Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice
Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice
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DOI：
10.1152/ajpendo.00466.2011
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发表时间：
2012-04-01
影响因子：
5.1
通讯作者：
Cushman, Samuel W.
中科院分区：
文献类型：
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作者：
Lizunov, Vladimir A.;Stenkula, Karin G.;Cushman, Samuel W.
胰岛素刺激HA-GLUT 4-GFP转基因小鼠骨骼肌中GLUT 4囊泡的融合，但不刺激束缚。Am J Physiol Endocrinol Metab 302：E950-E960，2012.首次发表于2012年1月31日; doi：10.1152/ajpendo.00466.2011。胰岛素通过调节GLUT 4在细胞表面和细胞内区室之间的亚细胞分布来调节葡萄糖摄取到脂肪和肌肉中。然而，通过经典的亚细胞分离技术定量肌肉中的这些易位过程被污染的微纤维蛋白混淆;在分子水平上的动态研究几乎是不可能的。在这项研究中，我们介绍了一种肌肉特异性转基因小鼠模型，其中HA-GLUT 4-GFP在MCK启动子的控制下表达。发现HA-GLUT 4-GFP在胰岛素刺激后易位至质膜和T-小管，从而模拟内源性GLUT 4。为了研究骨骼肌中GLUT 4运输的动力学，我们定量了肌膜和T小管附近含有HA-GLUT 4-GFP的囊泡，并通过全内反射荧光和共聚焦显微镜在单个囊泡水平上分析了胰岛素刺激的胞吐作用。我们发现，只有10%的细胞内GLUT 4池包括移动的囊泡，而大多数的GLUT 4结构保持静止或拴系在肌膜或T-小管。事实上，大多数胰岛素刺激的胞吐作用来自于预束缚的囊泡，而移动的GLUT 4囊泡的小池不受胰岛素的显著影响。我们的数据有力地表明，GLUT 4囊泡的移动的池不是胰岛素作用的主要部位，而是局部分布的。最有可能的是，预栓GLUT 4结构负责胰岛素刺激胞吐的初始阶段。
Insulin stimulates fusion, but not tethering, of GLUT4 vesicles in skeletal muscle of HA-GLUT4-GFP transgenic mice. Am J Physiol Endocrinol Metab 302: E950-E960, 2012. First published January 31, 2012; doi:10.1152/ajpendo.00466.2011.-Insulin regulates glucose uptake into fat and muscle by modulating the subcellular distribution of GLUT4 between the cell surface and intracellular compartments. However, quantification of these translocation processes in muscle by classical subcellular fractionation techniques is confounded by contaminating microfibrillar protein; dynamic studies at the molecular level are almost impossible. In this study, we introduce a muscle-specific transgenic mouse model in which HA-GLUT4-GFP is expressed under the control of the MCK promoter. HA-GLUT4-GFP was found to translocate to the plasma membrane and T-tubules after insulin stimulation, thus mimicking endogenous GLUT4. To investigate the dynamics of GLUT4 trafficking in skeletal muscle, we quantified vesicles containing HA-GLUT4-GFP near the sarcolemma and T-tubules and analyzed insulin-stimulated exocytosis at the single vesicle level by total internal reflection fluorescence and confocal microscopy. We found that only 10% of the intracellular GLUT4 pool comprised mobile vesicles, whereas most of the GLUT4 structures remained stationary or tethered at the sarcolemma or T-tubules. In fact, most of the insulin-stimulated exocytosis emanated from pretethered vesicles, whereas the small pool of mobile GLUT4 vesicles was not significantly affected by insulin. Our data strongly suggest that the mobile pool of GLUT4 vesicles is not a major site of insulin action but rather locally distributed. Most likely, pretethered GLUT4 structures are responsible for the initial phase of insulin-stimulated exocytosis.