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

目的GLUT4结合蛋白可能在GLUT4囊泡循环和胰岛素诱导的GLUT4移位过程中发挥重要作用。我们假设脂肪细胞中存在GLUT4“接头蛋白”，并鉴定出一种新的具有ANK结构和假定磷酸化位点的GLUT4结合蛋白“p61”，并对其功能进行了分析。方法用豚鼠免疫制备抗血清，进行亲和纯化。P61基因敲除对葡萄糖转运活性的影响：将shRNA导入3T3-L1脂肪细胞，将p61基因敲除，检测胰岛素诱导的GLUT4转位和葡萄糖摄取。细胞内定位：3T3-L1脂肪细胞固定和免疫染色后，在共聚焦显微镜下观察p61和GLUT4在细胞内的定位。结果在基础状态下，p61与GLUT4一起存在于3T3-L1脂肪细胞胞浆中，并存在部分共定位。胰岛素依赖的GLUT4转位和葡萄糖摄取均被p61基因敲除抑制。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。

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.