Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2.
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.
Division of Pharmacology, Indian Institute of Chemical Technology (IICT), Hyderabad 500607, India.
Abhinav
A
Kanwal
Shailendra Pratap
SP
Singh
Paramjit
P
Grover
Sanjay Kumar
SK
Banerjee
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D010695:Phlorhizin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051297:Sodium-Glucose Transporter 2 / Q000235:genetics / Q000378:metabolism; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D013997:Time Factors
D012333:RNA, Messenger; D051269:Sodium-Glucose Transporter 1; D051297:Sodium-Glucose Transporter 2; D000077203:Sodium-Glucose Transporter 2 Inhibitors; D003847:Deoxyglucose; D010695:Phlorhizin; D009327:4-Chloro-7-nitrobenzofurazan; D005947:Glucose; C098340:2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose