The bioactive dipeptide anserine is transported by human proton-coupled peptide transporters Stefanie Geissler 1 , Madlen Zwarg 1 , Ilka Knu ¨ tter 1 , Fritz Markwardt 2 and Matthias Brandsch 1 1 Membrane Transport Group, Biozentrum of Martin-Luther-University Halle-Wittenberg, Halle, Germany 2 Julius-Bernstein-Institute for Physiology, Martin-Luther-University Halle-Wittenberg, Halle, Germany Introduction The bioactive dipeptide anserine (b-alanyl-1-N-methyl- l-histidine) is found in considerable amounts in skele- tal muscle and brain of vertebrates [1,2]. It is formed as a secondary product through the methylation of the dipeptide carnosine (b-alanyl-l-histidine) [3]. The reac- tion is catalysed by carnosine N-methyltransferase [3,4]. Both anserine and carnosine exert antioxidative properties, pH buffering capacity and transglycating activity [5,6]. Anserine and carnosine are thought to inhibit lipid oxidation by a combination of free radical scavenging and metal chelation [7]. Furthermore, anserine and carnosine enhance postdenervation depolarization by the inhibition of NO production [8]. Because of the many recent reports on the endoge- nous biochemical effects of anserine on the one hand, and its presence in human diet on the other, the intestinal absorption of anserine has received increas- ing interest recently. In 2009, the intestinal absorption of anserine after the ingestion of an anserine-contain- ing diet, and its blood clearance, were studied [2]. Ingested anserine is absorbed intact into human blood and is then hydrolysed to p-methyl-l-histidine and Keywords carnosine; intestine; kidney; PEPT1; PEPT2 Correspondence M. Brandsch, Membrane Transport Group, Biozentrum of Martin-Luther-University Halle-Wittenberg, Weinbergweg 22, D-06120 Halle, Germany Fax: +49 345 5527258 Tel: +49 345 5521630 E-mail: matthias.brandsch@biozentrum. uni-halle.de (Received 4 November 2009, revised 2 December 2009, accepted 2 December 2009) doi:10.1111/j.1742-4658.2009.07528.x The bioactive dipeptide derivative anserine (b-alanyl-1-N-methyl-l-histidine) is absorbed from the human diet in intact form at the intestinal epithelium. The purpose of this study was to investigate whether anserine is a substrate of the H + ⁄ peptide cotransporters 1 and 2 (PEPT1 and PEPT2). We first assessed the effects of anserine on [ 14 C]glycylsarcosine ([ 14 C]Gly-Sar) uptake into Caco-2 cells expressing human PEPT1 and into spontaneous hyperten- sive rat kidney proximal tubule (SKPT) cells expressing rat PEPT2. Anser- ine inhibited [ 14 C]Gly-Sar uptake with K i values of 1.55 mm (Caco-2) and 0.033 mm (SKPT). In HeLa cells transfected with pcDNA3-hPEPT1 or pcDNA3-hPEPT2, K i values of 0.65 mm (hPEPT1) and 0.18 mm (hPEPT2) were obtained. We conclude from these data that anserine is recognized by PEPT1 and PEPT2. Carnosine also inhibited [ 14 C]Gly-Sar uptake. Using the two-electrode, voltage-clamp technique at Xenopus laevis oocytes, strong hPEPT1-specific inward transport currents were recorded for Gly-Sar, anserine and carnosine, but not for glycine. We conclude that anserine and carnosine interact with the human intestinal peptide transporter and are transported by hPEPT1 in an active, electrogenic H + symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. In addition, anserine might be useful for the design of new substrates of peptide transporters, such as prodrugs, that can be administered orally. Abbreviations Gly-Sar, glycylsarcosine; hPEPT, human PEPT; PEPT1, H + ⁄ peptide cotransporter 1; PEPT2, H + ⁄ peptide cotransporter 2; rPEPT, rat PEPT; SKPT, spontaneous hypertensive rat kidney proximal tubule. 790 FEBS Journal 277 (2010) 790–795 ª 2010 The Authors Journal compilation ª 2010 FEBS b-alanine by serum and tissue carnosinases. According to the authors, this was the first study to demonstrate the intestinal absorption of anserine [2]. It should be noted, however, that, as early as 1976, Hama et al. [9] concluded from their studies on the absorption of b-alanine, anserine and carnosine that physiological amounts of anserine (and carnosine) are absorbed from rat small intestine in intact form. Very recently, Yeum et al. [10] have investigated the metabolic stabil- ity of carnosine and anserine in human serum and their absorption kinetics in vivo. Again, the anserine concentration was increased significantly after diges- tion of anserine-rich food. The molecular mechanism of anserine uptake remains unknown. Based on the in vivo data and molecular structure of anserine, we hypothesized that anserine might be recognized by the intestinal peptide transporter. At the intestinal epithe- lium, di- and tripeptides are transported from the lumen into the enterocytes by H + ⁄ peptide cotransport- er 1 (PEPT1) (peptide transporter 1) (for a review, see [11,12]). At the renal epithelium, small peptides are reabsorbed from the glomerular filtrate into the cells by the subtypes PEPT1 and PEPT2. PEPT2 is also expressed in other tissues, such as lung and choroid plexus. In addition to peptides, both PEPT1 and PEPT2 also accept several pharmacologically relevant peptidomimetics as substrates, such as many b-lactam antibiotics, valacyclovir and d-aminolaevulinic acid [12]. The intestinal proton-coupled peptide transport system also accepts carnosine as substrate [13,14] (for a review, see [15]). To the best of our knowledge, the transport of anserine by H + ⁄ peptide cotransporters has not yet been studied. Interaction with these carri- ers would not only deliver new information on the substrate specificity of the transporters, but transport by PEPT1 would also explain the high oral availability of anserine. Results and discussion Inhibition of [ 14 C]glycylsarcosine ([ 14 C]Gly-Sar) uptake at Caco-2 and spontaneous hypertensive rat kidney proximal tubule (SKPT) cells by anserine Caco-2 and SKPT cell cultures are well-established systems for intestinal and renal peptide transport studies. Caco-2 cells express the human low-affinity, high- capacity (‘intestinal’)-type peptide transport system PEPT1, whereas SKPT cells express the rat high-affinity, low-capacity (‘renal’)-type system PEPT2, but not PEPT1 [16–18]. In the present investigation, we first determined the effect of anserine on [ 14 C]Gly-Sar uptake. Gly-Sar is used as reference substrate for peptide transport studies because of its relatively high enzymatic stability. At concentrations of 10 mm (Caco-2) and 2 mm (SKPT), anserine strongly inhibits the uptake of [ 14 C]Gly-Sar (10 lm) by 76% and 79%, respectively. With both cell lines, competition assays at increasing concentrations of Gly-Sar and anserine were performed. From the inhibition curves shown in Fig. 1, IC 50 values, i.e. the inhibitor concentration necessary to inhibit carrier-mediated [ 14 C]Gly-Sar uptake by 50%, were calculated and converted into K i values, as described previously [16–19]. Gly-Sar, a prototype substrate for PEPT1 and PEPT2, displayed K i values of 0.74 ± 0.01 mm and 0.11 ± 0.01 mm, respectively (Fig. 1, Table 1). Anserine inhibited [ 14 C]Gly-Sar uptake mediated by PEPT1 into Caco-2 cells with a K i value of 1.55 ± 0.02 mm. The K i value of anserine for the inhibition of [ 14 C]Gly-Sar uptake via PEPT2 into SKPT cells was 0.033 ± 0.001 mm (Table 1). The apparent affinity of anserine is thereby lower than that of Gly-Sar at PEPT1, but higher at PEPT2. As reviewed earlier, most dipeptides composed of natural amino acids display K i values in the range 0.07–0.7 mm at PEPT1 and 5–100 lm at PEPT2 [11,12]. According to our classification [12], anserine can be considered as a medium-affinity ligand for human PEPT1 and a high-affinity ligand for rat PEPT2. Effect of anserine on [ 14 C]Gly-Sar uptake in HeLa-hPEPT1 and HeLa-hPEPT2 cells Caco-2 and SKPT cells originate from different species, man and rat, respectively. To rule out the pos- Fig. 1. Inhibition of [ 14 C]Gly-Sar uptake into Caco-2 and SKPT cells by anserine. Uptake of 10 l M [ 14 C]Gly-Sar was measured for 10 min at pH 6.0 in the presence of increasing concentrations of anserine (n = 3–4). S. Geissler et al. Anserine transport by peptide transporters FEBS Journal 277 (2010) 790–795 ª 2010 The Authors Journal compilation ª 2010 FEBS 791 sibility that differences in substrate recognition between hPEPT1 and rPEPT2 reflect species’ differ- ences and to confirm the affinity constants obtained in Caco-2 and SKPT cells in a second, independent approach, we performed transport studies with cloned human PEPT1 and PEPT2 [17]. The interaction of anserine and, for comparison, carnosine and Gly-Sar with hPEPT1 and hPEPT2 was studied in competition assays after heterologous expression of the transporters in HeLa cells (Fig. 2). For anserine, K i values of 0.65 ± 0.02 mm and 0.18 ± 0.01 mm were determined at hPEPT1 and hPEPT2, respectively (Table 1). Carnosine inhibited [ 14 C]Gly-Sar uptake with K i values of 1.7 ± 0.1 mm (hPEPT1) and 0.06 ± 0.01 mm (hPEPT2). Unlabelled Gly-Sar inhibited [ 14 C]Gly-Sar uptake with K i values of 0.64 ± 0.02 mm (hPEPT1) and 0.24 ± 0.02 mm (hPEPT2). These results clearly show that anserine interacts specifically with hPEPT1 and hPEPT2 and that the compound inhibits the uptake of the prototype substrate Gly-Sar. Transport of anserine by hPEPT1 expressed in Xenopus laevis oocytes Inhibition of [ 14 C]Gly-Sar uptake at native intestinal or renal cells, or at transfected cells expressing peptide transporters heterologously, does not allow the conclu- sion to be drawn that the inhibiting, competing com- pound – in this case anserine – is indeed transported. Anserine could represent an inhibitor blocking directly the binding site of the carrier. Alternatively, the results obtained so far do not rule out an indirect effect, for example an effect on the proton gradient, as the driving force of [ 14 C]Gly-Sar uptake. Employing the two- electrode, voltage-clamp technique, we therefore inves- tigated whether anserine is able to generate currents at X. laevis oocytes expressing hPEPT1. These currents occur when a compound is cotransported by PEPT1 with H + in an electrogenic manner. As shown in Fig. 3, anserine (10 mm) generated inward currents (1254 ± 44 nA) comparable with those generated by Table 1. Inhibition constants (K i ) of Gly-Sar, anserine and carnosine at PEPT1 and PEPT2. Uptake of [ 14 C]Gly-Sar in Caco-2 and SKPT cells, or in HeLa cells transfected with hPEPT1- or hPEPT2-cDNA, was measured at pH 6.0 for 10 min at increasing concentrations of unlabelled dipeptides. K i values were derived from the competition curves shown in Figs 1 and 2 (n = 4). ND, not determined. K i (mM) Compound hPEPT1 Caco-2 rPEPT2 SKPT hPEPT1 HeLa hPEPT2 HeLa Gly-Sar 0.74 ± 0.01 0.11 ± 0.01 0.64 ± 0.02 0.24 ± 0.02 Anserine 1.55 ± 0.02 0.033 ± 0.001 0.65 ± 0.02 0.18 ± 0.01 Carnosine ND ND 1.7 ± 0.1 0.06 ± 0.01 Fig. 2. Inhibition of [ 14 C]Gly-Sar uptake into HeLa cells transfected with pcDNA3-hPEPT1 and pcDNA3-hPEPT2 constructs by anserine, carnosine and Gly-Sar. Uptake of 20 l M [ 14 C]Gly-Sar was measured for 10 min at pH 6.0 in the presence of increasing concentrations of the compounds for the determination of IC 50 values (n = 3–4). 10 mM Anserine 10 m M Carnosine 10 m M Gly-Sar 20 m M Glycine 500 nA 10 sec Fig. 3. Electrophysiological analysis of anserine transport in hPEPT1-cRNA-injected X. laevis oocytes (membrane potential, )60 mV; pH 6.5). Lower trace: currents induced by 10 m M anser- ine, carnosine and Gly-Sar, and 20 m M glycine. Upper trace: mea- surement in water-injected oocytes. Anserine transport by peptide transporters S. Geissler et al. 792 FEBS Journal 277 (2010) 790–795 ª 2010 The Authors Journal compilation ª 2010 FEBS the prototype transporter substrate Gly-Sar (1220 ± 42 nA) and by the structurally related sub- strate carnosine (1075 ± 24 nA). No currents were observed for either of the test compounds in water- injected oocytes (Fig. 3). Hence, indirect PEPT1- independent effects of anserine can be ruled out. PEPT1 does not accept free amino acids as substrates. Therefore, glycine was used as negative control in these experiments. No inward currents could be observed (Fig. 3). We conclude from these data that anserine is recog- nized by the proton-coupled peptide transporters PEPT1 and PEPT2 with medium affinity. Anserine is able to displace other substrates from the transport process. The experiments show that anserine and car- nosine are transported by hPEPT1 in an active, elec- trogenic manner by an H + symport. As PEPT1 is the predominant transport system for di- and tripeptides at the intestinal epithelium, this transporter is most probably responsible for the intestinal absorption of anserine after food intake. After entering the blood compartments and tissues, the hydrolysis of anserine – which is relatively resistant against intestinal dipeptid- ases – occurs in serum caused by the activity of carno- sinases [10,20,21]. With regard to the structural requirements for PEPT1 and PEPT2 substrates, it is surprising that anserine, with its N-terminal b-amino acid, displays such high affinity. Therefore, in addition to the physio- logical and biochemical aspects of anserine transport, this compound might also be useful for the design of new substrates of peptide transporters, such as pro- drugs, that can be administered orally. Experimental procedures Materials Caco-2 and HeLa cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braun- schweig, Germany). The renal cell line SKPT-0193 Cl.2, established from isolated cells of rat proximal tubules, was provided by U. Hopfer (Case Western Reserve University, Cleveland, OH, USA) [16]. Cell culture media, supplements and trypsin solution were purchased from Life Technolo- gies, Inc. (Karlsruhe, Germany) or PAA (Pasching, Austria). Fetal bovine serum was obtained from Biochrom (Berlin, Germany). [Glycine-1- 14 C]Gly-Sar (specific radioac- tivity, 56 mCiÆmmol )1 ) was custom synthesized by GE Healthcare (Little Chalfont, Buckinghamshire, UK). Anser- ine was purchased from Bachem (Weil am Rhein, Ger- many), and Gly-Sar and carnosine from Sigma-Aldrich (Deisenhofen, Germany). Culture of Caco-2 and SKPT cells Caco-2 cells were routinely cultured in 75 cm 2 culture flasks with minimum essential medium supplemented with 10% fetal bovine serum, gentamicin (50 lgÆmL )1 ) and 1% nones- sential amino acid solution [16–18]. Subconfluent cultures (80% of confluence) were treated for 5 min with Dulbecco’s phosphate-buffered saline, followed by a 2 min incubation with trypsin solution. For uptake experiments, cells were seeded in 35 mm disposable Petri dishes (Sarstedt, Nu ¨ mbr- echt, Germany) at a density of 0.8 · 10 6 cells per dish. The monolayers reached confluence the next day. The uptake measurements were performed on the seventh day after seed- ing. The protein content per dish was determined using a Pierce Ò 660 nm Protein Assay (Thermo Fisher Scientific, Bonn, Germany) according to the manufacturer’s protocol. The culture medium for SKPT cells was Dulbecco’s modified Eagle’s medium ⁄ F12 nutrient mixture (1 : 1, v ⁄ v) supplemented with fetal bovine serum (10%), gentamicin (50 lgÆmL )1 ), epidermal growth factor (10 ngÆmL )1 ), insulin (4 lgÆmL )1 ), dexamethasone (5 lgÆmL )1 ) and apo- transferrin (5 lgÆmL )1 ). SKPT cells were seeded in Petri dishes at a density of 0.8 · 10 6 cells per dish. The uptake measurements were performed on the fourth day after seeding [16–18]. Heterologous expression of human PEPT1 and human PEPT2 in HeLa cells HeLa cells were routinely cultured with Dulbecco’s modi- fied Eagle’s medium with Glutamax, supplemented with 10% fetal bovine serum and gentamicin (50 lgÆmL )1 ). The cDNA of human PEPT1 and PEPT2 was cloned into pcDNA3 using the pBluescript constructs as a template for PCR, and XhoI and BamHI as restriction sites [17]. The resulting pcDNA3-hPEPT1 ⁄ 2 constructs were confirmed by sequencing. Human PEPT1 and human PEPT2 were heter- ologously expressed in HeLa cells using pcDNA3-hPEPT1 or pcDNA3-hPEPT2 constructs (1 lg per well) and Turbo- fect (1.5 lL per well; Fermentas, St. Leon-Rot, Germany), according to the manufacturer’s protocol. Transfection was performed 1 h postseeding in 24-well plates [17]. [ 14 C]Gly-Sar uptake measurements The uptake of [ 14 C]Gly-Sar (10 lm) in Caco-2 and SKPT cells cultured on plastic dishes was measured at room tem- perature, as described previously [16–18]. The uptake buffer was 25 mm Mes ⁄ Tris (pH 6.0) containing 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl 2 , 0.8 mm MgSO 4 ,5mm glucose, [ 14 C]Gly-Sar (10 lm) and unlabelled Gly-Sar, anserine or carnosine (0–10 mm, pH readjusted if necessary). After incubation for 10 min, the monolayers were quickly washed four times with ice-cold uptake buffer, solubilized and pre- S. Geissler et al. Anserine transport by peptide transporters FEBS Journal 277 (2010) 790–795 ª 2010 The Authors Journal compilation ª 2010 FEBS 793 pared for liquid scintillation spectrometry [16–18]. The nonsaturable component of [ 14 C]Gly-Sar uptake (diffusion, adherent radioactivity), determined by measuring the uptake of [ 14 C]Gly-Sar in the presence of 50 mm (Caco-2) or 20 mm (SKPT) of unlabelled Gly-Sar, represented 8.0% and 12.3% of total uptake, respectively. This value was taken into account during nonlinear regression analysis of inhibition constants (IC 50 ). The uptake of [ 14 C]Gly-Sar into transfected HeLa-hPEPT1 and HeLa-hPEPT2 cells grown in 24-well plates was performed 20–24 h post-transfection at room temperature in the same manner, except that the tracer concentration was 20 lm. Construction of pNKS-hPEPT1 and in vitro cRNA synthesis The X. laevis oocyte expression vector pNKS was kindly provided by Professor G. Schmalzing (RWTH, Aachen, Germany). This vector contains the 5¢ and 3¢ UTRs of the X. laevis oocyte b-globin gene. To clone the transporter’s cDNA into pNKS, AatII and XbaI restriction sites were introduced at the 5¢ and 3¢ ends, respectively, by PCR. As template, the pBluescript-hPEPT1 vector was used. After restriction enzyme digestion, the PCR product was ligated into the digested pNKS vector. The insertion of the correct cDNA was verified by sequencing. The pNKS-hPEPT1 construct served as template for cRNA synthesis. After linearizing the plasmids with NotI, cRNAs were synthesized using the mMESSAGE mMACHINE Ò SP6 kit (Ambion, Huntingdon, Cambridgeshire, UK). The cRNAs were purified with the MEGAclearÔ kit (Ambion), and the concentration was determined by UV absorbance at 260 nm. The cRNAs were stored at )80 °C [22]. Xenopus laevis oocytes expressing hPEPT1 and electrophysiology Oocytes were surgically removed from anaesthetized X. laevis frogs, dissected and defolliculated as described by Riedel et al. [23]. In brief, for anaesthesia, tricaine methane sulfo- nate (Sigma-Aldrich) was used. The removed oocytes were separated by collagenase treatment (2 mgÆmL )1 ) for 2 h. Healthy-looking oocytes (stages V–VI) were manually selected and 23 nL (1.1 lgÆlL )1 ) of cRNA solution of hPEPT1 were injected per oocyte. Water-injected oocytes were used as controls. Injected oocytes were maintained at 19 °C in modified Barth’s medium (5 mm Hepes ⁄ NaOH, pH 7.4, 100 mm NaCl, 1 mm KCl, 1 mm CaCl 2 ,1mm MgCl 2 , 10 000 UÆmL )1 penicillin and 10 mgÆmL )1 streptomycin). Five days postinjection, electrophysiological measurements were performed. Oocytes were placed in a flow-through chamber and continuously superfused (75 lLÆs )1 ) with oocyte Ringer (ORi) buffer (10 mm Mes ⁄ Tris, pH 6.5, 100 mm NaCl, 1 mm MgCl 2 ,1mm CaCl 2 ,2mm KCl) in the absence or presence of anserine and carnosine at a concentra- tion of 10 mm. Quick and reproducible solution exchanges were achieved using a small tube-like chamber (0.1 mL) combined with fast superfusion [22–25]. Microelectrodes with resistances between 0.8 and 1.4 MX were made of borosili- cate glass and filled with 3 m KCl. Whole-cell currents were recorded and filtered at 100 Hz using a two-electrode, voltage-clamp amplifier (OC-725C, Hamden, USA) and sampled at 85 Hz. Oocytes were voltage clamped at a membrane potential of )60 mV. Data analysis Experiments were performed in duplicate or triplicate, and each experiment was repeated two to three times. Results are given as the means ± standard errors. The concentra- tion of the unlabelled compound necessary to inhibit 50% of [ 14 C]Gly-Sar carrier-mediated uptake was deter- mined by nonlinear regression using the logistical equation for an asymmetric sigmoid (allosteric Hill kinetics), y = Min + (Max – Min) ⁄ [1 + (X ⁄ IC 50 ) )P ], where Max is the initial Y value, Min is the final Y value and the power P represents Hills’ coefficient (sigmaplot program, Systat, Erkrath, Germany) [16–18]. Inhibitory constants (K i ) were calculated from the IC 50 values according to the method developed by Cheng and Prusoff [19]. Oocyte data were analysed using the superpatch 2000 program (Julius-Bernstein-Institute of Physiology, SP-Ana- lyzer by T. Bo ¨ hm, Halle, Germany). The statistical values of the oocyte experiments were taken from the measure- ments of three to seven oocytes each from two batches of oocyte preparation. 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The purpose of this study was to investigate whether anserine is