Inhibitory activity and selectivity assay against SGLT2 for C-glycosides in vitro

Human full-length SGLT2 cDNA ({"type":"entrez-nucleotide","attrs":{"text":"NM_003041.3","term_id":"164663744","term_text":"NM_003041.3"}}NM_003041.3) was synthesized by Invitrogen Co., Ltd (Shanghai, China) and amplified with a forward primer (5′-CCGCTCGAGGCCACCATGGACAGTAGCACCTGGAGC-3′) and reverse primer (5′-CCGGAATTCTCAGGCAAAATATGCATGGCAAAAG-3′). The PCR products were cloned into the retrovirus vector pMSCVpuro at the Xho I and EcoR I sites generating pMSCVpuro-SGLT2, and the sequences were confirmed by Sanger sequencing. To produce retrovirus, a mixture containing the plasmid pMSCVpuro-SGLT2 and the packing plasmids (Gag-Pol and pVSV-G) were transfected into 293T cells with Neofect (Neobiotech Co Ltd, Korea). Culture medium was changed after 16 h post transfection. The supernatant containing retrovirus was collected after 48 h post transfection and filtered through a 0.45-μm PVDF filter membrane. For generating the cells stably expressing SGLT2, HEK293 cells were transduced with retrovirus. After 48 h post infection, puromycin (2 μg mL^–1) was used to screen the positive clone cell lines for 2 days, and the SGLT2 expression status was validated by western blot analysis⁴⁶. To evaluate the selectivity of compounds, pMSCVpuro-SGLT1 stably transfected HEK293 cell line was also constructed in the same way. The full-length SGLT1 cDNA was obtained by PCR with forward primer: 5′-CCGCTCGAGGCCACCATGGACAGTAGCACCTGGAGC-3′ and reverse primer: 5′-CCGGAATTCTCAGGCAAAATATGCATGGCAAAAG-3′.

D-glucose transport was measured by performing 100 µM 1-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-1-deoxy-D-glucose (1-NBDG) uptakes in cells over-expressing SGLT1 or SGLT2. Cells were seeded in 24-well plates (10,0000 cells/well), grown to 90–95% confluency, and then the transport assays were performed. Cells were incubated in transport Na⁺ buffer containing 120 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 2.2 mM CaCl₂, 10 mM HEPES, pH 7.4 with various concentrations of C-glycosides. For nonspecific transport determination, cells were incubated in Na⁺-free buffer containing 140 mM choline chloride, 4.7 mM KCl, 1.2 mM MgCl₂, 2.2 mM CaCl₂, 10 mM HEPES, pH 7.4. After 4 h-incubation at 37 °C, cells were washed twice with ice-cold stop buffer (Na₊-free buffer containing 0.5 mM phlorizin), incubated at room temperature in cell lysis buffer (0.1 mM NaOH), and then neutralized with 0.1 mM HCl. Fluorescence intensity at 485/535 nm was then measured to determine the inhibitory activity of SGLT2 for C-glycosides. All experiments were repeated in triplicate. The IC₅₀ values were determined using nonlinear regression with Prism 5 software.