(A) Uptake of [¹⁴C]metformin by cell lines stably expressing human OCT1 and its variants. Cells expressing OCT1 and its variants were incubated with [¹⁴C]metformin (250 μM) for 10 minutes. Seven OCT1 variants exhibited reduced metformin uptake as compared with OCT1-reference. Data are expressed as mean ± SD for samples analyzed in quadruplicate. *P < 0.001 compared with the reference (2-tailed Student’s t test). (B) Metformin kinetics in cell lines expressing reduced function variants of OCT1. Four of the reduced function variants shown in A had enough activity to allow us to perform kinetic studies with metformin. The metformin uptake data at 8 different concentrations are plotted. The variants had significantly different V_max values, with a similar Michaelis-Menten constant (K_m) (Table 1). (C) OCT1-R61C tagged with GFP exhibits reduced membrane and enhanced cytoplasmic localization. GFP fusion constructs were generated for OCT1-reference and OCT1-R61C, which is common in human populations (13), and used to generate stable cell lines using Flp-In-293 cells. The plasma membrane was stained using Alexa Fluor 594 conjugated to wheat germ agglutinin, and cells were visualized by confocal microscopy. Original magnification, ×100. (D) Metformin-stimulated AMPK phosphorylation and ACC phosphorylation in cell lines stably overexpressing human OCT1 and its variants. The cells were treated with metformin (1 mM) for 1 hour, washed with blank medium, and then incubated for 5 hours before harvest. Immunoblots were performed against phospho-ACC (Ser79), phospho-AMPKα (Thr172), AMPKα, and β-actin.