The goals of this study were to determine the role of OCT3 in the pharmacologic action of metformin and to identify and functionally characterize genetic variants of OCT3 with respect to the uptake of metformin and monoamines.
For the pharmacologic studies, we evaluated metformin-induced activation of AMPK, a molecular target of metformin. We used quantitative PCR and immunostaining to localize the transporter and isotopic uptake studies in cells transfected with OCT3 and its nonsynonymous genetic variants for functional analyses.
Quantitative PCR and immunostaining showed that OCT3 was expressed high on the plasma membrane of skeletal muscle and liver, target tissues for metformin action. Both the OCT inhibitor, cimetidine, and OCT3-specific shRNA significantly reduced the activating effect of metformin on AMPK. To identify genetic variants in OCT3, we used recent data from the 1000 Genomes Project and the Pharmacogenomics of Membrane Transporters project. Six novel missense variants were identified. In functional assays, using various monoamines and metformin, 3 variants, T44M (c.131C>T), T400I (c.1199C>T) and V423F (c.1267G>T), showed altered substrate specificity. Notably, in cells expressing T400I and V423F, the uptakes of metformin and catecholamines were significantly reduced but the uptakes of metformin, MPP+ and histamine by T44M were significantly increased more than 50%. Structural modeling suggested that these two variants may be located in the pore-lining (T400) or proximal (V423) membrane-spanning helixes.
Our study suggests that OCT3 plays a role in the therapeutic action of metformin and that genetic variants of OCT3 may modulate metformin and catecholamine action.