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

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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.