Abstract
Monocarboxylate transporter 6 (MCT6; SLC16A5) has been recognized for its role as a xenobiotic transporter, with characterized substrates probenecid, bumetanide, and nateglinide. To date, the impact of commonly ingested dietary compounds on MCT6 function has not been investigated, and therefore, the objective of this study was to evaluate a variety of flavonoids for their potential MCT6-specific interactions. Flavonoids are a large group of polyphenolic phytochemicals found in commonly consumed plant-based products that have been recognized for their dietary health benefits. The uptake of bumetanide in human MCT6 gene-transfected Xenopus laevis oocytes was significantly decreased in the presence of a variety of flavonoids (e.g., quercetin, luteolin, phloretin, and morin), but was not significantly affected by flavonoid glycosides (e.g., naringin, rutin, phlorizin). The IC50 values of quercetin, phloretin, and morin were determined to be 25.3 ± 3.36, 17.3 ± 2.37, and 33.1 ± 3.29 μM, respectively. The mechanism of inhibition of phloretin was reversible and competitive, with a Ki value of 22.8 μM. Furthermore, typical MCT substrates were also investigated for their potential interactions with MCT6. Substrates of MCTs 1, 2, 4, 8, and 10 did not cause any significant decrease in MCT6-mediated bumetanide uptake, suggesting that MCT6 has distinct compound selectivity. In summary, these results suggest that dietary aglycon flavonoids may significantly alter the pharmacokinetics and pharmacodynamics of bumetanide and other MCT6-specific substrates, and may represent potential substrates for MCT6.
Keywords: transporters, diet, flavonoids, drug–food interactions, bumetanide, monocarboxylate transporter 6, slc16a5
Graphical abstract
INTRODUCTION
The monocarboxylate transporter family (SLC16A) is composed of 14 isoforms, with MCTs 1–4 being proton-dependent lactate transporters.1 Within the past decade, several previously uncharacterized MCT isoforms have been associated with a variety of diseases including cancer, atherosclerosis, cataract formation, and mental retardation.2 More specifically, monocarboxylate transporter 6 (MCT6; SLC16A5) has been reported to transport the organic anions bumetanide, nateglinide, and probenecid in a pH-dependent and membrane potential dependent manner;3,4 however no functional role or verified endogenous substrates have been characterized, making MCT6 one of the few remaining orphan transporters in a family of clinically significant isoforms. To date, limited MCT6 protein expression data is available in the literature for humans; however it has been noted in two reports that MCT6 is expressed in intestinal tissues (note that “MCT5” represents MCT6 in Gill et al.5 due to previous differences in isoform nomenclature).4,5 The high expression of MCT6 in the intestine suggests that MCT6 may play an important role in not just endogenous compound absorption and distribution but potentially the absorption of dietary compounds, such as flavonoids.
Flavonoids, a naturally occurring group of polyphenolic phytochemicals, are one of the most commonly ingested groups of compounds found in a plant-based diet. Responsible for a wide range of pharmacological activities, there are ~8,000 flavonoids that can be divided into several classes, including but not limited to flavanones, flavonols, flavones, and isoflavones, based on their chemical structure. Approximately 1 g of total flavonoids is ingested from the typical U.S. diet per day, and concentrations of these flavonoids have been shown to range in the low micromolar range in human plasma.6,7 In addition, flavonoids are also ingested as single-compound dietary supplements, resulting in potentially higher concentrations in humans. Overall, flavonoids continue to be extensively studied due to their potential treatment benefits in disorders such as obesity, diabetes, and cardiovascular disease, as well as cancer.8,9
Numerous studies have been performed investigating the effects of flavonoids as potent substrates/inhibitors of a wide variety of transporters such as glucose transporters (e.g., SGLT, GLUT),10,11 ATP-binding cassette (ABC) transporters (e.g., P-glycoprotein, BCRP),12,13 and a wide variety of phase I and phase II metabolizing enzymes.14,15 Flavonoids have been demonstrated to be substrates and potent inhibitors of many members of the organic anion transporter family, including MCTs,16,17 and cause significant drug–food interactions.18–21 More specifically, flavonoids have been reported to interact with organic anion transporters MCT1,16 OAT1,22 and OATP1B1.23 Flavonoids are eliminated predominantly by metabolism, resulting in high plasma concentrations of flavonoid metabolites. In the liver, flavonoids may undergo phase I and phase II metabolism into metabolic derivatives, with major phase II metabolites formed from methylation, sulfation, and glucuronidation reactions.7 It is important to note that these flavonoid metabolites vary significantly in structure and tissue distribution. Some of the conjugated metabolites of flavonoids have also been demonstrated to be substrates and potent inhibitors of many members of the organic anion transporter family,16,17 and may also be responsible for significant drug–food interactions.18–21
Considering that the structural similarity between flavonoids and estrone sulfate (which is a known inhibitor of MCT63) is relatively high, we hypothesize that some flavonoids may also inhibit MCT6. For this study, our laboratory investigated the inhibition of MCT6-mediated uptake of a probe substrate (bumetanide) using a variety of flavonoids of several structural classes in vitro using the Xenopus laevis oocyte expression system. In addition, we investigated whether other prototypical substrates/inhibitors of other MCT isoforms have any significant interactions with MCT6.
MATERIALS AND METHODS
Materials
Bumetanide and all of the other compounds (with the exception of AR-C155858) were purchased from Sigma-Aldrich (St. Louis, MO). AR-C155858 was purchased from Tocris Bioscience (Bristol, U.K.). [3H]Bumetanide (15–30 Ci/mmol) was purchased from American Radiolabeled Chemicals (St. Louis, MO). GIBCO Leibovitz’s L-15 medium with glutamine (Cat. No. 41300-039) and all Western blotting materials were supplied by Thermo Fisher Scientific (Rockford, IL). The pCR2.1-TOPO, TOPO TA cloning kit, TRIzol Reagent, and mMESSAGE mMACHINE T7 transcription kit were purchased from Thermo Fisher Scientific (Rockford, IL). pGH19 vector was kindly provided by Dr. Walter F. Boron (Case Western Reserve University, Department of Physiology and Biophysics, Cleveland, Ohio).24 The FlashGel System was purchased from Lonza (Portsmouth, NH). All enzymes were purchased from New England Biotechnology (Ipswich, MA). The gel extraction and PCR purification kits were purchased from Qiagen (Valencia, CA). DNA purity and concentration were verified using a NanoDrop 1000 instrument (Thermo Fisher Scientific, Rockford, IL). A ZOE fluorescent cell imager made by Bio-Rad (Hercules, CA) was used for fluorescent microscopy. The mouse anti-Egfp antibody (JL-8, Cat. No. 632381) was purchased from Clontech (Mountain View, CA).
Generation of the pGH19-hMCT6 Vector
For these studies, we generated injectable cRNA encoding for the human MCT6 protein from the in vitro transcription of a MCT6 genetic construct. Briefly, total RNA derived from human kidney cortical tissue (obtained from Ohio State University Tissue Procurement Services, National Cancer Institute Collaborative Human Tissue Network (CHTN), Columbus, OH) was isolated using TRIzol Reagent according to the manufacturer’s instructions. After checking the concentration, purity, and stability using the FlashGel System, cDNA was generated by performing RT-PCR using the BioRad CFX Connect RT System. Using this cDNA library, sequence specific primers were designed to amplify the specific cDNA fragment encoding for the MCT6 gene (SLC16A5) (forward primer, 5′-AAGAAAAGAGCACTTCCCTGC-3′; reverse primer, 5′-ACAGGGCACTCAGGTAGGGC-3). Following the successful subcloning of the MCT6 cDNA into an intermediate expression vector (pCR2.1-TOPO TA), the plasmid was linearized with BamHI, and PCR was performed using nested primers (FP, 5′-AGCGT CCCGGG GCCACC ATGCC-CCAGGCCCTGGAG-3′; RP, 5′-ATTA TCTAGA GGCAC-TCAGGTAGGGCTA-3′). Two restriction sites (XmaI and XbaI) were incorporated into the primers which are underlined, and a Kozak consensus sequence (in bold) was incorporated into the forward primer in order to enhance translational efficiency. The pGH19 plasmid was used as the oocyte expression vector due to its translation-enhancing features (5′/3′ untranslated regions of the X. laevis β-globin, polyadenosine tract, etc.).24
To insert the MCT6 cDNA into the construct, empty pGH19 vector and MCT6 PCR product were double-digested using XmaI and XbaI. The 5′ and 3′ phosphates of the digested vector were then removed using Antarctic Phosphatase. The digested PCR product was purified, and a ligation reaction was performed using a T4 DNA ligase reaction mixture. The construct was transformed into chemically competent TOP10 Escherichia coli cells, and the ligated product was isolated, purified, and confirmed via sequencing.
Preparation of cRNA and Verification of Protein Expression in X. laevis Oocytes
The cRNA encoding for MCT6 was in vitro transcribed from NotI-linearized pGH19-hMCT6 vector using the mMESSAGE mMACHINE T7 transcription kit. The oocytes were isolated from resected and collagenase-treated X. laevis ovaries. The oocytes that were injected ranged from Dumont stages IV–VI, and approximately 13.8 nL of cRNA or water was injected into each oocyte (~20 ng). The oocytes were then incubated in OR3 medium (which was prepared as described previously24) at 18 °C for 3 to 4 days. All cRNA was checked for purity, concentration, stability, and correct size utilizing a NanoDrop 1000 and FlashGel System. Due to the inability of a variety of antibodies against human MCT6 to verify positive protein expression 3–4 days postinjection in our oocytes, our lab developed an MCT6 Egfp-tagged construct using similar methods as described previously in order to verify the protein expression and localization to the membrane using a fluorescent imager and an anti-Egfp antibody. Briefly, 3 days postinjection, oocytes were washed with 200 mOsm Tris-buffered saline (TBS) and visualized using a fluorescence microscope. In addition, Western blotting was performed from water-injected, MCT6 cRNA-injected, Egfp cRNA-injected, and MCT6-Egfp cRNA-injected oocytes from day 1 to day 4 postinjection. Briefly, 5 oocytes from each group each day were washed three times in 200 mOsm TBS and homogenized using 500 μL of lysis buffer (200 mOsm TBS, 1% Triton X-100, with protease inhibitor). The insoluble fraction was removed via centrifugation at 4 °C at 3000g for 10 min. The soluble fraction was then spun in a Spin-X centrifuge tube filter (Corning Costar, 0.45 μm) at 4 °C at 16000g for 10 min. For each soluble fraction, 25 μL of lysate was added to 12.5 μL of LDS, 10 μL of lysis buffer, and 2.5 μL of 1 M DTT, and heated at 37 °C for 10 min. An aliquot of each of these samples (20 μL, equivalent to 0.25 oocytes) was loaded in a NuPAGE 4–12% Bis-Tris protein gel. The gel ran for 1.5 h at 100 V using 1× MOPS as running buffer. The gel was then transferred to a PVDF membrane at 100 V for 2.5 h. After the membrane was washed three times in 1× TBST, the membrane was blocked in 5% nonfat milk overnight. The membrane was then washed three times in 1× TBST and incubated in primary antibody (1:200, anti-Egfp, 5% nonfat milk) on a rocker overnight at 4 °C. After the membrane was washed an additional three times in 1× TBST, the membrane was incubated in secondary antibody (1:10,000 anti-mouse, HRP-linked, 5% nonfat milk) for 1 h at room temperature. After a final three washes in 1× TBST, the membrane was developed using ECL substrate (Bio-Rad) and visualized on a ChemiDoc XRS+ System (Bio-Rad).
Uptake Studies
The uptake studies were performed similarly to the methods published by Murakami et al.3 Briefly, the uptake studies were performed using groups of 4 to 10 oocytes that were transferred into 24-well multidishes and preincubated in uptake buffer for 30 min (15 mM HEPES, 82.5 mM NaCl, 2.5 mM KCl, 1 mM Na2HPO4, and 1 mM MgCl2, adjusted to pH 7.4 with Tris). The oocytes were then transferred to 400 μL of uptake buffer containing 0.1 μM radiolabeled bumetanide ([3H]bumetanide), and the oocytes were incubated at pH 7.4 at room temperature (~20–23 °C). The uptake was stopped by the addition of ice-cold uptake buffer, and the oocytes were washed three times. Individual oocytes were placed in separate scintillation vials and dissolved in 250 μL of 10% sodium lauryl sulfate by slowly shaking for 1.5 h. Radioactivity was determined by liquid scintillation counting following the addition of scintillation cocktail.
For these studies, a range of typical MCT substrates/inhibitors were investigated for the inhibitory effects on MCT6-mediated bumetanide uptake, as well as a variety of different flavonoids that we had previously investigated in our laboratory.13,16,22,23 Lastly, the mechanism of inhibition was investigated for a potent flavonoid inhibitor of MCT6, phloretin.
Data Analysis
The uptake by oocytes (μL/oocyte) was calculated as the ratio of radioactivity in each sample (dpm/oocyte) to the initial concentration in the uptake buffer (dpm/μL). MCT6-mediated uptake was calculated as the difference between the MCT6 cRNA-injected oocytes and the water-injected oocytes. Statistical analysis was performed using the one-way unpaired analysis of variance (ANOVA) followed by Dunnett’s test to test for multiple comparisons. Differences were considered statistically significant when p < 0.05. Data analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., San Diego CA). The MCT6-specific uptake rates of bumetanide were obtained from the uptake value at 30 min, which was previously shown to be in the linear range.3 The inhibition of bumetanide uptake by flavonoids was calculated by fitting with eq 1 using weighted nonlinear regression analysis (ADAPT 5; Biomedical Simulations Research, University of South California, Los Angeles, CA).
(1) |
where F is the percentage of uptake rate of bumetanide in the presence of flavonoids compared with the control, C is the concentration of flavonoids, Imax is the maximal inhibition, and IC50 is the inhibitor concentration at 50% inhibition. After comparisons of competitive, noncompetitive, and uncompetitive inhibition models, the mechanism of inhibition was best fitted to the competitive inhibition equation (eq 2):
(2) |
where J is the MCT6-mediated bumetanide uptake rate (pmol/oocyte/30 min), C is the concentration of bumetanide (μM), Jmax is the maximum uptake rate (pmol/oocyte/30 min), Kt is the substrate concentration at the half-maximal uptake rate (μM), and Ki is the inhibition constant (μM). The goodness of fit for all model fitting was determined by the sum of the squared derivatives, the residual plots, and the AIC values.
RESULTS
Verification of MCT6 Localization and Protein Expression in X. laevis Oocytes
In order to verify the positive protein expression of MCT6-Egfp in oocytes, a protein kinetics study was performed to evaluate the protein expression over 4 days postinjection in the oocytes. As shown in Figure 1, MCT6-Egfp protein expression was apparent in the membrane of the oocytes, demonstrating successful protein translation and trafficking. MCT6 cRNA- and water-injected oocytes demonstrated minimal background fluorescence in the oocytes. Egfp cRNA-injected oocytes demonstrated positive protein expression in the oocytes, however, this was mostly restricted to the cytosol of the cells. The Western blot results (Figure 1E) using an anti-Egfp antibody demonstrated apparent positive bands at the molecular weight corresponding to MCT6-Egfp protein (~85 kDa) and Egfp alone (~30 kDa). The MCT6-Egfp band was stable and did not appear to lose intensity for up to 4 days postinjection, in contrast to the Egfp cRNA-injected oocytes which appeared to be largely expressed at day 1 postinjection in comparison to day 4 postinjection.
Figure 1.
Immunofluorescence analysis of water-injected (A), MCT6 cRNA-injected (B), MCT6-Egfp cRNA-injected (C), and Egfp cRNA-injected (D) (3 days postinjected, 13.8 nL of respective cRNA or water injected per oocyte). A white arrow in panel C depicts the apparent positive MCT6-Egfp protein expression at the oocyte membrane. A Western blot using an anti-Egfp mouse antibody is shown in panel E. No bands were visualized for water and MCT6 cRNA-injected oocytes.
Effects of MCT Substrates and Inhibitors on MCT6
Previously, it has been demonstrated that bumetanide is a suitable substrate for measuring MCT6-mediated uptake.3 It was demonstrated that the uptake of bumetanide was shown to be in the linear range for 30 min, at a concentration of 0.1 μM; therefore, for the purpose of our studies we used the same concentration and uptake time. To investigate the effects of other MCT substrates and inhibitors on the activity of MCT6, cis-inhibition assays were performed as done previously using bumetanide as the probe substrate. The uptake of bumetanide in the absence of any other compounds was significantly higher in the MCT6 cRNA-injected oocytes in comparison to the water-injected control oocytes (Figure 2A), demonstrating that human MCT6 was actively transporting bumetanide. As shown in Figure 2B, probenecid inhibited MCT6-mediated bumetanide uptake (~75%), at the concentration previously used by Murakami et al., which was consistent with probenecid being a MCT6 substrate and inhibitor.3 Most inhibitors were evaluated at concentrations that were >10-fold higher than their reported plasma concentrations. Interestingly, taurocholate, an OAT/OATP substrate, also significantly inhibited bumetanide uptake (~50%). Substrates/inhibitors for other MCTs, namely, pyruvate (MCT1–4 substrate), L-tyrosine (MCT10 substrate), L-thyroxine (MCT8 substrate), and AR-C155858 (MCT1/2 inhibitor), did not significantly affect MCT6-mediated bumetanide uptake.
Figure 2.
Uptake of [3H]bumetanide by MCT6 in the absence (A) and presence of MCT substrates/inhibitors (B). Uptake of 0.1 μM [3H]bumetanide into MCT6-cRNA injected (red) or water-injected control oocytes (blue) was measured at room temperature and pH 7.4 (A and B) for 30 min. MCT6-mediated uptake (black) was calculated by subtracting the uptake amount of water-injected control oocytes from that of MCT6-expressing oocytes. Each bar represents the mean ± SEM with three replicates (n = 7–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs. One-way ANOVA followed by Dunnett’s test was used for statistical analysis. (A) ****p < 0.0001 versus water-injected oocytes; (B) *p < 0.05, ***p < 0.001 versus control.
Effects of Flavonoids on Bumetanide Uptake via MCT6
The effects of flavonoids (50 μM) on MCT6-mediated bumetanide uptake were investigated in this study. [3H]-Bumetanide (0.1 μM) uptake was measured for 30 min at pH 7.4 at room temperature in the presence and absence of a variety of flavonoids (Figure 3). Bumetanide uptake was significantly inhibited by the aglycon flavonoids: quercetin, morin, luteolin, and phloretin, but not naringenin, genistein, and biochanin a. The flavonoid glycosides tested did not have any effect on bumetanide uptake (i.e., naringin, rutin, and phlorizin).
Figure 3.
Uptake of [3H]bumetanide by MCT6 in the absence and presence of flavonoids (50 μM). Uptake of 0.1 μM [3H]bumetanide into MCT6-cRNA injected or water-injected control oocytes was measured at room temperature and pH 7.4 for 30 min. MCT6-mediated uptake was calculated by subtracting the uptake amount of water-injected control oocytes from that of MCT6-expressing oocytes. Flavonones (red), flavonols (green), flavone (brown), isoflavones (blue), and dihydrochalcones (purple) are plotted as mean ± SEM with three replicates (n = 8–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs. One-way ANOVA followed by Dunnett’s test was used for statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Concentration-Dependent Inhibition of Bumetanide Uptake by Flavonoids
Concentration-dependent inhibition of bumetanide (0.1 μM) uptake by three aglycon flavonoids (i.e., quercetin, morin, phloretin) is depicted in Figure 4. Luteolin was initially investigated; however, its limited solubility in aqueous uptake buffer restricted our analysis at higher concentrations. Using eq 1, the IC50 values were calculated for quercetin, morin, and phloretin (25.3, 33.1, and 17.3 μM, respectively; shown in Figure 4).
Figure 4.
Concentration-dependent inhibition of MCT6-mediated bumetanide transport by (left) quercetin, (center) morin, and (right) phloretin. The lines represent the fitted IC50 curves using eq 1. The results were normalized by the control uptake (absence of flavonoids). Uptake by water-injected oocytes has been subtracted from the uptake of MCT6-transfected oocytes. The studies were performed at pH 7.4, room temperature, and for 30 min. The concentration of bumetanide used was 0.1 μM. Data is plotted as mean ± SEM with three replicates (n = 4–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs.
Mechanism of Inhibition of Phloretin on MCT6-Mediated Bumetanide Uptake
The mechanism of phloretin inhibition on MCT6-mediated bumetanide uptake was investigated in this study. Concentration-dependent inhibition was examined in the absence and presence of phloretin (5 μM and 25 μM) using a range of bumetanide concentrations (0.3–100 μM) at pH 7.4, room temperature, for 30 min. Goodness of fitting parameters including the sum of the squared derivatives, the residual plots, and the AIC values were used to determine which inhibition mechanism model was most appropriate. The data best fit a model of competitive inhibition (equation displayed in eq 2). The Lineweaver–Burk plot is shown in Figure 5 to illustrate the inhibition mechanism. In addition, preincubation of MCT6-transfected oocytes with inhibitor and subsequent washing did not result in inhibition of bumetanide uptake; this inhibition was therefore deemed reversible (data not shown). Using the Michaelis–Menten equation in eq 2 for competitive inhibition yielded a Kt value of 60.5 ± 14.1 μM (estimate ± SD) for bumetanide, a Ki value of 22.8 ± 6.95 μM, and a Jmax value of 36.1 ± 6.82 pmol/oocyte/30 min.
Figure 5.
Lineweaver–Burk plot of the uptake of bumetanide in the absence and presence of phloretin. The concentration of substrate (S, bumetanide) was 0.3, 1.0, and 100 μM. Uptake by water-injected oocytes cells has been subtracted from the uptake of MCT6-transfected oocytes. The studies were performed at pH 7.4, room temperature, and for 30 min. Data is plotted as mean ± SEM with three replicates (n = 4–10 oocytes/replicate) and 2–3 separate ovaries from separate frogs.
DISCUSSION
In this study, we investigated the effects of a variety of flavonoids on the transport of bumetanide in human MCT6-transfected X. laevis oocytes. Flavonoids from five structurally distinct classes were investigated in this study, which included flavonones, flavonols, a flavone, isoflavones, and dihydrochalcones.
There are few compounds confirmed as MCT6 substrates or inhibitors, and there is little information regarding the structural characteristics and physiochemical motifs which govern their degree of interaction. As discussed previously, only two publications are available to date for the characterization of MCT6 function, and inhibitor/substrate identification.3,4 Interestingly in these studies, MCT6-mediated bumetanide uptake was not inhibited by prototypical MCT 1–4 ligands, such as the short-chain monocarboxylic acids L-lactic acid and pyruvic acid, which agrees with our experimental data. And unsurprisingly, various sulfur-containing loop diuretics that are structural analogues of bumetanide (i.e., furosemide, piretanide, torasemide, and azosemide) have also been reported to have significant interactions with MCT6-mediated bumetanide uptake, with IC50 values ranging from 13 to 163 μM.3 Thiazide diuretics and members of the meglitinide class of hypoglycemic agents have demonstrated MCT6 inhibition and affinity. Interestingly, prostaglandin F2α demonstrated significant MCT6-mediated uptake; however, further kinetic studies are necessary in order to characterize eicosanoids as physiological substrates for MCT6.
The degree of interaction of flavonoids with MCT6-mediated bumetanide uptake appears to be highly structure-dependent. While the majority of aglycon flavonoids appear to show inhibition of MCT6, specific aglycons of the isoflavone family (i.e., genistein and biochanin A), as well as naringenin of the flavonone family, did not significantly inhibit MCT6, at the high concentration of 50 μM. This may potentially be due to the steric hindrance caused by the phenyl group at the 3-position, resulting in the inability of this class of molecules to interact with the binding site. Also, the absence of any inhibition caused by glycoside moieties appears to be due to the same cause. However, considering that there is no information known regarding the binding pocket of MCT6, further studies are required to validate this theory. The aglycon flavonoids quercetin, morin, phloretin, and luteolin appeared to have the greatest inhibitory potential of MCT6, suggesting that these compounds may cause significant drug–flavonoid interactions. Physiological systemic concentrations of flavonoids from a typical U.S. diet of ≤1 g of total flavonoids per day25 range from 0.1–10 μM; however these estimates are highly variable.26 Intestinal concentrations of these flavonoids are expected to be much higher, especially if flavonoids are ingested as a dietary supplement, suggesting that these compounds could play a major role in governing MCT6–substrate interactions. Previous studies performed in our lab investigating the interactions of flavonoids on MCT1-mediated γ-hydroxybutyric acid (GHB) uptake in rat MCT1-transfected MDA-MB231 cells also demonstrated significant inhibition of MCT1 activity.16 However, in comparing inhibition of MCT1 and MCT6, aglycon isoflavones and flavonones were able to inhibit MCT1, but not MCT6. In another study in Caco-2 cells, Shin et al. demonstrated that aglycon flavonoids naringenin and silybin had a competitive inhibitory interaction on benzoic acid uptake, with Ki values ranging from 15 to 20 μM,17 similar to that seen for phloretin in this study where phloretin has been characterized to be a competitive inhibitor, with a Ki value of ~23 μM. Phloretin is a dihydrochalcone, commonly found in apples, that has been investigated for its antioxidant effects and inhibition of lipid peroxidation.27,28 It is unknown, however, whether MCT6 plays a role in the transport of these compounds, and more detailed quantitative data is required in order to determine the relevance of MCT6 for the membrane transport of flavonoids.
In summary, the results of this study provide further evidence that MCT6 is distinct from other MCT isoforms in that it does not seem to share overlapping ligand specificity. In contrast, our findings suggest that MCT6 could share more similarities to the OAT (SLC22A) family of transporters, in contrast to its MCT (SLC16A) family. Structurally specific classes of aglycon flavonoids (flavonols, flavones, dihydrochalcones) were shown to potently inhibit MCT6-mediated transport in contrast to flavonoid glycosides. IC50 values were obtained, and the mechanism of inhibition of a potent dihydrochalcone inhibitor (phloretin) was shown to be competitive and reversible in nature. This study was the first to investigate flavonoids and their inhibition potential on MCT6, and the first to report that taurocholate is an MCT6 inhibitor. To date, it is unclear what effect flavonoids have on MCT6-mediated transport in vivo and potential diet–drug interactions. With further characterization of MCT6 in future studies, we may be able to elucidate the contribution of flavonoids on overall drug exposure and tissue distribution.
Acknowledgments
The technical assistance from Evan Myers and Aniko Marshall in the training and assistance in preparation of the oocytes is acknowledged. Funding support was from the National Institutes of Health National Institute on Drug Abuse [Grant R01DA023223]. R.S.J. was supported in part by a PhRMA Pre-Doctoral Graduate Fellowship. M.D.P. was supported by startup funding from the Dean of the School of Medicine and Biomedical Sciences and the Department of Physiology and Biophysics as well as by a Carl W. Gottschalk Research Scholar Grant from the American Society of Nephrology Foundation for Kidney Researchr.
Footnotes
Notes
The authors declare no competing financial interest.
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