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Development of a Cell Based Non-radioactive Glucose Uptake Assay System for SGLT1 and SGLT2.
MedLine Citation:
PMID:  22796500     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Sodium glucose transporters (SGLT1 and SGLT2), which have a key role in the absorption of glucose in the kidney and/or gastrointestinal tract has been proposed as a novel therapeutic strategy for diabetes and cardiomyopathy. Here we developed a simple cell based, non-radioactive method for functional screening of SGLT1 and SGLT2 inhibitors. Stable cell lines expressing human SGLT1 and SGLT2 were established by transfecting HEK-293 cells with vectors (pCMV6-Neo) having full length of human hSGLT1 and hSGLT2, and selecting the positive clones following neomycin treatment. We confirmed the gene expression of SGLT1 and SGLT2 by RTPCR and immunoblotting. Further to analyze the function of SGLTs, we incubated stable cell lines with 2-deoxy glucose or fluorescent D-glucose analog (2-NBDG), and performed glucose uptake assay. Significant (p<0.001) increase in glucose uptake was observed in both the cell lines. The increased glucose uptake in both cell lines was completely inhibited when treated with nonspecific SGLT1/SGLT2 inhibitors, phlorizin (100μM) but not with non specific 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 transporter. This assay provides a simple and rapid method for identifying novel and selective SGLTs inhibitors.
Authors:
Abhinav Kanwal; Shailendra Pratap Singh; Paramjit Grover; Sanjay Kumar Banerjee
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-7-12
Journal Detail:
Title:  Analytical biochemistry     Volume:  -     ISSN:  1096-0309     ISO Abbreviation:  -     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-7-16     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Copyright Information:
Copyright © 2012. Published by Elsevier Inc.
Affiliation:
Division of Pharmacology, Indian Institute of Chemical Technology (IICT), Hyderabad, India.
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