顔写真

カンザキ マコト
神崎 展
Makoto Kanzaki
所属
大学院医工学研究科 医工学専攻 生体機械システム医工学講座(病態ナノシステム医工学分野)
職名
教授
学位
  • 博士(医学)(群馬大学)

  • 農学修士(新潟大学)

Researcher ID

学歴 2

  • 群馬大学 医学系研究科

    ~ 1995年3月31日

  • 新潟大学 農学研究科

    ~ 1991年3月

委員歴 4

  • American Society for Biochemistry and Molecular Biology Editorial Board(Journal of Biological Chemistry)

    2016年7月 ~ 継続中

  • American Society for Biochemistry and Molecular Biology 編集委員・Editorial Board(Journal of Biological Chemistry)

    2016年7月 ~ 継続中

  • PLOS ONE Scientific Editor

    2012年6月 ~ 継続中

  • PLOS ONE Scientific Editor

    2012年6月 ~ 継続中

所属学協会 4

  • 欧州糖尿病学会

  • 米国生化学・分子生物学会

  • 日本内分泌学会

  • 日本糖尿病学会

研究キーワード 5

  • GLUT4

  • 骨格筋

  • 運動

  • インスリン

  • 分子糖尿病学

研究分野 6

  • ライフサイエンス / 生理学 /

  • ライフサイエンス / 病態医化学 /

  • エネルギー / プラズマ科学 /

  • ライフサイエンス / 生体材料学 /

  • ライフサイエンス / 生体医工学 /

  • ライフサイエンス / 栄養学、健康科学 /

受賞 4

  1. プラズマエレクトロニクス賞 (共同受賞)

    2016年 応用物理学会

  2. American Diabetes Association (ADA) Junior Faculty Award

    2003年1月 American Diabetes Association (ADA)

  3. Juvenile Diabetes Foundation (JDF) Postdoctoral fellowship Award

    1998年6月 Juvenile Diabetes Foundation (JDF)

  4. 第17回日本内分泌学会研究奨励賞

    1997年5月 社団法人 日本内分泌学会 インスリン様成長因子(IGF-1)の作用の多様性とその情報伝達系に関する研究

論文 140

  1. Effects of CX3CR1 and CXCR2 antagonists on running-dependent intramuscular neutrophil recruitments and myokine upregulation. 国際誌

    Mazvita R Nyasha, Weijian Chen, Haopeng Wang, Fukie Yaoita, Masashi Aoki, Ryoichi Nagatomi, Makoto Kanzaki

    American journal of physiology. Endocrinology and metabolism 324 (5) E375-E389 2023年3月1日

    DOI: 10.1152/ajpendo.00196.2022  

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    Muscle contractile activity stimulates intramuscular recruitment of immune cells including neutrophils emerging to serve as a prerequisite for exerting proper muscular performance, although the underlying mechanisms and their contributions to myokine upregulation remain ill-defined. We previously reported that pharmacological inhibition of CX3CR1, a fractalkine receptor, dampens gnawing-dependent neutrophil recruitment into masseter muscles along with compromising their masticatory activity. By employing a running exercise model, we herein demonstrated that hindlimb muscles require collaborative actions of both CX3CR1- and CXCR2-mediated signals for achieving neutrophil recruitment, upregulation of myokines including interleukin (IL)-6, enhanced GLUT4 translocation, and adequate endurance capability. Mechanistically, we revealed that a combination of CX3CR1 and CXCR2 antagonists, i.e., AZD8797 and SB2205002, inhibits exercise-inducible ICAM-1 and fractalkine upregulations in the area of the endothelium and muscle-derived CXCL1 upregulation, both of which apparently contribute to the intramuscular neutrophil accumulation in working muscles. Intriguingly, we also observed that 2 h of running results in intramuscular augmentation of innate lymphoid type 2 cells (ILC2s) markers, i.e., Bcl11b mRNA levels and anti-GATA-3-antibody-positive signals, and that these effects are completely abolished by administration of the combination of CX3CR1 and CXCR2 antagonists. Taken together, our findings strongly suggest that the exercise-evoked regional interplay among working myofibers, the adjacent endothelium and recruited immune cells including neutrophils and possibly ILC2s, mediated through these local factors, plays a key role in organization of the intramuscular microenvironment supporting the performance of hindlimb muscles during running.

  2. A comparative study of the antibacterial properties of copper-based transparent oxides at the solid–liquid interface

    Takeru Okada, Kotaro Ohno, Makoto Kanzaki, Katsuyoshi Washio

    Japanese Journal of Applied Physics 61 (10) 108001-108001 2022年9月27日

    出版者・発行元:IOP Publishing

    DOI: 10.35848/1347-4065/ac9169  

    ISSN:0021-4922

    eISSN:1347-4065

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    Abstract This study investigates the antibacterial properties of CuCrO<sub>2</sub> (CCO) films fabricated by sputter deposition followed by calcination. The effects of the doped magnesium and the film fabrication in nitrogen atmosphere on both CCO crystallinity and antibacterial properties are compared. The antibacterial properties are evaluated using the plate counting method, and the crystallinity of the films are analyzed by X-ray diffraction. The results show the proliferation of Escherichia coli colonies that can be suppressed within 3 h, with magnesium-doped CCO showing the best antibacterial properties of all samples. This is explained by the formation of CuO byproduct during fabrication.

  3. Protocol for preparing sensor molecules and analyzing heterotypic endomembrane fusion in insulin-responsive cells using live-cell imaging. 国際誌

    Hiroyasu Hatakeyama, Makoto Kanzaki

    STAR protocols 3 (4) 101726-101726 2022年9月27日

    DOI: 10.1016/j.xpro.2022.101726  

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    Heterotypic endomembrane fusion between static GLUT4-containing vesicles and traveling transferrin receptor-containing endosomes triggers insulin-responsive translocation of the GLUT4 glucose transporter. Here, we provide a protocol for preparing BODIPY-based fluorescent sensor molecules allowing detection of heterotypic endomembrane fusion through dequenching via streptavidin-biotin binding and ratiometrically analyzing insulin-responsive events with live-cell imaging. Although this protocol is for evaluating specific fusion processes relating GLUT4 translocation, it is also applicable to assessing other processes so long as sensor molecules can properly label target molecules. For complete details on the use and execution of this protocol, please refer to Hatakeyama et al. (2022).

  4. RSPO3 is a novel contraction-inducible factor identified in an "in vitro exercise model" using primary human myotubes. 国際誌

    Tadahisa Takahashi, Yuqing Li, Weijian Chen, Mazvita R Nyasha, Kazumi Ogawa, Kazuaki Suzuki, Masashi Koide, Yoshihiro Hagiwara, Eiji Itoi, Toshimi Aizawa, Masahiro Tsuchiya, Naoki Suzuki, Masashi Aoki, Makoto Kanzaki

    Scientific reports 12 (1) 14291-14291 2022年8月22日

    DOI: 10.1038/s41598-022-18190-z  

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    The physiological significance of skeletal muscle as a secretory organ is now well known but we can only speculate as to the existence of as-yet-unidentified myokines, especially those upregulated in response to muscle contractile activity. We first attempted to establish an "insert-chamber based in vitro exercise model" allowing the miniature but high cell-density culture state enabling highly developed contractile human myotubes to be readily obtained by applying electric pulse stimulation (EPS). By employing this in vitro exercise model, we identified R-spondin 3 (RSPO3) as a novel contraction-inducible myokine produced by cultured human myotubes. Contraction-dependent muscular RSPO3 mRNA upregulation was confirmed in skeletal muscles of mice subjected to sciatic nerve mediated in situ contraction as well as those of mice after 2 h of running. Pharmacological in vitro experiments demonstrated a relatively high concentration of metformin (millimolar range) to suppress the contraction-inducible mRNA upregulation of human myokines including RSPO3, interleukin (IL)-6, IL-8 and CXCL1. Our data also suggest human RSPO3 to be a paracrine factor that may positively participate in the myogenesis processes of myoblasts and satellite cells. Thus, the "insert chamber-based in vitro exercise model" is a potentially valuable research tool for investigating contraction-inducible biological responses of human myotubes usually exhibiting poorer contractility development even in the setting of EPS treatment.

  5. Impact of habitual chewing on gut motility via microbiota transition

    Fukie Yaoita, Keita Watanabe, Ikuo Kimura, Masayuki Miyazawa, Shinobu Tsuchiya, Makoto Kanzaki, Masahiro Tsuchiya, Koichi Tan-No

    Scientific Reports 12 (1) 2022年8月15日

    出版者・発行元:Springer Science and Business Media LLC

    DOI: 10.1038/s41598-022-18095-x  

    eISSN:2045-2322

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    Abstract The gut environment, including the microbiota and its metabolites and short-chain fatty acids (SCFA), is essential for health maintenance. It is considered that functional recovery treatment for masticatory dysphagia affects the composition of the gut microbiota, indicating that habitual mastication, depending on the hardness of the food, may affect the gut microbiota and environment. However, the impact of chronic powdered diet feeding on the colonic condition and motility remains unclear. Here, we evaluated various colonic features in mice fed with powdered diets for a long-term and a mouse model with masticatory behavior. We observed a decreased abundance of the SCFA-producing bacterial genera in the ceca of the powdered diet-fed mice. Based on the importance of SCFAs in gut immune homeostasis and motility, interestingly, powdered diet feeding also resulted in constipation-like symptoms due to mild colitis, which were ameliorated by the administration of a neutrophil-depleting agent and neutrophil elastase inhibitors. Lastly, the suppressed colonic motility in the powdered diet-fed mice was significantly improved by loading masticatory activity for 2 h. Thus, feeding habits with appropriate masticatory activity and stimulation may play a key role in providing a favorable gut environment based on interactions between the gut microbiota and host immune system.

  6. Three live-imaging techniques for comprehensively understanding the initial trigger for insulin-responsive intracellular GLUT4 trafficking

    Hiroyasu Hatakeyama, Ko Kobayashi, Makoto Kanzaki

    iScience 25 (4) 104164-104164 2022年4月

    出版者・発行元:Elsevier {BV}

    DOI: 10.1016/j.isci.2022.104164  

    ISSN:2589-0042

  7. Tissue accumulation of neutrophil extracellular traps mediates muscle hyperalgesia in a mouse model. 国際誌

    Kazuaki Suzuki, Masahiro Tsuchiya, Shinichirou Yoshida, Kazumi Ogawa, Weijian Chen, Makoto Kanzaki, Tadahisa Takahashi, Ryo Fujita, Yuqing Li, Yutaka Yabe, Toshimi Aizawa, Yoshihiro Hagiwara

    Scientific reports 12 (1) 4136-4136 2022年3月9日

    DOI: 10.1038/s41598-022-07916-8  

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    Accumulation of uric acid (UA) during muscular trauma is a factor involved in the development of muscle hyperalgesia. Neutrophil extracellular traps (NETs), DNA-based reticular structures to capture UA, play a central role in the pain onset of gout attacks; however, the involvement of NETs via the elevation of local UA level in muscle hyperalgesia due to injuries from muscle overuse remains unknown. The triceps surae muscles (TSMs) in the unilateral hindlimb of mice were electrically stimulated to induce excessive muscle contraction. Mechanical withdrawal thresholds, tissue UA levels, neutrophil recruitment, and protein amount of citrullinated histone 3 (citH3), a major marker of NETs, were investigated. Furthermore, whether neutrophil depletion, extracellular DNA cleavage, and administration of the urate-lowering agent febuxostat improved muscle hyperalgesia caused by NET formation was examined. CitH3 expression upon neutrophil recruitment was significantly increased in the stimulated TSMs with increased tissue UA levels, whereas febuxostat administration improved muscle hyperalgesia with decreased citH3 and tissue UA levels, as observed in neutrophil depletion and extracellular DNA digestion. The underlying mechanism of muscle hyperalgesia associated with locally recruited neutrophils forming NETs due to increased tissue UA levels potentially plays a significant role in creating a vicious circle of muscle pain.

  8. Feeder-supported in vitro exercise model using human satellite cells from patients with sporadic inclusion body myositis. 国際誌

    Yuqing Li, Weijian Chen, Kazumi Ogawa, Masashi Koide, Tadahisa Takahashi, Yoshihiro Hagiwara, Eiji Itoi, Toshimi Aizawa, Masahiro Tsuchiya, Rumiko Izumi, Naoki Suzuki, Masashi Aoki, Makoto Kanzaki

    Scientific reports 12 (1) 1082-1082 2022年1月20日

    DOI: 10.1038/s41598-022-05029-w  

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    Contractile activity is a fundamental property of skeletal muscles. We describe the establishment of a "feeder-supported in vitro exercise model" using human-origin primary satellite cells, allowing highly-developed contractile myotubes to readily be generated by applying electrical pulse stimulation (EPS). The use of murine fibroblasts as the feeder cells allows biological responses to EPS in contractile human myotubes to be selectively evaluated with species-specific analyses such as RT-PCR. We successfully applied this feeder-supported co-culture system to myotubes derived from primary satellite cells obtained from sporadic inclusion body myositis (sIBM) patients who are incapable of strenuous exercise testing. Our results demonstrated that sIBM myotubes possess essentially normal muscle functions, including contractility development, de novo sarcomere formation, and contraction-dependent myokine upregulation, upon EPS treatment. However, we found that some of sIBM myotubes, but not healthy control myotubes, often exhibit abnormal cytoplasmic TDP-43 accumulation upon EPS-evoked contraction, suggesting potential pathogenic involvement of the contraction-inducible TDP-43 distribution peculiar to sIBM. Thus, our "feeder-supported in vitro exercise model" enables us to obtain contractile human-origin myotubes, potentially utilizable for evaluating exercise-dependent intrinsic and pathogenic properties of patient muscle cells. Our approach, using feeder layers, further expands the usefulness of the "in vitro exercise model".

  9. Blood glucose‐lowering effect of water and ethanolic extracts of Gynura procumbens (Lour.) Merr

    Cho L. Aung, Fumitaka Kawakami, Motoki Imai, Thet-Thet Lwin, Makoto Kanzaki, Ohn Mar, Khin P. Phyu, Mya M. Thwin, Hiroko Maruyama

    Traditional & Kampo Medicine 8 (2) 138-147 2021年8月

    出版者・発行元:Wiley

    DOI: 10.1002/tkm2.1277  

    ISSN:2053-4515

  10. Skeletal muscle-specific Keap1 disruption modulates fatty acid utilization and enhances exercise capacity in female mice. 国際誌

    Takahiro Onoki, Yoshihiro Izumi, Masatomo Takahashi, Shohei Murakami, Daisuke Matsumaru, Nao Ohta, Sisca Meida Wati, Nozomi Hatanaka, Fumiki Katsuoka, Mitsuharu Okutsu, Yutaka Yabe, Yoshihiro Hagiwara, Makoto Kanzaki, Takeshi Bamba, Eiji Itoi, Hozumi Motohashi

    Redox biology 43 101966-101966 2021年4月5日

    DOI: 10.1016/j.redox.2021.101966  

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    Skeletal muscle health is important for the prevention of various age-related diseases. The loss of skeletal muscle mass, which is known as sarcopenia, underlies physical disability, poor quality of life and chronic diseases in elderly people. The transcription factor NRF2 plays important roles in the regulation of the cellular defense against oxidative stress, as well as the metabolism and mitochondrial activity. To determine the contribution of skeletal muscle NRF2 to exercise capacity, we conducted skeletal muscle-specific inhibition of KEAP1, which is a negative regulator of NRF2, and examined the cell-autonomous and non-cell-autonomous effects of NRF2 pathway activation in skeletal muscles. We found that NRF2 activation in skeletal muscles increased slow oxidative muscle fiber type and improved exercise endurance capacity in female mice. We also observed that female mice with NRF2 pathway activation in their skeletal muscles exhibited enhanced exercise-induced mobilization and β-oxidation of fatty acids. These results indicate that NRF2 activation in skeletal muscles promotes communication with adipose tissues via humoral and/or neuronal signaling and facilitates the utilization of fatty acids as an energy source, resulting in increased mitochondrial activity and efficient energy production during exercise, which leads to improved exercise endurance.

  11. Imaging of muscle activity‐induced morphometric changes in fibril network of myofascia by two‐photon microscopy

    Chayanit Chaweewannakorn, Takashi Harada, Mazvita R. Nyasha, Masashi Koide, Yosuke Shikama, Yoshihiro Hagiwara, Keiichi Sasaki, Makoto Kanzaki, Masahiro Tsuchiya

    Journal of Anatomy 238 (3) 515-526 2021年3月19日

    出版者・発行元:Wiley

    DOI: 10.1111/joa.13339  

    ISSN:0021-8782

    eISSN:1469-7580

  12. Mitochondrial dysfunction underlying sporadic inclusion body myositis is ameliorated by the mitochondrial homing drug MA-5 国際誌

    Yoshitsugu Oikawa, Rumiko Izumi, Masashi Koide, Yoshihiro Hagiwara, Makoto Kanzaki, Naoki Suzuki, Koichi Kikuchi, Tetsuro Matsuhashi, Yukako Akiyama, Mariko Ichijo, Shun Watanabe, Takafumi Toyohara, Takehiro Suzuki, Eikan Mishima, Yasutoshi Akiyama, Yoshiaki Ogata, Chitose Suzuki, Hironori Hayashi, Eiichi N. Kodama, Ken-ichiro Hayashi, Eiji Itoi, Masashi Aoki, Shigeo Kure, Takaaki Abe

    PLOS ONE 15 (12) e0231064 2020年12月2日

    出版者・発行元:Public Library of Science ({PLoS})

    DOI: 10.1371/journal.pone.0231064  

    ISSN:1932-6203

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    <jats:p>Sporadic inclusion body myositis (sIBM) is the most common idiopathic inflammatory myopathy, and several reports have suggested that mitochondrial abnormalities are involved in its etiology. We recruited 9 sIBM patients and found significant histological changes and an elevation of growth differential factor 15 (GDF15), a marker of mitochondrial disease, strongly suggesting the involvement of mitochondrial dysfunction. Bioenergetic analysis of sIBM patient myoblasts revealed impaired mitochondrial function. Decreased ATP production, reduced mitochondrial size and reduced mitochondrial dynamics were also observed in sIBM myoblasts. Cell vulnerability to oxidative stress also suggested the existence of mitochondrial dysfunction. Mitochonic acid-5 (MA-5) increased the cellular ATP level, reduced mitochondrial ROS, and provided protection against sIBM myoblast death. MA-5 also improved the survival of sIBM skin fibroblasts as well as mitochondrial morphology and dynamics in these cells. The reduction in the gene expression levels of Opa1 and Drp1 was also reversed by MA-5, suggesting the modification of the fusion/fission process. These data suggest that MA-5 may provide an alternative therapeutic strategy for treating not only mitochondrial diseases but also sIBM.</jats:p>

  13. Continuous release of O 2 − /ONOO − in plasma‐exposed HEPES‐buffered saline promotes TRP channel‐mediated uptake of a large cation

    Shota Sasaki, Yuexing Zheng, Takayuki Mokudai, Hiroyasu Kanetaka, Masanori Tachikawa, Makoto Kanzaki, Toshiro Kaneko

    Plasma Processes and Polymers 17 (10) 2020年10月25日

    出版者・発行元:Wiley

    DOI: 10.1002/ppap.201900257  

    ISSN:1612-8850

    eISSN:1612-8869

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    Although the externally controllable extracellular supply of the short-lived reactive oxygen and nitrogen species, such as O-2(center dot-), (NO)-N-center dot, and ONOO-, could potentially manipulate cellular functions, their simple administration to cells is likely to be ineffective due to their rapid deactivation. In this study, we found a method of a continuous supply of O-2(center dot-)/ONOO- over a few minutes, which is triggered by irradiation of a nonequilibrium atmospheric pressure plasma to commonly used organic buffers (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES). In addition, a continuous low-dose O-2(center dot-)/ONOO- supply was shown to induce a physiologically relevant Ca2+ response and subsequently the uptake of a large cation mediated by transient receptor potential channel family member(s). Our results provide a novel approach to the continuous O-2(center dot-)/ONOO- supply, requiring controllable and mass-volume treatments.

  14. Development of optical resolution photoacoustic microscopy with sub-micron lateral resolution for visualization of cells and their structures 査読有り

    Ryo Shintate, Taisuke Morino, Kota Kawaguchi, Ryo Nagaoka, Kazuto Kobayashi, Makoto Kanzaki, Yoshifumi Saijo

    Japanese Journal of Applied Physics 59 2020年7月1日

    出版者・発行元:{IOP} Publishing

    DOI: 10.35848/1347-4065/ab840e  

    ISSN:0021-4922

    eISSN:1347-4065

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    © 2020 The Japan Society of Applied Physics. Photoacoustic (PA) imaging is a hybrid imaging technique with light-dependent contrast and ultrasound-dependent depth. In previous studies, the interest of PA imaging was mainly clinical research. However, in recent years, PA imaging that targets cells also has attracted attention. Cells are the smallest unit in our body, and cell abnormalities cause various diseases. Therefore, PA imaging of cells is important for elucidating the relationship between diseases and PA properties and linking the results to the clinical application of PA imaging. For PA imaging of cells and its internal structures, sub-micron lateral resolution is required. In this study, we have developed an optical resolution photoacoustic microscopy (OR-PAM) system using a high NA objective lens and a single-mode fiber to achieve sub-micron lateral resolution. With the OR-PAM, it was confirmed that the lateral resolution was better than 700 nm, and the characteristic shapes of red blood cells and melanoma cells could be visualized.

  15. TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca2+]i response. 国際誌 査読有り

    Masayoshi Kawase, Weijian Chen, Kota Kawaguchi, Mazvita R Nyasha, Shota Sasaki, Hiroyasu Hatakeyama, Toshiro Kaneko, Makoto Kanzaki

    Scientific reports 10 (1) 9687-9687 2020年6月16日

    DOI: 10.1038/s41598-020-66510-y  

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    Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca2+ ([Ca2+]i) concentration by eliciting Ca2+ influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca2+]i rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca2+ channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca2+]i elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca2+]i transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca2+]i responses.

  16. Characterization of middle-molecule introduction into cells using mm-scale discharge in saline 国際誌 査読有り

    Ryosuke Honda, Shota Sasaki, Keisuke Takashima, Makoto Kanzaki, Takehiko Sato, Toshiro Kaneko

    Japanese Journal of Applied Physics 59 (4) 2020年4月1日

    出版者・発行元:{IOP} Publishing

    DOI: 10.35848/1347-4065/ab7d7e  

    ISSN:0021-4922

    eISSN:1347-4065

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    While gas-phase atmospheric pressure plasma (APP) is a promising technology for highly-efficient and minimally-invasive gene/drug (molecule) introduction, the reduction of external gas supply and the expansion of the effective treatment area in biological fluids for in vivo treatment are still challenging. We developed a device for mm-scale plasma generation in saline without working gas supply and demonstrated the local introduction of YOYO-1 molecule (molecular weight : 1271) into cultured cells using the device. Unlike the conventional APP accounting for chemical stimuli with reactive species, plasma-induced mechanical stimulus was indicated as one of key factor(s) in the molecule introduction.

  17. LRRK2 Inhibition Ameliorates Dexamethasone-Induced Glucose Intolerance via Prevents Impairment in GLUT4 Membrane Translocation in Adipocytes.

    Motoki Imai, Fumitaka Kawakami, Makoto Kubo, Makoto Kanzaki, Hiroko Maruyama, Rei Kawashima, Tatsunori Maekawa, Yoshifumi Kurosaki, Fumiaki Kojima, Takafumi Ichikawa

    Biological & pharmaceutical bulletin 43 (11) 1660-1668 2020年

    DOI: 10.1248/bpb.b20-00377  

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    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson's disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5'-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.

  18. Exercise-evoked intramuscular neutrophil-endothelial interactions support muscle performance and GLUT4 translocation: a mouse gnawing model study. 国際誌 査読有り

    Chayanit Chaweewannakorn, Mazvita R Nyasha, Weijian Chen, Shigenori Sekiai, Masahiro Tsuchiya, Yoshihiro Hagiwara, Karim Bouzakri, Keiichi Sasaki, Makoto Kanzaki

    The Journal of physiology 598 (1) 101-122 2020年1月

    DOI: 10.1113/JP278564  

    ISSN:0022-3751

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    KEY POINTS: Fractalkine receptor antagonist inhibited neutrophil recruitment to masseter muscles and exacerbated fatigability during masticatory activity. Fractalkine-mediated neutrophil recruitment is required for both upregulation of myokines (CXCL1, interleukin-6) and enhanced GLUT4 translocation in response to masticatory activity. Fractalkine and intercellular adhesion molecule-1 expression in endothelial cells increased in response to masticatory activity. In vitro experiments demonstrated that contracting myotubes lack the ability to upregulate fractalkine but revealed that endothelial fractalkine upregulation is induced using a conditioned medium of contracting myotubes. ABSTRACT: Physical exercise stimulates neutrophil recruitment within working skeletal muscle, although its underlying mechanisms remain ill-defined. By employing a masticatory behaviour (gnawing) model, we demonstrate the importance of intramuscular paracrine and autocrine systems that are triggered by muscle contractile activity and reliant upon fractalkine/CX3CL1-mediated signals. These signals were revealed to be required for achieving proper GLUT4 translocation and glucose uptake to meet the glucose demands for fatigue alleviation. Specifically, fractalkine expression and neutrophil recruitment both increased in the masseter muscle tissues upon masticatory activity. Importantly, a fractalkine antagonist inhibited neutrophil accumulation and exacerbated fatigability during masticatory activity. We found that fractalkine-dependent neutrophil recruitment is required for both upregulation of myokines (i.e. CXCL1 and interleukin-6) and enhanced GLUT4 translocation in response to gnawing activity. Immunofluorescence analysis of masseter muscles demonstrated that fractalkine and intercellular adhesion molecule-1 expression are both upregulated in endothelial cells but not in myofibres. The in vitro exercise model further revealed that contractile activity failed to stimulate fractalkine upregulation in myotubes, implying that fractalkine is not a myokine (myofibre-derived factor). Nevertheless, endothelial fractalkine expression was markedly stimulated by a conditioned medium from the contracting myotubes. Moreover, intercellular adhesion molecule-1, a key adhesion molecule for neutrophils, was upregulated in endothelial cells by fractalkine. Taken together, our findings strongly suggest that endothelial fractalkine serves as a key factor for organizing a physiologically beneficial intramuscular microenvironment by recruiting neutrophils in response to relatively mild exercise (i.e. masticatory muscle activity).

  19. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis. 国際誌 査読有り

    Kobayashi J, Hasegawa T, Sugeno N, Yoshida S, Akiyama T, Fujimori K, Hatakeyama H, Miki Y, Tomiyama A, Kawata Y, Fukuda M, Kawahata I, Yamakuni T, Ezura M, Kikuchi A, Baba T, Takeda A, Kanzaki M, Wakabayashi K, Okano H, Aoki M

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 (9) 10240-10256 2019年9月

    DOI: 10.1096/fj.201802051R  

    ISSN:0892-6638

  20. In vitro exercise model using contractile human and mouse hybrid myotubes. 国際誌 査読有り

    Weijian Chen, Mazvita R Nyasha, Masashi Koide, Masahiro Tsuchiya, Naoki Suzuki, Yoshihiro Hagiwara, Masashi Aoki, Makoto Kanzaki

    Scientific reports 9 (1) 11914-11914 2019年8月15日

    DOI: 10.1038/s41598-019-48316-9  

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    Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses (i.e. myokine secretion). We herein describe an "in vitro exercise model", using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the "in vitro exercise model".

  21. Biomechanics of C2C12 Cells Observed with Cellular Resolution Scanning Acoustic Microscope Combined with Optical Microscope. 国際誌 査読有り

    Ryo Hirano, Makoto Kanzaki, Mototaka Arakawa, Norma Hermawan, Kazuto Kobayashi, Yoshifumi Saijo

    Conference proceedings : ... Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual Conference 2019 4828-4831 2019年7月

    DOI: 10.1109/EMBC.2019.8857008  

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    Biomechanics of the cell indicates the inner structure and viability of the cell. Mechanical properties are represented by acoustic properties such as speed of sound (SOS) or acoustic impedance. In the present study, cellular resolution scanning acoustic microscope combined with optical microscope (OptSAM) is developed to observe the change of mechanical properties in cell differentiation. Main part of the OptSAM was consisted of 350 MHz ultrasound transducer mechanically scanned by a piezo-actuator. Thickness, SOS, acoustic impedance, density and elastic bulk modulus of the cell were deduced by the ultrasound responses in both time domain and frequency domain. C2C12 cell changing its form from myoblast to myotube was observed by OptSAM. The value of bulk modulus slightly increased in response to differentiation process. OptSAM non-invasively provides important information on biomechanics of cells without contact or staining.

  22. Involvement of inflammasome activation via elevation of uric acid level in nociception in a mouse model of muscle pain 査読有り

    Yoshida, Shinichirou, Hagiwara, Yoshihiro, Tsuchiya, Masahiro, Shinoda, Masamichi, Koide, Masashi, Hatakeyama, Hiroyasu, Chaweewannakorn, Chayanit, Suzuki, Kazuaki, Yano, Toshihisa, Sogi, Yasuhito, Itaya, Nobuyuki, Sekiguchi, Takuya, Yabe, Yutaka, Sasaki, Keiichi, Kanzaki, Makoto, Itoi, Eiji

    MOLECULAR PAIN 15 1744806919858797 2019年7月

    出版者・発行元:SAGE PUBLICATIONS INC

    DOI: 10.1177/1744806919858797  

    ISSN:1744-8069

    eISSN:1744-8069

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    Muscle pain is a common condition in many diseases and is induced by muscle overuse. Muscle overuse induces an increase in uric acid, which stimulates the nucleotide-binding oligomerization domain-like receptor (NLR). This receptor contains the pyrin domain NLRP-3 inflammasome which when activated, results in the secretion of potent pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta). The aim of this study was to investigate the involvement of inflammasome activation via the elevation of uric acid level in nociception in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated as a control. Mechanical withdrawal thresholds, levels of uric acid, IL-1 beta, and NLRP3, caspase-1 activity, and the number of macrophages were investigated. Furthermore, the effects of xanthine oxidase inhibitors, such as Brilliant Blue G, caspase-1 inhibitor, and clodronate liposome, on pain were investigated. In the stimulated muscles, mechanical withdrawal thresholds decreased, and the levels of uric acid, NLRP3, and IL-1 beta, caspase-1 activity, and the number of macrophages increased compared to that in the non-stimulated muscles. Administration of the inhibitors attenuated hyperalgesia caused by excessive muscle contraction. These results suggested that IL-1 beta secretion and NLRP3 inflammasome activation in macrophages produced mechanical hyperalgesia by elevating uric acid level, and xanthine oxidase inhibitors may potentially reduce over-exercised muscle pain.

  23. Sparc, an EPS-induced gene, modulates the extracellular matrix and mitochondrial function via ILK/AMPK pathways in C2C12 cells 査読有り

    Aicha Melouane, Mayumi Yoshioka, Makoto Kanzaki, Jonny St-Amand

    Life Sciences 229 277 2019年7月

    出版者・発行元:Elsevier {BV}

    DOI: 10.1016/j.lfs.2019.05.070  

  24. Cooperative actions of Tbc1d1 and AS160/Tbc1d4 in GLUT4-trafficking activities 査読有り

    Hiroyasu Hatakeyama, Taisuke Morino, Takuya Ishii, Makoto Kanzaki

    Journal of Biological Chemistry jbc.RA118.004614 2019年1月

    出版者・発行元:American Society for Biochemistry {\&} Molecular Biology ({ASBMB})

    DOI: 10.1074/jbc.RA118.004614  

  25. Olfactory receptors are expressed in pancreatic β-cells and promote glucose-stimulated insulin secretion. 査読有り

    Munakata Y, Yamada T, Imai J, Takahashi K, Tsukita S, Shirai Y, Kodama S, Asai Y, Sugisawa T, Chiba Y, Kaneko K, Uno K, Sawada S, Hatakeyama H, Kanzaki M, Miyazaki JI, Oka Y, Katagiri H

    Scientific reports 8 (1) 1499 2018年12月1日

    出版者・発行元:None

    DOI: 10.1038/s41598-018-19765-5  

    ISSN:2045-2322

  26. Contractile Skeletal Muscle Cells Cultured with a Conducting Soft Wire for Effective, Selective Stimulation 査読有り

    Kuniaki Nagamine, Hirotaka Sato, Hiroyuki Kai, Hirokazu Kaji, Makoto Kanzaki, Matsuhiko Nishizawa

    Scientific Reports 8 (1) 2253 2018年12月1日

    出版者・発行元:Nature Publishing Group

    DOI: 10.1038/s41598-018-20729-y  

    ISSN:2045-2322

    eISSN:2045-2322

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    Contractile skeletal muscle cells were cultured so as to wrap around an electrode wire to enable their selective stimulation even when they were co-cultured with other electrically-excitable cells. Since the electrode wire was composed of the conducting polymer poly(3,4-ethylenedioxythiophene) (PEDOT) and polyurethane (PU), which is soft and highly capacitive (∼10 mF cm-2), non-faradaic electrical stimulation with charge/discharge currents could be applied to the surrounding cells without causing significant damage even for longer periods (more than a week). The advantage of this new culture system was demonstrated in the study of chemotactic interaction of monocytes and skeletal muscle cells via myokines.

  27. Effects of Acute Exercise Combined with Calorie Restriction Initiated Late-in-Life on Insulin Signaling, Lipids and Glucose Uptake in Skeletal Muscle from Old Rats. 査読有り

    Oki K, Arias EB, Kanzaki M, Cartee GD

    The journals of gerontology. Series A, Biological sciences and medical sciences 2018年10月

    DOI: 10.1093/gerona/gly222  

    ISSN:1079-5006

  28. Prior treatment with the AMPK activator AICAR induces subsequently enhanced glucose uptake in isolated skeletal muscles from 24-month-old rats. 査読有り

    Oki K, Arias EB, Kanzaki M, Cartee GD

    Applied physiology, nutrition, and metabolism = Physiologie appliquee, nutrition et metabolisme 43 (8) 795-805 2018年8月

    DOI: 10.1139/apnm-2017-0858  

    ISSN:1715-5312

  29. Roles of IL-1α/β in regeneration of cardiotoxin-injured muscle and satellite cell function 査読有り

    Chayanit Chaweewannakorn, Masahiro Tsuchiya, Masashi Koide, Hiroyasu Hatakeyama, Yukinori Tanaka, Shinichirou Yoshida, Shunji Sugawara, Yoshihiro Hagiwara, Keiichi Sasaki, Makoto Kanzaki

    American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 315 (1) R90-R103 2018年7月

    出版者・発行元:American Physiological Society

    DOI: 10.1152/ajpregu.00310.2017  

    ISSN:0363-6119

    eISSN:1522-1490

  30. Neutrophils Provide a Favorable IL-1-Mediated Immunometabolic Niche that Primes GLUT4 Translocation and Performance in Skeletal Muscles. 国際誌 査読有り

    Masahiro Tsuchiya, Shigenori Sekiai, Hiroyasu Hatakeyama, Masashi Koide, Chayanit Chaweewannakorn, Fukie Yaoita, Koichi Tan-No, Keiichi Sasaki, Makoto Watanabe, Shunji Sugawara, Yasuo Endo, Eiji Itoi, Yoshihiro Hagiwara, Makoto Kanzaki

    Cell reports 23 (8) 2354-2364 2018年5月22日

    DOI: 10.1016/j.celrep.2018.04.067  

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    Metabolic immunomodulation involving IL-1 has been investigated for unfavorable metabolic effects, including obesity, but a potentially favorable role for IL-1 remains unclear. Here, we find mechanistic interactions between working skeletal muscles and locally recruited neutrophils expressing IL-1β, which supports muscle performance through priming exercise-dependent GLUT4 translocation. Thus, during exercise, both IL-1α/β-deficient and neutrophil-depleted mice similarly exhibit increased fatigability associated with impaired muscle glucose homeostasis due to GLUT4 dysregulation. Deficiency of IL-1-producing neutrophils results in intrinsic abnormalities represented by aberrant Rac1 signaling and irregular GLUT4-storage vesicles, suggesting that these properties are maintained by local IL-1 produced by recruited neutrophils upon exercise, possibly on a daily basis. We propose that neutrophils are highly engaged in skeletal muscle performance via IL-1 regulation, which coordinates favorable inflammatory microenvironments supporting muscle glucose metabolism.

  31. Involvement of neutrophils and interleukin-18 in nociception in a mouse model of muscle pain 査読有り

    Yoshida, Shinichirou, Hagiwara, Yoshihiro, Tsuchiya, Masahiro, Shinoda, Masamichi, Koide, Masashi, Hatakeyama, Hiroyasu, Chaweewannakorn, Chayanit, Yano, Toshihisa, Sogi, Yasuhito, Itaya, Nobuyuki, Sekiguchi, Takuya, Yabe, Yutaka, Sasaki, Keiichi, Kanzaki, Makoto, Itoi, Eiji

    MOLECULAR PAIN 14 1744806918757286 2018年2月

    出版者・発行元:SAGE PUBLICATIONS INC

    DOI: 10.1177/1744806918757286  

    ISSN:1744-8069

    詳細を見る 詳細を閉じる

    Muscle pain is a common condition that relates to various pathologies. Muscle overuse induces muscle pain, and neutrophils are key players in pain production. Neutrophils also play a central role in chronic pain by secreting interleukin (IL)-18. The aim of this study was to investigate the involvement of neutrophils and IL-18 in a mouse model of muscle pain. The right hind leg muscles of BALB/c mice were stimulated electrically to induce excessive muscle contraction. The left hind leg muscles were not stimulated. The pressure pain threshold, number of neutrophils, and IL-18 levels were investigated. Furthermore, the effects of the IL-18-binding protein and Brilliant Blue G on pain were investigated. In stimulated muscles, pressure pain thresholds decreased, and neutrophil and IL-18 levels increased compared with that in non-stimulated muscles. The administration of IL-18-binding protein and Brilliant Blue G attenuated hyperalgesia caused by excessive muscle contraction. These results suggest that increased IL-18 secretion from larger numbers of neutrophils elicits mechanical hyperalgesia.

  32. Direct plasma stimuli including electrostimulation and OH radical induce transient increase in intracellular Ca2+ and uptake of a middle-size membrane-impermeable molecule 査読有り

    Shota Sasaki, Yutaro Hokari, Akiko Kumada, Makoto Kanzaki, Toshiro Kaneko

    Plasma Processes and Polymers 15 (1) e1700077-1-9 2018年1月1日

    出版者・発行元:Wiley-VCH Verlag

    DOI: 10.1002/ppap.201700077  

    ISSN:1612-8869 1612-8850

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    Despite the promising potential of medical treatments based on non-equilibrium atmospheric-pressure plasma, most of the underlying mechanisms remain unknown. Here we report that direct plasma irradiation (DPI) can induce transient increase in intracellular calcium (Ca2+) concentrations, thereby playing key roles in many cellular processes as well as the uptake of middle-size membrane-impermeable molecules, which did not occur with indirect plasma irradiation (IPI). Experimental measurements using a Pockels voltage probe and gelling chemical reagent showed that electric field of 150 kV/cm and hydroxyl radical (OH) supply to cell cultures were induced by DPI under a typical condition compared with IPI. In addition, significant inhibition of the OH scavenger D-mannitol indicated involvement of OH in those DPI-induced cellular responses. These results facilitate the elucidation of plasma-cell interaction and the development of plasma devices for medical treatments.

  33. Retained Myogenic Potency of Human Satellite Cells from Torn Rotator Cuff Muscles Despite Fatty Infiltration. 査読有り

    Masashi Koide, Yoshihiro Hagiwara, Masahiro Tsuchiya, Makoto Kanzaki, Hiroyasu Hatakeyama, Yukinori Tanaka, Takashi Minowa, Taro Takemura, Akira Ando, Takuya Sekiguchi, Yutaka Yabe, Eiji Itoi

    The Tohoku journal of experimental medicine 244 (1) 15-24 2018年1月

    DOI: 10.1620/tjem.244.15  

    ISSN:0040-8727

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    Rotator cuff tears (RCTs) are a common shoulder problem in the elderly that can lead to both muscle atrophy and fatty infiltration due to less physical load. Satellite cells, quiescent cells under the basal lamina of skeletal muscle fibers, play a major role in muscle regeneration. However, the myogenic potency of human satellite cells in muscles with fatty infiltration is unclear due to the difficulty in isolating from small samples, and the mechanism of the progression of fatty infiltration has not been elucidated. The purpose of this study was to analyze the population of myogenic and adipogenic cells in disused supraspinatus (SSP) and intact subscapularis (SSC) muscles of the RCTs from the same patients using fluorescence-activated cell sorting. The microstructure of the muscle with fatty infiltration was observed as a whole mount condition under multi-photon microscopy. Myogenic differentiation potential and gene expression were evaluated in satellite cells. The results showed that the SSP muscle with greater fatty infiltration surrounded by collagen fibers compared with the SSC muscle under multi-photon microscopy. A positive correlation was observed between the ratio of muscle volume to fat volume and the ratio of myogenic precursor to adipogenic precursor. Although no difference was observed in the myogenic potential between the two groups in cell culture, satellite cells in the disused SSP muscle showed higher intrinsic myogenic gene expression than those in the intact SSC muscle. Our results indicate that satellite cells from the disused SSP retain sufficient potential of muscle growth despite the fatty infiltration.

  34. Three-Dimensional Tracking of Quantum Dot-Conjugated Molecules in Living Cells. 査読有り

    Gardini L, Calamai M, Hatakeyama H, Kanzaki M, Capitanio M, Pavone FS

    Methods in molecular biology (Clifton, N.J.) 1814 425-448 2018年1月

    DOI: 10.1007/978-1-4939-8591-3_26  

    ISSN:1064-3745

  35. Neuronal signals regulate obesity induced β-cell proliferation by FoxM1 dependent mechanism. 査読有り

    Yamamoto J, Imai J, Izumi T, Takahashi H, Kawana Y, Takahashi K, Kodama S, Kaneko K, Gao J, Uno K, Sawada S, Asano T, Kalinichenko VV, Susaki EA, Kanzaki M, Ueda HR, Ishigaki Y, Yamada T, Katagiri H

    Nature communications 8 (1) 1930 2017年12月

    出版者・発行元:None

    DOI: 10.1038/s41467-017-01869-7  

    ISSN:2041-1723

  36. 3D electrochemical and ion current imaging using scanning electrochemical–scanning ion conductance microscopy 査読有り

    Yasufumi Takahashi, Hiroki Ida, Yoshiharu Matsumae, Hirokazu Komaki, Yuanshu Zhou, Akichika Kumatani, Makoto Kanzaki, Hitoshi Shiku, Tomokazu Matsue

    Phys. Chem. Chem. Phys. 19 (39) 26728-26733 2017年10月

    出版者・発行元:Royal Society of Chemistry ({RSC})

    DOI: 10.1039/c7cp05157c  

    ISSN:1463-9076

    eISSN:1463-9084

  37. Heterotypic endosomal fusion as an initial trigger for insulin-induced glucose transporter 4 (GLUT4) translocation in skeletal muscle 査読有り

    Hiroyasu Hatakeyama, Makoto Kanzaki

    JOURNAL OF PHYSIOLOGY-LONDON 595 (16) 5603-5621 2017年8月

    出版者・発行元:WILEY

    DOI: 10.1113/JP273985  

    ISSN:0022-3751

    eISSN:1469-7793

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    Skeletal muscle is the major systemic glucose disposal site. Both insulin and exercise facilitate translocation of the glucose transporter glucose transporter 4 (GLUT4) via distinct signalling pathways and exercise also enhances insulin sensitivity. However, the trafficking mechanisms controlling GLUT4 mobilization in skeletal muscle remain poorly understood as a resuly of technical limitations. In the present study, which employs various imaging techniques on isolated skeletalmyofibres, we showthat one of the initial triggers of insulin-inducedGLUT4translocation is heterotypic endomembrane fusion arising from very small static GLUT4-containing vesicles with a subset of transferrin receptor-containing endosomes. Importantly, pretreatment with exercise-mimetic stimuli potentiated the susceptibility to insulin responsiveness, as indicated by these acute endomembranous activities. We also found that AS160 exhibited stripe-like localization close to sarcomeric a-actinin and that insulin induced a reduction of the stripe-like localization accompanying changes in its detergent solubility. The results of the present study thus provide a conceptual framework indicating that GLUT4 protein trafficking via heterotypic fusion is a critical feature of GLUT4 translocation in skeletal muscles and also suggest that the efficacy of the endomembranous fusion process in response to insulin is involved in the benefits of exercise.

  38. Live-cell single-molecule labeling and analysis of myosin motors with quantum dots 査読有り

    Hiroyasu Hatakeyama, Yoshihito Nakahata, Hirokazu Yarimizu, Makoto Kanzaki

    MOLECULAR BIOLOGY OF THE CELL 28 (1) 173-181 2017年1月

    出版者・発行元:AMER SOC CELL BIOLOGY

    DOI: 10.1091/mbc.E16-06-0413  

    ISSN:1059-1524

    eISSN:1939-4586

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    Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.

  39. Gas-liquid interfacial plasmas producing reactive species for cell membrane permeabilization 査読有り

    Toshiro Kaneko, Shota Sasaki, Keisuke Takashima, Makoto Kanzaki

    JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 60 (1) 3-11 2017年1月

    出版者・発行元:JOURNAL CLINICAL BIOCHEMISTRY & NUTRITION

    DOI: 10.3164/jcbn.16-73  

    ISSN:0912-0009

    eISSN:1880-5086

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    Gas-liquid interfacial atmospheric-pressure plasma jets (GLI-APPJ) are used medically for plasma-induced cell-membrane permeabilization. In an attempt to identify the dominant factors induced by GLI-APPJ responsible for enhancing cell-membrane permeability, the concentration and distribution of plasma-produced reactive species in the gas and liquid phase regions are measured. These reactive species are classified in terms of their life-span: long-lived (e.g., H2O2), short-lived (e.g., O-2(center dot-)), and extremely-short-lived (e.g., (OH)-O-center dot). The concentration of plasma-produced (OHaq)-O-center dot, in the liquid phase region decreases with an increase in solution thickness (&lt;1 mm), and plasma-induced cell-membrane permeabilization is found to decay markedly as the thickness of the solution increases. Furthermore, the horizontally center-localized distribution of (OHaq)-O-center dot, resulting from the center-peaked distribution of (OH)-O-center dot in the gas phase region, corresponds with the distribution of the permeabilized cells upon APPJ irradiation, whereas the overall plasma produced oxidizing species such as H2O2aq, in solution exhibit a doughnut-shaped horizontal distribution. These results suggest that (OHaq)-O-center dot, is likely one of the dominant factors responsible for plasma-induced cell-membrane permeabilization.

  40. Characterization of plasma-induced cell membrane permeabilization: focus on OH radical distribution 査読有り

    Shota Sasaki, Ryosuke Honda, Yutaro Hokari, Keisuke Takashima, Makoto Kanzaki, Toshiro Kaneko

    JOURNAL OF PHYSICS D-APPLIED PHYSICS 49 (33) 2016年8月

    出版者・発行元:IOP PUBLISHING LTD

    DOI: 10.1088/0022-3727/49/33/334002  

    ISSN:0022-3727

    eISSN:1361-6463

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    Non-equilibrium atmospheric-pressure plasma (APP) is used medically for plasma-induced cell permeabilization. However, how plasma irradiation specifically triggers permeabilization remains unclear. In an attempt to identify the dominant factor(s), the distribution of plasma-produced reactive species was investigated, primarily focusing on OH radicals. A stronger plasma discharge, which produced more OH radicals in the gas phase, also produced more OH radicals in the liquid phase (OHaq), enhancing the cell membrane permeability. In addition, plasma irradiation-induced enhancement of cell membrane permeability decreased markedly with increased solution thickness (&lt;1 mm), and the plasma-produced OHaq decayed in solution (diffusion length on the order of several hundred micrometers). Furthermore, the horizontally center-localized distribution of OHaq corresponded with the distribution of the permeabilized cells by plasma irradiation, while the overall plasma-produced oxidizing species in solution (detected by iodine-starch reaction) exhibited a doughnut-shaped horizontal distribution. These results suggest that OHaq, among the plasma-produced oxidizing species, represents the dominant factor in plasma-induced cell permeabilization. These results enhance the current understanding of the mechanism of APP as a cell-permeabilization tool.

  41. Roles of charged particles and reactive species on cell membrane permeabilization induced by atmospheric-pressure plasma irradiation 査読有り

    Shota Sasaki, Makoto Kanzaki, Yutaro Hokari, Kanako Tominami, Takayuki Mokudai, Hiroyasu Kanetaka, Toshiro Kaneko

    JAPANESE JOURNAL OF APPLIED PHYSICS 55 (7) 2016年7月

    出版者・発行元:IOP PUBLISHING LTD

    DOI: 10.7567/JJAP.55.07LG04  

    ISSN:0021-4922

    eISSN:1347-4065

    詳細を見る 詳細を閉じる

    As factors that influence cell membrane permeabilization during direct and indirect atmospheric-pressure plasma irradiation, charged particle influx, superoxide anion radicals (O-2(-)center dot), and hydrogen peroxide (H2O2) in plasma-irradiated solution were evaluated. These are the three strong candidate factors and might multiply contribute to cell membrane permeabilization. In particular, a shorter plasma diffusion distance leads to the enhancement of the direct effects such as charged particle influx and further increase cell membrane permeability. In addition, O-2(-)center dot dissipates over time (a life span of the order of minutes) in plasma-irradiated water, and the deactivation of a plasma-irradiated solution in term of cell membrane permeabilization occurs in a life span of the same order. These results could promote the understanding of the mechanism of plasma-induced cell membrane permeabilization. (C) 2016 The Japan Society of Applied Physics

  42. The proneurotrophin receptor sortilin is required for Mycobacterium tuberculosis control by macrophages 査読有り

    Cristina L. Vazquez, Angela Rodgers, Susanne Herbst, Stephen Coade, Achim Gronow, Carlos A. Guzman, Mark S. Wilson, Makoto Kanzaki, Anders Nykjaer, Maximiliano G. Gutierrez

    SCIENTIFIC REPORTS 6 (29332) 2016年7月

    出版者・発行元:NATURE PUBLISHING GROUP

    DOI: 10.1038/srep29332  

    ISSN:2045-2322

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    Sorting of luminal and membrane proteins into phagosomes is critical for the immune function of this organelle. However, little is known about the mechanisms that contribute to the spatiotemporal regulation of this process. Here, we investigated the role of the proneurotrophin receptor sortilin during phagosome maturation and mycobacterial killing. We show that this receptor is acquired by mycobacteria-containing phagosomes via interactions with the adaptor proteins AP-1 and GGAs. Interestingly, the phagosomal association of sortilin is critical for the delivery of acid sphingomyelinase (ASMase) and required for efficient phagosome maturation. Macrophages from Sort1(-/-) mice are less efficient in restricting the growth of Mycobacterium bovis BCG and M. tuberculosis. In vivo, Sort1(-/-) mice showed a substantial increase in cellular infiltration of neutrophils in their lungs and higher bacterial burden after infection with M. tuberculosis. Altogether, sortilin defines a pathway required for optimal intracellular mycobacteria control and lung inflammation in vivo.

  43. Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution. 査読有り

    Sasaki S, Kanzaki M, Kaneko T

    Sci Rep. 6 (25728) 2016年5月12日

    DOI: 10.1038/srep25728.  

  44. Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution 査読有り

    Shota Sasaki, Makoto Kanzaki, Toshiro Kaneko

    SCIENTIFIC REPORTS 6 2016年5月

    出版者・発行元:NATURE PUBLISHING GROUP

    DOI: 10.1038/srep25728  

    ISSN:2045-2322

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    Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O-2 and N-2 in ambient air, uniquely produces multiple extremely reactive products, such as reactive oxygen species (ROS), in plasma-irradiated solution. We herein show that relatively short-lived unclassified reactive species (i.e., deactivated within approximately 10 min) generated by the He-APP irradiation can trigger physiologically relevant Ca2+ influx through ruthenium red-and SKF 96365-sensitive Ca2+-permeable channel(s), possibly transient receptor potential channel family member(s). Our results provide novel insight into understanding of the interactions between cells and plasmas and the mechanism by which cells detect plasma-induced chemically reactive species, in addition to facilitating development of plasma applications in medicine.

  45. PLASMA-IRRADIATED SOLUTION AS DRUG PERMEATION ENHANCER 査読有り

    Toshiro Kaneko, Kei Kikuchi, Shota Sasaki, Makoto Kanzaki

    2016 43RD IEEE INTERNATIONAL CONFERENCE ON PLASMA SCIENCE (ICOPS) 2016年

    出版者・発行元:IEEE

    DOI: 10.1109/PLASMA.2016.7534290   10.1109/plasma.2016.7534290  

  46. ATMOSPHERIC-PRESSURE PLASMA-INDUCED CELLULAR RESPONSES IN HUMAN COLORECTAL ADENOCARCINOMA CACO-2 CELLS: A STUDY OF COMPREHENSIVE QUANTITATIVE PROTEOMICS 査読有り

    Masanori Tachikawa, Daichi Sano, Shota Sasaki, Makoto Kanzaki, Tetsuya Terasaki, Toshiro Kaneko

    2016 43RD IEEE INTERNATIONAL CONFERENCE ON PLASMA SCIENCE (ICOPS) 2016年

    出版者・発行元:IEEE

    DOI: 10.1109/PLASMA.2016.7534130  

  47. Involvement of IL-1 in the Maintenance of Masseter Muscle Activity and Glucose Homeostasis 査読有り

    Ko Chiba, Masahiro Tsuchiya, Masashi Koide, Yoshihiro Hagiwara, Keiichi Sasaki, Yoshinori Hattori, Makoto Watanabe, Shunji Sugawara, Makoto Kanzaki, Yasuo Endo

    PLOS ONE 10 (11) e0143635 2015年11月

    出版者・発行元:PUBLIC LIBRARY SCIENCE

    DOI: 10.1371/journal.pone.0143635  

    ISSN:1932-6203

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    Physical exercise reportedly stimulates IL-1 production within working skeletal muscles, but its physiological significance remains unknown due to the existence of two distinct IL-1 isoforms, IL-1 alpha and IL-1 beta. The regulatory complexities of these two isoforms, in terms of which cells in muscles produce them and their distinct/redundant biological actions, have yet to be elucidated. Taking advantage of our masticatory behavior (Restrained/Gnawing) model, we herein show that IL-1 alpha/1 beta-double-knockout (IL-1-KO) mice exhibit compromised masseter muscle (MM) activity which is at least partially attributable to abnormalities of glucose handling (rapid glycogen depletion along with impaired glucose uptake) and dysfunction of IL-6 upregulation in working MMs. In wild-type mice, masticatory behavior clearly increased IL-1 beta mRNA expression but no incremental protein abundance was detectable in whole MM homogenates, whereas immunohistochemical staining analysis revealed that both IL-1 alpha- and IL-1 beta-immunopositive cells were recruited around blood vessels in the perimysium of MMs after masticatory behavior. In addition to the aforementioned phenotype of IL-1-KO mice, we found the IL-6 mRNA and protein levels in MMs after masticatory behavior to be significantly lower in IL-1-KO than in WT. Thus, our findings confirm that the locally-increased IL-1 elicited by masticatory behavior, although present small in amounts, contributes to supporting MM activity by maintaining normal glucose homeostasis in these muscles. Our data also underscore the importance of IL-1-mediated local interplay between autocrine myokines including IL-6 and paracrine cytokines in active skeletal muscles. This interplay is directly involved in MM performance and fatigability, perhaps mediated through maintaining muscular glucose homeostasis.

  48. Improvement of cell membrane permeability using a cell-solution electrode for generating atmospheric-pressure plasma. 査読有り

    Kaneko T, Sasaki S, Hokari Y, Horiuchi S, Honda R, Kanzaki M

    Biointerphases 10 (2) 029521 2015年7月21日

    DOI: 10.1116/1.4921278.  

  49. Hydrogel-based portable electrical stimulation culture film for skeletal muscle cells 査読有り

    Nagamine K, Hirata T, Kaji H, Kanzaki M, Nishizawa M

    MicroTAS 2015 - 19th International Conference on Miniaturized Systems for Chemistry and Life Sciences 627-629 2015年

  50. Insulin-Mimicking Bioactivities of Acylated Inositol Glycans in Several Mouse Models of Diabetes with or without Obesity 査読有り

    Susumu Suzuki, Chitose Suzuki, Yoshinori Hinokio, Yasushi Ishigaki, Hideki Katagiri, Makoto Kanzaki, Viatcheslav N. Azev, Nilanjana Chakraborty, Marc d'Alarcao

    PLOS ONE 9 (6) e100466 2014年6月

    出版者・発行元:PUBLIC LIBRARY SCIENCE

    DOI: 10.1371/journal.pone.0100466  

    ISSN:1932-6203

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    Insulin-mimetic species of low molecular weight are speculated to mediate some intracellular insulin actions. These inositol glycans, which are generated upon insulin stimulation from glycosylphosphatidylinositols, might control the activity of a multitude of insulin effector enzymes. Acylated inositol glycans (AIGs) are generated by cleavage of protein-free GPI precursors through the action of GPI-specific phospholipase C (GPI-PLC) and D (GPI-PLD). We synthesized AIGs (IG-1, IG-2, IG-13, IG-14, and IG-15) and then evaluated their insulin-mimicking bioactivities. IG-1 significantly stimulated glycogen synthesis and lipogenesis in 3T3-L1 adipocytes and rat isolated adipocytes dose-dependently. IG-2 significantly stimulated lipogenesis in rat isolated adipocytes dose-dependently. IG-15 also enhanced glycogen synthesis and lipogenesis in 3T3-L1 adipocytes. The administration of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with normal diets. The administration of IG-1 decreased plasma glucose in STZ-diabetic C57B6N mice. The treatment of IG-1 decreased plasma glucose, increased glycogen content in liver and skeletal muscles and improved glucose tolerance in C57B6N mice with high fat-diets and db/db mice. The long-term treatment of IG-1 decreased plasma glucose and reduced food intake and body weight in C57B6N mice with high fat-diets and ob/ob mice. Thus, IG-1 has insulin-mimicking bioactivities and improves glucose tolerance in mice models of diabetes with or without obesity.

  51. Effects of Plasma Irradiation Energy at Atmospheric Pressure on Gene Transfection Efficiency and Cell Viability 査読有り

    S. Sasaki, M. Kanzaki, T. Kaneko

    Proceedings of the 8th International Conference on Reactive Plasmas 31st Symposium on Plasma Processing (ICRP-8/SPP-31) (CD-ROM) 4A-PM-O6 2014年2月3日

  52. Highly efficient and minimally invasive transfection using time-controlled irradiation of atmospheric-pressure plasma 査読有り

    Shota Sasaki, Makoto Kanzaki, Toshiro Kaneko

    APPLIED PHYSICS EXPRESS 7 (2) 026202 2014年2月

    出版者・発行元:IOP PUBLISHING LTD

    DOI: 10.7567/APEX.7.026202  

    ISSN:1882-0778

    eISSN:1882-0786

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    To develop a highly efficient and minimally invasive gene transfection method, the cells after direct plasma irradiation under various conditions are evaluated by simultaneous analysis of transfection efficiency and cell viability. As a result, the efficiency has a maximal value at a short plasma irradiation time (3-5 s) while maintaining a very high cell viability, and the volume of irradiated cell suspension changes the time dependence of the efficiency, which could be caused by the competition between the synergetic effects of reactive oxygen species and electric field stimulation, and membrane transport such as exocytosis which is the process of excretion. (C) 2014 The Japan Society of Applied Physics

  53. Hydrogel film with skeletal muscle cell micropatterns to develop the soft fluidic tube of the perfusion culture system 査読有り

    Nagamine K, Okamoto K, Hirata T, Kaji H, Kanzaki M, Nishizawa M

    18th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2014 518-520 2014年

  54. Hydrogel-based bioassay sheets for in vitro evaluation of contraction-dependent metabolic regulation in skeletal muscle cells 査読有り

    Kuniaki Nagamine, Kohei Okamoto, Shingo Otani, Hirokazu Kaji, Makoto Kanzaki, Matsuhiko Nishizawa

    BIOMATERIALS SCIENCE 2 (2) 252-256 2014年

    出版者・発行元:ROYAL SOC CHEMISTRY

    DOI: 10.1039/c3bm60179j  

    ISSN:2047-4830

    eISSN:2047-4849

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    Two types of hydrogel-based bioassay sheets were developed for in vitro evaluation of contraction-dependent metabolic regulation in skeletal muscle cells: one is an oxygen sensor sheet and the other is an immunocapture sheet for a myokine, interleukin-6 (IL-6). These soft, molecularly permeable hydrogel-based bioassay sheets were directly laminated to another hydrogel on which myotubes were micro-patterned, and displayed usefully measurable changes in local oxygen consumption and IL-6 secretion of myotubes upon electrically-induced contraction.

  55. Expression, phosphorylation and function of the Rab-GTPase activating protein TBC1D1 in pancreatic beta-cells 査読有り

    Sabine Ruetti, Caroline Arous, Alexandra C. Nica, Makoto Kanzaki, Philippe A. Halban, Karim Bouzakri

    FEBS LETTERS 588 (1) 15-20 2014年1月

    出版者・発行元:ELSEVIER SCIENCE BV

    DOI: 10.1016/j.febslet.2013.10.050  

    ISSN:0014-5793

    eISSN:1873-3468

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    The Rab-GTPase activating protein TBC1D1 is a paralog of AS160/TBC1D4. AS160/TBC1D4, a downstream effector of Akt, has been shown to play a central role in beta-cell function and survival. The two proteins have overlapping function in insulin signalling in muscle cells. However, the expression and the potential role of TBC1D1 in beta-cells remain unknown. Therefore, the aim of this study is to investigate whether TBC1D1 is expressed in beta-cells and whether it plays, as AS160/TBC1D4, a role in beta-cell function and survival. Using human and rat beta-cells, this study shows for the first time that TBC1D1 is expressed and phosphorylated in response to glucose in these cells. Knockdown of TBC1D1 in beta-cells resulted in increased basal and glucose-stimulated insulin release, decreased proliferation but no change in apoptosis. Structured summary of protein interactions: TBC1D1, glucagon and insulin colocalize by fluorescence microscopy (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

  56. Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis 査読有り

    Yasuhiro Suzuki, Chandra Nath Roy, Warunya Promjunyakul, Hiroyasu Hatakeyama, Kohsuke Gonda, Junji Imamura, Biju Vasudevanpillai, Noriaki Ohuchi, Makoto Kanzaki, Hideo Higuchi, Mitsuo Kaku

    MOLECULAR AND CELLULAR BIOLOGY 33 (15) 3036-3049 2013年8月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.01717-12  

    ISSN:0270-7306

    eISSN:1098-5549

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    The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

  57. Regulatory mode shift of Tbc1d1 is required for acquisition of insulin-responsive GLUT4-trafficking activity 査読有り

    Hiroyasu Hatakeyama, Makoto Kanzaki

    Molecular Biology of the Cell 24 (6) 809-817 2013年3月15日

    DOI: 10.1091/mbc.E12-10-0725  

    ISSN:1059-1524 1939-4586

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    Tbc1d1 is key to skeletal muscle GLUT4 regulation. By using GLUT4 nanometry combined with a cell-based reconstitution model, we uncover a shift in the regulatory mode of Tbc1d1 by showing that Tbc1d1 temporally acquires insulin responsiveness, which triggers GLUT4 trafficking only after an exercise-mimetic stimulus such as aminoimidazole carboxamide ribonucleotide (AICAR) pretreatment. The functional acquisition of insulin responsiveness requires Ser-237 phosphorylation and an intact phosphotyrosine-binding (PTB) 1 domain. Mutations in PTB1, including R125W (a natural mutant), thus result in complete loss of insulinresponsiveness acquisition, whereas AICAR-responsive GLUT4-liberation activity remains intact. Thus our data provide novel insights into temporal acquisition/memorization of Tbc1d1 insulin responsiveness, relying on the PTB1 domain, possibly a key factor in the beneficial effects of exercise on muscle insulin potency. © 2013 Hatakeyama and Kanzaki.

  58. Characterization of contraction-induced IL-6 up-regulation using contractile C2C12 myotubes 査読有り

    Arta Farmawati, Yasuo Kitajima, Taku Nedachi, Masaaki Sato, Makoto Kanzaki, Ryoichi Nagatomi

    ENDOCRINE JOURNAL 60 (2) 137-147 2013年2月

    出版者・発行元:JAPAN ENDOCRINE SOC

    DOI: 10.1507/endocrj.EJ12-0316  

    ISSN:0918-8959

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    Muscle contractile activity functions as a potent stimulus for acute interleukin (IL)-6 expression in working skeletal muscles. Recently, we established an "in vitro contraction model" using highly-developed contractile C2C12 myotubes by applying electric pulse stimulation (EPS). Herein, we characterize the effects of PS-evoked contraction on IL-6 expression in contractile C2C12 myotubes. Both secretion and mRNA expression of IL-6 were significantly up-regulated by EPS in a frequency-dependent manner in contracting myotubes during a 24-h period, and the response was blunted by cyclosporine A, a calcineurin inhibitor. Longer time (similar to 12h) was required for the induction of IL-6 after the initiation of EPS as compared to that of other contraction-inducible CXC chemokines such as CXCL1/KC, which were induced in less than 3 hours. Furthermore, these acute inducible CXC chemokines exhibited no autocrine effect on IL-6 expression. Importantly, contraction-dependent IL-6 up-regulation was markedly suppressed in the presence of high levels of glucose along with increased glycogen accumulations. Experimental manipulation of intracellular glycogen contents by modulating available glucose or pyruvate during a certain EPS period further established the suppressive effect of glycogen accumulations on contraction-induced IL-6 up-regulation, which appeared to be independent of calcineurin activity. We also document that EPS-evoked contractile activity improved insulin-responsiveness in terms of intracellular glycogen accumulations. Taken together, these data provide important insights into the regulation of IL-6 expression in response to contractile activity of muscle cells, which is difficult to examine using in vivo experimental techniques. Our present results thus expand the usefulness of our "in vitro contraction model".

  59. Hydrogel-based imaging sensor for the assay of exercise-dependent metabolic regulation in skeletal muscle cells 査読有り

    Nagamine K, Okamoto K, Kaji H, Kanzaki M, Nishizawa M

    17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013 3 1505-1507 2013年

  60. Development of Dual-Color Simultaneous Single Molecule Imaging System for Analyzing Multiple Intracellular Trafficking Activities 査読有り

    Hiroyasu Hatakeyama, Makoto Kanzaki

    2013 35TH ANNUAL INTERNATIONAL CONFERENCE OF THE IEEE ENGINEERING IN MEDICINE AND BIOLOGY SOCIETY (EMBC) 2013 1418-1421 2013年

    出版者・発行元:IEEE

    DOI: 10.1109/EMBC.2013.6609776  

    ISSN:1557-170X

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    Intracellular trafficking is a critical process for cell physiology. Previous extensive studies employing biochemical and molecular biological approaches have provided qualitative information about intracellular trafficking, but we have little quantitative information due to technical limitations of these assays. We therefore developed a novel method for quantifying intracellular trafficking based on single molecule imaging with Quantum dot (QD) fluorescent nanocrystals and quantitatively described the trafficking properties of some recycling proteins. We herein first describe how to label intracellular molecules with QD which has no cell permeability and how to quantify intracellular trafficking, and then we detail the development of a novel experimental system allowing multi-color simultaneous single molecule imaging for analyzing the relationships of intracellular trafficking activities among multiple molecules having distinct trafficking properties. Finally, we document how we confirmed the reliability of our system by simultaneously analyzing the intracellular movements of two recycling protein, GLUT4 glucose transporter and transferrin receptor. Since impairment of intracellular trafficking has critical etiological roles in various late-onset diseases such as type 2 diabetes, our novel imaging system may be a powerful tool for developing next-generation biomedical devices for diagnostics and medical treatment based on intracellular trafficking.

  61. Sequential assembly of the functional material micropatterns on the hydrogel sheet for constructing skeletal muscle cell-based assay system 査読有り

    Nagamine K, Otani S, Ito S, Kaji H, Kanzaki M, Nishizawa M

    Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 1054-1056 2012年

  62. Exercise- And drug dose-dependent metabolic assay device using the hydrogel-supported skeletal muscle cells 査読有り

    Nagamine K, Kaji H, Kanzaki M, Nishizawa M

    Proceedings of the 16th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2012 383-385 2012年

  63. Hydrogel-supported skeletal muscle cell-based bioassay system 査読有り

    Kuniaki Nagamine, Shingo Otani, Mai Takeda, Makoto Kanzaki, Matsuhiko Nishizawa

    2011 Int. Symp. on Micro-NanoMechatronics and Human Science, Symp. on "COE for Education and Research of Micro-Nano Mechatronics", Symposium on "Hyper Bio Assembler for 3D Cellular System Innovation" 180-185 2012年

    DOI: 10.1109/MHS.2011.6102223  

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    Contractile C 2C 12 myotube line patterns supported by a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. Myotube line patterns cultured on dish were transferred with 100% efficiency to the surface of fibrin gel sheets. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (one week) than myotubes adhered on a conventional culture dish. The gel sheet-supported C 2C 12 myotube micropatterns were combined with a microelectrode array chip to fabricate a skeletal muscle cell-based bioassay system. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with the electropolymerized conducting polymer. We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell. © 2011 IEEE.

  64. Molecular Basis of Insulin-Responsive GLUT4 Trafficking Systems Revealed by Single Molecule Imaging 査読有り

    Hiroyasu Hatakeyama, Makoto Kanzaki

    TRAFFIC 12 (12) 1805-1820 2011年12月

    出版者・発行元:WILEY-BLACKWELL

    DOI: 10.1111/j.1600-0854.2011.01279.x  

    ISSN:1398-9219

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    Development of a &apos;static retention&apos; property of GLUT4, the insulin-responsive glucose transporter, has emerged as being essential for achieving its maximal insulin-induced surface exposure. Herein, employing quantum-dot-based nanometrology of intracellular GLUT4 behavior, we reveal the molecular basis of its systematization endowed upon adipogenic differentiation of 3T3L1 cells. Specifically, (i) the endosomes-to-trans-Golgi network (TGN) retrieval system specialized for GLUT4 develops in response to sortilin expression, which requires an intricately balanced interplay among retromers, golgin-97 and syntaxin-6, the housekeeping vesicle trafficking machinery. (ii) The Golgin-97-localizing subdomain of the differentiated TGN apparently serves as an intermediate transit route by which GLUT4 can further proceed to the stationary GLUT4 storage compartment. (iii) AS160/Tbc1d4 then renders the &apos;static retention&apos; property insulin responsive, i. e. insulin liberates GLUT4 from the static state only in the presence of functional AS160/Tbc1d4. (iv) Moreover, sortilin malfunction and the resulting GLUT4 sorting defects along with retarded TGN function might be etiologically related to insulin resistance. Together, these observations provide a conceptual framework for understanding maturation/retardation of the insulin-responsive GLUT4 trafficking system that relies on the specialized subdomain of differentiated TGN.

  65. Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not 査読有り

    Carlos M. Castorena, James G. MacKrell, Jonathan S. Bogan, Makoto Kanzaki, Gregory D. Cartee

    JOURNAL OF APPLIED PHYSIOLOGY 111 (4) 1106-1117 2011年10月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/japplphysiol.00631.2011  

    ISSN:8750-7587

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    Castorena CM, MacKrell JG, Bogan JS, Kanzaki M, Cartee GD. Clustering of GLUT4, TUG, and RUVBL2 protein levels correlate with myosin heavy chain isoform pattern in skeletal muscles, but AS160 and TBC1D1 levels do not. J Appl Physiol 111: 1106 -1117, 2011. First published July 28, 2011; doi:10.1152/japplphysiol.00631.2011.-Skeletal muscle is a heterogeneous tissue. To further elucidate this heterogeneity, we probed relationships between myosin heavy chain (MHC) isoform composition and abundance of GLUT4 and four other proteins that are established or putative GLUT4 regulators [Akt substrate of 160 kDa (AS160), Tre-2/Bub2/Cdc 16-domain member 1 (TBC1D1), Tethering protein containing an UBX-domain for GLUT4 (TUG), and RuvB-like protein two (RUVBL2)] in 12 skeletal muscles or muscle regions from Wistar rats [adductor longus, extensor digitorum longus, epitrochlearis, gastrocnemius (mixed, red, and white), plantaris, soleus, tibialis anterior (red and white), tensor fasciae latae, and white vastus lateralis]. Key results were 1) significant differences found among the muscles (range of muscle expression values) for GLUT4 (2.5-fold), TUG (1.7-fold), RUVBL2 (2.0-fold), and TBC1D1 (2.7-fold), but not AS160; 2) significant positive correlations for pairs of proteins: GLUT4 vs. TUG (R = 0.699), GLUT4 vs. RUVBL2 (R = 0.613), TUG vs. RUVBL2 (R = 0.564), AS160 vs. TBC1D1 (R = 0.293), and AS160 vs. TUG (R = 0.246); 3) significant positive correlations for %MHC-I: GLUT4 (R = 0.460), TUG (R = 0.538), and RUVBL2 (R = 0.511); 4) significant positive correlations for %MHC-IIa: GLUT4 (R = 0.293) and RUVBL2 (R = 0.204); 5) significant negative correlations for % MHC-IIb vs. GLUT4 (R = -0.642), TUG (R = -0.626), and RUVBL2 (R = -0.692); and 6) neither AS160 nor TBC1D1 significantly correlated with MHC isoforms. In 12 rat muscles, GLUT4 abundance tracked with TUG and RUVBL2 and correlated with MHC isoform expression, but was unrelated to AS160 or TBC1D1. Our working hypothesis is that some of the mechanisms that regulate GLUT4 abundance in rat skeletal muscle also influence TUG and RUVBL2 abundance.

  66. Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation 査読有り

    Tsubasa S. Matsui, Roland Kaunas, Makoto Kanzaki, Masaaki Sato, Shinji Deguchi

    INTERFACE FOCUS 1 (5) 754-766 2011年10月

    出版者・発行元:ROYAL SOC

    DOI: 10.1098/rsfs.2011.0031  

    ISSN:2042-8898

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    Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin-myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin-myosin crossbridges. Actin-myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin-myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

  67. Non-muscle myosin II induces disassembly of actin stress fibres independently of myosin light chain dephosphorylation 査読有り

    Tsubasa S. Matsui, Roland Kaunas, Makoto Kanzaki, Masaaki Sato, Shinji Deguchi

    INTERFACE FOCUS 1 (5) 754-766 2011年10月

    出版者・発行元:ROYAL SOC

    DOI: 10.1098/rsfs.2011.0031  

    ISSN:2042-8898

    詳細を見る 詳細を閉じる

    Dynamic remodelling of actin stress fibres (SFs) allows non-muscle cells to adapt to applied forces such as uniaxial cell shortening. However, the mechanism underlying rapid and selective disassembly of SFs oriented in the direction of shortening remains to be elucidated. Here, we investigated how myosin crossbridge cycling induced by MgATP is associated with SF disassembly. Moderate concentrations of MgATP, or [MgATP], induced SF contraction. Meanwhile, at [MgATP] slightly higher than the physiological level, periodic actin patterns emerged along the length of SFs and dispersed within seconds. The actin fragments were diverse in length, but comparable to those in characteristic sarcomeric units of SFs. These results suggest that MgATP-bound non-muscle myosin II dissociates from the individual actin filaments that constitute the sarcomeric units, resulting in unbundling-induced disassembly rather than end-to-end actin depolymerization. This rapid SF disassembly occurred independent of dephosphorylation of myosin light chain. In terms of effects on actin-myosin interactions, a rise in [MgATP] is functionally equivalent to a temporal decrease in the total number of actin-myosin crossbridges. Actin-myosin crossbridges are known to be reduced by an assisting load on myosin. Thus, the present study suggests that reducing the number of actin-myosin crossbridges promotes rapid and orientation-dependent disassembly of SFs after cell shortening.

  68. Spatiotemporally controlled contraction of micropatterned skeletal muscle cells on a hydrogel sheet 査読有り

    Kuniaki Nagamine, Takeaki Kawashima, Soichiro Sekine, Yuichiro Ido, Makoto Kanzaki, Matsuhiko Nishizawa

    LAB ON A CHIP 11 (3) 513-517 2011年

    出版者・発行元:ROYAL SOC CHEMISTRY

    DOI: 10.1039/c0lc00364f  

    ISSN:1473-0197

    eISSN:1473-0189

    詳細を見る 詳細を閉じる

    We have developed gel sheet-supported C(2)C(12) myotube micropatterns and combined them with a microelectrode array chip to afford a skeletal muscle cell-based bioassay system. Myotube line patterns cultured on a glass substrate were transferred with 100% efficiency to the surface of fibrin gel sheets. The contractile behavior of each myotube line pattern on the gel was individually controlled by localized electrical stimulation using microelectrode arrays that had been previously modified with electropolymerized poly(3,4-ethylenedioxythiophene) (PEDOT). We successfully demonstrated fluorescent imaging of the contraction-induced translocation of the glucose transporter, GLUT4, from intracellular vesicles to the plasma membrane of the myotubes. This device is applicable for the bioassay of contraction-induced metabolic alterations in a skeletal muscle cell.

  69. Palmitate-induced Down-regulation of Sortilin and Impaired GLUT4 Trafficking in C2C12 Myotubes 査読有り

    Yo Tsuchiya, Hiroyasu Hatakeyama, Natsumi Emoto, Fumie Wagatsuma, Shinichi Matsushita, Makoto Kanzaki

    JOURNAL OF BIOLOGICAL CHEMISTRY 285 (45) 34371-34381 2010年11月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M110.128520  

    ISSN:0021-9258

    eISSN:1083-351X

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    Elevated saturated FFAs including palmitate (C16:0) are a primary trigger for peripheral insulin resistance characterized by impaired glucose uptake/disposal in skeletal muscle, resulting from impaired GLUT4 translocation in response to insulin. We herein demonstrate that palmitate induces down-regulation of sortilin, a sorting receptor implicated in the formation of insulin-responsive GLUT4 vesicles, via mechanisms involving PKC theta and TNF-alpha-converting enzyme, but not p38, JNK, or mitochondrial reactive oxygen species generation, leading to impaired GLUT4 trafficking in C2C12 myotubes. Intriguingly, unsaturated FFAs such as palmitoleate (C16:1) and oleate (C18:1) had no such detrimental effects, appearing instead to effectively reverse palmitate-induced impairment of insulin-responsive GLUT4 recycling along with restoration of sortilin abundance by preventing aberrant PKC theta activation. On the other hand, shRNA-mediated reduction of sortilin in intact C2C12 myotubes inhibited insulin-induced GLUT4 recycling without dampening Akt phosphorylation. We found that the peroxisome proliferator-activated receptor gamma agonist troglitazone prevented the palmitate-induced sortilin reduction and also ameliorated insulin-responsive GLUT4 recycling without altering the palmitate-evoked insults on signaling cascades; neither highly phosphorylated PKC theta states nor impaired insulin-responsive Akt phosphorylation was affected. Taken together, our data provide novel insights into the pathogenesis of PKC theta-dependent insulin resistance with respect to insulin-responsive GLUT4 translocation, which could occur not only through defects of insulin signaling but also via a reduction of sortilin, which directly controls trafficking/sorting of GLUT4 in skeletal muscle cells. In addition, our data suggest the insulin-sensitizing action of peroxisome proliferator-activated receptor gamma agonists to be at least partially mediated through the restoration of proper GLUT4 trafficking/sorting events governed by sortilin.

  70. Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness 査読有り

    Hirokazu Kaji, Takeshi Ishibashi, Kuniaki Nagamine, Makoto Kanzaki, Matsuhiko Nishizawa

    BIOMATERIALS 31 (27) 6981-6986 2010年9月

    出版者・発行元:ELSEVIER SCI LTD

    DOI: 10.1016/j.biomaterials.2010.05.071  

    ISSN:0142-9612

    eISSN:1878-5905

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    A porous membrane-based cell culture device was developed to electrically stimulate a confluent monolayer of C2C12 myotubes. The device&apos;s cell culture substrate is a microporous alumina membrane-modified by attaching an atelocollagen membrane on the upperside and a hole-spotted poly(dimethylsiloxane) (PDMS) film on the underside. When electric current is generated between the device&apos;s Pt ring electrodes - one of which is placed above the cells and the other below the PDMS layer - the focused current at the PDMS hole can electrically stimulate the cells. C2C12 myoblasts were cultured on the substrate and differentiated into myotubes. When the electrical pulses were applied, myotubes started to contract slightly in and near the hole, and that the continuous stimulation increased both the number of stimuli-responding myotubes and the magnitude of the contraction considerably owing to the underlying atelocollagen membrane with muscle tissue-like stiffness. Also, the generation of contractile myotubes on a wider region of the membrane substrate was possible by applying the electrical pulses through the array of holes in the PDMS film. Using the present system, the glucose uptake by contractile myotubes was examined with fluorescence-labeled glucose, 2-NBDG, which displayed a positive correlation between the contractile activity of myotubes and the uptake of 2-NBDG. (C) 2010 Elsevier Ltd. All rights reserved.

  71. Identification of Three Distinct Functional Sites of Insulin-mediated GLUT4 Trafficking in Adipocytes Using Quantitative Single Molecule Imaging 査読有り

    Hideaki Fujita, Hiroyasu Hatakeyama, Tomonobu M. Watanabe, Masaaki Sato, Hideo Higuchi, Makoto Kanzaki

    MOLECULAR BIOLOGY OF THE CELL 21 (15) 2721-2731 2010年8月

    出版者・発行元:AMER SOC CELL BIOLOGY

    DOI: 10.1091/mbc.E10-01-0029  

    ISSN:1059-1524

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    Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be "static retention" in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.

  72. Role of p120-catenin in the morphological changes of endothelial cells exposed to fluid shear stress 査読有り

    Naoya Sakamoto, Kei Segawa, Makoto Kanzaki, Toshiro Ohashi, Masaaki Sato

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 (3) 426-432 2010年7月

    出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE

    DOI: 10.1016/j.bbrc.2010.06.092  

    ISSN:0006-291X

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    p120-Catenin is known to play important roles in cell-cell adhesion stability by binding to cadherin and morphological changes of cells by regulating small RhoGTPase activities. Although the expression and binding states of p120-catenin are thought to dynamically change due to morphological adaptation of endothelial cells (ECs) to fluid shear stress, these dynamics remain to be explored. In the present study, we examined the time course of changes in p120-catenin expression and its binding to vascular endothelial (VE)-cadherin in ECs exposed to shear stress. Human umbilical vein ECs began to change their morphologies at 3-6 h, and became elongated and oriented to the direction of flow at 24 h after exposure to a shear stress of 1.5 Pa. Binding and co-localization of p120-catenin with VE-cadherin at the foci of cell-cell adhesions were retained in ECs during exposure to shear stress, indicating that VE-cadherin was stabilized in the plasma membrane. In contrast, cytoplasmic p120-catenin that was dissociated from VE-cadherin was transiently increased at 3-6 h after the flow onset. These results suggest that the transient increase of cytoplasmic p120-catenin may stimulate RhoGTPase activities and act as a switch for the morphological changes in ECs in response to shear stress. (C) 2010 Elsevier Inc. All rights reserved.

  73. In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle 査読有り

    Katsuhiko Funai, George G. Schweitzer, Carlos M. Castorena, Makoto Kanzaki, Gregory D. Cartee

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 298 (5) E999-E1010 2010年5月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00758.2009  

    ISSN:0193-1849

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    Funai K, Schweitzer GG, Castorena CM, Kanzaki M, Cartee GD. In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle. Am J Physiol Endocrinol Metab 298: E999-E1010, 2010. First published February 23, 2010; doi: 10.1152/ajpendo.00758.2009.-The cellular mechanisms whereby prior exercise enhances insulin-stimulated glucose transport (GT) are not well understood. Previous studies suggested that a prolonged increase in phosphorylation of Akt substrate of 160 kDa (AS160) may be important for the postexercise increase in insulin sensitivity. In the current study, the effects of in vivo exercise and in vitro contraction on subsequent insulin-stimulated GT were studied separately and together. Consistent with results from previous studies, prior exercise resulted in an increase in AS160 (642)Thr phosphorylation immediately after exercise in rat epitrochlearis muscles, and this increase remained 3 h postexercise concomitant with enhanced insulin-stimulated GT. For experiments with in vitro contraction, isolated rat epitrochlearis muscles were electrically stimulated to contract in the presence or absence of rat serum. As expected, insulin-stimulated GT measured 3 h after electrical stimulation in serum, but not after electrical stimulation without serum, exceeded resting controls. Immediately after electrical stimulation with or without serum, phosphorylation of both AS160 (detected by phospho-Akt substrate, PAS, antibody, or phospho-(642)Thr antibody) and its paralog TBC1D1 (detected by phospho-(237)Ser antibody) was increased. However, both AS160 and TBC1D1 phosphorylation had reversed to resting values at 3 h poststimulation with or without serum. Increasing the amount of exercise (from 1 to 2 h) or electrical stimulation (from 5 to 10 tetani) did not further elevate insulin-stimulated GT. In contrast, the combination of prior exercise and electrical stimulation had an additive effect on the subsequent increase in insulin-stimulated GT, suggesting that these exercise and electrical stimulation protocols may amplify insulin-stimulated GT through distinct mechanisms, with a persistent increase in AS160 phosphorylation potentially important for increased insulin sensitivity after exercise, but not after in vitro contraction.

  74. Micropatterning Contractile C2C12 Myotubes Embedded in a Fibrin Gel 査読有り

    Kuniaki Nagamine, Takeaki Kawashima, Takeshi Ishibashi, Hirokazu Kaji, Makoto Kanzaki, Matsuhiko Nishizawa

    BIOTECHNOLOGY AND BIOENGINEERING 105 (6) 1161-1167 2010年4月

    出版者・発行元:WILEY-BLACKWELL

    DOI: 10.1002/bit.22636  

    ISSN:0006-3592

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    Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. The C2C12 myotube/fibrin gel system was prepared by transferring a myotube monolayer from a glass substrate to a fibrin gel while retaining the original line patterns of myotubes. To endow the myotubes with contractile activity, a series of electrical pulses was applied through a pair of carbon electrodes placed at either side of a fibrin gel separately. The frequency and magnitude of myotube contraction were functions of the pulse frequency and duration, respectively. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (1 week) than myotubes adhered on a conventional culture dish. Biotechnol. Bioeng. 2010; 105: 1161-1167. (C) 2009 Wiley Periodicals, Inc.

  75. Gel sheet based skeletal muscle cell culture system integrated with the microelectrode array device 査読有り

    Nagamine K, Kaji H, Kanzaki M, Nishizawa M

    14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 2010, MicroTAS 2010 1 175-177 2010年

  76. Topographic imaging of convoluted surface of live cells by scanning ion conductance microscopy in a standing approach mode 査読有り

    Yasufumi Takahashi, Yumi Murakami, Kuniaki Nagamine, Hitoshi Shiku, Shigeo Aoyagi, Tomoyuki Yasukawa, Makoto Kanzaki, Tomokazu Matsue

    PHYSICAL CHEMISTRY CHEMICAL PHYSICS 12 (34) 10012-10017 2010年

    出版者・発行元:ROYAL SOC CHEMISTRY

    DOI: 10.1039/c002607g  

    ISSN:1463-9076

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    Scanning ion conductance microscopy (SICM) using a nanopipette as a probe and ionic current as a feedback signal was introduced as a novel technique to study live cells in a physiological environment. To avoid contact between the pipette tip and cells during the conventional lateral scanning mode, we adopted a standing approach (STA) mode in which the probe was moved vertically to first approach and then retracted from the cell surface at each measurement point on an XY plane. The STA mode ensured non-contact imaging of the topography of live cells and for a wide range of uneven substrates (500 x 300 mu m to 5 x 5 mu m). We also used a field-programmable gate array (FPGA) board to enhance feedback distance regulation. FPGA dramatically increased the feedback speed and decreased the imaging time (450 s per image) with enhanced accuracy and quality of live cell images. To evaluate the potential of the STA mode for SICM, we carried out imaging of a convoluted surface of live cell in various scan ranges and estimated the spatial resolutions of these images.

  77. Different impacts of saturated and unsaturated free fatty acids on COX-2 expression in C2C12 myotubes 査読有り

    Akito Kadotani, Yo Tsuchiya, Hiroyasu Hatakeyama, Hideki Katagiri, Makoto Kanzaki

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 297 (6) E1291-E1303 2009年12月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00293.2009  

    ISSN:0193-1849

    eISSN:1522-1555

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    Kadotani A, Tsuchiya Y, Hatakeyama H, Katagiri H, Kanzaki M. Different impacts of saturated and unsaturated free fatty acids on COX-2 expression in C2C12 myotubes. Am J Physiol Endocrinol Metab 297: E1291-E1303, 2009. First published September 15, 2009; doi:10.1152/ajpendo.00293.2009.-In skeletal muscle, saturated free fatty acids (FFAs) act as proinflammatory stimuli, and cyclooxygenase-2 (COX-2) is a pro/anti-inflammatory enzyme induced at sites of inflammation, which contributes to prostaglandin production. However, little is known about the regulation of COX-2 expression and its responses to FFAs in skeletal muscle. Herein, we examined the effects of saturated and unsaturated FFAs, including a recently identified lipokine (lipid hormone derived from adipocytes), palmitoleate, on COX-2 expression in C2C12 myotubes as a skeletal muscle model. Exposure of myotubes to saturated FFAs [palmitate (16:0) and stearate (18:0)], but not to unsaturated FFAs [palmitoleate (16:1), oleate (18:1), and linoleate (18:2)], led to a slow-onset induction of COX-2 expression and subsequent prostaglandin E-2 production via mechanisms involving the p38 MAPK and NF-kappa B but not the PKC theta signaling cascades. Pharmacological modulation of mitochondrial oxidative function failed to interfere with COX-2 expression, suggesting the mitochondrial overload/excessive beta-oxidation contribution to this event to be minimal. On the contrary, unsaturated FFAs appeared to effectively antagonize palmitate-induced COX-2 expression with markedly different potencies (linoleate &gt; oleate &gt; palmitoleate), being highly associated with the suppressive profile of each unsaturated FFA toward palmitate-evoked intracellular signals, including p38, JNK, ERK1/2 MAPKs, and PKC theta, as well as I kappa B degradation. In addition, our data suggest little involvement of PPAR in the protective actions of unsaturated FFAs against palmitate-induced COX-2 expression. No direct contribution of the increased COX-2 activity in generating palmitate-induced insulin resistance was detected, at least in terms of insulin-responsive Akt phosphorylation and GLUT4 translocation. Taken together, our data provide a novel insight into the molecular mechanisms responsible for the FFA-induced COX-2 expression in skeletal muscle and raise the possibility that, in skeletal myocytes, COX-2 and its product prostaglandins may play an important role in the complex inflammation responses caused by elevated FFAs, for example, in the diabetic state.

  78. Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes 査読有り

    Taku Nedachi, Hiroyasu Hatakeyama, Tatsuyoshi Kono, Masaaki Sato, Makoto Kanzaki

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 297 (4) E866-E878 2009年10月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00104.2009  

    ISSN:0193-1849

    eISSN:1522-1555

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    Nedachi T, Hatakeyama H, Kono T, Sato M, Kanzaki M. Characterization of contraction-inducible CXC chemokines and their roles in C2C12 myocytes. Am J Physiol Endocrinol Metab 297: E866-E878, 2009. First published July 21, 2009; doi:10.1152/ajpendo.00104.2009.-Physicalexercise triggers the release of several cytokines/chemokines from working skeletal muscles, but the underlying mechanism(s) by which skeletal muscles decipher and respond to highly complex contractile stimuli remains largely unknown. In an effort to investigate the regulatory mechanisms of the expressions of two contraction-inducible CXC chemokines, CXCL1/KC and CXCL5/LIX, in contracting skeletal muscle cells, we took advantage of our in vitro exercise model using highly developed contractile C2C12 myotubes, which acquire properties similar to those of in vivo skeletal muscle via manipulation of Ca2+ transients with electric pulse stimulation (EPS). Production of these CXC chemokines was immediately augmented by EPS-evoked contractile activity in a manner dependent on the activities of JNK and NF-kappa B, but not p38, ERK1/2, or calcineurin. Intriguingly, exposure of myotubes to cyclic mechanical stretch also induced expression of these CXC chemokines; however, a much longer period of stimulation (similar to 12 h) was required, despite rapid JNK phosphorylation. We also demonstrate herein that CXCL1/KC and CXCL5/LIX have the ability to raise intracellular Ca2+ concentrations via CXCR2-mediated activation of pertussis toxin-sensitive G alpha(i) proteins in C2C12 myoblasts, an action at least partially responsible for their migration and differentiation. Although we revealed a possible negative feedback regulation of their own production in response to the contractile activity in differentiated myotubes, exogenous administration of these CXC chemokines did not acutely influence either insulin-induced Akt phosphorylation or GLUT4 translocation in C2C12 myotubes. Taken together, these data shed light on the fundamental characteristics of contraction-inducible CXC chemokine production and their potential roles in skeletal muscle cells.

  79. Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle 査読有り

    Katsuhiko Funai, George G. Schweitzer, Naveen Sharma, Makoto Kanzaki, Gregory D. Cartee

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 297 (1) E242-E251 2009年7月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00194.2009  

    ISSN:0193-1849

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    Funai K, Schweitzer GG, Sharma N, Kanzaki M, Cartee GD. Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle. Am J Physiol Endocrinol Metab 297: E242-E251, 2009. First published May 12, 2009; doi:10.1152/ajpendo.00194.2009.-A single exercise bout can increase insulin-independent glucose transport immediately postexercise and insulin-dependent glucose transport (GT) for several hours postexercise. Akt substrate of 160 kDa (AS160) and TBC1D1 are paralog Rab GTPase-activating proteins that have been proposed to contribute to these exercise effects. Previous research demonstrated greater AS160 and Akt threonine phosphorylation in rat skeletal muscle at 3-4 h postexercise concomitant with enhanced insulin-stimulated GT. To further probe whether these signaling events or TBC1D1 phosphorylation were important for the enhanced postexercise insulin-stimulated GT, male Wistar rats were studied using four experimental protocols (2-h swim exercise, differing with regard to timing of muscle sampling and whether food was provided postexercise) that were known to vary in their influence of insulin-independent and insulin-dependent GT postexercise. The results indicated that, in isolated rat epitrochlearis muscle, 1) elevated phosphorylation of AS160 (measured using anti-phospho-Akt substrate, PAS-AS160, and phosphospecific anti-Thr(642)-AS160, pThr(642)-AS160) consistently tracked with elevated insulin-stimulated GT; 2) PAS-TBC1D1 was not different from sedentary values at 3 or 27 h postexercise, when insulin sensitivity was increased; 3) insulin-stimulated Akt activity was not increased postexercise in muscles with increased insulin sensitivity; 4) PAS-TBC1D1 was increased immediately postexercise, when insulin-independent GT was elevated, and reversed at 3 and 27 h postexercise, when insulin-independent GT was also reversed; and 5) there was no significant effect of exercise or insulin on total abundance of AS160, TBC1D1, Akt, or GLUT4 protein with any of the protocols. The results are consistent with increased AS160 phosphorylation (PAS-AS160 or pThr(642)-AS160) but not increased PAS-TBC1D1 or Akt activity, which is important for increased postexercise insulin-stimulated GT in rat skeletal muscle. They also support the idea that increased TBC1D1 phosphorylation may play a role in the insulin-independent increase in GT postexercise.

  80. Localized electrical stimulation to C2C12 myotubes cultured on a porous membrane-based substrate 査読有り

    Takeshi Ishibashi, Yu Hoshino, Hirokazu Kaji, Makoto Kanzaki, Masaaki Sato, Matsuhiko Nishizawa

    BIOMEDICAL MICRODEVICES 11 (2) 413-419 2009年4月

    出版者・発行元:SPRINGER

    DOI: 10.1007/s10544-008-9247-7  

    ISSN:1387-2176

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    We report a porous membrane-based cell culture device that can conduct localized electrical stimulation of a cell monolayer. The device&apos;s cell culture substrate is a microporous alumina membrane with an underlying thin poly(dimethylsiloxane) (PDMS) film spotted with holes. When electric current is generated between the device&apos;s Pt ring electrodes-one of which is placed above the cells and the other below the PDMS layer-the current density condenses at the holes in the PDMS film, and cells located above the holes can be electrically stimulated. C2C12 cells were confluently cultured on the substrate and were differentiated to myotubes. To control the stimulated area in the substrate, we attempted to seal and reopen the holes of the PDMS film by using an air bubble. Since the current pulse could be effectively blocked at the sealed holes, fluorescent Ca(2+) transients, indicative of cellular excitation, were observed from the myotubes located above holes in the open state.

  81. Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle 査読有り

    Taku Nedachi, Hideaki Fujita, Makoto Kanzaki

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 295 (5) E1191-E1204 2008年11月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.90280.2008  

    ISSN:0193-1849

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    Nedachi T, Fujita H, Kanzaki M. Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle. Am J Physiol Endocrinol Metab 295: E1191-E1204, 2008. First published September 9, 2008; doi:10.1152/ajpendo.90280.2008.-Adequate exercise leads to a vast variety of physiological changes in skeletal muscle as well as other tissues/organs and is also responsible for maintaining healthy muscle displaying enhanced insulin-responsive glucose uptake via GLUT4 translocation. We generated highly developed contractile C2C12 myotubes by manipulating intracellular Ca2+ transients with electric pulse stimulation (EPS) that is endowed with properties similar to those of in vivo skeletal muscle in terms of 1) excitation-induced contractile activity as a result of de novo sarcomere formation, 2) activation of both the AMP kinase and stress-activated MAP kinase cascades, and 3) improved insulin responsiveness as assessed by GLUT4 recycling. Tbc1d1, a Rab-GAP implicated in exercise-induced GLUT4 translocation in skeletal muscle, also appeared to be phosphorylated on Ser(231) after EPS-induced contraction. In addition, a switch in myosin heavy-chain (MHC) expression from "fast type" to "slow type" was observed in the C2C12 myotubes endowed with EPS-induced repetitive contractility. Taking advantage of these highly developed contractile C2C12 myotubes, we identified myotube-derived factors responsive to EPS-evoked contraction, including the CXC chemokines CXCL1/KC and CXCL5/LIX, as well as IL-6, previously reported to be upregulated in contracting muscles in vivo. Importantly, animal treadmill experiments revealed that exercise significantly increased systemic levels of CXCL1/KC, perhaps derived from contracting muscle. Taken together, these results confirm that we have established a specialized muscle cell culture model allowing contraction-inducible cellular responses to be explored. Utilizing this model, we identified contraction-inducible myokines potentially linked to the metabolic alterations, immune responses, and angiogenesis induced by exercise.

  82. Erratum: Electric pulse stimulation induces NMDA glutamate receptor mRNa in NIH3T3 mouse fibroblasts (Tohoku Journal of Experimental Medicine (2008) vol. 215 (181-187))

    Saeko Okutsu, Hiroyasu Hatakeyama, Makoto Kanzaki, Hiroshi Tsubokawa, Ryoichi Nagatomi

    Tohoku Journal of Experimental Medicine 216 (2) 195 2008年10月24日

    DOI: 10.1620/tjem.216.195  

    ISSN:1349-3329 0040-8727

  83. Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes 査読有り

    Taku Nedachi, Akito Kadotani, Miyako Ariga, Hideki Katagiri, Makoto Kanzaki

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 294 (4) E668-E678 2008年4月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00640.2007  

    ISSN:0193-1849

    eISSN:1522-1555

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    Nutrition availability is one of the major environmental signals influencing cell fate, such as proliferation, differentiation, and apoptosis, often functioning in concert with other humoral factors, including insulin. Herein, we show that low-serum-induced differentiation of C2C12 myocytes is significantly hampered under low glucose (LG; 5 mM) compared with high glucose (HG; 22.5 mM) conditions, concurrently with nuclear accumulation of SIRT1, an NAD(+)-dependent deacetylase, and FoxO3a, both of which are implicated in the negative regulation of myogenesis. Intriguingly, insulin appears to exert opposite actions, depending on glucose availability, with regard to the regulation of SIRT1 and FoxO3a abundance, which apparently contributes to modulating the potency of insulin's myogenic action. Namely, insulin exerts a potent myogenic effect in the presence of sufficient glucose, whereas insulin is unable to exert its myogenic action under LG conditions, since insulin evokes massive upregulation of both SIRT1 and FoxO3a in the absence of sufficient ambient glucose. In addition, the hampered differentiation state under LG is significantly restored by sirtinol, a SIRT1 inhibitor, whereas insulin abolished this sirtinol-dependent restoration, indicating that insulin can function as a negative as well as a positive myogenic factor depending on glucose availability. Taken together, our data reveal the importance of ambient glucose levels in the regulation of myogenesis and also in the determination of insulin's myogenic potency, which is achieved, at least in part, through regulation of the cellular contents and

  84. Functional role of sortilin in myogenesis and development of insulin-responsive glucose transport system in C2C12 myocytes 査読有り

    Miyako Ariga, Taku Nedachi, Hideki Katagiri, Makoto Kanzaki

    JOURNAL OF BIOLOGICAL CHEMISTRY 283 (15) 10208-10220 2008年4月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M710604200  

    ISSN:0021-9258

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    Sortilin has been implicated in the formation of insulin-responsive GLUT4 storage vesicles in adipocytes by regulating sorting events between the trans-Golgi-network and endosomes. We herein show that sortilin serves as a potent myogenic differentiation stimulator for C2C12 myocytes by cooperatively functioning with p75NTR, which subsequently further contributes to development of the insulin-responsive glucose transport system in C2C12 myotubes. Sortilin expression was up-regulated upon C2C12 differentiation, and overexpression of sortilin in C2C12 cells significantly stimulated myogenic differentiation, a response that was completely abolished by either anti-p75NTR- or anti-nerve growth factor (NGF)-neutralizing antibodies. Importantly, small interference RNA-mediated suppression of endogenous sortilin significantly inhibited C2C12 differentiation, indicating the physiological significance of sortilin expression in the process of myogenesis. Although sortilin overexpression in C2C12 myotubes improved insulin-induced 2-deoxyglucose uptake, as previously reported, this effect apparently resulted from a decrease in the cellular content of GLUT1 and an increase in GLUT4 via differentiation dependent alterations at both the gene transcriptional and the post-translational level. In addition, cellular contents of Ubc9 and SUMO-modified proteins appeared to be increased by sortilin overexpression. Taken together, these data demonstrate that sortilin is involved not only in development of the insulin-responsive glucose transport system in myocytes, but is also directly involved in muscle differentiation via modulation of proNGF-p75NTR.

  85. Electric pulse stimulation induces NMDA glutamate receptor mRNA in NIH3T3 mouse fibroblasts 査読有り

    Saeko Okutsu, Hiroyasu Hatakeyama, Makoto Kanazaki, Hiroshi Tsubokawa, Ryoichi Nagatomi

    Tohoku Journal of Experimental Medicine 215 (2) 181-187 2008年

    DOI: 10.1620/tjem.215.181  

    ISSN:0040-8727 1349-3329

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    Excess glutamate and Ca2+ influx into neurons exacerbate brain damage such as ischemia. Astrocytes at the site of damage proliferate and attenuate the glutamate- and Ca2+-induced neuronal damage by removing excess glutamate and Ca2+ through the N-methyl-D-aspartate (NMDA) glutamate receptor and the L-type Ca2+ channel, respectively. Fibroblasts are commonly mobilized to the site of damage, probably supporting the restoration process. Notably, fibroblasts express the L-type voltage-sensitive Ca2+ channel, but not central nervous system-specific NMDA glutamate receptor. We examined if electric pulse stimulation (EPS) was capable of inducing NMDA receptor on fibroblasts by way of Ca2+ channel activation, so that they could potentially have a neuroprotective role. To activate L-type Ca2+ channel, we delivered electric pulse to cultured NIH3T3 mouse fibroblasts. EPS of 20 V with a pulse duration of 2 msec at a frequency of 1 Hz for more than 1 h up to 24 h successfully introduced Ca2+ into NIH3T3 fibroblasts as detected by Fluo-4AM calcium imaging, which was totally inhibited by a L-type Ca2+ channel inhibitor, verapamil. Remarkable expression of NMDA receptor mRNA in the fibroblasts after 24-h EPS was demonstrated by RT-PCR. Verapamil treatment during EPS totally abrogated the EPS-induced NMDA receptor mRNA expression. To the best of our knowledge, this is the first report showing that electric pulse is able to induce sustained Ca2+ influx via L-type Ca2+ channel in a non-excitatory fibroblast, which leads to the expression CNS-specific NMDA receptor mRNA. Neuroprotective role of NMDA receptor induced in fibroblasts needs to be further examined. © 2008 Tohoku University Medical Press.

  86. Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes 査読有り

    Hideaki Fujita, Taku Nedachi, Makoto Kanzaki

    EXPERIMENTAL CELL RESEARCH 313 (9) 1853-1865 2007年5月

    出版者・発行元:ELSEVIER INC

    DOI: 10.1016/j.yexcr.2007.03.002  

    ISSN:0014-4827

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    The assembly of sarcomeres, the smallest contractile units in striated muscle, is a complex and highly coordinated process that relies on spatio-temporal organization of sarcomeric proteins, a process requiring spontaneous Ca2+ transients. To investigate the relationship between Ca2+ transients and sarcomere assembly in C2C12 myotubes, we employed electric pulse stimulation (EPS), which allows the frequency of Ca2+ transients to be manipulated. We monitored contractile activity as a means of evaluating functional sarcomere establishment using the differential image subtraction (DIS) method. C2C12 myotubes initially displayed no contractility with EPS, due to a lack of sarcomere architecture. However, C2C12 myotubes showed remarkable contractile activity with EPS-induced repetitive Ca2+ transients (1 Hz) within only 2 h. This activity was concurrent with the development of sarcomere structure. Importantly, the period required for the acquisition of contractile activity in response to excitation was dependent upon the frequency of Ca2+ oscillations, but a sustained increase in intracellular Ca2+ (not oscillatory) by high-frequency EPS (10 Hz) was incapable of conferring either contractility or sarcomere assembly on the myotubes. The EPS-facilitated de novo functional sarcomere assembly appeared to require calpain-mediated proteolysis. In addition, modulation of integrin signals, by adding collagen IV or RGD-peptide, significantly affected the EPS-induced development of contractility. Taken together, these observations indicate that the frequency of the Ca2+ oscillation determines the time required to establish functionally active sarcomere assembly and also suggest that the Ca2+ oscillatory signal may be decoded through reorganization of the integrin-cytoskeletal protein complex via calpain-mediated proteolysis. (c) 2007 Elsevier Inc. All rights reserved.

  87. Subcellular compartmentalization of insulin signaling processes and GLUT4 trafficking events 査読有り

    Robert T. Watson, Alan R. Saltiel, Jeffrey E. Pessin, Makoto Kanzaki

    Mechanisms of Insulin Action: Medical Intelligence Unit 33-51 2007年

    出版者・発行元:Springer New York

    DOI: 10.1007/978-0-387-72204-7_2  

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    Skeletal muscle and adipose tissue are the major sites of postprandial glucose disposal. The insulin-regulated transport of glucose into these tissues is a multi-step process that begins with the binding of insulin to its cell surface receptor. Once activated, the insulin receptor generates multiple intracellular signaling cascades, some of which induce the rapid redistribution of the GLUT4 facilitative glucose transporter from intracellular compartments to the plasma membrane. Although probably best known for its role in glucose homeostasis, insulin regulates a variety of metabolic, mitogenic, and anti-apoptotic processes in specific tissues. Moreover, in addition to insulin several other hormones and growth factors can also activate signaling targets that function downstream of the insulin receptor. For example, phosphatidylinositol-3′-kinase (PI3K), a key enzyme in the signaling pathway leading to insulin-stimulated glucose uptake, can be activated by many extracellular signals. However, only insulin and highly related hormones such as IGF-I efficiently stimulate acute glucose transport. These observations suggest that cellular mechanisms have evolved for maintaining specificity among signaling pathways mediated by various hormones and growth factors. Elucidating the underlying mechanisms for this specificity is a current challenge engaging the attention of many researchers, and will require a thorough understanding of the signaling pathway responsible for each cellular response. To this end, recent work suggests that the coordination of multiple pathways occurs in part through the intracellular compartmentalization of key signaling molecules. Indeed, subcellular compartmentalization plays a critical role in maintaining the specificity of insulin signaling and the fidelity of GLUT4 vesicle trafficking. © 2007 Landes Bioscience and Springer Science+Business Media, LLC.

  88. Involvement of apolipoprotein E in excess fat accumulation and insulin resistance 査読有り

    Junhong Gao, Hideki Katagiri, Yasushi Ishigaki, Tetsuya Yamada, Takehide Ogihara, Junta Imai, Kenji Uno, Yutaka Hasegawa, Makoto Kanzaki, Tokuo T. Yamamoto, Shun Ishibashi, Yoshitomo Oka

    DIABETES 56 (1) 24-33 2007年1月

    出版者・発行元:AMER DIABETES ASSOC

    DOI: 10.2337/db06-0144  

    ISSN:0012-1797

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    Although apolipoprotein E (apoE) is well known to play a major role in lipid metabolism, its role in glucose and energy homeostasis remains unclear. Herein, we established apoE-deficient genetically obese Ay (apoE(-/-);Ay/+) mice. ApoE deficiency in Ay mice prevented the development of obesity, with decreased fat accumulation in the liver and adipose tissues. ApoE(-/-);Ay/+ mice exhibited better glucose tolerance than apoE(+/+);Ay/+ mice. Insulin tolerance testing and hyperinsulinemic-euglycemic clamp study revealed marked improvement of insulin sensitivity, despite increased plasma free fatty acid levels. These metabolic phenotypes were reversed by adenoviral replenishment of apoE protein, indicating circulating apoE to be involved in increased adiposity and obesity-related metabolic disorders. Uptake of apoE-lacking VLDL into the liver and adipocytes was markedly inhibited, but adipocytes in apoE(-/-);Ay/+ mice exhibited normal differentiation, suggesting that apoE-dependent VLDL transport is involved in the development of obesity, i.e., surplus fat accumulation. Interestingly, apoE(-/-);Ay/+ mice exhibited decreased food intake and increased energy expenditure. Pair-feeding experiments indicate these phenomena to both contribute to the obesity-resistant phenotypes associated with apoE deficiency. Thus, apoE is involved in maintaining energy homeostasis. ApoE-dependent excess fat accumulation is a promising therapeutic target for the metabolic syndrome.

  89. Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes 査読有り

    Taku Nedachi, Makoto Kanzaki

    AMERICAN JOURNAL OF PHYSIOLOGY-ENDOCRINOLOGY AND METABOLISM 291 (4) E817-E828 2006年10月

    出版者・発行元:AMER PHYSIOLOGICAL SOC

    DOI: 10.1152/ajpendo.00194.2006  

    ISSN:0193-1849

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    Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes. Am J Physiol Endocrinol Metab 291: E817 - E828, 2006. First published May 30, 2006; doi: 10.1152/ajpendo. 00194.2006. - It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C2C12 myocytes expressing exofacial-MycGLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C2C12 myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation (similar to 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C2C12 myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C2C12 myotubes can be achieved through surprisingly acute glucose- dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C2C12 myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.

  90. 2P164 Live cell imaging of GLUT4 molecules in adipocytes using fluorescent quantum dot and TIRF microscopy(34. Membrane protein,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

    Fujita Hideaki, Watanabe Tomonobu M., Nedachi Taku, Higuchi Hideo, Kanzaki Makoto

    生物物理 46 (2) S336 2006年

    出版者・発行元:一般社団法人 日本生物物理学会

    DOI: 10.2142/biophys.46.S336_4  

  91. 2P215 蛍光性ナノクリスタルを用いたGLUT4分子の脂肪細胞内1分子イメージング(細胞生物的課題(接着・運動・骨格・伝達・膜)))

    藤田 英明, 渡邉 朋信, 根建 拓, 樋口 秀男, 神崎 展

    生物物理 45 S173 2005年

    出版者・発行元:一般社団法人 日本生物物理学会

    DOI: 10.2142/biophys.45.S173_3  

  92. Phosphatidylinositol 4,5-bisphosphate regulates adipocyte actin dynamics and GLUT4 vesicle recycling 査読有り

    M Kanzaki, M Furukawa, W Raab, JE Pessin

    JOURNAL OF BIOLOGICAL CHEMISTRY 279 (29) 30622-30633 2004年7月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M401443200  

    ISSN:0021-9258

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    To investigate the potential role of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P-2) in the regulation of actin polymerization and GLUT4 translocation, the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) were expressed in 3T3L1 adipocytes. In preadipocytes (fibroblasts) PIP5K expression promoted actin polymerization on membrane-bound vesicles to form motile actin comets. In contrast, expression of PIP5K in differentiated 3T3L1 adipocytes resulted in the formation of enlarged vacuole-like structures coated with F-actin, cortactin, dynamin, and N-WASP. Treatment with either latrunculin B (an inhibitor for actin polymerization) or Clostridium difficile toxin B (a general Rho family inhibitor) resulted in a relatively slower disappearance of coated F-actin from these vacuoles, but the vacuoles themselves remained unaffected. Functionally, the increased PI(4,5) P2 levels resulted in an inhibition of transferrin receptor and GLUT4 endocytosis and a slow accumulation of these proteins in the PI(4,5)P-2-enriched vacuoles along with the non-clathrin-derived endosome marker (caveolin) and the AP-2 adaptor complex. However, these structures were devoid of early endosome markers (EEA1, clathrin) and the biosynthetic membrane secretory machinery markers p115 (Golgi) and syntaxin 6 (trans-Golgi Network). Taken together, these data demonstrate that PI(4,5) P2 has distinct morphologic and functional properties depending upon specific cell context. In adipocytes, altered PI(4,5) P2 metabolism has marked effects on GLUT4 endocytosis and intracellular vesicle trafficking due to the derangement of actin dynamics.

  93. Entry of newly synthesized GLUT4 into the insulin-responsive storage compartment is GGA dependent 査読有り

    RT Watson, AH Khan, M Furukawa, JCQ Hou, L Li, M Kanzaki, S Okada, KV Kandror, JE Pessin

    EMBO JOURNAL 23 (10) 2059-2070 2004年5月

    出版者・発行元:NATURE PUBLISHING GROUP

    DOI: 10.1038/sj.emboj.7600159  

    ISSN:0261-4189

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    Following biosynthesis, both GLUT1 and VSV-G proteins appear rapidly (2-3 h) at the plasma membrane, whereas GLUT4 is retained in intracellular membrane compartments and does not display any significant insulin responsiveness until 6-9 h. Surprisingly, the acquisition of insulin responsiveness did not require plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) had no effect on the insulin-stimulated GLUT4 translocation. Furthermore, expression of endocytosis-defective GLUT4 mutants or continuous surface labeling with an exofacial specific antibody demonstrated that GLUT4 did not transit the cell surface prior to the acquisition of insulin responsiveness. The expression of a dominant-interfering GGA mutant (VHS-GAT) had no effect on the trafficking of newly synthesized GLUT1 or VSV-G protein to the plasma membrane, but completely blocked the insulin-stimulated translocation of newly synthesized GLUT4. Furthermore, in vitro budding of GLUT4 vesicles but not GLUT1 or the transferrin receptor was inhibited by VHS-GAT. Together, these data demonstrate that following biosynthesis, GLUT4 directly sorts and traffics to the insulin-responsive storage compartment through a specific GGA-sensitive process.

  94. Atypical Protein Kinase C (PKCζ/λ) is a convergent downstream target of the insulin-stimulated phosphatidylinositol (PI) 3-kinase and TC10 signaling pathways. 査読有り

    Makoto Kanzaki, Silvia Mora Joseph, B. Hwang, Alan R. Saltiel Jeffrey E. Pessin

    J Cell Biol. 164 (2) 279-290 2004年1月

    出版者・発行元:None

    DOI: 10.1083/jcb.200306152  

    ISSN:0021-9525

  95. The exocytotic trafficking of TC10 occurs through both classical and nonclassical secretory transport pathways in 3T3L1 adipocytes 査読有り

    RT Watson, M Furukawa, SH Chiang, D Boeglin, M Kanzaki, AR Saltiel, JE Pessin

    MOLECULAR AND CELLULAR BIOLOGY 23 (3) 961-974 2003年2月

    出版者・発行元:AMER SOC MICROBIOLOGY

    DOI: 10.1128/MCB.23.3.961-974.2003  

    ISSN:0270-7306

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    To examine the structural determinants necessary for TC10 trafficking, localization, and function in adipocytes, we generated a series of point mutations in the carboxyl-terminal targeting domain of TC10. Wild-type TC10 (TC10/WT) localized to secretory membrane compartments and caveolin-positive lipid raft microdomains at the plasma membrane. Expression of a TC10/C206S point mutant resulted in a trafficking and localization pattern that was indistinguishable from that of TC10/WT. In contrast, although TC10/C209S or the double TC10/C206,209S mutant was plasma membrane localized, it was excluded from both the secretory membrane system and the lipid raft compartments. Surprisingly, inhibition of Golgi membrane transport with brefeldin A did not prevent plasma membrane localization of TOO or H-Ras. Moreover, inhibition of trans-Golgi network exit with a 19degreesC temperature block did not prevent the trafficking of TC10 or H-Ras to the plasma membrane. These data demonstrate that TC10 and H-Ras can both traffic to the plasma membrane by at least two distinct transport mechanisms in adipocytes, one dependent upon intracellular membrane transport and another independent of the classical secretory membrane system. Moreover, the transport through the secretory pathway is necessary for the localization of TC10 to lipid raft microdomains at the plasma membrane.

  96. A Crk-II/TC10 signaling pathway is required for osmotic shock-stimulated glucose transport 査読有り

    P Gual, S Shigematsu, M Kanzaki, T Gremeaux, T Gonzalez, JE Pessin, Y Le Marchand-Brustel, JF Tanti

    JOURNAL OF BIOLOGICAL CHEMISTRY 277 (46) 43980-43986 2002年11月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M203042200  

    ISSN:0021-9258

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    Osmotic shock stimulates the translocation of the glucose transporter Glut 4 to plasma membrane by a tyrosine kinase signaling pathway involving Gab-1 (the Grb2-associated binder-1 protein). We show here that, in response to osmotic shock, Gab-1 acts as a docking protein for phospholipase Cgammal, the p85 subunit of the phosphoinositide 3-kinase and Crk-II. It has been shown that the adapter Crk-II is constitutively associated with C3G, a GDP to GTP exchange factor for several small GTP-binding proteins. We found that inhibition of the activity of phosphoinositide 3-kinase or phospholipase C did not prevent the stimulation of glucose transport by osmotic shock, whereas inactivation of Rho proteins by Clostridium difficile toxin B severely inhibited glucose uptake. Among the Rho family members, overexpression of dominant-interfering TC10/T31N mutant inhibited osmotic shock-mediated Glut 4 translocation suggesting that TC10 is required for this process. Further, disruption of cortical actin integrity by latrunculin B or jasplakinolide severely impaired osmotic shock-induced glucose transport. In contrast, osmotic shock increased the amount of cortical actin associated with caveolin-enriched plasma membrane domains. These data provide the first evidence that activation of TC10 and remodeling of cortical actin, which could occur through the TC10 signaling, are required for osmotic shock-mediated Glut 4 translocation and glucose uptake.

  97. Small GTP-binding protein TC10 differentially regulates two distinct populations of filamentous actin in 3T3L1 adipocytes 査読有り

    M Kanzaki, RT Watson, JCQ Hou, M Stamnes, AR Saltiel, JE Pessin

    MOLECULAR BIOLOGY OF THE CELL 13 (7) 2334-2346 2002年7月

    出版者・発行元:AMER SOC CELL BIOLOGY

    DOI: 10.1091/mbc.01-10-0490  

    ISSN:1059-1524

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    TC10 is a member of the Rho family of small GTP-binding proteins that has previously been implicated in the regulation of insulin-stimulated GLUT4 translocation in adipocytes. In a manner similar to Cdc42-stimulated actin-based motility, we have observed that constitutively active TC10 (TC10/Q75L) can induce actin comet tails in Xenopus oocyte extracts in vitro and extensive actin polymerization in the perinuclear region when expressed in 3T3L1 adipocytes. In contrast, expression of TC10/Q75L completely disrupted adipocyte cortical actin, which was specific for TC10, because expression of constitutively active Cdc42 was without effect. The effect of TC10/Q75L to disrupt cortical actin was abrogated after deletion of the amino terminal extension (DeltaN-TC10/Q75L), whereas this deletion retained the ability to induce perinuclear actin polymerization. In addition, alteration of perinuclear actin by expression of TC10/Q75L, a dominant-interfering TC10/T31N mutant or a mutant N-WASP protein (N-WASP/DeltaVCA) reduced the rate of VSV G protein trafficking to the plasma membrane. Furthermore, TC10 directly bound to Golgi COPI coat proteins through a dilysine motif in the carboxyl terminal domain consistent with a role for TC10 regulating actin polymerization on membrane transport vesicles. Together, these data demonstrate that TC10 can differentially regulate two types of filamentous actin in adipocytes dependent on distinct functional domains and its subcellular compartmentalization.

  98. Caveolin-associated filamentous actin (Cav-actin) a novel F-actin structure in adipocytes 査読有り

    Makoto Kanzaki, Jeffrey E. Pessin

    J Biol Chem 277 (29) 25867-25869 2002年5月30日

    DOI: 10.1074/jbc.C200292200  

  99. Intracellular insulin-responsive glucose transporter (GLUT4) distribution but not insulin-stimulated GLUT4 exocytosis and recycling are microtubule dependent 査読有り

    S Shigematsu, AH Khan, M Kanzaki, JE Pessin

    MOLECULAR ENDOCRINOLOGY 16 (5) 1060-1068 2002年5月

    出版者・発行元:ENDOCRINE SOC

    DOI: 10.1210/me.16.5.1060  

    ISSN:0888-8809

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    To investigate the potential role of microtubules in the regulation of insulin-responsive glucose transporter (GLUT4) trafficking in adipocytes, we examined the effects of microtubule depolymerizing and stabilizing agents. In contrast to previous reports, disruption or stabilization of microtubule structures had no significant effect on insulin-stimulated GLUT4 translocation. However, consistent with a more recent study (Molero, J. C., J. P. Whitehead, T. Meerloo, and D. E. James, 2001, J Biol Chem 276:43829-43835) nocodazole did inhibit glucose uptake through a direct interaction with the transporter itself independent of the translocation process. In addition, the initial rate of GLUT4 endocytosis was not significantly affected by microtubule depolymerization. However, these internalized GLUT4 compartments are confined to regions just beneath the plasma membrane and were not exposed to the extracellular space. Furthermore, they were unable to undergo further sorting steps and trafficking to the perinuclear region. Nevertheless, these apparent early endocytic GLUT4 compartments fully responded to a second insulin stimulation with an identical extent of plasma membrane translocation. Together, these data demonstrate that although microtubular organization may play a role in the trafficking of GLUT4 early endocytic vesicles back to the perinuclear region, they do not have a significant role in insulin-stimulated GLUT4 exocytosis, initial endocytosis from the plasma membrane and/or recycling back to the plasma membrane.

  100. Functional dissection of lipid and protein kinase signals of PIKfyve reveals the role of PtdIns 3,5-P-2 production for endomembrane integrity 査読有り

    OC Ikonomov, D Sbrissa, K Mlak, M Kanzaki, J Pessin, A Shisheva

    JOURNAL OF BIOLOGICAL CHEMISTRY 277 (11) 9206-9211 2002年3月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M108750200  

    ISSN:0021-9258

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    PIKfyve enzymatic activity is required in maintaining late endocytic membrane integrity. PIKfyve is a dual specificity enzyme that phosphorylates phosphatidylinositol (PtdIns) and PtdIns 3-P at the 5-hydroxyl and unidentified endogenous protein substrate(s). To determine which of these activities (lipid versus protein kinase activity) is responsible for endomembrane homeostasis we analyzed a double mutant PIKfyve(K1999E/K2000E). These substitutions in the putative lipid-substrate activation loop nearly completely abrogated the lipid kinase activity without any significant effect on the protein kinase activity of PIKfyve(K1999FIC2000E). Expression of PlKfyve(K1999FJK2000E) in COS cells induced a dramatic dominant-negative effect in the form of endomembrane swelling and vacuolation. In addition, the lipid-substrate specificity of PIKfyve was modified by introducing single mutations in Lys-1999 or Lys-2000. This yielded proteins with preferentially abrogated synthesis of PtdIns 5-P (PIKfyve(K2000E)) or PtdIns 3,5-P-2 (PlKfyve(K1999E)), of which only the PIKfyve(K1999E) mutant induced the characteristic endomembrane defects upon cell transfection. Furthermore, phosphoinositide micro-injection into cells demonstrated a selective ability of PtdIns 3.5-P-2 to correct the endomembrane defects induced by the dominant-negative PlKfyve lipid kinase-deficient mutants. Thus, PtdIns 3,5-P-2 production by PlKfyve is crucial for endomembrane integrity, and Lys-1999 most likely directs the PIKfyve interactions with the 3-phosphate group in PtdIns 3-P.

  101. Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes. 査読有り

    M Kanzaki, RT Watson, AH Khan, JE Pessin

    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (52) 49331-49336 2001年12月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M109657200  

    ISSN:0021-9258

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    Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-[gamma-thio]triphosphate (GTP-gammaS) and sodium orthovanadate stimulation of actin comet tails. The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/DeltaVCA. Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTPgammaS and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments. Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles. In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein. Expression of N-WASP/DeltaVCA in intact cells had little effect on adipo-cyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation. Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.

  102. Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling 査読有り

    M Kanzaki, JE Pessin

    JOURNAL OF BIOLOGICAL CHEMISTRY 276 (45) 42436-42444 2001年11月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.M108297200  

    ISSN:0021-9258

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    Rhodamine-labeled phalloidin staining of morphologically differentiated 3T3L1 adipocytes demonstrated that F-actin predominantly exists juxtaposed to and lining the inner face of the plasma membrane (cortical actin) with a smaller amount of stress fiber and/or ruffling actin confined to the cell bottom in contact with the substratum. The extent of cortical actin disruption with various doses of either latrunculin B or Clostridium difficile toxin B (a Rho family small GTP-binding protein toxin) directly correlated with the inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. The dissolution of the cortical actin network had no significant effect on proximal insulin receptor signaling events including insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate and Cb1, or serine/threonine phosphorylation of Akt. Surprisingly, however, stabilization of F-actin with jasplakinolide also resulted in a dose-dependent inhibition of insulin-stimulated glucose uptake and GLUT4 translocation. In vivo time-lapse confocal fluorescent microscopy of actin-yellow fluorescent protein demonstrated that insulin stimulation initially results in cortical actin remodeling followed by an increase in polymerized actin in the peri-nuclear region. Importantly, the insulin stimulation of cortical actin rearrangements was completely blocked by treatment of the cells with latrunculin B, C. difficile toxin B, and jasplakinolide. Furthermore, expression of the dominant-interfering TC10/T31N mutant completely disrupted cortical actin and prevents any insulin-stimulated actin remodeling. Together, these data demonstrate that cortical actin, but not stress fibers, lamellipodia, or filopodia, plays an important regulatory role in insulin-stimulated GLUT4 translocation. In addition, cortical F-actin does not function in a static manner (e.g. barrier or scaffold), but insulin-stimulated dynamic cortical actin remodeling is necessary for the GLUT4 translocation process.

  103. Lipid raft microdomain compartmentalization of TC10 is required for insulin signaling and GLUT4 translocation 査読有り

    RT Watson, S Shigematsu, SH Chiang, S Mora, M Kanzaki, IG Macara, AR Saltiel, JE Pessin

    JOURNAL OF CELL BIOLOGY 154 (4) 829-840 2001年8月

    出版者・発行元:ROCKEFELLER UNIV PRESS

    DOI: 10.1083/jcb.200102078  

    ISSN:0021-9525

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    Recent studies indicate that insulin stimulation of glucose transporter (GLUT)4 translocation requires at least two distinct insulin receptor-mediated signals: one leading to the activation of phosphatidylinositol 3 (PI-3) kinase and the other to the activation of the small GTP binding protein TC10. We now demonstrate that TC10 is processed through the secretory membrane trafficking system and localizes to caveolin-enriched lipid raft microdomains. Although insulin activated the wildtype TC10 protein and a TC10/H-Ras chimera that were targeted to lipid raft microdomains, it was unable to activate a TC10/K-Ras chimera that was directed to the non-lipid raft domains. Similarly, only the lipid raft-localized TC10/H-Ras chimera inhibited GLUT4 translocation, whereas the TC10/K-Ras chimera showed no significant inhibitory activity. Furthermore, disruption of lipid raft microdomains by expression of a dominant-interfering caveolin 3 mutant (Cav3/DGV) inhibited the insulin stimulation of GLUT4 translocation and TC10 lipid raft localization and activation without affecting PI-3 kinase signaling. These data demonstrate that the insulin stimulation of GLUT4 translocation in adipocytes requires the spatial separation and distinct compartmentalization of the PI-3 kinase and TC10 signaling pathways.

  104. Identification of a Novel Chloride Channel Expressed in the Endoplasmic Reticulum, Golgi Apparatus, and Nucleus 査読有り

    Masahiro Nagasawa, Makoto Kanzaki, Yuichi Iino, Yasuo Morishita, Itaru Kojima

    Journal of Biological Chemistry 276 (23) 20413-20418 2001年6月8日

    DOI: 10.1074/jbc.M100366200  

    ISSN:0021-9258

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    MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. Using MID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mM KCI solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.

  105. Insulin-stimulated GLUT4 translocation requires the CAP-dependent activation of TC10 査読有り

    SH Chiang, CA Baumann, M Kanzaki, DC Thurmond, RT Watson, CL Neudauer, IG Macara, JE Pessin, AR Saltiel

    NATURE 410 (6831) 944-948 2001年4月

    出版者・発行元:MACMILLAN PUBLISHERS LTD

    DOI: 10.1038/35073608  

    ISSN:0028-0836

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    The stimulation of glucose uptake by insulin in muscle and adipose tissue requires translocation of the GLUT4 glucose transporter protein from intracellular storage sites to the cell surface(1-6). Although the cellular dynamics of GLUT4 vesicle trafficking are well described, the signalling pathways that link the insulin receptor to GLUT4 translocation remain poorly understood. Activation of phosphatidylinositol-3-OH kinase (PI(3)K) is required for this trafficking event, but it is not sufficient to produce GLUT4 translocation(7). We previously described a pathway involving the insulin-stimulated tyrosine phosphorylation of Cbl, which is recruited to the insulin receptor by the adapter protein CAP(8,9). On phosphorylation, Cbl is translocated to lipid rafts. Blocking this step completely inhibits the stimulation of GLUT4 translocation by insulin(10). Here we show that phosphorylated Cbl recruits the CrkII-C3G complex to lipid rafts, where C3G specifically activates the small GTP-binding protein TC10. This process is independent of PI(3)K, but requires the translocation of Cbl, Crk and C3G to the lipid raft. The activation of TC10 is essential for insulin-stimulated glucose uptake and GLUT4 translocation. The TC10 pathway functions in parallel with PI(3)K to stimulate fully GLUT4 translocation in response to insulin.

  106. CAP defines a second signalling pathway required for insulin-stimulated glucose transport 査読有り

    CA Baumann, Ribon, V, M Kanzaki, DC Thurmond, S Mora, S Shigematsu, PE Bickel, JE Pessin, AR Saltiel

    NATURE 407 (6801) 202-207 2000年9月

    出版者・発行元:MACMILLAN PUBLISHERS LTD

    DOI: 10.1038/35025089  

    ISSN:0028-0836

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    Insulin stimulates the transport of glucose into fat and muscle cells. Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates. One such substrate is the Cbl protooncogene product(1). Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP(2). Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction(3). Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin. Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane. Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase. Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

  107. Erratum: 'Molecular identification of a eukaryotic, stretch-activated nonselective cation channel' (Science (Aug. 6, 1999) (882)) 査読有り

    M. Kanzaki, M. Nagasawa, I. Kojima, C. Sato, K. Naruse, M. Sokabe, H. Iida

    Science 288 (5470) 1347 2000年5月26日

    DOI: 10.1126/science.288.5470.1347  

    ISSN:0036-8075

  108. The trimeric GTP-binding protein (Gq/G11) alpha subunit is required for insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes 査読有り

    M Kanzaki, RT Watson, NO Artemyev, JE Pessin

    JOURNAL OF BIOLOGICAL CHEMISTRY 275 (10) 7167-7175 2000年3月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.275.10.7167  

    ISSN:0021-9258

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    To investigate the potential role of trimeric GTP-binding proteins regulating GLUT4 translocation in adipocytes, wild type and constitutively active G(q) (G(q)/Q209L), G(i) (G(i)/Q205L), and G(s) (G(s)/Q227L) alpha subunit mutants were expressed in 3T3L1 adipocytes, Although expression of neither the wild type nor G(i)/Q205L and G(s)/Q227L alpha subunit mutants had any effect on the basal or insulin-stimulated translocation of a co-expressed GLUT4-enhanced green fluorescent protein (EGFP) fusion protein, expression of G(q)/Q209L resulted in GLUT4-EGFP translocation in the absence of insulin. In contrast, microinjection of an inhibitory G(q)/G(11) alpha subunit-specific antibody but not a G(i) or G(s) alpha subunit antibody prevented insulin-stimulated endogenous GLUT4 translocation, Consistent with a required role for GTP-bound G(q)/G(11), expression of the regulators of G protein signaling (RGS4 and RGS16) also attenuated insulin-stimulated GLUT4-EGFP translocation. To assess the relationship between G(q)/G(11) function with the phosphatidylinositol 3-kinase dependent pathway, expression of a dominant-interfering p85 regulatory subunit, as well as wortmannin treatment inhibited insulin-stimulated but not G(q)/Q209L-stimulated GLUT4-EGFP translocation, Furthermore, G(q)/Q209L did not induce the in vivo accumulation of phosphatidylinositol-3,4,5-trisphosphate (PIP3), whereas expression of the RGS proteins did not prevent the insulin-stimulated accumulation of PIP3. Together, these data demonstrate that insulin stimulation of GLUT4 translocation requires at least two independent signal transduction pathways, one mediated through the phosphatidylinositol 3-kinase and another through the trimeric GTP-binding proteins G(q) and/or G(11).

  109. Translocation of a calcium-permeable cation channel induced by insulinlike growth factor-I. 査読有り

    Kojima, I, M Kanzaki

    CONTROL AND DISEASES OF SODIUM DEPENDENT TRANSPORT PROTEINS AND ION CHANNELS 1208 243-246 2000年

    出版者・発行元:ELSEVIER SCIENCE BV

    ISSN:0531-5131

  110. Munc18e function is required for insulin-stimulated plasma membrane fusion of GLUT4 and insulin-responsive amino peptidase storage vesicles 査読有り

    DC Thurmond, M Kanzaki, AH Khan, JE Pessin

    MOLECULAR AND CELLULAR BIOLOGY 20 (1) 379-388 2000年1月

    出版者・発行元:AMER SOC MICROBIOLOGY

    ISSN:0270-7306

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    To examine the functional role of the interaction between Munc18c and syntaxin 4 in the regulation of GLUT4 translocation in 3T3L1 adipocytes, we assessed the effects of introducing three different peptide fragments (20 to 24 amino acids) of Munc18c from evolutionarily conserved regions of the Sec1 protein family predicted to be solvent exposed. One peptide, termed 18c/pep3, inhibited the binding of full-length Munc18c to syntaxin 4, whereas expression of the other two peptides had no effect. In parallel, microinjection of 18c/pep3 but not a control peptide inhibited the insulin-stimulated translocation of endogenous GLUT4 and insulin-responsive amino peptidase (IRAP) to the plasma membrane. In addition, expression of 18c/pep3 prevented the insulin-stimulated fusion of endogenous and enhanced green fluorescent protein epitope-tagged GLUT4- and IRAP-containing vesicles into the plasma membrane, as assessed by intact cell immunofluorescence. However, unlike the pattern of inhibition seen with full-length Munc18c expression, cells expressing 18c/pep3 displayed discrete clusters of GLUT4 abd IRAP storage vesicles at the cell surface which were not contiguous with the plasma membrane, Together, these data suggest that the interaction between Munc18c and syntaxin 4 is required for the integration of GLUT4 and IRAP storage vesicles into the plasma membrane but is not necessary for the insulin-stimulated trafficking to and association with the cell surface.

  111. Molecular identification of a eukaryotic, stretch-activated nonselective cation channel 査読有り

    M Kanzaki, M Nagasawa, Kojima, I, C Sato, K Naruse, M Sokabe, H Iida

    SCIENCE 285 (5429) 882-886 1999年8月

    出版者・発行元:AMER ASSOC ADVANCEMENT SCIENCE

    DOI: 10.1126/science.285.5429.882  

    ISSN:0036-8075

    eISSN:1095-9203

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    Calcium-permeable, stretch-activated nonselective cation (SA Cat) channels mediate cellular responses to mechanical stimuli. However, genes encoding such channels have not been identified in eukaryotes. The yeast MID1 gene product (Mid1) is required for calcium influx in the yeast Saccharomyces cerevisiae. Functional expression of Midi in Chinese hamster ovary cells conferred sensitivity to mechanical stress that resulted in increases in both calcium conductance and the concentration of cytosolic free calcium. These increases were dependent on the presence of extracellular calcium and were reduced by gadolinium, a blocker of SA Cat channels. Single-channel analyses with cell-attached patches revealed that Midi acts as a calcium-permeable, cation-selective stretch-activated channel with a conductance of 32 picosiemens at 150 millimolar cesium chloride in the pipette. Thus, Midi appears to be a eukaryotic, SA Cat channel.

  112. Involvement of Smad proteins in the differentiation of pancreatic AR42J cells induced by activin A 査読有り

    YQ Zhang, M Kanzaki, M Furukawa, H Shibata, M Ozeki, Kojima, I

    DIABETOLOGIA 42 (6) 719-727 1999年6月

    出版者・発行元:SPRINGER VERLAG

    ISSN:0012-186X

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    Aims/hypothesis. Activin A induces differentiation of amylase-secreting pancreatic AR42J cells into endocrine cells. This study assesses the role of Smad proteins in the actions of activin A in AR42J cells. Methods. The expression of Smad proteins was determined by northern blotting. Phosphorylation and translocation of Smad2 was measured by transfecting flag-tagged Smad2. Involvement of Smad2 was examined by transfecting cDNA encoding N-terminal and C-terminal domains of Smad2. Results. The mRNAs for Smad2 and Smad4 were abundantly expressed whereas the expression of mRNA for Smad1 and Smad3 was very low. Activin A induced serine-phosphorylation and the subsequent accumulation of the Smad2 in nuclei. Transfection of the N-terminal domain of Smad2, which acts as a dominantly negative mutant (Smad2-N), blocked the morphological changes induced by activin A whereas the C-terminal domain of Smad2, which acts as a constitutively active mutant (Smad2-C), reproduced the activin-induced morphological changes. Similarly, Smad2-N blocked apoptosis induced by activin A and Smad2-C induced apoptosis. Activin A converted AR42J into insulin-secreting cells in the presence of hepatocyte growth factor and introduction of Smad2-N inhibited the differentiation. Smad2-C, however, did not induce differentiation in the presence of hepatocyte growth factor. Conclusions/interpretation. Activation of the Smad2 pathway is necessary and sufficient to induce apoptosis and morphological changes. Although activation of the Smad2 pathway is required, it is not solely sufficient for the differentiation of AR42J into endocrine cells.

  113. Synip: A novel insulin-regulated syntaxin 4-binding protein mediating GLUT4 translocation in adipocytes 査読有り

    J Min, S Okada, M Kanzaki, JS Elmendorf, KJ Coker, BP Ceresa, LJ Syu, Y Noda, AR Saltiel, JE Pessin

    MOLECULAR CELL 3 (6) 751-760 1999年6月

    出版者・発行元:CELL PRESS

    DOI: 10.1016/S1097-2765(01)80007-1  

    ISSN:1097-2765

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    Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.

  114. Involvement of Smad proteins in the differentiation of pancretic AR42J cells induced by activin A 査読有り

    YQ Zhang, M Kanzaki, M Furukawa, H Shibata, Kojima, I

    DIABETES 48 A3-A3 1999年

    出版者・発行元:AMER DIABETES ASSOC

    DOI: 10.1007/s001250051220  

    ISSN:0012-1797

  115. Translocation of a calcium-permeable cation channel induced by insulin-like growth factor-I 査読有り

    Makoto Kanzaki, You-Qing Zhang, Hirosato Mashima, Lu Li, Hiroshi Shibata, Itaru Kojima

    Nature Cell Biology 1 (3) 165-170 1999年

    出版者・発行元:Macmillan Magazines Ltd

    DOI: 10.1038/11086  

    ISSN:1465-7392

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    Calcium plays a critical part in the regulation of cell growth, and growth factors stimulate calcium entry into cells through calcium-permeable channels. However, the molecular nature and regulation of calcium-permeable channels are still unclear at present. Here we report the molecular characterization of a calcium-permeable cation channel that is regulated by insulin-like growth factor-I (IGF-I). This channel, which we name growth-factor-regulated channel (GRC), belongs to the TRP-channel family and localizes mainly to intracellular pools under basal conditions. Upon stimulation of cells by IGF-I, GRC translocates to the plasma membrane. Thus, IGF-I augments calcium entry through GRC by regulating trafficking of the channel.

  116. Studies on the betacellulin receptor in pancreatic AR42J cells 査読有り

    N Ishiyama, M Kanzaki, M Seno, H Yamada, Kobayashi, I, Kojima, I

    DIABETOLOGIA 41 (6) 623-628 1998年6月

    出版者・発行元:SPRINGER VERLAG

    DOI: 10.1007/s001250050959  

    ISSN:0012-186X

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    Betacellulin is a member of the epidermal growth factor family and converts pancreatic AR42J cells into insulin-producing cells. This study was conducted to characterise the receptor for betacellulin in AR42J cells. AR42J cells expressed two classes of binding sites for radioactive iodine labelled betacellulin, with Kd values of 4.6 x 10(-11) mol/l and 3.0 x 10(-10) mol/l. The binding of [I-125]betacellulin was inhibited by unlabelled betacellulin in a dose-dependent manner, but epidermal growth factor was 50 fold less effective than betacellulin. Affinity cross-linking showed a [I-125]betacellulin-binding protein with a molecular weight of approximately 180 KDa. When this protein was immunoprecipitated with antibody against epidermal growth factor receptors ErbB-1, ErbB-2, ErbB-3 or ErbB-4, it was immunoprecipitated only by the anti-ErbB-1 antibody. When the [I-125]betacellulin-labelled proteins were immunoprecipitated with a combination of the four ErbB antibodies, and the unprecipitated proteins were then immunoprecipitated with anti-phosphotyrosine antibody, a 190 KDa protein was observed. Betacellulin induced the tyrosine phosphorylation of ErbB-1, ErbB-2 and ErbB-4. Finally, while 100 pmol/l betacellulin converted all of the AR42J into insulin-producing cells in the presence of activin A, 10 nmol/l epidermal growth factor induced differentiation in only about 30% of the cells. Higher concentrations of epidermal growth factor were less effective. Neu differentiation factor in the presence or absence of epidermal growth factor was ineffective. These results indicate that betacellulin binds to ErbB-1 and possibly another protein with a molecular weight of 190 KDa. The latter betacellulin-binding protein may be involved in the differentiation-inducing activity of betacellulin.

  117. Activation of calcium-permeable cation channel by insulin in Chinese hamster ovary cells expressing human insulin receptors 査読有り

    L Nie, M Kanzaki, H Shibata, Kojima, I

    ENDOCRINOLOGY 139 (1) 179-188 1998年1月

    出版者・発行元:ENDOCRINE SOC

    ISSN:0013-7227

    eISSN:1945-7170

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    The present study was conducted to examine the ability of insulin receptor to activate the calcium signaling system in Chinese hamster ovary (CHO) cells expressing human insulin receptor (CHO-IR cells). In these cells, insulin evoked the elevation of cytoplasmic free calcium concentration, [Ca2+](c), measured by using fura-2. Insulin-induced increase in [Ca2+](c) was blocked by reducing the extracellular calcium concentration to 1 mu M or by adding nickel chloride, an inorganic inhibitor of calcium entry. Insulin did not elevate [Ca2+](c) in parental CHO cells or in CHO cells expressing mutant insulin receptor lacking an ATP-binding site. When the transmembrane calcium current was measured by perforated whole-cell patch clamp, adding insulin to the bath solution markedly augmented the inward calcium current. In a cell-attached patch, a single channel activity appeared when insulin was included in the pipette. In contrast, insulin added outside the patch was ineffective. The current/voltage relationship demonstrated that insulin activated a voltage-independent calcium-permeable cation channel with a single-channel conductance of 10 pS. Exposing CHO-IR cells to pertussis toxin abolished the subsequent insulin effect on [Ca2+](c) and activation of the calcium-permeable channel. Mastoparan activated the 10-pS calcium-permeable cation channel. In an inside-out patch, insulin activated the calcium permeable channel when the bath solution contained both GTP and ATP. Nonhydrolyzable ATP could substitute for ATP. These results indicate that in CHO-IR cells, insulin elevates [Ca2+](c) by activating the 10-pS calcium-permeable cation channel. Activation by the insulin receptor involves pertussis toxin-sensitive G protein.

  118. Regulation of the expression of follistatin in rat hepatocytes 査読有り

    YQ Zhang, M Kanzaki, H Shibata, Kojima, I

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1354 (3) 204-210 1997年11月

    出版者・発行元:ELSEVIER SCIENCE BV

    DOI: 10.1016/S0167-4781(97)00085-7  

    ISSN:0167-4781

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    To elucidate the regulation of follistatin production in the liver, we studied changes in steady-state follistatin mRNA levels in cultured rat hepatocytes. Activin A stimulated follistatin mRNA levels in a time- and concentration-dependent manner. The stimulatory effect of activin A on follistatin mRNA was significant at 2 h, maximal at 6 h and declined thereafter. Incubating the cells with EGF increased follistatin mRNA levels at 48 h and later. The EGF-induced increase in follistatin mRNA was markedly inhibited by exogenous follistatin in the culture medium, which blocks the action of activin A synthesized in hepatocytes, suggesting that endogenous activin A at least partly mediated the effect of EGF. We also examined the effects of transforming growth factor-beta (TGF-beta), glucagon and alpha-adrenergic agonist, phenylephrine, on follistatin mRNA levels. TGF-beta increased the follistatin mRNA to levels similar to those caused by activin A. Phenylephrine and glucagon also increased follistatin mRNA levels but the effects were transient and weaker than those caused by activin A. Finally, follistatin mRNA levels were markedly increased in remnant liver 3 h after 70% hepatectomy. The mRNA remained elevated for up to 72 h. These results indicate that the expression of mRNA for follistatin is positively controlled by activin A, TGF-beta and other hormones or neurotransmitters. The stimulatory effect of EGF on follistatin mRNA is mediated by activin A released from hepatocytes. (C) 1997 Elsevier Science B.V.

  119. Assessment of the role of activin A and transforming growth factor beta in the regulation of AML12 cell growth 査読有り

    YQ Zhang, H Mashima, M Kanzaki, H Shibata, Kojima, I

    HEPATOLOGY 25 (6) 1370-1375 1997年6月

    出版者・発行元:W B SAUNDERS CO

    DOI: 10.1002/hep.510250612  

    ISSN:0270-9139

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    The present study was conducted to determine the role of two autocrine factors, activin A and transforming growth factor beta (TGF-beta), in the growth regulation of AML12 hepatocytes, me overexpressed truncated type II activin and/or TGF-beta receptors in AML12 cells, In AML12 cells overexpressing truncated type II activin receptors (AML-tAR cells), the inhibitory effect of activin A on DNA synthesis was completely blocked, AML-tAR cells proliferated faster than parental cells, both in the presence and absence of epidermal growth factor (EGF). However, AML-tAR cells could not grow in soft agar. Fol listatin augmented EGF-induced DNA synthesis in AML12 cells, whereas it was ineffective in AML-tAR cells, In AML12 cells overexpressing truncated type II TGF-beta receptor (AML-tTR cells), the inhibitory effect of TGF-beta on DNA synthesis was blocked. AML-tTR cells proliferated faster than parental cells, both in the presence and absence of EGF, but at a slower rate than that of AML-tAR cells, AML-tTR cells did not grow in soft agar. The growth rate of cells overexpressing both types of truncated receptors was identical to that of AML-tAR cells, and these cells did not grow in soft agar, These results indicate that both activin A and TGF-beta act as autocrine inhibitors of DNA synthesis in AML12 cells, and that the blocking of the actions of two factors does not lead to transformation. Activin A is a predominant autocrine factor in these cells.

  120. Activation of the calcium-permeable cation channel CD20 by alpha subunits of the G(i) protein 査読有り

    M Kanzaki, MA Lindorfer, JC Garrison, Kojima, I

    JOURNAL OF BIOLOGICAL CHEMISTRY 272 (23) 14733-14739 1997年6月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.272.23.14733  

    ISSN:0021-9258

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    When the calcium-permeable cation channel CD20 is expressed in Balb/c 3T3 cells, it is activated by insulin-like growth factor-I (IGF-I) via the IGF-I receptor (Kanzaki, M., Nie, L., Shibata, H., and Kojima, I. (1997) J. Biol. Chem. 272, 4964-4969). The present study was conducted to investigate the role of G proteins in the regulation of the CD20 channel. In the excised patch clamp mode, activation of the CD20 channel by IGF-I required GTP, Mg2+, and ATP in the bath solution, and removal of either GTP or ATP attenuated the activation. Non-hydrolyzable ATP could substitute for ATP, and guanyl-5'-yl thiophosphate blocked the activation of the channel by IGF-I. The CD20 channel was also activated by guanosine 5'-3-O-(thio)triphosphate, and ATP was not required for the activation. Addition of a preparation of G(i)/G(o) holoprotein purified from bovine brain activated the CD20, and the beta-adrenergic receptor kinase peptide did not affect the number of channel openings induced by the G protein. The CD20 channel was stimulated by the GTP-bound form of recombinant G(i2) alpha subunit purified from Sf9 cells. The G(i1) alpha subunit was less effective, and the G(i1) alpha subunit had no effect. Purified recombinant beta(1) gamma(2) subunits did not affect the activity of the channel. Finally, IGF-I-induced activation of CD20 was inhibited by an antibody against G(i2) alpha subunit. These findings indicate that the CD20 channel expressed in Balb/c 3T3 cells is activated by the IGF-I receptor via the alpha subunits of heterotrimeric G proteins.

  121. Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-1 査読有り

    M Kanzaki, L Nie, H Shibata, Kojima, I

    JOURNAL OF BIOLOGICAL CHEMISTRY 272 (8) 4964-4969 1997年2月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.272.8.4964  

    ISSN:0021-9258

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    CD20 functions as a calcium-permeable cation channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the G(1) progression induced by insulin-like growth factor-I (IGF-I). To further characterize how CD20 modulates the action of IGF-I, we investigated whether the activity of CD20 channel was affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased cytoplasmic free calcium concentration, [Ca2+](c), which was reversed by the removal of extracellular calcium. In contrast, IGF-I did not increase [Ca2+](c) in cells that did not express CD20. In perforated patch clamp recordings, addition of IGF-I to the bath solution augmented the Ca2+ permeability, which was reversed by anti CD20 antibody. In cell-attached patch, calcium-permeable channel activity with unitary conductance of 7 picosiemens was detected, which was abolished by anti-CD20 antibody. The single channel activities were markedly enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was blocked. Transfection of cDNA for Gip2, a constitutive active form of alpha(i2), activated the CD20 channel. These results indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.

  122. Characterization of the activin receptor in cultured rat hepatocytes 査読有り

    YQ Zhang, M Kanzaki, H Mashima, T Mine, Kojima, I

    HEPATOLOGY 24 (2) 446-450 1996年8月

    出版者・発行元:W B SAUNDERS CO

    DOI: 10.1002/hep.510240225  

    ISSN:0270-9139

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    Activin A is an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes. The present study was conducted to characterize the cell-surface receptors for activin A in cultured rat hepatocytes by measuring I-125-activin A binding, Scatchard analysis of I-125-activin A binding indicated the existence of two classes of binding sites with apparent Ma values of 3 x 10(-10) mol/L and 3.5 x 10(-9) mol/L. Pretreatment of the cells with heparitinase reduced the number of low-affinity binding sites, whereas pretreatment with excess exogenous follistatin increased the number of low-affinity binding sites. Affinity cross-linking of I-125-activin A to hepatocytes-revealed distinct protein complexes with molecular weights of approximately 48, 65, and 85 kd, which may represent cross-linked cell-bound follistatin, type I and type II activin receptors, respectively, Another band with a molecular weight of 180 kd was also found, which may represent the type III activin receptor. When hepatocytes were cultured with epidermal growth factor (EGF), both high- and low-affinity binding sites increased at 12 hours without altering their affinities, At 60 hours of the incubation with EGF, the high-affinity binding sites decreased while the number of low-affinity binding sites increased slightly. These results indicate that two classes of I-125-activin A binding sites exist in cultured hepatocytes: the high-affinity binding site may represent oligomeric complex of the type I and type II receptors, and at least part of the low-affinity binding site may represent cell-bound follistatin. The number of activin receptors in hepatocytes is increased after the stimulation with EGF.

  123. Intravenous administration of follistatin: Delivery to the liver and effect on liver regeneration after partial hepatectomy 査読有り

    K Kogure, YQ Zhang, M Kanzaki, W Omata, T Mine, Kojima, I

    HEPATOLOGY 24 (2) 361-366 1996年8月

    出版者・発行元:W B SAUNDERS CO

    DOI: 10.1002/hep.510240212  

    ISSN:0270-9139

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    When 1 mu g I-125-follistatin was administered into a rat intravenously, radioactivity levels in serum decreased rapidly, Analysis with a biexponential equation showed that the initial half-life and the terminal half-life were 4.0 and 130.8 minutes, respectively, After 2 hours of infusion, approximately 9% of the follistatin infused remained in the liver, which was much more than that in kidney, spleen, pancreas, intestine, or lung. Autoradiography of the liver obtained at 24 hours of infusion revealed that numerous grains were located in parenchymal cells, Radioactivity of I-125-follistatin in the liver remained elevated until 72 hours and declined markedly thereafter, When a booster shot of I-125-follistatin was administered at 72 hours, radioactivity in the liver at 120 hours was markedly increased compared with that in rats that received a single shot of I-125-follistatin. We then examined the effect of intravenous infusion of follistatin on liver regeneration after hepatectomy of 70%. Immediately after the hepatectomy, either 1 mu g follistatin or saline was infused intravenously. In some rats, a booster shot was infused at 72 hours. After 120 hours of hepatectomy of 70%, remnant liver weight, liver regeneration rate, and DNA content were significantly (P &lt;.0.5) higher in rats that received a booster shot of follistatin at 72 hours than those in control rats, These results indicate that follistatin administered intravenously accumulates in the liver and promotes liver regeneration after partial hepatectomy.

  124. Two distinct signaling pathways activated by activin A in glucose-responsive pancreatic beta-cells lines 査読有り

    H Shibata, M Kanzaki, T Takeuchi, J Miyazaki, Kojima, I

    JOURNAL OF MOLECULAR ENDOCRINOLOGY 16 (3) 249-258 1996年6月

    出版者・発行元:J ENDOCRINOLOGY LTD

    DOI: 10.1677/jme.0.0160249  

    ISSN:0952-5041

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    Activin A stimulates insulin secretion in pancreatic beta-cells by a calcium-dependent mechanism. The present study was conducted to further characterize the effects of activin A in two glucose-responsive insulinoma cell lines, MIN6 and HIT-T15 cells. In HIT-T15 cells, activin A evoked an increase in cytoplasmic free calcium concentration, stimulated insulin secretion, maintained glucose responsiveness of the cells and inhibited DNA synthesis. However, activin A did not have any effect in MIN6 cells. Measurement of I-125-labeled activin A binding in MIN6 cells revealed that the number of binding sites was markedly reduced, suggesting that the refractoriness was due, at least partly, to the reduced numbers of the activin receptor. Stable transfectants of MIN6 cells that overexpressed the type II activin receptor were then developed. The transfected cells (MIN6-ActR cells) expressed ten times more I-125-labeled activin A-binding sites than parental cells and the apparent K-d was 1.15 nM, which was nearly identical to that in parental cells. Affinity cross-linking in MIN6-ActR cells showed that a 90 kDa type II receptor as well as a 52 kDa protein, presumably follistatin, was markedly labeled with I-125-labeled activin A. Although MIN6-ActR cells expressed significant numbers of activin receptors, activin A did not induce immediate calcium-dependent responses in these cells. In contrast, activin A was capable of inducing long-term effects in MIN6-ActR cells; thus, reduction of the glucose concentration in culture medium from 25 to 5.5 mM for 4 days resulted in a remarkable loss of insulin response to glucose stimulation but his decline in response to glucose was prevented by the addition of activin A during culture. In addition, activin A inhibited DNA synthesis in MIN6-ActR cells. Hence, although activin A did not induce calcium-dependent responses, it evoked some calcium-independent effects in MIN6-ActR cells. Taken together, activin A elicits various effects in beta-cells by both calcium-dependent and -independent mechanism.

  125. Calcium as a second messenger of the action of transforming growth factor-beta on insulin secretion 査読有り

    N Ishiyama, H Shibata, M Kanzaki, S Shiozaki, J Miyazaki, Kobayashi, I, Kojima, I

    MOLECULAR AND CELLULAR ENDOCRINOLOGY 117 (1) 1-6 1996年3月

    出版者・発行元:ELSEVIER SCI IRELAND LTD

    DOI: 10.1016/0303-7207(95)03726-8  

    ISSN:0303-7207

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    In MIN6 insulinoma cells, transforming growth factor-beta (TGF-beta) induced the oscillatory elevation of the cytoplasmic free calcium concentration, [Ca2+](c), in the presence of 5.5 mM glucose. The increase in [Ca2+](c) induced by TGF-beta was totally dependent on calcium entry and attenuated by nifedipine or nickel chloride. In contrast, carbachol elevated [Ca2+](c) in the presence of nickel chloride. When the plasma membrane was hyperpolarized by diazoxide, TGF-beta did not raise [Ca2+](c), whereas both carbachol and depolarizing concentration of potassium elevated [Ca2+](c) under the same conditions. TGF-beta did not affect either the cellular cyclic AMP or inositol trisphosphate levels. In the presence of 5.5 mM glucose, TGF-beta induced a 3-fold increase in insulin secretion and the effect of TGF-beta was blocked by either nifedipine or nickel chloride. TGF-beta did not stimulate insulin secretion in the presence of 100 mu M diazoxide, whereas both carbachol and 40 mM potassium chloride significantly increased insulin secretion. These results suggest that TGF-beta induces the oscillatory elevation of [Ca2+](c) in MIN6 cells by stimulating calcium entry via voltage-dependent calcium channels. Calcium is an intracellular messenger of the action of TGF-beta on insulin secretion.

  126. Growth or differentiation: Determination by FSH of the action of insulin-like growth factor-I in cultured rat granulosa cells 査読有り

    M Kanzaki, M Hattori, Kojima, I

    ENDOCRINE JOURNAL 43 (1) 15-23 1996年2月

    出版者・発行元:JAPAN ENDOCRINE SOCIETY

    DOI: 10.1507/endocrj.43.15  

    ISSN:0918-8959

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    Insulin-like growth factor-I (IGF-I) is a potent mitogen in many cell systems. In cultured rat granulosa cells, however, IGF-I is known to be an inducer of differentiation. The present study was conducted to identify the factor which determines the direction of IGF-I action: either DNA synthesis or LH receptor expression. When granulosa cells were incubated with IGF-I in the presence of various concentrations of follicle-stimulating hormone (FSH), DNA synthesis as assessed by [H-3]thymidine incorporation was increased only in the presence of low doses of FSH. The stimulatory effect of FSH on DNA synthesis was observed in a very narrow range of FSH concentration between 2 and 10 ng/ml. At higher concentrations, FSH had little effect on DNA synthesis but instead induced expression of receptors for luteinizing hormone (LH), a marker of granulosa cell differentiation. At 5 ng/ml, FSH elicited maximal stimulation of DNA synthesis and simultaneously induced LH receptor expression to some extent. In these cells, DNA synthesis peaked at 36 h but expression of LH receptor occurred later than 36 h, peaking at 60 h. The ability of IGF-I to stimulate DNA synthesis was enhanced by the long term pretreatment with FSH: when FSH was added from the beginning and IGF-I was added after 36 h or later, IGF-I-mediated DNA synthesis was approximately twice as great, and was accompanied by a two-fold increase in the number of bromodeoxyuridine-labeled nuclei. Under these conditions, LH receptor expression was reduced to approximately 50%. Finally when cells were incubated for 12 h with or without FSH, washed extensively with the medium and then IGF-I was added, DNA synthesis was augmented only in FSH-primed cells. Forskolin, an activator of adenylate cyclase, reproduced the effect of FSH. These results indicate that, in the presence of FSH, IGF-I has the ability to induce both DNA synthesis and differentiation and that FSH determines the action of IGF-I on promotion of either growth or differentiation. Furthermore, priming with FSH renders granulosa cells responsive to IGF-I in terms of DNA synthesis.

  127. Norepinephrine reverses the effects of activin A on DNA synthesis and apoptosis in cultured rat hepatocytes 査読有り

    YQ Zhang, M Kanzaki, H Mashima, T Mine, Kojima, I

    HEPATOLOGY 23 (2) 288-293 1996年2月

    出版者・発行元:W B SAUNDERS CO

    DOI: 10.1002/hep.510230214  

    ISSN:0270-9139

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    Activin A, an autocrine factor produced by hepatocytes, inhibits mitogen-stimulated DNA synthesis and induces apoptotic death of cultured rat hepatocytes. Several lines of evidence indicate that norepinephrine (NE), as a comitogenic growth factor, alters the balance between growth stimulation and inhibition and acts as a trigger for the initiation of hepatocyte proliferation. In the present study, we examined whether NE modulated the effects of activin A on rat hepatocytes in primary culture. Activin A, at a concentration of 10(-9) mol/L, blocked the effect of epidermal growth factor (EGF) on DNA synthesis, that was assessed by measuring [H-3] thymidine incorporation and nuclear labeling, almost completely, and NE reversed the inhibitory effect of activin A on DNA synthesis. This effect of NE was dose-dependent, being significant at concentrations of 10(-6) mol/L and above, but was overcome by higher concentrations of activin A, and was attenuated by prazosin, but not by yohimbine or propranolol. NE exerted its effect during the first 24 hours of culture, but was ineffective when added after 24 hours. EGF augmented the release of follistatin, an activin-binding protein known to block the action of activin A, by hepatocytes and NE did not affect the amount of follistatin they released In addition to inhibiting DNA synthesis by hepatocytes cultured with EGF, activin A induced death of hepatocytes cultured in the absence of EGF. The nuclear morphology of cells cultured with activin A alone was strikingly changed compared with untreated control cells and marked identation of the nuclear membranes and moderate chromatin condensation were observed. Fragmentation of DNA was also observed, suggesting that activin A induced apoptosis, and activin-mediated cell death was prevented significantly by NE. These results indicate that NE, acting on alpha(1)-adrenergic receptors, attenuates the effects of activin A on DNA synthesis by and apoptosis of cultured rat hepatocytes.

  128. A sensitive, non-isotopic immunoassay for progesterone using the avidin-biotin system 査読有り

    M Kanzaki, A Iwasawa

    BIOMEDICAL RESEARCH-TOKYO 16 (6) 381-386 1995年12月

    出版者・発行元:BIOMED RES FOUND

    DOI: 10.2220/biomedres.16.381  

    ISSN:0388-6107

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    A non-isotopic immunoassay for progesterone has been developed using streptavidin-biotin system and time-resolved fluorometry. A biotinylated progesterone derivative and a progesterone standard or sample was allowed to react competitively with rabbit anti-progesterone in microtiter wells coated with anti-rabbit gamma-globulin (second antibody). A fluorescent product was then generated on the solid-phase by adding streptavidin labeled with europium (Eu, a lantanide element). The fluorescent complex, (second antibody)-(anti-progesterone)-(biotinylated progesterone derivative)-(Eu-labeled streptavidin) was quantified using a time-resolved fluorometer. This assay for progesterone was highly sensitive, precise and accurate. The lower detection limit was 5 pg/ml. The intra-assay coefficients of variation (CV) were 3.70-5.20% and the inter-assay CV were 7.31-8.23%. The correlation with radioimmunoassay was good (r = 0.965), This assay was used to measure the amount of progesterone that accumulated in the culture medium of rat granulosa cells. The accumulation increased with the amount of insulin-like growth factor-I added to the culture in the presence of follicle-stimulating hormone. The present assay with streptavidin-biotin system can be applied to various tracers such as enzymes and fluorescent materials. This approach should also be widely applicable to antigens and haptens that can be labeled with biotin.

  129. Blockade of DNA synthesis induced by platelet-derived growth factor by tranilast, an inhibitor of calcium entry, in vascular smooth muscle cells. 査読有り

    Nie L Mogami, H, Kanzaki M, Shibata, H, Kojima I

    Mol. Pharmacol. 50 (4) 763-769 1995年10月

  130. PRODUCTION OF ACTIVIN-A IN HUMAN INTESTINAL EPITHELIAL-CELL LINE 査読有り

    N KAWAMURA, R NOBUSAWA, H MASHIMA, M KANZAKI, H SHIBATA, KOJIMA, I

    DIGESTIVE DISEASES AND SCIENCES 40 (10) 2280-2285 1995年10月

    出版者・発行元:PLENUM PUBL CORP

    ISSN:0163-2116

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    Production of activin was studied in four cell lines of epithelial cells: FRTL-5, JCT-12, GH(4)C(1), and FHs74Int cells. Bioactivity of activin was detected in conditioned media of FRTL-5, JCT-12 and FHs74Int cells. Among these three cell lines, FHs74Int cells, which were derived from human embryonic intestine, released a relatively large amount of bioactive activin. In these cells, serum and epidermal growth factor (EGF), which were capable of stimulating DNA synthesis, augmented release of bioactive activin in middle to late G(1) phase. In addition, basic FGF (bFGF), which had no effect on DNA synthesis in these cells, also increased release of activin. In bFGF-treated FHs74Int cells, bioactive activin was released within 4 hr of the addition of bFGF. The reverse-transcription polymerase chain reaction reveals that mRNA for only the beta(A) subunit of activin is expressed in these cells. Immunoblotting of lysate from serum-treated cells using anti-human activin A antibody indicated the existence of a 12.5-kDa protein under a reducing condition. FHs74Int cells did not express binding site for [I-125]activin A and exogenous activin A did not affect DNA synthesis in these cells. These results indicate that FHs74Int cells derived from human embryonic intestine synthesize and release activin A. Activin A released from intestinal epithelial cells might be a modulatory factor in cells in intestinal mucosa.

  131. MODULATION OF ADENOSINE TRIPHOSPHATE-SENSITIVE POTASSIUM CHANNEL AND VOLTAGE-DEPENDENT CALCIUM-CHANNEL BY ACTIVIN-A IN HIT-T15 CELLS 査読有り

    H MOGAMI, M KANZAKI, R NOBUSAWA, YQ ZHANG, M FURUKAWA, KOJIMA, I

    ENDOCRINOLOGY 136 (7) 2960-2966 1995年7月

    出版者・発行元:ENDOCRINE SOC

    ISSN:0013-7227

    eISSN:1945-7170

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    The ATP-sensitive potassium channel(K-ATP channel) determines the membrane potential of pancreatic beta-cells and plays a critical role in the regulation of insulin secretion. The present study was conducted to investigate the effect of activin A, a member of the transforming growth factor-beta supergene family, on the K-ATP channel in HIT-T15 clonal hamster insulinoma cells. In an excised inside-out patch, ATP-sensitive currents with a single channel conductance of approximately 20 picosiemens were observed. In an outside-out-patch, currents with identical unitary conductance were also observed. In either case, the currents were augmented by diazoxide and blocked by glibenclamide, verifying that they were K-ATP channel currents. When K-ATP channel currents were monitored in an outside-out patch, activin A added to the bath solution inhibited K-ATP channel currents. Upon removal of activin A, the K-ATP channel currents were restored, suggesting that the inhibition was not due simply to spontaneous disappearance of channel activity (run-down). The K-ATP channel activity was markedly reduced after the addition of activin A and was reversed by diazoxide. Besides the inhibition of K-ATP channel, activin A increased, in a perforated patch, the amplitude of the inward Ba2+ current in response to a depolarizing pulse from -40 to +10 mV. Under the current clamp condition, activin A induced gradual depolarization, followed by a burst of action potentials. Activin-mediated action potentials were accompanied by an elevation of the cytoplasmic free calcium concentration. These results indicate that activin A causes depolarization of the plasma membrane by inhibiting the activity of the K-ATP channel. In addition, activin A directly modulates the voltage-dependent calcium channel and augments calcium entry.

  132. EXPRESSION OF CALCIUM-PERMEABLE CATION CHANNEL CD20 ACCELERATES PROGRESSION THROUGH THE G(1) PHASE IN BALB/C 3T3 CELLS 査読有り

    M KANZAKI, H SHIBATA, H MOGAMI, KOJIMA, I

    JOURNAL OF BIOLOGICAL CHEMISTRY 270 (22) 13099-13104 1995年6月

    出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

    DOI: 10.1074/jbc.270.22.13099  

    ISSN:0021-9258

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    CD20 is a transmembrane protein that functions as a Ca2+-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. D., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the cDNA encoding a Ca2+-permeable cation channel, CD20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing cDNA encoding CD20 expressed the CD20 protein, which was detected by assaying the binding of a monoclonal antibody against CD20. Calcium-permeable cation channel activity was detected in CDaO-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of CD20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF-I). IGF-I induced DNA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA synthesis by 30% of the quiescent CD20 expressing cells was initiated in response to IGF-I in the absence of priming with PDGF and EGF. When control quiescent cells were primed with PDGF and EGF, the addition of IGF-I led to the initiation of DNA synthesis after 14 h or more, whereas it induced DNA synthesis by CD20 expressing cells primed with PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent on extracellular Ca2+, and expression of CD20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, DNA synthesis by approximately 25% of the CD20-expressing cells was initiated after priming with PDGF and EGF, even in the absence of the progression factor IGF-I. These results indicate that CD20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca2+-permeable cation channel and that expression of CD20 accelerates G(1) progression in a Ca2+-dependent manner.

  133. A SINGLE INTRAPORTAL ADMINISTRATION OF FOLLISTATIN ACCELERATES LIVER-REGENERATION IN PARTIALLY HEPATECTOMIZED RATS 査読有り

    K KOGURE, W OMATA, M KANZAKI, YQ ZHANG, H YASUDA, T MINE, KOJIMA, I

    GASTROENTEROLOGY 108 (4) 1136-1142 1995年4月

    出版者・発行元:W B SAUNDERS CO

    ISSN:0016-5085

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    Background/Aims: Activin A is an autocrine negative regulator of DNA synthesis in rat hepatocytes and is expressed in remnant liver after partial hepatectomy. To determine the role of activin A in liver regeneration, the effects of exogenous follistatin, which blocks the action of activin A, were examined. Methods: Human recombinant follistatin was infused into the portal Vein immediately after 70% hepatectomy. Changes in body weight, remnant liver weight, liver regeneration rate, and nuclear bromodeoxyuridine labeling were measured. Results: In control rats, nuclear labeling was observed at 24 hours and peaked at 36 hours after the hepatectomy. In follistatin-treated rats, nuclear labeling was first observed after 18 hours and was significantly (P &lt; 0.05) greater than that in control rats at 24 hours. In follistatin-treated rats, both remnant liver weight and river regeneration rate were significantly greater at 120 hours. Serum concentrations of albumin and glucose remained reduced for up to 120 hours in control rats but recovered in follistatin-treated rats. Conclusion: A single administration of follistatin accelerates the initial round of DNA synthesis after partial hepatectomy. Activin A produced in remnant liver may exert tonic inhibitory effect on liver regeneration. Follistatin may be useful as a potential therapeutic agent to promote liver regeneration.

  134. DERANGEMENTS IN THE ACTIVIN-FOLLISTATIN SYSTEM IN HEPATOMA-CELLS 査読有り

    H MASHIMA, M KANZAKI, R NOBUSAWA, YQ ZHANG, M SUZUKI, T MINE, KOJIMA, I

    GASTROENTEROLOGY 108 (3) 834-840 1995年3月

    出版者・発行元:W B SAUNDERS CO

    ISSN:0016-5085

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    Background/Aims: The growth of normal hepatocytes is regulated by the activin-follistatin system. The aim of this study was to investigate the activin-follistatin system in hepatoma cells. Methods: The production and action of activin and follistatin in human hepatoma cell lines were examined. Activin A and follistatin were measured by bioassay and protein-binding assay, respectively. Results: Activin A inhibited cell growth in HepG2 cells but not in either PLC/PRF/5 or HLE cells. However, the effect of activin A in HepG2 cells was attenuated at high cell density. In HepG2 cells, two classes of activin-binding sites were expressed, and affinity cross-linking showed that I-125-activin A bound specifically to three proteins with molecular weights of 48, 67, and 94 kilodaltons. In PLC/PRF/5 cells, a single class of binding site was observed, and the binding capacity was approximately 60% of the capacity in HepG2 cells. Virtually no I-125-activin A binding was detected in HLE cells. Bioactivity and messenger RNA for activin A were undetectable in three cell lines. In contrast, follistatin was released from three cell lines. Conclusions: Multiple alterations in the activin-follistatin system were found in three hepatoma cell lines. The accelerated growth observed in hepatoma cells may be caused, at least partly, by the attenuation of the action of activin A.

  135. PRODUCTION OF ACTIVIN-A AND FOLLISTATIN IN CULTURED RAT VASCULAR SMOOTH-MUSCLE CELLS 査読有り

    M KANZAKI, R NOBUSAWA, H MOGAMI, H YASUDA, N KAWAMURA, KOJIMA, I

    MOLECULAR AND CELLULAR ENDOCRINOLOGY 108 (1-2) 11-16 1995年2月

    出版者・発行元:ELSEVIER SCI PUBL IRELAND LTD

    DOI: 10.1016/0303-7207(94)03451-X  

    ISSN:0303-7207

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    Activin A, a member of the transforming growth factor beta supergene family, modulates DNA synthesis in cultured rat vascular smooth muscle cells (VSMC) (Kopma et al. (1993) Exp. Cell. Res. 206, 152-156). In the present study, we studied the production of activin A and follistatin in VSMC. When VSMCs cultured in a 24-well plate were cultured with 10% fetal calf serum (FCS) for 24 h, 0.94 +/- 0.20 pmol/well (mean +/- SE, n = 6) of bioactive activin was released into the culture media. Reverse-transcription polymerase chain-reaction revealed the expression of mRNA for the beta(A) subunit of inhibin but not for either the beta(B) or alpha subunit. Bioactivity of activin was increased in quiescent cells treated with FCS or platelet-derived growth factor (PDGF) but not with angiotensin II (Ang II) or insulin-like growth factor-I (IGF-I). Ang II or IGF-I did not stimulate DNA synthesis by itself but, when these two agents were combined, they increased nuclear labeling by 16.4% and release of bioactive activin by 170% of basal. The dose-response relationship and time course study indicated that PDGF-mediated release of activin correlated with initiation of DNA synthesis. Steady state expression of mRNA for the beta(A) subunit was markedly elevated 12 h after the addition of PDGF and was reduced thereafter. To assess the significance of autocrine activin, the effect of PDGF was determined in the presence and absence of excess of exogenous follistatin. The PDGF-mediated DNA synthesis was enhanced by the addition of excess follistatin. In addition to the production of activin, PDGF also enhanced release of follistatin from VSMC. These results indicate that VSMC synthesize and release both activin and follistatin and that the activin-follistatin system may modulate DNA synthesis in VSMC.

  136. Stimulation of Follistatin Production by Epidermal Growth Factor in Cultured Rat Hepatocytes 査読有り

    Makoto Kanzaki, Zhang You-Qing, Tetsuya Mine, Itaru Kojima

    Biochemical and Biophysical Research Communications 202 (1) 422-428 1994年7月15日

    DOI: 10.1006/bbrc.1994.1945  

    ISSN:1090-2104 0006-291X

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    Production of follistatin in cultured rat hepatocytes was studied by measuring follistatin release with a protein-binding assay using [125I]activin A. Follistatin was detected in conditioned medium of cultured hepatocytes. Ligand blotting using [125I]activin revealed that follistatin released into the medium consisted of two different forms with molecular weight of approximately 40 K-Da. Epidermal growth factor (EGF) elicited dose-dependent increases in DNA synthesis and follistatin release. Dose-response relationship for EGF-induced follistatin release correlated well with that for EGF-induced DNA synthesis. In EGF-stimulated cells, a marked increase in DNA synthesis occurred after 48 hrs. Similarly, follistatin release was markedly augmented after 48 hrs. Amount of cell-bound follistatin was not changed by the treatment with EGF. These results indicate that cultured hepatocytes synthesize and release follistatin. The activin-follistatin system operates in cultured rat hepatocytes and may modulate DNA synthesis by altering the action of activin A. © 1994 Academic Press, Inc.

  137. HUMAN GROWTH-HORMONE AUGMENTATION OF EPIDERMAL GROWTH-FACTOR BINDING-SITES ON RAT GRANULOSA-CELLS 査読有り

    MA HATTORI, Y SHINOHARA, E YOSHINO, M KANZAKI, KOJIMA, I, R HORIUCHI

    JOURNAL OF ENDOCRINOLOGY 142 (1) 69-75 1994年7月

    出版者・発行元:J ENDOCRINOLOGY LTD

    DOI: 10.1677/joe.0.1420069  

    ISSN:0022-0795

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    The effect of human GH (hGH) on the regulation of epidermal growth factor (EGF) receptor was investigated during differentiation of FSH-treated rat granulosa cells, which has been reported to be mediated by a cAMP-dependent mechanism. By measuring the binding of [I-125]iodo-EGF to the intact cells, FSH was shown to cause increases in the number of EGF binding sites after culture for 72 h. When granulosa cells were cultured with hGH, the number of FSH-induced EGF binding sites was augmented, with a half-maximal effect at about 10 mu g hGH/l and a maximal stimulatory concentration of 100 mu g/l. The stimulatory effect of hGH was absolutely dependent on insulin which by itself showed stimulatory effects on EGF binding sites. Scatchard analysis of EGF binding sites indicated that treatment with hGH increased the number of EGF binding sites (17 200 sites/cell after treatment with FSH; 31 700 sites/cell after FSH plus hGH), but did not alter the binding affinity. The augmentation was observed after culturing for 48 h and increased progressively with time, reaching 280% of the level after FSH treatment by 120 h. Although progesterone synthesis was increased by hGH, the markers of cell differentiation such as cAMP synthesis and LH binding sites were suppressed, indicating hGH inhibition of the cAMP-mediated signal. The action of hGH on the EGF binding sites was not accompanied by cell proliferation. These findings indicate that hGH has a novel action on the regulation of rat granulosa cell EGF binding sites and that the granulosa cell may possess both cAMP-dependent and -independent mechanisms for expression of EGF binding sites.

  138. COORDINATE ACTIONS OF FSH AND INSULIN-LIKE GROWTH-FACTOR-I ON LH RECEPTOR EXPRESSION IN RAT GRANULOSA-CELLS 査読有り

    M KANZAKI, MA HATTORI, R HORIUCHI, KOJIMA, I

    JOURNAL OF ENDOCRINOLOGY 141 (2) 301-308 1994年5月

    出版者・発行元:J ENDOCRINOLOGY LTD

    DOI: 10.1677/joe.0.1410301  

    ISSN:0022-0795

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    The actions of FSH and Insulin-like growth factor-I (IGF-I) were studied in cultured rat ovarian granulosa cells. Cells became differentiated and expressed LH receptors when they were incubated for 72 h with 200 mu g FSH/1 (high FSH) but not 20 mu g FSH/1 (low FSH). Treatment with high but not low FSH increased the release of both immunoreactive and bioactive IGF-I into the medium. A combination of low FSH and IGF-I reproduced the effect of high FSH on LH receptor expression. We then examined the critical time when low FSH and IGF-I exerted their effects. In the presence of continuous low FSH, IGF-I was capable of inducing LH receptor expression even when added 24 h after the addition of low FSH. However, when IGF-I was added at 36 h, LH receptor expression measured at 72 h was greatly reduced. In contrast to the action of IGF-I, continuous exposure to low FSH was required for LH receptor expression, and IGF-I had no effect when FSH was not included for the entire 72 h of culture. DNA synthesis as assessed by both [H-3]thymidine incorporation and nuclear bromodeoxyuridine labelling was moderate at the beginning of culture and markedly reduced at 24 h both in the presence and absence of either high FSH or low FSH plus IGF-I. In the presence of either high FSH or a combination of low FSH plus IGF-I, DNA synthesis remained decreased for up to 72 h whereas it began to increase in the absence of either high FSH or a combination of low FSH plus IGF-I. A similar increase in DNA synthesis was observed after 48 h when granulosa cells were treated with low FSH alone, which did not induce LH receptor expression. These results indicate that 1) growth and differentiation of granulosa cells are regulated inversely; 2) FSH and IGF-I act together to induce LH receptor expression; and 3) action of IGF-I is dependent on the presence of FSH.

  139. BASIC FIBROBLAST GROWTH-FACTOR INDUCES LUTEINIZING-HORMONE RECEPTOR EXPRESSION IN THE PRESENCE OF INSULIN-LIKE GROWTH-FACTOR-I IN OVARIAN GRANULOSA-CELLS 査読有り

    M KANZAKI, M HATTORI, R HORIUCHI, KOJIMA, I

    MOLECULAR AND CELLULAR ENDOCRINOLOGY 101 (1-2) 95-99 1994年5月

    出版者・発行元:ELSEVIER SCI IRELAND LTD

    DOI: 10.1016/0303-7207(94)90223-2  

    ISSN:0303-7207

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    Effect of basic fibroblast growth factor (bFGF) on the expression of receptors for luteinizing hormone (LH), a marker of differentiation, was studied using estrogen-primed rat ovarian granulosa cells in primary culture. bFGF had no effect by itself but dose-dependently induced expression of functional LH receptors in the presence of insulin-like growth factor-I (IGF-I). The effect of a combination of bFGF and IGF-I was delayed in onset and the magnitude of the response was smaller when compared to the action of follicle-stimulating hormone (FSH). Scatchard analysis revealed that dissociation constant (K-d) and number of LH receptors induced by bFGF and IGF-I were 0.47 nM and 6.48 fmol/10(6) cells, respectively. Unlike FSH, bFGF plus IGF-I did not cause an immediate increase in cAMP release, however, considerable amount of cAMP release was observed in cells incubated for 72 h with bFGF plus IGF-I. Indomethacin, an inhibitor of cyclooxygenase, attenuated both LH receptor expression and cAMP release induced by bFGF plus IGF-I but had little effect on the action of FSH. Finally, a combination of bFGF and IGF-I increased production of prostaglandin E(2) in granulosa cells. These results indicate that bFGF is capable of inducing LH receptor in the presence of IGF-I by a mechanism involving production of prostaglandin E(2).

  140. GRANULOSA-CELL LUTEINIZING-HORMONE RECEPTOR EXPRESSION IS MODULATED BY GANGLIOSIDE-SPECIFIC LIGANDS 査読有り

    M HATTORI, M KANZAKI, KOJIMA, I, R HORIUCHI

    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 1221 (1) 47-53 1994年3月

    出版者・発行元:ELSEVIER SCIENCE BV

    ISSN:0167-4889

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    The ganglioside GM1 (Gal beta 1 --&gt; 3GalNAc beta 1 --&gt; 4[NeuAc alpha 2 --&gt; 3]Gal beta 1 --&gt; 4Glc beta 1 --&gt; 1Cer) was synthesized during granulosa cell development in vitro, and the effect of the interaction between cell-surface GM1 and its ligands on the luteinizing hormone (LH) receptor expression was investigated. GM1 synthesis, demonstrated by metabolic labeling of glycosphingolipids with [H-3]galactose and binding studies using the I-125-B-subunit of cholera toxin, a specific ligand for GM1, was increased in follicle-stimulating hormone (FSH)-treated granulosa cells. When granulosa cells were cultured for 72 h in a medium containing the B-subunit of cholera toxin, FSH-induced LH-receptor contents determined by measuring the binding of I-125-deglycosylated human chorionic gonadotropin to intact cells, was augmented. The stimulatory effect of the B-subunit was dependent on the FSH concentration and culture duration. The augmentation was observed after culture for 48 h, and marked increases were evident after 72 h, which coincided with an increase of the I-125-B-subunit binding capacity. Scatchard analysis of the LH-receptor binding indicated that treatment with the B-subunit increased the number of LH-binding sites (6580 sites/cell after treatment with 20 ng/ml FSH; 11290 sites/cell after FSH plus 100 ng/ml B-subunit), but did not alter the binding affinity. A specific antibody against GM1 mimicked the stimulatory effect of the B-subunit. The augmentation was not accompanied by granulosa cell proliferation. These findings suggest that binding of exogenous or possible endogenous ligands to cell-surface GM1 produces signals and modulates the cellular behavior during granulosa cell development.

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  1. 【健康寿命の鍵を握る骨格筋 代謝・内分泌を介した全身性制御の分子基盤から運動による抗老化まで】(第7章)骨格筋と他臓器の連関 好中球が骨格筋の糖代謝および運動能力におよぼす作用 免疫機能との連関

    神崎 展

    実験医学 40 (2) 336-344 2022年2月

    出版者・発行元:(株)羊土社

    ISSN:0288-5514

    詳細を見る 詳細を閉じる

    最近われわれは、運動中の骨格筋組織の毛細血管網内には好中球が一過性に集積する微小領域(運動筋ニッチ)が存在し、この局所における骨格筋線維(筋細胞)と好中球や血管内皮細胞を含む非筋細胞間の連携が骨格筋機能の保持に不可欠であることを見出している。驚くべきことに、この運動筋ニッチでは生活習慣病の増悪因子として有名なIL-1が良性作用を発揮し、運動骨格筋の糖取込亢進とマイオカイン発現を誘導して結果的に運動持久力を高める働きがあることが明らかになってきた。(著者抄録)

  2. 液中プラズマ・パルス電場複合法による細胞内高効率遺伝子導入

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 69th 2022年

    ISSN:2436-7613

  3. ヒト収縮型培養筋細胞系で見出した新規マイオカインRspo3の作用機序

    高橋 忠久, 萩原 嘉廣, 神崎 展, 土谷 昌広, 小出 将志, 関口 拓矢, 小川 和美, 李 雨晴, 井樋 栄二, 相澤 俊峰

    日本整形外科学会雑誌 95 (8) S1720-S1720 2021年8月

    出版者・発行元:(公社)日本整形外科学会

    ISSN:0021-5325

  4. 筋・筋膜痛における好中球細胞外トラップの関与

    鈴木 一瑛, 土谷 昌広, 神崎 展, 吉田 新一郎, 綿貫 宗則, 矢部 裕, 藤田 涼, 高橋 忠久, 萩原 嘉廣, 井樋 栄二, 相澤 俊峰

    日本整形外科学会雑誌 95 (8) S1778-S1778 2021年8月

    出版者・発行元:(公社)日本整形外科学会

    ISSN:0021-5325

  5. 液相中プラズマを活用した遺伝子導入法開発に関する研究

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 68th 2021年

    ISSN:2436-7613

  6. 運動筋ニッチが司る運動免疫ネットワーク制御の解明

    神崎 展

    上原記念生命科学財団研究報告集 34 1-5 2020年12月

    出版者・発行元:(公財)上原記念生命科学財団

    eISSN:2433-3441

  7. GLUT4細胞膜移行を制御するインスリン初期作用過程の高精度可視化解析

    神崎 展, 畠山 裕康

    糖尿病 63 (Suppl.1) S-348 2020年8月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  8. 高効率な細胞内分子導入における液相中プラズマの活用

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    プラズマ・核融合学会年会(Web) 37th 2020年

  9. 液相中プラズマ直接照射による薬剤模擬分子導入の作用機序に関する研究

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 67th 2020年

    ISSN:2436-7613

  10. 液相中プラズマを活用した中分子・遺伝子の高効率導入

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 81st 2020年

    ISSN:2436-7613

  11. 免疫伝達物質cGAMPのトランスポーターSLC19Aの同定

    神﨑 展

    実験医学1月号 (2020) 38 (1) 60-61 2020年1月

  12. 脂肪組織における糖代謝に対するLRRK2の生理的役割の解析

    今井 基貴, 川上 文貴, 井阪 勇輝, 川島 麗, 前川 達則, 神崎 展

    日本生化学会大会プログラム・講演要旨集 92回 [1P-300] 2019年9月

    出版者・発行元:(公社)日本生化学会

  13. 長期粉末食飼育マウスにおける結腸機能と免疫細胞の関連性

    八百板富紀枝, 宮澤将之, 土谷昌広, 土谷忍, 神崎展

    日本薬理学会北部会 2019年9月

  14. 筋機能を制御する異種細胞連関 運動骨格筋内微小領域(運動筋ニッチ)における好中球の役割

    神崎 展

    日本筋学会学術集会プログラム・抄録集 5回 40-40 2019年8月

    出版者・発行元:日本筋学会

    ISSN:2433-975X

  15. ヒト筋衛星細胞を用いた「in vitro Exerciseモデル」の構築

    神崎展

    月刊メディカル・サイエンス・ダイジェスト 45 (6) 358‐361 2019年6月25日

    ISSN:1347-4340

  16. 炎症性筋疾患におけるミトコンドリア機能異常の検討とその治療法開発

    及川善嗣, 井泉瑠美子, 小出将志, 萩原嘉廣, 神崎展, 鈴木直輝, 青木正志, 阿部高明, 阿部高明, 阿部高明

    日本NO学会学術集会プログラム抄録集 19th 67 2019年6月1日

  17. CuCrO<sub>2</sub>薄膜の抗菌効果の検討

    大野航太朗, 岡田健, 川島知之, 神崎展, 鷲尾勝由

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 66th ROMBUNNO.11p‐S011‐17 2019年2月25日

  18. 2型糖尿病治療薬メトホルミンの肝臓における真なる薬理機序

    神﨑 展

    実験医学1月号 (2019) 37 (1) 60-61 2019年1月

  19. 液相中プラズマによる薬剤模擬分子導入の作用機序の探索

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 79th ROMBUNNO.21a‐144‐3 2018年9月5日

    ISSN:2436-7613

  20. イタコン酸によるマクロファージ免疫代謝制御系の解明

    神﨑 展

    実験医学7月号 (2018) 36 (11) 1878-1879 2018年7月

  21. マウス筋機械痛覚過敏に対するIL-18の関与

    土谷 昌広, 吉田 新一郎, 萩原 嘉廣, 篠田 雅路, 小出 将志, 畠山 裕康, チャウイワンナコン・チャヤニ, 矢野 利尚, 曽木 靖仁, 板谷 信行, 関口 拓矢, 矢部 裕, 土谷 忍, 佐々木 啓一, 神崎 展, 井樋 栄二

    PAIN RESEARCH 33 (2) 160-160 2018年6月

    出版者・発行元:日本疼痛学会

    ISSN:0915-8588

  22. マウス筋機械痛覚過敏に対するIL-18の関与

    土谷 昌広, 吉田 新一郎, 萩原 嘉廣, 篠田 雅路, 小出 将志, 畠山 裕康, チャウイワンナコン・チャヤニ, 矢野 利尚, 曽木 靖仁, 板谷 信行, 関口 拓矢, 矢部 裕, 土谷 忍, 佐々木 啓一, 神崎 展, 井樋 栄二

    PAIN RESEARCH 33 (2) 160-160 2018年6月

    出版者・発行元:日本疼痛学会

    ISSN:0915-8588

  23. 多角的光学イメージング計測により解明したマウス単離骨格筋線維におけるGLUT4輸送制御とインスリン初期作用

    畠山 裕康, 神崎 展

    糖尿病 61 (Suppl.1) S-132 2018年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  24. 薬剤分子導入へ向けた液相中プラズマの電極構造の最適化

    本田竜介, 佐々木渉太, 高島圭介, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 65th ROMBUNNO.19p‐C201‐11 2018年3月5日

    ISSN:2436-7613

  25. プラズマ分子導入の高効率化に向けた短寿命活性種の測定

    ZHENG Yuexing, 佐々木渉太, 神崎展, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 65th ROMBUNNO.19p‐C201‐10 2018年3月5日

    ISSN:2436-7613

  26. 気液界面プラズマ生成複合刺激による細胞膜輸送促進の機構解明

    金子俊郎, 佐々木渉太, 神崎展

    日本光医学・光生物学会 40th (Web) 2018年

  27. 自然免疫DNAセンサー(cGAS)による細胞老化の制御

    神﨑 展

    実験医学1月号 (2018) 36 (1) 60-61 2018年1月

  28. 気液界面プラズマによる細胞機能制御 -低侵襲高効率プラズマ薬剤分子導入にむけて- 査読有り

    金子俊郎, 佐々木渉太, 本田竜介, 佐藤岳彦, 神崎展

    表面と真空 61 (3) 143-149 2018年

    DOI: 10.1380/vss.61.143  

    ISSN:2433-5843

  29. 気液界面プラズマによる細胞機能制御 -低侵襲高効率プラズマ薬剤分子導入にむけて-

    金子俊郎, 佐々木渉太, 本田竜介, 佐藤岳彦, 神崎展

    表面と真空 61 (3) 143‐149(J‐STAGE) 2018年

    DOI: 10.1380/vss.61.143  

    ISSN:2433-5835

  30. 大気圧プラズマ反応性Ca<sup>2+</sup>透過性チャンネルの同定

    川瀬将義, 川口航汰, 畠山裕康, 佐々木渉太, 金子俊郎, 神崎展

    日本分子生物学会年会プログラム・要旨集(Web) 41st ROMBUNNO.2P‐0335 (WEB ONLY) 2018年

  31. プラズマ照射緩衝液中で活性化されるラジカル連鎖反応の解明

    佐々木渉太, ZHENG Yuexing, 本田竜介, 高島圭介, 神崎展, 金子俊郎

    プラズマ・核融合学会年会(Web) 35th ROMBUNNO.5Cp04 (WEB ONLY) 2018年

  32. 腱板断裂における脂肪変性を伴う筋組織中のヒト筋衛星細胞の分化

    小出 将志, 萩原 嘉廣, 安藤 晃, 関口 拓矢, 金澤 憲治, 土谷 昌広, 神崎 展, 井樋 栄二

    日本肩関節学会抄録集 44回 130-130 2017年10月

    出版者・発行元:(一社)日本肩関節学会

  33. プラズマ由来複合刺激を用いた細胞膜輸送の能動的制御

    佐々木渉太, ZHENG Yuexing, 本田竜介, 高島圭介, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 78th ROMBUNNO.6a‐S22‐2 2017年8月25日

    ISSN:2436-7613

  34. 薬剤導入に寄与するプラズマ照射溶液中活性種の探究

    ZHENG Yuexing, 佐々木渉太, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 78th ROMBUNNO.7p‐PA5‐2 2017年8月25日

    ISSN:2436-7613

  35. 電気パルス刺激を用いた収縮型培養筋細胞の創製

    神﨑 展

    高度物理刺激と生体応答 (佐藤岳彦・大橋俊郎・川野聡恭・白樫了 編著) 2017年8月1日

    出版者・発行元:養賢堂

  36. タンパク質の品質管理と寿命決定-CHIPによるインスリン受容体の恒常性制御-

    神﨑 展

    実験医学7月号 (2017) 35 (11) 1841-1842 2017年7月1日

    出版者・発行元:羊土社

  37. プラズマ・メカノバイオロジーの医用応用に向けた科学的基盤の構築 疾患治療からドラッグデリバリーまで 大気圧低温プラズマによるTRPチャネル活性化と遺伝子導入

    金子 俊郎, 佐々木 渉太, 鄭 悦星, 神崎 展

    日本薬学会年会要旨集 137年会 (1) 160-160 2017年3月

    出版者・発行元:(公社)日本薬学会

    ISSN:0918-9823

  38. 異種気液界面プラズマによる細胞膜透過性促進の比較

    金子俊郎, 佐々木渉太, 保苅雄太郎, ZHENG Yuexing, 高島圭介, 神崎展

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 64th ROMBUNNO.16a‐313‐4 2017年3月1日

    ISSN:2436-7613

  39. 動脈硬化症と老化細胞除去療法の可能性

    神﨑 展

    実験医学1月号 (2017) 35 (1) 57-58 2017年1月1日

    出版者・発行元:羊土社

  40. 大気圧プラズマ照射溶液によるヒト大腸がんCaco‐2細胞の細胞膜輸送変化

    佐野大知, 立川正憲, 佐々木渉太, 神崎展, 寺崎哲也, 金子俊郎

    生体膜と薬物の相互作用シンポジウム講演要旨集 38th 140 2016年11月17日

    ISSN:0919-2131

  41. 細胞膜輸送制御に向けたプラズマ誘起活性種・電位分布計測

    佐々木渉太, 保苅雄太郎, 高島圭介, 熊田亜紀子, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 77th ROMBUNNO.15p‐B7‐15 2016年9月1日

    ISSN:2436-7613

  42. プラズマ照射溶液中短寿命活性種に対するTRPチャネル介在細胞内カルシウム応答

    佐々木渉太, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 77th ROMBUNNO.16p‐P4‐10 2016年9月1日

    ISSN:2436-7613

  43. 細胞膜輸送に対する液相中プラズマの物理的・化学的刺激の効果

    保苅雄太郎, 佐々木渉太, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 77th ROMBUNNO.15p‐B7‐14 2016年9月1日

    ISSN:2436-7613

  44. 廃用萎縮・脂肪変性した筋組織中の筋・脂肪前駆細胞の分化能の評価

    小出 将志, 萩原 嘉廣, 矢部 裕, 安藤 晃, 金澤 憲治, 関口 拓矢, 板谷 信行, 吉田 新一郎, 神崎 展, 畠山 裕康, 土谷 昌広, 井樋 栄二

    日本整形外科学会雑誌 90 (8) S1543-S1543 2016年8月

    出版者・発行元:(公社)日本整形外科学会

    ISSN:0021-5325

  45. インスリンシグナルに関わるメンブレントラッフィック

    畠山裕康, 神﨑 展

    DOJIN BIOSCIENCE:メンブレントラッフィック・膜/小胞による細胞内輸送ネットワーク 170-183 2016年7月15日

    出版者・発行元:化学同人

  46. 微小管の脱チロシン化修飾と心筋メカノバイオロジー

    神﨑 展

    実験医学7月号 (2016) 34 (11) 1767-1768 2016年7月1日

    出版者・発行元:羊土社

  47. 大気圧プラズマ照射によるヒト大腸がん細胞の細胞膜輸送変動機構の解明

    立川 正憲, 佐野 大知, 佐々木 渉太, 神崎 展, 寺崎 哲也, 金子 俊郎

    日本薬剤学会年会講演要旨集 31年会 171-171 2016年5月

    出版者・発行元:(公社)日本薬剤学会

  48. 骨格筋における糖輸送体GLUT4挙動のライブイメージングと運動効果の基盤解析

    畠山 裕康, 堰合 茂智, 神崎 展

    糖尿病 59 (Suppl.1) S-305 2016年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  49. 膵内副交感神経節と膵島の組織学的関係

    高橋 啓範, 今井 淳太, 井泉 知仁, 山本 淳平, 川名 洋平, 遠藤 彰, 菅原 裕人, 洲崎 悦生, 畠山 裕康, 澤田 正二郎, 山田 哲也, 上田 泰己, 神崎 展, 片桐 秀樹

    糖尿病 59 (Suppl.1) S-158 2016年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  50. 嗅覚受容体は膵β細胞に発現しグルコース応答性インスリン分泌を促進する

    宗像 佑一郎, 山田 哲也, 今井 淳太, 突田 壮平, 高橋 圭, 白井 勇太, 児玉 慎二郎, 浅井 洋一郎, 椙澤 貴志, 千葉 弓子, 高橋 広延, 穂坂 真一郎, 井泉 知仁, 高 俊弘, 宇野 健司, 澤田 正二郎, 畠山 裕康, 神崎 展, 宮崎 純一, 片桐 秀樹

    糖尿病 59 (Suppl.1) S-138 2016年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  51. プラズマ由来液中活性種が誘導する細胞内カルシウム濃度上昇・振動

    佐々木渉太, 保苅雄太郎, 神崎展, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 63rd ROMBUNNO.21A-W621-2 2016年3月3日

    ISSN:2436-7613

  52. 液相微小プラズマを用いた接着細胞への薬剤分子導入

    保苅雄太郎, 佐々木渉太, 神崎展, 佐藤岳彦, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 63rd ROMBUNNO.21A-W621-6 2016年3月3日

    ISSN:2436-7613

  53. プラズマ刺激による細胞膜輸送制御

    金子俊郎, 佐々木渉太, 神崎 展

    機械の研究 68 (2) 151-154 2016年2月1日

    出版者・発行元:養賢堂

  54. プラスマ刺激による細胞応答と応用

    佐藤岳彦, 横山茉代, 城倉浩平, 金子俊郎, 佐々木渉太, 神崎展, 太田貴之

    機械の研究 68 (2) 147-157 2016年2月1日

    出版者・発行元:養賢堂

  55. 高度物理刺激と生体応答(7)―第5章 プラズマ刺激による細胞応答と応用 その1―

    佐藤岳彦, 横山茉代, 城倉浩平, 金子俊郎, 佐々木渉太, 神崎展, 太田貴之

    機械の研究 68 (2) 147-157 2016年2月1日

    ISSN:0368-5713

  56. 細胞膜透過性促進に寄与する液相中プラズマ生成活性種の同定

    保苅雄太郎, 佐々木渉太, 神崎展, 佐藤岳彦, 金子俊郎

    プラズマ・核融合学会年会(Web) 33rd 2016年

  57. プラズマ照射溶液による細胞内への薬剤模擬分子導入の時間発展計測

    ZHENG Yuexing, 佐々木渉太, 神崎展, 金子俊郎

    プラズマ・核融合学会年会(Web) 33rd 2016年

  58. 電気パルス刺激を用いた収縮型培養筋細胞の創製

    神﨑 展

    機械の研究 68 (1) 56-58 2016年1月1日

    出版者・発行元:養賢堂

  59. アミロイド前駆タンパク質の新規プロセッシング経路の発見

    神﨑 展

    実験医学1月号 (2016) 34 (1) 66-67 2016年1月1日

    出版者・発行元:羊土社

  60. 嗅覚受容体は膵β細胞に発現しグルコース応答性インスリン分泌を促進する

    宗像佑一郎, 山田哲也, 今井淳太, 突田壮平, 高橋圭, 白井勇太, 児玉慎二郎, 浅井洋一郎, 椙澤貴志, 千葉弓子, 高橋広延, 穂坂真一郎, 井泉知仁, 高俊弘, 宇野健司, 澤田正二郎, 畠山裕康, 神崎展, 宮崎純一, 片桐秀樹

    糖尿病(Web) 59 (Suppl) S.138(J‐STAGE) 2016年

    ISSN:1881-588X

  61. 膵内副交感神経節と膵島の組織学的関係

    高橋啓範, 今井淳太, 井泉知仁, 山本淳平, 川名洋平, 遠藤彰, 菅原裕人, 洲崎悦生, 畠山裕康, 澤田正二郎, 山田哲也, 上田泰己, 神崎展, 片桐秀樹

    糖尿病(Web) 59 (Suppl) S.158(J‐STAGE) 2016年

    ISSN:1881-588X

  62. 骨格筋における糖輸送体GLUT4挙動のライブイメージングと運動効果の基盤解析

    畠山裕康, 畠山裕康, 堰合茂智, 神崎展

    糖尿病(Web) 59 (Suppl) S.305(J‐STAGE) 2016年

    ISSN:1881-588X

  63. 高度物理刺激と生体応答(6)―第4章 電気刺激による細胞応答と応用―

    白樫了, 神崎展, 高木浩一, 勝木淳

    機械の研究 68 (1) 53-64 2016年1月1日

    ISSN:0368-5713

  64. 非平衡大気圧プラズマ刺激による新作用機序遺伝子導入

    金子俊郎, 佐々木渉太, 保苅雄太郎, 神崎展

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 76th ROMBUNNO.14P-1F-7 2015年8月31日

    ISSN:2436-7613

  65. 大気圧プラズマ照射液中化学活性種に対する細胞感受性

    佐々木渉太, 保苅雄太郎, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 76th ROMBUNNO.16P-2V-11 2015年8月31日

    ISSN:2436-7613

  66. 脂質ラフト形成において細胞膜内外が共役するしくみ

    神﨑 展

    実験医学7月号 (2015) 33 (11) 1759-1760 2015年7月1日

    出版者・発行元:羊土社

  67. 非平衡プラズマによる物理的・化学的刺激の細胞膜輸送に対する相乗効果

    金子俊郎, 佐々木渉太, 神崎展

    日本物理学会講演概要集(CD-ROM) 70 (1) ROMBUNNO.21PAP-9 2015年3月24日

    ISSN:2189-079X

  68. 大気圧プラズマ照射による水溶性高分子の細胞膜透過性促進

    佐々木渉太, 保苅雄太郎, 神崎展, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 62nd ROMBUNNO.13A-A28-7 2015年2月26日

    ISSN:2436-7613

  69. ゴルジ複合体における輸送小胞の選択的な繫留とその意義

    神﨑 展

    実験医学1月号(2015) 33 (1) 57-58 2015年1月1日

    出版者・発行元:羊土社

  70. 筋細胞アッセイのためのハイドロゲルデバイスの開発

    岡本滉平, 島崎立彬, 長峯邦明, 梶弘和, 神崎展, 西澤松彦

    電気化学秋季大会講演要旨集 2014 211 2014年9月27日

  71. W271003 細胞膜輸送に対する非平衡プラズマ刺激の効果([W27100]部門協議会直属分科会:高度物理刺激と生体応答に関する研究分科会(P-SCC12),環境工学部門,バイオエンジニアリング部門,流体工学部門,熱工学部門企画,高度物理刺激と生体応答)

    金子 俊郎, 佐々木 渉太, 神崎 展

    年次大会 : Mechanical Engineering Congress, Japan 2014 "W271003-1"-"W271003-2" 2014年9月7日

    出版者・発行元:一般社団法人日本機械学会

    詳細を見る 詳細を閉じる

    The time-controlled atmospheric pressure plasma flow is generated and irradiated to the living-cell suspended solution for clarifying the transfection mechanism toward developing highly-efficient and minimally-invasive gene transfection system. It is observed that the transfection efficiency is significantly increased by the short-time (< 4 sec) and short-distance (< 40 mm) plasma irradiation, and the high transfection efficiency of 53% is realized together with the high cell viability (> 90%). This result indicates that the physical stimulation such as the electric field caused by the charged particles arriving at the surface of the cell membrane, and chemical stimulation associated with plasma-activated products in solution act synergistically to enhance the cell-membrane transport with low-damage.

  72. 細胞膜輸送に対する非平衡プラズマ刺激の効果

    金子俊郎, 佐々木渉太, 神崎展

    日本機械学会年次大会講演論文集(CD-ROM) 2014 ROMBUNNO.W271003 2014年9月6日

    ISSN:2424-2667

  73. 遺伝子細胞膜透過性に対するプラズマ照射起因電気的ストレスと酸化ストレスの効果

    佐々木渉太, 神崎展, 金子俊郎

    応用物理学会秋季学術講演会講演予稿集(CD-ROM) 75th ROMBUNNO.17A-PB2-15 2014年9月1日

    ISSN:2436-7613

  74. 細胞容量調節の「鍵」分子SWELL1の同定

    神崎 展

    実験医学 32 (11) 1750-1751 2014年7月

    出版者・発行元:羊土社

  75. Submissive Role of AS160 in TBC1D1-mediated GLUT4 Trafficking Activation in Response to Ca2+and Insulin

    Hiroyasu Hatakeyama, Makoto Kanzaki

    DIABETES 63 A97-A97 2014年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

    eISSN:1939-327X

  76. インスリン応答性GLUT4輸送制御におけるTBC1D family Rab GTPase活性化タンパク質群が司る運動効果の発現機序

    畠山 裕康, 神崎 展

    糖尿病 57 (Suppl.1) S-419 2014年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  77. ハイドロゲル間接着法による筋細胞電気刺激デバイスの構築

    長峯邦明, 岡本滉平, 梶弘和, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 81st 237 2014年3月29日

  78. 細胞膜輸送に対する非平衡プラズマ生成ラジカルの効果

    金子俊郎, 佐々木渉太, 神崎展

    日本物理学会講演概要集 69 (1) 279 2014年3月5日

    ISSN:1342-8349

  79. 遺伝子導入効率に対するプラズマ直接照射の効果

    佐々木渉太, 神崎展, 金子俊郎

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 61st ROMBUNNO.19P-F2-9 2014年3月3日

    ISSN:2436-7613

  80. 2C33 筋の運動効果とインスリン感受性の亢進(OS4-1:メカノバイオロジー(1))

    神崎 展

    バイオエンジニアリング講演会講演論文集 2014 (26) 367-367 2014年1月10日

    出版者・発行元:一般社団法人日本機械学会

  81. 微小管アセチル化とエンドサイトーシスが細胞進路を決める

    神崎 展

    実験医学 32 (1) 65-66 2014年1月

  82. 医工連携のための医療・工学技術者Co‐education事業の構築と実践

    神崎展

    医工連携のための医療・工学技術者Co-education事業の構築と実践 平成25年度 総括・分担研究報告書 104-105 2014年

  83. ハイドロゲル間接着技術を用いた筋細胞アッセイデバイスの構築

    岡本滉平, 長峯邦明, 梶弘和, 神崎展, 西澤松彦

    化学とマイクロ・ナノシステム学会研究会講演要旨集 28th 15 2013年12月5日

  84. 物理刺激に対する筋骨格系細胞の形態的および機能的応答解析

    佐藤 正明, 安達 泰治, 上岡 寛, 神崎 展

    運動器リハビリテーション 24 (4) 404-408 2013年12月

    出版者・発行元:日本運動器科学会

    ISSN:2187-8420

  85. センサ分子修飾ハイドロゲルによる骨格筋細胞の代謝活性イメージング

    長峯邦明, 岡本滉平, 梶弘和, 神崎展, 西澤松彦

    Chemical Sensors 29 (Supplement B) 58-60 2013年9月27日

    出版者・発行元:電気化学会化学センサ研究会

  86. 非平衡プラズマ照射による細胞膜界面電位形成と遺伝子導入への効果

    金子俊郎, 高橋祥平, 佐々木渉太, 小西秀明, 神崎展

    日本物理学会講演概要集 68 (2) 183 2013年8月26日

    ISSN:1342-8349

  87. 細胞融合にかかわるアクチン制御ナノシステムとその可視化解析

    神崎 展

    実験医学 31 (11) 1749-1750 2013年7月

    出版者・発行元:羊土社

  88. 物理刺激に対する筋骨格系細胞の形態的および機能的応答解析

    佐藤 正明, 安達 泰治, 上岡 寛, 神崎 展

    運動器リハビリテーション 24 (2) 172-172 2013年6月

    出版者・発行元:日本運動器科学会

    ISSN:2187-8420

  89. 筋細胞パターンゲルを用いた細胞の代謝活性計測

    岡本滉平, 伊藤俊太郎, 長峯邦明, 梶弘和, 神崎展, 西澤松彦

    化学とマイクロ・ナノシステム学会研究会講演要旨集 27th 63 2013年5月23日

  90. 放電プラズマ中ラジカル照射による低侵襲遺伝子導入法の開発

    金子俊郎, 佐々木渉太, 小西秀明, 石田裕康, 神崎展

    電子情報通信学会技術研究報告 113 (18(OME2013 1-22)) 75-79 2013年4月18日

    ISSN:0913-5685

  91. Tbc1d1が示すインスリン不応性およびインスリン応答性GLUT4輸送制御とその遷移による「インスリン応答性獲得」の分子基盤

    畠山 裕康, 神崎 展

    糖尿病 56 (Suppl.1) S-374 2013年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  92. センサ分子修飾ハイドロゲルによる筋細胞分泌物の計測

    長峯邦明, 岡本滉平, 梶弘和, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 80th 279 2013年3月29日

  93. 筋細胞パターンゲルを用いた細胞の代謝活性センシング

    岡本滉平, 長峯邦明, 梶弘和, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 80th 458 2013年3月29日

  94. センサ分子修飾ハイドロゲルによる筋細胞分泌物の計測

    長峯邦明, 岡本滉平, 梶弘和, 神崎展, 西澤松彦

    Chemical Sensors 29 (Supplement A) 147-149 2013年3月29日

    出版者・発行元:電気化学会化学センサ研究会

  95. 水酸基ラジカル制御プラズマ生成と遺伝子導入への応用

    金子俊郎, 佐々木渉太, 小西秀明, 石田裕康, 神崎展

    応用物理学会春季学術講演会講演予稿集(CD-ROM) 60th ROMBUNNO.29P-B7-4 2013年3月11日

    ISSN:2436-7613

  96. 遺伝子導入効率に対する大気圧プラズマ照射距離の影響

    佐々木渉太, 神崎展, 金子俊郎

    プラズマ・核融合学会年会(Web) 30th 2013年

  97. オルガネラ膜の直接パッチクランプ解析によるTPC1/2機能の解明

    神崎 展

    実験医学 31 (1) 59-60 2013年1月

    出版者・発行元:羊土社

  98. 医工連携のための医療・工学技術者Co‐education事業の構築と実践

    神崎展

    医工連携のための医療・工学技術者Co-education事業の構築と実践 平成24年度 総括・分担研究報告書 69-70 2013年

  99. アストロサイト由来のglypicansと中枢神経ネットワークの構築

    神崎 展

    実験医学 30 (16) 2600-2601 2012年10月

    出版者・発行元:羊土社

  100. ハイドロゲルを用いた骨格筋細胞アッセイ系の開発

    長峯邦明, 大谷真吾, 伊藤俊太郎, 梶弘和, 神崎展, 西澤松彦

    化学系学協会東北大会プログラムおよび講演予稿集 2012 138 2012年9月15日

  101. Characterization of Unique Regulatory Mechanism of Tbc1d1 in GLUT4 Trafficking by Single Molecule Analysis of GLUT4 Behavior

    Hiroyasu Hatakeyama, Makoto Kanzaki

    DIABETES 61 A7-A8 2012年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

    eISSN:1939-327X

  102. 細胞移動を支配するメカノセンシングのしくみ

    神崎 展

    実験医学 30 (6) 936-937 2012年4月

    出版者・発行元:羊土社

  103. GLUT4輸送調節におけるAS160とTbc1d1の作用点とその分子機序

    畠山 裕康, 神崎 展

    糖尿病 55 (Suppl.1) S-302 2012年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  104. センサ微粒子パターンゲルを用いた細胞の代謝活性イメージング

    長峯邦明, 大谷真吾, 伊藤俊太郎, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 79th 47 2012年3月29日

  105. Sortilinとソーティング障害,そして生活習慣病

    神崎展

    生化学 83 (11) 1035-1039 2011年11月25日

    ISSN:0037-1017

  106. カベオリンによる力学的微細環境構築とガンの浸潤

    神崎 展

    実験医学 29 (16) 2655-2656 2011年10月

    出版者・発行元:羊土社

  107. ハイドロゲルを用いた細胞培養法の開発

    大谷真吾, 武田舞, 三宅丈雄, 長峯邦明, 神崎展, 西澤松彦

    化学系学協会東北大会プログラムおよび講演予稿集 2011 191 2011年9月17日

  108. 筋肉細胞アッセイゲルシートの構築と評価

    長峯邦明, 武田舞, 大谷真吾, 神崎展, 西澤松彦

    電気化学秋季大会講演要旨集 2011 117 2011年9月9日

  109. 細胞内輸送の新規定量解析系を用いたインスリン応答性GLUT4輸送システムの分子基盤解析

    畠山 裕康, 神崎 展

    日本生化学会大会プログラム・講演要旨集 84回 2T15a-8 2011年9月

    出版者・発行元:(公社)日本生化学会

    ISSN:0037-1017

  110. 温度センサーとして機能するロドプシンとTRPA1

    神崎 展

    実験医学 29 (9) 2011年6月

    出版者・発行元:羊土社

  111. 一分子動態計測系に基づくインスリン応答性GLUT4輸送システムの分子基盤解析

    畠山 裕康, 神崎 展

    糖尿病 54 (Suppl.1) S-87 2011年4月

    出版者・発行元:(一社)日本糖尿病学会

    ISSN:0021-437X

    eISSN:1881-588X

  112. ハイドロゲルを基板とする筋肉細胞アッセイシステムの構築と評価

    長峯邦明, 武田舞, 大谷真吾, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 78th(CD-ROM) ROMBUNNO.1D08 2011年3月29日

  113. 生活習慣病とsorting disorder

    神崎 展

    実験医学 29 (3) 430-431 2011年2月

    出版者・発行元:羊土社

  114. Sortilinとソーティング障害、そして生活習慣病

    神崎 展

    生化学 83 (11) 1035-1039 2011年

    出版者・発行元:日本生化学会

    ISSN:0037-1017

  115. 収縮活動可能な培養筋管細胞系の構築とその代謝研究への応用

    神崎 展, 長峯邦明, 西澤松彦

    内分泌・糖尿病・代謝内科( 31 (5) 325-328 2010年12月

    出版者・発行元:科学評論社

  116. 収縮活動可能な培養筋管細胞系の構築とその代謝研究への利用

    神崎展, 長峯邦明, 西澤松彦

    月刊内分泌・糖尿病・代謝内科 31 (5) 464-471 2010年11月28日

    ISSN:1884-2917

  117. ゲルシート培養法を用いた神経・筋マイクロパターン共培養系の構築

    武田舞, 大谷真吾, 長峯邦明, 三宅丈雄, 神崎展, 西澤松彦

    化学とマイクロ・ナノシステム研究会講演要旨集 22nd 30 2010年11月17日

  118. 焦点接着斑における「ちから」の視覚化解析

    神崎 展

    実験医学 28 (16) 2627-2628 2010年10月

    出版者・発行元:羊土社

  119. ゲル培養法を用いた収縮型筋管細胞の代謝活性計測

    長峯邦明, 梶弘和, 神崎展, 西澤松彦

    電気化学秋季大会講演要旨集 2010 147 2010年9月2日

  120. 運動刺激によって活性化される細胞内シグナル伝達系とマイオカインの分泌制御

    神崎展

    運動療法と物理療法 21 (2) 114 2010年6月10日

    ISSN:1342-7776

  121. Molecular Basis of Static GLUT4 Storage Compartment Formation in 3T3-L1 Adipocytes Revealed by Quantum Dot-Based Quantitative Single Molecular Imaging

    Hiroyasu Hatakeyama, Makoto Kanzaki

    DIABETES 59 A11-A12 2010年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  122. マクロファージの貪食作用におけるTRPV2チャネルの役割

    神崎 展

    実験医学6月号 28 (9) 1400-1401 2010年6月1日

    出版者・発行元:羊土社

  123. Quantum Dot(Qdot)を用いたGLUT4細胞内小胞輸送系の定量的解析

    畠山裕康, 神崎展

    ナノ学会大会講演予稿集 8th 101 2010年5月13日

  124. GLUT4‐分子動態追跡系を用いたGLUT4 storage compartment形成機構の定量解析

    畠山裕康, 神崎展

    糖尿病 53 (Supplement 1) S.164 2010年4月25日

    ISSN:0021-437X

  125. 多角バレルスパッタリング法を利用した低環境負荷操作による収縮型筋管細胞培養系の構築

    長峯邦明, 梶弘和, 神崎展, 西澤松彦, 阿部孝之

    電気化学会大会講演要旨集 77th 382 2010年3月26日

  126. Polycystin-1、-2による伸展活性化(Stretch-activated: SA)チャネルの制御

    神崎展

    実験医学2月号 27 (16) 422-423 2010年2月

    出版者・発行元:羊土社

  127. Metabolic Assay System for Micropatterned Contractile Myotubes

    K. Nagamine, Y. Ido, S. Sekine, T. Miyake, M. Kanzaki, M. Nisizawa

    CHEMICAL SENSORS 9 -AND- MEMS/NEMS 9 33 (8) 35-39 2010年

    出版者・発行元:ELECTROCHEMICAL SOC INC

    DOI: 10.1149/1.3484104  

    ISSN:1938-5862

    詳細を見る 詳細を閉じる

    Contractile C2C12 myotube line patterns embedded in a fibrin gel have been developed to afford a physiologically relevant and stable bioassay system. We found that the myotubes supported by an elastic fibrin gel maintained their line patterns and contractile activities for a longer period of time (one week) than myotubes adhered on a conventional culture dish. Now, we are investigating contraction-mediated translocation of the GLUT4 glucose transporter protein in myotubes using this system.

  128. 転写因子TFEBによるリソソームの制御

    神崎展

    実験医学10月号 27 (16) 2596-2597 2009年10月

    出版者・発行元:羊土社

  129. 局所電気刺激を用いた培養筋管細胞のグルコース代謝活性に関する研究

    石橋毅之, 長峰邦明, 梶弘和, 神崎展, 佐藤正明, 西澤松彦

    電気化学秋季大会講演要旨集 2009 107 2009年9月10日

  130. 収縮型筋管細胞アレイの3次元ゲル培養系の構築

    長峯邦明, 石橋毅之, 川島丈明, 梶弘和, 安部隆, 神崎展, 西澤松彦

    電気化学秋季大会講演要旨集 2009 94 2009年9月10日

  131. Protective Effects of Lipokine (Falmitoleate) and Other Unsaturated Fatty Acids on Palmitate-Induced COX-2 Expression in Skeletal Muscle Cells

    Akito Kadotani, Hideki Katagiri, Makoto Kanzaki

    DIABETES 58 A393-A393 2009年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  132. 「前」初期エンドソームとその成熟

    神崎展

    実験医学6月号 27 (9) 1364-1365 2009年6月

  133. 筋・脂肪にけるインスリン作用

    神崎 展

    糖尿病 52 (5) 325-328 2009年5月30日

    出版者・発行元:日本糖尿病学会

    DOI: 10.11213/tonyobyo.52.325  

    ISSN:0021-437X

  134. エネルギー消費調節 4.筋肉におけるエネルギー代謝調節とインスリン抵抗性

    神崎展

    実験医学 27 (7) 1050-1057 2009年4月20日

    ISSN:0288-5514

  135. インスリン応答性アッセイに向けた3次元培養筋管細胞モデルの検討

    川島丈明, 横井丈誌, 梶弘和, 安部隆, 神崎展, 西澤松彦

    電気化学会大会講演要旨集 76th 289 2009年3月29日

  136. 培養骨格筋細胞の高効率培養システムの検討

    石橋毅之, 星野佑, 梶弘和, 安部隆, 神崎展, 佐藤正明, 西澤松彦

    電気化学会大会講演要旨集 76th 290 2009年3月29日

  137. F-15 Electrical pulse stimulation of C2C12 cultured myotubes induces IL-6 production(Free communication (Slide),8^<TH> INTERNATIONAL SOCIETY OF EXERCISE AND IMMUNOLOGY SYMPOSIUM,ISEI2007 INFLAMMATION IN EXERCISE FRIEND OR FOE?)

    Farmawati Arta, Nedachi Taku, Kanzaki Makoto, Nagatomi Ryoichi

    体力科學 58 (1) 190-190 2009年2月1日

    出版者・発行元:日本体力医学会

    ISSN:0039-906X

  138. インスリン受容体シグナルとGLUT4小胞輸送系との接点 —モーター蛋白質Myo1cの活性化—

    神崎展

    実験医学2月号 27 (3) 407-408 2009年2月

    出版者・発行元:羊土社

  139. 筋肉におけるエネルギー代謝調節とインスリン抵抗性

    神崎 展

    実験医学増刊号 27 (7) 78-85 2009年

    出版者・発行元:羊土社

  140. 容量依存性カルシウム流入の分子基盤の解明

    神崎展

    実験医学12月号 26 (19) 3046-3047 2008年12月

    出版者・発行元:羊土社

  141. Electric Pulse Stimulation Induces NMDA Glutamate Receptor mRNA in NIH3T3 Mouse Fibroblasts (vol 215, pg 181, 2008)

    Saeko Okutsu, Hiroyasu Hatakeyama, Makoto Kanzaki, Hiroshi Tsubokawa, Ryoichi Nagatomi

    TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE 216 (2) 195-195 2008年10月

    出版者・発行元:TOHOKU UNIV MEDICAL PRESS

    ISSN:0040-8727

    eISSN:1349-3329

  142. PPARδとAMPキナーゼを活性化する薬剤は運動効果をもたらす

    神崎展

    実験医学10月号 26 (16) 2592-2593 2008年10月

    出版者・発行元:羊土社

  143. リソソームにおけるClC-1の働きと酸性オルガネラの成熟

    神崎展

    実験医学8月号 26 (13) 2092-2093 2008年8月

    出版者・発行元:羊土社

  144. 培養骨格筋細胞への電気刺激システムの検討

    石橋毅之, 星野佑, 梶弘和, 神崎展, 安部隆, 西澤松彦

    電気学会バイオ・マイクロシステム研究会資料 BMS-08 (6-15) 47-50 2008年6月12日

  145. Identification of contraction-inducible myokines and their impact on insulin responsive GLUT4 translocation in C2C12 myotubes

    Taku Nedachi, Hideaki Fujita, Makoto Kanzaki

    DIABETES 57 A308-A308 2008年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  146. 糖尿病基磯研究の進歩 II.糖尿病と対糖能低下の成因分類と発症機序 2型糖尿病 インスリン抵抗性にかかわる因子 GLUT4小胞輸送とインスリン抵抗性

    有賀美也子, 根建拓, 神崎展

    日本臨床 66 432-436 2008年5月28日

    ISSN:0047-1852

  147. 培養筋管細胞の収縮能を発揮させる基板に関する研究

    星野佑, 石橋毅之, 神谷崇志, 梶弘和, 神崎展, 西澤松彦

    化学とマイクロ・ナノシステム研究会講演要旨集 17th 14 2008年5月20日

  148. 分子糖尿病学の進歩―基礎から臨床まで―2008 2.インスリン作用の分子機構 2 Live cellイメージング技術を用いたインスリン反応性:GLUT4トランスロケーションの解析

    藤田英明, 神崎展

    分子糖尿病学の進歩 2008 48-55 2008年4月10日

    ISSN:1344-0861

  149. 細胞遊走進路のガイダンスとサイレント受容体CXCR7の役割

    神崎展

    実験医学4月号 26 (6) 901-902 2008年4月

    出版者・発行元:羊土社

  150. 走査型イオンコンダクタンス顕微鏡を用いた細胞膜表面観察と局所電気浸透流インジェクションへの応用

    高橋康史, 村上有美, 安川智之, 珠玖仁, 神崎展, 末永智一

    電気化学会大会講演要旨集 75th 435 2008年3月29日

  151. 培養筋管細胞への均一な電気刺激の検討

    石橋毅之, 星野佑, 梶弘和, 神崎展, 安部隆, 西澤松彦

    電気化学会大会講演要旨集 75th 184 2008年3月29日

  152. 鶏初代骨格筋細胞グルコース輸送における一酸化窒素(NO)のシグナル調節

    錦ゆりか, 河野龍義, 神崎展, 佐藤幹, 高橋和昭, 豊水正昭, 秋葉征夫

    日本畜産学会大会講演要旨 109th 9 2008年3月27日

    ISSN:1342-4688

  153. mTORによるミトコンドリア機能の制御機構

    神崎展

    実験医学2月号 26 (3) 402-403 2008年2月

    出版者・発行元:羊土社

  154. Live cellイメージング技術を用いたインスリン反応性GLUT4トランスローケーションの解析

    藤田英明, 神崎

    分子糖尿病学の進歩2008 48-55 2008年

    出版者・発行元:金原出版

  155. GLUT4小胞輸送とインスリン抵抗性

    有賀 美也子, 根建, 拓, 神崎 展

    新時代の糖尿病学1 432-436 2008年

    出版者・発行元:日本臨床社

  156. 培養筋細胞を運動させる

    神崎展

    日本比較生理生化学会大会予稿集 30th 20 2008年

  157. 培養C2C12筋管の電気パルス刺激によるIL-6産生(IL-6 production in C2C12 cultured myotube by electrical pulse stimulation)

    Farmawati Arta, 根建 拓, 神崎 展, 永富 良一

    体力科学 56 (6) 621-621 2007年12月

    出版者・発行元:(一社)日本体力医学会

    ISSN:0039-906X

  158. 脂質膜の構造変形に関わるMechano-enzyme EHDs

    神崎展

    実験医学12月号 25 (19) 3005-3005 2007年12月

    出版者・発行元:羊土社

  159. 生細胞内におけるGLUT4分子動態ナノイメージングとインスリン作用機構

    藤田英明, 渡邉朋信, 根建拓, 樋口秀男, 神崎展

    日本学術会議材料工学連合講演会講演論文集 51st 107 2007年11月27日

  160. 3P325 高度発達型培養筋細胞におけるGLUT4分子動態イメージング(バイオイメージング,ポスター発表,第45回日本生物物理学会年会)

    藤田 英明, 渡邉 朋信, 根建 拓, 樋口 秀男, 神崎 展

    生物物理 47 (1) S284 2007年11月20日

    出版者・発行元:一般社団法人日本生物物理学会

    DOI: 10.2142/biophys.47.S284_2  

    ISSN:0582-4052

  161. 電気・磁気刺激によるリハビリテーションへの応用 電気刺激による骨格筋収縮運動とその2型糖尿病治療への応用

    神崎展, 片桐秀樹

    Monthly Book Medical Rehabilitation (86) 79-85 2007年11月15日

    ISSN:1346-0773

  162. 隔膜型培養基板用いた筋管細胞への電気刺激

    星野佑, 石橋毅之, 梶弘和, 神崎展, 安部隆, 西澤松彦

    化学とマイクロ・ナノシステム研究会講演要旨集 16th 58 2007年10月29日

  163. 隔膜型培養基板を用いた骨格筋細胞への局所電気刺激

    石橋毅之, 星野佑, 梶弘和, 神崎展, 安部隆, 西澤松彦

    電気化学秋季大会講演要旨集 2007 179 2007年9月19日

  164. 隔膜型培養基板を用いた骨格筋細胞への局所電気刺激

    石橋毅之, 星野佑, 梶弘和, 神崎展, 安部隆, 西澤松彦

    生体機能関連化学シンポジウム講演要旨集 22nd 244-245 2007年9月10日

  165. SUMO化修飾によるカイニン酸受容体チャネルのエンドサイトーシス

    神崎展

    実験医学8月号 25 (12) 1834-1835 2007年8月

    出版者・発行元:羊土社

  166. Single molecule analysis of insulin-induced GLUT4 behavior in 3T3L1 adipocyte using Qdot nano-crystals

    Hideaki Fujita, Tomonobu Watanabe, Taku Nedachi, Hideo Higuchi, Makoto Kanzaki

    DIABETES 56 A330-A330 2007年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  167. Functional role of sortilin in myogenesis and the development of insulin-responsive glucose transport system in C2C12 myocytes

    Miyako Ariga, Taku Nedachi, Hideki Katagiri, Makoto Kanzaki

    DIABETES 56 A36-A37 2007年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  168. ナノ蛍光粒子を用いたインスリン反応性GLUT4分子挙動変化の解析

    神崎展

    糖尿病 50 (Supplement 1) S.41 2007年4月25日

    ISSN:0021-437X

  169. 擬似的運動刺激によるインスリン依存的なGLUT4膜移行増強機構の解析~高度発達型筋管細胞の作製と応用~

    根建拓, 藤田英明, 神崎展

    糖尿病 50 (Supplement 1) S.259 2007年4月25日

    ISSN:0021-437X

  170. TRPA1チャネルは痛み物質の共有結合によって活性化される

    神崎展

    実験医学4月号 26 (6) 838-839 2007年4月

    出版者・発行元:羊土社

  171. インスリンによる糖の取り込み促進作用とatypical PKCλ/ζの役割―脂肪細胞分化に伴うカベオラ構造の発達とその生理的意義―

    神崎展

    医科学応用研究財団研究報告 24 186-189 2007年2月

    ISSN:0914-5117

  172. 電気刺激による骨格筋収縮運動とその2型糖尿病治療への応用

    神崎 展, 片桐, 秀

    Medical Rehabilitation 86 79-85 2007年

  173. Qdotを用いた細胞膜表面でのGLUT4分子動態TIRF観察

    藤田英明, 渡邉朋信, 根建拓, 樋口秀男, 神崎展

    日本生体医工学会大会プログラム・論文集(CD-ROM) 46th ROMBUNNO.PS3-4-12 2007年

  174. 高度発達型培養筋管細胞の作製技術とその糖尿病研究への利用

    根建拓, 藤田英明, 神崎展

    日本生体医工学会大会プログラム・論文集(CD-ROM) 46th ROMBUNNO.PS1-1-4 2007年

  175. 肝再生時におけるカベオリンの新たな役割

    神崎展

    実験医学12月号 24 (19) 2982-2983 2006年12月

    出版者・発行元:羊土社

  176. 脂肪細胞の肥大とマトリックスメタロプロテアーゼ

    神崎展

    実験医学8月号 24 (12) 1768-1769 2006年8月

    出版者・発行元:羊土社

  177. Development of insulin-responsive GLUT4 trafficking machinery by forced Ca2+ transients in C2C12 myotubes

    Taku Nedachi, Hideaki Fujita, Makoto Kanzaki

    DIABETES 55 (Suppl. 1) A242-A242 2006年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  178. Insulin receptor signals regulating GLUT4 translocation and act in dynamics

    Makoto Kanzaki

    ENDOCRINE JOURNAL 53 (3) 267-293 2006年6月

    出版者・発行元:JAPAN ENDOCRINE SOCIETY

    DOI: 10.1507/endocrj.KR-65  

    ISSN:0918-8959

    詳細を見る 詳細を閉じる

    In skeletal muscle and adipose tissue, insulin-stimulated glucose uptake is dependent upon translocation of the insulin-responsive glucose transporter GLUT4 from intracellular storage compartments to the plasma membrane. This insulin-induced redistribution of GLUT4 protein is achieved through a series of highly organized membrane trafficking events, orchestrated by insulin receptor signals. Recently, several key molecules linking insulin receptor signals and membrane trafficking have been identified, and emerging evidence supports the importance of subcellular compartmentalization of signaling components at the right time and in the right place. In addition, the translocation of GLUT4 in adipocytes requires insulin stimulation of dynamic actin remodeling at the inner surface of the plasma membrane (cortical actin) and in the perinuclear region. This results from at least two independent insulin receptor signals, one leading to the activation of phosphatidylinositol (PI) 3-kinase and the other to the activation of the Rho family small GTP-binding protein TC10. Thus, both spatial and temporal regulations of actin dynamics, both beneath the plasma membrane and around endomembranes, by insulin receptor signals are also involved in the process of GLUT4 translocation.

  179. 新規高度発達型培養筋細胞系におけるGLUT4膜移行を指標とした擬似的運動刺激効果の検討

    根建拓, 神崎展

    糖尿病 49 (Supplement 1) S.111 2006年4月25日

    ISSN:0021-437X

  180. pHセンサーとしてのV-ATPaseの役割と細胞内小胞輸送制御

    神崎展

    実験医学4月号 24 (6) 827-828 2006年4月

    出版者・発行元:羊土社

  181. カルシウム透過性チャネルPolycystin-2による転写と細胞増殖の制御

    神崎展

    実験医学2月号 24 (3) 381-382 2006年2月

    出版者・発行元:羊土社

  182. カルシウム流入の新たな制御機構

    神崎展

    実験医学12月号 23 (19) 2927-2928 2005年12月

    出版者・発行元:羊土社

  183. 成体でも骨髄細胞から卵母細胞が形成される

    神崎展

    実験医学10月号 26 (16) 2465-2466 2005年10月

    出版者・発行元:羊土社

  184. コネクチンが筋活動を感知する新機構の発見

    神崎展

    実験医学8月号 23 (13) 2028 2005年8月

    出版者・発行元:羊土社

  185. Possible involvement of G alpha-interacting protein (GAIP) in the sorting process of insulin-responsive glucose transporter GLUT4 at the trans-Golgi network (TGN)

    Chongxia Zhong, Hideaki Fujita, Hideki Katagiri, Makoto Kanzaki

    CELL STRUCTURE AND FUNCTION 30 21-21 2005年6月

    出版者・発行元:JAPAN SOC CELL BIOLOGY

    ISSN:0386-7196

    eISSN:1347-3700

  186. Formation of periodic integrin-β1 alignment serves as a scaffold of the initial sarcomere assembly in C2C12 myotubes.

    Hideaki Fujita, Taku Nedachi, Makoto Kanzaki

    Cell Structure and Function 30 (Suppl.) 111-111 2005年6月

    出版者・発行元:JAPAN SOC CELL BIOLOGY

    ISSN:0386-7196

    eISSN:1347-3700

  187. Cytoplasmic sequestration of Sir2 that depends upon metabolic states is directly engaged in the differentiation of C2C12 myoblasts

    Taku Nedachi, Hideaki Fujita, Makoto Kanzaki

    CELL STRUCTURE AND FUNCTION 30 (Suppl.) 85-85 2005年6月

    出版者・発行元:JAPAN SOC CELL BIOLOGY

    ISSN:0386-7196

    eISSN:1347-3700

  188. SUMO化によるリークK+チャネルK2P1の新規活性制御機構

    神崎展

    実験医学6月号 23 (10) 1547-1548 2005年6月

    出版者・発行元:羊土社

  189. マウス筋細胞株C2C12の分化およびインスリン依存性糖取り込み(GLUT4トランスロケーション)におよぼす細胞外糖濃度の影響

    根建拓, 神崎展

    糖尿病 48 (Supplement 2) S.261 2005年4月25日

    ISSN:0021-437X

  190. インスリン反応性グルコース輸送担体GLUT4の細胞内輸送に関与するタンパクGGA1

    古川恵, 神崎展

    糖尿病 48 (Supplement 2) S.234 2005年4月25日

    ISSN:0021-437X

  191. 興奮毒性時におけるNa+/Ca2+ exchangerのカルパイン依存性分解

    神崎展

    実験医学4月号 23 (7) 1094-1095 2005年4月

    出版者・発行元:羊土社

  192. 特集 インスリンによる臓器作用の調節 脂肪細胞におけるインスリン作用

    神崎展, 片桐秀樹

    月刊内分泌・糖尿病科 20 (2) 117-124 2005年2月28日

    ISSN:1341-3724

  193. ポリADPリボシル化酵素PPRPによるクロマチン構造と転写活性の新規制御機構

    神崎展

    実験医学2月号 23 (3) 392-393 2005年2月

    出版者・発行元:羊土社

  194. Spatial reorganization of integrin beta 1 and talin is required for Cav-actin/caveolae-rosette formation and adipogenesis

    M Kanzaki, T Nedachi, J Pessin

    DIABETES 54 A72-A72 2005年

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  195. Calcium oscillation facilitates assembly of sarcomere structure in C2C12 myotubes

    Hideaki Fujita, Taku Nedachi, Makoto Kanzaki

    The Japanese Journal of Physiology 55 (Suppl.) S122 2005年

  196. 低分子量G蛋白質によるインスリン依存性糖輸送の制御 先端医療シリーズ32

    神崎 展

    糖尿病の最新医療 100-107 2005年

    出版者・発行元:先端医療技術研究所

  197. 脂肪細胞におけるインスリン作用

    神崎 展, 片桐, 秀

    内分泌・糖尿病科 20 (2) 117-124 2005年

  198. ユビキチン-プロテアソーム系による筋組織の衰弱とIKKb/NFkB経路を介したその新規制御機構

    神崎展

    実験医学12月号 22 (18) 2639 2004年12月

    出版者・発行元:羊土社

  199. Spatial segregation of integrin beta 1 and Talin in the Caveolae-rosette structure defines unique cortical actin organization (Cav-actin) in differentiated 3T3L1 adipocytes

    M Kanzaki, T Nedachi, JE Pessin

    MOLECULAR BIOLOGY OF THE CELL 15 439A-439A 2004年11月

    出版者・発行元:AMER SOC CELL BIOLOGY

    ISSN:1059-1524

  200. TRPチャネルのトランスロケーションと限局したCa2+流入の生理的重要性

    神崎展

    実験医学10月号 22 (15) 2160 2004年10月

    出版者・発行元:羊土社

  201. リンカーヒストンH1bによるMyoD遺伝子の発現調節と筋分化の制御

    神崎展

    実験医学8月号 22 (12) 1720 2004年8月

    出版者・発行元:羊土社

  202. Altered phosphatidylinositol 4,5-bisphosphate (PI4,5P2) metabolism severely affects GLUT4 endocytosis and intracellular vesicle trafficking in 3T3L1 adipocytes

    M Kanzaki, JE Pessin

    DIABETES 53 A15-A15 2004年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  203. 1,4ベンゾジアゼピン誘導体(JTV519)による心突然死の予防効果

    神崎展

    実験医学6月号 22 (9) 1720 2004年6月

    出版者・発行元:羊土社

  204. 小胞輸送システムを介した増殖因子受容体の新しいシグナル伝達機構

    神崎展

    実験医学4月号 22 (6) 852 2004年6月

    出版者・発行元:羊土社

  205. Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocytes

    RT Watson, M Kanzaki, JE Pessin

    ENDOCRINE REVIEWS 25 (2) 177-204 2004年4月

    出版者・発行元:ENDOCRINE SOC

    DOI: 10.1210/er.2003-0011  

    ISSN:0163-769X

    詳細を見る 詳細を閉じる

    Since the discovery of insulin roughly 80 yr ago, much has been learned about how target cells receive, interpret, and respond to this peptide hormone. For example, we now know that insulin activates the tyrosine kinase activity of its cell surface receptor, thereby triggering intracellular signaling cascades that regulate many cellular processes. With respect to glucose homeostasis, these include the function of insulin to suppress hepatic glucose production and to increase glucose uptake in muscle and adipose tissues, the latter resulting from the translocation of the glucose transporter 4 (GLUT4) to the cell surface membrane. Although simple in broad outline, elucidating the molecular intricacies of these receptor-signaling pathways and membrane-trafficking processes continues to challenge the creative ingenuity of scientists, and many questions remain unresolved, or even perhaps unasked. The identification and functional characterization of specific molecules required for both insulin signaling and GLUT4 vesicle trafficking remain key issues in our pursuit of developing specific therapeutic agents to treat and/or prevent this debilitating disease process. To this end, the combined efforts of numerous research groups employing a range of experimental approaches has led to a clearer molecular picture of how insulin regulates the membrane trafficking of GLUT4.

  206. ROLE OF THE ACTIN DYNAMICS IN INSULIN-INDUCED GLUT4 TRANSLOCATION

    KANZAKI Makoto, PESSIN Jeffrey E.

    Proceedings of the Japan Society for Comparative Endocrinology (18) 9-9 2003年8月8日

    ISSN:0913-9036

  207. Insulin signaling: GLUT4 vesicles exit via the exocyst

    M Kanzaki, JE Pessin

    CURRENT BIOLOGY 13 (14) R574-R576 2003年7月

    出版者・発行元:CELL PRESS

    DOI: 10.1016/S0960-9822(03)00478-0  

    ISSN:0960-9822

    詳細を見る 詳細を閉じる

    The localization of the GTP-binding protein TC10 to lipid raft microdomains has been suggested to play a role in the stimulation of GLUT4 translocation. The exocyst has now been identified as a downstream target for TC10, directing GLUT4-containing vesicles to the site of fusion.

  208. 細胞骨格とインスリン依存性糖輸送

    神崎 展, Pessin.JE

    分子糖尿病学の進歩2003 51-57 2003年

    出版者・発行元:金原出版

  209. インスリンによるGLUT4のトランスロケーション誘導機構

    神崎展, PESSIN Jeffrey E

    日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨 28th 23 2003年

  210. Recruitment of atypical PKC lambda/zeta to the lipid raft microdomain through the small GTPase TC10 in 3T3L1 adipocytes

    M Kanzaki, S Mora, AR Saltiel, JE Pessin

    MOLECULAR BIOLOGY OF THE CELL 13 233A-233A 2002年11月

    出版者・発行元:AMER SOC CELL BIOLOGY

    ISSN:1059-1524

  211. Identification of the TC10 amino terminal extension as a disruptor of adipocyte cortical actin and inhibitor of GLUT4 translocation

    JC Hou, SH Chiang, RT Watson, M Kanzaki, AR Saltiel, JE Pessin

    DIABETES 51 A309-A309 2002年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  212. Identification of cav-actin (caveolae-associated actin), a novel cortical actin structure regulated by TC10 in differentiated 3T3L1 adipocyte

    M Kanzaki, S Mora, SH Chiang, AR Saltiel, JE Pessin

    DIABETES 51 A44-A44 2002年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  213. Insulin-stimulated GLUT 4 translocation requires the CAP-Dependent activation of the small GTP binding protein TC10

    SH Chiang, CA Baumann, M Kanzaki, MacAra, I, JE Pessin, AR Saltiel

    DIABETES 50 A278-A278 2001年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  214. Plasma membrane lipid raft compartmentalization of TC10 is required for insulin-stimulated GLUT 4 translocation

    RT Watson, S Shigematsu, M Kanzaki, SH Chiang, IG Macara, AR Saltiel, JE Pessin

    DIABETES 50 A79-A79 2001年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  215. The small GTPase TC10 regulates actin dynamics necessary for insulin-stimulated GLUT4 translocation in adipocytes

    M Kanzaki, AR Saltiel, JE Pessin

    DIABETES 50 A19-A19 2001年6月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  216. G蛋白を介するインスリン依存性糖輸送

    神崎 展, Pessin.JE

    分子糖尿病学の進歩2001 30-36 2001年

    出版者・発行元:金原出版

  217. カルシウムによる細胞増殖の調節

    神崎 展

    カルシウムと骨 2001年

    出版者・発行元:朝倉出版

  218. Signal integration and the specificity of insulin action

    M Kanzaki, JE Pessin

    CELL BIOCHEMISTRY AND BIOPHYSICS 35 (2) 191-209 2001年

    出版者・発行元:HUMANA PRESS INC

    ISSN:1085-9195

    詳細を見る 詳細を閉じる

    Insulin is a potent metabolic hormone essential for the maintenance of normal circulating blood glucose level in mammals. The physiologic control of glucose homeostasis results from a balance between hepatic glucose release (glycogenolysis and gluconeogenesis) and dietary glucose absorption versus skeletal muscle and adipose tissue glucose uptake and disposal. Disruption of this delicate balance either through defects in insulin secretion, liver glucose output, or peripheral tissue glucose uptake results in pathophysiological states of insulin resistance and diabetes. In particular, glucose transport into skeletal muscle and adipose tissue is the rate-limiting step in glucose metabolism and reduction in the efficiency of this process (insulin resistance) is one of the earliest predictors for the development of Type II diabetes. Importantly, recent studies have directly implicated an impairment in insulin receptor signal transduction as the prime mechanism for peripheral tissue insulin resistance. In this review, we have focused on recent developments in our understanding of the molecular mechanisms and signal transduction pathways that insulin utilizes to specifically regulate glucose uptake. The detailed understanding of these events will provide a conceptual framework for the development of new therapeutic targets to treat this chronic and debilitating disease process.

  219. カルシウム透過性チャネルGRCの調節機構

    小島至, 神崎展

    生化学 72 (8) 675 2000年8月25日

    ISSN:0037-1017

  220. Report clarification

    M Kanzaki, M Nagasawa, Kojima, I, C Sato, K Naruse, M Sokabe, H Iida

    SCIENCE 288 (5470) 1347-1347 2000年5月

    出版者・発行元:AMER ASSOC ADVANCEMENT SCIENCE

    ISSN:0036-8075

  221. c-Cbl defines a second signaling pathway required for insulin-stimulated glucose transport independent of PI-3 kinase

    CA Baumann, Ribon, V, PE Bickel, DC Thurmond, M Kanzaki, JE Pessin, AR Saltiel

    DIABETES 49 A82-A82 2000年5月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  222. Role of trimeric GTP-binding protein Gq/11 on vesicle trafficking in 3T3L1 adipocytes

    M Kanzaki, JE Pessin

    DIABETES 49 A331-A331 2000年5月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  223. IGF‐Iにより活性化されるカルシウム透過性チャネルGRCの組織発現

    小和瀬貴律, 張有青, 神崎展, 小島至

    日本内分泌学会雑誌 76 (1) 111 2000年4月20日

    ISSN:0029-0661

  224. 糖尿病を治す遺伝子治療の新戦略

    神崎展

    実験医学4月号 18 (6) 778-779 2000年4月

    出版者・発行元:羊土社

  225. 増殖因子によって活性化されるCa<sup>2+</sup>透過性チャネル

    小島至, 神崎展, 長沢雅裕

    生理学研究所年報 20 379-380 1999年12月1日

  226. インスリンの作用機構の解析 (上原記念生命科学財団S)

    神崎展

    上原記念生命科学財団研究報告集 13 238-240 1999年11月30日

  227. Potential role of the trimeric G protein (Gq-alpha) in the regulation of insulin-stimulated GLUT4 translocation

    M Kanzaki, N Artemyev, JE Pessin

    DIABETES 48 A11-A12 1999年

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  228. Synip, a novel Syntaxin4 binding protein that mediates insulin-stimulated GLUT4 translocation

    J Min, S Okada, M Kanzaki, JS Elmendorf, KJ Coker, BP Ceresa, LJ Syu, Y Noda, AR Saltiel

    DIABETES 48 A11-A11 1999年

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  229. 真核生物のCa<sup>2+</sup>透過性機械受容チャネル その発見,性質,機能

    飯田秀利, 神崎展, 長沢雅裕, 小島至, 佐藤主税, 成瀬恵治, 曽我部正博

    日本細胞生物学会大会講演要旨集 52nd 17 1999年

  230. 増殖因子受容体研究の進歩

    神崎 展, 小島

    日本臨床 56 (7) 1791-1797 1998年7月

  231. 受容体研究の進歩と臨床 ReceptorとSignal Transduction 増殖因子受容体研究の進歩

    神崎展, 小島至

    日本臨床 56 (7) 1791-1797 1998年7月

    ISSN:0047-1852

  232. 酵母細胞内Ca<sup>2+</sup>動態を支えるCa<sup>2+</sup>透過性陽イオンチャネルMid1の構造と機能

    飯田秀利, 佐藤主税, 神崎展, 小島至

    日本植物生理学会年会要旨集 38th(1998) 221 1998年5月

  233. 成長因子受容体

    神崎 展, 小島

    生命の科学 48 (5) 489-494 1997年9月

    出版者・発行元:日本医学雑誌

    DOI: 10.11477/mf.2425901258  

    ISSN:0370-9531

  234. Characterization of the betacellulin receptor in pancreatic AR42J-B20 cells

    N Ishiyama, M Kanzaki, M Furukawa, J Miyagawa, T Hanafusa, M Seno, H Yamada, Kobayashi, I, Kojima, I

    DIABETOLOGIA 40 91-91 1997年6月

    出版者・発行元:SPRINGER VERLAG

    ISSN:0012-186X

  235. Activation of calcium-permeable cation channel by insulin in CHO-IR cells.

    L Nie, M Kanzaki, H Shibata, Kojima, I

    DIABETES 46 799-799 1997年5月

    出版者・発行元:AMER DIABETES ASSOC

    ISSN:0012-1797

  236. 酵母におけるカルシウムシグナルと新規のCa<sup>2+</sup>透過性陽イオンチャネル候補者Mid1

    飯田秀利, 中村寛夫, 小野智子, 奥村万樹子, 嶋田淳子, 坂口修一, 安楽泰宏, 佐藤主税, 神崎展

    日本分子生物学会年会プログラム・講演要旨集 19th 30 1996年7月

  237. ステロイド合成のカルシウム感受性 (厚生省S)

    小島至, 柴田宏, 神崎展

    副腎ホルモン産生異常症調査研究班 平成7年度研究報告書 46-51 1996年

  238. IGFの情報伝達系

    小島 至, 神崎 展

    内分泌・糖尿病科 1 (5), 516-522 1995年

  239. インスリン様成長因子のシグナル伝達機構

    神崎 展, 小島

    内分泌糖尿病科 1 (5) 516-522 1995年

  240. ホルモンと神経伝達物質のシグナル伝達機構

    神崎 展, 小島

    臨床検査 38 (11) 25-30 1994年10月

    DOI: 10.11477/mf.1542902165  

    ISSN:0485-1420

  241. 卵巣か粒膜細胞の増殖と分化 〈IGF‐1作用と細胞外カルシウムについて〉

    神崎展, 服部真彰, 小島至

    日本畜産学会大会講演要旨 89th 122 1994年9月

    ISSN:1342-4688

  242. 下垂体関連成長因子

    神崎 展, 小島

    日本臨床 51 (10) 2555-2560 1993年10月

︎全件表示 ︎最初の5件までを表示

書籍等出版物 1

  1. Mechanisms of Insulin Action

    Alan Saltiel, Jeffrey Pessin ed, Watson RT, Saltiel AR, Pessin JE, Kanzaki M

    Landes Bioscience / Eurekah.com 2005年4月

産業財産権 9

  1. 遺伝子導入装置および遺伝子導入方法

    金子 俊郎, 佐々木 渉太, 神崎 展, 加藤 俊顕

    産業財産権の種類: 特許権

  2. 運動に応答して筋肉細胞において発現変動するタンパク質及びそれらの受容体、それらをコードする遺伝子、並びにそれらを用いたスクリーニング方法

    神崎 展, 根建 拓

    特許第5804389号

    産業財産権の種類: 特許権

  3. 細胞検査用バイオアッセイ用キット

    西澤 松彦, 神崎 展, 長峯 邦明

    特許第5544474号

    産業財産権の種類: 特許権

  4. 運動に応答して筋肉細胞において発現変動するタンパク質及びそれらの受容体、それらをコードする遺伝子、並びにそれらを用いたスクリーニング方法

    神崎 展, 根建 拓

    特許第5246740号

    産業財産権の種類: 特許権

  5. フィーダー細胞を利用した高度発達型培養筋細胞の作製方法

    神崎 展, 根建 拓

    特許第5140827号

    産業財産権の種類: 特許権

  6. 電気刺激による脚運動装置

    神崎 展, 三澤 裕

    特許第4839163号

    産業財産権の種類: 特許権

  7. インスリン反応性糖輸送担体の膜移行活性が測定可能な培養筋細胞

    神崎 展, 根建 拓

    特許第4769935号

    産業財産権の種類: 特許権

  8. 高代謝能を有する培養筋細胞の作製方法

    神崎 展, 藤田 英明, 根建 拓

    特許第4710008号

    産業財産権の種類: 特許権

  9. Cultured Muscle Cells with High Metabolic Activity and Methods for Production of the Cultured Muscle Cells

    Makoto Kanzaki, Taku Nedachi, Hideaki Fujita

    7829334

    産業財産権の種類: 特許権

︎全件表示 ︎最初の5件までを表示

共同研究・競争的資金等の研究課題 31

  1. 皮膚イオントロニクス医工学の開拓

    西澤 松彦, 山崎 研志, 神崎 展, 中川 敦寛

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (S)

    研究機関:Tohoku University

    2022年4月27日 ~ 2027年3月31日

  2. アミノペプチダーゼによる骨格筋量制御機構の解明

    永富 良一, 村山 和隆, 神崎 展, 鈴木 直輝, 長名 シオン

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (A)

    研究機関:Tohoku University

    2021年4月5日 ~ 2024年3月31日

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    日常生活において不自由なく身体を動かすためには骨格筋の働きが不可欠である。スポーツ競技者だけでなく、高齢者においても骨格筋の量や機能について最大の関心事の一つである。世界的な高齢化の進行に伴い、フレイル(虚弱)やサルコペニア(加齢性筋減弱症)、ロコモティブシンドローム(運動器症候群)などの運動機能の低下による要介護者の増加が問題となっており、骨格筋の量・機能低下を予防することは重要な健康課題であると言える。 我々はこれまでに、骨格筋特異的プロテアソーム機能不全マウスでは骨格筋の量・機能が低下することや筋組織中のアミノ酸量が著しく減少することを明らかにしプロテアソームがタンパク質分解を介してオリゴペプチド・アミノ酸を供給することで骨格筋量を維持する可能性を示してきたが、その分子メカニズムについては不明であった。そこで本研究では、タンパク質分解経路であるプロテアソーム・アミノペプチダーゼに着目し、タンパク質分解経路であるアミノペプチダーゼによる骨格筋量を調節機構の解明することを目指す。 今年度はロイシンアミノペプチダーゼ(LAP3)が筋分化に与える影響を検証した。その結果、C2C12マウス筋芽細胞におけるLAP3遺伝子発現抑制は筋分化に顕著な影響を与えることが明らかとなった。さらなる解析を進めるためにLAP3遺伝子欠損マウスの作出に取り組み、現在までに第3世代のマウス繁殖が完了した。次年度にはこのLAP3遺伝子欠損マウスの解析に取り組んでいく予定である。

  3. 運動骨格筋内微小環境と免疫代謝制御に関する研究

    神崎 展

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    研究種目:Grant-in-Aid for Scientific Research (B)

    研究機関:Tohoku University

    2020年4月1日 ~ 2023年3月31日

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    咬筋咀嚼運動モデルに加えて、強制走行運動モデルにおいても、その運動刺激により骨格筋組織内に好中球が一過性に集積する「運動筋ニッチ」が存在し、運動依存性のマイオカイン類の発現亢進と、筋の運動能力(筋持久力)に不可欠であることを明らかにしている。これらの成果をもとに、本年度は、座骨神経の電気パルス刺激(EPS)により任意の収縮運動刺激を後肢骨格筋組織に負荷する実験系を確立して「運動筋ニッチ」における異種細胞間の機能連携性に関する解析を行った。座骨神経へのEPS条件を確定し、ヒラメ筋や長指伸筋などの後肢各骨格筋組織において筋収縮活動に依存して好中球が集積する「運動筋ニッチ」を確認した。また、薬理的実験により、本EPSモデルにおいても強制走行運動モデルと同様にCX3CR1とCXCR2シグナル経路の両方が、「運動筋ニッチ」の形成と各種マイオカイン発現亢進に寄与することを確認した。 運動依存性の好中球動員は筋損傷後の修復過程における重要性が知られていたが、我々の研究により、この運動依存性に一過性に整備される筋内微小領域が各種の骨格筋組織の多岐にわたる運動モダリティーにおいても重要な役割を果たしており、より一般的な運動生理学的事象として普遍的であることを示唆された。さらに麻酔下にあるマウス後肢骨格筋を任意に運動負荷可能なこのEPSモデルのメリットを活用して、多光子顕微鏡をもちいてEPS依存性の後肢骨格筋(大腿四頭筋)の収縮活動時の生体イメージング観察系を確立した。 また、好適な微小領域としての「運動筋ニッチ」から、過度の運動による筋損傷を伴ういわゆる炎症応答としての好中球動員や免疫応答反応への遷移状況を掌握するために、上述した比較的軽微な各種骨格筋運動モデルに加えて、EPSによる筋強収縮負荷に伴う筋損傷と筋痛を惹起する筋炎症モデルでの好中球集積とその生理的意義の解析を行った。

  4. 好中球融合による筋線維の炎症性形質転換 ー慢性筋痛の新規発症メカニズムの解明ー

    土谷 昌広, 神崎 展, 萩原 嘉廣, 四釜 洋介

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    2019年4月1日 ~ 2023年3月31日

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    顎関節症に伴う慢性筋痛の多くは筋・筋膜性疼痛疾患(MPS)と同様の症状を含み,異常な線維性構造(索状硬結)の存在と遷延化した炎症性サイトカイン産生を特徴とする.しかしながら,その発症メカニズムは不明であり,治療法も確立されていない.運動後の筋の超回復では細胞融合/取り込みが活発化し,障害(壊死)細胞をも取り込むことが報告されている.本研究ではその現象に着目し,運動後に浸潤する好中球の取込み(細胞融合のエラー)が炎症性筋線維の形質転換に繋がることを明らかとすることを目的として行う. これまで①運動時の筋結合織内への好中球遊走とIL-1産生、および②炎症性サイトカインIL-1の筋衛星細胞(筋の幹細胞)の増殖/分化の誘導能について示してきた.本年度、①についてはマウス(Balb/cマウス,オス,5週齢)を用い,咀嚼様運動に伴う筋疲労を誘導し,筋膜内における好中球の動態について検討を行い、2光子顕微鏡によるライブイメージングを行った.また、好中球エラスターゼの発現と筋膜エラスチンネットワーク動態を指標とした形態学的検討を行った.その後,シベルスタット投与(ELANE阻害剤)により,筋膜構造の機能適応が抑制されることを明らかとした。②については筋衛星細胞の培養モデルにより、増殖/分化に対する電気刺激収縮の作用について検討を行った。また,筋衛星細胞と血管内皮細胞との共培養による分化促進が示された. 以上の結果から,軽度な筋組織外傷といえる運動疲労時において,好中球には筋機能を維持する機能があり,それらの作用はエラスターゼ産生を介している可能性が示された.エラスターゼは好中球の主要な酵素としての働きが知られる一方で、生理的な意義については不明な点が多かったが、我々の結果は筋機能維持における好中球の機能的役割を示す所見であることが明らかである.

  5. 新概念高速液流気液界面プラズマによる短寿命活性種バイオサイエンスの基盤確立

    金子 俊郎, 高島 圭介, 宮本 浩一郎, 神崎 展, 立川 正憲

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (A)

    研究機関:Tohoku University

    2018年4月1日 ~ 2022年3月31日

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    本研究では,気液界面プラズマで生成される活性種を寿命で区別して,特に1秒未満の時間で反応・自己分解してしまう「極短寿命活性種」の生成・消滅過程を明らかにすると同時に,生体分子・細胞への作用を実験的に明らかにすることを目的として,「高速液流導入大気圧プラズマ装置」を製作して研究を推進した. その結果,高速液流中での極短寿命活性種(OHラジカルおよび亜硝酸前駆体)の高時間分解計測に初めて成功し,さらにOHラジカルの表面局在分布と長寿命活性種による消費を考慮した反応拡散モデルを構築するとともに,極短寿命活性種の生体分子(アミノ酸)への作用を明らかにした.

  6. 筋運動ニッチにおける免疫代謝微小環境の生体イメージング解析

    神崎 展

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Challenging Research (Exploratory)

    研究機関:Tohoku University

    2018年6月29日 ~ 2020年3月31日

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    骨格筋組織は運動活動により多彩な液性因子(マイオカイン)を分泌する。このマイオカインとして多数のサイトカイン類やインターロイキン類が存在することが知られているが、運動と免疫系との連携制御の詳細は不明な点が多い。本研究課題では運動時の骨格筋組織内における微小血管系のライブイメージング解析系を構築し、運動刺激により遊走してくる免疫系細胞(特に好中球)と血管内皮細胞への接着凝集状態を観察することを目的としている。さらに、このライブイメージング系を活用することにより、活動筋組織中への好中球の動員メカニズムとその生理的重要性を探索する。 H30年度は、多光子共焦点レーザー顕微鏡を用いたin vivoライブイメージング観察を構築し、予め染色した好中球(量子ドット標識)が「筋運動ニッチ」領域へと動員される様子(特に動員頻度と滞在時間/ローリング動態)を可視化解析することに成功した。これは、麻酔下にあるマウスの大腿四頭筋に対して電気パルス刺激(EPS)を付与することにより、顕微鏡下で筋収縮運動を任意に負荷できる独自の実験系を新規に構築することにより達成することができた。さらに、このin vivoライブイメージング観察系を用いることにより、骨格筋特異的GLUT4-EGFP発現トランスジェニックマウス(GLUT4-EGFP-Tgマウス)の、この好中球動員部位(賦活化された筋運動ニッチ領域)局所における筋線維(筋膜およびT-tubuleへ)のGLUT4膜移行量を毛細血管(および動員好中球)との位置関係を把握した上で定量解析することに成功した。

  7. ヒト由来筋細胞を用いた「収縮型培養筋細胞作製システム」の構築とその応用

    神崎 展, 萩原 嘉廣

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    研究種目:Grant-in-Aid for Scientific Research (B)

    研究機関:Tohoku University

    2017年4月1日 ~ 2020年3月31日

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    本研究課題では、これまでマウス骨格筋細胞系を用いて開発してきた電気パルス刺激(EPS)特殊培養システムに改良を加えることにより、健常者のみならず、高齢者や腱板断裂患者から採取単離した筋衛星細胞を用いた「運動できる培養ヒト筋細胞系」を構築することを目的としている。 昨年度はヒト筋細胞の脆弱性を補強する目的でフィーダー細胞との混合培養系を構築することに成功した。H30年度は混合培養系をさらに発展させたマウス筋芽細胞株と初代ヒト筋芽細胞(筋衛星細胞)とのハイブリッド筋管細胞系を新たに構築した。これは筋分化にともない筋芽細胞同士が融合して多核の筋管細胞となる特性を利用したものであり、この異種ハイブリッド筋管細胞を作製してEPS特殊培養系へと導入することにより極めて高い収縮能力を獲得させることに成功した。さらに、このハイブリッド筋管細胞の運動刺激応答性の生物反応(各種マイオカイン遺伝子群の発現亢進)について、種差を考慮したRT-PCR手法を開発することにより、ヒト由来およびマウス由来の各々の遺伝子発現変動を正確に評価することを可能にした。さらに、種差を考慮したBioPlexアッセイ法によりヒト由来マイオカインを正確に計測することに成功し、収縮活動負荷によってインターロイキン類 (IL-6, IL-8, IL-10, and IL16), CXC ケモカイン類 (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CCケモカイン類 (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), さらに IFN-γの分泌増加が誘導されることを見出した。また、EPS特殊培養系へと適応できる領域限局導電型のインサートチェンバ-を新たに開発した。

  8. Fusion Errorによる筋痛の慢性化

    土谷 昌広, 神崎 展, 渡邉 誠, 萩原 嘉廣

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Tohoku Fukushi University

    2016年4月1日 ~ 2020年3月31日

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    損傷後の骨格筋の再生において、炎症を含めて複雑な過程を経る。IL-1は主要な炎症性サイトカインであり、炎症のみならず多面的な影響を及ぼすことが報告されている。我々は骨格筋の再生過程におけるIL-1の役割について検討を行った。IL-1欠損マウスでは筋再生過程の遅延が認められ、IL-6などの発現低下や、局所への免疫細胞の浸潤、筋衛星細胞の活性化などの遅延を特徴として確認した。また、インフラマゾームを介したIL-1βの発現が筋痛発症を誘導することについても明らかとした。以上の結果から、IL-1が筋再生において非常に重要な役割を果たす一方で、筋痛の発症因子ともなることが示された。

  9. 超高感度細胞センサアレイによる極微量異種プラズマ刺激同時計測の挑戦

    金子 俊郎, 神崎 展, 佐々木 渉太

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Challenging Exploratory Research

    研究機関:Tohoku University

    2016年4月1日 ~ 2019年3月31日

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    大気圧非平衡プラズマによる細胞への遺伝子導入等の細胞活動に作用する極微量のプラズマ刺激の時空間分布を測定できる『超高感度細胞センサアレイ』の作製に挑戦した.大気圧プラズマジェットおよび液中マイクロプラズマを照射した溶液中に生成される極微量の活性種による酸化刺激,荷電粒子による電気刺激,衝撃波による圧力刺激の2次元分布を計測した.さらに,各々の刺激に個別に反応するチャネルを有する細胞をアレイ状に配置し,細胞内のカルシウムイオン濃度および遺伝子模擬蛍光分子濃度に比例する蛍光強度の2次元分布を計測することで,酸化刺激,電気刺激,圧力刺激に対するセンサアレイとして動作することを実証した.

  10. 脂肪変性したヒト筋組織の再生能評価法の確立

    萩原 嘉廣, 神崎 展, 土谷 昌広

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Challenging Exploratory Research

    研究機関:Tohoku University

    2016年4月1日 ~ 2018年3月31日

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    腱板断裂修復術において、術前の腱板筋の脂肪変性は術後成績の不良因子とされている。また、腱板断裂修復術後に脂肪変性が改善するかどうかは不明で、患者は治療後も日常生活動作の制限を強いられ、そのためにリハビリテーション期間も長くなっている。医療費抑制の観点からも早急に筋肉の脂肪変性の病因を解明し、効果的な治療法の開発が望まれる。本研究では、廃用萎縮・脂肪変性した筋組織から筋衛星細胞を単離し、筋再生能力の評価を行った。廃用萎縮し、脂肪変性した筋肉から得られた筋衛星細胞でも、十分な筋分化能をもっていることが示され、筋委縮・脂肪変性の予防には、筋収縮刺激などが必要であることが示唆された。

  11. インスリン標的細胞に対するプラズマ効果とその治療的応用

    神崎 展, 金子 俊郎

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    研究種目:Grant-in-Aid for Challenging Exploratory Research

    研究機関:Tohoku University

    2016年4月1日 ~ 2018年3月31日

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    大気圧プラズマジェットを生体に照射するとさまざまな生体応答が惹起されるが、その詳細は未解明な点が多い。 本研究では、我々が発見した「プラズマ誘導性の[Ca2+]i応答現象」についてより深く探索することにより,生細胞に対するプラズマ作用の分子機構を解明することを目標とした。特に、インスリン反応性を有する細胞を研究対象とした。そして3T3L1細胞が発現するTRPチャネルがプラスマ作動性カルシウム透過性チャネルであること、さらに、極めて短寿命のプラズマ生成活性種がTRPチャネルの直接的な活性化に関わることを見出した。さらに、プラズマによるインスリン標的細胞の機能制御が可能であることを明らかにした。

  12. ナノプラズマ制御技術の創成と局所照射による生体機能制御

    金子 俊郎, 佐藤 岳彦, 加藤 俊顕, 神崎 展, 立川 正憲, 金高 弘恭, 高島 圭介

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    研究機関:Tohoku University

    2012年6月28日 ~ 2017年3月31日

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    本研究では,高効率・低侵襲に遺伝子・薬剤を細胞内に導入する手法の開発を目的として,液中で微小プラズマを生成・制御する技術を創製し,プラズマ局所照射により生体機能を制御する実験を行った.まず,曲率半径を数100 nmで制御した極細電極に高電圧パルスを印加することで,微小プラズマを生成することに成功し,薬剤及び遺伝子を導入する効果を持つことを実証した.また,プラズマの作用を分離した実験を通して,プラズマ生成に由来する短寿命活性種が薬剤導入に寄与することを示した.さらに,プラズマ照射が液中に生成した短寿命活性種が,細胞膜上のイオンチャネルを介在した薬物輸送を促進することを明らかにした.

  13. 単離骨格筋線維内における小胞輸送動態の超解像ナノ計測手法の確立

    神崎 展

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Exploratory Research

    研究種目:Grant-in-Aid for Challenging Exploratory Research

    研究機関:Tohoku University

    2014年4月1日 ~ 2016年3月31日

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    本研究では,マウスより単離/培養した骨格筋線維内において、空間分解能6 nmというナノ計測を単一分子レベルで実現し,GLUT4およびトランスフェリン受容体分子の輸送動態を定量的に計測することに世界で初めて成功した.また,これまで電子顕微鏡に依存してきた微細構造の観察が超解像顕微鏡により達成できることを確認した.これらの研究成果は,構造的に特殊なためライブイメージング解析が困難であった骨格筋のオルガネラ膜や輸送小胞の細胞生物学的研究に大きく貢献するものである.

  14. Vps10pファミリーのソーティング障害と生活習慣病に関する研究

    神崎 展, 畠山 裕康

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    研究種目:Grant-in-Aid for Scientific Research (B)

    研究機関:Tohoku University

    2013年4月1日 ~ 2016年3月31日

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    Vps10p受容体:分泌性リガンド類:複数回膜貫通型蛋白質(GLUT4)からなるTripartite Complex形成が,GLUT4量およびその輸送制御に重要な役割を果たすことを明らかにした.この過渡的分子複合体は、エンドソーム上で一時的に形成されるため既存方法論では解析が困難であったが、本研究では単一分子視覚化技術により,その高次複合体の形成過程を観察することに成功した.さらに,それらの輸送動態はTbc1dファミリーRabGAP(AS160およびTbc1d1)により調節されることも明らかにした.

  15. インスリン抵抗性とGLUT4トランスロケーションの障害に関する研究

    神崎 展

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)

    研究種目:Grant-in-Aid for Scientific Research (C)

    研究機関:Tohoku University

    2010年 ~ 2012年

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    インスリン抵抗性病態では、インスリン応答性のGLUT4トランスロケーションが著しく減弱しているが、その病態機序の全貌については未解明である。生化学的およびナノ計測学的な解析により、Vps10p受容体ファミリーに属するSortilinがインスリン応答性GLUT4貯蔵小胞の形成に必須であること、さらにインスリン抵抗性病態ではSortilin減少に伴うGLUT4のソーティング障害が惹起されていることを明らかにした。既知の「シグナル伝達系の減弱」に加えて、「ソーティング障害」に起因したインスリン抵抗性病態の発症機序が存在することを示す新知見である。

  16. 細胞の力覚機構の解明

    佐藤 正明, 大橋 俊朗, 神崎 展, 出口 真次, 坂元 尚哉, 安達 泰治, 小椋 利彦, 大橋 一正, 安達 泰治

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Specially Promoted Research

    研究機関:Tohoku University

    2008年 ~ 2012年

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    我々の身体には多くの力を感じる細胞があり、組織や器官が力に応じて機能を発揮するよう制御されているが、その機構は不明であった。本研究では、血管壁、骨、骨格筋の細胞を主たる対象にした。その結果、細胞が接着している部位や細胞の形を決める役目をしている細胞骨格などがセンサの働きをして、シグナルの機能を果たしているタンパク質も分かった。また、生体の発生過程の器官形成において力が重要な役割を果たしていることが明らかとなってきた。

  17. 運動に伴う免疫系の変動のメカニズムとその意義に関する研究

    永富 良一, 神崎 展

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (B)

    研究機関:Tohoku University

    2008年 ~ 2010年

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    運動やストレスに伴う腸管防御的HSP70の発現増強は副腎皮質ホルモンとTLRによる腸内細菌認識の双方に依存していることを明らかにした。逆に腸管TLRによる腸内細菌認識は免疫担当細胞非依存的に腸管運動を抑制するとともに損傷骨格筋の回復過程に強く影響し、筋力トレーニングの効果が腸内環境に依存する可能性が示唆された。一方、骨格筋線維は収縮時に免疫系非依存的にサイトカインIL-6およびケモカインCXCL1,5を分泌すること、IL-6分泌はグリコーゲン減少条件においてカルシニューリン、NFATc1を介して促進的調節が行われていることをin vitro で明らかにした。またin vivo でも骨格筋収縮にともないケモカインCXCL5の転写レベルが増加するものの、蛋白レベルでの増加は筋損傷時だけであり、epigenetic な調節の関与が示唆された。

  18. 生体材料・医用材料として有用な培養細胞由来骨格筋モデルの創製

    根建 拓, 神崎 展

    2008年 ~ 2009年

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    本年度は、研究実施計画に基づき、伸展による培養筋細胞制御についての研究を行った。すなわち、培養筋細胞C2C12をシリコン膜上に播種し、分化させた後、ストレックス社製ストレッチ負荷装置を用いて伸展刺激を行い、細胞形態、細胞機能について解析を行った。まず、10%程度の伸展率でサイクリック伸展刺激を負荷することにより、ストレス応答性MAPキナーゼ、AMPキナーゼなどの酵素が活性化することがわかった。また、伸展刺激を付加することにより、以前申請者らが発見した運動に応答して筋からの分泌が促進される運動因子(CXCケモカイン群)の発現が促進されることが明らかとなった。さらに、伸展刺激負荷時にストレス応答性MAPキナーゼのひとつであるJNKの阻害剤を添加したところ、この運動因子の産生は完全に抑制されることも明らかとなった(Nedachi他、AJPEM, 2009)。2年間の研究成果をまとめると、継代可能な均質な培養筋細胞を用いて、高度発達型培養筋細胞系、擬似的運動刺激系、伸展刺激系の開発に成功した。これらのモデルは、生体筋でみられる運動効果やストレッチ効果を少なくとも一部再現しており、特に運動効果やストレッチ効果を正あるいは負に制御する新規物質の探索など、様々な分野に有用な生体材料・医用材料であることが強く示唆された。

  19. 収縮筋細胞における糖輸送担体GLUT4の分子挙動解析

    神崎 展, 藤田 英明, 根建 拓

    2007年 ~ 2008年

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    筋肉の主要なエネルギー源であるグルコースの取り込みに関わるGLUT4が、筋収縮活動やインスリンの刺激によってどのような分子挙動制御を受けるかについて、収縮能力を獲得した培養筋細胞と蛍光ナノ粒子(Qdot)を用いて1分子レベルで解析することを目的としている。 【計画】Qdotで標識可能なGLUT4分子(myc-GLUT4-ECFP)を恒常発現するマウスC2C12筋芽細胞を作製し、人為的な電気パルス刺激により収縮能力(サルコメア構造)を発達させることにより、本研究に最適化した条件を確立する。さらに、サルコメア構造の発達(有無)によりGLUT4分子挙動制御がどのように変化するのか調べることを目的とした。 【実績】サルコメア構造の発達した収縮型培養筋細胞を作製し、GLUT4分子をQdotにて特異的に標識することに成功した。独自に構築した共焦点顕微鏡を用いてGLUT4分子挙動を解析したところ、刺激のない状態ではGLUT4は何らかの機構により停留されており挙動が著しく制限されていた。一方、インスリンの刺激は、この停留機構を解除することにより、より活発に挙動できる分子群の総数を有意に増加させることが明らかとなった。この停留-解除の強度は、サルコメア構造の発達構築とともに変動することが観察されていることから、アクチン細胞骨格系の規則的な構築体がGLUT4の挙動制御に関与していることが示唆された。 【まとめ】生体筋では解析が難しかったGLUT4分子の挙動解析を、独自に開発した高度発達型の培養筋細胞を利用することによりはじめて可能した。筋構造の発達によりGLUT4の分子(およびGLUT4小胞)の挙動制御は大きく変化することが明らかとなった。

  20. GLUT4膜移行を制御するマシーナリー分子群の網羅的同定

    神崎 展, 根建 拓, 藤田 英明

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (B)

    研究種目:Grant-in-Aid for Scientific Research (B)

    研究機関:Tohoku University

    2006年 ~ 2008年

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    インスリン反応性GLUT4膜移行機構に関わる制御蛋白質の同定およびそれらの機能について、生化学的手法および生細胞のイメージング技術を組み合わせることにより解析した。GLUT4小胞のインスリン反応性獲得には、GLUT4に直接会合する3種類の蛋白(Sortilin-p75NTR-proNGF)による複合体形成とそれに伴うGLUT4の蛋白寿命制御が重要な役割を果たしていることを明らかにした。さらに、このGLUT4を含む蛋白複合体形成は、細胞内シグナル伝達系を活性化して積極的に筋分化にも関与することを明らかにした。

  21. ナノテクノロジーを駆使したインスリン依存性糖輸送システムの統合的解析

    神崎 展, 藤田 英明, 根建 拓

    2005年 ~ 2006年

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    蛍光ナノ粒子Q-dotで標識可能な遺伝子改変型GLUT4 (myc-GLUT4-ECFP)を恒常的に発現する培養脂肪細胞を用いて、Q-dotによりGLUT4分子を標識し、インスリン刺激に依存したGLUT4分子挙動の変化を1分子レベルで正確にとらえ生物物理学的に解析した。 共焦点顕微鏡を用いて、GLUT4貯蔵エンドソームからの運搬過程について、インスリンの効果と細胞分化に伴う変化を解析した。 【実績1】(1)インスリン刺激がないとGLUT4分子自体が「何らかの未知の機構」によりGLUT4貯蔵エンドソームに抑留されていること。(2)一方インスリン刺激により、活発に挙動するGLUT4分子(およびGLUT4含有小胞)が増加すること(GLUT4分子の抑留からの解放)。(3)そして、これらのGLUT4分子挙動制御系は細胞分化に伴い成熟していくこと(インスリン反応性ナノシステムの細胞分化に伴う成熟)をはじめて明らかにした。 全反射顕微鏡をもちいて、GLUT4小胞が最終的に膜融合する過程について、インスリンの効果を解析した。 【実績2】インスリンの刺激により、(1)GLUT4小胞の細胞膜への接近の確率が約3.75倍に増加すること、(2)さらに飛来したGLUT4小胞が細胞膜直下(あるいは細胞膜に)停留する時間が数倍に延長されることを明らかにした。これまでとらえることができなかった微細な生命反応(GLUT4分子挙動の制御)を最先端のナノ材料と視覚化技術により高精度で解析することに成功した。そして、インスリン受容体シグナルの作用点として、少なくとも2カ所(GLUT4貯蔵庫からの放出と開口放出前の繋ぎ止め過程)存在することをはじめて明らかにした。

  22. 糖代謝能研究に適した高度分化型培養筋管細胞系の確立

    神崎 展, 藤田 英明, 根建 拓

    2005年 ~ 2006年

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    電気パルス刺激により培養筋細胞に収縮活動を負荷できる特殊培養系を作製し、代謝能の研究に適した高度発達型培養筋管細胞系を確立することを目的としている。昨年度までの成果をさらに発展させ、電気パルスにより誘導されるサルコメア構造の新規構築の分子機構と細胞内カルシウムの影響について基礎的な検討を行うことを本年度の目的とした。 【実績1】電気パルス刺激に完全に依存して細胞内カルシウム濃度が変動(Ca2+オシレーション)することを確認し、このCa2+オシレーションの周期(周波数)がサルコメア構造の新規構築に重要な役割を果たすことを明らかにした。さらに、Ca2+オシレーションにより、カルシウム依存性蛋白分解酵素であるカルパインの活性化が誘導され、このカルパインの活性化がサルコメア構造の新規構築に必須であることを明らかにした。この過程において、活性化されたカルパインは、細胞膜のIntegrinとアクチン細胞骨格を連結する役割を果たすTALINを分解すること、integrinを修飾するコラーゲン添加によりサルコメア構築が影響受けることから、筋管細胞の細胞接着班様領域を構成する蛋白複合体のCa2+-カルパイン依存性の分解と再構築が、新規サルコメア構造の形成に関与していることが示唆された。 【実績2】本特殊培養系で作製された収縮型培養筋管細胞では、収縮に伴い(1)エネルギー代謝の亢進(2)糖の取り込み亢進(3)インスリン反応性GLUT4膜移行の改善が確認され、生体に非常に近似な高度発達型培養細胞系を確立することに成功した。

  23. カルシウム透過性チャネルを標的とした血管平滑筋細胞増殖制御法の確立

    小島 至, 神崎 展

    1998年 ~ 1998年

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    細胞増殖にはカルシウムイオンの流入が増加することが必須で,これが抑制されると増殖因子はその増殖促進作用を発揮することが出来ない。細胞周期を進行させるプログレッション因子はカルシウム透過性チャネルを活性化して持続的なカルシウム流入増加を惹起するが,そのチャネルの分子実体はこれまでまったく明らかではなかった。本研究ではPCRを用いたクローニング法によりインスリン様成長因子(IGF-I)によって活性化されるカルシウム透過性チャネルの遺伝子クローニングを行いそのチャネルの調節機構について検討を行った。クローニングされたチャネルGRC(growth factor-regulated channel)は756個のアミノ酸からなる膜貫通蛋白で,疎水性の検討から6個の膜貫通部位を持つものと推定された。 その一次構造はTRPに類似し,とくにVR-1とはアミノ酸レベルで40%の相同性を示した。GRCをCHO細胞に発現させるとカルシウム透過性チャネルとして機能した。生理的なカルシウムが存在する場合には,このチャネルは主にカルシウムなど二価陽イオンを透過した。GRCは非刺激時には細胞内プールに存在するが,IGF-Iにより細胞膜上にトランスローケーションした。IGF-Iを除去するとGRCチャネルは再びエンドサイトーシスにより細胞内プールにもどった。このようにGRCチャネルはその細胞内局在が変化することによって調節されるというまったく新しい調節機構をもっていた。

  24. 細胞増殖を調節するカルシウム透過性チャネルの研究

    小島 至, 神崎 展

    1998年 ~ 1998年

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    細胞増殖にはカルシウムイオンの流入が増加することが必須で,これが抑制されると増殖因子はその増殖促進作用を発揮することが出来ない。細胞周期を進行させるプログレッション因子はカルシウム透過性チャネルを活性化して持続的なカルシウム流入増加を惹起するが,そのチャネルの分子実体はこれまでまったく明らかではなかった。本研究ではPCRを用いたクローニング法によりインスリン様成長因子(IGF-I)によって活性化されるカルシウム透過性チャネルの遺伝子クローニングを行いそのチャネルの調節機構について検討を行った。クローニングされたチャネルGRC(growth factor-regulated channel)は756個のアミノ酸からなる膜貫通蛋白で,疎水性の検討から6個の膜貫通部位を持つものと推定された。 その一次構造はTRPに類似し,とくにVR-1とはアミノ酸レベルで40%の相同性を示した。GRCをCHO細胞に発現させるとカルシウム透過性チャネルとして機能した。生理的なカルシウムが存在する場合には,このチャネルは主にカルシウムなど二価陽イオンを透過した。GRCは非刺激時には細胞内プールに存在するが,IGF-Iにより細胞膜上にトランスローケーションした。IGF-Iを除去するとGRCチャネルは再びエンドサイトーシスにより細胞内プールにもどった。このようにGRCチャネルはその細胞内局在が変化することによって調節されるというまったく新しい調節機構をもっていた。

  25. 新たな増殖因子作動性カルシウムチャネルの探索とその活性制御機構の解析

    神崎 展

    1997年 ~ 1998年

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    昨年度の検討により、マウス胸腺cDNAライブラリーから新規チャネル様蛋白の遺伝子の単離に成功し、その機能解析を遺伝子工学的、電気生理学的手法を用いて行った。 この遺伝子は全長2268bpで、アミノ酸756残基からなり細胞膜を6回貫通する新規チャネル様蛋白をコードしていた。trp familyに属するカプサイシン受容体(VRl)と約43%の相同性が認められた。ノーザン解析により種々の組織でその発現が認められ、特に胸腺、肺、脳で強く発現していた。発現ベクターにcDNAを組み込みCHO細胞などに強制発現させたところ、インスリン様成長因子-1(IGF-I)に反応し細胞内カルシウム濃度が著明に増加した。C末端に対する抗体を作製し細胞内における局在、各組織におけるこの蛋白の発現についても検討した。IGF-I刺激によりこのチャネル蛋白の細胞内から細胞膜上へのトランスローケションが惹起されることが免疫染色により確認された。。電気生理学的検討から、IGF-Iで前処理した強制発現細胞では陽イオンの透過性が大きく増加しており、このチャネル蛋白がnon-selective Ca2+透過性チャネルとして機能することが確認された。 結論:増殖因子作動性の新規カルシウム透過性チャネルを同定した。

  26. 増殖因子によって活性化されるカルシウム透過性チャネルの研究

    小島 至, 神崎 展

    1997年 ~ 1997年

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    1)MID1チャネルの機能解析: MID1はフェロモンによる酵母の分化誘導に関与する分子としてクローニングされた遺伝子である。MID1遺伝子は細胞内へのカルシウム流入機構に関与すると考えられており,その遺伝子のコードする蛋白は膜貫通蛋白であることから,我々はこれを哺乳動物細胞に導入して機能を解析した。その結果,MID1の発現により細胞膜のカルシウム透過性が増すことが明らかになり,MID1蛋白はカルシウム透過性チャネルであると考えられた。MID1チャネルはカルシウムイオンだけでなく,一価の陽イオンも透過させることから,非選択性の陽イオンチャネルである。また通常のチャネルが透過させないセシウムを効率よく透過させる点が特徴である。MID1チャネルは膜電位に依存しない電位非依存性陽イオンチャネルである。このチャネルのもっとも大きな特徴は,細胞膜に陰圧をかけることにより活性化される点である。すなわち細胞膜に静水圧をかけるとそれに応じてチャネルの開口確率が増加するのである。この結果はMID1チャネルが機械受容チャネルであることを示している。実際,機会受容チャネルを抑制するガドリニウムの存在によりこのMID1チャネルは抑制された。MID1は真核細胞で初めて見出された機械受容チャネル遺伝子であることが明らかになった。

  27. 増殖因子によって活性化されるカルシウム透過性チャネルの研究

    小島 至, 神崎 展

    1997年 ~ 1997年

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    1)MID1チャネルの機能解析: MID1はフェロモンによる酵母の分化誘導に関与する分子としてクローニングされた遺伝子である。MID1遺伝子は細胞内へのカルシウム流入機構に関与すると考えられており,その遺伝子のコードする蛋白は膜貫通蛋白であることから,我々はこれを哺乳動物細胞に導入して機能を解析した。その結果,MID1の発現により細胞膜のカルシウム透過性が増すことが明らかになり,MID1蛋白はカルシウム透過性チャネルであると考えられた。MID1チャネルはカルシウムイオンだけでなく,一価の陽イオンも透過させることから,非選択性の陽イオンチャネルである。また通常のチャネルが透過させないセシウムを効率よく透過させる点が特徴である。MID1チャネルは膜電位に依存しない電位非依存性陽イオンチャネルである。このチャネルのもっとも大きな特徴は,細胞膜に陰圧をかけることにより活性化される点である。すなわち細胞膜に静水圧をかけるとそれに応じてチャネルの開口確率が増加するのである。この結果はMID1チャネルが機械受容チャネルであることを示している。実際,機会受容チャネルを抑制するガドリニウムの存在によりこのMID1チャネルは抑制された。MID1は真核細胞で初めて見出された機械受容チャネル遺伝子であることが明らかになった。

  28. ペプチド黄学を駆使し新たな肝再生法の開発より広範な肝切除を可能にする方法、移植肝の再生を促進する治療法を目指して

    小暮 公孝, 神崎 展, 柴田 宏, 小島 至

    提供機関:Japan Society for the Promotion of Science

    制度名:Grants-in-Aid for Scientific Research

    研究種目:Grant-in-Aid for Scientific Research (A)

    研究機関:Gunma University School of Medicine

    1996年 ~ 1997年

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    肝硬変を伴った肝癌に対し有効な肝再生促進法あれば広範な肝切除が可能になり根治手術の幅が広がる、また、生体肝移植においても小さな移植肝グラフトの再生を促進させることで手術の安全性を高めることが可能となる。我々はTGR-βファミリーに属する肝細胞のオートクリン増殖抑制因子であるアクチビンAの作用を特異的にブロックするアクチビンA特異的結合蛋白のフォリスタチンを用いてラットの70%肝切除のみならず90%肝切除に対しても著明な肝再生促進効果を認めている。平成9年度はこのフォリスタチンの薬物としての臨床応用を展望して1)活性をもち安定で有効内濃度を持つフォリスタチンのアナログペプチドの開発、2)90%肝切除モデルを作成し合成ペプチドの有効性を検討する、の2点の研究計画を立てて実験を行った。1)に関してはフォリスタチンの作用を発現すると考えられるアミノ酸配列4カ所(1.Heparin binding side.2.3.follistatin module,4.c-terminal)を選定してアナログを作成した。現在、その合成ペプチドの精度をHPLCを用いて検討中である。また、新たにアミノ酸315個と288個の2種類のフォリスタチンを分子生物学的に作成しその肝再生促進効果をin vitro、in vivoで比較検討を行っている。2)に関しては術式、飼育条件などを検討した結果、絶食状態で手術を行うことにより確立した90%肝切除モデルを用いてフォリスタチンの肝再生促進効果の検討を行ったところ、著名な肝再生促進効果を確認することができた。また、絶食状態とフォリスタチンの作用機序の相関に関しても検討を行った(投稿中)。以上、平成9年度の2点の研究目的に関してはほぼ、計画どおりの成果を得ることができた。

  29. 増殖因子によって活性化されるカルシウム透過性チャネルの研究

    小島 至, 神崎 展

    1996年 ~ 1996年

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    Bリンパ球の発現するカルシウム(Ca^<2+>)透過性チャネルCD20を線維芽細胞に発現させると,インスリン様成長因子(IGF-I)により惹起させる細胞周期のG1期のプログレッションが加速すること,IGF-IがCD20チャネルを活性化することなどをこれまで明らかにしてきた。今回,IGF-IがCD20チャネルを活性化する機構,とくにG蛋白の関与を検討した。その結果,IGF-I受容体によるCD20チャネル活性化には細胞質にGTPだけでなく,ATPが必要であることが明らかになった。またこのATPは非水解性のアナログAMP-PNPによって代替え可能であった。CD20チャネルは細胞内のGTP-γSによって活性化され,この作用にはATPは必要でなかった。したがってATPはGTP-γSによりG蛋白が直接活性化される際には不要であり,受容体によるG蛋白活性化の過程で必要であろうと思われた。excised modeのパッチクランプ法によりCD20チャネルの活性はG_<i2>蛋白のαサブユニットにより活性化されること,G_<i3>蛋白のαサブユニットは弱いながらも活性化作用をもつが,G_<i1>のαサブユニットやβγサブユニットには活性化作用がないことが明らかになった。さらにIGF-IによるCD20チャネル活性化は抗α_<i2>抗体を投与することにより抑制されたことから,IGF-I受容体が活性化されるとG_<i2>蛋白が活性化され,そのαサブユニットによりCD20チャネルが活性化されることが明らかになった。

  30. 増殖因子によって活性化されるカルシウム透過性チャネルの制御機構

    小島 至, 神崎 展

    1996年 ~ 1996年

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    Bリンパ球の発現するカルシウム(Ca^<2+>)透過性チャネルCD20を線維芽細胞に発現させると,インスリン様成長因子(IGF-I)により惹起させる細胞周期のG1期のプログレッションが加速すること,IGF-IがCD20チャネルを活性化することなどをこれまで明らかにしてきた。今回,IGF-IがCD20チャネルを活性化する機構,とくにG蛋白の関与を検討した。その結果,IGF-I受容体によるCD20チャネル活性化には細胞質にGTPだけでなく,ATPが必要であることが明らかになった。またこのATPは非水解性のアナログAMP-PNPによって代替え可能であった。CD20チャネルは細胞内のGTP-γSによって活性化され,この作用にはATPは必要でなかった。したがってATPはGTP-γSによりG蛋白が直接活性化される際には不要であり,受容体によるG蛋白活性化の過程で必要であろうと思われた。excised modeのパッチクランプ法によるCD20チャネルの活性はG_<i2>蛋白のαサブユニットにより活性化されること,G_<i3>蛋白のαサブユニットは弱いながらも活性化作用をもつが,G_<i1>のαサブユニットやβγサブユニットには活性化作用がないことが明らかになった。さらにIGF-IによるCD20チャネル活性化は抗α_<i2>抗体を投与することにより抑制されたことから,IGF-I受容体が活性化されるとG_<i2>蛋白が活性化され,そのαサブユニットによりCD20チャネルが活性化されることが明らかとなった。

  31. GTP結合蛋白による増殖因子作動性カルシウム透過性チャネルの活性化機構の解析

    神崎 展

    1996年 ~ 1996年

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    増殖因子の作用発現においてカルシウムイオンは必須の役割を果たしているが,その流入経路すなわちカルシウムチャネルの本体は不明で,その活性調節機構も明らかでない.本研究では細胞増殖に関与することが確かめられたカルシウムチャネル分子CD20を遺伝子導入により過剰発現させたBalb/c3T3細胞を作製し,今まで解析が困難であった増殖因子受容体によるチャネル活性化の分子機構を分子生物学的および電気生理学的手法を用いて明らかにすることを目的とした. その結果,CD20チャネルはインスリン様増殖因子-1(IGF-1)によって活性化されそのカルシウム透過性が亢進するが百日咳毒素(PTX)処理によりこのIGF-1の活性化作用は完全に消失すること,一方GTP結合蛋白を直接的に活性化するマストパランやconstitutively active G蛋白変異体であるGip2の共発現によりこのチャネルが強く活性化されることなどから,CD20がPTX感受性GTP結合蛋白を介して活性化されていることを明らかにした(Kanzaki et al. J.Biol. Chem. 272:4964-4969).またIGF-1によるCD20チャネルの活性化には細胞内にGTP,Mg2+に加えATPが必要であるが,一方GTP結合蛋白を直接活性化するマストパランやconstitutively active変異体Gip2の共発現によるCD20の活性化にはATPは必要でなかった.これらのことからATPはIGF-1受容体がGTP結合蛋白を活性化するステップに重要であることを明らかにした.またこのATPは非水解性ATPアナログであるAMP-PNPによって置き換えることができることから蛋白のリン酸化は関与しないと考えられた(投稿中).

︎全件表示 ︎最初の5件までを表示

担当経験のある科目(授業) 2

  1. 基礎生命工学 東北大学

  2. 基礎生物科学 東北大学