東 森生

The adenohypophysis is composed of the anterior and intermediate lobes (AL and IL, respectively), and secretes hormones that play an important role in reproduction. CD9- and SOX2-double (CD9/SOX2) positive cells located in the marginal cell layer (MCL) facing the Rathke's cleft in the AL and IL form the primary stem cell niche in the adult adenohypophysis of rats. In this study, we successfully obtained 3-dimensional (3D) cell aggregates that closely resembled the primary niche of MCL in vivo. After incubation in a Matrigel containing several growth factors, approximately 20% of the cells in the CD9/SOX2-positive cell aggregates were differentiated into hormone-producing cells. The cell aggregates generated in this study may provide insight into the regulation of the pituitary stem/progenitor cell niche and the turnover of hormone-producing cells.

Close contact between lactating rodent mothers and their infants is essential for effective nursing. Whether the mother's effort to retrieve the infants to their nest requires the vasopressin-signaling via V1b receptor has not been fully defined. To address this question, V1b receptor knockout (V1bKO) and control mice were analyzed in pup retrieval test. Because an exploring mother in a new test cage randomly accessed to multiple infants in changing backgrounds over time, a computer vision-based deep learning analysis was applied to continuously calculate the distances between the mother and the infants as a parameter of their relationship. In an open-field, a virgin female V1bKO mice entered fewer times into the center area and moved shorter distances than wild-type (WT). While this behavioral pattern persisted in V1bKO mother, the pup retrieval test demonstrated that total distances between a V1bKO mother and infants came closer in a shorter time than with a WT mother. Moreover, in the medial preoptic area, parts of the V1b receptor transcripts were detected in galanin- and c-fos-positive neurons following maternal stimulation by infants. This research highlights the effectiveness of deep learning analysis in evaluating the mother-infant relationship and the critical role of V1b receptor in pup retrieval during the early lactation phase.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

During the development of analgesic tolerance to morphine, the V1b vasopressin receptor has been proposed to bind to β-arrestin 2 and the µ-opioid receptor to enable their interaction. However, direct evidence of such a high-order complex is lacking. Using bioluminescent resonance energy transfer between a split Nanoluciferase and the Venus fluorescent protein, the NanoBit-NanoBRET system, we found that β-arrestin 2 closely located near the heteromer µ-V1b receptor in the absence of an agonist and moved closer to the receptor carboxyl-termini upon agonist stimulation. An additive effect of the two agonists for opioid and vasopressin receptors was detected on the NanoBRET between the µ-V1b heteromer and β-arrestin 2. To increase the agonist response of NanoBRET, the ratio of the donor luminophore to the acceptor fluorophore was decreased to the detection limit of luminescence. In the first phase of access, β-arrestin 2 was likely to bind to the unstimulated V1b receptor in both its phosphorylated and unphosphorylated forms. In contrast, the second-phase access of β-arrestin 2 was agonist dependent, indicating a possible pharmacological intervention strategy. Therefore, our efficient method should be useful for evaluating chemicals that directly target the vasopressin binding site in the µ-V1b heteromer to reduce the second-phase access of β-arrestin 2 and thereby to alleviate tolerance to morphine analgesia.

Hypertension leads to structural remodeling of cerebral blood vessels, which has been implicated in the pathophysiology of cerebrovascular diseases. The remodeling and progression of arteriolosclerosis under hypertension involve fibrosis along with the production of type I collagen around cerebral arterioles. However, the source and regulatory mechanisms of this collagen production remain elusive. In this study, we examined if perivascular macrophages (PVMs) are involved in collagen production around cerebral small vessels in hypertensive SHRSP/Izm rats. Immunoreactivity for type I collagen around cerebral small vessels in 12-week-old hypertensive rats tended to higher than those in 4-week-old hypertensive and 12-week-old control rats. In ultrastructural analyses using transmission electron microscopy, the substantial deposition of collagen fibers could be observed in the intercellular spaces around PVMs near the arterioles of rats with prolonged hypertension. In situ hybridization analyses revealed that cells positive for mRNA of Col1a1, which comprises type I collagen, were observed near cerebral small vessels. The Col1a1-positive cells around cerebral small vessels were colocalized with immunoreactivity for CD206, a marker for PVMs, but not with those for glial fibrillary acidic protein or desmin, markers for other perivascular cells such as astrocytes and vascular smooth muscle cells. These results demonstrated that enhanced production of type I collagen is observed around cerebral small vessels in rats with prolonged hypertension and Col1a1 is expressed by PVMs, and support the concept that PVMs are involved in collagen production and vascular fibrosis under hypertensive conditions.

Leukemias are refractory hematopoietic malignancies, for which the development of new therapeutic agents requires in vivo studies using tumor-bearing mouse models. Although several organs are commonly examined in such studies to evaluate the disease course, the effectiveness of interventions and the localization of tumor cells in the affected organs are still unclear. In this study, we histologically examined the distribution of leukemia cells in several organs using two leukemic mouse models produced by the administration of two cell lines (THP-1, a human myelomonocytic leukemia, and A20, a mouse B cell leukemia/lymphoma) to severe immunodeficient mice. Survival of the mice depended on the tumor burden. Although A20 and THP-1 tumor cells massively infiltrated the parenchyma of the liver and spleen at 21 days after transplantation, A20 cells were hardly found in connective tissues in Glisson's capsule in the liver as compared with THP-1 cells. In the bone marrow, there was more severe infiltration of A20 cells than THP-1 cells. THP-1 and A20 cells were widely spread in the lungs, but were rarely observed in the small intestine. These findings suggest that each leukemia model has a unique localization of tumor cells in several affected organs, which could critically affect the disease course and the efficacy of therapeutic agents, including cellular immunotherapies.

Extracellular matrix (ECM) is essential in tissue physiology and pathologic conditions such as tumorigenesis. It affects tumor cell behavior, proliferation, and metastasis. Pituitary adenomas differ in their clinical characteristics, including ECM deposition, and we recently reported that the characteristics of collagen-producing cells differed between control human anterior pituitary gland and pituitary adenomas. ECM deposition is not defined solely by production; degradation and maintenance are also important. Tissue inhibitors of metalloproteinases (TIMPs) help maintain ECM by inhibiting degradation caused by matrix metalloproteases. The present study attempted to characterize TIMP-expressing cells in the human anterior pituitary. Specimens of human pituitary adenomas and control pituitary were obtained during surgery, and in situ hybridization for TIMP1, TIMP2, TIMP3, and TIMP4, followed by immunohistochemistry, was used to characterize TIMP-expressing cells. TIMP expression exhibited a distinct pattern in the human anterior pituitary. Azan staining showed that fibrous matrix deposition varied among pituitary adenomas and that the area of fibrosis was associated with the number and number of types of TIMP3-expressing cells. These results suggest that TIMPs are important in the maintenance of ECM in human pituitary and that TIMP expressions are altered in fibrosis associated with pituitary adenoma.

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10(-6) M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

The rat anterior pituitary is composed of hormone-producing cells, non-hormone-producing cells (referred to as folliculostellate cells) and marginal layer cells. In the adult rat, progenitor cells of hormone-producing cells have recently been reported to be maintained within this non-hormone-producing cell population. In tissue, non-hormone-producing cells construct homophilic cell aggregates by the differential expression of the cell adhesion molecule E-cadherin. We have previously shown that Notch signaling, a known regulator of progenitor cells in a number of organs, is activated in the cell aggregates. We now investigate the relationship between Notch signaling and E-cadherin-mediated cell adhesion in the pituitary gland. Immunohistochemically, Notch signaling receptor Notch2 and the ligand Jagged1 were localized within E-cadherin-positive cells in the marginal cell layer and in the main part of the anterior lobe, whereas Notch1 was localized in E-cadherin-positive and -negative cells. Activation of Notch signaling within E-cadherin-positive cells was confirmed by immunostaining of the Notch target HES1. Notch2 and Jagged1 were always co-localized within the same cells suggesting that homologous cells have reciprocal effects in activating Notch signaling. When the E-cadherin function was inhibited by exposure to a monoclonal antibody (DECMA-1) in primary monolayer cell culture, the percentage of HES1-positive cells among Notch2-positive cells was less than half that of the control. The present results suggest that E-cadherin-mediated cell attachment is necessary for the activation of Notch signaling in the anterior pituitary gland but not for the expression of the Notch2 molecule.

After publication of reports describing the presence of stem/progenitor cells among non-hormone-producing cells in the pituitary, the mechanism responsible for proliferation and differentiation generated considerable interest. Several studies have suggested that Notch signaling is involved. In the present study, we examined the histochemical relationship between Notch signaling molecules and the transcription factor SOX2 in rat pituitary. Combined in situ hybridization and immunohistochemistry showed that Notch2 mRNA and SOX2 were co-expressed at embryonic day 14.5 in most cells in the adenohypophyseal primordium. In adult rat pituitary, double immunohistochemistry showed that SOX2 and either Notch2 or the Notch signaling target HES1 were co-localized within cells with large oval nuclei in both the marginal cell layer and cell aggregates in the main part of the anterior lobe, which are believed to be stem cell niches. Furthermore, when the Notch signaling inhibitor DAPT was added to a primary culture of adult rat anterior pituitary cells, the proportion of SOX2-expressing cells within Notch2-positive cells was approximately 30% lower. These findings suggest that Notch signaling has a role in maintaining the stemness of precursor cells in the adult rat pituitary gland.

Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the G(s)-proteinicAMP/CRE, G(12/13)-protein/Rho/SRE, and G(q)-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174th position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. (C) 2015 Elsevier Inc. All rights reserved.

The extracellular matrix (ECM) is important in creating cellular environments in tissues. Recent studies have demonstrated that ECM components are localized in anterior pituitary cells and affect cell activity. Thus, clarifying the mechanism responsible for ECM maintenance would improve understanding of gland function. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of matrix metalloproteinases and participate in ECM degradation. In this study, we investigated whether cells expressing TIMPs are present in rat anterior pituitary gland. Reverse transcription polymerase chain reaction was used to analyze expression of the TIMP family (TIMP1-4), and cells producing TIMPs in the gland were identified by using in situ hybridization. Expression of TIMP1, TIMP2, and TIMP3 mRNAs was detected, and the TIMP-expressing cells were located in the gland. The TIMP-expressing cells were also investigated by means of double-staining with in situ hybridization and immunohistochemical techniques. Double-staining revealed that TIMP1 mRNA was expressed in folliculostellate cells. TIMP2 mRNA was detected in folliculostellate cells, prolactin cells, and thyroid-stimulating hormone cells. TIMP3 mRNA was identified in endothelial cells, pericytes, novel desmin-immunopositive perivascular cells, and folliculostellate cells. These findings indicate that TIMP1-, TIMP2-, and TIMP3-expressing cells are present in rat anterior pituitary gland and that they are involved in maintaining ECM components.

Midkine (MK) belongs to a family of secreted heparin-binding growth factors and is highly expressed in various tissues during development. MK has multiple functions, such as regulation of cell proliferation, migration, survival and differentiation. We recently reported that MK mRNA is strongly expressed in the developing rat pituitary gland. In the adult pituitary, however, expression of MK and its receptor and the characteristics of the cells that produce them, have not been determined. Therefore, in this study, we investigate whether MK and its receptor, protein tyrosine phosphatase receptor-type Z (Ptprz1), are present in the adult rat pituitary. In situ hybridization, real-time reverse transcription-PCR and immunoblotting were performed to assess MK and Ptprz1 expression. We also characterize MK- and Ptprz1-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for each pituitary hormone or S100 protein [a marker of folliculostellate (FS) cells]. MK-expressing cells were located in the anterior and posterior lobes but not in the intermediate lobe. Double-staining and immunoblotting revealed that MK mRNA and protein were only expressed in FS cells in the anterior pituitary. Regarding Ptprz1 expression, Ptprz1 mRNA was detected in adrenocorticotropic hormone (ACTH) cells and growth hormone (GH) cells but not in prolactin cells, thyroid-stimulating hormone cells, luteinizing hormone cells, or FS cells. These findings suggest that MK produced in FS cells acts locally on ACTH cells and GH cells via Ptprz1 in the adult rat anterior pituitary.

Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OCR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NEAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. (C) 2015 Elsevier Inc. All rights reserved.

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke's pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.

Cell-matrix interaction is required for tissue development. Laminin, a major constituent of the basement membrane, is important for structural support and as a ligand in tissue development. Laminin has 19 isoforms, which are determined by combinational assembly of five α, three β, and three γ chains (eg, laminin 121 is α1, β2, and γ1). However, no report has identified the laminin isoforms expressed during pituitary development. We used in situ hybridization to investigate all laminin chains expressed during rat anterior pituitary development. The α5 chain was expressed during early pituitary development (embryonic day 12.5-15.5). Expression of α1 and α4 chains was noted in vasculature cells at embryonic day 19.5, but later diminished. The α1 chain was re-expressed in parenchymal cells of anterior lobe from postnatal day 10 (P10), while the α4 chain was present in vasculature cells from P30. The α2 and α3 chains were transiently expressed in vasculature cells and anterior lobe, respectively, only at P30. Widespread distribution of β and γ chains was also observed during development. These findings suggest that numerous laminin isoforms are involved in anterior pituitary gland development and that alteration of the expression pattern is required for proper development of the gland.

The anterior pituitary gland is organized tissue comprising hormone-producing cells and folliculostellate (FS) cells. FS cells interconnect to form a meshwork, and their cytoplasmic processes are anchored by a basement membrane containing laminin. Recently, we developed a three-dimensional (3D) cell culture that reproduces this FS cell architecture. In this study of the novel function of FS cells, we used transgenic rats that express green fluorescent protein in FS cells for the 3D culture. Anterior pituitary cells were cultured with different proportions of FS cells (0%, 5%, 10%, and 20%). Anterior pituitary cells containing 5-20% FS cells formed round/oval cell aggregates, whereas amorphous cell aggregates were formed in the absence of FS cells. Interestingly, immunohistochemistry showed laminin-immunopositive cells instead of extracellular laminin deposition in FS cell-deficient cell aggregates. Double-immunostaining revealed that these laminin-immunopositive cells were gonadotrophs. Laminin mRNA expression did not differ in relation to the presence or absence of FS cells. When anterior pituitary cells with no FS cells were cultured with FS cell-conditioned medium, the proportion of laminin-immunopositive cells was lower than in control. These results suggest that a humoral factor from FS cells is required for laminin release from gonadotrophs.

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide implicated in appetite regulation in mammals. However, except for teleost fish such as the goldfish and zebrafish, the involvement of NPY in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and pro-metamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in pre- and pro-metamorphic larvae. We investigated the role of NPY in food intake using bullfrog larvae including pre- and pro-metamorphic stages, and examined the effect of feeding status on the expression level of the NPY transcript in the hypothalamus. NPY mRNA levels in hypothalamus specimens obtained from larvae that had been fasted for 3 days were higher than those in larvae that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of NPY on food intake in the larvae. Cumulative food intake was significantly increased by ICV administration of NPY (5 and 10 pmol/g body weight, BW) during a 15-min observation period. The NPY-induced orexigenic action (10 pmol/g BW) was blocked by treatment with a NPY Y1 receptor antagonist, BIBP-3226 (100 pmol/g BW). These results indicate that NPY acts as an orexigenic factor in bullfrog larvae. (C) 2013 Elsevier Inc. All rights reserved.

The present study aimed to investigate the distribution of the octadecaneuropeptide (ODN) in the goldfish brain and to look for a possible effect of ODN on somatolactin (SL) release from pituitary cells. A discrete population of ODN-immunoreactive neurones was localised in the lateral part of the nucleus lateralis tuberis. These neurones sent projections through the neurohypophyseal tract towards the neurohypophysis, and nerve fibres were seen in the close vicinity of SL-producing cells in the pars intermedia. Incubation of cultured goldfish pituitary cells with graded concentrations of ODN (109105m) induced a dose-dependent stimulation of SL-, but not SL-, release. ODN-evoked SL release was blocked by the metabotrophic endozepine receptor antagonist cyclo18[DLeu5]OP but was not affected by the central-type benzodiazepine receptor antagonist flumazenil. ODN-induced SL release was suppressed by treatment with the phospholipase C (PLC) inhibitor U-73122 but not with the protein kinase A (PKA) inhibitor H-89. These results indicate that, in fish, ODN produced by hypothalamic neurones acts as a hypophysiotrophic neuropeptide stimulating SL release. The effect of ODN is mediated through a metabotrophic endozepine receptor positively coupled to the PLC/inositol 1,4,5-trisphosphate/protein kinase C-signalling pathway.

Food intake is a fundamental for animals to surviving and keeping offspring. The hypothalamic region of the brain and the brain stem in vertebrates are a center that plays an important role in the control of feeding and its related behaviors including locomotor and psychomotor activities. Pituitary adenylate cyclase-activating polypeptide (PACAP) has firstly been identified as a hypophysiotropic hormone involved in adenohypophyseal hormone release, and subsequently has been considered as a neuropeptide exerting multifunctional roles in the central and peripheral nervous systems and several tissues in vertebrates. For example, PACAP is involved in the neuroendocrine control of food intake and acts as an anorexigenic peptide to regulate satiety. Recent works on animal models such as rodents and goldfish which are both excellent animal models for investigating the neuroendocrinological roles of PACAP have been extensively examined and considerable information has been accumulated. In addition, psychophysiological effects of PACAP on emotional behavior have recently been found. Therefore, this review article provides an overview of the neuroendocrine regulation of feeding behavior and psyphysiological activity by PACAP in vertebrates. (C) 2012 Asian Oceanian Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Neuropeptide Y (NPY) is a neuropeptide distributed widely among vertebrates. In mammals, NPY and its related peptides such as pancreatic polypeptide and peptide YY (PYY) are distributed throughout the brain and gastrointestinal tissues, and are centrally involved in many physiological functions such as the regulation of food intake, locomotion and psychomotor activities through their receptors. With regard to non-mammalian vertebrates, there has also been intensive study aimed at the identification and functional characterization of NPY, PYY and their receptors, and recent investigations of the role of NPY have revealed that it exerts several behavioral effects in goldfish and zebrafish. Both of these species are excellent teleost fish models, in which it has been demonstrated that NPY increases food consumption as an orexigenic factor and reduces locomotor activity, as is the case in mammals. This paper reviews current knowledge of NPY derived from studies of teleost fish, as representative non-mammals, focusing particularly on the role of the NPY system, and examines its significance from a comparative viewpoint. (C) 2012 Elsevier Ltd. All rights reserved.

Orexin is a neuropeptide distributed widely among vertebrates. In mammals, orexin and its receptor system are involved in the regulation of food intake, locomotion, and psychomotor activities including the sleep/wakefulness cycle. With regard to nonmammalian vertebrates, there has also been intensive study aimed at the identification and functional characterization of orexin and its receptor, and recent investigations of the role of orexin have revealed that it exerts behavioral effects in teleost fish. Goldfish and zebrafish are excellent teleost fish models, and in these species it has been demonstrated that orexin increases food consumption as an orexigenic factor and enhances locomotor activity, as well as being involved in the regulation of active and rest status (circadian rhythmicity and the sleep/wakefulness cycle), as is the case in mammals. This chapter reviews current knowledge of orexin derived from studies of teleost fish, as representative nonmammals, focusing particularly on the role of the orexin system, and examines its significance from a comparative viewpoint. (C) 2012 Elsevier Inc.

Gonadotropin-releasing hormone (GnRH) is an evolutionarily conserved neuropeptide with 10 amino acid residues, of which several structural variants exist. A molecular form known as GnRH2 ([His5 Trp7 Tyr8]GnRH, also known as chicken GnRH II) is widely distributed in vertebrates except for rodents, and has recently been implicated in the regulation of feeding behavior in goldfish. However, the influence of GnRH2 on feeding behavior in other fish has not yet been studied. In the present study, therefore, we investigated the role of GnRH2 in the regulation of feeding behavior in a zebrafish model, and examined its involvement in food intake after intracerebroventricular (ICV) administration. ICV injection of GnRH2 at 0.1 and 1 pmol/g body weight (BW) induced a marked decrease of food consumption in a dose-dependent manner during 30 min after feeding. Cumulative food intake was significantly decreased by ICV injection of GnRH2 at 1 pmol/g BW during the 30-min post-treatment observation period. The anorexigenic action of GnRH2 was completely blocked by treatment with the GnRH type I receptor antagonist Antide at 25 pmol/g BW. We also examined the effect of feeding condition on the expression level of the GnRH2 transcript in the hypothalamus. Levels of GnRH2 mRNA obtained from fish that had been provided excess food for 7 days were higher than those in fish that had been fed normally. These results suggest that, in zebrafish, GnRH2 acts as an anorexigenic factor, as is the case in goldfish. © 2012 Nishiguchi, Azuma, Yokobori, Uchiyama and Matsuda.

Although spice compounds have several pharmacological and biochemical actions such as antioxidant activity, their physiological effects on neuropeptides related to feeding regulation are not well known. The aim of the present study was to identify the pharmacological activities of spice compounds on appetite regulation using a goldfish (Carassius auratus) model with emphasis on the role of neuropeptides. The spice compounds used in this study were curcumin, piperine, and ursolic acid. Goldfish were injected intraperitoneally with test solutions containing each spice or vehicle (including 10% dimethyl sulfoxide in saline), and the changes in food intake were measured every 15 min for 60 min. Among the tested spice compounds, curcumin was found to reduce cumulative food intake and was thus selected for further experiments. Pretreatment with capsaicin, a neurotoxin of afferent nerves, abolished the curcumin-induced decrease of food intake. Curcumin-induced anorexigenic action was also attenuated by intracerebroventricular injection of the corticotropin-releasing hormone (CRH) receptor antagonist alpha-helical CRH((9-41)). We also examined the expression levels of mRNA for CRH, which is a potent anorexigenic neuropeptide in goldfish, in the diencephalon at 1 h after treatment with curcumin, and found that they were increased. Therefore, the reduction of appetite induced by curcumin treatment in goldfish was suggested to be mediated by the vagal afferent and subsequently through the CRH/CRH receptor pathway.

Orexin is a potent orexigenic neuropeptide implicated in feeding regulation of mammals. However, except for the case of goldfish, the involvement of orexin in the feeding behavior of teleost fish has not well been studied. Therefore, we investigated the role of orexin on food intake using a zebrafish (Danio redo) model. We examined the effect of feeding status on orexin-like immunoreactivity and the expression level of orexin transcript in the brain. The number of neuronal cells showing orexin-like immunoreactivity in the hypothalamic region, including the posterior tuberal nucleus, was significantly increased in fish fasted for 7 days. Orexin precursor mRNA levels in the brain obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of intracerebroventricular (ICV) administration of orexin A on food intake. Cumulative food intake was significantly increased by ICV administration of orexin A (at 0.3 and 3 pmol/g body weight, BW) during a 60-min observation period after treatment. The orexin A-induced orexigenic action (at 0.3 pmol/g BW) was blocked by treatment with an orexin receptor antagonist, SB334867, at 10 pmol/g BW. These results indicate that orexin A acts as feeding regulator in the zebrafish. (C) 2011 Elsevier Inc. All rights reserved.

I.c.v. administration of the octadecaneuropeptide (ODN), a peptide derived from diazepam-binding inhibitor (DBI), induces anorexigenic and anxiogenic-like actions in rodents. We have recently shown that, in goldfish, i.c.v. injection of ODN also reduces food consumption via the metabotropic endozepine receptor. However, there is little information regarding the structure of DBI and the psychophysiological roles of endozepines in fish. Therefore, in the present study, we isolated and cloned a cDNA encoding goldfish DBI. The deduced sequence exhibits high similarity with non-mammalian DBIs, and we investigated the effect of homologous ODN on psychomotor activity in goldfish. i.c.v. injection of synthetic goldfish ODN at 10 pmol/g body weight (BW) stimulated locomotor activity. Since intact goldfish placed in a tank with both black and white background areas prefers the black compartment, we developed a method for measuring the time taken for fish to move from the black to the white area. I.c.v. administration of diazepam (35 and 350 pmol/g BW) decreased, whereas i.c.v. administration of ODN (10 pmol/g BW) or the central-type benzodiazepine receptor inverse agonist FG-7142 (9 pmol/g BW) increased the time taken to move from the black to the white background area. The anxiogenic-like effect of ODN was blocked by the central-type benzodiazepine receptor antagonist flumazenil (100 pmol/g BW), but was not affected by the metabotropic endozepine receptor antagonist cyclo1-8[D-Leu(5)]octapeptide (100 pmol/g BW). These data indicate that ODN can potently affect locomotor and psychomotor activities in goldfish and that this action is mediated via the central-type benzodiazepine receptor-signaling pathway. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

We have been extensively investigating the mechanisms by which neuropeptides regulate feeding behavior by using a goldfish (Carassius auratus) model In this species the anorexigenic action of melanocortin peptide is centrally mediated via the corticotropin-releasing hormone (CRH)/CRH receptor neuronal system whereas sulfated cholecystokinin octapeptide (CCK-8s) is Involved in the appetite regulation as a peripheral anorexigenic factor The aim of the present study was to identify the mechanism of the anorexigenic effect of peripherally injected CCK-8s which has not yet been identified in goldfish Co-administration of capsaicin a neurotoxin that destroys primary sensory afferents at 100 nmol/g BW blocked the anorexigenic action of intraperitoneally injected CCK-8s (100 pmol/g BW) whereas the anorexigenic action of intracerebroventricularly injected CCK-8s (5 pmol/g BW) was not blocked by co-administration of capsaicin Pre-treatment with a specific CRH receptor antagonist alpha-helical CRH((9-41)) attenuated the anorexigenic action of CCK-8s The expression level of CRH mRNA in the diencephalic tissue of the CCK-8s-injected group was not changed but the level of proopiomelanocortin mRNA was significantly Increased at 1 h after treatment Therefore we have identified for the first time that the reduction of appetite induced by peripherally injected CCK-8s in goldfish appears to be mediated by the vagal afferent and subsequently through the melanocortin- and corticotropin-releasing hormone-signaling pathways (C) 2010 Elsevier Inc All rights reserved

Melanin-concentrating hormone (MCH)-containing neurons directly innervate the adenohypophysis in the teleost pituitary. We examined immunohistochemically the relationship between MCH-containing nerve fibres or endings and somatolactin (SL)-producing cells in the goldfish pituitary. Nerve fibres or endings with MCH-like immunoreactivity were identified in the neurohypophysis in close proximity to the adenohypophysial cells showing SL-like immunoreactivity. We also examined the effect of MCH on SL release from Cultured goldfish pituitary cells and SL synthesis using a cell immunoblot and a real-time PCR method. Treatment of individually dispersed pituitary cells with MCH 10(-7) M for 3 h decreased the area of SL-like immunoreactivity on immunoblots, and MCH-induced reductions in SL release were blocked by treatment with the mammalian MCH receptor (MCHR) antagonist, compound-30, at a concentration of 10(-5) M. Treatment with 10(-7) M MCH for 3 h did not affect sl-alpha and -beta (smtla and -b as given in the Zfin Database) mRNA expression levels. These led us to explore the signal transduction mechanism leading to the inhibition of SL release, for which we examined whether MCH-induced reductions in SL release are mediated by the G(i) or G(q) protein-coupled signalling pathway. The MCH-induced reductions in SL release were abolished by treatment with the G(i/o) protein inhibitors, NF023 (10(-5) M) or pertussis toxin (260 ng/ml), but not by the phospholipase C inhibitor, U-73122 (3 x 10(-6) M). These results indicate that MCH can potentially function as a hypothalamic factor suppressing SL release via the MCHR, and subsequently through the G(i) protein to inhibit the adenylate cyclase/cAMP/protein kinase A-signalling pathway in goldfish pituitary cells. journal of Endocrinology (2009) 203, 389-398

In the goldfish pituitary, nerve fibers containing pituitary adenylate cyclase-activating polypeptide (PACAP) are located in close proximity to somatolactin (SL)-producing cells, and PACAP enhances SL release from cultured pituitary cells. However, there is little information about the mechanism of PACAP-induced SL release. In order to elucidate this issue, we used the cell immunoblot method. Treatment with PACAP at 10(-8) and 10(-7) M, but not with vasoactive intestinal polypeptide (VIP) at the same concentrations, increased the immunoblot area for SL-like immunoreactivity from dispersed pituitary cells, and PACAP-induced SL release was blocked by treatment with the PACAP selective receptor (PAC(1)R) antagonist, PACAP((6-38)), at 10(-6) M, but not with the PACAP/VIP receptor antagonist, VIP((6-28)). PACAP-induced SL release was also attenuated by treatment with the calmodulin inhibitor, calmidazolium at 10(-6) M. This led us to explore the signal transduction mechanism up to SL release, and we examined whether PACAP-induced SL release is mediated by the adenylate cyclase (AC)/cAMP/protein kinase A (PKA)- or the phospholipase C (PLC)/inositol 1,4,5-trisphosphate (IP(3))/protein kinase C (PKC)-signaling pathway. PACAP-induced SL release was attenuated by treatment with the AC inhibitor, MDL-12330A, at 10(-5) M or with the PKA inhibitor, H-89, at 10(-5) M. PACAP-induced SL release was suppressed by treatment with the PLC inhibitor, U-73122, at 3 x 10(-6) M or with the PKC inhibitor, GF109203X, at 10(-6) M. These results suggest that PACAP can potentially function as a hypophysiotropic factor mediating SL release via the PAC(1)R and subsequently through perhaps the AC/cAMP/PKA- and the PLC/IP(3)/PKC-signaling pathways in goldfish pituitary cells. (C) 2009 Elsevier Inc. All rights reserved.