Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2

Abhinav Kanwal, Shailendra Pratap Singh, Paramjit Grover, Sanjay Kumar Banerjee

Research output: Contribution to journalArticle

26 Citations (Scopus)

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 (100 μM), but not when treated with nonspecific sodium-independent facilitative glucose transporter (GLUT) inhibitors (100 μM). 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.

Original languageEnglish
Pages (from-to)70-75
Number of pages6
JournalAnalytical Biochemistry
Volume429
Issue number1
DOIs
Publication statusPublished - 01-10-2012

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Assays
Glucose
Phlorhizin
Cell Line
Cells
Facilitative Glucose Transport Proteins
Sodium-Glucose Transport Proteins
Screening
Sodium
Neomycin
HEK293 Cells
Deoxyglucose
Cardiomyopathies
Immunoblotting
Polymerase chain reaction
Reverse Transcription
Transcription
Gastrointestinal Tract
Medical problems
Gene expression

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Kanwal, Abhinav ; Singh, Shailendra Pratap ; Grover, Paramjit ; Banerjee, Sanjay Kumar. / Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2. In: Analytical Biochemistry. 2012 ; Vol. 429, No. 1. pp. 70-75.
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Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2. / Kanwal, Abhinav; Singh, Shailendra Pratap; Grover, Paramjit; Banerjee, Sanjay Kumar.

In: Analytical Biochemistry, Vol. 429, No. 1, 01.10.2012, p. 70-75.

Research output: Contribution to journalArticle

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