Identification of three distinct functional sites of insulin-mediated GLUT4 trafficking in adipocytes using quantitative single molecule imaging.
Identification of three distinct functional sites of insulin-mediated GLUT4 trafficking in adipocytes using quantitative single molecule imaging.
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DOI：
10.1091/mbc.e10-01-0029
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发表时间：
2010-08-01
影响因子：
3.3
通讯作者：
Kanzaki M
中科院分区：
文献类型：
--
作者：
Fujita H;Hatakeyama H;Watanabe TM;Sato M;Higuchi H;Kanzaki M
我们开发了一种新的方法，允许在单分子水平上使用量子点直接分析细胞内GLUT4动力学，以定量确定GLUT4的行为性质。通过这种方法，我们定义了胰岛素信号直接收敛并影响动态GLUT4运输事件过程的实际步骤。胰岛素刺激葡萄糖摄取是通过胰岛素应答的葡萄糖转运蛋白GLUT4从脂肪细胞和肌肉细胞的细胞内储存室重新分配到质膜来实现的。尽管利用各种方法研究了GLUT4易位，但GLUT4在细胞内的运输特性在很大程度上是未知的。我们的新方法允许使用量子点技术在单分子水平上直接分析细胞内GLUT4的动力学，定量地确定GLUT4的行为性质。我们的数据表明，在完全分化的3T3L1脂肪细胞中，细胞内GLUT4在基础状态下的主要隔离机制是“静态保留”。我们还直接定义了三种不同的胰岛素刺激的GLUT4运输过程：1)从存储室中假定的GLUT4锚定系统释放，2)运输含GLUT4的囊泡移动的速度，以及3)在质膜上的系缚/对接步骤。有趣的是，用磷脂酰肌醇3-激酶抑制剂预处理或过表达显性干扰型AS160突变体（AS160/T642A）似乎可以消除胰岛素诱导的静态状态下的GLUT4释放。此外，我们的新方法揭示了在某些胰岛素抵抗状态下，GLUT4行为紊乱可能损害胰岛素反应性GLUT4易位的可能性。
We developed a novel approach allowing intracellular GLUT4 dynamics to be analyzed directly at the single molecule level using Quantum dot to quantitatively establish the behavioral nature of GLUT4. With this approach, we defined the actual steps at which insulin signals directly converge and impact the process of dynamic GLUT4 trafficking events. Insulin stimulation of glucose uptake is achieved by redistribution of insulin-responsive glucose transporters, GLUT4, from intracellular storage compartment(s) to the plasma membrane in adipocytes and muscle cells. Although GLUT4 translocation has been investigated using various approaches, GLUT4 trafficking properties within the cell are largely unknown. Our novel method allows direct analysis of intracellular GLUT4 dynamics at the single molecule level by using Quantum dot technology, quantitatively establishing the behavioral nature of GLUT4. Our data demonstrate the predominant mechanism for intracellular GLUT4 sequestration in the basal state to be “static retention” in fully differentiated 3T3L1 adipocytes. We also directly defined three distinct insulin-stimulated GLUT4 trafficking processes: 1) release from the putative GLUT4 anchoring system in storage compartment(s), 2) the speed at which transport GLUT4-containing vesicles move, and 3) the tethering/docking steps at the plasma membrane. Intriguingly, insulin-induced GLUT4 liberation from its static state appeared to be abolished by either pretreatment with an inhibitor of phosphatidylinositol 3-kinase or overexpression of a dominant-interfering AS160 mutant (AS160/T642A). In addition, our novel approach revealed the possibility that, in certain insulin-resistant states, derangements in GLUT4 behavior can impair insulin-responsive GLUT4 translocation.
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影响因子：
5.3
作者：
Govers, R;Coster, ACF;James, DE
通讯作者：
James, DE
影响因子：
4.8
作者：
Larance, M;Ramm, G;James, DE
通讯作者：
James, DE
影响因子：
3.3
作者：
Karylowski, O;Zeigerer, A;McGraw, TE
通讯作者：
McGraw, TE
影响因子：
29
作者：
Bai, Li;Wang, Yan;Xu, Tao
通讯作者：
Xu, Tao
影响因子：
3.3
作者：
El-Jack, AK;Kandror, KV;Pilch, PF
通讯作者：
Pilch, PF