Division of Pharmacology, Indian Institute of Chemical Technology (IICT), Hyderabad 500607, India.
Anal Biochem. 2012 Oct 1;429(1):70-5. doi: 10.1016/j.ab.2012.07.003. Epub 2012 Jul 14.
Sodium-dependent glucose cotransporters (SGLT1 and SGLT2), which have a key role in the absorption of glucose in the kidney and/or gastrointestinal tract, have been proposed as a novel therapeutic strategy for diabetes and cardiomyopathy. Here we developed a simple cell-based, nonradioactive method for functional screening of SGLT1 and SGLT2 inhibitors. Stable cell lines expressing human SGLT1 and SGLT2 were established by transfecting HEK293 cells with vectors (pCMV6-Neo) having full-length human SGLT1 and SGLT2 and selecting the positive clones following neomycin treatment. We confirmed the gene expression of SGLT1 and SGLT2 by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. Furthermore, to analyze the function of SGLTs, we incubated stable cell lines with 2-deoxyglucose or fluorescent d-glucose analog (2-NBDG) and performed glucose uptake assay. A significant (P<0.001) increase in glucose uptake was observed in both cell lines. The increased glucose uptake in both cell lines was completely inhibited when treated with nonspecific SGLT1/SGLT2 inhibitors and phlorizin (100μM), but not when treated with nonspecific sodium-independent facilitative glucose transporter (GLUT) inhibitors (100μM). Taken together, our data suggest that cell-based methods developed for screening SGLT1/SGLT2 inhibitors are phlorizin sensitive and specific for respective glucose transporters. This assay provides a simple and rapid method for identifying novel and selective SGLT inhibitors.
钠依赖型葡萄糖共转运蛋白（SGLT1 和 SGLT2）在肾脏和/或胃肠道葡萄糖吸收中起关键作用，被提议作为糖尿病和心肌病的一种新的治疗策略。在这里，我们开发了一种简单的基于细胞的、非放射性方法，用于筛选 SGLT1 和 SGLT2 抑制剂的功能。通过用载体（pCMV6-Neo）转染 HEK293 细胞来建立稳定表达人 SGLT1 和 SGLT2 的细胞系，该载体具有全长人 SGLT1 和 SGLT2，并在新霉素处理后选择阳性克隆。我们通过逆转录聚合酶链反应（RT-PCR）和免疫印迹证实了 SGLT1 和 SGLT2 的基因表达。此外，为了分析 SGLTs 的功能，我们用 2-脱氧葡萄糖或荧光 d-葡萄糖类似物（2-NBDG）孵育稳定细胞系，并进行葡萄糖摄取测定。在两种细胞系中均观察到葡萄糖摄取的显著（P<0.001）增加。当用非特异性 SGLT1/SGLT2 抑制剂和根皮苷（100μM）处理时，两种细胞系中的葡萄糖摄取增加均被完全抑制，但当用非特异性钠非依赖性易化葡萄糖转运体（GLUT）抑制剂（100μM）处理时则没有抑制。总之，我们的数据表明，为筛选 SGLT1/SGLT2 抑制剂而开发的基于细胞的方法对各自的葡萄糖转运体是根皮苷敏感和特异的。该测定法提供了一种识别新型和选择性 SGLT 抑制剂的简单而快速的方法。
