Nesfatin-1, corticotropin-releasing hormone, thyrotropin-releasing hormone, and neuronal histamine interact in the hypothalamus to regulate feeding behavior

Authors


Address correspondence and reprint requests to Koro Gotoh, Department of Internal Medicine 1, Faculty of Medicine, Oita University, Yufu, Oita 879-5593, Japan. E-mail: gotokoro@oita-u.ac.jp

Abstract

Nesfatin-1, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), and hypothalamic neuronal histamine act as anorexigenics in the hypothalamus. We examined interactions among nesfatin-1, CRH, TRH, and histamine in the regulation of feeding behavior in rodents. We investigated whether the anorectic effect of nesfatin-1, α-fluoromethyl histidine (FMH; a specific suicide inhibitor of histidine decarboxylase that depletes hypothalamic neuronal histamine), a CRH antagonist, or anti-TRH antibody affects the anorectic effect of nesfatin-1, whether nesfatin-1 increases CRH and TRH contents and histamine turnover in the hypothalamus, and whether histamine increases nesfatin-1 content in the hypothalamus. We also investigated whether nesfatin-1 decreases food intake in mice with targeted disruption of the histamine H1 receptor (H1KO mice) and if the H1 receptor (H1-R) co-localizes in nesfatin-1 neurons. Nesfatin-1-suppressed feeding was partially attenuated in rats administered with FMH, a CRH antagonist, or anti-TRH antibody, and in H1KO mice. Nesfatin-1 increased CRH and TRH levels and histamine turnover, whereas histamine increased nesfatin-1 in the hypothalamus. Immunohistochemical analysis revealed H1-R expression on nesfatin-1 neurons in the paraventricular nucleus of the hypothalamus. These results indicate that CRH, TRH, and hypothalamic neuronal histamine mediate the suppressive effects of nesfatin-1 on feeding behavior.

Abbreviations used
ARC

arcuate nucleus

BBB

blood–brain barrier

CRH

corticotropin-releasing hormone

FMH

α-fluoromethyl histidine

HDC

histidine decarboxylase

MCH

melanin-concentrating hormone

NUCB2

nucleobindin-2

PBS

phosphate-buffered saline

PVN

paraventricular nucleus

TMN

tuberomammillary nucleus

TRH

thyrotropin-releasing hormone

VMH

ventromedial nucleus

The gene encoding nucleobindin-2 (NUCB2) in the hypothalamus can be cleaved into the novel peptides, nesfatin-1, -2, and -3 (Oh-I et al. 2006). Nesfatin-1, but neither nesfatin-2 nor nesfatin-3, reduces dark-phase food intake in rats when injected into the third ventricle (Oh-I et al. 2006). Although a nesfatin-1 receptor has not been identified, several studies have demonstrated abundant NUCB2/nesfatin-1 expression in the hypothalamic and medullary sites involved in feeding regulation in rats, including the paraventricular nucleus (PVN), the arcuate nucleus (ARC), and the nucleus of the solitary tract (NTS) (Brailoiu et al. 2007; Berthoud and Morrison 2008; Elmquist et al. 2009; Goebel et al. 2009). These findings support the notion that nesfatin-1 is involved in the regulation of food intake. Levels of NUCB2 mRNA and nesfatin-1 are significantly decreased in the PVN of rats deprived of food for 24 h compared with that of rats fed ad libitum, suggesting that nesfatin-1 in the PVN plays a role in satiety, and possibly energy homeostasis, after food intake (Oh-I et al. 2006). Conversely, daily food consumption increased and body weight cumulatively increased after endogenous NUCB2 was blocked by administering an anti-NUCB2 antisense oligonucleotide into the third ventricle (Oh-I et al. 2006).

Hypothalamic histamine neurons originating from the tuberomammillary nucleus (TMN) distributed in the posterior hypothalamus project diffusely throughout the brain, including the PVN and the ventromedial nucleus (VMH), which is known as the satiety center (Kennedy 1950). Other study showed that hypothalamic histamine suppresses food intake via histamine H1-receptor (H1-R) in VMH and PVN (Fukagawa et al. 1989), several distinct endogenous peptides in the PVN such as corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH), inhibit food intake (Kow and Pfaff 1991; Richard et al. 2000). Moreover, CRH and TRH directly activate histamine neurons (Gotoh et al. 2005, 2007). Based on these findings, we hypothesized that nesfatin-1, CRH, TRH, and hypothalamic neuronal histamine constitute a neuronal network within the hypothalamus that regulates energy metabolism. The aim of this study was to examine whether nesfatin-1 affects the expressions of CRH or TRH and histamine turnover in the hypothalamus, whether a central infusion of histamine activates nesfatin-1 neurons and whether nesfatin-1 expression is altered in H1KO mice.

Methods

Animals

Male Sprague-Dawley rats weighting 250–280 g (Seac Yoshitomi, Fukuoka, Japan), male C57BL/6N mice weighting 25–30 g (Seac Yoshitomi), and male H1KO mice weighting 25–30 g (Kyushu University, Fukuoka, Japan) were used. They were housed in a room with daily illumination from 07:00 to 19:00 (12/12-h light/dark cycle) and maintained at 21 ± 1°C with 55 ± 5% humidity. H1-R knockout (H1KO) mice that lack the H1-R neuronal histamine receptor for backcrossing were maintained at Oita University (Yufu, Japan). Backcrossing H1-R−/− homozygous mice with the C57BL/6N strain (Kyudo, Fukuoka, Japan) for six generations resulted in incipient congenital N5 mice with two genotypes (H1-R−/− and H1-R+/+). All genotypes were confirmed by Southern blotting as described (Inoue et al. 1996). Animals were fed standard chow (Clea chow, Clea, Japan), allowed access to and tap water ad libitum, and handled for 5 min each on four successive days to habituate arousal levels before the experiment. On test days, all animals had recovered to at least their pre-experiment body weight. All experiments proceeded in accordance with the Oita University Guidelines, which are based on the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.

Surgery

Rats were placed in a stereotaxic apparatus under intraperitoneal (i.p.) anesthesia with 100 mg/kg of sodium pentobarbital (Narishige, Tokyo, Japan). The implantation of a chronic cannula (a 23-gauge stainless steel; length, 15 mm) into the third cerebroventricle (3vt) was performed at least 10 days before starting infusions. A 30-gauge stainless steel wire stylet was inserted into the guide cannula to prevent cerebrospinal fluid (CSF) leakage and cannula obstruction. A chronic cannula was inserted into the 3vt along the midline, 6.0-mm anterior to the zero ear bar coordinate, to a depth of 7.8 mm from the cortical surface according to the atlas of Paxinos and Watson (1986).

At least 1 week before starting infusions, a cannula was similarly implanted into the lateral cerebroventricle (lvt) of the mice, 1.0-mm lateral to the midline, and 0.22-mm posterior to the bregma at a depth of 2.0 mm from the cortical surface, according to the atlas of Franklin and Paxinos (1997).

Reagents

On the day of the experiment, l-histamine (0.1 μmol/μL), recombinant nesfatin-1 (5 ng/μL; Sigma Chemical Co., St Louis, MO, USA), α-helical CRH9-41 (α-helical CRH, 1 μg/μL; Sigma) which is a competitive CRH receptor antagonist that blocks both CRH types 1 (CRH1-R) and 2 (CRH2-R) receptors, rabbit anti-TRH serum that neutralizes endogenous TRH of rat (Antibodies-online GmbH, Atlanta, GA, USA), pargyline hydrochloride (1 mmol/mL), and α-fluoromethylhistidine (FMH), a suicide inhibitor of histidine decarboxylase (HDC) (100 μg/μL; Research Biochemical International, Natick, MA, USA) were dissolved in a fresh phosphate-buffered saline (PBS) and the pH was adjusted to 6.5–7.5.

Experimental protocol

Experiment 1: A total of 108 rats were divided into 18 groups (= 6 per group) as follows: the PBS/PBS (4 groups, n = 6 in each), FMH/PBS (2 groups, n = 6 in each), PBS/nesfatin-1(3vt, 3 groups, n = 6 in each), PBS/nesfatin-1(i.p.), FMH/nesfatin-1(3vt), FMH/nesfatin-1(i.p.), α-helical CRH (10 or 50 μg)/PBS (n = 6 in each), α-helical CRH (50 μg)/nesfatin-1(3vt), anti-TRH serum/PBS, and anti-TRH serum/nesfatin-1(3vt) groups. They were pre-treated with an 3vt injection of FMH (1 mg/10 μL/10 min; this dose depletes most neuronal histamine in the hypothalamus), α-helical CRH (10 or 50 μg/10 μL/10 min), anti-TRH serum (10 μL/10 min, this dose decreases rectal temperature by approximately 1°C; Prasad et al. 1980) or PBS (10 μL/10 min) at 2 h before a further 3vt or i.p. injection of nesfatin-1 (30 ng/10 μL/10 min), or 3vt infusion of PBS (10 μL/10 min).

Experiment 2: Twelve wild-type and 12 H1KO mice were divided into the nesfatin-1 (5 ng/1 μL/1 min; lvt) and PBS (1 μL/1 min; lvt) groups, respectively (= 6 in each).

Measurement of food intake in rats and mice

Food intake was measured in rats and mice fed ad libitum for 4 h during the dark phase (between 19:00 and 23:00 h) after the administration of each agent as described above, using an indirect calorimetry system (Columbus Instruments, Columbus, OH, USA).

CRH, TRH, and tele-methylhistamine contents, and histamine-regulated changes in hypothalamic and plasma nesfatin-1 levels

Forty-two rats were divided into seven equal groups (= 6 per group) as follows: the PBS/PBS, α-helical CRH/PBS, anti-TRH serum/PBS, PBS/nesfatin-1(3vt), PBS/nesfatin-1(i.p.), α-helical CRH/nesfatin-1 (50 ng, 3vt), and anti-TRH serum/nesfatin-1(3vt) and PBS/histamine (1 μmol/10 μL/10 min) groups. Each group was pre-treated by i.p. administration of pargyline hydrochloride (0.33 mmol/kg), which inhibits monoamine oxidase B and induces the extraneuronal accumulation of tele-methylhistamine (t-MH), a major metabolite of released neuronal histamine. All rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and then given a transcardiac perfusion of 100 mL of saline containing 200 U heparin. Each rat was decapitated, and then sections containing the hypothalamus were cut according to the rat brain atlas of Paxinos and Watson (1986) to measure t-MH content. Tissue blocks were homogenized in 400 μL of 0.5 M acetic acid, boiled for 10 min, and then protein was assayed (Bio-Rad, Hercules, CA, USA) in 50 μL aliquots. Blood from all rats obtained by cardiac puncture was collected into tubes containing EDTA (7.5%, 10 μL/0.5 mL blood; Sigma Chemical Co.) and aprotinin (0.6 U trypsin inhibitor/0.5 mL blood; ICN Pharmaceuticals, Costa Mesa, CA, USA) and then separated by centrifugation at 4000 g for 10 min at 4°C. Plasma was immediately frozen and stored at −80°C. The CRH and TRH contents were measured in PVN-specific hypothalamus section punches dissected with a frozen blade, using CRH (Yanaihara Co. Shizuoka, Japan) and a TRH (Life Science Inc., Houston, TX, USA) ELISA kits. Plasma and hypothalamic nesfatin-1 levels were measured in punches from LH-, PVN-, VMH-, and TMN-specific sections using a nesfatin-1 EIA kit (Phoenix Pharmaceuticals Inc., Burlingame, CA, USA). The t-MH content in the LH, PVN, VMH, and TMN was assayed by HPLC using deproteinized supernatants containing amine extracts. Details of the amine assays are described elsewhere (Sakata et al. 1981).

Immunohistochemistry

Rats were anesthetized using 100 mg/kg sodium pentobarbital i.p., and killed by transcardiac perfusion with 50 mL PBS that contained 50 U heparin, followed by 50 mL 4% p-formaldehyde in PBS. The brains were removed and divided into forebrain, diencephalon, and brainstem segments. The specimens were rapidly frozen at −80°C and sectioned at a thickness of 40 μm using a cryostat at −20°C. The sections were incubated overnight at 4°C with polyclonal rabbit antiserum against rat HDC (1 : 2000; Chemicon Inc., Temecula, CA, USA), followed by biotin-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate-conjugated streptavidin (ABC reagent; Vector Laboratories, Burlingame, CA, USA). Next, they were incubated with a specific polyclonal rabbit antiserum against rat nesfatin-1 (1 : 2000; Phoenix Pharmaceuticals Inc.) in 0.3% Triton X-100 containing 1% normal rabbit serum. Biotin-conjugated secondary IgG antibodies (ABC reagent; Vector Laboratories) were added, followed by rhodamine-conjugated streptavidin (ABC reagent; Vector Laboratories).

Other sections were incubated overnight at 4°C with a specific polyclonal rabbit antiserum against rat nesfatin-1 (1 : 2000; Phoenix Pharmaceuticals Inc.) and a specific polyclonal rabbit antiserum against rat H1-R (1 : 1000; Millipore, Billerica, MA, USA). The sections were then incubated with biotin-conjugated secondary IgG antibody (ABC reagent; Vector Laboratories), fluorescein isothiocyanate-conjugated streptavidin for H1-R and rhodamine-conjugated streptavidin (ABC reagent; Vector Laboratories) for nesfatin-1. The negative controls in each experiment were incubated with normal serum instead of the primary antibody (nesfatin-1 or H1-R), followed by both secondary antibodies. We determined the specificity of the nesfatin-1 and H1-R antisera, after incubating each with recombinant nesfatin-1 (Sigma) or H1-R (Gene Tex Inc., Irvine, CA, USA) protein. Increasing amounts of protein were added to a fixed concentration of nesfatin-1 and H1-R antiserum at a 1 : 1, 1 : 5, and 1 : 10 molar ratio to the concentration of each recombinant protein. Stained sections were analyzed using a confocal immunofluorescence microscope (Olympus, Tokyo, Japan) and imaging software (Lumina Vision; Mitsutani Corp., Tokyo, Japan). The resultant digital images of the same section were not adjusted or altered, except for an occasional change in brightness. Nesfatin-1 or H1-R immunoreactivity was determined by drawing areas of PVN in hypothalami and applying an optical density scale using automated counting tools from the imaging software (Lumina Vision; Mitsutani Corp.). Ratios of co-expressed signals were also assessed by counting single-labeled nesfatin-1 and double-labeled nesfatin-1 and H1 receptor-positive neurons. Every fifth section of serial hypothalamus samples from six rats (seven sections per rat) was analyzed.

Western blotting

We examined the molecular weight of proteins recognized by the nesfatin-1 and H1-R antibodies by western blotting recombinant nesfatin-1 (Sigma), and H1-R (Gene Tex Inc.) protein in the hypothalamus of rats. These proteins were diluted to final concentrations in sample buffer (50 mM Tris-HCl, pH 8.0, 10% glycerol, 1 mM EDTA, 2 mM dithiothreitol, and 1 mM o-vanadate), heated at 94°C for 4 min, and then separated by centrifugation (12 000 g) for 5 min. The supernatants were resolved by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene difluoride membranes. After blocking non-specific binding with 0.5% non-fat milk, the membranes were incubated overnight at 4°C with the same anti-nesfatin-1 and anti-H1-R antisera. The membrane was repeatedly washed and then incubated with IgG secondary antibodies. Immunopositive bands were visualized on Hyperfilm (GE Healthcare Bioscience, Piscataway, NJ, USA) using enhanced chemiluminescence.

In mice, hypothalamic preparations were homogenized in sample buffer, separated by centrifugation and the supernatant was boiled for 4 min. The total protein content of the tissue was measured using the Bradford method. Equal amounts of total protein were resolved by electrophoresis on 8% sodium dodecyl sulfate–polyacrylamide gels and then electrophoretically transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories). Non-specific binding on the membranes was blocked with 5% non-fat milk for 1 h, and then the membranes were incubated overnight with primary antibodies at 4°C followed by secondary antibody for 1 h at 20°C. The primary antibody was polyclonal rabbit anti-human nesfatin-1 (with specificity for rat and mouse nesfatin-1; LifeSpan Bioscience Inc., Seattle, WA, USA). Nesfatin-1 was detected using enhanced chemiluminescence (Amersham Life Sciences, Arlington Heights, IL, USA). Each protein was measured using Quantity One imaging software (Bio-Rad).

Statistics

Data are expressed as means ± SEM. Statistical significance was evaluated using a two-way analysis of variance (anova) followed by the Scheffé test for post hoc comparisons. The level of significance for all tests was established at < 0.05.

Results

Effect of FMH on subsequent nesfatin-1-induced suppression of food intake and effect of nesfatin-1 on t-MH content in rat hypothalamus

The i.v.t. administration of nesfatin-1 significantly decreased food intake for 4 h compared with PBS and FMH partially attenuated this decrease (both = 6 per group, < 0.05). In contrast, FMH alone did not affect cumulative food intake compared with the control (Fig. 1a). Nesfatin-1 increased the pargyline-induced accumulation of t-MH in the PVN, VMH, and TMN, but not in the LH, compared with PBS (= 6 per group, < 0.05; Fig. 1b). However, nesfatin-1 administered intraperitoneally did not affect either appetite (Fig. 1c) or t-MH accumulation (Fig. 1d). Moreover, plasma levels of nesfatin-1 did not significantly differ after either i.v.t. or i.p. administration (Fig. 1e). Immunohistochemical analysis of double-labeled neurons revealed very few nesfatin-1-positive fibers projecting into to histamine neurons in the TMN (2.4 ± 0.5%; Fig. 1f), whereas nesfatin-1-positive neurons were detected in the PVN (Fig. 1g). These findings suggested that few nesfatin-1 neurons localized in the PVN directly activate histamine neurons in the TMN.

Figure 1.

Food intake at 4 h after intracerebroventricular (i.v.t.; a) and intraperitoneal (i.p.; c) administration of phosphate-buffered saline (PBS), α-fluoromethyl histidine (FMH), nesfatin-1, or FMH followed by nesfatin-1. Hypothalamic tele-methylhistamine content after i.v.t. (b) or i.p. (d) infusion of PBS or nesfatin-1, FMH + nesfatin-1, and FMH followed by nesfatin-1. Plasma nesfatin-1 levels after i.v.t. or i.p. administration of nesfatin-1 (e). *< 0.05 versus PBS and FMH; < 0.05 versus nesfatin-1 (i.v.t.). Immunohistochemical staining for nesfatin-1 (red) in histaminergic neurons (green; yellow arrows) in the tuberomammillary nucleus (TMN) (f). Very few histamine neurons are co-localized with nesfatin-1-positive fibers (blue arrow). Immunohistochemical staining for nesfatin-1 in the paraventricular nucleus (PVN) (g). Scale bar, 10 μm.

Effect of α-helical CRH pre-treatment on nesfatin-1-induced suppression of food intake and effect of nesfatin-1 infusion on CRH and t-MH contents in rat hypothalamus

Both 10 and 50 ng of α-helical CRH attenuated the nesfatin-1-induced suppression of food intake (= 6 per group, < 0.05), whereas α-helical CRH alone did not alter feeding behavior compared with PBS (Fig. 2a). Nesfatin-1 significantly increased CRH content in the PVN compared with PBS (n = 6 per group, < 0.05; Fig. 2b). Nesfatin-1 also increased pargyline-induced t-MH accumulation in the PVN, VMH, and TMN, but not in the LH, and α-helical CRH attenuated this effect (= 6 per group, < 0.05; Fig. 2c).

Figure 2.

Food intake at 4 h after central administration of phosphate-buffered saline (PBS), α-helical corticotropin-releasing hormone (CRH), nesfatin-1, or α-helical CRH followed by nesfatin-1 (a). *< 0.05 versus PBS and α-helical CRH; < 0.05 versus nesfatin-1. Contents of CRH in hypothalamus after central infusion of PBS or nesfatin-1 (b). *< 0.05 versus PBS. Hypothalamic t-MH content after central administration of PBS, α-helical CRH, nesfatin-1, or α-helical CRH followed by nesfatin-1(c). *< 0.05 versus PBS and α-hCRH; < 0.05 versus nesfatin-1. Food intake at 4 h after central administration of PBS, anti-thyrotropin-releasing hormone (TRH), nesfatin-1, or anti-TRH followed by nesfatin-1 (d). *< 0.05 versus PBS and anti-TRH, < 0.05 versus nesfatin-1. Hypothalamic TRH content after central infusion of PBS or nesfatin-1 (e). *< 0.05 versus PBS. Hypothalamic t-MH content after central administration of PBS, anti-TRH, nesfatin-1, or anti-TRH followed by nesfatin-1 (f). *< 0.05 versus PBS and anti-TRH; < 0.05 versus nesfatin-1. α-hCRH, α-helical CRH.

Anti-TRH antiserum diminished the subsequent nesfatin-1-induced appetite loss like CRH (= 6 per group, < 0.05), but did not affect food intake alone (Fig. 2d). Nesfatin-1 increased the TRH content in the PVN compared with PBS (n = 6 per group, < 0.05; Fig. 2e). Moreover, nesfatin-1 elevated pargyline-induced t-MH accumulation in the PVN, VMH, and TMN, but not in the LH, and prior loading with anti-TRH antiserum attenuated this effect (= 6 per group, < 0.05; Fig. 2f).

Attenuation of the anorexic effect of nesfatin-1 in H1KO mice

The lvt administration of nesfatin-1 led to approximate 40% and 25% reductions in food intake by wild-type and H1KO mice, respectively, within 4 hours (both = 6 per group, < 0.05; Fig. 3).

Figure 3.

Food intake 4 h after central administration of phosphate-buffered saline (PBS) or nesfatin-1 in wild-type and H1KO mice. *< 0.05 versus PBS (wild type and H1KO); < 0.05 versus nesfatin-1 (wild type).

Effect of histamine infusion on nesfatin-1 content in rat hypothalamus

Histamine significantly increased nesfatin-1 contents in the PVN, but not in the LH, VMH, or TMN of the rat hypothalamus compared with PBS (= 6 per group, < 0.05; Fig. 4a). The effects of histamine and PBS on plasma nesfatin-1 levels did not significantly differ (Fig. 4b).

Figure 4.

(a and b): Nesfatin-1 content in the hypothalamus (a) and plasma nesfatin-1 levels (b) after central infusion of phosphate-buffered saline (PBS) or histamine. *< 0.05 versus PBS. (c) Nesfatin-1 expression in the hypothalamus in wild type and H1KO mice. *< 0.05 versus wild-type mice.

Differential hypothalamic nesfatin-1 expression in wild-type and H1KO mice

Nesfatin-1 expression in the hypothalamus was significantly decreased in H1KO, compared with wild-type mice (= 6 per group, < 0.05; Fig. 4c).

Immunohistochemical staining for nesfatin-1 and H1-R in the PVN

Immunohistochemical analysis of double-labeled neurons revealed several nesfatin-1-positive neurons expressing H1-R in the PVN (Fig. 5a–c). We pre-absorbed the antibodies with purified nesfatin-1 and H1-R protein to examine their specificity. The immunohistochemical findings showed significantly less antibody labeling compared with the absence of absorption (Fig. 5d–g). Moreover, labeling specificity was controlled by omitting the nesfatin-1 and H1-R antibodies (Fig. 5h and j). Figure 5k shows the semi-quantitative findings of nesfatin-1 immunolabeling after pre-absorption with nesfatin-1 protein. With increasing concentrations of recombinant nesfatin-1 protein relative to primary antibody (1 : 1, 1 : 5, and 1 : 10 molar ratio of nesfatin-1 antibody concentration to recombinant protein concentration), nesfatin-1 immunolabeling was decreased significantly compared with immunolabeling using the primary antibody alone. The staining intensity of the anti-nesfatin-1 antibody signal for nesfatin-1 protein was linearly reduced up to fivefold the molar ratio of antigen to antibody. Pre-absorption with H1-R peptide yielded similar results (Fig. 5l). Moreover, an immunopositive band was evident at the predicted molecular weight of full length NUCB2 (50 kDa), the nesfatin-1 (∼10 kDa) and H1-R (∼57 kDa) proteins, indicating that the respective primary antibodies recognized nesfatin-1 and H1-R (Fig. 5m).

Figure 5.

Nesfatin-1 neurons (red) express H1-Rs (green) (a–c). Nesfatin-1 or H1-R antibody pre-absorbed without (d and e) or with (f and g) 1× nesfatin-1 or H1-R protein. Signals are undetectable after substituting nesfatin-1 or H1-R antibodies with normal serum and incubation with secondary antibody for staining with primary antibodies (h and j). Scale bar, 10 μm. Bar graph shows intensity of nesfatin-1 and H1-R labeling with and without nesfatin-1 and H1-R recombinant protein at 1 : 1 (×1 pro.), 1 : 5 (×5 pro.), and 1 : 10 (×10 pro.) molar concentrations of nesfatin-1 or H1-R antibodies (k and l). Values in graph represent mean total immunolabeling; *< 0.05 versus nesfatin-1 or H1-R; < 0.05 versus nesfatin-1 + 1 × 1 pro. or H1-R + 1 × 1 pro. Nes-1 means nesfatin-1. Pro. means protein. (m) Western blots show that nesfatin-1 and H1-R antibodies detected NUCB2 (~50 kDa), nesfatin-1 (~10 kDa), and H1-R (~57 kDa) proteins, respectively.

Discussion

Nesfatin-1, CRH, TRH, and hypothalamic neuronal histamine exert anorexigenic effects in the hypothalamus, but their functional relationships are not clear, particularly those between nesfatin-1 and histamine. Our results showed that FMH-induced depletion of neuronal histamine or H1-R deficiency partially attenuated the subsequent nesfatin-1-induced suppression of food intake. These findings indicate that endogenous neuronal histamine and associated H1-R partially mediate the anorexigenic effect of nesfatin-1. Thus, understanding how nesfatin-1 affects hypothalamic neuronal histamine levels is important. After release from nerve terminals, histamine is rapidly converted into the metabolite, t-MH, which is then deaminated, indicating that more information is generated about histamine by measuring metabolite concentrations than by measuring histamine itself. Pargyline, a monoamine oxidase B inhibitor, induces t-MH accumulation in extraneuronal spaces (Oishi et al. 1987). We showed that nesfatin-1 increased t-MH levels in the PVN, VMH, and TMN, but not in the LH. However, very few nesfatin-1-positive fibers projected into histamine neurons, suggesting that nesfatin-1 indirectly activates histamine neurons. Furthermore, we found that nesfatin-1 increased the CRH and TRH contents in the PVN, suggesting that nesfatin-1 increases histamine turnover, synthesis and release in the hypothalamus via CRH and/or TRH neurons in the PVN.

A recent study has shown that nesfatin-1 influences the activity of a large proportion of PVN neurons, including those containing CRH and TRH in vitro (Price et al. 2008). Because the activation of brain CRH and TRH signaling pathways inhibits food intake (Kow and Pfaff 1991; Richard et al. 2000), we investigated whether endogenous CRH or TRH activation mediated the central action of nesfatin-1 on food intake. We demonstrated that a central nesfatin-1 infusion increased CRH and TRH levels in the PVN of the hypothalamus. The central administration of α-helical CRH and anti-TRH attenuated the nesfatin-1-induced reduction in food intake, suggesting that CRH and TRH mediate the anorectic effect of nesfatin-1 in the hypothalamus.

Our results provide insight into the relationships among CRH, TRH, and neuronal histamine. We previously demonstrated that CRH- and TRH-induced increases in histamine turnover are suppressed by α-helical CRH and anti-TRH pre-treatment, respectively. Moreover, CRH1-Rs and TRH type 2 receptors (TRH2-Rs) are expressed on the cell bodies of histamine neurons in the TMN (Gotoh et al. 2005, 2007). Thus, we speculated that nesfatin-1 signaling regulates neuronal histamine via CRH and/or TRH. Our results support this hypothesis because α-helical CRH and anti-TRH attenuated the subsequent nesfatin-1-induced increase in hypothalamic histamine turnover. We demonstrated that nesfatin-1 suppressed food intake by activating histamine neurons in the TMN via CRH and TRH neurons, which supports the finding that 20–30% of PVN nesfatin-1 cells synthesize TRH and that a subpopulation of about 20% of nesfatin-1 PVN neurons co-express CRH, which inhibits food intake (Stengel et al. 2010). Nesfatin-1 was found to act centrally to reduce dark-phase food intake through CRH pathways, using the non-selective CRH antagonist, astressin B; however, α-helical CRH diminished nesfatin-1-induced hypophagia (Stengel et al. 2009a). These data support our finding that the nesfatin-1-induced appetite loss involves TRH neurons. The mechanism underlying this discrepancy is not clear, but one possible explanation is that it is due to differences in pharmacological mechanism between these CRH antagonists.

Here, essential questions can be raised as to why hyperphagia was not evident in rats depleted of histamine by FMH and why food intake did not differ between wild-type and H1KO mice. A definite answer to the query has not yet been proposed. However, these results are compatible with the findings that histamine depletion by FMH does not affect daily food intake, and that H1KO mice tended to consume more basal food than in wild-type mice; but this effect was not significant (Yoshimatsu et al. 1999; Mollet and Lutz 2001). One possible explanation is that the differences in mean daily food intake between PBS and FMH administration as well as between wild-type and H1KO mice are relatively small compared with the deviation of daily food intake in both groups. In addition, neither α-helical CRH nor anti-TRH alone increased food intake. These results are also compatible with those of others showing that α-helical CRH attenuates the leptin-induced reduction of food intake although a single administration of α-helical CRH does not significantly affect food consumption, and TRH as well as a TRH-R deficiency does not affect feeding behavior (Yamada et al. 1997; Uehara et al. 1998; Zeng et al. 2007; Sun et al. 2009). A higher dose of α-helical CRH and anti-TRH might have resulted in significantly less food intake compared with a control group as well as a more sustained attenuation of the anorexigenic effect of nesfatin-1.

Finally, we examined whether neuronal histamine activated nesfatin-1 neurons. We showed that histamine activates nesfatin-1 neurons in the PVN, suggesting that histamine neurons may activate some nesfatin-1 neurons via CRH and TRH release. Others have shown that central leptin administration activates CRH and TRH neurons and increases histamine turnover in the hypothalamus (Yoshimatsu et al. 1999; Nillni et al. 2000). Leptin-induced suppression of food intake is attenuated in rats pre-treated with FMH, which depletes neuronal histamine, and in H1KO mice that lack H1-R (Yoshimatsu et al. 1999). Moreover, we previously demonstrated that CRH directly mediated the effect of leptin signaling on neuronal histamine turnover and TRH stimulated histamine neurons (Gotoh et al. 2005, 2007). Furthermore, the results of this study showed that nesfatin-1 administration activates CRH, TRH, and histamine neurons, all of which are involved in the regulation of leptin in the hypothalamus. However, leptin does not appear to be a modulator of NUCB2 and nesfatin-1 expression in hypothalamic nuclei (Oh-I et al. 2006; Garcia-Galiano et al. 2010). This observation confirms the suggestion that nesfatin-1 has a leptin-independent mode of action (Shimizu et al. 2009). Thus, we speculated that the anorexigenic action of nesfatin-1 is mediated by CRH, TRH, and neuronal histamine via leptin-independent pathways, although a leptin to CRH, TRH, and neuronal histamine signaling cascade exists (Gotoh et al. 2005, 2007).

Nesfatin-1 is synthesized in several areas of the hypothalamus, including the PVN, VMH, ARC, and LH, where it co-localizes with neurotransmitters/neuropeptides (Oh-I et al. 2006; Brailoiu et al. 2007; Foo et al. 2008; Fort et al. 2008). These hypothalamic nuclei play important roles in the regulation of energy metabolism, and they contain receptors that signal nutritional status, including leptin levels (Mercer et al. 1996). Far less is known about the role of nesfatin-1 in the LH. The LH has often been referred to as a feeding center because lesions in this region result in hypophagia and weight loss (Anand and Brobeck 1951). The LH contains cells that synthesize melanin-concentrating hormone (MCH) and orexin, both of which are orexigenic factors (Date et al. 1999). We found that the central administration of histamine increased nesfatin-1 content in the PVN, but not in the LH, suggesting that nesfatin-1 does not act in the LH to regulate feeding behavior. This finding is not consistent with a previous study showing that about 80% of the nesfatin-1-positive neurons were double labeled with MCH (Fort et al. 2008). Co-expression of nesfatin-1 and MCH suggests a complex physiological relationship because nesfatin-1 induces satiety (Oh-I et al. 2006), whereas MCH stimulates the appetite (Pissios et al. 2006). Moreover, the central administration of nesfatin-1 increases orexin mRNA expression in fed goldfish, but decreases it in unfed goldfish, suggesting that the effect of nesfatin-1 on brain expression of peptides that regulate appetite might may depend on feeding status (Kerbel and Unniappan 2012). Considering the opposing anorectic and orexigenic actions of nesfatin-1 and MCH, nesfatin-1 might modulate the effect of MCH in integrative feeding behavior.

Intraperitoneal and subcutaneous routes of nesfatin-1 administration suppress food intake and thus, peripheral nesfatin-1 probably crosses the blood–brain barrier (BBB) and acts in the hypothalamus to regulate energy metabolism and appetite (Shimizu et al. 2009). Nesfatin-1 has been detected in rat serum (but its origin remains unknown) and in rat gastric mucosa (Stengel et al. 2009b). Furthermore, nesfatin-1-immunoreactive cells co-localize with insulin in pancreatic islets (Gonzalez et al. 2009) implying that centrally infused nesfatin-1 might leak into the periphery via the venous system. Our results showed that the central administration of a single dose of nesfatin-1 at a concentration too low to exert effects when delivered peripherally immediately suppressed feeding behavior. We found no significant difference in plasma nesfatin-1 levels after the i.v.t. and i.p. administration of nesfatin-1. These findings support that the dose of peripherally administered nesfatin-1 required to reduce food intake is about 1000-fold higher than effective dose of centrally treated nesfatin-1 (Stengel et al. 2010).

We developed a working model of the feeding and satiety neuronal network via signaling between nesfatin-1 and histamine (Fig. 6). Nesfatin-1 might act on CRH and TRH neurons in the PVN, and then activated CRH and TRH neurons might in turn regulate histamine neurons in the TMN of the hypothalamus through CRH1-R and TRH2-R, respectively. Furthermore, we suggest that neuronal histamine stimulates nesfatin-1 neurons in the PVN directly via H1-R. This study provides novel insight into the action of nesfatin-1 in the hypothalamic regulation of energy metabolism.

Figure 6.

Proposed model for interactions among nesfatin-1, corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), and histamine neurons in regulation of food intake. Nesfatin-1 neurons in paraventricular nucleus (PVN) regulate tuberomammillary nucleus (TMN) histamine neurons via CRH and/or TRH neurons for which receptors are expressed on the histamine neurons. Neuronal histamine directly suppresses food intake via H1-R, which is expressed on neurons (including nesfatin-1 neurons) in the ventromedial nucleus (VMH) and PVN that are related to feeding. Such neurons produce peptides such as BDNF and NPY or co-localize with H1-R and influence feeding behavior.

Acknowledgments

This study was supported by a grant for Research on Measures for Intractable Diseases from Japan's Ministry of Health, Labour, and Welfare. The authors do not have any conflicting or competing interests.

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