[A new fluorescence method for determination of sodium-glucose cotransporter 2 activity].
OBJECTIVE
To develop a new fluorescence method for the determination of human sodium glucose cotransporter 2 (SGLT2) activity.


METHODS
Firstly full length human SGLT2 cDNA was cloned and the recombined plasmid pIRES2-EGFP-SGLT2 was constructed. Then the construct was subjected to restriction enzyme digestion analysis. In addition, SGLT2 insert clones were fully sequenced to confirm its nucleotide sequence, and then the recombined plasmid was transfected into HEK293 cells. The expression of green fluoresscent protein (GFP) was detected by confocal and flow cytometry (FCM), respectively. The protein expression of SGLT2 was determined by Western Blot assay. The transport activity of SGLT2 was determined by FCM choosing 2-NBDG as the detection target.


RESULTS
Both restriction enzyme digestion and DNA sequencing assays showed that the recombined plasmid was constructed successfully. After transient transfection into HEK293 cells, the GFP expression analysis displayed high transfection efficiency and transcription activity, and the fluorescence intensity of the transfected cells was much higher than that of the untransfected cells (P<0.01). SGLT2 was more highly expressed in pIRES2-EGFP-SGLT2 transfected cells as compared with the empty vector (pIRES2-EGFP) transfected cells (P<0.05); the expression of SGLT2 in the untransfected cells was similar to that in the empty vector transfected cells. The Na(+) dependent 2-NBDG uptake was significantly increased in the transfected cells compared with that in the untransfected cells (P<0.01).


CONCLUSIONS
A new fluorescence method for determination of SGLT2 activity has been developed with a eukaryotic expression vector of human SGLT2.
Department of Pharmacology, Chinese Academy of Medical Sciences, Beijing, China.
Xiao-lin
XL
Zhang
Xiao-meng
XM
Yang
Juan
J
Li
Jian-yang
JY
Fu
Jin-ying
JY
Tian
Fei
F
Ye
D003847:Deoxyglucose / Q000737:chemistry; D005434:Flow Cytometry; D005453:Fluorescence; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D051297:Sodium-Glucose Transporter 2 / Q000378:metabolism*; D014162:Transfection
C089180:SLC5A2 protein, human; D051297:Sodium-Glucose Transporter 2; D003847:Deoxyglucose