Involvement of melanin-concentrating hormone 2 in background color adaptation of barfin flounder Verasper moseri

https://doi.org/10.1016/j.ygcen.2014.07.008Get rights and content

Highlights

  • Amino acid sequence of MCH2 from barfin flounder was determined.

  • Synthesized MCH2 aggregates pigment in the skin chromatophores.

  • MCH2 interacts with MCH-R2 that is expressed in the skin.

  • The expression of mch2 is up-regulated under white background.

  • MCH2 might play an important role in the regulation of skin color change, to the same extent as MCH1.

Abstract

In teleosts, melanin-concentrating hormone (MCH) plays a key role in skin color changes. MCH is released into general circulation from the neurohypophysis, which causes pigment aggregation in the skin chromatophores. Recently, a novel MCH (MCH2) precursor gene, which is orthologous to the mammalian MCH precursor gene, has been identified in some teleosts using genomic data mining. The physiological function of MCH2 remains unclear. In the present study, we cloned the cDNA for MCH2 from barfin flounder, Verasper moseri. The putative prepro-MCH2 contains 25 amino acids of MCH2 peptide region. Liquid chromatography-electrospray ionization mass spectrometry with a high resolution mass analyzer were used for confirming the amino acid sequences of MCH1 and MCH2 peptides from the pituitary extract. In vitro synthesized MCH1 and MCH2 induced pigment aggregation in a dose-dependent manner. A mammalian cell-based assay indicated that both MCH1 and MCH2 functionally interacted with both the MCH receptor types 1 and 2. Mch1 and mch2 are exclusively expressed in the brain and pituitary. The levels of brain mch2 transcript were three times higher in the fish that were chronically acclimated to a white background than those acclimated to a black background. These results suggest that in V. moseri, MCH1 and MCH2 are involved in the response to changes in background colors, during the process of chromatophore control.

Introduction

Melanin-concentrating hormone (MCH) was originally identified from the chum salmon, Oncorhynchus keta, as a pituitary peptide that concentrates melanin granules in the melanophores of the skin (Kawauchi et al., 1983). Later studies indicated that teleost MCH is synthesized in the hypothalamus, transported to the nerve terminal in the pituitary neural lobe, and released into the blood (Amano and Takahashi, 2009). MCH has subsequently been identified in the mammalian brain, and shown to act as a neuromodulator regulating feeding behavior, energy homeostasis, stress, reproduction, sensory perception, and neuroendocrine responses (Griffond and Baker, 2002, Nahon, 2006, Saito and Nagasaki, 2008, Sherwood et al., 2012, Wu et al., 2009). The functions of the original teleost MCH (designated as MCH1 in this text) have been well investigated, especially pigment aggregation and cooperation with various pituitary hormones (Kawauchi, 2006). MCH1 has also been implicated in feeding behavior in teleosts, but this function is inconsistent amongst different species. Intracerebroventricular injection studies have suggested that MCH1 has an anorexic function in the goldfish, Carassius auratus (Matsuda et al., 2006, Matsuda et al., 2007); whereas, white background color enhances mch1 expression and feeding behavior in the barfin flounder, Verasper moseri, suggesting a possible orexigenic function in this fish (Sunuma et al., 2009).

Recently, a gene encoding another MCH (termed MCH2 in this text) has been identified as an ortholog of mammalian mch, in teleosts (zebrafish, Danio rerio; medaka, Oryzias latipes; three-spined stickleback, Gastierosteus aculeatus; torafugu, Takifugu rubripes; winter flounder, Pseudopleuronectes americanus; and starry flounder, Platichthys stellatus) (Berman et al., 2009, Tuziak and Volkoff, 2012, Kang and Kim, 2013). Very few studies have been conducted to elucidate the physiological functions of MCH2 as compared to MCH1. In D. rerio, mch2 is expressed in the lateral tuberal nucleus (NLT) within the hypothalamus where mch1 is also expressed (Berman et al., 2009). In D. rerio and P. americanus, the up-regulated expression of mch2 in the hypothalamus is induced by fasting (Berman et al., 2009, Tuziak and Volkoff, 2012), suggesting the possible involvement of mch2 in the regulation of feeding in teleosts. The elevated expression of mch2 upon exposure to white background in D. rerio and P. stellatus suggests that MCH2 acts on background color-adaptation (Berman et al., 2009, Zhang et al., 2010, Kang and Kim, 2013). However, to date the MCH2 peptide has not been identified, and the physiological functions of MCH2 involved in body color changes remain unknown.

V. moseri is an interesting model organism for investigating the molecular mechanisms of MCH systems because the roles of MCH1 in the regulation of skin color changes (Mizusawa et al., 2011) and feeding/growth behavior (Sunuma et al., 2009, Takahashi et al., 2004, Yamanome et al., 2005) have been thoroughly investigated. In addition, two MCH receptors MCH-R1 and MCH-R2 have also been characterized. MCH-R1 is exclusively expressed in the brain, whereas MCH-R2 is expressed in the brain as well as several peripheral tissues including the skin (Takahashi et al., 2007). MCH-R2 is expressed in skin melanophores and xanthophores (but not in other dermal cells) where MCH1 was shown to induce pigment aggregation (Mizusawa et al., 2011). The present study was undertaken to elucidate the properties of MCH2 using V. moseri as to the following five items; (i) molecular cloning of the prepro-MCH2 cDNA, (ii) identification of the MCH2 peptide derived from prepro-MCH2, (iii) characterization of the pigment-aggregation activities of MCH2, (iv) characterization of the pharmacological properties of MCH2, and (v) characterization of the expression levels of MCH2 in response to background color.

Section snippets

Fish

Immature V. moseri were purchased from the Iwate Cultivating Fishery Association (Iwate, Japan), or kindly provided by the Hokkaido National Fisheries Research Institute (Hokkaido, Japan). All experiments were conducted in accordance with the Kitasato University guidelines for the care and use of animals. The photoperiods and water temperatures were maintained at natural conditions. Tissue sampling was performed on fish that were anesthetized by immersion, in 0.05% 2-phenoxyethanol for

Structure of prepro-MCH2

Sequential cloning of PCR- and RACE-amplified cDNA revealed a 635 bp full-length sequence of prepro-MCH2, excluding the poly-A tail. The sequence consists of 150 amino acid (AA) residues, with a signal peptide at AA positions 1–25, and the MCH2 sequence spanning AA positions 126–150 (Fig. 1). The putative MCH2 in V. moseri is composed of 25 AA, similar to the P. americanus MCH2 (Fig. 2). The putative prepro-MCH2 has a higher AA sequence identity with P. americanus prepro-MCH2 (87%), but lower

Discussion

In this study, we demonstrated the existence of the MCH2 peptide for the first time, using V. moseri as a model organism. The presence of MCH2 was first indicated by molecular cloning of the prepro-MCH2 cDNA from a single brain. A multi-species alignment and a phylogenetic analysis of prepro-MCH determined the amino acid sequence as that of prepro-MCH2. Processing of pro-proteins and pro-hormones often occurs at specific single or pairs of basic amino acids in the precursors (Seidah et al., 1998

Conclusion

In the present study, we identified the MCH2 peptide from V. moseri by cDNA cloning and LC–ESI-MS, followed by database searches. Results of in vitro and pharmacological studies revealed that both MCH1 and MCH2 have a pigment-aggregating activity in chromatophores, and this activity is mediated by MCH-R2. Furthermore, the expression of both, MCH1 and MCH2 in the brain increases under bright background conditions. These results suggest that MCH2 might play an important role as much as MCH1 in

Acknowledgments

The authors would like to thank the following people for their cooperation: Tadashi Andoh and Naoto Murakami at Hokkaido National Fisheries Research Institute; Takashi Sunada and Tomoaki Mikawa at Iwate Cultivating Fishery Association; Hoshito Tomizawa and Daisuke Saito at Kitasato University. This study was supported by Grants from the Japan Society for the Promotion of Science – Japan to A.T. (22248023).

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    1

    Present address: RIKEN Center for Integrative Medical Sciences, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan.

    2

    Present address: Fisheries Agency, Ministry of Agriculture, Forestry and Fisheries, 1-2-1 Kasumigaseki, Chiyoda-ku, Tokyo 100-8950, Japan.

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