Characterization of Insulin-Responsive GLUT4 Storage Vesicles Isolated from 3T3-L1 Adipocytes

Insulin regulates glucose transport in muscle and adipose tissue by triggering the translocation of a facilitative glucose transporter, GLUT4, from an intracellular compartment to the cell surface. It has previously been suggested that GLUT4 is segregated between endosomes, the trans-Golgi network (TGN), and a postendosomal storage compartment. The aim of the present study was to isolate the GLUT4 storage compartment in order to determine the relationship of this compartment to other organelles, its components, and its presence in different cell types. A crude intracellular membrane fraction was prepared from 3T3-L1 adipocytes and subjected to iodixanol equilibrium sedimentation analysis. Two distinct GLUT4-containing vesicle peaks were resolved by this procedure. The lighter of the two peaks (peak 2) was comprised of two overlapping peaks: peak 2b contained recycling endosomal markers such as the transferrin receptor (TfR), cellubrevin, and Rab4, and peak 2a was enriched in TGN markers (syntaxin 6, the cation-dependent mannose 6-phosphate receptor, sortilin, and sialyltransferase). Peak 1 contained a significant proportion of GLUT4 with a smaller but significant amount of cellubrevin and relatively little TfR. In agreement with these data, internalized transferrin (Tf) accumulated in peak 2 but not peak 1. There was a quantitatively greater loss of GLUT4 from peak 1 than from peak 2 in response to insulin stimulation. These data, combined with the observation that GLUT4 became more sensitive to ablation with Tf-horseradish peroxidase following insulin treatment, suggest that the vesicles enriched in peak 1 are highly insulin responsive. Iodixanol gradient analysis of membranes isolated from other cell types indicated that a substantial proportion of GLUT4 was targeted to peak 1 in skeletal muscle, whereas in CHO cells most of the GLUT4 was targeted to peak 2. These results indicate that in insulin-sensitive cells GLUT4 is targeted to a subpopulation of vesicles that appear, based on their protein composition, to be a derivative of the endosome. We suggest that the biogenesis of this compartment may mediate withdrawal of GLUT4 from the recycling system and provide the basis for the marked insulin responsiveness of GLUT4 that is unique to muscle and adipocytes.

胰岛素可通过触发易化葡萄糖转运蛋白GLUT4（facilitative glucose transporter）从细胞内区室向细胞膜表面转位，调控肌肉与脂肪组织的葡萄糖转运。此前已有研究提出，GLUT4可分选定位于内体、反式高尔基体网络（trans-Golgi network, TGN）以及胞后储存区室中。本研究旨在分离GLUT4储存区室，以明确该囊泡区室与其他细胞器的关联、其组分构成，以及在不同细胞类型中的分布情况。
研究人员从3T3-L1脂肪细胞中制备粗制细胞内膜组分，并通过碘克沙醇平衡沉降分析进行分离。该方法可分离得到两个截然不同的含GLUT4囊泡峰。两个峰中密度较轻的一个（峰2）包含两个重叠的亚峰：其中2b峰含有转铁蛋白受体（transferrin receptor, TfR）、胞裂蛋白（cellubrevin）与Rab4等循环内体标记物，而2a峰则富集了反式高尔基体网络标记物，包括突触融合蛋白6（syntaxin 6）、阳离子依赖型甘露糖6-磷酸受体、sortilin以及唾液酸转移酶。峰1中含有大量GLUT4，同时伴随少量但可检测到的cellubrevin，且转铁蛋白受体含量相对较低。
与上述结果一致的是，内吞转铁蛋白（transferrin, Tf）仅在峰2中积累，而未在峰1中检出。经胰岛素刺激后，峰1中GLUT4的减少量显著高于峰2。结合胰岛素处理后GLUT4对Tf-辣根过氧化物酶介导的消融敏感性升高这一观察结果，上述数据表明峰1富集的囊泡对胰岛素具有高度响应性。
对其他细胞类型分离得到的膜组分进行碘克沙醇梯度分析结果显示，在骨骼肌细胞中，大部分GLUT4定向分布至峰1；而在CHO细胞中，绝大多数GLUT4则定位于峰2。
以上结果表明，在胰岛素敏感细胞中，GLUT4被靶向至一类囊泡亚群；根据其蛋白质组构成来看，该亚群属于内体的衍生物。我们推测，该储存区室的生物发生过程可能介导GLUT4从循环内体系统中脱离，并为肌肉与脂肪细胞所特有、且具有显著胰岛素响应性的GLUT4转运机制提供了分子基础。