Pregnane X Receptor agonist Increases the Expression Levels of the Plasma Membrane Monoamine Transporter

Organic cation drugs are distributed in the body via a specific transport system consisting of organic cation transporters (OCTs) of the solute carrier 22 (SLC22) family and multidrug and toxin extrusion proteins (MATEs) from the SLC47 family. [1, 2] Recently, Engel et al. reported a new polyspecific organic cation transporter known as the plasma membrane monoamine transporter (PMAT, SLC29A4), which is an Na⁺-independent and membrane potential-sensitive transporter that transports monoamine neurotransmitters and neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺).[3] Functionally, PMAT shares its substrate and the specificity of its inhibitors with OCTs.[3, 4, 5] Metformin, an organic cation drug, is also transported by PMAT.[6] PMAT is expressed in the small intestine, kidney, heart, and brain.[6, 7] Zhou et al. reported localization of PMAT in the mucosal epithelial layer of the human small intestine.[6] Considering that the Km value of metformin for PMAT, hOCT1, and hOCT2 is 1320, 1470, and 1380 µM, respectively,[6] metformin transportation by PMAT is comparable to that by OCTs. Since the expression level of PMAT in the small intestine is considerable, PMAT may play an important role in metformin absorption in the small intestine. Interestingly, our previous study has indicated that the absorption of metformin was increased in healthy volunteers after administration of rifampin, a pregnane X receptor (PXR) agonist.[8] Similarly, the PXR agonist pregnenolone 16 alpha-carbonitrile (PCN) has demonstrated potential effects of inducing some drug transporters in animal models. PCN increased the mRNA levels of OCTs, but not of the multidrug and toxic extrusion (MATE) transporter.[9, 10] Therefore, we hypothesized that PCN increases PMAT's expression levels in the small intestine and may contribute to increased absorption of metformin. We determined the protein levels of PMAT in the presence and absence of PCN treatment in mice and compared PMAT expression levels.

Consistent with the results of a previous report,[6] PMAT was expressed in the small intestine of the mice in our study. Our results demonstrated that PCN treatment increased the expression levels of PMAT in the small intestine. These result are in line with our previous clinical study that the PXR agonist rifampin enhanced the absorption of metformin in healthy volunteers.[8] PMAT is a known transporter of metformin in the small intestine.[6] Thus, the enhanced absorptive activity of metformin may be mediated by the PCN-induced increase in PMAT expression. The effect of the transactivator PXR has been well described in several studies on metformin transport. PXR has not been shown to affect the expression level of MATEs, but has demonstrated effects on OCTs.[9, 10] However, the effect of PXR on PMAT expression has been unknown. To the best of our knowledge, this is the first study to report that a PXR agonist increases the expression levels of PMAT in the small intestine.

OCT1 and OCT3 are also localized in the intestine[13] but the expression level was much lower than liver. Although the relative intestinal or hepatic expression level of PMAT compared to OCT1 or OCT3 has been unknown in human, increased levels of PMAT may have contributed to the enhanced uptake of PMAT substrate such as metformin and possibly its effect. PMAT in other organs including liver and brain would be induced by PCN treatment. We also obtained liver samples and observed an increased expression in western blot analysis but immunohistochemistry finding was not definite (data not shown). Monoamine neurotransmitters (e.g. dopamine, serotonin) are known PMAT substrates and induced brain PMAT would. affect these neurotransmitters' activities.

There are some limitations in this study. First, no quantitative PMAT mRNA analysis was performed. PCN treatment is expected to increase PMAT mRNA by transcriptional up-regulation. However, it is rational to infer that the increased PMAT protein expression was mediated by increased transcription because PCN is a specific PXR agonist activating transcription. Second, other transactivators may contribute to the increased levels of PMAT. For example, the peroxisome proliferator agonist receptor-alpha and receptor-gamma have been shown to affect the transcriptional regulation of OCT1.[14] Moreover, despite the lack of available data on its effects, constitutive androstane receptor (CAR) might contribute to the transcription of this transporter.[15] In addition, the pharmacokinetics of PMAT substrates such as metformin before and after PCN treatment may contribute to the interpretation of our results. Third, the small number of samples and no statistical analysis may be not sufficient to warrant our conclusion, but the increased staining results in immunohistochemistry were consistent in all of the samples from different tissues. Finally, our results cannot be readily extrapolated to humans; it is necessary to measure the changes in PMAT expression levels in the human small intestine before and after rifampin treatment, which will require a more invasive endoscopic biopsy.

In summary, we found that the PXR agonist PCN increased the expression levels of PMAT in the small intestine. The results of this study imply that induction of PMAT may contribute to increased transport activity of PMAT substrates in the small intestine and probably enhance its absorption.