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To clarify the relationships between growth, endocrine status and habitat characteristics in Japanese eel (Anguilla japonica), plasma and stomach mRNA levels of ghrelin were examined in wild eels captured in the river and the bay, and in cultured eels during and after experimental fasting. Wild juvenile eels captured in freshwater habitats within the river showed significantly higher plasma and stomach mRNA levels of ghrelin than did fish obtained from brackish-water habitats within the bay. In cultured eels experimentally fasted for 4 weeks, plasma and stomach mRNA levels of ghrelin increased. After refeeding, the both parameters returned to the levels observed in continuously feeding control fish. In pigmented elvers, 2 months of feed restriction resulted in a significant increase in whole-body ghrelin mRNA. It is suggested that interaction between ghrelin and feeding is related to their habitats through differential food acquisition in fresh and brackish water environments.

DOI： 10.1016/j.cbpa.2020.110700

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Neuromedin U (NMU) shows circadian expression in the rat pars tuberalis (PT), and is known to be suppressed by melatonin. Here we examined the involvement of adenosine in the regulation of Nmu expression. We found that the rat PT expressed adenosine receptor A2b and that an adenosine receptor agonist, NECA, stimulated Nmu expression in brain slice cultures. In vitro promoter assays revealed that NECA stimulated Nmu promoter activity via a cAMP response element (CRE) in the presence of adenosine receptor A2b. NECA also increased the levels of phosphorylated CRE-binding protein. These findings suggest that adenosine stimulates Nmu expression by activating the cAMP signaling pathway through adenosine receptor A2b in the rat PT. This is the first report to demonstrate that Nmu expression in the PT is regulated by adenosine, which acts as an intravital central metabolic signal, in addition to melatonin, which acts as an external photoperiodic environmental signal.

DOI： 10.1016/j.mce.2019.110518

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We previously demonstrated that female Runx3 knockout (Runx3-/-) mice were anovulatory and their uteri were atrophic and that Runx3 mRNA was expressed in granulosa cells. To clarify how Runx3 regulates folliculogenesis and ovulation, we examine the effects of Runx3 knockout on the gene expression of growth factors associated with folliculogenesis and enzymes associated with steroidogenesis. In Runx3-/- mouse ovaries, the numbers of primary and antral follicles were lower than those in wild-type (wt) mice at 3 weeks of age, indicating that the loss of Runx3 affects folliculogenesis. The expression of genes encoding activin and inhibin subunits (Inha, Inhba and Inhbb) was also decreased in ovaries from the Runx3-/- mice compared with that in wt mice. Moreover, the expression of the genes Cyp11a1 and Cyp19a1 encoding steroidogenic enzymes was also decreased. In cultured granulosa cells from 3-week-old mouse ovaries, Cyp19a1 mRNA levels were lower in Runx3-/- mice than those in wt mice. Follicle-stimulating hormone (FSH) treatment increased Cyp19a1 mRNA levels in both wt and Runx3-/- granulosa cells in culture but the mRNA level in Runx3-/- granulosa cells was lower than that in wt ones, indicating that granulosa cells could not fully function in the absence of Runx3. At 3 weeks of age, gonadotropin α subunit, FSHβ subunit and luteinizing hormone (LH) β subunit mRNA levels were decreased in Runx3-/- mice. These findings suggest that Runx3 plays a key role in female reproduction by regulating folliculogenesis and steroidogenesis in granulosa cells.

DOI： 10.1007/s00441-018-2947-2

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Chicken early (EF) and late feathering (LF) are sex-linked phenotypes conferred by wild-type k+ and dominant K alleles on chromosome Z, respectively. Besides prolactin (PRL) receptor (PRLR) and sperm flagellar 2 (SPEF2) genes, the K allele contains a fusion gene in which partially duplicated PRLR (dPRLR) and SPEF2 (dSPEF2) genes are linked in a tail-to-tail manner. The causative dPRLR gene encodes a C-terminal truncated receptor. LF chickens have short or no primaries at hatching; however, their feather growth rate is higher than that of EF chickens. This study aimed to elucidate the molecular basis of the K allele's biphasic effect on feather development. By 3'RACE and RT-PCR analyses, we demonstrated that dSPEF2 gene transcription occurred beyond all coding exons of the dPRLR gene on the opposite strand and that dPRLR mRNA was less abundant than PRLR mRNA. In addition, a 5'UTR splice variant (SPV) of PRL receptor mRNAs was increased in LF chickens. In vitro expression analysis of 5'UTR linked to the luciferase reporter gene revealed higher translation efficiency of SPV. RT-qPCR showed that the dPRLR mRNA level was higher in embryos; conversely, SPV was higher in hatched chickens, as was dSPEF2 mRNA. These findings suggest that the K allele inhibits feather development at the fetal stage by expressing dPRLR to attenuate PRLR function and promotes feather growth after hatching by increasing PRLR through dSPEF2 mRNA expression. Increased SPV may cause greater feather growth than that in EF chickens by increasing the availability of PRLR homodimers and enhancing PRL signaling.

DOI： 10.1016/j.ygcen.2018.12.011

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We previously demonstrated that the Runx3 transcription factor is expressed in the hypothalami, pituitaries, and ovaries of mice, and that Runx3 knockout (Runx3-/-) mice are anovulatory and their uteri are atrophic. Runx3 mRNA expression was detected in the granulosa cells of ovarian follicles, and in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC). In the present study, we examined the effects of Runx3 knockout on the gene expression of enzymes associated with steroidogenesis. We found decreased Cyp11a1 mRNA expression in Runx3-/- mouse ovaries compared with that in wild-type (wt) mouse ovaries at the age of 8 weeks. In situ hybridization analysis showed that the percentages of Cyp11a1 mRNA-expressing theca cells in follicles of Runx3-/- mice were decreased compared with those of wt mice. In accord with the alterations in Runx3-/- mouse ovaries, Kiss1 mRNA levels in ARC were increased, whereas mRNA levels of kisspeptin in AVPV were decreased, and gonadotropin-releasing hormone in the preoptic area and follicle-stimulating hormone β subunit gene were increased in Runx3-/- mice. Following an ovarian transplantation experiment between Runx3-/- mice and wt mice, corpora lutea were observed when ovaries from Runx3-/- mice were transplanted into wt mice, but not when those from wt mice were transplanted into Runx3-/- mice, suggesting that Runx3 in the hypothalamo-pituitary system may drive gonadotropin release to induce ovulation in the ovary. These findings indicate that Runx3 plays a crucial role in the hypothalamo-pituitary-gonadal axis.

DOI： 10.1262/jrd.2016-005

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Melanocortin receptor 3 (MC3R) is expressed in the hypothalamus and pituitary in humans and rodents, and is involved in the control of feeding, energy metabolism, and pituitary function. In the mouse pituitary, MC3R is detected in mammotrophs. This study aimed to clarify the regulatory mechanism for Mc3r expression in the mouse pituitary. The promoter activities of reporter constructs for the MC3R gene 5'-flanking region up to -4000 bp (transcription initiation site designated as +1) were analyzed. The promoter activity significantly increased in the -86/+109 construct, but decreased in the -38/+109 construct, indicating that the minimal promoter required for basal expression of Mc3r is located in the -86/+109 region. Putative binding sites for transcription factors AP-1 and ATF4 were found in the 5'-flanking region of Mc3r. Site-directed mutation or deletion of these sites affected the promoter activities. In gel-shift assays with a nuclear extract of mouse anterior pituitary cells, band-shifts were detected for both sites after the addition of the nuclear extract, and were decreased in the presence of excess unlabeled probe competitors. These results indicated that both sites were involved in the regulation of Mc3r expression in anterior pituitary cells. Estradiol-17β treatment increased the Mc3r promoter activity, indicating that the gene is regulated by estradiol-17β. In conclusion, we have demonstrated the minimum promoter region required for Mc3r expression, and identified two binding sites for AP-1 and ATF4 and in the 5' upstream-flanking region of Mc3r that are essential for Mc3r expression.

DOI： 10.1016/j.gene.2015.02.043

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

INTRODUCTION: The adrenomedullins (AMs) comprise a hormonal family in mammals and teleost fishes, with five members (AM1-5) found or predicted in most of the teleosts including Japanese medaka (Oryzias latipes). AM1 is known to have cardiovascular and osmoregulatory functions in mammals, but the roles of most AMs are yet to be determined. RESULTS: Using medaka, we first analyzed the tissue distribution of all five AM genes and found detectable expression in all tissues examined, with relatively high levels of AM3 and AM5 in the liver and kidney. To assess the osmoregulatory roles of these AMs, mRNA levels were examined in the brain (including the eyes), gill, liver, kidney and spleen of medaka one week after transfer from isotonic saline (11 ppt) to freshwater (0 ppt) or seawater (33 ppt). Expression of AM1 in the brain-eye increased in freshwater. The central level of AM4 (the paralog of AM1) decreased in seawater; the branchial level of AM4 decreased in freshwater and seawater, but the renal level increased in freshwater. The branchial level of AM2 increased in seawater, whereas the renal level decreased in freshwater and seawater. Expression of AM3, the AM2 paralog, decreased in the brain-eye of seawater-acclimated fish. Expression of AM5 in the brain-eye and kidney decreased in seawater. CONCLUSIONS: Except for branchial AM2, the members of AM family tend to be involved in promotion of hyper-osmoregulation and/or inhibition of hypo-osmoregulation, although each AM may play a distinct role during adaptation to different salinities.

DOI： 10.1186/s40851-015-0012-5

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Feathers are elaborate skin appendages shared by birds and theropod dinosaurs that have hierarchical branching of the rachis, barbs, and barbules. Feather filaments consist of β-keratins encoded by multiple genes, most of which are located in tandem arrays on chromosomes 2, 25, and 27 in chicken. The expansion of the genes is thought to have contributed to feather evolution; however, it is unclear how the individual genes are involved in feather formation. The aim of the present study was to identify feather keratin genes involved in the formation of barbules. Using a combination of microarray analysis, reverse-transcription polymerase chain reaction, and in situ hybridization, we found an uncharacterized keratin gene on chromosome 7 that was expressed specifically in barbule cells in regenerating chicken feathers. We have named the gene barbule specific keratin 1 (BlSK1). The BlSK1 gene structure was similar to the gene structure of previously characterized feather keratin genes, and consisted of a non-coding leader exon, an intron, and an exon with an open reading frame (ORF). The ORF was predicted to encode a 98 aa long protein, which shared 59% identity with feather keratin B. Orthologs of BlSK1 were found in the genomes of other avian species, including turkey, duck, zebra finch, and flycatcher, in regions that shared synteny with chromosome 7 of chicken. Interestingly, BlSK1 was expressed in feather follicles that generated pennaceous barbules but not in follicles that generated plumulaceous barbules. These results suggested that the composition of feather keratins probably varies depending on the structure of the feather filaments and, that individual feather keratin genes may be involved in building different portions and/or types of feathers in chicken.

DOI： 10.1016/j.gene.2014.03.027

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Vertebrates have expanded their habitats from aquatic to terrestrial environments, which has accompanied the evolution of cardiovascular and osmoregulatory hormones. Specifically, mammals have developed mechanisms to maintain high blood pressure and blood volume, while extant fishes have developed hypotensive and Na-extruding mechanisms to adapt to the marine environment where they underwent a vast diversification. The CGRP family is one of the hormone systems that decrease blood pressure and blood volume. Within the CGRP family of teleost fishes, we found that adrenomedullins (AMs) have diversified and five paralogs (AM1-5) form an independent subfamily. Based on this discovery in fishes, we found AM2 and AM5 in mammals. In mammalian species that have AM2 and/or AM5, the peptides assume greater importance in the case of pathophysiological disturbances in pressure and fluid balance such as hypertension and cardiac and renal failure. In addition, novel functions of AM peptides have been suggested by the discovery of AM2 and AM5 in mammals. Current research on the CGRP family is focused on the identification of new receptors for AM2/AM5 and the establishment of AM2 knockout mice, which will enable new developments in the basic and clinical research on this intriguing hormone family. Importantly, comparative fish studies can contribute to new developments in our understanding of the function of the AM peptides.

DOI： 10.2174/13892037113149990048

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We examined growth of euryhaline Japanese medaka (Oryzias latipes) after transfer to freshwater or seawater from isotonic saline. Growth was unaffected by the different salinities for 1 week, but the body weight increase and BMI of fish kept in freshwater for 2-3 weeks were significantly higher than those in the isotonic controls. These results may reflect the usual habitat of this species. To assess the basis for the difference in growth, energetics and the hepatic stress axis were evaluated 1 week after the transfer. Unexpectedly, despite the higher growth rate, the rate of routine oxygen consumption was significantly higher in freshwater. Plasma cortisol levels in freshwater were significantly higher than those in seawater, and the mRNA levels of the glucocorticoid receptor (GR1) in the liver were significantly lower in freshwater and seawater, compared to that in isotonic saline. Branchial Na(+)/K(+)-ATPase activities were also reduced significantly in freshwater and seawater, compared to that in isotonic saline. The higher levels of hepatic GR1 expression and branchial Na(+)/K(+)-ATPase activity in isotonic salinity than those in freshwater and seawater for 1 week may account for the lower growth rate under the isotonic condition. After 3 weeks, however, the Na(+)/K(+)-ATPase activity in seawater was significantly higher than that in freshwater. No significant difference in growth rate between freshwater and seawater groups indicates that medaka is a good model for studies of hypo- and hyperosmotic adaptations, since osmoregulation is not strongly associated with size and growth.

DOI： 10.1016/j.ygcen.2012.05.001

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In cartilaginous fish, two cDNAs encoding calcitonin-family receptors were isolated for the first time from the stingray brain. The open reading frame of one receptor cDNA coded a 525-amino acid protein. The amino acid identity of this receptor to human calcitonin-receptor-like receptor (CRLR) is 64.5%, frog CRLR is 64.7%, and flounder CRLR is 61.2% and this was higher than to human calcitonin receptor (CTR) (46.1%), frog CTR (54.7%), and flounder CTR (48.9%). We strongly suggested that this receptor is a ray CRLR based on phylogenetic analysis. In case of the second receptor, amino acid identity among CRLRs (human 50.5%, frog 50.7%, flounder 48.0%) and CTRs (human 43.2%, frog 49.1%, flounder 41.8%) was similar. From phylogenetic analysis of both CRLRs and CTRs, we believe that this receptor is ray CTR. The expression of ray CRLR mRNA was predominantly detected in the nervous system (brain) and vascular system (atrium, ventricle, and gill), which reflects the similar localization of CGRP in the nervous and vascular systems as mammals. It was observed that the second receptor was expressed in several tissues, namely cartilage, brain, pituitary gland, gill, atrium, ventricle, pancreas, spleen, liver, gall bladder, intestine, rectal gland, kidney, testis and ovary. This localization pattern was very similar to flounder CTR. Both receptor mRNAs were strongly expressed in the gill. This suggests that the calcitonin-family members are involved in the osmoregulation of stingray as this fish is known to be euryhaline. When a stingray was transferred to diluted seawater (20% seawater), the expression of both receptors significantly decreased in the gill. Similar results were obtained in the kidney of the stingray. Thus, our cloning and isolation of both receptors in the stingray will be helpful for elucidation of their physiological role(s) such as osmoregulation including calcium metabolism of cartilaginous fish.

DOI： 10.1016/j.gene.2012.03.042

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Steroid hormones easily cross the blood-brain barrier because of their physicochemical lipid solubility. The hormones act through nuclear receptor-mediated mechanisms and modulate gene transcription. In contrast to their genomic actions, the non-genomic rapid action of steroid hormones, acting via various types of membrane-associated receptors, reveals pharmacological properties that are distinct from the actions of the intracellular nuclear receptors. As a result, non-genomic rapid actions have gained increased scientific interest. However, insight into the phylogenic and/or comparative actions of steroids in the brain is still poorly understood. In this review, we summarize recent findings concerning the rapid, non-genomic signaling of steroid hormones in the vertebrate central nervous system, and we discuss (using a comparative view from fish to mammals) recently published data regarding the mechanism underlying physiology and behavior.

DOI： 10.2741/3970

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Authorship：Lead author   Language：Japanese   Publisher：Japan Society for Comparative Endocrinology  

DOI： 10.5983/nl2008jsce.38.209

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Other Link： https://jlc.jst.go.jp/DN/JALC/10013529337?from=CiNii

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The swimbladder volume is regulated by O(2) transfer between the luminal space and the blood In the swimbladder, lactic acid generation by anaerobic glycolysis in the gas gland epithelial cells and its recycling through the rete mirabile bundles of countercurrent capillaries are essential for local blood acidification and oxygen liberation from hemoglobin by the "Root effect." While O(2) generation is critical for fish flotation, the molecular mechanism of the secretion and recycling of lactic acid in this critical process is not clear. To clarify molecules that are involved in the blood acidification and visualize the route of lactic acid movement, we analyzed the expression of 17 members of the H(+)/monocarboxylate transporter (MCT) family in the fugu genome and found that only MCT1b and MCT4b are highly expressed in the fugu swimbladder. Electrophysiological analyses demonstrated that MCT1b is a high-affinity lactate transporter whereas MCT4b is a low-affinity/high-conductance lactate transporter. Immunohistochemistry demonstrated that (i) MCT4b expresses in gas gland cells together with the glycolytic enzyme GAPDH at high level and mediate lactic acid secretion by gas gland cells, and (ii) MCT1b expresses in arterial, but not venous, capillary endothelial cells in rete mirabile and mediates recycling of lactic acid in the rete mirabile by solute-specific transcellular transport. These results clarified the mechanism of the blood acidification in the swimbladder by spatially organized two lactic acid transporters MCT4b and MCT1b.

DOI： 10.1371/journal.pone.0034579

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DOI： 10.1186/2008-6970-4-18

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It has long been held that cortisol, a glucocorticoid in many vertebrates, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. However, 11-deoxycorticosterone (DOC) has been identified as a specific endogenous ligand for the teleostean mineralocorticoid receptor (MR). Furthermore, the expressions of MR mRNA are modest in the osmoregulatory organs, but considerably higher in the brain of most teleosts. These recent findings suggest that the mineralocorticoid system (DOC/MR) may carry out some behavioral functions in fish. To test this possibility, we examined the effects of cortisol and DOC administration in the amphibious behavior in mudskipper (Periophthalmus modestus) in vivo. It was found that mudskippers remained in the water for an increased period of time when they were immersed into 5 μM DOC or cortisol for 8h. Additionally, an exposure to 25 μM DOC for 4 to 8 h caused a decreased migratory frequency of mudskippers to the water, reflected a tendency to remain in the water. It was further observed that after 8 h of intracerebroventricular (ICV) injection with 0.3 pmol DOC or cortisol the staying period in the water increased in fish. The migratory frequency was decreased after ICV DOC injection which indicated that fishes stayed in the water. Concurrent ICV injections of cortisol with RU486 [a specific glucocorticoid-receptor (GR) antagonist] inhibited only the partial effects of cortisol. Together with no changes in the plasma DOC concentrations under terrestrial conditions, these results indicate the involvement of brain MRs as cortisol receptors in the preference for an aquatic habitat of mudskippers. Although the role of GR signaling cannot be excluded in the aquatic preference, our data further suggest that the MR may play an important role in the brain dependent behaviors of teleost fish.

DOI： 10.1016/j.physbeh.2011.06.002

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The process of NaCl reabsorption in the distal nephron allows freshwater fishes to excrete hypotonic urine and seawater fishes to excrete urine containing high concentrations of divalent ions; the relevant transporters, however, have not yet been identified. In the mammalian distal nephron, NaCl absorption is mediated by Na(+)-K(+)-Cl(-) cotransporter 2 (NKCC2, Slc12a1) in the thick ascending limb, Na(+)-Cl(-) cotransporter (NCC, Slc12a3) in the distal convoluted tubule, and epithelial sodium channel (ENaC) in the collecting duct. In this study, we compared the expression profiles of these proteins in the kidneys of euryhaline and seawater pufferfishes. Mining the fugu genome identified one NKCC2 gene and one NCC gene, but no ENaC gene. RT-PCR and in situ hybridization analyses demonstrated that NKCC2 was highly expressed in the distal tubules and NCC was highly expressed in the collecting ducts of euryhaline pufferfish (mefugu, Takifugu obscurus). On the other hand, the kidney of seawater pufferfish (torafugu, Takifugu rubripes), which lacked distal tubules, expressed very low levels of NCC, and, in the collecting ducts, high levels of NKCC2. Acclimation of mefugu to seawater resulted in a 2.7× decrease in NCC expression, whereas NKCC2 expression was not markedly affected. Additionally, internalization of NCC from the apical surface of the collecting ducts was observed. These results suggest that NaCl reabsorption in the distal nephron of the fish kidney is mediated by NCC and NKCC2 in freshwater and by NKCC2 in seawater.

DOI： 10.1152/ajpregu.00725.2009

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© Springer Science+Business Media B.V. 2010. All rights reserved. Adrenomedullin (AM) comprises a unique family of five paralogous peptides (AM1, 2, 3, 4 and 5) in teleost fish, of which AM1 is an ortholog of mammalian AM, and AM1/4 and AM2/3 were produced at the teleost-specific whole genome duplication. Therefore, CGRP, amylin, AM1, AM2 and AM5 existed when ray-finned fish and lobe-finned fish (leading to tetrapods) were diverged. Based on this finding, we discovered novel AM2 and AM5 in mammals. In addition, comparative genomic analyses based on fish studies delineated an evolutionary history of the CGRP family of peptides in vertebrates. As a first chapter of this volume, we initially propose an idea of how the CGRP family, including multiple AM peptides, have been organized during the course of vertebrate evolution. We will also show how comparative fish studies can contribute to general and clinical endocrinology by providing new insights into the molecule and function of the CGRP family throughout vertebrate species.

DOI： 10.1007/978-90-481-2909-6_1

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Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

DOI： 10.1152/ajpregu.00228.2009

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER PHYSIOLOGICAL SOC  

The teleost adrenomedullin (AM) family consists of three groups, AM1/AM4, AM2/AM3, and AM5. In the present study, we examined the effects of homologous AM1, AM2, and AM5 on drinking and renal function after peripheral or central administration in conscious freshwater eels. AM2 and AM5, but not AM1, exhibited dose-dependent (0.01-1 nmol/kg) dipsogenic and antidiuretic effects after intra-arterial bolus injection. The antidiuretic effect was significantly correlated with the degree of associated hypotension. To avoid the potential indirect osmoregulatory effects of AM-induced hypotension, infusion of AMs was also performed at nondepressor doses. Drinking was enhanced dose-dependently at 0.1-3 pmol.kg(-1).min(-1) of AM2 and AM5, matching the potency and efficacy of angiotensin II (ANG II), the most potent dipsogenic hormone known thus far. AM2 and AM5 infusion also induced mild antidiuresis, while AM1 caused antinatriuresis. Additionally, AMs were injected into the third and fourth ventricles of conscious eels to assess their site of dipsogenic action. However, none of the AMs at 0.05-0.5 nmol induced drinking, while ANG II was highly dipsogenic. AM2 and ANG II injected into the third ventricle increased arterial pressure while AM5 decreased it in a dose-dependent manner, and both AM2 and AM5 decreased blood pressure when injected into the fourth ventricle. These data suggest that circulating AM2 and AM5 act on a target site in the brain that lacks the blood-brain barrier. Collectively, the present study showed that AM2 and AM5 are potent osmoregulatory hormones in the eel, and their actions imply involvement in seawater adaptation of this euryhaline species.

DOI： 10.1152/ajpregu.90688.2008

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Adrenomedullin 5 (AM5) is a new member of the calcitonin gene-related peptide (CGRP) family idetitified in teleost fish. Although its presence was suggested in the genome database of mammals, molecular identity and biological function of AM5 have not been examined yet. In this study, we cloned a cDNA encoding AM5 in the pig and examined its cardiovascular and renal effects. Putative mature AM5 was localized in the middle of prohormone and had potential signals for intermolecular ring formation and C-terminal amidation. The AM5 gene was expressed most abundantly in the spleen and thymus. Several AM5 genes were newly identified in the database of mammals, which revealed that the AM5 gene exists in primates, carnivores, and undulates but could not be identified in rodents. In primates, nucleotide deletion occurred in the mature AM5 sequence in anthropoids (human and chimp) during transition from the rhesus monkey. Synthetic mature AM5 injected intravenously into rats induced dose-dependent decreases in arterial pressure at 0.1-1 nmol/kg without apparent changes in heart rate. The decrease was maximal in 1 min and AM5 was approximately half as potent as AM. AM5 did not cause significant changes in urine flow and urine Na+ concentration at any dose. In contrast to the peripheral vasodepressor action, AM5 injected into the cerebral ventricle dose-dependently increased arterial pressure and heart rate at 0.1-1 nmol. The increase reached maximum more quickly after AM5 (5 min) than AM (15-20 min). AM5 added to the culture cells expressing calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of the receptor activity-modifying proteins (RAMPs), the combination of which forms major receptors for the CGRP family, did not induce appreciable increases in cAMP production in any combination, although AM increased it at 1010 -10-9 M when added to the CLR and RAMP2/3 combination. These data indicate that AM5 seems to act on as yet unknown receptor(s) for AM5, other than CLP/CTR+RAMP, to exert central and peripheral cardiovascular actions in mammals. © 2008 Society for Endocrinology.

DOI： 10.1677/JOE-07-0541

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Adrenomedullin (AM), known as a multifunctional hormone in mammals, forms a unique family of five paralogous peptides in teleost fish. To examine their cardiovascular effects using homologous AMs in eels, we isolated cDNAs encoding four eel AMs, and named AM1 (ortholog of mammalian AM), AM2, AM3 (paralog of AM2 generated only in teleost lineage), and AM5 according to the known teleost AM sequences. Unlike pufferfish, not only AM1 but AM2/3 and AM5 were expressed ubiquitously in various eel tissues. Synthetic mature AM1, AM2, and AM5 exhibited vasodepressor effects after intra-arterial injections, and the effects were more potent at dorsal aorta than at ventral aorta. This indicates that AMs preferentially act on peripheral resistance vessels rather than on branchial arterioles. The potency was in the order of AM2 = AM5 > AM1 in both freshwater (FW) and seawater (SW) eels, which is different from the result of mammals in which AM1 is as potent as, or more potent than, AM2 when injected peripherally. The minimum effective dose of AM2 and AM5 in eels was 1/10 that of AM1 in mammals. The hypotension reached 50% at 1.0 nmol/kg of AM2 and AM5, which is much greater than atrial natriuretic peptide (20%), another potent vasodepressor hormone. Even with such hypotension, AMs did not change heart rate in eels. In addition, AM1 increased blood pressure at ventral aorta and dorsal aorta immediately after an initial hypotension at 5.0 nmol/kg, but not with AM2 and AM5. These data strongly suggest that specific receptors for AM2 and AM5 exist in eels, which differ from the AM1 receptors identified in mammals.

DOI： 10.1152/ajpregu.00707.2007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Vertebrates expanded their habitats from aquatic to terrestrial environments during the course of evolution. In parallel, osmoregulatory and cardiovascular systems evolved to counter the problems of desiccation and gravity on land. In our physiological studies on body fluid and blood pressure regulation in various vertebrate species, we found that osmoregulatory and cardiovascular hormones have changed their structure and function during the transition from aquatic to terrestrial life. In fact, Na(+)-regulating and vasodepressor hormones play essential roles in fishes, while water-regulating and vasopressor hormones are dominant in tetrapods. Accordingly, Na(+)-regulating and vasodepressor hormones, such as natriuretic peptide (NP) and adrenomedullin (AM), are much diversified in teleost fishes compared with mammals. Based on this finding, new NPs and AMs were identified in mammals and other tetrapods. These hormones have only minor roles in the maintenance of normal blood volume and pressure in mammals, but their importance seems to increase when homeostasis is disrupted. Therefore, such hormones can be used for diagnosis and treatment of body fluid and cardiovascular disorders such as cardiac/renal failure and hypertension. In this review, we introduce a new approach for identification of novel Na(+)-regulating and vasodepressor hormones in mammals based on fish studies. Until recently, new hormones were first discovered in mammals, and then identified and applied in fishes. However, chances are increasing in recent years to identify new hormones first in fishes then in mammals, based on the difference in the regulatory systems between fishes and tetrapods. As the direction is opposite from the traditional phylogenetic approach, we added 'reverse' to its name. The 'reverse' phylogenetic approach offers a typical example of how comparative fish studies can contribute to the general and clinical endocrinology.

DOI： 10.1016/j.yfrne.2007.05.001

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Calcitonin (CT), a 32-amino acid peptide, was initially isolated from fish. Fish CT has higher affinity to mammalian CT receptor (CTR). and has activity on calcium homeostasis. Therefore, fish CT has been used as a drug for the treatment of human bone diseases. However, the physiological roles of CT in fish as well as the characteristics of the fish CTR have not been clarified. Here, we cloned and characterized CTR from mefugu (Takfiigu obscurus). Full-length cDNA sequencing revealed that mfCTR (mf, mefugu) consists of N-terminal four tandem putative hormone-binding domains (HBDs). Database mining showed that the multiple HBD-containing CTR is a common feature for some other fishes. Detailed pharmacological studies revealed that mfCTR generated cAMP in response to (1) fish CT (2) calcitonin gene-related peptide (CGRP) in combinations with receptor activity-modifying proteins (mfRAMPs) I and 4, and (3) amylin in combinations with mfRAMPs 1-5. Unlike mammalian CTR. mfCTR showed dual affinity sites. Corresponding EC50 values of those are in close proximity of the in vivo concentration of CT in fish. Analyses of the deletion mutants of mfCTR demonstrated that only the nearmost HBD to the first transmembrane region is functional to the ligands. Although, fish CT has higher affinity to the human CTR. human CT did not bind to the mfCTR. This is the first report that demonstrates the structure and property of fish receptor for CT. CGRR and amylin. Fish CTR is the first example that has multiple HBD-like sequences. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ygcen.2007.06.017

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

The calcitonin gene-related peptide (CGRP) family is composed of CGRP, amylin and adrenomedullin (AM) in mammals. In teleost fish, AM forms an independent subfamily of five members (AM1-5), which inspired us to trace the evolutionary history of the CGRP family throughout vertebrates by comparative genomic approach. Linkage mapping and synteny analyses of the CGRP family genes in medaka, Oryzias latipes, revealed that AM1/CGRP, AM2/amylin, and AM5 genes were located on respective proto-chromosomes before the divergence of teleost lineage. In teleost fish, additional whole genome duplication generated AM1/4, CGRP1/2, AM2/3, but one of the duplicated amylin and AM5 genes was silenced. In mammals, the amylin or AM2 gene was translocated to different chromosomes, while the CGRP gene was multiplied in tandem to generate CGRP-alpha,beta, and recently identified calcitonin receptor-stimulating peptide genes. Based on these data, we identified a novel AM5 gene in several mammalian species as we previously did for AM2.

DOI： 10.1016/j.peptides.2006.09.011

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Authorship：Lead author  

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Language：English   Publishing type：Research paper (scientific journal)  

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.

DOI： 10.1002/jez.a.309

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DOI： 10.1002/jez.a.309

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have identified cDNA encoding a new member of the adrenomedullin (AM) family, AM2, for the first time in mammals (mouse, rat and human). The predicted precursor carried mature AM2 in the C-terminus, which had an intramolecular ring formed by an S-S bond and a possibly amidated C-terminus. Phylogenetic analyses clustered AM2 and AM into two distinct but closely related groups. Similarity of exon-intron structure and synteny of neighboring genes showed that mammalian AM2 is an ortholog of pufferfish AM2 and a paralog of mammalian AM. AM2 mRNA was expressed in submaxillary gland, kidney, stomach, ovary, lymphoid tissues and pancreas of mice, but not in adrenal and testis. Intravenous injection of synthetic mature AM2 decreased arterial pressure more potently than AM, and induced antidiuresis and antinatriuresis in mice. These results show that at least two peptides, AM and AM2, comprise an adrenomedullin family in mammals, and that AM2 may play pivotal roles in cardiovascular and body fluid regulation.

DOI： 10.1016/S0014-5793(03)01368-1

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Adrenomedullin (AM) is a multifunctional peptide known to form a hormone family with calcitonin gene-related peptide (CGRP) and amylin. We have cloned five distinct AM cDNAs from the pufferfish, Takifugu rubripes, and named them TrAM-1, -2, -3, -4, and -5. Judging from the deduced precursor sequences and processing pattern of the C-terminal mature peptides, TrAMs may be divided into at least two groups; AM-2 and -3, and AM-1, -4, and possibly -5. Phylogenetic analysis of the mature peptides, exon-intron structure of their genes, and tissue distribution of their mRNA also support this classification. TrAM-1 and -4 were ubiquitously expressed in various tissues including the kidney and interrenal (adrenal homolog) as in the case of mammalian AM, while TrAM-2 and -3 were expressed most abundantly in the brain followed by the vascular tissues. Synteny of the genes around AM gene showed that TrAM-1 is the ortholog of mammalian AM. The presence of a PAMP-like sequence in the prosegment of TrAM-1 also supports this notion. Multiple AMs were also detected in another pufferfish, Tetraodon nigroviridis, and in zebrafish, Danio rerio. The present study shows for the first time the presence of a novel AM family in teleost fish that is independent from CGRP and amylin, which further suggests the possible existence of multiple AMs in mammals.

DOI： 10.1016/j.bbrc.2003.10.111

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Total pages：185p   Language：Japanese Book type：Textbook, survey, introduction

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Book type：Scholarly book

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ENDOCRINE SOC  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

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Language：Japanese   Publisher：Japan Society for Comparative Endocrinology  

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Language：Japanese   Publisher：日本比較内分泌学会  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：WILEY-LISS  

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DOI： 10.11477/mf.2425100299

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：English   Publisher：Zoological Society of Japan  

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Other Link： http://id.nii.ac.jp/1141/00038656/