School of Pharmacy, National Taiwan University, Taipei, Taiwan.
J Food Drug Anal. 2021 Sep 15;29(3):521-532. doi: 10.38212/2224-6614.3371.
Glucose is an important energy source for cells. Glucose transport is mediated by two types of glucose transporters: the active sodium-coupled glucose cotransporters (SGLTs), and the passive glucose transporters (GLUTs). Development of an easy way to detect glucose uptake by the cell can be valuable for research. 1-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-1-deoxy-d-glucose (1-NBDG) is a newly synthesized fluorescent glucose analogue. Unlike 2-NBDG, which is a good substrate of GLUTs but not SGLTs, 1-NBDG can be transported by both GLUTs and SGLTs. Thus, 1-NBDG is useful for the screening of SGLT1 and SGLT2 inhibitors. Here we further characterized 1-NBDG and compared it with 2-NBDG. The fluorescence of both 1-NBDG and 2-NBDG was quenched under alkaline conditions, but only 1-NBDG fluorescence could be restored upon neutralization. HPLC analysis revealed that 2-NBDG was decomposed leading to loss of fluorescence, whereas 1-NBDG remained intact in a NaOH solution. Thus, after cellular uptake, 1-NBDG fluorescence can be detected on a plate reader simply by cell lysis in a NaOH solution followed by neutralization with an HCl solution. The fluorescence stability of 1-NBDG was stable for up to 5 h once cells were lysed; however, similar to 2-NBDG, intracellular 1-NBDG was not stable and the fluorescence diminished substantially within one hour. 1-NBDG uptake could also be detected at the single cell level and inhibition of 1-NBDG uptake by SGLT inhibitors could be detected by flow cytometry. Furthermore, 1-NBDG was successfully used in a high-throughput cell-based method to screen for potential SGLT1 and SGLT2 inhibitors. The SGLT inhibitory activities of 67 flavonoids and flavonoid glycosides purified from plants were evaluated and several selective SGLT1, selective SGLT2, as well as dual SGLT1/2 inhibitors were identified. Structure-activity relationship analysis revealed that glycosyl residues were crucial since the aglycon showed no SGLT inhibitory activities. In addition, the sugar inter-linkage and their substitution positions to the aglycon affected not only the inhibitory activities but also the selectivity toward SGLT1 and SGLT2.
葡萄糖是细胞的重要能量来源。葡萄糖的转运是由两种类型的葡萄糖转运蛋白介导的：主动钠偶联葡萄糖协同转运蛋白（SGLTs）和被动葡萄糖转运蛋白（GLUTs）。开发一种简单的方法来检测细胞摄取葡萄糖的情况，对于研究可能是有价值的。1-（N-（7-硝基苯并-2-氧杂-1,3-二唑-4-基）氨基）-1-去氧-D-葡萄糖（1-NBDG）是一种新合成的荧光葡萄糖类似物。与 2-NBDG 不同，2-NBDG 是 GLUTs 的良好底物，但不是 SGLTs 的底物，1-NBDG 可以被 GLUTs 和 SGLTs 转运。因此，1-NBDG 可用于筛选 SGLT1 和 SGLT2 抑制剂。在这里，我们进一步对 1-NBDG 进行了表征，并与 2-NBDG 进行了比较。在碱性条件下，1-NBDG 和 2-NBDG 的荧光都会被猝灭，但只有 1-NBDG 荧光可以在中和后恢复。HPLC 分析表明，2-NBDG 分解导致荧光丧失，而 1-NBDG 在 NaOH 溶液中保持完整。因此，细胞摄取后，只需在 NaOH 溶液中裂解细胞，然后用 HCl 溶液中和，就可以在平板读数器上检测到 1-NBDG 荧光。一旦细胞裂解，1-NBDG 的荧光稳定性可稳定长达 5 小时；然而，与 2-NBDG 类似，细胞内的 1-NBDG 并不稳定，一个小时内荧光显著减弱。1-NBDG 的摄取也可以在单细胞水平上检测到，并且可以通过流式细胞术检测 SGLT 抑制剂对 1-NBDG 摄取的抑制作用。此外，1-NBDG 成功地用于高通量基于细胞的方法来筛选潜在的 SGLT1 和 SGLT2 抑制剂。评估了从植物中分离得到的 67 种类黄酮和类黄酮糖苷的 SGLT 抑制活性，鉴定了几种选择性 SGLT1、选择性 SGLT2 以及双重 SGLT1/2 抑制剂。构效关系分析表明，糖基残基是至关重要的，因为糖苷配基没有 SGLT 抑制活性。此外，糖基之间的连接以及它们与糖苷配基的取代位置不仅影响抑制活性，还影响对 SGLT1 和 SGLT2 的选择性。
