GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor
GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor
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DOI：
10.1016/j.bbamem.2007.11.015
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发表时间：
2008-04-01
影响因子：
3.4
通讯作者：
Frommer, Wolf B.
中科院分区：
文献类型：
--
作者：
Takanaga, Hitomi;Chaudhuri, Bhavna;Frommer, Wolf B.
基因编码的FRET葡萄糖纳米传感器已被证明可用于成像HepG 2细胞中的葡萄糖流量。然而，原始传感器的动态范围是有限的，因此对于用于鉴定参与糖通量调节的蛋白质的siRNA群体的高通量筛选，它似乎不是最佳的。在这里，我们描述了一种混合的方法，结合连接子缩短与荧光团插入，以减少自由度的荧光定位，从而改善纳米传感器的动态。我们能够开发出一种新型的高灵敏度FRET纳米传感器，其在体内显示出10倍更高的比率变化和动态范围（0.05-11 mM），允许在生理相关范围内进行分析。作为该传感器可用于筛选在糖通量及其控制中起作用的蛋白质的概念证明，我们使用GLUT家族成员的siRNA抑制，并表明GLUT 1是HepG 2细胞中的主要葡萄糖转运蛋白，GLUT 9也有贡献，但程度较低。GFP融合表明GLUT 1和9优先定位于质膜，因此可以解释运输活性。新型葡萄糖纳米传感器的灵敏度提高了体内葡萄糖通量分析的可靠性，并为使用高含量筛选筛选siRNA集合以及药物提供了新的手段。(C)2007 Elsevier B. V.保留所有权利。
Genetically encoded FRET glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05-11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens. (C) 2007 Elsevier B.V. All rights reserved.