Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2.
Anal Biochem
; 429(1): 70-5, 2012 Oct 01.
Article
en En
| MEDLINE
| ID: mdl-22796500
ABSTRACT
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Evaluación Preclínica de Medicamentos
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Transportador 1 de Sodio-Glucosa
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Inhibidores del Cotransportador de Sodio-Glucosa 2
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Glucosa
Tipo de estudio:
Prognostic_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2012
Tipo del documento:
Article
País de afiliación:
India