Mechanism of insulin exocytosis in pancreatic β cells by analyzing single insulin granule motion with evanescent wave microscopy
| Project/Area Number |
14570130
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Kyorin University |
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Principal Investigator |
OHARA Mica Kyorin University, School of Medicine, Lecturer, 医学部, 講師 (40201941)
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| Co-Investigator(Kenkyū-buntansha) |
NAGAMATSU Shinya Kyorin University, School of Medicine, Professor, 医学部, 教授 (80231489)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | insulin / exocytosis / pancreatic beta cell / evanescentwave microscopy / type 2 diaetes / secretory granule / インスリン分泌 / エバネッセント顕微鏡 / 分泌顆粒 / 2型糖尿病 |
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| Research Abstract |
It is essential to use primary β cell for revealing the molecular mechanism of insulin exocytosis and the machinery behind impaired insulin release in type 2 diabetes, nevertheless, technical difficulties prevented us to analyze such a process. We have recently succeeded in monitoring the dynamics of insulin granules in primary β cell prepared from rat pancreas using TIRF imaging system with high time resolution. In the 1^<st>-phase of insulin release, the fusing granules occurred mostly from previously docked granules, but fusion granules in 2^<nd>-phase originated from newcomers. TIRF images could reveal that the docking time of newcomers on the plasma membrane was extremely short within 50 ms, in contrast to relatively long time in insulinoma MIN6 cells (37±11s), thus the exocytotic mechanism in 1^<st>-phase is quite different from that in the 2^<nd>-phase in primary β cells. We have then addressed the question if insulin exocytotic site is associated with SNAREs or related proteins. To study the interaction between t-SNAREs and the docking and fusion of insulin granules, we labeled endogenous syntaxin 1A and SNAP-25 with Cy3-labeled anti-syntaxin 1A and anti SNAP-25 antibodies by conjugating antibodies with HIV-1 TAT, that was rapidly transduced into the living β cells. TIRF image showed that t-SNAREs are distributed in numerous separated clusters (<400 nm) on the plasma membrane. Insulin granules were preferentially docked (about 80%) to sites on syntaxin 1A clusters, colocalizing with SNAP-25 clusters. The fusion was seen at the syntaxin 1A clusters. Now, we are investigating the relationship between site of insulin exocytosis and CAZ (cytomatrix at the active zone) proteins. Thus, our data revealed the molecular mechanism of insulin exocytosis in primary β cells.
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Report
(4 results)
Research Products
(52 results)
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[Journal Article] Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.2005
Author(s)
Kasai, K., Ohara-Imaizumi, M., Takahashi, N., Mizutani, S., Zhao, S., Kikuta, T., Kasai, H., Nagamatsu, S., Gomi, H., Izumi, T.
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Journal Title
J.Clin.Invest. 115
Pages: 388-396
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy.2005
Author(s)
Kikuta, T., Ohara-Imaizumi, M., Nakazaki, M., Nishiwaki, C., Nakamichi, Y, Tei, C., Aguilar-Bryan, L., Bryan, J., Nagamatsu, S.
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Journal Title
FEBS Lett. 579
Pages: 1602-1606
Description
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[Journal Article] Site of docking and fusion of insulin secretory granules in live MIN6 β cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.2004
Author(s)
Ohara-Imaizumi, M., Nishiwaki, C., Kikuta, T., Kumakura, K., Nakamichi, Y., Nagamatsu, S.
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Journal Title
J.Biol.Chem. 279
Pages: 8403-8408
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells : different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.2004
Author(s)
Ohara-Imaizumi, M., Nishiwaki, C., Kikuta, T., Nagai, S., Nakamichi, Y., Nagamatsu, S.
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Journal Title
Biochem.J. 381
Pages: 13-18
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Functions of pancreatic beta cells and adipocytes in bombesin receptor subtype-3-deficient mice.2004
Author(s)
Nakamichi, Y., Wada, E., Aoki, K., Ohara-Imaizumi, M., Kikuta, T., Nishiwaki, C., Matsushima, S., Watanabe, T., Wada, K., Nagamatsu, S.
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Journal Title
Biochem.Biophys.Res.Commun. 318
Pages: 698-703
Description
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[Journal Article] The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis2004
Author(s)
Ohara-Imaizumi, M., Cardozo, A.K., Kikuta, T., Eizirik, D.L., Nagamatsu, S.
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Journal Title
J.Biol.Chem. 279
Pages: 41271-41274
Description
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[Journal Article] Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analyzed by total internal reflection fluorescence microscopy.2004
Author(s)
Ohara-Imaizumi, M., Nishiwaki, C., Nakamichi, Y., Kikuta, T., Nagai, S., Nagamatsu, S.
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Journal Title
Diabetologia. 47(12)
Pages: 2200-2207
Description
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[Journal Article] PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells.2003
Author(s)
Nakamichi, Y., Kikuta, T., Ito, E., Ohara-Imaizumi.M., Nishiwaki, C., Ishida, H., Nagamatsu, S.
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Journal Title
Biochem.Biophys.Res.Commun. 306
Pages: 832-836
Description
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[Journal Article] Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.2002
Author(s)
Ohara-Imaizumi, M., Nakamichi, Y., Tanaka, T., Katsuta, H., Ishida, H., Nagamatsu, S.
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Journal Title
Biochem.J. 363
Pages: 73-80
Description
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[Journal Article] Decreased marble burying behavior in female mice lacking neuromedin-B receptor (NMB-R) implies the involvement of NMB/NMB-R in 5-HT neuron function.2002
Author(s)
Yamada, K., Wada, E., Yamano, M., Sun, Y.J., Ohara-Imaizumi, M., Nagamatsu, S., Wada, K.
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Journal Title
Brain Res. 942
Pages: 71-78
Description
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[Journal Article] Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells.2002
Author(s)
Sasakawa, N., Ohara-Imaizumi, M., Okubo, S., Hosaka, S., Hayashi, M., Kumakura, K.
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Journal Title
Ann N Y Acad.Sci. 971
Pages: 273-274
Description
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