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. 2015 Mar;352(3):519-28.
doi: 10.1124/jpet.114.220350. Epub 2015 Jan 6.

Four cation-selective transporters contribute to apical uptake and accumulation of metformin in Caco-2 cell monolayers

Tianxiang Kevin Han  1 , William R Proctor  1 , Chester L Costales  1 , Hao Cai  1 , Ruth S Everett  1 , Dhiren R Thakker  2
Affiliations

Four cation-selective transporters contribute to apical uptake and accumulation of metformin in Caco-2 cell monolayers

Tianxiang Kevin Han et al. J Pharmacol Exp Ther. 2015 Mar.

Abstract

Metformin is the frontline therapy for type II diabetes mellitus. The oral bioavailability of metformin is unexpectedly high, between 40 and 60%, given its hydrophilicity and positive charge at all physiologic pH values. Previous studies in Caco-2 cell monolayers, a cellular model of the human intestinal epithelium, showed that during absorptive transport metformin is taken up into the cells via transporters in the apical (AP) membrane; however, predominant transport to the basolateral (BL) side occurs via the paracellular route because intracellular metformin cannot egress across the BL membrane. Furthermore, these studies have suggested that the AP transporters can contribute to intestinal accumulation and absorption of metformin. Transporter-specific inhibitors as well as a novel approach involving a cocktail of transporter inhibitors with overlapping selectivity were used to identify the AP transporters that mediate metformin uptake in Caco-2 cell monolayers; furthermore, the relative contributions of these transporters in metformin AP uptake were also determined. The organic cation transporter 1, plasma membrane monoamine transporter (PMAT), serotonin reuptake transporter, and choline high-affinity transporter contributed to approximately 25%, 20%, 20%, and 15%, respectively, of the AP uptake of metformin. PMAT-knockdown Caco-2 cells were constructed to confirm the contribution of PMAT in metformin AP uptake because a PMAT-selective inhibitor is not available. The identification of four intestinal transporters that contribute to AP uptake and potentially intestinal absorption of metformin is a significant novel finding that can influence our understanding of metformin pharmacology and intestinal drug-drug interactions involving this highly prescribed drug.

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Figures

Fig. 1.
Fig. 1.
Expression levels of cation-selective transporter genes in Caco-2 cell monolayers (A) and human intestine tissue (B). Data represent mean ± S.D., n = 3.
Fig. 2.
Fig. 2.
Relative contributions of cation-selective transporters to the AP uptake of metformin into Caco-2 cell monolayers determined using a novel chemical inhibition scheme. Inhibition of metformin uptake (10 µM, 5 minutes) into OCT1-, 2-, and 3-expressing CHO cells by mitoxantrone (A), corticosterone (B), cimetidine (C), and desipramine (D). Data represent mean ± S.D., n = 3. Inhibition curves were fit to corrected uptake rate in the presence of varying concentrations of each inhibitor. (E) Chemical inhibition scheme to determine the contributions of transporters to metformin AP uptake in Caco-2 cell monolayers. (F) Inhibition of metformin AP uptake (10 µM, 5 minutes) in Caco-2 cell monolayers in the presence of chemical inhibitors shown in (E). Data represent mean ± S.D., n = 3. **P < 0.01, ***P < 0.001 compared with the control; #P < 0.05 compared with each other.
Fig. 3.
Fig. 3.
SERT is a metformin transporter and contributes to the AP uptake of metformin in Caco-2 cell monolayers. (A) Uptake of metformin (1 µM) as a function of time in SERT- transfected HEK 293 cells (closed symbols) and HEK 293 control cells (open symbols). (B) Uptake of metformin (2 minutes) as a function of concentration in SERT-HEK 293 cells, corrected for surface binding and uptake of metformin in control HEK 293 cells. A Michaelis-Menten equation with one saturable component was fit to the corrected uptake rate data and the estimated Km and Vmax values are presented. (C) Inhibition curves depicting metformin uptake (10 µM, 5 minutes) by OCTs and SERT in the presence of paroxetine. (D) Modified chemical inhibition scheme to determine the contributions of OCT1, PMAT, and SERT to metformin uptake into Caco-2 cell monolayers. (E) Inhibition of metformin AP uptake (10 µM, 5 minutes) in the presence of inhibitors considered in (D). Data represent mean ± S.D., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control; #P < 0.05 compared with each other.
Fig. 4.
Fig. 4.
Inhibition by HC3 reduced the initial AP uptake rate of metformin. (A) Inhibition of metformin uptake by HC3 into OCT1-, 2-, 3- and SERT-expressing cells. (B) Modified chemical inhibition scheme to determine the relative contribution of OCT1, PMAT, SERT, and CHT to metformin AP uptake into Caco-2 cell monolayers. (C) Inhibition of metformin AP uptake (10 µM, 5 minutes) by inhibitors considered in (B). Data represent mean ± S.D., n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control; #P < 0.05 compared with each other. (D) Chemical structures of metformin and choline. (E and F) Protein expression levels of four metformin transporters in Caco-2 cell monolayers (E), and in human intestinal tissue (F) as determined by Western blot analysis.
Fig. 5.
Fig. 5.
Metformin AP uptake into PMAT- and OCT1-knockdown Caco-2 cells. (A–C) Expression levels of the PMAT gene (A) and protein (B) in PMAT-knockdown clones, and the OCT1 gene (C) in OCT1-knockdown clones of Caco-2 cells relative to the control (wild-type Caco-2 cells); gene expression data represent mean ± S.D., n = 3. *P < 0.05, **P < 0.01 compared with the control, and protein expression data are from n = 1. (D and E) Metformin AP uptake (10 µM, 5 minutes) into (D) PMAT- and (E) OCT1-knockdown clones relative to the control (wild-type Caco-2 cells). Data represent mean ± S.D., n = 3. *P < 0.05, **P < 0.01 compared with the control.
Fig. 6.
Fig. 6.
Schematic representation of the contributions of cation-selective transporters to metformin AP uptake in Caco-2 cell monolayers. OCT1, PMAT, SERT, and CHT are responsible for 25%, 20%, 20%, and 15% of the AP uptake of metformin in Caco-2 cell monolayers, respectively. Passive processes and nonspecific binding likely contribute to the remaining ∼20% of the apparent uptake measured.
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