hypothalamic origins of sympathetic nerve efferent to the BAT (48). Furthermore, direct monosynaptic pathway from the PVN in contrast to polysynaptic pathway from the VMH, projected to sympathetic preganglionic neurons in the spinal cord (49). Thus, we may say that the leptin-CRH signaling pathway in the PVN, more so than in the VMH, regulates energy expenditure by affecting sympathetic nerve activity and BAT UCP1 function.

As a consequence of the effects of leptin-CRH signaling on feeding behavior and energy expenditure, we found that several parameters related with adiposity were also influenced by treatment with leptin and {alpha}CRH. The icv leptin treatment reduced fat weight, cell size of adipocytes, triglyceride content, and ob gene expression in WAT. Treatment with {alpha}CRH attenuated all of these regulatory effects of leptin in WAT. It is unlikely that a leakage of centrally infused leptin acts directly in the peripheral tissues in the present study because the serum concentration of leptin was not increased, compared with controls, and ip administration of leptin at the icv dose did not effect the above physiological changes (Masaki, T., and H. Yoshimatsu, unpublished data). A previous study (50) demonstrated that transgenic mice with a deficiency of BAT, because of UCP1 promoter-driven diphtheria toxin A expression, develop obesity that is due initially to decreased energy expenditure and later accompanied by hyperphagia. These results suggest that it is likely that reduction of food intake and up-regulation of BAT UCP1 by leptin-CRH signaling contributed to changes in adiposity. Irrespective of the mechanism involved, our observations indicate that leptin-CRH signaling in the PVN and/or the VMH may function to prevent the development of obesity by affecting energy intake and expenditure.

The antiobese and antidiabetic actions of CRH signaling have been observed in a previous study (51). We have also shown in rodent models an antiobese and antidiabetic effect of neuronal histamine acting downstream of leptin (39). These experiments in db/db and diet-induced obese mice found that neuronal histamine, one of the targets of leptin in the hypothalamus, decreased adiposity by reducing food intake and increasing BAT UCP1 (38). Because CRH has been shown to activate hypothalamic neuronal histamine (52), it is possible that the leptin-CRH signaling pathway examined in our study may act to connect these histaminergic functions. Further study is necessary to clarify the neuronal network regulated by leptin and/or CRH and gain a greater understanding of these systems’ role in the pathogenesis and treatment of human obesity.


   Footnotes
 
This work was supported by Grant-in-Aid 10470233 from the Japanese Ministry of Education, Science, and Culture; Research Grants for Intractable Diseases from the Japanese Ministry of Health and Welfare, 1998–2000; and Research Grants from the Japanese Fisheries Agency for Research into Efficient Exploitation of Marine Products for Promotion of Health, 1998–2000.

Abbreviations: ARC, Arcuate nucleus of the hypothalamus; BAT, brown adipose tissue; c-FLI, c-fos-like immunoreactivity; {alpha}CRH, {alpha}-helical CRH 8–41; DMH, dorsomedial nucleus of the hypothalamus; LHA, lateral hypothalamic area; PVN, paraventricular nucleus of the hypothalamus; UCP, uncoupling protein; VMH, ventromedial nucleus of the hypothalamus; WAT, white adipose tissue.

Received March 7, 2003.

Accepted for publication April 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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