Researcher Database

Munetaka Shimizu
Field Science Center for Northern Biosphere

Researcher Profile and Settings


  • Field Science Center for Northern Biosphere

Job Title

  • Professor


Research funding number

  • 90431337

J-Global ID

Research Interests

  • 機能解析   スモルト   成長・ストレス指標   増養殖   生活史   成長   サケ科魚類   内分泌   組換えタンパク   測定系   インスリン様成長因子と結合タンパク   

Research Areas

  • Life sciences / Aquaculture
  • Life sciences / Marine/Aquatic life sciences

Educational Organization

Academic & Professional Experience

  • 2023/04 - Today 北海道大学北方生物圏フィールド科学センター 七飯淡水実験所 所長(兼任)
  • 2021/04 - Today Hokkaido University Field Science Center for Northern Biosphere
  • 2017/03 - 2021/03 Hokkaido University Faculty of Fisheries Sciences
  • 2006/08 - 2017/02 Hokkaido University Faculty of Fisheries Sciences
  • 1999/09 - 2005/12 米国ワシントン大学海洋水産学部・NOAA Fisheries ポスドク
  • 1997/04 - 1999/08 日本学術振興会 特別研究員(PD)
  • 1995/04 - 1997/03 日本学術振興会 特別研究員(DC2)


  • 1993/04 - 1997/03  Hokkaido University
  • 1991/04 - 1993/03  Hokkaido University
  • 1987/04 - 1991/03  Hokkaido University  School of Fisheries Sciences

Association Memberships


Research Activities

Published Papers

  • Yuki Ugachi, Haruka Kitade, Eisuke Takahashi, Shotaro Suzuki, Mizuki Hayashi, Taiga Yamada, Wenda Cui, Munetaka Shimizu
    Scientific reports 13 (1) 16643 - 16643 2023/10/03 [Refereed]
    Anadromous salmonids exhibit partial migration, where some individuals within a population migrate down to the ocean through complex interactions between body size and photoperiod. This study aimed to integrate the ontogenetic and seasonal patterns of smoltification, a series of changes for future marine life, in a strain of masu salmon (Oncorhynchus masou). Spring smoltification, as evidenced by the activation of gill Na+,K+-ATPase (NKA), was induced during winter under an advanced photoperiod. In addition, juveniles showed an additional peak in gill NKA activity in August regardless of the photoperiod. When juvenile masu salmon were subjected to feeding manipulations during the first spring/summer, only fish exceeding a fork length of 12 cm exhibited an increased gill NKA activity. We tested whether size-driven smoltification required a long-day period by exposing juveniles to a constant short-day length (9-h light and 15-h dark) from January to November. Juveniles under short-day conditions exceeded 12 cm in June but showed no signs of smoltification. Thus, masu salmon undergo photoperiod-limited, size-driven smoltification during the first summer and size-limited, photoperiod-driven smoltification the following spring. The findings of the present study provide a framework for further elucidation of the physiological mechanisms underlying partial migration in salmonids.
  • Momoka Arimoto, Ayaka Izutsu, Akihiko Hara, Munetaka Shimizu
    Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 284 111488 - 111488 2023/10 [Refereed]
    Insulin-like growth factor-binding proteins (IGFBPs) regulate the activity of insulin-like growth factor (IGF)-1. Among the three major circulating IGFBPs in salmonids, IGFBP-1b is an inhibitor of IGF activity induced under catabolic conditions. IGFBP-1b is considered to quickly sequester IGF-1 from the circulation. However, the level of circulating IGFBP-1b present in its unoccupied free form is unknown. Here, we aimed to develop a non-equilibrium ligand immunofunctional assay (LIFA) to evaluate IGF-binding capacity of circulating intact IGFBP-1b. Purified Chinook salmon IGFBP-1b, its antiserum, and europium-labeled salmon IGF-1 were used as the assay components. In the LIFA, IGFBP-1b was first captured by the antiserum, allowed to bind to the labeled IGF-1 for 22 h at 4 °C, and quantified its IGF-binding capacity. Serial dilutions of the standard and serum were prepared simultaneously within a certain concentration range (1.1-12.5 ng/ml). In underyearling masu salmon, IGF-binding capacity of intact IGFBP-1b was higher in fasted fish than in fed fish. Transferring Chinook salmon parr to seawater also increased IGF-binding capacity of IGFBP-1b, most likely due to osmotic stress. In addition, there was a strong relationship between total IGFBP-1b levels and its IGF-binding capacity. These results suggest that IGFBP-1b expressed under stress is mostly present in the free form. On the contrary, during smoltification of masu salmon, IGF-binding capacity of IGFBP-1b in the serum was relatively low and less related to the total IGFBP-1b level, suggesting its functional difference under certain physiological conditions. These results indicate that estimating both total IGFBP-1b level and its IGF-binding capacity is useful for evaluating the catabolic status and unraveling the regulation of IGF-1 activity by IGFBP-1b.
  • Ayaka Izutsu, Shiori Habara, Nobuto Kaneko, Daiji Tadokoro, Akihiko Hara, Munetaka Shimizu
    General and comparative endocrinology 340 114305 - 114305 2023/09/01 [Refereed]
    Insulin-like growth factor (IGF)-1 promotes the growth of vertebrates, and its binding proteins (IGFBPs) regulate the activity of circulating IGF-1. Three IGFBPs, IGFBP-2b, -1a, and -1b, were consistently detected in the circulatory system of salmonids. IGFBP-2b is thought to be the main carrier of IGFs and promoter of IGF-1-mediated growth in salmonids. Currently, there are no immunoassays for detecting IGFBP-2b. In this study, we developed a time-resolved fluoroimmunoassay (TR-FIA) for IGFBP-2b detection in salmonid fishes. To establish TR-FIA, we produced two recombinant trout (rt) IGFBP-2bs expressed, one with thioredoxin (Trx) and a histidine (His) tag, and the other with His-tag only. We labeled both recombinant proteins with europium (Eu). Only Eu-Trx.His.rtIGFBP-2b cross-reacted with anti-IGFBP-2b, and the addition of increasing amounts of Trx.His.rtIGFBP-2b replaced the binding, indicating its utility as a tracer and assay standard. The addition of unlabeled salmon IGF-1 did not affect the binding of the standard or sample. Serial dilution curves of sera from rainbow trout, Chinook salmon, and chum salmon were parallel to those of the standard. The assay range (ED80-ED20) of the TR-FIA was 60.4 to 251.3 ng/ml, and its minimum detection limit of this assay was 21 ng/ml. The intra- and inter-assay coefficients of variation were 5.68% and 5.65%, respectively. Circulating IGFBP-2b levels in fed rainbow trout were higher than those in fasted fish and were correlated with individual growth rates. This TR-FIA is useful for further exploring the physiological responses of circulating IGFBP-2b and evaluating the growth status of salmonids.
  • Wenda Cui, Eisuke Takahashi, Bernat Morro, Pablo Balseiro, Amaya Albalat, Cindy Pedrosa, Simon Mackenzie, Tom O Nilsen, Harald Sveier, Lars O Ebbesson, Sigurd O Handeland, Munetaka Shimizu
    Comparative biochemistry and physiology. Part A, Molecular & integrative physiology 268 111205 - 111205 2022/06 [Refereed]
    Smoltification in salmonids occurs during spring in response to increasing photoperiod to prepare for marine life. Smoltification is associated with increased hypo-osmoregulatory ability and enhanced growth potential, mediated by growth hormone and insulin-like growth factor (IGF)-1. Rainbow trout is uniquely insensitive to the induction of smoltification-associated changes by photoperiod, such as the activation of gill Na+,K+-ATPase (NKA). We measured the circulating IGF-1 and IGF-binding protein (IGFBP)-2b levels in yearling rainbow trout exposed to natural and manipulated photoperiods during spring and correlated these with gill NKA activity and body size. Although the effect of photoperiod manipulation on body size and circulating IGF-1 and IGFBP-2b was negligible, they were positively correlated with gill NKA activity in fish under simulated natural photoperiod. We next pit-tagged yearling rainbow trout and fed them a restricted ration or to satiation under a natural photoperiod. In April, gill NKA activity was higher in the satiation group than in the restricted group and positively correlated with body size and growth rate. In addition, circulating IGFBP-2b was positively correlated with gill NKA, size and growth, whereas circulating IGF-1 was correlated only with size and growth. The relationship between circulating IGF-1 and growth intensified from May to June, suggesting that the IGF-1-growth relationship was disrupted in April when gill NKA was activated. Two additional IGFBPs were related to growth parameters but not to gill NKA activity. The present study suggests that circulating IGFBP-2b and IGF-1 mediate the size-dependent activation of gill NKA in yearling rainbow trout during spring.
  • Ayaka Izutsu, Daiji Tadokoro, Shiori Habara, Yuki Ugachi, Munetaka Shimizu
    General and comparative endocrinology 320 114008 - 114008 2022/05/01 [Refereed]
    Circulating insulin-like growth factor (IGF)-I has been proposed as a growth index in several teleosts, including salmonids, and its level in circulation is stabilized by multiple IGF-binding proteins (IGFBPs). Three IGFBPs, IGFBP-2b, -1a, and -1b, are consistently detected in salmonid blood and are suggested to be indices of positive or negative growth, although their applicability to rainbow trout (Oncorhynchus mykiss) is unclear. The present study examined the usefulness of IGFBPs along with IGF-I as a physiological indicator of growth rate in rainbow trout through a rearing experiment. Two groups of underyearling rainbow trout were pit-tagged and either fed or fasted for 33 days. A third group was fasted for 22 days, followed by refeeding for 11 days. Serum IGF-I levels were reduced after fasting for 22 days, but refeeding did not retore its levels to those of the fed control. Nevertheless, there was a positive relationship between serum IGF-I levels and individual growth rates over 33 days of experimentation, confirming its validity as a growth index. Ligand blotting using labeled human IGF-I revealed two IGFBP bands at 43 and 32 kDa, which corresponded to IGFBP-2b and an unidentified form, respectively. In contrast, bands corresponding to IGFBP-1a and -1b, which usually increase after fasting, were hardly detected, even in the fasted fish. The responses of circulating IGFBP-2b to fasting and refeeding were similar to those of circulating IGF-I and positively correlated with growth rate and IGF-I levels. The intensity of the serum 32-kDa IGFBP band was higher in constantly fed fish than in the fasted fish; however, its correlation with growth rate was weaker than those of IGF-I and IGFBP-2b. The present study shows that IGF-I and IGFBP-2b can be used as growth indices for rainbow trout. In contrast, circulating IGFBP-1a and -1b may not serve as negative growth indices in rainbow trout under regular aquaculture conditions because they are rarely detected by ligand blotting or respond to fasting/refeeding.
  • Mizuki Hayashi, Shu Maruoka, Jin Oikawa, Yuki Ugachi, Munetaka Shimizu
    Zoological science 38 (6) 513 - 522 2021/12 [Refereed]
    We examined the effects of environmental salinity and feeding status on the growth and metabolic parameters of underyearling masu salmon. Fish were first acclimated to salinities of 0 (< 0.1), 11, or 22 psu for 10 days, after which time 50% of the fish in each group were fasted for 5 days followed by refeeding for 5 days. No effects on body length/weight were observed over the 20 days from the beginning of the experiment. Gill Na+, K+-ATPase (NKA) activity increased 20 and 10 days after transfer to water at 11 and 22 psu, respectively. Serum Na+ and Cl- levels were high in fish at 22 psu on day 20 but much lower than those in the environmental water, suggesting that fish at this salinity were able to hypo-osmoregulate. However, acclimation to 22 psu resulted in a reduction in feeding rate on day 20. Serum insulin-like growth factor (IGF)-I levels and liver glycogen content were reduced by fasting and restored after 5 days of refeeding, except in the fish at 22 psu. Intensities of serum IGFBP-1a and -1b bands were increased at higher salinities, whereas fasting/refeeding affected only IGFBP-1b. The present study suggests that acclimating masu salmon parr to 11 psu had no effect on metabolic and growth parameters, while 22 psu presumably suppressed their growth potential due to the possible energy cost or stress for osmoregulation. The disparate responses of circulating IGFBP-1a and -1b to higher salinity and fasting highlight their utility as indices of various catabolic statuses.
  • Bernat Morro, Richard Broughton, Pablo Balseiro, Sigurd O Handeland, Simon Mackenzie, Mary K Doherty, Phillip D Whitfield, Munetaka Shimizu, Marnix Gorissen, Harald Sveier, Amaya Albalat
    BMC genomics 22 (1) 824 - 824 2021/11/16 [Refereed]
    BACKGROUND: Rainbow trout (Oncorhynchus mykiss) is a salmonid species with a complex life-history. Wild populations are naturally divided into freshwater residents and sea-run migrants. Migrants undergo an energy-demanding adaptation for life in seawater, known as smoltification, while freshwater residents display these changes in an attenuated magnitude and rate. Despite this, in seawater rainbow trout farming all fish are transferred to seawater. Under these circumstances, weeks after seawater transfer, a significant portion of the fish die (around 10%) or experience growth stunting (GS; around 10%), which represents an important profitability and welfare issue. The underlying causes leading to GS in seawater-transferred rainbow trout remain unknown. In this study, we aimed at characterising the GS phenotype in seawater-transferred rainbow trout using untargeted and targeted approaches. To this end, the liver proteome (LC-MS/MS) and lipidome (LC-MS) of GS and fast-growing phenotypes were profiled to identify molecules and processes that are characteristic of the GS phenotype. Moreover, the transcription, abundance or activity of key proteins and hormones related to osmoregulation (Gill Na+, K + -ATPase activity), growth (plasma IGF-I, and liver igf1, igfbp1b, ghr1 and ctsl) and stress (plasma cortisol) were measured using targeted approaches. RESULTS: No differences in Gill Na+, K + -ATPase activity and plasma cortisol were detected between the two groups. However, a significant downregulation in plasma IGF-I and liver igf1 transcription pointed at this growth factor as an important pathomechanism for GS. Changes in the liver proteome revealed reactive-oxygen-species-mediated endoplasmic reticulum stress as a key mechanism underlying the GS phenotype. From the lipidomic analysis, key observations include a reduction in triacylglycerols and elevated amounts of cardiolipins, a characteristic lipid class associated with oxidative stress, in GS phenotype. CONCLUSION: While the triggers to the activation of endoplasmic reticulum stress are still unknown, data from this study point towards a nutritional deficiency as an underlying driver of this phenotype.
  • Jin Oikawa, Shu Nakamura, Nobuto Kaneko, Mitsuru Torao, Yosuke Koshino, Munetaka Shimizu
    FISHERIES SCIENCE 87 (4) 579 - 588 0919-9268 2021/07 [Refereed]
    We examined the combined effects of fasting in freshwater and water temperature during the transition from freshwater to seawater on growth/metabolic parameters in juvenile chum salmon Oncorhynchus keta. Juveniles of 1 g in freshwater were first subjected to a 5-day fasting period at 10 or 5 degrees C, acclimated to either 10 or 5 degrees C seawater and fed ad libitum for 30 days. Control fish were transferred from 10 degrees C freshwater to 10 degrees C seawater and fed ad libitum throughout the experimental period. Serum insulin-like growth factor (IGF)-I was measured to evaluate growth status/potential and liver glycogen as an index of energy storage. Fasting in freshwater for 5 days negatively affected body size. Growth of juveniles kept at colder temperatures was retarded in seawater for at least 20 days, which may partly be explained by a lower feeding rate in cold seawater. Serum IGF-I levels were lower in fasted fish in freshwater at both temperatures and colder seawater had a negative effect on restoring serum IGF-I levels after refeeding for 20 days. Liver glycogen content was low in fish fasted in freshwater for 5 days. After refeeding in seawater for 10 days, liver glycogen content increased significantly in juveniles kept at colder temperatures. Colder water temperatures in both salinities positively affected glycogen content for 30 days after transfer to seawater, suggesting that juveniles allocated energy stores to the liver rather than growth under suboptimal feeding and temperature conditions. The findings of the present study suggested that relatively cold freshwater could negatively affect juvenile chum salmon growth soon after sea entry.
  • Ginnosuke Yamaguchi, Shiori Habara, Shotaro Suzuki, Yuki Ugachi, Hisashi Kawai, Takuro Nakajima, Munetaka Shimizu
    In this study, we examined the effects of porcine growth hormone (GH) and cortisol on plasma insulin-like growth factor binding proteins (IGFBPs) in juveniles of three subspecies of Oncorhynchus masou (masu, amago, and Biwa salmon). Ligand blotting using digoxigenin-labeled human IGF-I was used to detect and semiquantify three major circulating IGFBP bands at 41, 28, and 22 kDa, corresponding to IGFBP-2b, -1a, and -1b, respectively. GH increased plasma IGFBP-2b concentration in masu and Biwa salmon but suppressed it in amago salmon. Plasma IGFBP-2b levels were increased by cortisol in the three subspecies. Cortisol induced plasma IGFBP-1a in the three subspecies, whereas GH had a suppressive effect in masu and Biwa salmon. Sham and cortisol injections increased plasma IGFBP-1b levels after 1 day in masu and amago salmon, suggesting that IGFBP-1b is induced following exposure to stressors via cortisol. Increased IGFBP-lb levels were restored to basal levels when co-injected with GH in Biwa salmon, and the same trend was seen in masu and amago salmon. However, the suppressive effect of GH disappeared 2 days after injection in the three subspecies. Despite some differences among subspecies, the findings suggest that cortisol is a primary inducer of plasma IGFBP-1b; however, GH counteracts it in the short term. Therefore, GH has the potential to modulate the degree of increase in circulating IGFBP-1b levels during acute stress.
  • Beth M Cleveland, Shiori Habara, Jin Oikawa, Lisa M Radler, Munetaka Shimizu
    Genes 11 (12) 2020/12/10 [Refereed]
    Rainbow trout with gene editing-induced reductions in serum insulin-like growth factor binding protein (IGFBP)-2b exhibit similar growth performance compared to fish without IGFBP-2b gene disruption. The objective of this study is to determine how the components of the insulin-like growth factor (IGF)/IGFBP system respond to a reduction in serum IGFBP-2b abundance. Editing the IGFBP-2b genes in rainbow trout resulted in an 83% decrease in serum IGFBP-2b in mutants. This resulted in a 35% reduction in serum IGF-I, which was offset by reduced expression of hepatic igfbp-1a2 and increased muscle igfr-1a; these responses suggest that an increased IGF-I signaling capacity offset reductions in serum IGF-I. During feed deprivation, the differential expression of igfbp genes supports the attenuation of the growth inhibitory response, likely due to the further reduction in serum IGF-I that alleviated the need for an IGF-inhibitory response. Unique igfbp expression patterns occurred during refeeding, suggesting an enhanced IGF-I signaling capacity in controls. Collectively, these findings support that the role of IGFBP-2b is to regulate serum IGF-I concentrations. The compensatory regulation of IGF/IGFBP system genes indicates that adjustments in other IGFBP, both circulating and at the local level, maintain IGF-I signaling at a level appropriate for the nutritional state of the fish.
  • Ryuya Hasegawa, Takuto Miura, Nobuto Kaneko, Ryousuke Kizaki, Gakuto Oishi, Hanae Tanaka, Moe Sato, Munetaka Shimizu
    General and comparative endocrinology 299 113606 - 113606 2020/12/01 [Refereed]
    Salmonids have four subtypes of insulin-like growth factor binding protein (IGFBP)-1, termed -1a1, -1a2, -1b1 and 1b2, owing to teleost- and a lineage-specific whole-genome duplications. We have previously produced recombinant proteins of masu salmon IGFBP-1a1 and -1b2 and conducted functional analysis. To further characterize salmonid-specific IGFBP-1s, we cloned cDNAs encoding mature proteins of IGFBP-1a2 and -1b1 from the liver of masu salmon (Oncorhynchus masou). IGFBP-1a2 and -1b1 shared a 56% amino acid sequence homology whereas their homologies with their counterparts (i.e. -1a1 and -1b2) were 77% and 82%, respectively. We next expressed recombinant masu salmon (rs) IGFBP-1a2 and -1b1 with fusion partners thioredoxin (Trx) and a His-tag using the pET-32a(+) vector system in Escherichia coli. Trx.His.rsIGFBP-1s were detected in the insoluble faction, solubilized in a buffer containing urea, and isolated by Ni-affinity chromatography. They were refolded by dialysis and cleaved from the fusion partners by enterokinase. rsIGFBP-1a2 and -1b1 were purified by reversed-phase high performance liquid chromatography. Purified rsIGFBP-1a2 and -1b1 had the ability to bind digoxigenin-labeled human IGF-I on ligand blotting. We then examined the effects of rsIGFBP-1a1, -1a2, -1b1 and -1b2 in combination with human IGF-I on growth hormone (GH) release from cultured pituitary cells of masu salmon. IGF-I alone reduced GH release while the addition of rsIGFBP-1a1, -1b1 or -1b2, but not rsIGFBP-1a2, diminished the suppressive effect of IGF-I. Addition of rsIGFBP-1s without IGF-I had no effect on GH release. These results show that rsIGFBP-1b1, along with rsIGFBP-1a1 and -1b2, inhibits IGF-I action on the pituitary in masu salmon. The lack of the effect by rsIGFBP-1a2 suggests that salmon IGFBP-1 subtypes underwent subfunction partitioning and have different degrees of IGF-inhibitory action.
  • Shotaro Suzuki, Eisuke Takahashi, Tom Ole Nilsen, Nobuto Kaneko, Hirokazu Urabe, Yuki Ugachi, Etsuro Yamaha, Munetaka Shimizu
    AQUACULTURE 526 0044-8486 2020/09 [Refereed][Not invited]
    We induced season independent smolts in masu salmon (Oncorhynchus masou) by rearing juveniles under an advanced photoperiod (AP) in which LD15:9 was interrupted by LD9:15 from June for 4 months. AP fish showed higher degree of skin slivering from November than fish reared under a simulated natural photoperiod (SNP) and became larger in length and lower in condition factor in January. Gill Na+,K+-ATPase in AP fish started increasing in November and peaked in December to typical spring smolt levels while it remained low in SNP fish. In addition, AP fish had higher hypo-osmoregulatory ability than SNP fish, as judged by the lower serum sodium and chloride concentrations 48 h after seawater transfer. Serum levels of insulin-like growth factor (IGF)-I and IGF-binding protein (IGFBP)-1b, components of the somatotropic axis, in AP fish peaked in November and January, respectively. Serum IGFBP-1b levels were increased 48 h after transfer to 70% seawater in both groups, suggesting that it is not a quantitative index of hypo-osmoregulatory ability in the short-term but a marker of osmotic stress. The present study showed that off-season smolts could be induced in underyearling masu salmon by a square-wave photoperiod, and off-season smolts showed morphological and physiological changes comparable to yearling spring smolts. Increased levels of circulating IGF-I and IGFBP-1b in smolting fish suggest that these endocrine parameters are involved in the smoltification process.
  • Bernat Morro, Pablo Balseiro, Amaya Albalat, Simon Mackenzie, Cindy Pedrosa, Tom O. Nilsen, Shotaro Suzuki, Munetaka Shimizu, Harald Sveier, Marnix Gorissen, Lars O. E. Ebbesson, Sigurd O. Handeland
    AQUACULTURE 519 0044-8486 2020/03 [Refereed][Not invited]
  • Tharmini Kalananthan, Koji Murashita, Ivar Ronnestad, Mitsumasa Ishigaki, Kota Takahashi, Marta S. Silva, Yuki Wakabayashi, Floriana Lai, Munetaka Shimizu, Tom O. Nilsen, Enrique Pino Martinez, Ana S. Gomes
    FRONTIERS IN PHYSIOLOGY 11 1664-042X 2020/02 [Refereed][Not invited]
    The orexigenic agouti-related protein (AgRP) and the anorexigenic pro-opiomelanocortin (POMC) are crucial players in the control of feed intake in vertebrates, yet their role in teleosts has not been fully established. Triplicate groups of Atlantic salmon (Salmo salar) post smolts were subjected to (1) fasting for 3 days (fast) and (2) normal feeding (fed), resulting in a significant (p < 0.05) upregulation of hypothalamic agrp1 transcripts levels in the fast group. Moreover, the mRNA abundance of agrp1 was significantly (p < 0.05) correlated with the stomach dry weight content. Corresponding inverse patterns were observed for pomca2, albeit not statistically significant. No significant differences were found for the other paralogues, agrp2 and pomca1 and b, between fed and fast groups. The significant correlation between stomach fullness and agrp1 mRNA expression suggests a possible link between the stomach filling/distension and satiety signals. Our study indicates that hypothalamic agrp1 acts as an orexigenic signal in Atlantic salmon.
  • Nobuto Kaneko, Tom Ole Nilsen, Hanae Tanaka, Akihiko Hara, Munetaka Shimizu
    Insulin-like growth factor binding protein (IGFBP)-1a is one of three major circulating forms in salmon and induced under catabolic conditions. However, there is currently no immunoassay available for this form because of a lack of standard and specific antibodies. We developed a time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1a using recombinant protein for labeling, an assay standard, and production of antiserum. The TR-FIA had a low cross-reactivity (3.6%) with IGFBP-1b, another major form in the circulation. Fasting for 4 wk had no effect on serum immunoreactive (total) IGFBP-1a levels in yearling masu salmon, whereas 6-wk fasting significantly increased it. There was a significant, but weak, negative relationship between serum total IGFBP-1a level and individual growth rate (r(2) = 0.12, P = 0.01). We next developed a ligand immuno-functional assay (LIFA) using europium-labeled IGF-I to quantify intact IGFBP-1a. In contrast to total IGFBP-1a, serum intact IGFBP-1a levels increased after 4 wk of fasting, and refeeding for 2 wk restored it to levels similar to those of the fed control. Serum intact IGFBP-1a levels showed a significant negative correlation with individual growth rate (r(2) = 0.52, P < 0.001), which was as good as that of IGFBP-1b. Our findings using newly developed TR-FIA and LIFA suggest that regulation of intact IGFBP-1a levels has an important effect on growth in salmon and that intact IGFBP-1a is a negative index of salmon growth.
  • Nobuto Kaneko, Meredith L. Journey, Chrys M. Neville, Marc Trudel, Brian R. Beckman, Munetaka Shimizu
    FISH PHYSIOLOGY AND BIOCHEMISTRY 45 (6) 1867 - 1878 0920-1742 2019/12 [Refereed][Not invited]
    Monitoring the growth of salmon during their early marine phase provides insights into prey availability, and growth rates may be linked to risks of size-dependent mortality. However, the measurement of growth rate is challenging for free-living salmon in the ocean. Insulin-like growth factor (IGF)-I is a growth-promoting hormone that is emerging as a useful index of growth in salmon. In addition, laboratory-based studies using coho salmon have shown that one of circulating IGF-binding proteins (IGFBPs), IGFBP-1b, is induced by fasting and thus could be used as an inverse index of growth and/or catabolic state in salmon. However, few studies have measured plasma levels of IGFBP-1b in salmon in the wild. We measured plasma IGFBP-1b levels for postsmolt coho salmon collected in the Strait of Georgia and surrounding waters, British Columbia, Canada, and compared regional differences in IGFBP-1b to ecological information such as seawater temperature and stomach fullness. Plasma IGFBP-1b levels were the highest in fish from Eastern Johnstone Strait and relatively high in Queen Charlotte Strait and Western Johnstone Strait, which was in good agreement with the poor ocean conditions for salmon hypothesized to occur in that region. The molar ratio of plasma IGF-I to IGFBP-1b, a theoretical parameter of IGF-I availability to the receptor, discriminated differences among regions better than IGF-I or IGFBP-1b alone. Our data suggest that plasma IGFBP-1b reflects catabolic status in postsmolt coho salmon, as highlighted in fish in Eastern Johnston Strait, and is a useful tool to monitor negative aspects of salmon growth in the ocean.
  • Shu Nakamura, Nobuto Kaneko, Tatsuhiro Nonaka, Daiki Kurita, Yasuyuki Miyakoshi, Munetaka Shimizu
    1444-2906 2019/04/03 [Refereed][Not invited]
  • Effect of different photoperiod regimes on the smoltification and seawater adaptation of seawater-farmed rainbow trout (Oncorhynchus mykiss): Insights from Na+,K+-ATPase activity and transcription of osmoregulation and growth regulation genes
    Morro B, Balseiro P, Albalat A, Pedrosa C, Mackenzie S, Nakamura S, Shimizu M, Nilsen TO, Sveier H, Ebbesson LO, Handeland SO
    Aquaculture 507 282 - 292 2019/04 [Refereed][Not invited]
  • Kaneko N, Torao M, Koshino Y, Fujiwara M, Miyakoshi Y, Shimizu M
    General and comparative endocrinology 274 50 - 59 0016-6480 2019/01 [Refereed][Not invited]
    This study aimed to utilize circulating insulin-like growth factor binding protein (IGFBP)-1b as a negative index of growth to evaluate the growth status of juvenile chum salmon (Oncorhynchus keta) in the ocean. First, rearing experiments using PIT-tagged juveniles were conducted to examine the relationship of circulating IGFBP-1b with growth rate of the fish in May and in June. The serum IGFBP-1b level negatively correlated with fish growth rate in both months, suggesting its utility as a negative index of growth. Next, the growth status of out-migrating juveniles in northeastern Hokkaido, Japan, was monitored for 3 years using the growth indices. Serum levels of IGF-I, a positive index of growth, in fish collected from the nearshore zone were low in May and high in June of all years. Levels of serum IGFBP-1b showed a trend opposite to that of serum IGF-I. However, the IGF-I/IGFBP-1b molar ratios well reflected the seasonal and regional trends. These findings suggest that the juveniles in June left the nearshore area under better growth conditions. The present study also suggests that the use of multiple growth indices would improve the sensitivity and accuracy to evaluate the current growth status of out-migrating juvenile chum salmon.
  • Cleveland Beth M, Yamaguchi Ginnosuke, Radler Lisa M, Shimizu Munetaka
    SCIENTIFIC REPORTS 8 2045-2322 2018/10/30 [Refereed][Not invited]
  • Inatani Yu, Ineno Toshinao, Sone Shiori, Matsumoto Naoto, Uchida Katsuhisa, Shimizu Munetaka
    JOURNAL OF FISH BIOLOGY 93 (3) 490 - 500 0022-1112 2018/09 [Refereed][Not invited]
  • Balseiro Pablo, Moe Oyvind, Gamlem Ingrid, Shimizu Munetaka, Sveier Harald, Nilsen Tom O, Kaneko Nobuto, Ebbesson Lars, Pedrosa Cindy, Tronci Valentina, Nylund Are, Handel, Sigurd O
    JOURNAL OF FISH BIOLOGY 93 (3) 567 - 579 0022-1112 2018/09 [Refereed][Not invited]
  • Hanae Tanaka, Gakuto Oishi, Yusuke Nakano, Hiroko Mizuta, Yuta Nagano, Naoshi Hiramatsu, Hironori Ando, Munetaka Shimizu
    General and comparative endocrinology 257 184 - 191 0016-6480 2018/02/01 [Refereed][Not invited]
    Insulin-like growth factor (IGF)-I is a growth promoting hormone that exerts its actions through endocrine, paracrine and autocrine modes. Local IGF-I is essential for normal growth, whereas circulating IGF-I plays a crucial role in regulating the production and secretion of growth hormone (GH) by the pituitary gland. These actions of IGF-I are modulated by six insulin-like growth factor binding proteins (IGFBPs). In teleosts, two subtypes of each IGFBP are present due to an extra round of whole-genome duplication. IGFBP-1 is generally inhibitory to IGF-I action under catabolic conditions such as fasting and stress. In salmon, IGFBP-1a and -1b are two of three major circulating IGFBPs and assumed to affect growth through modulating IGF-I action. However, exact functions of salmon IGFBP-1 subtypes on growth regulation are not known due to the lack of purified or recombinant protein. We expressed recombinant salmon (rs) IGFBP-1a and -1b with a fusion protein (thioredoxin, Trx) and a His-tag using the pET-32a(+) vector expression system in Escherichia coli. Trx.His.rsIGFBP-1s were isolated by Ni-affinity chromatography, enzymatically cleaved by enterokinase to remove the fusion partners and further purified by reversed-phase HPLC. We next examined effects of rsIGFBP-1a and -1b in combination with human IGF-I on GH release from cultured masu salmon (Oncorhynchus masou) pituitary cells. Unexpectedly, IGF-I increased GH release and an addition of rsIGFBP-1a, but not rsIGFBP-1b, restored GH levels. The results suggest that IGFBP-1a can inhibit IGF-I action on the pituitary in masu salmon. Availability of recombinant salmon IGFBP-1s should facilitate further functional analyses and assay development.
  • Munetaka Shimizu, Walton W. Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 252 150 - 161 0016-6480 2017/10 [Refereed][Not invited]
    Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating the availability of IGFs to receptors and prolong the half-lives of IGFs. There are six IGFBPs present in the mammalian circulation with IGFBP-3 being most abundant. In mammals IGFBP-3 is the major carrier of circulating IGFs, facilitated by forming a ternary complex with IGF and an acid-labile subunit (ALS). IGFBP-1 is generally inhibitory to IGF action by preventing it from interacting with its receptors. In teleosts, the third-round of vertebrate whole genome duplication created paralogs of each IGFBP, except IGFBP-4. In the fish circulation, three major IGFBPs are typically detected at molecular ranges of 20-25, 28-32 and 40-50 kDa. However, their identities are not well established. Three major circulating IGFBPs in Chinook salmon have been identified through protein purification and cDNA cloning. Salmon 28- and 22-kDa IGFBPs are coorthologs of IGFBP-1, termed IGFBP-1a and-1b, respectively. They are induced under catabolic conditions such as stress and fasting but their responses are somewhat different, with IGFBP-1b being the most sensitive of the two. Cortisol stimulates production and secretion of these IGFBP-1 subtypes while, unlike in mammals, insulin may not be a primary suppressor. Salmon 41-kDa IGFBP, a major carrier of IGF-I, is not IGFBP-3, as might be expected extrapolating from mammals, but is in fact IGFBP-2b. Salmon IGFBP-2b levels in plasma are high when fish are fed, and GH treatment increases its circulating levels similar to mammalian IGFBP-3. These findings suggest that salmon IGFBP-2b acquired the role and regulation similar to mammalian IGFBP-3. Multiple replications of fish IGFBPs offer a unique opportunity to investigate molecular evolution of IGFBPs. (C) 2017 Published by Elsevier Inc.
  • Natsumi Taniyama, Nobuto Kaneko, Yu Inatani, Yasuyuki Miyakoshi, Munetaka Shimizu
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 236 146 - 156 0016-6480 2016/09 [Refereed][Not invited]
    Insulin-like growth factor (IGF)-I, IGF-binding protein (IGFBP)-1 and RNA/DNA ratio are endocrine and biochemical parameters used as growth indices in fish, however, they are subjected to environmental modulation. Chum salmon (Oncorhynchus keta) migrate from freshwater (FW) to seawater (SW) at fry/juvenile stage weighing around 1 g and suffer growth-dependent mortality during the early phase of their marine life. In order to reveal environmental modulation of the IGF/IGFBP system and establish a reliable growth index for juvenile chum salmon, we examined effects of SW transfer and fasting on IGF-I, IGFBP-1 and RNA/DNA ratio, and correlated them to individual growth rate. Among serum IGF-I, liver and muscle igf-1, igfbp-1a, igfbp-1b and RNA/DNA ratio examined, muscle RNA/DNA ratio and muscle igfbp-1a responded to SW transfer. Serum IGF-I, liver igf-1 and liver RNA/DNA ratio were sensitive to nutritional change by being reduced in 1 week in both FW and SW while muscle igf-1 was reduced 2 weeks after fasting. In contrast, igfbp-1a in both tissues was increased by 2 weeks of fasting and igfbp-1b in the liver of SW fish was increased in 1 week. These results suggest that the sensitivity of igf-1 and igfbp-1s to fasting differs between tissues and subtypes, respectively. When chum salmon juveniles in SW were marked and subjected to feeding or fasting, serum IGF-I showed the highest correlation with individual growth rate. Liver igfbp-1a and -1b, and muscle igf-1 exhibited moderate correlation coefficients with growth rate. These results show that serum IGF-I is superior to the other parameters as a growth index in juvenile chum salmon in term of its stability to salinity change, high sensitivity to fasting and strong relationship with growth rate. On the one hand, when collecting blood from chum salmon fry/juveniles is not practical, measuring liver igfbp-1a and -1b, or/and muscle igf-1 is an alternative. (C) 2016 Elsevier Inc. All rights reserved.
  • Tomonori Kuwada, Tetsuya Tokuhara, Munetaka Shimizu, Goro Yoshizaki
    FISHERIES SCIENCE 82 (1) 59 - 71 0919-9268 2016/01 [Refereed][Not invited]
    The present study retrospectively examined relationships between growth trajectory and smolting in order to identify the key regulators in commencement of smolting using groups of all-female homozygous clonal amago salmon. We found that (1) regardless of specific growth rate, the majority of fish above the threshold size (12-13 g) by the end of the decision window (early fall) became smolt in the future; (2) even if fish smaller than the threshold size during the decision window exceeded that size afterwards, they were not recruited to smolt; (3) smolting of fish that attained sufficient size during the decision window could not be suppressed by subsequent restriction of growth rate; (4) smaller fish that had not attained the threshold size during the decision window could not smoltify, even though they showed faster growth rates than that of future smolt during/after the decision window. Taken together, these findings indicated that fish mass by the end of the decision window was responsible for the determination of whether individual amago salmon could smoltify or not and that the faster growth rates observed in future smolt after the decision window occurred as a result of commitment of smoltification.
  • Ernst M. Hevroy, Christian K. Tipsmark, Sofie C. Remo, Tom Hansen, Miki Fukuda, Thomas Torgersen, Vibeke Vikesa, Pal A. Olsvik, Rune Waagbo, Munetaka Shimizu
    A comparative experiment with Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) postsmolts was conducted over 35 days to provide insight into how growth, respiration, energy metabolism and the growth hormone (GH) and insulin-like growth factor 1 (IGF-1) system are regulated at elevated sea temperatures. Rainbow trout grew better than Atlantic salmon, and did not show reduced growth at 19 degrees C. Rainbow trout kept at 19 degrees C had increased blood hemoglobin concentration compared to rainbow trout kept at 13 degrees C, while salmon did not show the same hemoglobin response due to increased temperature. Both species showed reduced length growth and decreased muscle glycogen stores at 19 degrees C. Circulating IGF-1 concentration was higher in rainbow trout than in Atlantic salmon, but was not affected by temperature in either species. Plasma IGF-binding protein 1b (IGFBP-1b) concentration was reduced in Atlantic salmon reared at 19 degrees C after 15 days but increased in rainbow trout at 19 degrees C after 35 days. The igfbp1b mRNA level in liver showed a positive correlation to plasma concentrations of glucose and IGFBP-1b, suggesting involvement of this binding protein in carbohydrate metabolism at 19 degrees C. At this temperature muscle igfbp1a mRNA was down-regulated in both species. The muscle expression of this binding protein correlated negatively with muscle igf1 and length growth. The plasma IGFBP-1b concentration and igfbp1b and igfbp1a expression suggests reduced muscle igf1 signaling at elevated temperature leading to glucose allostasis, and that time course is species specific due to higher thermal tolerance in rainbow trout. (C) 2015 Elsevier Inc. All rights reserved.
  • Miki Fukuda, Nobuto Kaneko, Kohei Kawaguchi, Ernst M. Hevroy, Akihiko Hara, Munetaka Shimizu
    In salmon plasma/serum, three major insulin-like growth factor binding proteins (IGFBPs) are consistently detected at 22-, 28- and 41-kDa. The 22-kDa form has been identified as IGFBP-1b and shown to increase under catabolic conditions. We developed a competitive time-resolved fluoroimmunoassay (TR-FIA) for salmon IGFBP-1b. Purified salmon IGFBP-1b was used for biotin-labeling, assay standard and antiserum production. The TR-FIA did not cross-react with the 41-kDa form (IGFBP-2b) but showed 3% cross-reactivity with the 28-kDa form (IGFBP-1a). It measured IGFBP-1b levels as low as 0.4 ng/ml, and ED80 and ED20 were 0.9 and 24.6 ng/ml, respectively. There appears to be little interference by IGF-I. Using the TR-FIA, serum IGFBP-1b levels were measured in individually-tagged underyearling masu salmon fed or fasted for 5 weeks, or fasted for 3 weeks followed by refeeding for 2 weeks. Fasting for 3 weeks significantly increased circulating IGFBP-1b levels, while it returned to the basal levels after prolonged fasting for additional 2 weeks. Serum IGFBP-1b level negatively correlated with body weight, condition factor, specific growth rate and serum IGF-I level. During parr-smolt transformation of masu salmon, average circulating IGFBP-1b levels were the highest in May. There was a positive correlation between serum IGFBP-1b and IGF-I, which is in contrast to that in the fasting/feeding experiment. IGFBP-1b also showed a positive relationship with gill Na+, K+-ATPase activity. These results suggest that the relationship between circulating IGFBP-1b and IGF-I during smoltification differs from that during fasting and IGFBP-1b may play a role in the development of hypoosmoregulatory ability. (C) 2015 Elsevier Inc. All rights reserved.
  • Nobuto Kaneko, Natsumi Taniyama, Yu Inatani, Yuta Nagano, Makoto Fujiwara, Mitsuru Torao, Yasuyuki Miyakoshi, Munetaka Shimizu
    FISH PHYSIOLOGY AND BIOCHEMISTRY 41 (4) 991 - 1003 0920-1742 2015/08 [Refereed][Not invited]
    Chum salmon (Oncorhynchus keta) migrate to the ocean in their first spring, and growth during early marine life is critical for survival. We examined the validity of circulating IGF-I and muscle RNA/DNA ratio as indices of growth rate using individually tagged juvenile chum salmon fed or fasted for 10 days. Serum IGF-I level was highly, positively correlated with individual growth rate. Muscle RNA/DNA ratio also showed a positive correlation, but its relation was not as high as that of IGF-I. We next measured these physiological parameters in chum salmon juveniles caught at river, estuary, port and nearshore of the northeastern Hokkaido, Japan, from May to June in 2013 and 2014, respectively. In both years, there was a trend that serum IGF-I levels were high in nearshore fish and low in river/estuarine fish in June. In contrast, muscle RNA/DNA ratio showed no clear temporal and spatial patterns. The present study shows that circulating IGF-I can be used as a growth index in juvenile chum salmon. Monitoring growth status using serum IGF-I suggests that growth of juvenile chum salmon in the survey area was activated when they left the coast.
  • Nobuto Kaneko, Anai Iijima, Takahiro Shimomura, Takuro Nakajima, Haruka Shimura, Hajime Oomori, Hirokazu Urabe, Akihiko Hara, Munetaka Shimizu
    FISHERIES SCIENCE 81 (4) 643 - 652 0919-9268 2015/07 [Refereed][Not invited]
    We compared profiles of serum insulin-like growth factor (IGF)-I levels during smoltification of masu salmon reared under different environments, hatcheries and growth histories. Masu salmon from the Kenichi River in Hokkaido showed a sharp increase in serum IGF-I from April to May, followed by a peak of gill Na+,K+-ATPase (NKA) activity. Fish at Kumaishi Hatchery had an IGF-I profile similar to that of the river fish, while the increase in gill NKA was lower. At Shimamaki Hatchery, interval feeding during winter appeared to suppress the spring IGF-I peak. At Kumaishi Hatchery, a difference in size during smoltification affected IGF-I levels at release, but the numbers of adults that returned to the release site were not significantly different. In the following year, three release groups differing in winter size and/or spring growth (Large-High, Large-Low and Small-High) were created. Large-High and Small-High fish showed a higher IGF-I peak than Large-Low fish in April, while smolt-to-adult return of Large-High fish was highest. These results suggest that in smolting masu salmon in freshwater, circulating IGF-I level alone is not a predictor of long-term survival in seawater. However, since growth history in freshwater affected the smolt-to-adult return, optimizing rearing conditions is a critical component of hatchery releases for masu salmon.
  • Takuro Nakajima, Haruka Shimura, Miyuki Yamazaki, Yasuhiro Fujioka, Kazuhiro Ura, Akihiko Hara, Munetaka Shimizu
    Landlocking of salmon relaxes selective pressures on hypoosmoregulatory ability (seawater adaptability) and may lead to the abandonment of its physiological system. However, little is known about the mechanism and consequence of the process. Biwa salmon is a strain/subspecies of Oncorhynchus masou that has been landlocked in Lake Biwa for an exceptionally long period (about 500,000 years) and has low ability to adapt to seawater. We compared activity of gill Na+, K+-ATPase (NKA) of Biwa salmon with those of anadromous strains of the same species (masu and amago salmon) during downstream migration periods and after exogenous hormone treatment. Gill NKA activity in anadromous strains increased during their migration periods, while that in Biwa salmon remained low. However, treatments of Biwa salmon with growth hormone (GH) and cortisol increased gill NKA activity. Cortisol treatment also improved the whole body seawater adaptability of Biwa salmon. Receptors for GH and cortisol responded to hormonal treatments, whereas their mRNA levels during downstream migration period were essentially unchanged in Biwa salmon. Circulating levels of cortisol in masu salmon showed a peak during downstream migration period, while no such increase was seen in Biwa salmon. The present results indicate that Biwa salmon can improve its seawater adaptability by exogenous hormonal treatment, and hormone receptors are capable of responding to the signals. However, secretion of the endogenous hormone (cortisol) was not activated during the downstream migration period, which explains, at least in part, their low ability to adapt to seawater.
  • Kohei Kawaguchi, Nobuto Kaneko, Miki Fukuda, Yusuke Nakano, Shizuo Kimura, Akihiko Hara, Munetaka Shimizu
    Comparative Biochemistry and Physiology - A Molecular and Integrative Physiology 165 (2) 191 - 198 1095-6433 2013/06 [Refereed][Not invited]
    Two subtypes of insulin-like growth factor binding protein (IGFBP)-1 are present in salmon blood and they are both up-regulated under catabolic conditions such as stress. The present study examined effects of fasting and re-feeding on IGFBP-1a (28-kDa form) and IGFBP-1b (22-kDa form) both at mRNA and protein levels along with IGF-I and RNA/DNA ratio in yearling masu salmon. Fish were individually tagged and assigned to one of three treatments: Fed, Fasted or Re-fed. Circulating IGF-I levels significantly decreased after fasting for 5. weeks and were positively correlated with individual growth rates. Liver igf-1 mRNA levels were not affected by the treatment. Muscle RNA/DNA ratio did not respond to fasting nor showed correlations with growth rates. Circulating IGFBP-1a and IGFBP-1b increased during fasting and decreased after re-feeding. Both serum levels were inversely correlated with growth rates, while IGFBP-1b had consistent negative relationships with growth rates. Fasting/re-feeding also affected their mRNA levels in the liver. These results suggest that circulating IGF-I and IGFBP-1b could serve as positive and negative indices of growth, respectively, in masu salmon. Different sensitivities of IGBP-1a and IGFBP-1b may be useful to assess a broad range of catabolic conditions when they are combined. © 2013 Elsevier Inc.
  • Ernst M. Hevroy, Christine Hunskar, Stefan de Gelder, Munetaka Shimizu, Rune Waagbo, Olav Breck, Harald Takle, Sissel Sussort, Tom Hansen
    Growth regulation in adult Atlantic salmon (1.6 kg) was investigated during 45 days in seawater at 13, 15, 17, and 19 A degrees C. We focused on feed intake, nutrient uptake, nutrient utilization, and endocrine regulation through growth hormone (GH), insulin-like growth factors (IGF), and IGF-binding proteins (IGFBP). During prolonged thermal exposure, salmon reduced feed intake and growth. Feed utilization was reduced at 19 A degrees C after 45 days compared with fish at lower temperatures, and body lipid storage was depleted with increasing water temperature. Although plasma IGF-1 concentrations did not change, 32-Da and 43-kDa IGFBP increased in fish reared at a parts per thousand currency sign17 A degrees C, and dropped in fish reared at 19 A degrees C. Muscle igf1 mRNA levels were reduced at 15 and 45 days in fish reared at 15, 17, and 19 A degrees C. Muscle igf2 mRNA levels did not change after 15 days in response to increasing temperature, but were reduced after 45 days. Although liver igf2 mRNA levels were reduced with increasing temperatures after 15 and 45 days, temperature had no effect on igf1 mRNA levels. The liver igfbp2b mRNA level, which corresponds to circulating 43-kDa IGFBP, exhibited similar responses after 45 days. IGFBP of 23 kDa was only detected in plasma in fish reared at 17 A degrees C, and up-regulation of the corresponding igfbp1b gene indicated a time-dependent catabolic response, which was not observed in fish reared at 19 A degrees C. However, higher muscle ghr mRNA levels were detected in fish at 17 and 19 A degrees C than in fish at lower temperatures, indicating lipolytic regulation in muscle. These results show that the reduction of muscle growth in large salmon is mediated by decreased igf1 and igf2 mRNA levels in addition to GH-associated lipolytic action to cope with prolonged thermal exposure. Accordingly, 13 A degrees C appears to be a more optimal temperature for the growth of adult Atlantic salmon at sea.
  • David C. Metzger, J. Adam Luckenbach, Munetaka Shimizu, Brian R. Beckman
    Feeding, fasting and re-feeding is a common experimental paradigm for studying growth endocrinology. Herein we demonstrate dynamic changes in the livers of coho salmon under these conditions and how changes in liver composition can influence quantification and interpretation of liver gene expression data. A three-week fast resulted in decreases in hepatosomatic index (liver size), liver glycogen content, and liver DNA concentration. In addition, significant differences were found in liver transcript levels from fed and fasted fish for the reference genes, arp and ef1a, when these were normalized to total RNA. We took the additional step of normalizing reference gene transcript levels to the liver homogenate RNA/DNA ratio to account for differences in RNA yield/cell and the number of cells sampled, normalizing to transcript number per cell rather than transcript number per unit RNA. After this additional step no significant differences in liver transcript levels of reference genes were found. The significance of these results was illustrated by normalizing liver transcript levels of insulin-like growth factor 1 (igf1) to ef1a transcript levels or ef1a transcript levels by RNA/DNA. The different normalization strategies resulted in differing patterns of change in igf1 transcript levels between fed and fasted fish. The novelty of this work rests in a two-step normalization process, attempting to account for both 1) technical errors in reverse transcription and qPCR reactions, and 2) biological variance in liver samples. Published by Elsevier Inc.
  • Takahiro Shimomura, Takuro Nakajima, Moeri Horikoshi, Anai Iijima, Hirokazu Urabe, Shinya Mizuno, Naoshi Hiramatsu, Akihiko Hara, Munetaka Shimizu
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 2 178 (2) 427 - 435 0016-6480 2012/09 [Refereed][Not invited]
    We established profiles of insulin-like growth factor (IGF)-I mRNA in the liver, gill and white muscle and circulating IGF-I during smoltification of hatchery-reared masu salmon, and compared with that of gill Na+,K+-ATPase (NKA) activity. Gill NKA activity peaked in May and dropped in June. Liver igf1 mRNA was high in March and decreased to low levels thereafter. Gill igf1 increased from March, maintained its high levels during April and May and decreased in June. Muscle igf1 mRNA levels were relatively high during January and April when water temperature was low. Serum IGF-I continuously increased from March through June. Serum IGF-I during March and May showed a positive correlation with NKA activity, although both were also related to fish size. These parameters were standardized with fork length and re-analyzed. As a result, serum IGF-I and gill igf1 were correlated with NKA activity. On the other hand, samples from desmoltification period (June) that had high serum IGF-I levels and low NKA activity disrupted the relationship. Expression of two IGF-I receptor (igf1r) subtypes in the gill decreased in June, which could account for the disruption by preventing circulating IGF-I from acting on the gill and retaining it in the blood. The present study suggests that the increase in gill NKA activity in the course of smoltification of masu salmon was supported by both endocrine and local IGF-I, and the decrease during desmoltification in freshwater was due at least in part to the down-regulation of gill IGF-I receptors. (C) 2012 Elsevier Inc. All rights reserved.
  • Munetaka Shimizu, Keisuke Kishimoto, Teppei Yamaguchi, Yusuke Nakano, Akihiko Hara, Walton W. Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 174 (2) 97 - 106 0016-6480 2011/11 [Refereed][Not invited]
    Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20-25, 28-32 and 40-45 kDa. However, their identity is poorly established and often confused. We previously identified salmon 22- and 41-kDa forms as IGFBP-1 and -2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon (Oncorhynchus tshawytscha) as well as rainbow trout (Oncorhynchus mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1 a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28- and 22-kDa forms as IGFBP-la and -1b, respectively. Salmon IGFBP-la contains a potential PEST region involved in rapid protein turnover and phosphorylation sites typically found in mammalian IGFBP-1, although the PEST and phosphorylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-la has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other teleosts, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth. (C) 2011 Elsevier Inc. All rights reserved.
  • Munetaka Shimizu, Seira Suzuki, Moeri Horikoshi, Akihiko Hara, Walton W. Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 171 (3) 326 - 331 0016-6480 2011/05 [Refereed][Not invited]
    In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150 kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50 kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates. (C) 2011 Elsevier Inc. All rights reserved.
  • E. M. Hevrøy, C. Azpeleta, M. Shimizu, A. Lanzén, H. Kaiya, M. Espe, P. A. Olsvik
    Fish Physiology and Biochemistry 37 (1) 217 - 232 0920-1742 2011 [Refereed][Not invited]
    The effects of short-time fasting on appetite, growth, and nutrient were studied in Atlantic salmon (Salmo salar) smolts. Feed deprivation did change the energy metabolism with reduced plasma protein and muscle indispensible amino acid levels. Plasma levels of ghrelin were significantly higher in starved salmon tion in the stomach. Lower ghrelin-1 mRNA levels were detected at day 2 in starved fish compared with fed fish. The mRNA levels of GHSR-LR1a were not affected by starvation. Fasting reduced the phenotypic growth and the transcription of insulin-like growth factor (IGF)-II together with IGF-IIR, but IGF-I mRNA were not regulated in fasted salmon after 14 days. Three IGF-binding proteins (IGFBP) at 23, 32, and 43 kDa were found in salmon, and circulating 23 kDa was significantly increased after 14 days of starvation compared with fed fish, indicating increased catabolism. The levels of IGFBP-1 mRNA were significantly higher in fed and starved fish after 14 days compared to those at the start of the experiment, but no significant difference was observed between the treatments. In conclusion, we have shown that circulating ghrelin and ghrelin-1 mRNA is related to changes in energy metabolism in Atlantic salmon. © 2010 Springer Science+Business Media B.V.
  • Munetaka Shimizu, Kathleen A. Cooper, Walton W. Dickhoff, Brian R. Beckman
    Shimizu M, Cooper KA, Dickhoff WW, Beckman BR. Postprandial changes in plasma growth hormone, insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins in coho salmon fasted for varying periods. Am J Physiol Regul Integr Comp Physiol 297: R352-R361, 2009. First published May 27, 2009; doi:10.1152/ajpregu.90939.2008.-We examined postprandial changes in circulating growth hormone (GH), insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins (IGFBPs) in yearling coho salmon under different feeding regimes. Fish were initially fasted for 1 day, 1 wk, or 3 wk. Fasted fish were then fed, and blood was collected at 4-h intervals over 26 h. After the various periods of fasting, basal levels of insulin were relatively constant, whereas those of IGF-I, IGFBPs and GH changed in proportion to the duration of the fast. A single meal caused a rapid, large increase in the circulating insulin levels, but the degree of the increase was influenced by the fasting period. IGF-I showed a moderate increase 2 h after the meal but only in the regularly fed fish. Plasma levels of 41-kDa IGFBP were increased in all groups within 6 h after the single meal. The fasting period did not influence the response of 41-kDa IGFBP to the meal. IGFBP-1 and GH decreased after the meal to the same extent among groups regardless of the fasting period. The present study shows that insulin and IGF-I respond differently to long (weeks)- and short (hours)-term nutritional changes in salmon; insulin maintains its basal level but changes acutely in response to food intake, whereas IGF-I adjusts its basal levels to the long-term nutritional status and is less responsive to acute nutritional input. IGFBPs maintain their sensitivity to food intake, even after prolonged fasting, suggesting their critical role in the nutritional regulation of salmon growth.
  • Toshiaki Fujita, Haruhisa Fukada, Munetaka Shimizu, Naoshi Hiramatsu, Akihiko Hara
    FISHERIES SCIENCE 74 (5) 1198 - 1200 0919-9268 2008/10 [Refereed][Not invited]
  • Toshiaki Fujita, Haruhisa Fukada, Munetaka Shimizu, Naoshi Hiramatsu, Akihiko Hara
    MOLECULAR REPRODUCTION AND DEVELOPMENT 75 (7) 1217 - 1228 1040-452X 2008/07 [Refereed][Not invited]
    Three cDNAs, each encoding a different choriogenin (Chg), were isolated from a female masu salmon (Oncorhynchus masou) liver cDNA library. Two of the cDNA clones, Chg H alpha and Chg H beta, showed a close relationship and contained the typical domains of zona pellucida (ZP) B genes in fish, namely proline and glutamine rich repeats, a trefoil factor family domain, and a ZP domain. Specific antibodies against recombinant Chg H products (rmH alpha and rmH beta) were generated to elucidate the relationship between the Chg H cDNAs and two types of serum Chg H protein, which were previously purified and characterized, and designated as very-high -molecular-weight vitelline envelope-related protein (vhVERP) and Chg H of masu salmon. The immunobiochemical analyses revealed that the Chg H alpha and Chg H beta clones encoded vhVERP and Chg H proteins, respectively. The third cDNA clone (Chg L) appeared to be a ZPC gene and, by mapping the N-terminal sequence of purified Chg L, was shown to encode serum Chg L protein. Various types of heteromultimer of the three Chgs were identified immunologically as high molecular weight chorion components, indicating the involvement of complex heterodimerization of multiple Chgs in the construction of chorion architecture in masu salmon.
  • Munetaka Shimizu, Haruhisa Fukada, Akihiko Hara, Walton W. Dickhoff
    AQUACULTURE 273 (2-3) 320 - 328 0044-8486 2007/12 [Refereed]
    We compared the response of plasma insulin-like growth factor-I (IGF-I) to growth hormone (GH) administration under two different salinities to test the hypothesis that environmental salinity alters the "activity" of the GH-IGF-I axis. In July, postsmolt coho salmon reared in fresh water (FW) were transferred to either FW or half seawater (1/2 SW) (15 ppt) tank. During the experiment, water temperature was maintained at 10 degrees C for both salinities; photoperiod was adjusted to that of Seattle (48 degrees N), and fish were not fed. Two days after transfer, fish were injected once with porcine GH (pGH) at a dose of 2 or 8 mu g/g body weight. Liver and blood samples were collected 1, 2 and 3 days after injection. Liver GH receptor (GHR) mRNA expression was analyzed by quantitative real-time RT-PCR, and plasma IGF-I, 41-kDa IGF-binding protein (main carrier of IGF-I) and pGH were quantified by radioinummoassays. Transfer to 1/2 SW resulted in transient increases in basal levels of liver GHR mRNA and 41 kDa IGF-binding protein (IGFBP) but not IGF-I. The GH injection increased liver GHR mRNA, plasma IGF-I and 41-kDa IGFBP in fish in both FW and 1/2 SW However, the time course and magnitude of the response differed between salinities. Fish in FW receiving 8 mu g/g pGH had the highest IGF-I levels (63.7 +/- 6.8 ng/ml) one day after injection, whereas fish in 1/2 SW showed a peak (88.8 +/- 14.3 ng/ml) two days after injection of the same dose. It is speculated that the prolonged response to GH by fish in 1/2 SW may be due to slower disappearance of pGH from the circulation in fish in 1/2 SW. The transient increase in basal liver GHR mRNA may also contribute to a greater response for fish in 1/2 SW These results suggest that salinity is capable of altering the "activity" of the GH-IGF-I axis in salmon. (C) 2007 Elsevier B.V. All rights reserved.
  • Haruna Amano, Toshiaki Fujita, Naoshi Hiramatsu, Munetaka Shimizu, Sayumi Sawaguch, Takahiro Iatsubara, Hirohiko Kagawa, Masaki Nagae, Craig V. Sullivan, Akihiko Hara
    JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-ECOLOGICAL AND INTEGRATIVE PHYSIOLOGY 307A (6) 324 - 341 2471-5646 2007/06 [Refereed][Not invited]
    Seven yolk proteins (YPs), four large lipoproteins (YPs1-4) and three minor yolk components (YPs5-7) including one phosphoprotein (YP7), were purified from extracts of vitellogenic ovaries of gray mullet (Mugil cephalus) by combinations of hydroxylapatite, ion exchange, immunoadsorbent, and gel filtration chromatography. The molecular masses of native Y-P1, YP2, YP3, and YP4 were estimated to be 330, 325, 335, and 570 kDa, respectively. The tertiary structures of YP1, YP2, and YP3 revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis were typical of teleost lipovitellins (Lvs), consisting of a heavy chain (similar to 110, similar to 99, and similar to 97 kDa, respectively) and a light chain (similar to 30, similar to 29, and similar to 21.5 kDa, respectively), while YP4 exhibited a heavy chain (similar to 110 kDa) and two more polypeptide bands (similar to 70 and similar to 54kDa). Mapping of N-terminal peptide sequences of the purified YPs to the primary structure of multiple mullet vitellogenins (Vgs) deduced from their respective complete cDNAs, which were cloned and sequenced, conclusively identified YP1, YP2, and YP3 as Lvs derived from mullet VgA, VgB, and VgC, respectively. The fourth YP (YP4) appeared to be a proteolytic variant consisting of Lv and phosvitin components of VgA. Two other YPs (YP5 and YP6) were identified as beta'-components derived from VgA and VgB based on their structures and common, but not identical, antigenicity to salmonid beta'-component, while purified YP7, a phosphoprotein with a high content of serine residues, was identified as a phosvitin derived from VgB. This is the first report, of which we are aware, on purification and molecular classification of three distinct forms of Lv from any oviparous vertebrate.
    Environ Sci (Tokyo) 14 (2) 95 - 108 0915-955X 2007 [Refereed][Not invited]
  • A. L. Pierce, M. Shimizu, L. Felli, P. Swanson, W. W. Dickhoff
    JOURNAL OF ENDOCRINOLOGY 191 (2) 379 - 386 0022-0795 2006/11 [Refereed][Not invited]
    IGF-binding proteins (IGFBPs) modulate the effects of the IGFs, major stimulators of vertebrate growth and development. In mammals, IGFBP-1 inhibits the actions of IGF-I. Rapid increases in circulating IGFBP-1 occur during catabolic states. Insulin and glucocorticoids are the primary regulators of circulating IGFBP-1 in mammals. Insulin inhibits and glucocorticoids stimulate hepatocyte IGFBP-1 gene expression and production. A 22 kDa IGFBP in salmon blood also increases during catabolic states and has recently been identified as an IGFBP-1 homolog. We examined the hormonal regulation of salmon IGFBP-1 mRNA levels and protein secretion in primary cultured salmon hepatocytes. The glucocorticoid agonist dexamethasone progressively increased hepatocyte IGFBP-1 mRNA levels (eightfold) and medium IGFBP-1 immunoreactivity over concentrations comparable with stressed circulating cortisol levels (10(-9)-10(-6) M). GH progressively reduced IGFBP-1 mRNA levels (0.3-fold) and medium IGFBP-1 immunoreactivity over physiological concentrations (5x10(-11)-5x10(-9) M). Unexpectedly, insulin slightly increased hepatocyte IGFBP-1 mRNA (1.4-fold) and did not change medium IGFBP-1 immunoreactivity over physiological concentrations and above (10(-9)-10(-6) M). Triiodothyronine had no effect on hepatocyte IGFBP-1 mRNA, whereas glucagon increased IGFBP-1 mRNA (2.2-fold) at supraphysiological concentrations (10(-6) M). This study suggests that the major inhibitory role of insulin in the regulation of liver IGFBP-1 production in mammals is not found in salmon. However, regulation of salmon liver IGFBP-1 production by other metabolic hormones is similar to what is found in mammals.
  • M Shimizu, BR Beckman, A Hara, WW Dickhoff
    JOURNAL OF ENDOCRINOLOGY 188 (1) 101 - 110 0022-0795 2006/01 [Refereed][Not invited]
    Fish plasma/serum contains multiple IGF binding proteins (IGFBPs), although their identity and physiological regulation are poorly understood. In salmon plasma, at least three IGFBPs with molecular masses of 22, 28 and 41 kDa are detected by Western ligand blotting. The 22 kDa IGFBP has recently been identified as a homolog of mammalian IGFBP-1. In the present study, an RIA for salmon IGFBP-l was established and regulation of IGFBP-1 by food intake and temperature, and changes in IGFBP-1 during smoltification, were examined. Purified IGFBP-1 from serum was used for as a standard, for tracer preparation and for antiserum production. Cross-linking I-125-labelled IGFBP-1 with salmon IGF-I eliminated interference by IGFs. The RIA had little cross-reactivity with salmon 28 and 41 kDa IGFBPs (<0.5%) and measured IGFBP-1 levels as low as 0.1 ng/ml. Fasted fish had significantly higher IGFBP-1 levels than fed fish (21.6 +/- 4.6 vs 3.0 +/- 2.2 ng/ml). Plasma IGFBP-1 was measured in individually tagged 1-year-old coho salmon reared for 10 weeks under four different feeding regimes as follows: high constant (2% body weight/day), medium constant (1% body weight/day), high variable (2% to 0.5% body weight/day) and medium variable (1% to 0.5% body weight/day). Fish fed with the high ration had lower IGFBP-1 levels than those fed with the medium ration. Circulating IGFBP-1 increased following a drop in feeding ration to 0.5% and returned to the basal levels when feeding ration was increased. Another group of coho salmon were reared for 9 weeks under different water temperatures (11 or 7 degrees C) and feeding rations (1.75, 1 or 0.5% body weight/day). Circulating IGFBP-1 levels were separated by temperature during the first 4 weeks; a combined effect of temperature and feeding ration was seen in week 7; only feeding ration influenced IGFBP-1 level thereafter. These results indicate that IGFBP-1 is responsive to moderate nutritional and temperature changes. There was a clear trend that circulating IGFBP-1 levels were negatively correlated with body weight, condition factor (body weight/body length(3) x 100), growth rates and circulating 41 kDa IGFBP levels but not IGF-I levels. During parr-smolt transformation of coho salmon, IGFBP-1 levels showed a transient peak in late April, which was opposite to the changes in condition factor. Together, these findings suggest that salmon IGFBP-1 is inhibitory to IGF action. In addition, IGFBP-1 responds to moderate changes in dietary ration and temperature, and shows a significant negative relationship to condition factor.
  • Y Fujiwara, H Fukada, M Shimizu, A Hara
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 143 (3) 267 - 277 0016-6480 2005/09 [Refereed][Not invited]
    Two distinct yolk proteins (YP1 and YP2) were purified from the ovary of medaka, and specific antisera against YPs were generated to characterize YPs and reveal their relation to two vitellogenins (Vg1 and Vg2). The molecular masses of purified YPI and YP2 on gel filtration were 270 and 380 kDa, respectively. YPs were confirmed to be lipoproteins by staining with Sudan black. Amino acid compositions of YPI and YP2 were similar to those of Vgl and Vg2, respectively. In double immunodiffusion using anti-Vg1, a precipitin line of YPI formed a spur against the Vg1 line. YP2 and Vg2 were reacted with anti-Vg2, and a precipitin line of YP2 formed a fuse against the Vg2 line. These biochemical and immunological analyses of purified YPs revealed that YPI is lipovitellin 1 (Lv1) derived from Vg1 and YP2 is lipovitellin 2 (Lv2) derived from Vg2. Using specific antibodies against Lvs and Vgs, specific, high sensitivity chemiluminescent immunoassays (CLIAs) for two Vgs were developed to reveal basal Vg levels and response to exogenous estradiol-17 beta (E-2). The measurable range of both CLIAs was from 0.975 to 1000 ng/ml. The cross-reactivity to the alternative Vg in each CLIA was extremely low (<= 0.57%). When immature fish were immersed in water containing E2 for I h, both Vgs were induced by 0.5 mu g/L of E-2 at 24 h after treatment. Vg1 increased in a concentration-dependant manner up to 100 mu g/L E2, while Vg2 reached a plateau at 10 mu g/L of E2. The ratio of Vg1:Vg2 in E2-treated fish changed in a concentration-dependent manner from 1.5:1 to 8.5: 1. The results from E-2-treatment suggest that differential regulation may control the expression of medaka Vgs. (c) 2005 Elsevier Inc. All rights reserved.
  • T Fujita, H Fukada, M Shimizu, N Hiramatsu, A Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 141 (2) 211 - 217 1096-4959 2005/06 [Refereed][Not invited]
    Annual changes in serum levels of two chorion precursors, choriogenin H (Chg H) and choriogenin L (Chg L), vitellogenin (Vg) and estradiol-17 beta (E2) were quantified in masu salmon, Oncorhynchus masou, using specific immunoassays. Serum Chg levels were higher than Vg during the previtellogenic growth phase when circulating E2 levels were low (similar to 0.1 ng/mL), suggesting higher sensitivity of Chg to E2. When oocyte growth shifted to the vitellogenic phase, Vg levels increased and became the most abundant in serum coincident with elevations of E2 and GSI. Chg H, Chg L and Vg peaked 1 month prior to ovulation at 0.61 +/- 0.08, 0.98 +/- 0.18 and 10.93 +/- 3.24 mg/mL, respectively. These results suggest that chorion formation by Chgs occurs prior to vitellogenesis and that the sensitivity of Chgs to low circulating E2 is closely related to the sequential events of oocyte growth. (c) 2005 Elsevier Inc. All rights reserved.
  • AL Pierce, M Shimizu, BR Beckman, DM Baker, WW Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 140 (3) 192 - 202 0016-6480 2005/02 [Refereed][Not invited]
    Body growth in vertebrates is chiefly regulated by the GH/IGF axis. Pituitary growth hormone (GH) stimulates liver insulin-like growth factor-I (IGF-I) production. During fasting, plasma IGF-I levels decline due to the development of liver GH resistance, while GH levels generally increase. In mammals., decreased insulin during fasting is thought to cause liver GH resistance. However, the sequence of events in the GH/IGF axis response to fasting is not well characterized, especially in non-mammalian vertebrates. We assessed the time course of the GH/IGF axis response to fasting and increased ration in chinook salmon. Fish were placed on Fasting, Increased, or Control rations, and sampled daily for 4 days and at more widely spaced intervals through 29 days. Plasma IGF-I, GH, insulin, and 41 kDa IGF binding protein (putative salmon IGFBP-3), and liver IGF-I gene expression were measured. Control and Increased ration fish did not differ strongly. Plasma IGF-I and 41 kDa IGFBP were significantly lower in Fasted versus Control fish from day 4 onward, and liver IGF-I gene expression was significantly lower from day 6 onward. Liver IGF-I gene expression and plasma IGF-I levels were correlated. Plasma insulin was lower in Fasted fish from day 6 onward. There was a trend toward increased GH in Fasted fish on days 1-2, and GH was significantly increased Fasted fish from day 3 onward. Fasted GH first increased (days 1-3) to a plateau of 10-20 ng/ml (days 4-12) and then increased dramatically (days 15-29), suggesting that the GH response to fasting had three phases. The early increase in GH, followed by the decrease in plasma IGF-I after 4 days, suggests that GH resistance developed within 4 days. (C) 2004 Elsevier Inc. All rights reserved.
  • Munetaka Shimizu, Munetaka Shimizu, J. T. Dickey, J. T. Dickey, H. Fukada, H. Fukada, W. W. Dickhoff, W. W. Dickhoff
    Journal of Endocrinology Society for Endocrinology 184 (1) 267 - 276 0022-0795 2005/01/01 [Refereed][Not invited]
    Western ligand blotting of salmon serum typically reveals three insulin-like growth factor (IGF) binding proteins (IGFBPs) at 22, 28 and 41 kDa. Physiologic regulation of the 22 kDa IGFBP is similar to that of mammalian IGFBP-1; it is increased in catabolic states such as fasting and stress. On the other hand, its molecular mass on Western ligand blotting is closest to mammalian IGFBP-4. The conflict between physiology and molecular mass makes it difficult to determine the identity of the 22 kDa IGFBP. This study therefore aimed to identify the 22 kDa IGFBP from protein and cDNA sequences. The 22 kDa IGFBP was purified from chinook salmon serum by a combination of IGF-affinity chromatography and reverse-phase chromatography. The N-terminal amino-acid sequence of the purified protein was used to design degenerate primers. Degenerate PCR with liver template amplified a partial IGFBP cDNA, and full-length cDNA was obtained by 5′- and 3-pm-rapid amplification of cDNA ends (RACE). The 1915-bp cDNA clone encodes a 23.8 kDa IGFBP, and its N-terminal amino-acid sequence matched that of purified 22 kDa IGFBP. Sequence comparison with six human IGFBPs revealed that it is most similar to IGFBP-1 (40% identity and 55% similarity). These findings indicate that salmon 22 kDa IGFBP is IGFBP-1. Salmon IGFBP-1 mRNA is predominantly expressed in the liver, and its expression levels appear to reflect circulating levels. The 3′-untranslated region of salmon IGFBP-1 mRNA contains four repeats of the nucleotide sequence ATTTA, which is involved in selective mRNA degradation. In contrast, amino-acid sequence analysis revealed that salmon IGFBP-1 does not have an Arg-Gly-Asp (RGD) integrin recognition sequence nor a Pro, Glu, Ser and Thr (PEST)-rich domain (a segment involved in rapid turnover of protein), both of which are characteristic of mammalian IGFBP-1. These findings suggest that association with the cell surface and turnover rate may differ between salmon and mammalian IGFBP-1. © 2005 Society for Endocrinology.
  • BR Beckman, M Shimizu, BA Gadberry, PJ Parkins, KA Cooper
    AQUACULTURE 241 (1-4) 601 - 619 0044-8486 2004/11 [Refereed][Not invited]
    The effect of temperature change on the relations between growth and plasma levels of insulin-like growth factor-I (IGF-I) and 41-kDa insulin-like growth factor binding protein (41-kDa IGFBP) were assessed in postsmolt coho salmon. An experiment was initiated by reducing the temperature from 11 to 7 degreesC for two groups of fish that were subsequently fed at either 0.5% or 1.0% body weight per day. Two additional groups of fish were maintained at 11 degreesC and fed either 1.0% or 1.75% body weight per day. Fish were weighed and measured, and plasma samples were obtained on four dates: 12, 27, 47, and 63 days after the temperature change. Plasma IGF-I values were significantly lower in cool water groups than warm water groups for the first sample, by the last sampling, there were no significant differences between groups. There were no significant differences between treatment groups for plasma 41-kDa IGFBP levels for the first sampling. Subsequently, significant differences in plasma 41-kDa IGFBP levels between fish fed at different levels, regardless of temperature, were found. On all dates, significant associations were found between growth, plasma IGF-1, and 41-kDa IGFBP for fish held in warm water; however, no such relations were found for fish held in cool water for the first two sampling dates. After 47 and 63 days in cool water, significant relations between growth, plasma IGF-I, and 41-kDa IGFBP were reestablished. These data suggest that associations between growth, plasma IGF-I, and 41-kDa IGFBP were disrupted by a temperature decrease for at least 4 weeks. However, significant relations were reestablished after 47 days of temperature acclimation. These results support further investigation of plasma IGF-I and 41-kDa IGFBP as growth indicators in fish. (C) 2004 Elsevier B.V. All rights reserved.
  • DA Larsen, M Shimizu, KA Cooper, P Swanson, WW Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 139 (1) 29 - 37 0016-6480 2004/10 [Refereed][Not invited]
    Among many species of salmonids, fast growing fish mature earlier than slow growing fish, and maturing males grow faster than non-maturing ones. To study the potential endocrine basis for this reciprocal relationship we examined the in vivo effects of the androgens, testosterone (T) and 11-ketotestosterone (11-KT), on plasma growth hormone (GH), insulin-like growth factor-I (IGF-I) and 41-kDa IGF binding protein (41-kDa IGFBP) (putative IGFBP-3) in coho salmon, Oncorhynchus kisutch. Immature male and female, two-year old fish (avg. wt. 31.7 +/- 0.63 g) were injected with coconut oil containing T or 11-KT at a dose of 0. 1, 0.25, or 1 mug/g body weight. Blood samples were taken 1 and 2 weeks postinjection, and analyzed by immunoassay for T, 11-KT, GH, IGF-I, and 41-kDa IGFBP. Steroid treatments elevated the plasma T and 11-KT levels to physiological ranges typical of maturing fish. Plasma IGF-I and 41-kDa IGFBP levels increased in response to both T and 11-KT in a significant and dose-dependent manner after 1 and 2 weeks, but GH levels were not altered. These data suggest that during reproductive maturation, in addition to the previously demonstrated effects of the IGFs on steroidogenesis, the gonadal steroids may in turn play a significant role in regulating IGF-I and its binding proteins in fish. The interaction between the reproductive and growth axes may provide a regulatory mechanism for bringing about the dimorphic growth patterns observed between maturing and non-maturing salmonids and other species of fish. (C) 2004 Elsevier Inc. All rights reserved.
  • T Fujita, H Fukada, M Shimizu, N Hiramatsu, A Hara
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 136 (1) 49 - 57 0016-6480 2004/03 [Refereed][Not invited]
    Previously two precursors to vitelline envelope proteins, choriogenin H (Chg H) and choriogenin L (Chg L), were identified in masu salmon, Oncorhynchus masou, and specific antisera against these two proteins were generated in rabbits. In this study, two methods of immunoassay have been developed using these specific antibodies: single radial immunodiffusion (SRID) and enzyme-linked immunosorbent assay (ELISA). Non-competitive sandwich ELISAs for Chg H and Chg L were designed using digoxigenin-labeled antibodies and purified Chgs as assay components. The working range of the ELISAs was 1-128 and 2-256 ng/ml for Chg H and Chg L, respectively. Using these immunoassays and a chemiluminescent immunoassay for vitellogenin (Vg), the changes in these three estrogen-responsive proteins were measured in the serum of masu salmon after treatment with various doses of estradiol-17beta (E-2). The changes in serum levels of Chgs and Vg in male fish differed according to the E-2 dose. When fish were given a 5 mg/kg body weight (BW) of E-2, Vg was induced to a greater extent than Chgs. By contrast, Chg levels were higher than that of Vg after a 10 tg/ kg BW of E-2 injection. A similar trend was seen in the response time to exogenous E-2. Serum Chgs were induced from 8 h after E-2 injection and reached a peak of about 5 mug/ml at 24 h. Although Vg was not detected until 8 h after E-2 injection, its levels remained considerably low at around 0.03 mug/ml, even after 24 h. Chg H was more sensitive than was Chg L to I mug/kg BW of estrogen: the long-term exposure of fish to E-2 showed that Chg H could be induced from a lower dose of E-2 than could Chg L. Taken together, these results suggest that the serum levels of Chg H, Chg L, and Vg in masu salmon are regulated by circulating levels of E-2. (C) 2003 Elsevier Inc. All rights reserved.
  • BR Beckman, M Shimizu, BA Gadberry, KA Cooper
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 135 (3) 334 - 344 0016-6480 2004/02 [Refereed][Not invited]
    The effect of different feeding levels on plasma levels of insulin-like growth factor-I (IGF-I), 41 kDa insulin-like growth factor binding protein (41 kDa IGFBP), and growth hormone (GH) were assessed in post-smolt coho salmon. Fish were fed at either stable (1 and 2% body weight/day) or varying (1-0.5-1%, 2-0.5-2% body weight/day) feeding rates and plasma was sampled from 10 fish/treatment at 2-3 week intervals over five dates from June to September, resulting in a total of 200 samples. Fish fed at higher rates grew faster and had higher plasma IGF-I and 41 kDa IGFBP levels. Plasma GH levels were variable but generally showed an inverse relationship to feeding rate. Both plasma IGF-I and 41 kDa IGFBP increased seasonally, average IGF-I levels doubled from June to September, regardless of feeding rate. On any one date both IGF-I and 41 kDa IGFBP were highly related to growth rate with regression coefficients ranging from 0.36 to 0.68 (IGF-I) and from 0.33 to 0.70 (41 kDa IGFBP). No relationship was found between IGF-I:41 kDa IGFBP ratio and individual growth rate. Overall, both feeding rate and date were important in explaining variation in IGF-I and 41 kDa IGFBP levels. Published by Elsevier Inc.
  • Immunochemical comparison between sera from the primary and secondary circulations in a salmonid fish, Sakhalin taimen (Hucho Perryi)
    Yuki Tokushima, Yuki Ito, Munetaka Shimizu, Akihiko Hara
    Fish Physiology and Biochemistry 30 179 - 188 2004 [Refereed]
  • J Johnson, J Silverstein, WR Wolters, M Shimizu, WW Dickhoff, BS Shepherd
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 134 (2) 122 - 130 0016-6480 2003/11 [Refereed][Not invited]
    Vertebrate growth is principally controlled by growth hormone (GH) and, its intermediary, insulin-like growth factor-I (IGF-I). The actions of IGF-I are modulated by high-affinity binding proteins called insulin-like growth factor binding-proteins (IGFBPs). Channel catfish exhibit atypical responses (increased percentage body fat and reduced percentage protein) to GH treatment, despite GH-dependent IGF-I production. Among possible explanations for this atypical response to GH treatment is an unusual regulation of blood IGFBPs. In this species, there has been one report of a single 33-kDa plasma binding protein. To examine the occurrence and regulation of plasma IGFBPs in this species, two strains of channel catfish (Norris and USDA-103) were treated with weekly injections of recombinant bovine GH at different temperatures (21 degreesC versus 26 degreesC). In a separate experiment involving catfish of a different strain, endogenous GH levels were altered via injection of the GH secretogogue, bGHRH(1-29)-amide, and held in fresh water or transferred to brackish water (12 ppt). Following these treatments, the type and regulation of plasma IGFBPs in these catfish strains were examined by Western ligand blotting. We have identified five IGFBPs (19, 35, 44, 47, and >80 kDa) in catfish plasma that are differentially altered by experimental treatment and genetic lineage. Levels of the 19-kDa IGFBP were elevated in catfish of Norris and USDA-103 strains that were exposed to a higher environmental temperature (26 degreesC versus 21 degreesC), but was not seen in those animals used for the GH secretogogue/salinity study. In most vertebrates, treatment with GH increases levels of plasma IGFBP-3 (similar to40-50 kDa). In the USDA-103 and Norris catfish strains, bGH injection reduced plasma levels of the 44- and 47-kDa IGFBPs. Similarly, elevations in plasma GH levels in GH secretogoge-treated and brackish water-adapted catfish resulted in reductions of the 44- and 47-kDa IGFBPs as well as a reduction in presence of a 35-kDa IGFBP that was not detected in the Norris or USDA-103 strains. Reduced levels of the 35, 44, and 47 kDa IGFBPs, seen in the plasma of the GH secretogogue-treated and brackish water-adapted animals, suggests that the atypical response of channel catfish to GH treatment is not attributed to the use of heterologous (bovine) GH. This negative response of the 35-47 kDa IGFBPs to GH has not been reported in any teleost or vertebrate (healthy) and may be partly responsible for the atypical physiological responses of channel catfish to GH treatment. (C) 2003 Elsevier Inc. All rights reserved.
  • M Shimizu, A Hara, WW Dickhoff
    JOURNAL OF ENDOCRINOLOGY 178 (2) 275 - 283 0022-0795 2003/08 [Refereed][Not invited]
    Salmon plasma contains at least three IGF-binding proteins (IGFBPs) with molecular masses of 41, 28 and 22 kDa. The 41 kDa IGFBP is similar to mammalian IGFBP-3 in size, type of glycosylation and physiological responses. In this study, we developed an RIA for the 41 kDa IGFBP. The 41 kDa IGFBP purified from serum was used for antibody production and as an assay standard. Binding of three different preparations of tracer were examined: I-125-41 kDa IGFBP, I-125-41 kDa IGFBP cross-linked with IGF-I and 41 kDa IGFBP cross-linked with I-125- IGF-I (41 kDa IGFBP/I-125-IGF-I). Only binding of 41 kDa IGFBP/I-125-IGF-I was not affected by added IGFs, and therefore it was chosen for the tracer in the RIA. Plasma 41 kDa IGFBP levels measured by RIA were increased by GH treatment (178.9 +/- 4.9 ng/m) and decreased after fasting (95.0 +/- 7.0 ng/ml). The molarities of plasma 41 kDa IGFBP and total IGF-I were comparable, and they were positively correlated, suggesting that salmon 41 kDa IGFBP is a main carrier of circulating IGF-I in salmon, as is mammalian IGFBP-3 in mammals. During the parr-smolt transformation (smoltification) of coho salmon, plasma 41 kDa IGFBP levels showed a transient peak (182.5 +/- 10.3 ng/ml) in March and stayed relatively constant thereafter, whereas IGF-I showed peak levels in March and April. Differences in the molar ratio between 41 kDa IGFBP and IGF-I possibly influence availability of IGF-I in the circulation during smoltification.
  • M Shimizu, P Swanson, A Hara, WW Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 132 (1) 103 - 111 0016-6480 2003/06 [Refereed][Not invited]
    In salmon, at least three insulin-like growth factor binding proteins (IGFBPs) with molecular masses of 41, 28, and 22 kDa exist in serum. The 41-kDa IGFBP is up-regulated by growth hormone treatment and down-regulated by fasting, suggesting that it is a homolog of IGFBP-3. We purified the 41-kDa IGFBP from chinook salmon serum by IGF-I affinity chromatography followed by reversed-phase high pressure liquid chromatography. Purified IGFBP appeared as doublet bands on electrophoresis and was N-glycosylated. Analysis of partial N-terminal amino acid sequence revealed that salmon 41-kDa IGFBP has the cysteine rich domain conserved among IGFBP family. In a binding assay using I-125-salmon IGF-I, purified 41-kDa IGFBP specifically bound salmon IGF-I, human IGF-I and human IGF-II, but neither Long R 3 IGF-I nor salmon insulin, showing that binding characteristics of the salmon IGFBP are similar to those of mammalian IGFBPs. Although the partial amino acid sequence of 41-kDa IGFBP showed highest homologies with zebrafish and seabream IGFBP-2, the highly conserved nature of the N-terminus makes it impossible to identify the type of IGFBP from partial sequence data. However, based on physiological responses, molecular weight and type of glycosylation, the 41-kDa IGFBP is most similar to mammalian IGFBP-3. (C) 2003 Elsevier Science (USA). All rights reserved.
  • M Shimizu, Y Fujiwara, H Fukada, A Hara
    JOURNAL OF EXPERIMENTAL ZOOLOGY 293 (7) 726 - 735 0022-104X 2002/12 [Refereed][Not invited]
    Estrogen treatment of medaka leads to accumulation of ascites, in which vitellogenin (Vg) and choriogenins (precursors to vitelline envelope) are abundant. Besides those female-specific proteins, we detected a new component in ascites that cross-reacts with antiserum against egg yolk proteins. We tentatively named it egg yolk-related protein (YRP). YRP was purified from ascites by hydroxylapatite chromatography followed by gel filtration. Purified YRP had a molecular mass of 460 kDa in intact state while 570 kDa for Vg. The molecular weight of purified YRP on SDS-PAGE under both reducing and nonreducing conditions was 130 kDa. YRP was confirmed to be a lipoglycophosphoprotein by staining with Sudan black, periodic acid-Schiff (PAS) and methyl green. Amino acid composition of YRP resembled that of Vg except for a relatively low content of serine. A specific antiserum against YRP was raised in a rabbit. Antiserum against YRP specifically immunostained its antigen but not Vg or choriogenins. YRP was detected as a female-specific protein in serum of breeding medaka. The antiserum also cross-reacted with a band at 29 kDa in egg extracts, which is not immunoreactive to antiserum against Vg. These data show that YRP is a precursor to some egg yolk proteins with differing antigenicity from Vg (Hamazaki et al. '87). We thus conclude that YRP is a second form of medaka Vg and rename YRP as Vg 2 while formerly reported Vg as Vg 1. (C) 2002 Wiley-Liss, Inc.
  • T Fujita, M Shimizu, N Hiramatsu, H Fukada, A Hara
    COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 132 (3) 599 - 610 1096-4959 2002/07 [Refereed][Not invited]
    Three vitelline envelope-related proteins (VERPs), very-high-molecular-weight VERP (vhVERP), high-molecular-weight VERP (hVERP) and low-molecular-weight VERP (IVERP) were purified from female masu salmon serum. The apparent molecular weights of vhVERP, hVERP and IVERP, in their native state, were 520, 88 and 54 kDa, respectively, by gel-filtration chromatography Very-high-molecular-weight VERP comprises two subunits, corresponding to 175 and 126 kDa. On SDS-PAGE, hVERP and IVERP migrate at 53 and 47 kDa, respectively. Amino acid analysis of vhVERP and hVERP showed that they share a high content of glutamic acid and proline. By contrast, IVERP is rich in glutamic acid and asparatic acid. These features are in good agreement with the amino acid composition of the vitelline envelope. Immuno-biochemical analysis suggested that vhVERP is derived from hVERP by polymerization and/or aggregation. Antibodies against hVERP and IVERP specifically immunostained the vitelline envelope and liver of female masu salmon. In addition, both hVERP and IVERP were induced in the serum of estrogen-treated male fish. Taken together, it is suggested that hVERP and IVERP are homologous molecules with choriogenin H and choriogenin L in medaka, respectively. These results indicate that hVERP and IVERP are precursor proteins to the vitelline envelope (choriogenins) in masu salmon. (C) 2002 Elsevier Science Inc. All rights reserved.
  • HIRAMATSU Naoshi, FUKADA Haruhisa, KITAMURA Makiko, SHIMIZU Munetaka, FUDA Hirotoshi, KOBAYASHI Kunihiko, HARA Akihiko
    水産増殖 日本水産増殖学会 49 (3) 347 - 355 0371-4217 2001/09/20 [Refereed][Not invited]
    Immunoglobulin M (IgM) was purified from the serum of Sakhalin taimen (Hucho perryi) by salting-out, ion-exchange chromatography on DEAF-cellulose, and gel filtration on Sepharose 6B. The intact, tetrameric taimen IgM has a mass of 750 kDa with molecular architecture typical of IgM from other salmonids. The molecular weights of heavy (μ) chain and light chains (L) of the IgM monomer were estimated to be 68 kDa and 23 kDa, respectively. Purified taimen IgM was used to raise a specific rabbit antiserum and to develop a single radial immunodiffusion assay for measuring circulating IgM levels. The serum IgM levels in captive, immature or maturing female taimen varied between 1 and 5 mg/ml, showing seasonal changes regardless of fish age, with relatively low levels in spring and conversely high levels in autumn. Production of specific serum IgM to a parasite, Salmincola stellatus, was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The parasitized taimen serum could react with protein components of aqueous extracts from the parasite that were blotted on a nitrocellulose membrane after SDS-PAGE, but normal taimen serum did not, indicating that the parasitized fish produced the specific IgM to S. stellatus in the serum.
  • JT Silverstein, WR Wolters, M Shimizu, WW Dickhoff
    AQUACULTURE 190 (1-2) 77 - 88 0044-8486 2000/10 [Refereed][Not invited]
    Channel catfish from two strains, USDA-103 and Norris, were reared in circular 800-1 tanks and injected once weekly with 2.5 mu g recombinant bovine growth hormone (rbGH) per gram body weight, or the saline vehicle. In addition to the rbGH, and strain treatments, half the tanks were supplied with well water of 26.0 degrees C and the other half received water of 21.7 degrees C. Growth rate, plasma insulin-like growth factor I (IGF-I) levels, feed consumption and body fatness of the fish injected with rbGH were higher than in saline-injected controls. Strain and temperature effects were also significant. Feed consumption was significantly greater with growth hormone treatment, higher temperature, and in the USDA-103 strain of catfish. Feed efficiency was significantly better in rbGH-injected fish. The effect of temperature on feed efficiency was also significant, higher temperature treatments performed better. Growth hormone enabled channel catfish to grow better at lower and higher temperatures than saline-treated counterparts. (C) 2000 Elsevier Science B.V. All rights reserved.
  • Munetaka Shimizu, Penny Swanson, Haruhisa Fukada, Akihiko Hara, Walton W. Dickhoff
    General and Comparative Endocrinology 119 (1) 26 - 36 1095-6840 2000 [Refereed][Not invited]
    Insulin-like growth factor-binding proteins (IGFBPs) may interfere with accurate measurement of plasma IGFs in radioimmunoassay (RIA). Although several simplified extraction methods for IGFs have been developed, these methods are not always validated for differing physiological states, developmental stages, and animal species. For teleost fish, neither the necessity of plasma extraction nor the validity of extraction methods for IGF RIA is widely established. We systematically examined the validity of acid-ethanol (AE) extraction, AE extraction followed by cryoprecipitation (AEC extraction), and SP-Sephadex extraction in RIA for salmon IGF-I using commercially available components (GroPep Pty Ltd). Displacement curves of plasma extracted by AE, AEC, and SP-Sephadex were parallel to those of the standard. Measured IGF-I levels in plasma from several developmental stages and under different physiological and experimental conditions were significantly increased by the extractions and comparable to those after acid-size exclusion chromatography (SEC). On Western ligand blotting using digoxigenin-labeled human IGF-I, the intensity of IGFBP bands remaining in plasma were reduced after extraction, although some IGFBPs remained. However, these residual IGFBPs did not interfere measurably with the RIA based on quantitative comparison of IGF-I levels with acid-SEC. We conclude that with this RIA extraction is necessary for measurement of salmon IGF-I in plasma since measured values were routinely lower in unextracted samples, and AE, AEC, and SP-Sephadex extractions are applicable to the IGF-I RIA using the commercially available components. Using the validated RIA for IGF-I, plasma IGF-I levels in nonmaturing and precociously maturing chinook salmon in spring were measured after AE extraction. During spring, nonmaturing and maturing fish fed and grew well, and plasma IGF-I level was significantly correlated with body weight in both fish. This result indicates that circulating IGF-I plays a key role in controlling growth in precociously maturing chinook salmon in spring as in nonmaturing fish. (C) 2000 Academic Press.
  • M Shimizu, P Swanson, WW Dickhoff
    GENERAL AND COMPARATIVE ENDOCRINOLOGY 115 (3) 398 - 405 0016-6480 1999/09 [Refereed][Not invited]
    Total and free insulin-like growth factor-I (IGF-I) levels were quantified in plasma from growth hormone (GH)-treated and fasted coho salmon. Total IGF-I was measured by radioimmunoassay after acid-ethanol extraction and free IGF-I was separated from protein-bound IGF-I using ultrafiltration by centrifugation. Total and free TGF-I increased in plasma after GH treatment and decreased after fasting. The level of free IGF-I, however, was maintained at approximately 0.3% in both experiments. Unsaturated binding activity in plasma for IGF-I was assessed by incubation with I-125-recombinant salmon IGF-I (I-125-sIGF-I). Although there was no difference in binding activity between GH-treated and control fish, fasted fish showed higher binding activity than did fed fish, suggesting induction of unsaturated binding protein by fasting. IGF-binding protein (IGFBP) bands were observed in plasma of coho salmon by Western ligand blotting using I-125-sIGF-I. A low-molecular-weight (22 kDa) band was clear in fasted fish but not detectable in fed fish. The IGFBP band, which has molecular weight similar to that of human IGFBP-3 (41 kDa), was more intense in GH-treated fish than in controls. The molecular distribution of IGF-1 in plasma was examined by gel filtration under neutral conditions. Most IGF-I was eluted around 40 kDa. This result suggests that the major form of bound IGF-I in the circulation of coho salmon may be in a 40-kDa binary complex rather than in a 150-kDa ternary complex, as in mammals.
  • Changes in serum choriogenin and vitellogenin levels in masu salmon (Oncorhynchus masou) during sexual maturation and after estrogen treatment.
    Fujita T, Hiramatsu N, Shimizu M, Sullivan CV, Hara A
    Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish 314  1999 [Refereed][Not invited]
  • Changes of serum growth hormone and vitellogenin levels in chtthroat trout during sexual maturation.
    Fukada H, Hiramatsu N, Shimizu M, Sullivan CV, Hara A
    Proceedings of the 6th International Symposium on the Reproductive Physiology of Fish 314  1999 [Refereed][Not invited]
  • Munetaka Shimizu, Toshiaki Fujita, Akihiko Hara
    Journal of Experimental Zoology 282 (3) 385 - 395 0022-104X 1998/10/15 [Refereed][Not invited]
    High and low molecular weight vitelline envelope-related proteins (hVERP and lVERP) were purified from serum of vitellogenic female Sakhalin taimen (Hucho perryi) by a combination of ion-exchange, hydroxylapatite and gel filtration chromatography. The molecular weight of hVERP was estimated to be 83 kDa by gel filtration, and 48 kDa and 54 kDa in SDS-PAGE under non-reduced and reduced conditions, respectively. The molecular weight of lVERP was 56 kDa by gel filtration, and 42 kDa and 46 kDa on SDS-PAGE (non-reduced and reduced, respectively). Amino acid composition of hVERP was characterized by high content of proline (15.9%) and glutamic acid (13.8%). The lVERP had high contents of glutamic acid (10.8%) and aspartic acid (10.5%). Specific antibodies against hVERP and against lVERP were prepared by immunizing rabbits. The antiserum to hVERP stained bands corresponding to 98 kDa and 48 kDa of vitelline envelope (VE) in SDS-PAGE without reduction, whereas the antiserum to lVERP immunostained 98 kDa and 42 kDa bands. Both specific antibodies recognized the vitelline envelope of vitellogenic oocytes immunocytochemically. Thus, hVERP and lVERP are precursors to vitelline envelope proteins in this species.
  • Munetaka Shimizu, Toshiaki Fujita, Naoshi Hiramatsu, Akihiko Hara
    Fisheries Science 64 (4) 600 - 605 0919-9268 1998 [Refereed][Not invited]
    Vitelline envelope-Related proteins (VERPs) were immunologically detected in serum and liver of female Sakhalin taimen Hucho perryi, but not in males, using an antiserum against vitelline envelope. Two VERPs were observed in serum as female-specific proteins with molecular weights of 48 kDa and 42 kDa on SDS-PAGE under non-reduced conditions. VERPs were induced in serum and the liver of immature fish by injection of 10 μg estradiol-17β (E2) per kg of body weight. These results suggest that the origin of VERPs is the liver. During vitellogenesis, one VERP band first appeared in July in the serum of five-year-old fish. Two VERP bands were observed during winter and became undetectable in June after ovulation. The changes in VERPs were parallel to those in vitellogenin.
  • N Hiramatsu, M Shimizu, H Fukada, M Kitamura, K Ura, H Fuda, A Hara
    A specific and sensitive enzyme linked immunosorbent assay (ELISA) and a single radial immunodiffusion (SRID) were developed for measurement of serum vitellogenin (Vg) levels in Sakhalin taimen (Hucho perryi). Regarding specificity for serum Vg, an antiserum raised against lipovitellin of taimen (a-Lv) was adequate for both assays. ELISA and SRID could detect Vg in serum at concentrations as low as 10 ng/ml and 25 mu g/ml, respectively. In estrogen administration experiments, the level of serum Vg began clearly increasing within 12 to 24 hr after injection of immature females with estradiol-17 beta (E-2) The appearance and levels of Vg in males treated with E-2 were delayed and smaller, respectively, than for females. Vg levels varied throughout natural vitellogenesis from 0-4 mu g/ml (3 years old) to approximately 30 mg/ml (5-6 years old). We observed an early transitory peak of serum Vg levels (primary reaction) at the time of early vitellogenesis and chronic high Vg levels (for 6-7 months) in winter period before ovulation. Changes of serum E-2 levels were correlated with Vg levels. However, E-2 levels decreased a month earlier than Vg levels near ovulation. It appears that the duration of vitellogenesis in taimen is considerably longer than that in other salmonids, lasting more than 2 years. (C) 1997 Elsevier Science Inc.
  • Shimizu M, Fujita T, Hara A
    ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 1493 - 1496 1997 [Refereed][Not invited]
  • M Shimizu, H Ueda, H Kawamura, K Shimazaki, K Yamauchi
    JOURNAL OF FISH BIOLOGY 47 (6) 1044 - 1054 0022-1112 1995/12 [Refereed][Not invited]
    Changes in cytosolic proteins of the olfactory system (olfactory epithelium, olfactory nerve and olfactory bulb) and the telencephalon of masu salmon Oncorhynchus masou were analysed by two-dimensional polyacrylamide gel electrophoresis during parr-smelt transformation; parr, pre-smelt and full-smelt stages. In the olfactory system, several protein spots appeared and disappeared in the course of smelting. One protein spot in particular with an estimated molecular weight of 27 kDa and isoelectric point of 5.6 (M27) disappeared in common with the olfactory system during smelting. The disappearance of M27 was also observed in the telencephalon. These proteins, which appeared and disappeared, may reflect the changes in olfactory function during smelting. Simultaneously, the present study confirmed a salmonid olfactory specific protein of 24 kDa (N24), which existed in the olfactory system but not in the telencephalon, as a single neutral protein spot in masu salmon. (C) 1995 The Fisheries Society of the British Isles
  • Ueda Hiroshi, Shimizu Munetaka, Kudo Hideaki, Hara Akihiko, Hiroi Osamu, Kaeriyama Masahide, Tanaka Hisao, Kawamura Hiroshi, Yamauchi Kohei
    Fisheries science 公益社団法人 日本水産学会 60 (2) 239 - 240 0919-9268 1994 [Refereed][Not invited]
    ZOOLOGICAL SCIENCE 10 (4) 685 - 690 0289-0003 1993/08 [Refereed][Not invited]
    An olfactory system-specific protein (N24; molecular weight 24 kDa) was examined for immunological similarity to proteins from various organs (heart, intestine, kidney, muscle, ovary and testis) of mature kokanee salmon (Oncorhynchus nerka) by means of Western blotting analysis using a polyclonal antibody to N24. The antibody recognized one 24 kDa protein in the testis but none in the other organs examined. Immunoelectron microscopic analysis revealed that immunoreactive gold particles were mainly concentrated on the nuclei of spermatids and spermatozoa, while no specific gold particles were observed on the nuclei and the cytoplasm of spermatogonia, spermatocytes, Sertoli, Leydig and peritubular cells in maturing and mature testes of kokanee salmon. This is the first description of an antigenic similarity between an olfactory system-specific protein and a testicular germ cell protein in the animal kingdom. This corresponding protein may prove to be a useful molecular marker for studying the mechanism of sperm chemotaxis during fertilization.
    ZOOLOGICAL SCIENCE 10 (2) 287 - 294 0289-0003 1993/04 [Refereed][Not invited]
    An olfactory system-specific protein of 24 kDa (N24) was identified in kokanee salmon (Oncorhynchus nerka) by the comparison of proteins restricted to the olfactory system (olfactory epithelium, olfactory nerve and olfactory bulb) with those found in other parts of the brain (telencephalon, optic tectum, cerebellum, hypothalamus) and hypophysis. A polyclonal antibody to N24 was raised in a rabbit, and the specificity of the antiserum was examined by Western blotting analysis; the antiserum recognized only one 24 kDa band in the olfactory system but not in other parts of the brain in kokanee salmon, sockeye salmon (O. nerka), masu salmon (O. masou) and chum salmon (O. keta). Immunocytochemical analysis revealed that positive immunoreactivity occurred exclusively in the olfactory nerve and in some olfactory neuroepithelial cells. Both at the time of imprinting of maternal stream odorants and at the time of homing to the maternal stream, the immunoreactivity of N24 in fish in the maternal stream was stronger than that in seawater fish. The present study indicates that an olfactory system-specific protein may be importantly related to both imprinting and homing mechanisms in salmonids.

Conference Activities & Talks

  • ニジマスのインスリン様成長因子(IGF)-IとIGF結合蛋白の絶食・再給餌に対する反応  [Not invited]
    井筒彩歌, 田所大二, 羽原史織, 宇賀地優希, 清水宗敬
  • サクラマスにおける光周期に依存的・非依存的なスモルト化の検討  [Not invited]
    宇賀地優希, 鈴木章太郎, 高橋英佑, 崔聞達, 清水宗敬
  • 北海道産サクラマスの光周期操作に対するスモルト化時期の応答比較  [Not invited]
    宇賀地優希, 鈴木章太郎, 高橋英佑, 崔聞達, 清水宗敬
  • ジマスにおける生理学的な成長指標の評価  [Not invited]
    井筒彩歌, 田所大二, 羽原史織, 宇賀地優希, 清水宗敬
  • Assessing growth of salmon in the wild by using hormones and proteins  [Invited]
    Munetaka Shimizu
    Society for Experimental Biology Annual Meeting 2021  2021/06
  • Physiological tools for evaluating growth status of migrating salmon  [Invited]
    Munetaka Shimizu
    The Third NPAFC Virtual Workshop on Linkages between Pacific Salmon Production and Environmental Changes  2021/05
  • シロザケ稚魚の成長に及ぼす降海前後の摂餌状態と低水温の影響  [Not invited]
    及川 仁, 中村 周, 金子信人, 虎尾 充, 越野陽介, 清水宗敬


Awards & Honors

  • 2017/03 日本水産学会 平成29年度日本水産学会論文賞
    受賞者: 桑田知宣;徳原哲也;清水宗敬;吉崎悟朗

Research Grants & Projects

  • サケ科魚類の成長調節メカニズムの理解に基づいた養殖魚診断法の実用化
    Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)
    Date (from‐to) : 2019/04 -2023/03 
    Author : 清水 宗敬, 棟方 有宗, 森山 俊介, 内田 勝久
  • 海水移行時の低水温と栄養状態に着目したサケ稚魚の減耗機構に関する実験的検証
    Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2018/04 -2021/03 
    Author : 宮腰 靖之, 虎尾 充, 清水 宗敬
    異なる栄養状態と水温条件が海水移行時のサケ稚魚の初期減耗に与える影響を調べるため,2018年,水温条件と給餌・絶食の条件を変えた以下の試験群を設定してサケ稚魚の飼育試験を行った。給餌率をそれぞれ0%(絶食),1%,および3%で5日間飼育した後,それぞれの群を低水温区(4℃),中水温区(7℃),高水温区(10℃)の試験水槽に収容し,3日間の馴致期間を経て5日間の海水飼育を行った。 これらの試験群について,栄養状態の指標として筋肉中のトリグリセリド(TG)含量と肝臓中のグリコーゲン含量を測定した。成長率の指標として血中のインスリン様成長因子-I(IGF-I)量を測定した。海水飼育後のTG含量はいずれの水温区でも淡水飼育時の給餌率が高い群でTG量は高く,肝臓中グリコーゲン含量は高水温・絶食群で高い傾向がみられた。血中IGF-I量は淡水での絶食には差は無かったが,海水馴致時にはいずれの水温でも絶食群でIGF-I濃度が低かった。海水飼育後は絶食による違いはなく,高水温群でIGF-Iが高くなった。 遊泳力の指標として臨界遊泳速度(Ucrit)を測定した。海水飼育後には,低水温試験群の遊泳速度は高水温試験群より有意に低かった。サケ稚魚の海水移行後の遊泳速度は水温に大きく影響を受け,低水温では遊泳速度が低下することが示された。また,淡水生活期の絶食が海水移行後の遊泳速度低下につながることも示されたが,水温が高い場合はその影響が軽減される可能性がある。
  • サケ科魚類の新しい成長・ストレス診断法の開発と海面養殖への応用
    Date (from‐to) : 2016/04 -2020/03 
    Author : 清水宗敬
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2013/04 -2016/03 
    Author : MIYAKOSHI Yasuyuki, SHIMIZU Munetaka, TORAO Mitsuru
    To evaluate the growth rates of chum and pink salmon in their early marine life, we analyzed the otolith daily increments of juvenile chum and pink salmon and examined the effective physiological indicators for growth rates. By otolith increment analyses, the dates of ocean entry and daily growth rates can be estimated. From examining the physiological indicators, the serum insulin-like growth factor (IGF)-I can be used as a most useful indicator to evaluate the growth rates of juvenile salmon. Serum IGF-I levels in chum and pink salmon estuarine area were lower and fish in the coastal area were higher, indicating that growths of juvenile salmon was activated when they left the estuarine area and migrated to the offshore while fish of low growth stay in the estuary. Until May, IGF-I levels in pink salmon were lower than chum salmon but were higher in June, which likely reflects a higher growth rate of this species.
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2012 -2012 
    Author : 清水 宗敬
  • 文部科学省:科学研究費補助金(基盤研究(C))
    Date (from‐to) : 2009 -2011 
    Author : 清水 宗敬
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(C))
    Date (from‐to) : 2007 -2008 
    Author : Munetaka SHIMIZU
    魚類を含む脊椎動物の成長には、インスリン様成長因子(insulin-like growth factor, IGF)-Iが重要な役割を果たしている。しかし、IGF-Iの活性は6種類のIGF結合蛋白(IGF-binding protein, IGFBP)によって厳密に調節されている。これらのIGFBPの機能はタイプにより異なるが、大まかにIGF-Iを介した成長の「促進型」と「阻害型」に分けられる。サケ科魚類の血中には少なくとも3種類のIGFBPが検出されるが、それぞれがどのタイプに相当するのかは非常に混乱している。本研究はこれらのIGFBPの同定を行って上述の混乱を解決することを目的としている。まず、サケ科魚類の血中IGF-Iの主要な運搬役であり、成長の「促進型」と考えられている41 kDa IGFBP(41K BP)およびIGFBP-3のcDNAクローニングを行った。クローニングにより得られた配列を解析したところ、41K BPはIGFBP-2に最も高い相同性を示した。一方、IGFBP-3の配列は、41K BPのものとは異なっていた。このことから、41K BPはIGFBP-3ではなくIGFBP-2であることが明らかになった。さらにもう一種類のIGFBPをクローニングすると共に、これらの発現解析を行った。新たに得られたIGFBPの演繹アミノ酸配列はIGFBP-2に最も近かった。ま...
  • サケ科魚類の河川生活期における相分化機構に関する免疫生化学的研究
    日本学術振興会:科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 1999 -1999 
    Author : 清水 宗敬

Educational Activities

Teaching Experience

  • Advanced Course in Marine Biosphere Science II (Marine Environmental Biology)
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 魚類、海産無脊椎動物、環境、生理学、生活史、適応、生物生産(増養殖) fishes, marine invertebrates, environment, physiology, life-history, adaptation, aquaculture and resource enhancement
  • Fundamental Course in Marine Biological Processes
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : 海洋学、海洋生物科学 Oceanography、Marine Biological Science
  • Special Lecture in Biosphere Science II
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 環境科学院
    キーワード : Aquatic Biology, Marine Ecology, Oceanography
  • Environment and People
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : サケ、回遊、人工ふ化放流、分類、気候変化、資源変動、生活史、個体群密度効果、生物多様性、バイオロギング、母川回帰、嗅覚、物質循環、海水適応、ゲノム、性統御、始原生殖細胞、流通、食品,機能性素材、アスタキサンチン,考古学、先住民族、市民運動
  • Marine Biological Science Ⅰ
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 海洋生物,遺伝,分子生物学,機能,生理学,構造,細胞生物学,解剖組織学
  • Field Training on Aquatic Biological Science
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 生物相・分類・生態・物質循環・行動・組織
  • English for Fisheries Sciences I
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 海洋生物学、資源生物学、海洋環境化学、海洋共生学
  • Comparative Physiology
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 繁殖,ホルモン,免疫系,生理機能,生体防御
  • Marine Science Biology II - Laboratory
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : サケ マス ベントス 貝類 甲殻類 魚類 頭足類 分類 成長曲線 資源量推定 生物測定 外部形態 稚魚 耳石
  • Functional Biology in the Northern Biosphere
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 水産学部
    キーワード : 水圏環境、水圏生物機能、水圏生物資源、養殖生産、生物改変手法
  • Environment and People
    開講年度 : 2021
    課程区分 : 学士課程
    開講学部 : 全学教育
    キーワード : 海洋物理,海洋化学、海洋生態系,気候変化,プランクトン,ベントス,カレイ,サケ、深海魚、イカ・タコ類,鯨類,海鳥類,海獣類,衛星海洋学,人間活動,地球温暖化,海洋動物の保全・保護

Social Contribution

Social Contribution

Social Contribution

  • マリンラーニングセミナー
    Date (from-to) : 2021/10/02
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本財団・海と日本Project
  • マリンラーニングセミナー
    Date (from-to) : 2018/08
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本財団・海と日本Project
  • マリンラーニングセミナー
    Date (from-to) : 2017/07
    Role : Lecturer
    Sponser, Organizer, Publisher  : 日本財団・海と日本Project

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