Targeted Disruption of Organic Cation Transporter 3 Attenuates the Pharmacologic Response to Metformin S
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Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2015/04/29/mol.114.096776.DC1 1521-0111/88/1/75–83$25.00 http://dx.doi.org/10.1124/mol.114.096776 MOLECULAR PHARMACOLOGY Mol Pharmacol 88:75–83, July 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Targeted Disruption of Organic Cation Transporter 3 Attenuates the Pharmacologic Response to Metformin s Eugene C. Chen, Xiaomin Liang, Sook Wah Yee, Ethan G. Geier,1 Sophie L. Stocker,2 Ligong Chen, and Kathleen M. Giacomini Department of Bioengineering and Therapeutic Sciences (E.C.C., X.L., S.W.Y., E.G.G, S.L.S., K.M.G.) and Institute for Human Genetics (K.M.G.),University of California, San Francisco, California; and Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, China (L.C.) Received November 9, 2014; accepted April 28, 2015 Downloaded from ABSTRACT Metformin, the most widely prescribed antidiabetic drug, P , 0.001). Importantly, metformin’s effect on the plasma requires transporters to enter tissues involved in its pharmaco- glucose concentration-time curve was reduced in knockout logic action, including liver, kidney, and peripheral tissues. compared with wild-type mice (12 versus 30% reduction, re- Organic cation transporter 3 (OCT3, SLC22A3), expressed spectively, P , 0.05) along with its accumulation in skeletal ubiquitously, transports metformin, but its in vivo role in muscle and adipose tissue (P , 0.05). Furthermore, the effect of molpharm.aspetjournals.org metformin response is not known. Using Oct3 knockout mice, metformin on phosphorylation of AMP activated protein kinase, the role of the transporter in metformin pharmacokinetics and and expression of glucose transporter type 4 was absent in the pharmacodynamics was determined. After an intravenous dose adipose tissue of Oct32/2 mice. Additional analysis revealed of metformin, a 2-fold decrease in the apparent volume of that an OCT3 39 untranslated region variant was associated with distribution and clearance was observed in knockout compared reduced activity in luciferase assays and reduced response to with wild-type mice (P , 0.001), indicating an important role of metformin in 57 healthy volunteers. These findings suggest that OCT3 in tissue distribution and elimination of the drug. After oral OCT3 plays an important role in the absorption and elimination doses, a significantly lower bioavailability was observed in of metformin and that the transporter is a critical determinant of knockout compared with wild-type mice (0.27 versus 0.58, metformin bioavailability, clearance, and pharmacologic action. at ASPET Journals on June 1, 2017 Introduction AMP/ATP ratios (Pernicova and Korbonits, 2014). One of the outcomes from the altered energy status of the cell is In 2012, approximately 20 million people in the United activation of AMP-activated protein kinase, AMPK. At least States had been diagnosed with type 2 diabetes mellitus, in part due to activation of AMPK, metformin decreases resulting in a substantial toll on health in the country glucose output by the liver, which is widely considered to be (Centers for Disease Control and Prevention, 2014). Among the primary site of metformin action. In addition, the drug the array of pharmacological agents aimed at the treatment of enhances glucose utilization in peripheral tissues, particu- type 2 diabetes, the biguanide metformin is recommended for larly skeletal muscle and adipose tissues (Kirpichnikov et al., first-line oral therapy and is particularly beneficial for overweight diabetic patients (Boyle et al., 2010). Although 2002). These changes ultimately contribute to the therapeutic metformin has been used for decades, its mechanism of action effect of metformin: improved insulin sensitivity and glycemic is not completely understood. However, the drug clearly control. reduces the rate of ATP synthesis and results in higher Because of its low hydrophobicity, metformin requires membrane transporters to cross biologic membranes and This work was supported by the National Institutes of Health National enter and exit from cells. Previously, studies from our Institute of General Medical Sciences [Grant U19-GM61390] and the laboratory and others have shown that organic cation trans- Burroughs Wellcome Fund Innovation in Regulatory Science Awards [Grant porters 1 and 2, OCT1 (SLC22A1) and OCT2 (SLC22A2), BWF ID 1012485]; E. C. Chen and X. Liang are supported in part by the National Institutes of Health Training Grant [Grant T32-GM007175]. The transport metformin in the liver and kidney, respectively genotyping using the Illumina HumanOmniExpress array is supported by the (Jonker et al., 2001, 2003; Wang et al., 2002; Chen et al., – National Institutes of Health Pharmacogenomics Research Network RIKEN 2014). For example, in Oct1 knockout mice, the accumulation Global Alliance. 1Current affiliation: Apricity Therapeutics, Inc, Atherton, CA. of metformin is significantly reduced in the liver compared 2Current affiliation: GlaxoSmithKline, Victoria, Australia. with wild-type mice (Shu et al., 2007). Furthermore, healthy dx.doi.org/10.1124/mol.114.096776. s This article has supplemental material available at molpharm. volunteers with reduced function variants of OCT1 exhibit aspetjournals.org. reduced pharmacologic response to metformin compared to ABBREVIATIONS: AMPK, AMP activated protein kinase; AUC, area under the concentration-time curve; CL, clearance; F, bioavailability; HBSS, Hank’s balanced salt solution; MATE, multidrug and toxin extrusion; OCT, organic cation transporter; SNP, single nucleotide polymorphism; UTR, untranslated region; V, apparent volume of distribution. 75 76 Chen et al. those with reference OCT1 (Shu et al., 2007). On the other streptomycin (100 mg/ml), and 10% fetal bovine serum. For the hand, significantly altered systemic exposure and renal uptake studies, cells were cultured on poly-D-lysine–coated 24-well clearance were observed in healthy volunteers with genetic plates to 95% confluence. The cells were washed once with warm ’ variants of OCT2 (Song et al., 2008; Chen et al., 2009). OCT2- Hank s balanced salt solution (HBSS) and then incubated in the mediated drug-drug interactions have also been described uptake buffer with various concentration of unlabeled metformin and [14C]metformin (American Radiolabeled Chemicals, St. Louis, MO) in (Somogyi and Muirhead, 1987; Somogyi et al., 1987), although HBSS. The uptake was performed at 37°C for 3 minutes, then the cells recent studies suggest that transporters in the multidrug and were washed three times with ice-cold HBSS. The cells were lysed toxin extrusion protein family, MATE (SLC47A), play more with lysis buffer containing 0.1 N NaOH and 0.1% SDS, and the important roles in these interactions. Because of their radioactivity in the lysate was determined by liquid scintillation interactions with many drugs, OCT1 and OCT2 along with counting. The Km and Vmax were calculated by fitting the data to MATEs have been included in drug-drug interaction guidances Michaelis-Menten equations using GraphPad Prism software from regulatory authorities in Europe and the U.S. (La Jolla, CA). In contrast to our knowledge of OCT1 and OCT2, much less Real-Time Reverse Transcription Polymerase Chain Re- is known about the third member of the SLC22 family, OCT3. action Analysis of mRNA Level in Tissues. Total RNA from The transporter exhibits overlapping substrate specificity various C57BL/6J mouse tissues was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. Two with OCT1 and OCT2 (Koepsell et al., 2003) and in particular micrograms of total RNA from each sample were reverse transcribed is an excellent transporter for metformin. However, unlike into cDNA using SuperScript VILO cDNA Synthesis kit (Life Downloaded from OCT1 and OCT2, OCT3 is expressed ubiquitously in most Technologies) according to the manufacturer’s protocol. Quantitative tissues. Originally thought to be a major component of the real-time polymerase chain reaction was carried out in 384-well extraneuronal monoamine transporter system, scavenging reaction plates using 2X Taqman Fast Universal Master Mix (Applied neurotransmitters that escaped reuptake in the central Biosystems, Foster City, CA), 20X Taqman specific gene expression nervous system, OCT3 has been shown to play an important probes, and 10 ng of the cDNA template. The reactions were carried role in the homeostasis and neuropharmacology of mono- out on an Applied Biosystems 7500 Fast Real-Time Polymerase Chain amines (Zwart et al., 2001; Wultsch et al., 2009; Zhu et al., Reaction System (Applied Biosystems). The relative expression level molpharm.aspetjournals.org 2010; Horton et al., 2013). Although OCT3 is widely expressed of each mRNA transcript was calculated by the comparative method (DDCt method) normalized to the housekeeping gene glyceraldhyde-3- in many tissues and has been shown to be the most highly phosphate dehydrogenase (Gapdh). expressed organic cation transporter in skeletal muscle and Pharmacokinetic Study and Tissue Distribution of Metfor- adipose tissue (Bleasby et al., 2006), little is known about its min. Mice were fasted for 16 hours and then given a dose of 50 mg/kg biologic or pharmacologic roles in peripheral tissues. A recent metformin in saline with 0.2 mCi/g of [14C]metformin via oral gavage study shows that OCT3 is important in the salivary or tail vein