Cellular metformin uptake in H4IIE and HEK293 cells. (a,b) The effect of KU-55933 and OCT inhibitors on the uptake of metformin in H4IIE cells (a) and HEK293 cells stably expressing empty vector or human OCT1 (b). Cells were pretreated with DMSO or KU-55933 or other OCT1 inhibitors for 30 min and then treated with various concentrations of metformin (with 10 mM [¹⁴C]-metformin) for 1 h. The amount of [¹⁴C]-metformin in cells was measured. All the compounds except para-aminohippuric acid (PAH; a negative control) significantly inhibited metformin uptake (***P < 0.001, **P < 0.01, *P < 0.05; unpaired Student’s t test) compared to cells preincubated with DMSO alone. All data are expressed as mean ± s.d. from two independent uptake experiments. HEK293 cells stably expressing human OCT1 showed greater OCT1 expression on the plasma membrane compared to cells expressing empty vector⁶.