Metformin transport by a newly cloned proton-stimulated organic cation transporter (plasma membrane monoamine transporter) expressed in human intestine

Abstract

Metformin is a widely used oral antihyperglycemic drug for the treatment of type II diabetes mellitus. The intestinal absorption of metformin is dose-dependent and involves an active, saturable uptake process. Metformin has been shown to be transported by the human organic cation transporters 1 and 2 (hOCT1-2). We recently cloned and characterized a novel proton-activated organic cation transporter, plasma membrane monoamine transporter (PMAT). We previously showed that PMAT transports many classic organic cations (e.g., monoamine neurotransmitters, 1-methyl-4-phenylpyridinium) in a pH-dependent manner and its mRNA is expressed in multiple human tissues. The goal of this study is to investigate whether metformin is a substrate of PMAT and whether PMAT plays a role in the intestinal uptake of metformin. Using Madin-Darby canine kidney cells stably expressing human PMAT, we showed that metformin is avidly transported by PMAT, with an apparent affinity (K(m) = 1.32 mM) comparable to those reported for hOCT1-2. Interestingly, the concentration-velocity profile of PMAT-mediated metformin uptake is sigmoidal, with a Hill coefficient of 2.64. PMAT-mediated metformin transport is greatly stimulated by acidic pH, with the uptake rate being approximately 4-fold higher at pH 6.6 than at pH 7.4. Using a polyclonal antibody against PMAT, we showed that the PMAT protein (58 kDa) was expressed in human small intestine and concentrated on the tips of the mucosal epithelial layer. Taken together, our results suggest that PMAT transports metformin, is expressed in human intestine, and may play a role in the intestinal absorption of metformin and possibly other cationic drugs.

Fig. 1

Structures of biguanides (a) and inhibition of PMAT-specific MPP⁺ uptake by biguanides (b). Transport was measured in PMAT-expressing cells and vector-transfected cells (control) at 1 min with 1 μM [³H]MPP⁺. The PMAT-specific uptake was calculated by subtracting the transport activity in control cells. Each value represents mean ± S.D. (n = 3).

Fig. 2

Influence of pH on PMAT-mediated [¹⁴C]metformin (1 μM). Vector-transfected cells and PMAT-transfected cells were incubated for 1 min at 37°C. Each value represents mean ± S.D. (n = 3).

Fig. 3

Influence of membrane potential on PMAT-mediated [¹⁴C]metformin (1 μM). Vector-transfected cells and PMAT-transfected cells were incubated for 1 min at 37°C. Ba²⁺ was added to block the potassium channels. To avoid the precipitation of barium by sulfate and phosphate in the KRH buffer, a chloride salt-based buffer (5 mM glucose, 145 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, and 5 mM HEPES, pH 7.4) with different compositions of potassium and sodium was used. Each value represents mean ± S.D. (n = 3).

Fig. 4

Time courses of [¹⁴C]metformin uptake at 1 μM (a) and 4 mM (b) mediated by PMAT. Vector-transfected cells (open circles) and PMAT-transfected cells (solid circles) were incubated at 37°C. Inset, a plot of initial phase with linear fitting of PMAT-specific metformin uptake calculated by subtracting the transport activity in the control cells. Each value represents mean ± S.D. (n = 3).

Fig. 5

Concentration-dependent transport of [¹⁴C]metformin by PMAT at 1 min (a) or 3 min (b) of incubation. The PMAT-specific uptake was measured at 37°C at pH 7.4 and calculated by subtracting the transport activity in control cells. Inset, Eadie-Hofstee plots. Each value represents mean ± S.D. (n = 3).

Fig. 6

Determination of PMAT expression in human small intestine by RT-PCR (a), Western blot (b), and immunohistochemistry (c). a, human small intestine was analyzed for PMAT mRNA expression by semiquantitative RT-PCR. GAPDH primers were used as an internal control. b, immunoblotting was carried out with the anti-PMAT antibody at 1:1000 dilution. The same blots were stripped and blotted with an antibody against an internal standard, GAPDH. c, immunoblotting was carried out with the anti-PMAT antibody at 1:500 dilution. Slides containing human small intestine tissue were permeabilized and stained with either anti-PMAT antibody or prebleed serum (control).