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The analysis of a GLUT4 binding ANK structure protein, which may facilitate insulin induced-glucose uptake.

GLUT4 结合 ANK 结构蛋白的分析，该蛋白可能促进胰岛素诱导的葡萄糖摄取。

基本信息

批准号：
17590935
负责人：
OKUYA Shigeru
金额：
$ 2.24万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590935/
关键词：
GLUT4 translocation recycling adaptor protein ANK structure glucose uptake insulin adipocyte

项目摘要

PurposeIt has been supposed that a protein binding to GLUT4 could play an important role during the recycling of GLUT4 vesicle and insulin-induced GLUT4 translocation. We assumed the existence of GLUT4 "adaptor protein" in adipocytes, and we have identified a novel GLUT4 binding protein "p61" that has ANK structure and presumed phosphorylated sites, and analyzed its function.MethodsAnit-p61 antibody: After antiserum was made with guinea pig immunity, affinity purification was performed. Influence of p61 knockdown on glucose transport activity: p61 was knocked down by introducing shRNA into 3T3-L1 adipocytes, and then insulin-induced GLUT4 translocation and glucose uptake were examined. Intracellular localization: After the fixation and immunostaining 3T3-L1 adipocytes were observed with a confocal microscope, and the intracellular localization of p61 and the GLUT4 was examined.ResultsIn the basal state, p61 existed in the 3T3-L1 adipocyte cytoplasm as well as GLUT4, and the partial co-localization was suggested. Both the insulin-dependent GLUT4 translocation and glucose uptake were suppressed by p61 knockdown. On the other hand, an obvious change in the p61 localization was not recognized under the insulin stimulation.ConclusionIt is not probable that the p61 localization in the adipocyte was changed in insulin-dependent manner. It is suggested that a novel ANK structure protein p61 facilitates GLUT4 translocation, and then activates insulin-induced glucose uptake.

目的推测一种与GLUT4结合的蛋白可能在GLUT4囊泡循环和胰岛素诱导的GLUT4易位过程中发挥重要作用。我们假设在脂肪细胞中存在GLUT4“适配蛋白”，并鉴定了一种新的GLUT4结合蛋白“p61”，该蛋白具有ANK结构和推测的磷酸化位点，并分析了其功能。方法sanit -p61抗体：用豚鼠免疫制备抗血清后，进行亲和纯化。p61敲低对葡萄糖转运活性的影响：通过在3T3-L1脂肪细胞中引入shRNA敲低p61，然后检测胰岛素诱导的GLUT4易位和葡萄糖摄取。细胞内定位：3T3-L1脂肪细胞固定免疫染色后，共聚焦显微镜下观察，检测p61和GLUT4的细胞内定位。结果在基础状态下，p61存在于3T3-L1脂肪细胞质和GLUT4中，且存在部分共定位。p61敲低抑制了胰岛素依赖性GLUT4易位和葡萄糖摄取。另一方面，在胰岛素刺激下，p61定位未发生明显变化。结论p61在脂肪细胞中的定位不可能以胰岛素依赖的方式发生改变。提示一种新的ANK结构蛋白p61促进GLUT4易位，进而激活胰岛素诱导的葡萄糖摄取。