LEPTIN IS A CRUCIAL factor in the regulation of energy

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1 /03/$15.00/0 Endocrinology 144(6): Printed in U.S.A. Copyright 2003 by The Endocrine Society doi: /en Neuronal Histamine Regulates Food Intake, Adiposity, and Uncoupling Protein Expression in Agouti Yellow (A y /a) Obese Mice TAKAYUKI MASAKI, SEIICHI CHIBA, GO YOSHIMICHI, TOHRU YASUDA, HITOSHI NOGUCHI, TETSUYA KAKUMA, TOSHIIE SAKATA, AND HIRONOBU YOSHIMATSU Department of Internal Medicine, School of Medicine, Oita Medical University, Oita , Japan LEPTIN IS A CRUCIAL factor in the regulation of energy intake and expenditure (1 3). There is increasing evidence that the effects of leptin are governed by a number of hypothalamic mediators including orexigenic substances such as neuropeptide Y (4), melanin-concentrating hormone (5), and agouti-related protein (6) in addition to anorexigenic substances, such as CRH (7), cocaine-amphetamine-regulated transcript (8), proopiomelanocortin (POMC), and melanocortin-4 receptor (MC-4R) (9 17). This latter receptor has been shown to be one of the major pathways for leptin signaling in the hypothalamus (9 17). Disruption of MC-4R signaling that occurs in knockout mice or following administration of a receptor antagonist results in leptin-resistant obesity and diabetes (12, 13). Mutations of POMC and MC-4R signaling have also been reported to induce this phenotypic change in both rodents and humans (12 14, 18). Agouti yellow (A y /a) obese mice develop mature-onset obesity and diabetes because of blockade of hypothalamic MC-4R caused by overexpression of agouti protein (19 21). Agouti protein antagonizes the action of -melanocyte-stimulating hormone on the MC-1R to inhibit pigmentation in the skin (19). Ectopic overexpression of agouti protein in A y /a obese mice results in Abbreviations: ARC, Arcuate nucleus; A y /a, agouti yellow; BAT, brown adipose tissue; c-fli, c-fos-like immunoreactivity; H1KO, histamine H 1 receptor knockout; H 1 -R, histamine H 1 receptor; icv, intracerebroventricular; MC4, melanocortin-4; MC-4R, melanocortin-4 receptor; POMC, proopiomelanocortin; PVN, paraventricular; UCP, un-coupling protein; VMH, ventromedial hypothalamic; WAT, white adipose tissue. Hypothalamic neuronal histamine and its H 1 receptor (H 1 -R) form a part of the leptin-signaling pathway in the brain and have been shown to regulate body weight and adiposity in diabetic (db/db) and diet-induced obese mice by affecting food intake and uncoupling protein mrna expression. The proopiomelanocortin (POMC) melanocortin-4 receptor (MC-4R) is also important for leptin signaling. The present study had two aims: first, to clarify the antiobesity action of neuronal histamine in agouti yellow (A y /a) obese mice, a model of obesity in which POMC/MC-4R signaling is disrupted by blockade of MC-4R and second, to investigate the functional relationship between neuronal histamine and POMC/MC-4R signaling. Central administration of histamine into the lateral cerebroventricle decreased cumulative food intake and body weight in A y /a obese mice. Histamine treatment also decreased mrna expression of ob gene in epididymal white adipose tissue and up-regulated uncoupling protein 1 mrna expression in brown adipose tissue. These effects were attenuated in A y /a obese mice with histamine H 1 -receptor (H 1 -R) knockout. Histamine treatment induced c-fos-like immunoreactivity in both paraventricular and arcuate nucleus. There was no significant difference in histamine-induced c-fos-like immunoreactivity in the hypothalamus between A y /a obese mice and lean littermates, indicating histamine signaling was not disrupted at the hypothalamic level in A y /a obese mice. These results suggest that neuronal histamine have an antiobese action, even in A y /a obese mice despite a deficiency in POMC/MC-4R signaling. In addition, it appears that the histamine H 1 -R signaling pathway may be independent or downstream of the POMC/MC-4R signaling. (Endocrinology 144: , 2003) increased food intake and body weight because of antagonism of MC-4R (22, 23), leading to marked abdominal obesity and ultimately leptin-resistant diabetes (21). Central administration of leptin suppressed food intake in wild-type mice but not in MC-4R knockout obese (13) or A y /a obese mice (24, 25). Hypothalamic neuronal histamine has been implicated in the regulation of feeding behavior and body adiposity through the postsynaptic histamine H 1 -receptor (H 1 -R) in the ventromedial hypothalamic (VMH) and paraventricular (PVN) nucleus (26, 27). Recent studies have shown that hypothalamic neuronal histamine is one of the targets for leptin action in the brain (28, 29). Central administration of leptin increased histamine turnover in the hypothalamus (28). Leptin-induced suppression of food intake and up-regulation of uncoupling protein (UCP) expression measured as a marker of energy expenditure were both attenuated in H 1 -R knockout mice (29). Furthermore, long-term central infusion of histamine was observed to decrease food intake and fat deposition and increase UCP family expression in both dietinduced obese mice and db/db obese mice, animals with defective leptin receptors (30). It is generally accepted that MC-4R and H 1 -R contribute to the regulation of energy homeostasis in the brain downstream from the action of leptin. However, little is known regarding the relationship among neuronal histamine, H 1 -R, and POMC/MC-4R system. The aim of the present study was to determine whether neuronal histamine reduced body weight and adiposity in A y /a obese mice that have a disturbance of POMC/MC-4R signaling. In addition, we investigated how neuronal histamine regulated 2741

2 2742 Endocrinology, June 2003, 144(6): Masaki et al. Histamine Regulation in A y /a Obese Mice food intake and UCP mrna expression and studied the interaction between neuronal histamine and the POMC/ MC4 system in the regulation of energy homeostasis. These objectives were achieved by measuring changes in food intake, body weight, adiposity, UCP mrna, and c-fos like immunoreactivity (c-fli) in A y /a obese mice after infusion of histamine. Animals Materials and Methods Mature male C57Bl/6J, A y /a obese mice littermates (Seac Yoshitomi Ltd., Fukuoka, Japan) and H 1 -R-deficient mice (Kyushu Bioregulation Medical Institute, Fukuoka, Japan; Ref. 31), at wk of age, were housed in a room illuminated daily from h (a 12-h light, 12-h dark cycle) with temperature at 21 1 C and humidity 55% 5%. The mice were allowed free access to standard pellet mice chow (CLEA Japan Ltd., Tokyo, Japan) and tap water. Food intake in mice was monitored after 7-d adaptation. Implantation of a cannula, acute and chronic infusion methods 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 [intracerebroventricularly (icv), 0.5 mm posterior, 1.0 mm lateral, and 2.0 mm ventral to the bregma]. Following the procedure, a stainless wire stylet was inserted into the cannula to prevent coagulation. The mice had 1 wk of postoperative recovery after which they were handled daily to equilibrate their arousal levels. After the completion of the experiments, the animals were decapitated, and the cannula location was verified histologically. In infusion study, leptin was administered icv by bolus infusion at a dose of 1 g and histamine was infused icv at a dose of mol/g body weight once a day for 7 consecutive days. Cross-breeding of H 1 -R knockout and A y /a obese mice A y /a and H 1 -R gene ( / ) mice were backcrossed to the C57Bl/6 strain (N4 8). C57Bl/6-A y /a and C57Bl/6-H 1 -R gene ( / ) breeder pairs were also cross-bred to create C57Bl/6-agouti yellow H 1 -R knockout models (A y /a: / ). H 1 -R gene carriers were identified by Southern blotting analysis with genomic DNA followed by allele-specific hybridization to identify the presence or absence of the H 1 -R mutation. Details of the gene locus of H 1 -R mutation were described in a previous report (31). Four phenotypes were used in experiments: agouti yellow H 1 -R null (A y /a: / ), H 1 -R null (a/a: / ), agouti yellow (A y /a: / ), and wild type (a/a: / ). Blood and tissue samples Either histamine or PBS was injected into C57Bl/6, A y /a, ora y /a-h 1 - R-deficient mice. Serum was separated from blood samples and immediately frozen at 20 C until the measurement. Serum leptin was measured by a commercially available kit (Sandwich enzyme immunoassay, Immune Biological Laboratory, Gunma, Japan.). Brown adipose tissue (BAT) and white adipose tissue (WAT) were surgically removed after the treatment. Measurement and experimental procedures of food consumption Matched on the basis of the initial body weight at start of the experiment, the mice were divided equally into the histamine, PBS control, and pair fed with histamine subgroups (n 4 6 for each). Food intake and body weight were measured using an analytical balance for rodents (Mettlar Co. Ltd., Osaka, Japan). To evaluate the effects of histamine on food intake and body weight in H 1 -R knockout (H1KO) mice, the mice were equally divided into the PBS and histamine groups, respectively (n 4 6 for each). To exclude difference in food intake between the histamine and PBS control groups, the histamine group was pair fed daily with the appropriate histamine-treated mice. After the feeding evaluation, tissues were surgically removed according to the procedures as mentioned above and analyzed as to fat accumulation and/or gene expression. Histology Adipose tissue sections (20- m thick) were cut in a cryostat and mounted on glass slides. Some sections after formalin fixation were stained with hematoxylin and eosin. Determinations of triglyceride in adipose tissues For the determination of triglyceride in adipose tissues, 100 mg WAT were homogenized in 10 ml of a solution containing 150 mm sodium chloride, 0.1% Triton X-100, and 10 mm Tris (ph 8.0), at C. Homogenization was performed using a homogenizer (NS-310E, Micro Tech Nichion, Chiba, Japan) at high speed for 60 sec; 100 l of this homogenized solution were used for triglyceride determination using a determination kit (Eiken Chemical Co. Ltd., Tokyo, Japan). Preparation of cdna probe PCR primers of were designed to the UCP1 gene (GenBank accession no. D28561). Reverse transcription of 10 g total RNA from C57Bl/6 mice was performed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). PCR was carried out with Taq DNA polymerase (Amersham International, Buckinghamshire, UK) and 20 pmol primers. The reaction profiles were as follows: 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 Corp., San Diego, CA) and nucleotide sequence of amplified cdna was 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 Corp., Norwalk, CT). The ob (GenBank accession no. X03894) probe was generated in an analogous fashion. RNA extraction and Northern blot analysis Total cellular RNA was prepared from various mice tissues with the use of TRIzol (Life Technologies, Inc.) according to the manufacturer s protocol. Total RNA (20 g) was electrophoresed on 1.2% formaldehyde-agarose gel, and the separated RNA was transferred onto a Biodyne B membrane (Pall Canada Co. Ltd., Toronto, Ontario, Canada) in 20 saline sodium citrate by capillary blotting and immobilized by exposure to UV light (0.80 J). Prehybridization and hybridization were carried out according to the manufacturer s protocol. Membranes were washed under high-stringency conditions. After washing the membranes, the hybridization signals were analyzed with a BIO-image analyzer BAS 2000 (Fuji Photo Film Co., Ltd., Tokyo, Japan). The membranes were stripped by exposure to boiling 0.1% sodium dodecyl sulfate and an ethidium-bromide that was used to quantify the amounts of RNA species on the blots. c-fli immunohistochemistry To prevent stress-induced c-fos expression on the test day, mice were regularly handled during recovery from surgery. On the test day, mice were given icv infusion of histamine or PBS (n 4 6). Then 1.5 h after injection, mice were anesthetized with Nembutal (3.3 ml/kg ip) and transcardially perfused with isotonic PBS followed by 4% paraformaldehyde in 0.1 m phosphate buffer. Brains were removed and postfixed for 24 h and then processed 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 central nucleus of the various nuclei of the hypothalamus [the PVN, VMH, arcuate nucleus (ARC), and lateral hypothalamic area[. Tissues were rinsed (3 PBS), incubated for 1 h in 0.3% H 2 O 2 in absolute methanol to quench endogenous peroxidase, and rinsed (3 PBS). Slices were then transferred without rinsing

3 Masaki et al. Histamine Regulation in A y /a Obese Mice Endocrinology, June 2003, 144(6): to the primary antibody solution, consisting of g/ml polyclonal rabbit anti-serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), which recognizes residues 3 16 of the c-fos protein. After 24 h of incubation on ice, slices were rinsed (3 PBS) and processed with the ABC method (Vector Laboratories, Burlingame, CA). Slices were transferred to biotinylated goat antirabbit antibody for 1 h, rinsed, transferred to avidin-biotinylated peroxidase for 1 h, rinsed, and developed with diaminobenzidine substrate (6 min). Slices were rinsed, mounted on slides, and coverslipped with Permount. Camera lucida drawings of c-fospositive brain structures were prepared by an experimenter naive to group treatments. Care was taken so that structures were scored in approximately the same plane. Drawings were scored by blinded raters who recorded the number and location of c-fos-positive nuclei (Olympus Corp. Optical Co. Ltd., Tokyo, Japan). Scores across raters were averaged for statistical analyses. Statistical analysis All the data were expressed as the mean sem. The unpaired t test or ANOVA analysis with corresponding control group assessed the statistical analysis of difference between mean values. The animals used were treated in accordance with the Oita Medical University Guidelines for the Care and Use of Laboratory Animals. Results Effects of acute central administration of leptin or histamine on body weight and food intake in A y /a obese mice Acute icv infusion of leptin decreased daily food intake in C57Bl/6 (vehicle g vs. leptin g; P 0.01) but not in A y /a obese mice (PBS g vs. leptin g; P 0.1). Acute icv infusion of leptin also decreased body weight in C57Bl/6 (PBS g vs. leptin g; P 0.05) but not in A y /a obese mice (PBS g vs. leptin g; P 0.1). In contrast, icv infusion of histamine significantly (P 0.05) decreased body weight and food intake in both C57Bl/6 (food intake: PBS g vs. histamine g; body weight change: PBS g vs. histamine g) and A y /a obese mice (food intake: PBS g vs. histamine g; body weight change: PBS g vs. histamine g). Effects of chronic central administration histamine on body weight and food intake in A y /a obese mice Central icv administration of histamine for 7datadose of 0.05 mol/g body weight caused a 6.6% and 9.1% decrease in body weight (P 0.01 for each) and a 22.0% and 27.1% decrease of cumulative food intake (P 0.01 for each) in C57Bl/6 and A y /a obese mice, respectively, compared with PBS-treated A y /a controls (Fig. 1, A and B). Central icv administration of histamine also caused a decrease in body weight (P 0.05) in A y /a obese mice, respectively, compared with pair-fed A y /a controls (Fig. 1B). Effects of chronic central administration of histamine on adiposity and triglyceride contents of WAT and/or BAT in A y /a obese mice Compared with PBS control animals, histamine treatment caused a 20% decrease in WAT tissue weight comprised of epididymal, mesenteric, retroperitoneal fat. This reduction occurred in visceral fat with a 36%, 33%, and 23% decrease in the weight of mesenteric, retroperitoneal, and epididymal fat, respectively, compared with PBS-treated A y /a obese mice (P 0.01, P 0.05; Fig. 2-A). Morphological changes of BAT and WAT were shown in Fig. 3. No significant change in the weight of other organs such as heart and kidney was observed following treatment with histamine. This treatment was, however, associated with a 19%, 25%, and 12% reduction in the triglyceride content of 100-mg sections of epididymal WAT (Fig. 2B), liver, and skeletal muscle, respectively (all P 0.05). The above effects of histamine were also shown, compared with pair-fed A y /a obese mice (P 0.05; Fig. 2). Effects of chronic central administration of histamine on WAT ob gene mrna expression and serum leptin concentration in A y /a obese mice The effects of neuronal histamine on WAT ob gene mrna expression and serum leptin concentration in A y /a obese mice FIG. 1. Central effects of chronic histamine infusion on cumulative food intake (A) and body weight change (B) in A y /a obese mice treated with histamine (A y /a-ha), PBS (A y /a-pbs), or C57Bl/6 control mice treated with either histamine (C57Bl/6-HA) or PBS (C57Bl/6-PBS). Each HA-treated mice pair fed with the corresponding control animal (PF). The columns represent the mean SEM, with statistical significance marked in parentheses. **, P 0.01 vs. the corresponding PBS controls., P 0.05 vs. the corresponding PF control.

4 2744 Endocrinology, June 2003, 144(6): Masaki et al. Histamine Regulation in A y /a Obese Mice FIG. 2. Central effects of chronic histamine infusion on fat weight (A), triglyceride content (B), ob gene expression (C), and serum leptin concentration (D) in A y /a obese mice treated with either histamine (HA) or PBS. Each HA-treated mice pair fed with the corresponding control animal (PF). Mes, Ret, and Epi represent mesenteric, retroperitoneal, and epididymal fat, respectively, and r.a.u. signifies relative arbitrary units. Each value and vertical bar represents the mean SEM. The columns represent the mean SEM with each value s expressed as percentage of PBS controls. Statistical significance is marked in parentheses. *,P 0.05; **, P 0.01 vs. the corresponding PBS controls;, P 0.05 vs. the corresponding PF control. are summarized in Fig. 2, C and D, which shows histamine treatment for 7 d decreased the expression of ob gene mrna in epididymal WAT and serum leptin concentration by 55% and 38%, compared with A y /a controls fed ad libitum (P 0.01 for each). WAT ob gene mrna and serum leptin concentration also decreased by 45% and 20% in A y /a obese mice, compared with pair-fed A y /a controls matched according to histamine treatment (P 0.05 for each; Fig. 2, C and D). Effects of central administration of histamine on BAT UCP1 expression in A y /a obese mice After 7dofhistamine treatment, expression of BAT UCP1 mrna increased to 146% in A y /a obese mice, compared with A y /a obese mice fed ad libitum and 168% in A y /a obese mice, compared with pair-fed A y /a controls (P 0.01 for each; Fig. 4). Representative photomicrographs of UCP1 mrna expression in BAT are presented in Fig. 4B. Effects of chronic central administration of histamine on food intake and BAT UCP1 gene expression in H1KO-A y /a obese mice Figure 5A shows the effects of neuronal histamine on food intake in A y /a obese mice with H1KO. Although this treatment markedly decreased cumulative food intake by 24% in A y /a obese mice, compared with PBS-treated A y /a controls (P 0.01), this effect was partially but significantly attenuated in the A y /a obese mice with the disrupted H 1 -R gene (P 0.01). Histamine treatment resulted in a marked increase in BAT UCP1 mrna expression of 166% in Ay/a obese mice, compared with the pair-fed A y /a controls (P 0.01), an effect that was also attenuated in the H1KO A y /a obese mice (P 0.05; Fig. 5B). c-fli in the hypothalamus Data of the number of c-fli-positive cell in discrete hypothalamic nuclei are shown in Fig. 6 and representative photomicrographs of PVN and ARC are presented in Fig. 7. In the hypothalamus, administration of histamine to A y /a obese and lean littermates caused induction of c-fli after 1.5 h in the PVN (Fig. 6A; P 0.01) and ARC (Fig. 6B; P 0.01) but not the VMH (Fig. 6C; P 0.1) and lateral hypothalamic area (Fig. 6D; P 0.1). There was, however, no significant change in the numbers of c-fli-positive cells between A y /a obese animals and lean littermates (P 0.1; Fig. 6). Discussion Previous studies have demonstrated that A y /a obese mice are highly resistant to the effects of leptin on body weight and food intake (24, 25), suggesting that POMC/MC4 systems play a key role in mediating these effects (9 17). The findings of the present study are in agreement with these earlier studies in that central administration of leptin did not affect

5 Masaki et al. Histamine Regulation in A y /a Obese Mice Endocrinology, June 2003, 144(6): FIG. 3. Effects of histamine on histology of WAT and BAT in A y /a obese mice treated with either histamine (HA) or PBS. Each HA-treated mice pair fed with the corresponding control animal (pair-fed). Scale bar, 100 m. FIG. 4. Central effects of chronic histamine infusion on BAT UCP1 gene expression in A y /a obese mice treated with either histamine (HA) or PBS. Each HA-treated mice pair fed with the corresponding control animal (pair-fed). The columns represent the mean SEM. Each value is expressed as percentage of PBS controls with statistical significance marked in parentheses. *, P 0.05; **, P 0.01 vs. the corresponding PBS controls; 2, P 0.01 vs. the corresponding pairfed control. r.a.u. corresponds to relative arbitrary units. Representative photomicrographs of (B) UCP1 mrna expression in BAT. food intake and body weight in A y /a obese mice. The novel findings in the present study were that both acute and chronic central administration of histamine reduced food intake and body weight even in leptin-resistant A y /a obese mice. We have previously demonstrated the same in dietinduced obese mice with acquired leptin resistance and db/db mice, a model with a leptin receptor mutation (30). The findings that the antiobesity action of histamine occur in all three leptin-resistant models, histamine induces c-fos in the PVN, and the effects depend partly on the H 1 -R suggest that neuronal hypothalamic histamine regulates feeding behavior and body weight by a mechanism that bypasses the POMC/MC-4R signaling pathway. Our study also demonstrated histamine treatment significantly decreased adiposity of WAT, possibly as a consequence of suppression of food intake. It is highly probable that the effect of histamine on adiposity and triglyceride contents in WAT is due, in part, to activation of lipolysis of WAT through the descending neuronal pathway. This possibility is supported by the observation that activation of hypothalamic neuronal histamine increases activity of sympathetic nerves innervating WAT, leading to enhanced lipolysis in this tissue (32). Another possible explanation for histamine-induced reduction of adiposity may be increased energy expenditure through the activation of BAT UCP1. In our study, chronic histamine treatment induced an increase in BAT UCP1 mrna expression. Furthermore, this histamine-induced up-regulation of BAT UCP1 was also significantly greater than in the pair-fed animals. Central administration of histamine has been shown to increase activity of sympathetic nerve innervating BAT (Yasuda, T., T. Masaki, and H. Yoshimatsu, unpublished observations), a finding that indicates histamine increases energy expenditure by

6 2746 Endocrinology, June 2003, 144(6): Masaki et al. Histamine Regulation in A y /a Obese Mice FIG. 5. Central effects of chronic histamine infusion on food intake (A) and gene expression (B) of UCP1 in BAT in A y /a obese mice with and without H1KO. The groups include H1KO-A y /a mice treated with histamine (A y /a-h 1 ( / )-HA), wild-type A y /a mice treated with HA (A y /a-h 1 ( / )-HA), H1KO-A y /a mice treated with PBS (A y /a-h 1 ( / )-PBS), and wild-type A y /a mice with PBS (A y /a-h 1 ( / )-PBS). Statistical significance marked as *, P 0.05; **, P 0.01 vs. the corresponding PBS control;, P 0.05; 2, P 0.01 vs. the corresponding wild-type control. FIG. 6. Effects of histamine on c-fli in A y /a obese mice treated with either histamine (A y /a-ha) or PBS (A y /a-pbs) and C57Bl/6 control mice treated with either histamine (C57Bl/6-HA) or PBS (C57Bl/6-PBS). The columns represent the mean SEM. Statistical significance marked as **, P 0.01 vs. the corresponding PBS controls. up-regulation of BAT UCP1. This effect appears to be mediated through sympathetic nervous activity independent of histamine s effect of suppressing food intake. From these observations, it is likely that the effect of the BAT induced by histamine treatment also protect against the development of obesity in A y /a mice.

7 Masaki et al. Histamine Regulation in A y /a Obese Mice Endocrinology, June 2003, 144(6): FIG. 7. Representative photomicrographs of c-fli in PVN and ARC in A y /a obese and lean littermates treated with either histamine (A y /a-ha) or PBS (A y /a-pbs) and C57Bl/6 control mice treated with either histamine (C57Bl/6-HA) or PBS (C57Bl/6-PBS). To further confirm the involvement of histamine H 1 -R in the antiobesity action of histamine in A y /a obese mice, the effects of histamine treatment on feeding behavior and BAT UCP1 expression were investigated using A y /a H1KO double-mutant mice. It was found that suppression of food intake and up-regulation of BAT UCP1 by histamine was partially attenuated in these animals. Our observation that histamine treatment was effective even in A y /a obese mice implies that deficiency of POMC/MC-4R signaling does not affect histamine action. However, because these effects of histamine were blocked in A y /a H1KO mice, it can be concluded that histamine treatment affects food intake and UCP mrna expression through the H 1 -R rather than by POMC/ MC-4R signaling. Here a question can be raised as to why the effect of the H 1 -R knockout was partial, not complete, in the present study. This result indicates the involvement of other histamine receptors than H 1 -R. The previous studies showed that H 3 -R but not H 2 -R regulated feeding behavior (26, 27, 33). It is well known that H 3 -R mediates the release of serotonin and norepinephrine (34, 35), which have potent effects in food intake. Taken together, the possibility that these neurotransmitters are involved in the component of histamine action independent of H 1 -R cannot be excluded. In this study, we also measured the expression of c-fli in the hypothalamus of A y /a mice to investigate the histaminesignaling pathway to peripheral tissues. Compatible with a previous study (36), we observed that histamine induced expression of c-fli in the PVN. It has been shown previously that H 1 -Rs are abundantly distributed in PVN and VMH (26, 27), raising the possibility that the PVN may be a major histamine-signaling pathway to peripheral tissues. The other hypothalamic regions such as the lateral hypothalamic area, which are not activated by histamine, may be upstream of this pathway or are independent of histamine signaling. In extrahypothalamic areas, we observed marked c-fli expression in hippocampus in which histamine H 1 -Rs were richly distributed (Masaki, T., and H. Yoshimatsu, unpublished observations). However, the functional role of H 1 -R in hippocampus in the regulation of adiposity is still unknown. The final series of experiments in this study investigated the expression of c-fli in the hypothalamus to compare histamine signaling between A y /a obese mice and controls. Histamine induced the expression of c-fli in nuclei in both the PVN and ARC, regions that constitute the major neuronal network regulating feeding behavior and peripheral energy metabolism. Although this suggests that both these nuclei are important targets for the histamine-signaling pathway, we found no significant difference in histamine-induced c-fli expression in the hypothalamus between A y /a obese mice and lean littermates. This result indicates that histamine signaling was not disrupted at the hypothalamic level in A y /a obese mice, leading us to conclude that disruption of POMC/ MC-4R signaling does not impair histamine signaling. With regard to the functional correlation between neuronal histamine H 1 -R and POMC/MC-4R signaling, both of which are targets of leptin, our results suggest the two pathways act independently of each other. In contrast to the observations of the present study, this result implies POMC/MC4 signaling can function in animals that have a deficiency of histamine H 1 -R signaling and also supports our hypothesis that histamine H 1 -R and POMC/MC-4R signaling act independently in the regulation of feeding and body weight. In summary, we have demonstrated central administration of histamine decreases body weight and adiposity in A y /a obese mice by affecting food intake and UCP mrna expression. It is suggested that the specific reduction of visceral fat mass induced by histamine and associated prevention of adipocyte hypertrophy may contribute to an amelioration of obesity-related metabolic disorders. We contend that understanding the actions of hypothalamic neuronal histamine may provide strategies for further research of the hypothalamic neuronal circuit that regulates feeding behavior and UCP mrna expression as downstream of leptin signaling. Acknowledgments We thank Professor Takeshi Watanabe for the gift of H1KO mice. Received January 7, Accepted February 21, 2003.

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