| Project/Area Number |
08671165
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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 |
内分泌・代謝学
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| Research Institution | UNIVERSITY OF TOKUSHIMA |
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Principal Investigator |
NOMA Yoshihiko Medical School Hospital, UNIVERSITY OF TOKUSHIMA assistant professor, 医学部・附属病院, 講師 (10218349)
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| Co-Investigator(Kenkyū-buntansha) |
MIZUNO Akira Medical School Hospital, UNIVERSITY OF TOKUSHIMA research assistant, 医学部・附属病院, 助手 (80219641)
KUWAJIMA Masamichi Scool of Medicine, UNIVERSITY OF TOKUSHIMA associate Professor, 医学部, 助教授 (00205262)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1997: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1996: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | glucokinase / islet B cell / insulin secretion / グルコカイネース |
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| Research Abstract |
This project were planned to identify the place of the glucokinase localization in islet cell and to make clear whether the glucokinase activity is related to the place of the enzyme localization in the cell.
At first, isolated isltets cells were cultured in normal or high glucose condition mediums and the islet cell components were fractionated into 4, nucleus rich, mitochondria rich, pellet, and supernatant. The amounts of glucokinase in each fraction were analyzed with western blotting. Some experiments showed that the glucokinase amount of mitochondria rich fraction increased after glucose stimulation, but another experiment showed reversed result or no change. We could not conclude any with this method. We suspected that the glucokinase binding to some organella were liberated in the solution for the fractionation. Several kinds of solution for the fractionation were tested but not good result.
At second, we analyzed with immunofluorescent staining of the pancreatic tissues. High ma
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gnification iamges using confocal microscope of double staining for glucokinase and mitochondria showed the co-localization of glucokinase and mitochondria after glucose stimulation. Hexokinase is already known to bind to mitochondria. To confirm whether the images that the glucokinase binding to mitochondria, we compared the double stain images of hexokinase and mitochondria to that of glucokinase and mitochondria. The position of hexokinase on mitochondria was equivalent to the position of glucokinase on mitochondria. Glucokinase was suggested to move to mitochondria and to access ATP more efficiently after glucose stimulation . At third, to know the peri-nuclear organell where glucokinase locate without glucose stimulation, we stained Golgi apparatus with glucokinase. Those images did not match. Then, we stained glucokinase with Gold particle and tried to observe glucokinase location with electron-microscopy Gold staining seemed to be good with confocal microscope but most organella were destroyed during the following fixation and dehydration. We are trying to find proper condition. Less
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