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Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs
Accepted Manuscript Title: Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs Authors: Satomi Sonoda, Mitsuhiro Yoshimura, Chikara Abe, Hironobu Morita, Hiromichi Ueno, Yasuhito Motojima, Reiko Saito, Takashi Maruyama, Hirofumi Hashimoto, Yoshiya Tanaka, Yoichi Ueta PII: DOI: Reference:
S0196-9781(18)30102-5 https://doi.org/10.1016/j.peptides.2018.05.004 PEP 69971
To appear in:
Peptides
Received date: Revised date: Accepted date:
6-3-2018 30-4-2018 6-5-2018
Please cite this article as: Sonoda Satomi, Yoshimura Mitsuhiro, Abe Chikara, Morita Hironobu, Ueno Hiromichi, Motojima Yasuhito, Saito Reiko, Maruyama Takashi, Hashimoto Hirofumi, Tanaka Yoshiya, Ueta Yoichi.Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs.Peptides https://doi.org/10.1016/j.peptides.2018.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs
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Satomi Sonodaa,d,*, Mitsuhiro Yoshimuraa,*, Chikara Abeb, Hironobu Moritab,
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Hiromichi Uenoa, Yasuhito Motojimaa, Reiko Saitoa, Takashi Maruyamaa, Hirofumi Hashimotoc, Yoshiya Tanakad, and Yoichi Uetaa
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Department of Physiology and dFirst Department of Internal Medicine, School of
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a
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Medicine, University of Occupational and Environmental Health, Kitakyushu
Department of Physiology, Gifu University Graduate School of Medicine, Gifu
501-1194, Japan
Department of Regulatory Physiology, Dokkyo Medical University, Tochigi 321-0293,
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Japan
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c
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b
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807-8555, Japan
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*These authors equally contributed to this work.
Corresponding author: Yoichi Ueta, M.D., Ph.D. Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
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Tel.: +81-93-691-7420; Fax: +81-93-692-1711 e-mail: [email protected]
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Highlights: ・Involvement of hypergravity and vestibular system on feeding regulation was
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examined ・Vestibular dysfunction was generated by vestibular lesion (VL)
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・Hypothalamic gene expression was examined by in situ hybridization histochemistry
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・ CRH and POMC were increased significantly in Sham but not in VL in 2g
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environment
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Abstract
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・CRH and POMC may be responsible for feeding regulation in different gravity
The effects of hypergravity on the gene expression of the hypothalamic
feeding-related neuropeptides in sham-operated (Sham) and vestibular-lesioned (VL)
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mice were examined by in situ hybridization histochemistry. Corticotrophin-releasing hormone (CRH) in the paraventricular nucleus was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2 g environment compared with a 1g environment. Significant decreases in pro-opiomelanocortin (POMC) and cocaine2
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and amphetamine-regulated transcript and significant increases in neuropeptide Y, agouti-related protein in the arcuate nucleus and orexin in the lateral hypothalamic area were observed in both Sham and VL mice. After 2 weeks of exposure, CRH and
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POMC were increased significantly in Sham but not in VL mice. After 8 weeks of
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exposure, the hypothalamic feeding-related neuropeptides were comparable between Sham and VL mice. These results suggest that the hypothalamic feeding-related neuropeptides may be affected during the exposed duration of hypergravity via
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vestibular inputs.
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histochemistry, vestibular lesion.
neuropeptides, in situ hybridization
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Keywords: hypergravity, hypothalamus,
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Abbreviations
ACTH, adrenocorticotropic hormone; AgRP, agouti-related protein; Arc, arcuate
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nucleus; CART, cocaine- and amphetamine-regulated transcript; CRH, corticotrophin-releasing hormone; CRH-R1, the type 1 CRH receptor; CRH-R2, the type 2 CRH receptor; HPA, hypothalamic-pituitary-adrenal; LHA, lateral hypothalamic area; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; OD,
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optical densities; POMC, pro-opiomelanocortin; PVN, paraventricular nucleus; SEM, standard error of the mean; Sham, sham-operated; VL, vestibular lesion
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1. Introduction
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Centrifugation is often used to examine biological responses to the artificial
hypergravity environment in rodents [1, 2]. Transient hypophagia followed by reducing the body mass is a common biological response to centrifugation-induced
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hypergravity [3]. Since the reduced food intake is fully or partially ameliorated by the
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vestibular lesion (VL), the vestibular system is, at least in part, involved in the
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hypergravity-induced hypophagia.
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The feeding behavior is controlled by a complex, central signaling network of neuropeptides in the hypothalamus [4, 5]. The hypothalamic neural circuits, including
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various neuropeptide-containing neurons are involved in the regulation of the feeding
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behavior and body mass [4-6]. Abe et al. reported that the immunoreactivity of corticotrophin-releasing hormone (CRH) in the paraventricular nucleus (PVN) after
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being exposed to a 2 g environment for 90 min was significantly increased but was attenuated by VL in rats [2]. However, to our knowledge, how the hypothalamic feeding-related neuropeptides change in the hypergravity environment remains unveiled. We hypothesized that vestibular function may be involved in the modulation
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of neuropeptides expressions in the hypergravity environment. To examine the involvement of hypothalamic feeding-related neuropeptides in the hypergravity-induced hypophagia, the gene expression of the hypothalamic
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feeding-related neuropeptides was compared between Sham and VL mice. We
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examined these neuropeptides at 3 days, 2 weeks, and 8 weeks after being exposed to a 2 g environment. Since hypergravity-induced hypophagia is transient and ameliorated by VL, neuropeptides, which are altered only in Sham but not in VL mice, may be a
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candidate neuropeptide responsible for hypergravity-induced hypophagia. The
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neuropeptides studied in the present study included 3 anorexigenic neuropeptides
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(CRH, pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated
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transcript (CART)) and 4 orexigenic neuropeptides (neuropeptide Y (NPY),
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agouti-related protein (AgRP), melanin-concentrating hormone (MCH) and orexin).
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2. Materials and methods
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2.1. Animals
Adult male C57BL/6J mice (weighing 19.0–27.6 g each) were group housed (6
mice per cage) and maintained in temperature-controlled (23-25°C) conditions under a 12/12-h light/dark cycle (lights on at 07:00 h). All mice had free access to food and
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water throughout the experiment. The food was consisted of 4.6% fat, 48.6% carbohydrate, 25.5% protein (total 14.2 kJ/g) (CLEA Rodent diet CE-2, CLEA Japan, Inc., Tokyo, Japan). Animals used in the present study were maintained in accordance
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with the “Guiding Principles for the Care and Use of Animals in the Field of
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Physiological Science” set by the Physiological Society of Japan. The experiments
were approved by the Animal Research Committees of Gifu University, University of
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Occupational and Environmental Health, and Japan Aerospace Exploration Agency.
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2.2. Surgical procedures
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All mice received Sham or VL surgery before the experiment. Six- to 7-week-old
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mice were anesthetized with isoflurane (Escain, Pfizer, Tokyo, Japan) inhalation via a face mask (2%), and the VL surgery was performed through an external auditory
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meatus approach as described previously in detail [7-9]. Penicillin G potassium (3000
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U/kg, Meiji Seika Pharma, Tokyo, Japan) and buprenorphine (3 µg/kg, Lepetan, Otsuka, Tokyo, Japan) were administered to the Sham and VL mice subcutaneously
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prior to returning the animals to their home cages (a group-housing cage, 6 mice per cage). Five to 7 days after the Sham or VL surgery, the success of the VL was confirmed by observing the swimming behavior of the mice [7-9]. During the recovery from Sham or VL surgery and during centrifugation, food was placed on the cage floor
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instead of on a wire bar lid [7].
2.3. Experimental procedures
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After recovery, the mice were divided into 4 groups: Sham-1g, VL-1g, Sham-2g,
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and VL-2g (n=6, each). They were exposed to a 1 g (Sham-1g, VL-1g) or 2 g
(Sham-2g, VL-2g) environment for 3 days, 2 weeks, and 8 weeks. The 2 g environment was developed by centrifugation of a custom-made gondola-type rotating
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box with a 1.5 m long-arm (Shimadzu, Kyoto, Japan) rotating at 32 rpm. Cages for 2 g
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groups were set in the rotating box. All the mice had access to food and water ad
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libitum, and the room temperature was maintained at 24 ± 1°C with a 12:12 h
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light-dark cycle (lights on at 07:00 h). For cage cleaning and water/food refreshment, rotation was stopped for 30 min once a week. At the end of each time point, the mice
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were decapitated immediately after being anesthetized with isoflurane (Escain, Pfizer,
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Tokyo, Japan) inhalation. Brains were removed, put promptly onto dry ice, and then
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stored at -80°C.
2.4. In situ hybridization histochemistry The brains were cut into 12 μm thickness coronal brain sections by using a cryostat (OTF5000, Bright Instrument Co Ltd, England) at –20˚C and thaw mounted
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on gelatin/chrome alum-coated slides. The locations of the PVN, arcuate nucleus (Arc) and lateral hypothalamic area (LHA), were determined according to the coordinates given in the mouse brain atlas [10].
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S 3’-end-labeled deoxyoligonucleotides
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complementary to the transcripts encoding CRH, pro-opiomelanocortin (POMC),
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cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) and orexin
were used. The specificity of these probes was confirmed by previous studies [11-13].
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The sequences of these probes used in situ hybridization histochemistry was
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summarized in the Table 1. The in situ hybridization protocol has been described exposed to
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previously in detail [14]. Hybridized sections which were
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autoradiography film (BioMax XAR film, KODAK, USA) for 1 week was analyzed by using an MCID imaging analyzer (Imaging Research, Inc., Ontario, Canada). The
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mean optical densities (OD) of the autoradiographs were measured by comparison
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with simultaneously exposed 14C-labeled microscale samples (Amersham, Bucks, UK)
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and represented in arbitrary units setting the mean OD obtained from control mice.
2.5. Statistical analysis In the results of the in situ hybridization, the gene expression of the hypothalamic feeding-related neuropeptides was expressed as a percentage of Sham-1g. All data
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were analyzed by one-way ANOVA followed by a Bonferroni-type adjustment for multiple comparisons (Origin Pro version 8.5J, Lightstone, Tokyo, Japan). Data are
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presented as the mean ± SEM. Statistical significance was set at P<0.05.
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3. Results
3.1. The body mass change after 3 days, 2 weeks, and 8 weeks exposure to
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hypergravity
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The body mass in each group (n=6, each) was measured after 3 days of exposure
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to a 1 g or 2 g environment: –0.03 ± 0.09 g in Sham-1g, 0.05 ± 1.15 g in VL-1g, –3.13
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± 0.32 g in Sham-2g, and –0.5 ± 1.03 g in VL-2g (Fig. 1A). The body mass gain of Sham-2g was significantly lower compared to other groups, whereas, those were
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comparable between Sham-1g and VL-1g. The body mass gain of VL-2g was
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markedly higher than Sham-2g. These data indicated that VL caused the attenuation of hypergravity-induced body mass loss. The body mass change after 2 weeks exposure
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to hypergravity (Fig. 1B) and after 8 weeks exposure to hypergravity (Fig. 1C) are shown in Fig. 1B and 1C, respectively. There were no significant differences in body mass changes among the groups after 2 or 8 weeks exposure to hypergravity.
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3.2. The gene expression of the hypothalamic feeding-related neuropeptides after 3 days exposure to hypergravity All examined gene expression levels of the hypothalamic feeding-related
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neuropeptides were comparable between Sham-1g and VL-1g at 3 days (Fig. 3). The
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gene expression of the CRH in the PVN was significantly increased in Sham-2g compared to Sham-1g (Fig. 3A). That of VL-2g was not increased compared to
Sham-1g (Fig. 3A). Those of the POMC and CART in the Arc were markedly
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decreased in Sham-2g and VL-2g compared to Sham-1g (Fig. 3Ba, b). NPY and AgRP
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in the Arc and orexin in the LHA were significantly increased in the groups that were
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exposed to 2 g but were comparable between these two groups (Fig. 3Ca, b and 3Db).
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MCH in the LHA were comparable among all the groups (Fig. 3Da). The representative digital images obtained from autoradiographs are shown in Fig. 2. CRH
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was expressed in the parvocellular divisions of the PVN (Fig. 2A). CRH was densely
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expressed only in Sham-2g but not in other groups (Fig. 2Aa-d). POMC/CART were expressed in the Arc (Fig. 2Ba-d, 2Ca-d). They were expressed lower in Sham-2g and
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VL-2g than in Sham-1g (Fig. 2Bc and d, 2Cc and d). NPY/AgRP (Fig. 2Da-d, 2Ea-d), which were also expressed in Arc, were expressed higher in Sham-2g and VL-2g than in Sham-1g (Fig. 2Dc and d, 2Ec and d). MCH and orexin were expressed in the LHA (Fig. 2Fa-d, 2Ga-d). The mean OD of MCH was to the same extent in all groups (Fig.
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2Fa-d). Orexin was expressed higher in Sham-2g and VL-2g than in Sham-1g (Fig. 2Gc and d).
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3.3. The gene expression of the hypothalamic feeding-related neuropeptides after 2
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weeks exposure to hypergravity
All examined gene expression levels of the hypothalamic feeding-related neuropeptides were comparable between Sham-1g and VL-1g at 2 weeks (Fig. 4). The
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gene expression of CRH in PVN was significantly increased in Sham-2g compared to
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Sham-1g (Fig. 4A). POMC in the Arc was markedly increased in Sham-2g compared
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to Sham-1g as well, while those changes were not observed in VL-2g (Fig. 4Ba).
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There were no significant differences in the gene expression of CART, NPY, AgRP,
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MCH, and orexin among the groups (Fig. 4Bb, Ca and b and Da and b).
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3.4. The gene expression of the hypothalamic feeding-related neuropeptides after 8 weeks exposure to hypergravity
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No statistical significance was seen in all the gene expression levels of the
neuropeptides among the groups (Fig. 5A-D).
4. Discussion
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We clarified that the gene expression of the hypothalamic feeding-related neuropeptides was affected by centrifugation-induced hypergravity. The gene expression of CRH in the PVN and POMC in the Arc, which is altered only in
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Sham-2g but not in VL-2g, may be a potent candidate of neuropeptides, which are
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responsible for the feeding regulation in different gravity environments mediated by a vestibular input.
CRH is known to be involved in the stress response and suppression of feeding
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anterior
pituitary,
resulting
in
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activation
of
the
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(ACTH)
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behavior [5, 15, 16]. It stimulates the secretion of the adrenocorticotropic hormone
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hypothalamic-pituitary-adrenal (HPA) axis. The type 1 CRH receptor (CRH-R1) and
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the type 2 CRH receptor (CRH-R2) have been cloned in rodents [17]. The 'fight-or-flight' response, which is characterized by the activation of the HPA axis, is
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mainly mediated by CRH-R1, whereas the stress-coping responses are mediated by
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CRH-R2 [18]. Hypophagia, which is induced by emotional stress, is partially mediated by CRH-R1 [19]. It is indicated that the increased gene expression of CRH may be a
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result of a stress response induced by a hypergravity environment. Although it is unclear which types of receptors are responsible for hypophagia developed by hypergravity, CRH-R1 may be strongly involved as CRH-R1 is activated in the earlier phase of the stress response [18].
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There are linkages between the vestibular system and hypothalamus. For example, there are neural inputs from the vestibular system to the hypothalamus [20]. Morita et al. demonstrated that in mice, there is a marked increase of Fos immunoreactivity in
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the PVN after 90 min exposure to 2 g, which was abolished by VL [1]. In het mice,
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which lack macular otoconia, Fos expression in the PVN was significantly suppressed after 2 g loading [21]. Abe et al. showed that the concentrations of plasma ACTH and corticosterone increase after 90 min exposure to 2 g. They also demonstrated that, after
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being exposed to hypergravity, the CRH in the PVN was markedly decreased in VL
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compared to Sham by using immunohistochemical method in rats [2]. Thus,
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CRH-producing neurons in the PVN may receive neuronal input from the vestibular
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systems. These reports are consistent with our results that increased the expression of CRH in the 2 g environment, which was completely abolished by VL. Whereas, they
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did not refer to the synthesis of CRH. Our results suggest that CRH synthesis increased
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after exposure to hypergravity as these results were obtained by using in situ hybridization histochemistry. The species of animals were also different, they used rats
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we used mice. We observed for longer duration after being exposed to hypergravity, up to 8 weeks, than previous reports [1, 2, 6, 21] to elucidate more chronic effects of hypergravity on the hypothalamic feeding regulated neuropeptides gene expression. Acute hypophagia-induced starvation, which was seen after 3 days exposure to a 2
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g environment, may cause the downregulation of the anorexigenic neuropeptides, such as POMC and CART, and the upregulation of the orexigenic neuropeptides such as NPY, AgRP and orexin. The starved state induced both the upregulation of NPY/AgRP
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and orexin neurons and the downregulation of the POMC/CART neuron [4, 13, 22].
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These alterations are thought to be a homeostatic response [4]. The results obtained
from the present study at 3 days of exposure to 2 g suggest that the changes in the feeding-related neuropeptides, except CRH, are due to the results of starvation. The
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reason why MCH at 3 days of exposure did not change was unclear. The gene
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expression of MCH was upregulated by fasting in rats or in hyperleptinemic ob/ob
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mice [23]. However, Nara-ashizawa et al. reported that the gene expression of MCH
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was not increased in cancer cachexia-anorexia syndrome [24]. The pathophysiological mechanisms of hypergravity-induced hypophagia and cancer cachexia-anorexia
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syndrome may be similar regarding the MCH upregulation.
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After being exposed to hypergravity for 2 weeks, the gene expression of the CRH and POMC showed dramatic increases in Sham-2g but not in VL-2g mice. We
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revealed hypergravity increased the gene expression of the POMC, which is a novel finding. We consider that the changes in the gene expression of the hypothalamic feeding-related neuropeptides have different meanings at each time point. For example, being exposed to hypergravity for 3 days reflects starvation. However, a previous
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study reported that rats being exposed to 3g ate the same amount of food as those being exposed to 1g after 2 weeks from the start of the experiment, compared to 3g [6]. We speculate that it takes over 2 weeks for the vestibular system plasticity to adapt to
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the different gravity. The results of 2 weeks of exposure may indicate inadequate
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adaptation for the different gravity rather than starvation or fasting. POMC in the Arc
is known to be regulated by several factors including leptin, insulin, and fasting. It is shown that leptin and insulin stimulates POMC neuron in the Arc in the previous
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studies by using electrophysiological methods [25, 26]. Although it is possible that the
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change of gravity caused the upregulation of the gene expression of POMC directly or
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indirectly, we could not explain the reason why the gene expression of POMC
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increased after hypergravity in our study. In addition, as CART neurons are known to be co-expressed with POMC neurons, they might fluctuate as POMC do. We would
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like to seek the mechanism in further study.
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Hypergravity may be sensed not only by the vestibular system but also by the somatosensory, proprioceptive and visceral sensors, which remain intact in VL animals.
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For example, in human astronauts, fluid redistribution, neurovestibular effects, muscle changes, bone demineralization, psychosocial effects, and immune dysregulation are elicited in different gravity environments [27]. The mice in the 2 g environment may be adapted to the hypergravity environment at 8 weeks. We considered that the
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vestibular system is accommodated to the new gravity at 8 weeks, as the vestibular system is known to be highly plastic and able to adapt to the new gravitational
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environment [1, 28].
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5. Conclusion
In conclusion, we clarified that the gene expression of the hypothalamic feeding-related neuropeptides is altered after exposure to a hypergravity environment.
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The gene expression of CRH and POMC may be a potent candidate of neuropeptides,
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Author contributions
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which are responsible for feeding regulation in different gravity environments.
The experiments were designed by S.S., M.Y., H.M., Y.T., and Y.U. The
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experiments were performed by S.S., M.Y., H.H., A.C., H.M., H.U., Y.M., R.S., and
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T.M. Data were analyzed by S.S. and M.Y. The draft and figures were prepared by S.S., M.Y., and Y.U. Final approval was made by Y.U. All authors approved the final version
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of the manuscript and agreed to be accountable for all aspects of the work regarding questions related to the accuracy. All authors designated as authors qualify for authorship, and all those who qualify for authorship are listed.
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Conflict of interest The authors declare that they have no conflict of interest.
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Acknowledgements
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We would like to thank Ms. Yuki Nonaka for her technical assistance.
Funding
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This study was funded by Grant-in-Aid for Young Scientists (B) (17K15575) for
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M.Y., a Grant-in-Aid for Scientific Research (B) (17H04027) for Y.U. and a
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Grant-in-Aid for Scientific Research on Innovative Areas (15H05940) for Y.U. from
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［20］Balaban CD, Thayer JF. Neurological bases for balance-anxiety links. J Anxiety Disord. 2001 Jan-Apr;15(1-2):53-79. PubMed PMID: 11388358. ［21］Fuller PM, Jones TA, Jones SM, Fuller CA. Neurovestibular modulation of
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circadian and homeostatic regulation: vestibulohypothalamic connection? Proc
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Natl Acad Sci USA. 2002 Nov;99(24):15723-8 PubMed PMID: 12434016; PubMed Central PMCID: PMC137783.
［22］Diano S, Horvath B, Urbanski HF, Sotonyi P, Horvath TL. Fasting activates the
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nonhuman primate hypocretin (orexin) system and its postsynaptic targets.
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Endocrinology. 2003 Sep;144(9):3774-8. PubMed PMID: 12933647.
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［23］Presse F, Sorokovsky I, Max JP, Nicolaidis S, Nahon JL. Melanin-concentrating
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hormone is a potent anorectic peptide regulated by food-deprivation and glucopenia in the rat. Neuroscience. 1996 Apr;71(3):735-45. PubMed PMID:
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8867046
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［24］Nara-ashizawa N, Tsukada T, Maruyama K, Akiyama Y, Kajimura N, Nagasaki K, et al. Hypothalamic appetite-regulating neuropeptide mRNA levels in
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cachectic nude mice bearing human tumor cells. Metabolism. 2001 Oct;50(10):1213-9. PubMed PMID: 11586496.
［25］Cowley MA, Smart JL, Rubinstein M, Cerdan MG, Diano S, Horvath TL, et al. Leptin activates anorexigenic POMC neurons through a neural network in the
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arcuate nucleus. Nature. 2001 May 24;411(6836):480-4. PubMed PMID: 11373681 ［26］Williams KW, Margatho LO, Lee CE, Choi M, Lee S, Scott MM, et al.
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Segregation of acute leptin and insulin effects in distinct populations of arcuate
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proopiomelanocortin neurons. J Neurosci. 2010 Feb17;30(7):2472-9. PubMed PMID: 20164331; PubMed Central PMCID: PMC2836776.
［27］Williams D, Kuipers A, Mukai C, Thirsk R. Acclimation during space flight:
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effects on human physiology. CMAJ. 2009 Jun;180(13):1317-23. PubMed
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PMID: 19509005; PubMed Central PMCID: PMC2696527.
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［28］Abe C, Tanaka K, Awazu C, Chen H, Morita H. Plastic alteration of
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vestibulo-cardiovascular reflex induced by 2 weeks of 3-G load in conscious
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rats. Exp Brain Res. 2007 Aug;181(4):639-46. PubMed PMID: 17492278.
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Figure Legends Fig. 1
The body mass change after 3 days (A), 2 weeks (B), and 8 weeks (C)
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exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. Data are presented as mean ± SEM (n=6, each). *P<0.05 vs. Sham-1g. #P<0.05 vs. Sham-2g.
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Fig. 2 The gene expression of the feeding-related neuropeptides after 3 days of exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC (Ba) and CART in the Arc
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(Bb), NPY (Ca) and AgRP in the Arc (Cb), and MCH (Da) and orexin in the LHA (Db)
SEM (n=6, each). *P<0.05 and **P<0.01 vs. Sham-1g.
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are shown. Signals for Sham-1g were set at 100%. Date are presented as the mean ±
35
S-labeled oligonucleotide probes complementary to CRH in the PVN
A
section with
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Fig. 3 Representative digital images obtained from autoradiographs of hybridized
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(Aa-d), POMC in the Arc (Ba-d), CART in the Arc (Ca-d), NPY in the Arc (Da-d),
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AgRP in the Arc (Ea-d), MCH in the LHA (Fa-d), and orexin in the LHA (Ga-d) after 3 days exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and
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vestibular-lesioned (VL) mice. Analyzed areas are surrounded by red dotted lines (Aa,
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Ba, Ca, Da, Ea, Fa and Ga). Scale bars=1 mm. Signal intensity ranges from high
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(black) to low (white).
Fig. 4 The gene expression of the feeding-related neuropeptides after 2 weeks of exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC in the Arc (Ba), CART in
23
24
the Arc (Bb), NPY in the Arc (Ca), AgRP in the Arc (Cb), MCH in the LHA (Da), and orexin in the LHA (Db) are shown. Signals for Sham-1g were set at 100%. Date are
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presented as the mean ± SEM (n=6, each). **P<0.01 vs. Sham-1g.
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Fig. 5 The gene expression of the feeding-related neuropeptides after 8 weeks of exposure to different gravity (1 g or 2 g) in the sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC in the Arc (Ba), CART in
N
U
the Arc (Bb), NPY in the Arc (Ca), AgRP in the Arc (Cb), MCH in the LHA (Da) and
A
orexin in the LHA (Db) are shown. Signals for Sham-1g were set at 100%. Date are
A
CC E
PT
ED
M
presented as the mean ± SEM (n=6, each).
24
4
4
#
−2 −3 −4
Fig. 1
*
3
3
2
2
1 0 −1 −2
Body mass change (g)
−1
CC E
1
Body mass change (g)
ED PT
2
A
Body mass change (g)
3
0
C
M
4
N U SC
B
A
A
1 0 −1 −2
−3
−3
−4
−4
Sham-1g VL-1g Sham-2g VL-2g
Sham-2g
B a
b
c
C a
b
c
D a
b
VL-2g d
d
d
M
A
c
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VL-1g b
N U SC R
Sham-1g A a
d
c
d
b
c
d
b
c
d
TE
D
c
E a
A
EP
CC
F a
b
G a
High
Low
Fig. 2
a
NPY
b
200
200
150
150
A
probe binding (Sham-1g as 100 %)
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C
* *
100
100
50
50
0
0
Fig. 3
100
150 100
** *
** *
50
50
0
0
AgRP ** *
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
ED
100
0
150
b
POMC
M
**
probe binding (Sham-1g as 100 %)
150
200
PT
probe binding (Sham-1g as 100 %)
200
a
N U SC
B
CRH
A
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin ** **
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NPY
200
150
150
100
100
50
50
0
0
Fig. 4
N U SC
A
100
b
200
A
probe binding (Sham-1g as 100 %)
a
150 100
50
50
0
0
AgRP
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
ED
100
C
150
b
POMC **
M
150
0
a
200
PT
probe binding (Sham-1g as 100 %)
200
B
CRH **
probe binding (Sham-1g as 100 %)
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin
a
NPY
200
150
150
100
100
50
50
0
0
Fig. 5
100
b
200
A
probe binding (Sham-1g as 100 %)
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C
150 100
50
50
0
0
AgRP
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
ED
100
0
150
b
POMC
M
150
probe binding (Sham-1g as 100 %)
200
PT
probe binding (Sham-1g as 100 %)
200
a
N U SC
B
CRH
A
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin
25
Table 1. The sequences of the probes used in in situ hybridization histochemistry Oligonucleotide sequence
CRH
5’-CAG TTT CCT GTT GCT GTG AGC TTG CTG AGC TAA CTG CTC TGC CCT GGC-3’
POMC
5’- CTT CTT GCC CAG CGG CTT GCC CCA GCA GAA GTG CTC CAT GGA CTA GGA-3’
CART
5’-TGG GGA CTT GGC CGT ACT TCT TCT CAT AGA TCG GAA TGC-3’
NPY
5’-GGA GTA GTA TCT GGC CAT GTC CTC TGC TGG CGC GTC-3’
AgRP
5’-CGA CGC GGA GAA CGA GAC TCG CGG TTC TGT GGA TCT AGC ACC TCT GCC-3’
MCH
5’-CCA ACA GGG TCG GTA GAC TCG TCC CAG CAT-3’
orexin
5’-TTC GTA GAG ACG GCA GGA ACA CGT CTT CTG GCG ACA-3’
A
CC E
PT
ED
M
A
N
U
SC R
IP T
mRNA
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S0196-9781(18)30102-5 https://doi.org/10.1016/j.peptides.2018.05.004 PEP 69971
To appear in:
Peptides
Received date: Revised date: Accepted date:
6-3-2018 30-4-2018 6-5-2018
Please cite this article as: Sonoda Satomi, Yoshimura Mitsuhiro, Abe Chikara, Morita Hironobu, Ueno Hiromichi, Motojima Yasuhito, Saito Reiko, Maruyama Takashi, Hashimoto Hirofumi, Tanaka Yoshiya, Ueta Yoichi.Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs.Peptides https://doi.org/10.1016/j.peptides.2018.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of hypergravity on the gene expression of the hypothalamic feeding-related neuropeptides in mice via vestibular inputs
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Satomi Sonodaa,d,*, Mitsuhiro Yoshimuraa,*, Chikara Abeb, Hironobu Moritab,
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Hiromichi Uenoa, Yasuhito Motojimaa, Reiko Saitoa, Takashi Maruyamaa, Hirofumi Hashimotoc, Yoshiya Tanakad, and Yoichi Uetaa
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Department of Physiology and dFirst Department of Internal Medicine, School of
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a
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Medicine, University of Occupational and Environmental Health, Kitakyushu
Department of Physiology, Gifu University Graduate School of Medicine, Gifu
501-1194, Japan
Department of Regulatory Physiology, Dokkyo Medical University, Tochigi 321-0293,
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Japan
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c
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b
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807-8555, Japan
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*These authors equally contributed to this work.
Corresponding author: Yoichi Ueta, M.D., Ph.D. Department of Physiology, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan
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Tel.: +81-93-691-7420; Fax: +81-93-692-1711 e-mail: [email protected]
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Highlights: ・Involvement of hypergravity and vestibular system on feeding regulation was
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examined ・Vestibular dysfunction was generated by vestibular lesion (VL)
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・Hypothalamic gene expression was examined by in situ hybridization histochemistry
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・ CRH and POMC were increased significantly in Sham but not in VL in 2g
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environment
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Abstract
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・CRH and POMC may be responsible for feeding regulation in different gravity
The effects of hypergravity on the gene expression of the hypothalamic
feeding-related neuropeptides in sham-operated (Sham) and vestibular-lesioned (VL)
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mice were examined by in situ hybridization histochemistry. Corticotrophin-releasing hormone (CRH) in the paraventricular nucleus was increased significantly in Sham but not in VL mice after 3 days of exposure to a 2 g environment compared with a 1g environment. Significant decreases in pro-opiomelanocortin (POMC) and cocaine2
3
and amphetamine-regulated transcript and significant increases in neuropeptide Y, agouti-related protein in the arcuate nucleus and orexin in the lateral hypothalamic area were observed in both Sham and VL mice. After 2 weeks of exposure, CRH and
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POMC were increased significantly in Sham but not in VL mice. After 8 weeks of
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exposure, the hypothalamic feeding-related neuropeptides were comparable between Sham and VL mice. These results suggest that the hypothalamic feeding-related neuropeptides may be affected during the exposed duration of hypergravity via
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vestibular inputs.
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histochemistry, vestibular lesion.
neuropeptides, in situ hybridization
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Keywords: hypergravity, hypothalamus,
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Abbreviations
ACTH, adrenocorticotropic hormone; AgRP, agouti-related protein; Arc, arcuate
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nucleus; CART, cocaine- and amphetamine-regulated transcript; CRH, corticotrophin-releasing hormone; CRH-R1, the type 1 CRH receptor; CRH-R2, the type 2 CRH receptor; HPA, hypothalamic-pituitary-adrenal; LHA, lateral hypothalamic area; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; OD,
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optical densities; POMC, pro-opiomelanocortin; PVN, paraventricular nucleus; SEM, standard error of the mean; Sham, sham-operated; VL, vestibular lesion
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1. Introduction
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Centrifugation is often used to examine biological responses to the artificial
hypergravity environment in rodents [1, 2]. Transient hypophagia followed by reducing the body mass is a common biological response to centrifugation-induced
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hypergravity [3]. Since the reduced food intake is fully or partially ameliorated by the
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vestibular lesion (VL), the vestibular system is, at least in part, involved in the
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hypergravity-induced hypophagia.
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The feeding behavior is controlled by a complex, central signaling network of neuropeptides in the hypothalamus [4, 5]. The hypothalamic neural circuits, including
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various neuropeptide-containing neurons are involved in the regulation of the feeding
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behavior and body mass [4-6]. Abe et al. reported that the immunoreactivity of corticotrophin-releasing hormone (CRH) in the paraventricular nucleus (PVN) after
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being exposed to a 2 g environment for 90 min was significantly increased but was attenuated by VL in rats [2]. However, to our knowledge, how the hypothalamic feeding-related neuropeptides change in the hypergravity environment remains unveiled. We hypothesized that vestibular function may be involved in the modulation
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5
of neuropeptides expressions in the hypergravity environment. To examine the involvement of hypothalamic feeding-related neuropeptides in the hypergravity-induced hypophagia, the gene expression of the hypothalamic
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feeding-related neuropeptides was compared between Sham and VL mice. We
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examined these neuropeptides at 3 days, 2 weeks, and 8 weeks after being exposed to a 2 g environment. Since hypergravity-induced hypophagia is transient and ameliorated by VL, neuropeptides, which are altered only in Sham but not in VL mice, may be a
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candidate neuropeptide responsible for hypergravity-induced hypophagia. The
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neuropeptides studied in the present study included 3 anorexigenic neuropeptides
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(CRH, pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated
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transcript (CART)) and 4 orexigenic neuropeptides (neuropeptide Y (NPY),
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agouti-related protein (AgRP), melanin-concentrating hormone (MCH) and orexin).
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2. Materials and methods
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2.1. Animals
Adult male C57BL/6J mice (weighing 19.0–27.6 g each) were group housed (6
mice per cage) and maintained in temperature-controlled (23-25°C) conditions under a 12/12-h light/dark cycle (lights on at 07:00 h). All mice had free access to food and
5
6
water throughout the experiment. The food was consisted of 4.6% fat, 48.6% carbohydrate, 25.5% protein (total 14.2 kJ/g) (CLEA Rodent diet CE-2, CLEA Japan, Inc., Tokyo, Japan). Animals used in the present study were maintained in accordance
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with the “Guiding Principles for the Care and Use of Animals in the Field of
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Physiological Science” set by the Physiological Society of Japan. The experiments
were approved by the Animal Research Committees of Gifu University, University of
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Occupational and Environmental Health, and Japan Aerospace Exploration Agency.
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2.2. Surgical procedures
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All mice received Sham or VL surgery before the experiment. Six- to 7-week-old
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mice were anesthetized with isoflurane (Escain, Pfizer, Tokyo, Japan) inhalation via a face mask (2%), and the VL surgery was performed through an external auditory
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meatus approach as described previously in detail [7-9]. Penicillin G potassium (3000
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U/kg, Meiji Seika Pharma, Tokyo, Japan) and buprenorphine (3 µg/kg, Lepetan, Otsuka, Tokyo, Japan) were administered to the Sham and VL mice subcutaneously
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prior to returning the animals to their home cages (a group-housing cage, 6 mice per cage). Five to 7 days after the Sham or VL surgery, the success of the VL was confirmed by observing the swimming behavior of the mice [7-9]. During the recovery from Sham or VL surgery and during centrifugation, food was placed on the cage floor
6
7
instead of on a wire bar lid [7].
2.3. Experimental procedures
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After recovery, the mice were divided into 4 groups: Sham-1g, VL-1g, Sham-2g,
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and VL-2g (n=6, each). They were exposed to a 1 g (Sham-1g, VL-1g) or 2 g
(Sham-2g, VL-2g) environment for 3 days, 2 weeks, and 8 weeks. The 2 g environment was developed by centrifugation of a custom-made gondola-type rotating
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box with a 1.5 m long-arm (Shimadzu, Kyoto, Japan) rotating at 32 rpm. Cages for 2 g
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groups were set in the rotating box. All the mice had access to food and water ad
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libitum, and the room temperature was maintained at 24 ± 1°C with a 12:12 h
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light-dark cycle (lights on at 07:00 h). For cage cleaning and water/food refreshment, rotation was stopped for 30 min once a week. At the end of each time point, the mice
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were decapitated immediately after being anesthetized with isoflurane (Escain, Pfizer,
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Tokyo, Japan) inhalation. Brains were removed, put promptly onto dry ice, and then
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stored at -80°C.
2.4. In situ hybridization histochemistry The brains were cut into 12 μm thickness coronal brain sections by using a cryostat (OTF5000, Bright Instrument Co Ltd, England) at –20˚C and thaw mounted
7
8
on gelatin/chrome alum-coated slides. The locations of the PVN, arcuate nucleus (Arc) and lateral hypothalamic area (LHA), were determined according to the coordinates given in the mouse brain atlas [10].
35
S 3’-end-labeled deoxyoligonucleotides
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complementary to the transcripts encoding CRH, pro-opiomelanocortin (POMC),
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cocaine- and amphetamine-regulated transcript (CART), neuropeptide Y (NPY), agouti-related protein (AgRP), melanin-concentrating hormone (MCH) and orexin
were used. The specificity of these probes was confirmed by previous studies [11-13].
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The sequences of these probes used in situ hybridization histochemistry was
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summarized in the Table 1. The in situ hybridization protocol has been described exposed to
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previously in detail [14]. Hybridized sections which were
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autoradiography film (BioMax XAR film, KODAK, USA) for 1 week was analyzed by using an MCID imaging analyzer (Imaging Research, Inc., Ontario, Canada). The
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mean optical densities (OD) of the autoradiographs were measured by comparison
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with simultaneously exposed 14C-labeled microscale samples (Amersham, Bucks, UK)
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and represented in arbitrary units setting the mean OD obtained from control mice.
2.5. Statistical analysis In the results of the in situ hybridization, the gene expression of the hypothalamic feeding-related neuropeptides was expressed as a percentage of Sham-1g. All data
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were analyzed by one-way ANOVA followed by a Bonferroni-type adjustment for multiple comparisons (Origin Pro version 8.5J, Lightstone, Tokyo, Japan). Data are
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presented as the mean ± SEM. Statistical significance was set at P<0.05.
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3. Results
3.1. The body mass change after 3 days, 2 weeks, and 8 weeks exposure to
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hypergravity
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The body mass in each group (n=6, each) was measured after 3 days of exposure
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to a 1 g or 2 g environment: –0.03 ± 0.09 g in Sham-1g, 0.05 ± 1.15 g in VL-1g, –3.13
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± 0.32 g in Sham-2g, and –0.5 ± 1.03 g in VL-2g (Fig. 1A). The body mass gain of Sham-2g was significantly lower compared to other groups, whereas, those were
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comparable between Sham-1g and VL-1g. The body mass gain of VL-2g was
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markedly higher than Sham-2g. These data indicated that VL caused the attenuation of hypergravity-induced body mass loss. The body mass change after 2 weeks exposure
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to hypergravity (Fig. 1B) and after 8 weeks exposure to hypergravity (Fig. 1C) are shown in Fig. 1B and 1C, respectively. There were no significant differences in body mass changes among the groups after 2 or 8 weeks exposure to hypergravity.
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3.2. The gene expression of the hypothalamic feeding-related neuropeptides after 3 days exposure to hypergravity All examined gene expression levels of the hypothalamic feeding-related
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neuropeptides were comparable between Sham-1g and VL-1g at 3 days (Fig. 3). The
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gene expression of the CRH in the PVN was significantly increased in Sham-2g compared to Sham-1g (Fig. 3A). That of VL-2g was not increased compared to
Sham-1g (Fig. 3A). Those of the POMC and CART in the Arc were markedly
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decreased in Sham-2g and VL-2g compared to Sham-1g (Fig. 3Ba, b). NPY and AgRP
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in the Arc and orexin in the LHA were significantly increased in the groups that were
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exposed to 2 g but were comparable between these two groups (Fig. 3Ca, b and 3Db).
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MCH in the LHA were comparable among all the groups (Fig. 3Da). The representative digital images obtained from autoradiographs are shown in Fig. 2. CRH
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was expressed in the parvocellular divisions of the PVN (Fig. 2A). CRH was densely
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expressed only in Sham-2g but not in other groups (Fig. 2Aa-d). POMC/CART were expressed in the Arc (Fig. 2Ba-d, 2Ca-d). They were expressed lower in Sham-2g and
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VL-2g than in Sham-1g (Fig. 2Bc and d, 2Cc and d). NPY/AgRP (Fig. 2Da-d, 2Ea-d), which were also expressed in Arc, were expressed higher in Sham-2g and VL-2g than in Sham-1g (Fig. 2Dc and d, 2Ec and d). MCH and orexin were expressed in the LHA (Fig. 2Fa-d, 2Ga-d). The mean OD of MCH was to the same extent in all groups (Fig.
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2Fa-d). Orexin was expressed higher in Sham-2g and VL-2g than in Sham-1g (Fig. 2Gc and d).
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3.3. The gene expression of the hypothalamic feeding-related neuropeptides after 2
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weeks exposure to hypergravity
All examined gene expression levels of the hypothalamic feeding-related neuropeptides were comparable between Sham-1g and VL-1g at 2 weeks (Fig. 4). The
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gene expression of CRH in PVN was significantly increased in Sham-2g compared to
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Sham-1g (Fig. 4A). POMC in the Arc was markedly increased in Sham-2g compared
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to Sham-1g as well, while those changes were not observed in VL-2g (Fig. 4Ba).
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There were no significant differences in the gene expression of CART, NPY, AgRP,
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MCH, and orexin among the groups (Fig. 4Bb, Ca and b and Da and b).
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3.4. The gene expression of the hypothalamic feeding-related neuropeptides after 8 weeks exposure to hypergravity
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No statistical significance was seen in all the gene expression levels of the
neuropeptides among the groups (Fig. 5A-D).
4. Discussion
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We clarified that the gene expression of the hypothalamic feeding-related neuropeptides was affected by centrifugation-induced hypergravity. The gene expression of CRH in the PVN and POMC in the Arc, which is altered only in
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Sham-2g but not in VL-2g, may be a potent candidate of neuropeptides, which are
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responsible for the feeding regulation in different gravity environments mediated by a vestibular input.
CRH is known to be involved in the stress response and suppression of feeding
from
the
anterior
pituitary,
resulting
in
the
activation
of
the
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(ACTH)
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behavior [5, 15, 16]. It stimulates the secretion of the adrenocorticotropic hormone
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hypothalamic-pituitary-adrenal (HPA) axis. The type 1 CRH receptor (CRH-R1) and
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the type 2 CRH receptor (CRH-R2) have been cloned in rodents [17]. The 'fight-or-flight' response, which is characterized by the activation of the HPA axis, is
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mainly mediated by CRH-R1, whereas the stress-coping responses are mediated by
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CRH-R2 [18]. Hypophagia, which is induced by emotional stress, is partially mediated by CRH-R1 [19]. It is indicated that the increased gene expression of CRH may be a
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result of a stress response induced by a hypergravity environment. Although it is unclear which types of receptors are responsible for hypophagia developed by hypergravity, CRH-R1 may be strongly involved as CRH-R1 is activated in the earlier phase of the stress response [18].
12
13
There are linkages between the vestibular system and hypothalamus. For example, there are neural inputs from the vestibular system to the hypothalamus [20]. Morita et al. demonstrated that in mice, there is a marked increase of Fos immunoreactivity in
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the PVN after 90 min exposure to 2 g, which was abolished by VL [1]. In het mice,
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which lack macular otoconia, Fos expression in the PVN was significantly suppressed after 2 g loading [21]. Abe et al. showed that the concentrations of plasma ACTH and corticosterone increase after 90 min exposure to 2 g. They also demonstrated that, after
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being exposed to hypergravity, the CRH in the PVN was markedly decreased in VL
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compared to Sham by using immunohistochemical method in rats [2]. Thus,
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CRH-producing neurons in the PVN may receive neuronal input from the vestibular
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systems. These reports are consistent with our results that increased the expression of CRH in the 2 g environment, which was completely abolished by VL. Whereas, they
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did not refer to the synthesis of CRH. Our results suggest that CRH synthesis increased
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after exposure to hypergravity as these results were obtained by using in situ hybridization histochemistry. The species of animals were also different, they used rats
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we used mice. We observed for longer duration after being exposed to hypergravity, up to 8 weeks, than previous reports [1, 2, 6, 21] to elucidate more chronic effects of hypergravity on the hypothalamic feeding regulated neuropeptides gene expression. Acute hypophagia-induced starvation, which was seen after 3 days exposure to a 2
13
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g environment, may cause the downregulation of the anorexigenic neuropeptides, such as POMC and CART, and the upregulation of the orexigenic neuropeptides such as NPY, AgRP and orexin. The starved state induced both the upregulation of NPY/AgRP
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and orexin neurons and the downregulation of the POMC/CART neuron [4, 13, 22].
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These alterations are thought to be a homeostatic response [4]. The results obtained
from the present study at 3 days of exposure to 2 g suggest that the changes in the feeding-related neuropeptides, except CRH, are due to the results of starvation. The
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reason why MCH at 3 days of exposure did not change was unclear. The gene
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expression of MCH was upregulated by fasting in rats or in hyperleptinemic ob/ob
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mice [23]. However, Nara-ashizawa et al. reported that the gene expression of MCH
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was not increased in cancer cachexia-anorexia syndrome [24]. The pathophysiological mechanisms of hypergravity-induced hypophagia and cancer cachexia-anorexia
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syndrome may be similar regarding the MCH upregulation.
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After being exposed to hypergravity for 2 weeks, the gene expression of the CRH and POMC showed dramatic increases in Sham-2g but not in VL-2g mice. We
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revealed hypergravity increased the gene expression of the POMC, which is a novel finding. We consider that the changes in the gene expression of the hypothalamic feeding-related neuropeptides have different meanings at each time point. For example, being exposed to hypergravity for 3 days reflects starvation. However, a previous
14
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study reported that rats being exposed to 3g ate the same amount of food as those being exposed to 1g after 2 weeks from the start of the experiment, compared to 3g [6]. We speculate that it takes over 2 weeks for the vestibular system plasticity to adapt to
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the different gravity. The results of 2 weeks of exposure may indicate inadequate
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adaptation for the different gravity rather than starvation or fasting. POMC in the Arc
is known to be regulated by several factors including leptin, insulin, and fasting. It is shown that leptin and insulin stimulates POMC neuron in the Arc in the previous
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studies by using electrophysiological methods [25, 26]. Although it is possible that the
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change of gravity caused the upregulation of the gene expression of POMC directly or
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indirectly, we could not explain the reason why the gene expression of POMC
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increased after hypergravity in our study. In addition, as CART neurons are known to be co-expressed with POMC neurons, they might fluctuate as POMC do. We would
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like to seek the mechanism in further study.
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Hypergravity may be sensed not only by the vestibular system but also by the somatosensory, proprioceptive and visceral sensors, which remain intact in VL animals.
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For example, in human astronauts, fluid redistribution, neurovestibular effects, muscle changes, bone demineralization, psychosocial effects, and immune dysregulation are elicited in different gravity environments [27]. The mice in the 2 g environment may be adapted to the hypergravity environment at 8 weeks. We considered that the
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vestibular system is accommodated to the new gravity at 8 weeks, as the vestibular system is known to be highly plastic and able to adapt to the new gravitational
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environment [1, 28].
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5. Conclusion
In conclusion, we clarified that the gene expression of the hypothalamic feeding-related neuropeptides is altered after exposure to a hypergravity environment.
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The gene expression of CRH and POMC may be a potent candidate of neuropeptides,
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Author contributions
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which are responsible for feeding regulation in different gravity environments.
The experiments were designed by S.S., M.Y., H.M., Y.T., and Y.U. The
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experiments were performed by S.S., M.Y., H.H., A.C., H.M., H.U., Y.M., R.S., and
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T.M. Data were analyzed by S.S. and M.Y. The draft and figures were prepared by S.S., M.Y., and Y.U. Final approval was made by Y.U. All authors approved the final version
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of the manuscript and agreed to be accountable for all aspects of the work regarding questions related to the accuracy. All authors designated as authors qualify for authorship, and all those who qualify for authorship are listed.
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Conflict of interest The authors declare that they have no conflict of interest.
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Acknowledgements
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We would like to thank Ms. Yuki Nonaka for her technical assistance.
Funding
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This study was funded by Grant-in-Aid for Young Scientists (B) (17K15575) for
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M.Y., a Grant-in-Aid for Scientific Research (B) (17H04027) for Y.U. and a
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Grant-in-Aid for Scientific Research on Innovative Areas (15H05940) for Y.U. from
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Figure Legends Fig. 1
The body mass change after 3 days (A), 2 weeks (B), and 8 weeks (C)
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exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. Data are presented as mean ± SEM (n=6, each). *P<0.05 vs. Sham-1g. #P<0.05 vs. Sham-2g.
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Fig. 2 The gene expression of the feeding-related neuropeptides after 3 days of exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC (Ba) and CART in the Arc
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(Bb), NPY (Ca) and AgRP in the Arc (Cb), and MCH (Da) and orexin in the LHA (Db)
SEM (n=6, each). *P<0.05 and **P<0.01 vs. Sham-1g.
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are shown. Signals for Sham-1g were set at 100%. Date are presented as the mean ±
35
S-labeled oligonucleotide probes complementary to CRH in the PVN
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section with
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Fig. 3 Representative digital images obtained from autoradiographs of hybridized
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(Aa-d), POMC in the Arc (Ba-d), CART in the Arc (Ca-d), NPY in the Arc (Da-d),
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AgRP in the Arc (Ea-d), MCH in the LHA (Fa-d), and orexin in the LHA (Ga-d) after 3 days exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and
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vestibular-lesioned (VL) mice. Analyzed areas are surrounded by red dotted lines (Aa,
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Ba, Ca, Da, Ea, Fa and Ga). Scale bars=1 mm. Signal intensity ranges from high
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(black) to low (white).
Fig. 4 The gene expression of the feeding-related neuropeptides after 2 weeks of exposure to different gravity (1 g or 2 g) in sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC in the Arc (Ba), CART in
23
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the Arc (Bb), NPY in the Arc (Ca), AgRP in the Arc (Cb), MCH in the LHA (Da), and orexin in the LHA (Db) are shown. Signals for Sham-1g were set at 100%. Date are
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presented as the mean ± SEM (n=6, each). **P<0.01 vs. Sham-1g.
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Fig. 5 The gene expression of the feeding-related neuropeptides after 8 weeks of exposure to different gravity (1 g or 2 g) in the sham-operated (Sham) and vestibular-lesioned (VL) mice. CRH in the PVN (A), POMC in the Arc (Ba), CART in
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the Arc (Bb), NPY in the Arc (Ca), AgRP in the Arc (Cb), MCH in the LHA (Da) and
A
orexin in the LHA (Db) are shown. Signals for Sham-1g were set at 100%. Date are
A
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presented as the mean ± SEM (n=6, each).
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4
4
#
−2 −3 −4
Fig. 1
*
3
3
2
2
1 0 −1 −2
Body mass change (g)
−1
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1
Body mass change (g)
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2
A
Body mass change (g)
3
0
C
M
4
N U SC
B
A
A
1 0 −1 −2
−3
−3
−4
−4
Sham-1g VL-1g Sham-2g VL-2g
Sham-2g
B a
b
c
C a
b
c
D a
b
VL-2g d
d
d
M
A
c
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VL-1g b
N U SC R
Sham-1g A a
d
c
d
b
c
d
b
c
d
TE
D
c
E a
A
EP
CC
F a
b
G a
High
Low
Fig. 2
a
NPY
b
200
200
150
150
A
probe binding (Sham-1g as 100 %)
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C
* *
100
100
50
50
0
0
Fig. 3
100
150 100
** *
** *
50
50
0
0
AgRP ** *
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
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100
0
150
b
POMC
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**
probe binding (Sham-1g as 100 %)
150
200
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probe binding (Sham-1g as 100 %)
200
a
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B
CRH
A
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin ** **
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NPY
200
150
150
100
100
50
50
0
0
Fig. 4
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A
100
b
200
A
probe binding (Sham-1g as 100 %)
a
150 100
50
50
0
0
AgRP
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
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100
C
150
b
POMC **
M
150
0
a
200
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probe binding (Sham-1g as 100 %)
200
B
CRH **
probe binding (Sham-1g as 100 %)
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin
a
NPY
200
150
150
100
100
50
50
0
0
Fig. 5
100
b
200
A
probe binding (Sham-1g as 100 %)
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C
150 100
50
50
0
0
AgRP
D probe binding (Sham-1g as 100 %)
50
Sham-1g VL-1g Sham-2g VL-2g
CART
200
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100
0
150
b
POMC
M
150
probe binding (Sham-1g as 100 %)
200
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probe binding (Sham-1g as 100 %)
200
a
N U SC
B
CRH
A
A
a
MCH
b
200
200
150
150
100
100
50
50
0
0
Orexin
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Table 1. The sequences of the probes used in in situ hybridization histochemistry Oligonucleotide sequence
CRH
5’-CAG TTT CCT GTT GCT GTG AGC TTG CTG AGC TAA CTG CTC TGC CCT GGC-3’
POMC
5’- CTT CTT GCC CAG CGG CTT GCC CCA GCA GAA GTG CTC CAT GGA CTA GGA-3’
CART
5’-TGG GGA CTT GGC CGT ACT TCT TCT CAT AGA TCG GAA TGC-3’
NPY
5’-GGA GTA GTA TCT GGC CAT GTC CTC TGC TGG CGC GTC-3’
AgRP
5’-CGA CGC GGA GAA CGA GAC TCG CGG TTC TGT GGA TCT AGC ACC TCT GCC-3’
MCH
5’-CCA ACA GGG TCG GTA GAC TCG TCC CAG CAT-3’
orexin
5’-TTC GTA GAG ACG GCA GGA ACA CGT CTT CTG GCG ACA-3’
A
CC E
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ED
M
A
N
U
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mRNA
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