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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS
Department of Clinical Pharmacy (Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 2706) and Institut Fédératif de Recherche-141, Faculty of Pharmaceutical Sciences Paris XI, Châtenay-Malabry, France (P.H., R.F., M.B.); and Institut National de la Santé et de la Recherche Médicale-U773, Faculty of Medicine Xavier Bichat University Paris VII, Paris, France (A.B.)
Received May 16, 2007; accepted July 9, 2007.
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Abstract |
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). They are efficiently absorbed by the brush-border H+-coupled peptide cotransporter 1 (PepT1, SLC15A1), which is primarily expressed in the small intestine and less so in the kidney (Ogihara et al., 1996). Because of its broad substrate specificity, PepT1 can transport several peptidomimetic drugs, such as 
-lactam antibiotics (Inui et al., 1992), angiotensin-converting enzyme inhibitors (Thwaites et al., 1995), sulpirid (Watanabe et al., 2002), midodrine (Tsuda et al., 2006), and antiviral and cytostatic prodrugs (valacyclovir, valganciclovir, and floxuridine amino acid ester prodrugs) (Ganapathy et al., 1998; Sugawara et al., 2000; Landowski et al., 2005). Thus, PepT1 functions as a nutrient transporter involved in the handling of body nitrogen and also as a drug transporter. Several studies demonstrated that PepT1 activity and expression are regulated by nutritional status and by its own substrates (Shiraga et al., 1999), pharmacological agents (Berlioz et al., 2000; Watanabe et al., 2004), hormones such as insulin (Gangopadhyay et al., 2002), epidermal growth factor (Nielsen et al., 2001), and leptin. Indeed, we have reported previously in vivo that leptin can rapidly increase PepT1-mediated intestinal absorption of peptides and peptidomimetic drugs such as cephalexin (Buyse et al., 2001). This short-term action of leptin was shown to involve a recruitment of PepT1 molecules from an intracellular preformed pool to the apical membrane of intestinal Caco-2 cells. This acute short-term effect of leptin occurred when applied to the apical side, presumably by interacting with the brush-border membrane receptors, but not on the basolateral side. It is noteworthy that this short-term effect is consistent with rapid meal-induced gastric leptin release (Bado et al., 1998) and with the fact that leptin can be found in very large amounts in intestinal juices (i.e., available on the apical side of the enterocytes) (Guilmeau et al., 2003). Together, these data suggest that under physiological conditions, stomach-derived leptin acting from the apical side regulates the function of PepT1. In addition, the major source of systemic leptin is adipocytes and leptin receptors was shown to be expressed in enterocytes in both the apical and basolateral sides (Barrenetxe et al., 2002). This raises the possibility of a key role for systemic leptin in the maintenance of intestinal and energy homeostasis, including drug transporters such as PepT1. Likewise, other nutrient transporters such as the glucose transporter sodium-glucose transporter 1 (SGLT-1) (Ducroc et al., 2005) and the intestinal fatty acid-binding protein intestinal fatty acid binding protein (I-FABP) (Dubé et al., 2001) have been shown to be controlled by leptin. In this study, we questioned whether chronic leptinemia (reflecting adipocyte production of leptin) could regulate PepT1 expression. We demonstrated that chronic sustained hyperleptinemia is associated with an increase in PepT1 activity and expression. Alternatively, leptin-deficient ob/ob mice exhibited an impaired function of PepT1, which can be completely restored by peripheral administration of leptin. Moreover, using intestinal human enterocyte-like Caco-2 cells, we showed that the action of leptin is likely to be direct, at least in part.
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Materials and Methods |
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Experimental Protocol in Animals. Rats and mice were anesthetized with isoflurane (inhalation route; Abbott, Rungis, France), and they were surgically implanted subcutaneously with an Alzet mini-osmotic pump (model 2ML1 for rats and model 1007D for mice; L'Arbresle, Lyon, France). The pump delivered either leptin or phosphate-buffered saline (PBS) (vehicle) for 7 days.
Rats were divided into two groups: a leptin-treated group (group 1) receiving 1 µg/g/day recombinant leptin (R&D Systems, Minneapolis, MN) and a vehicle-treated pair-fed group, with the amount of chow matched to the leptin-treated group (group 2). Mice were divided into three groups: leptin-treated ob/ob mice (group 1) receiving 10 µg/day recombinant leptin (R&D Systems), vehicle-treated ob/ob mice (group 2), and vehicle-treated wild type-mice (group 3).
Body weight and food intake were monitored daily. At day 8, rats and mice were anesthetized after an 18-h period of food deprivation. Blood samples were collected and centrifuged; the plasma was stored at –20°C until the leptin and insulin levels were determined. Glycemia was measured in mice with the ACCU-CHEK Compact Plus System (Roche Diagnostics, Meylan, France).
Animals were sacrificed, and the jejunums were removed and flushed with 1 ml of ice-cold saline. The jejunums were then gently pressed from top to bottom to collect the total jejunal fluid contents. These fluids were centrifuged and stored at –20°C until leptin determination.
Rats leptin levels in plasma and jejunal fluids were determined by RIA using a murine leptin RIA kit following the manufacturer's instructions (Linco Research Inc., St. Charles, MO). Rat insulin levels in plasma were measured using a murine RIA kit (Linco Research Inc.). Mice leptin and insulin levels were determined using murine (Crystal Chem Inc., Downers Grove, IL).
Another set of experiments was performed in 18-h fasted rats in which acute hyperleptinemia was induced by intrafemoral bolus injection of 0.50 mg/kg leptin (31 nmol/kg). The control group received i.v. PBS. Plasma leptin and insulin levels were measured as indicated above.
Transport of [3H]Gly-Sar across the Rat Jejunum in Situ. The transport and uptake of a specific nonhydrolyzable PepT1 substrate, glycyl-sarcosine ([3H]Gly-Sar; Isobio, Fleurus, Belgium; specific activity 1 Ci/mmol) was monitored using the in situ jejunal loop method. In brief, 18-h fasted rats were anesthetized with ethylurethane (13 mmol/kg i.m.; Prolabo, VWR, Fontenay-sous-Bois, France), and the left carotid artery was cannulated to collect blood samples at designated times. After laparotomy, a 10-cm loop of jejunum was isolated 3 cm below the Treitz ligament. The loop was cannulated at both ends, and it was cleared by slowly passing warmed KrebsRinger bicarbonate buffer (119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl2, 1.19 mM KH2PO4, 1.19 MgCl2, and 25 mM NaHCO3, pH 7.4). The loop was returned to the abdominal cavity, and the abdominal wall was sutured. The distal cannula was clamped, and 1 ml of [3H]Gly-Sar solution in Krebs-Ringer bicarbonate buffer [0.2 µM [3H]Gly-Sar and 20 µM Gly-Sar (Sigma-Aldrich, St. Louis, MO)] was introduced in the loop at time 0 (t0). Blood samples were withdrawn from the carotid artery at t = 5, 10, 15, 30, 60, 120, 180, 240, 300 min, and animals were injected with the equivalent volume of a gelatin plasma expander (modified fluid gelatin, Plasmion; Fresenius Kabi France, Sevres, France). Samples were immediately centrifuged at 10,000 rpm for 5 min at 4°C, and radioactivity was measured on the plasma using a beta counter (LS 6000 TA liquid scintillation counter; Beckman Coulter, Fullerton, CA). Transport of Gly-Sar was followed by the measurement of Gly-Sar concentration in the peripheral blood. The area under the curve from 0 to 300 min (AUC0–30) values were then calculated via the linear trapezoidal rule. Gly-Sar uptake was evaluated at 300 min: the mucosa of the jejunum loop was scraped, homogenized in lysis buffer, and the sample was centrifuged. Radioactivity was measured on the supernatant. The results were normalized to protein concentration in the supernatant.
Transport of [3H]Gly-Sar ex Vivo in Mice. Transport of GlySar was monitored in mice using the ex vivo jejunal loop method. In brief, a 6-cm segment of jejunum was rinsed with a Krebs' modified buffer at pH 6 (5.4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 0.3 mM NaHPO4, 137 mM NaCl, 0.3 mM KH2PO4, and 10 mM MES). Segments were then filled with 100 µl/cm Krebs' modified buffer at pH 6 containing [3H]Gly-Sar [1 µM [3H]Gly-Sar (Isobio) and 20 µM Gly-Sar (Sigma-Aldrich)] and 500 mg/l phenol red as a test of paracellular permeability. An intestinal segment was ligated at both ends, and it was put in a 37°C thermostated bath of Krebs' modified buffer at pH 7.4 (5.4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 0.3 mM NaHPO4, 137 mM NaCl, 0.3 mM KH2PO4, and 10 mM HEPES). In another set of experiments, PepT1 Gly-Sar transport specificity was tested by adding known dipeptide competitors to the Krebs' modified buffer at pH 6 containing Gly-Sar [final concentration of competitors: 170 mM containing 94% glycyl-glycine (Gly-Gly) and 6% glycylproline (Gly-Pro); both Sigma-Aldrich]. In transport or competition studies, samples were withdrawn from the bath at t = 5, 10, 15, 20, 25, and 30 min, and radioactivity was measured using a beta counter (LS 6000 TA liquid scintillation counter; Beckman Coulter). Apparent percentage of transport of Gly-Sar was estimated by the following equation: Papp = (1/Q0)(dQ/dt), where Q0 is the total amount of radiolabeled Gly-Sar introduced in the loop and dQ/dt is the flux across the intestinal loop.
Protein Extraction in Animals. All procedures were done on ice or at 4°C to minimize proteolysis. After euthanasia, the upper small intestine of rats or mice was removed and flushed with ice-cold saline. Mucosa of rats containing enterocytes was then scraped with a glass microscope slide. Mice intestine and kidney were removed in the same experiment.
For total protein extraction, mucosa of rats or kidneys were homogenized in TENTS lysis buffer [10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 126 mM NaCl, 1% (v/v) Triton X-100, 0.1% (v/v) SDS, and 1 mM phenylmethanesulfonyl fluoride (PMSF), with 5 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml leupeptin], and tissue was incubated for 15 min. The homogenates were then centrifuged at 12,000g for 20 min. Total proteins were contained in the supernatant. Protein concentration was quantified using the bicinchoninic acid kit protein determination (Sigma-Aldrich).
For membrane protein extraction, mouse intestines were homogenized in lysis buffer (250 mM sucrose, 50 mM Tris-HCl, pH 7.4, and 1 mM PMSF, with 5 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml leupeptin), and tissue was centrifuged at 3,000g for 10 min. Supernatants were removed and centrifuged at 15,000g for 30 min. Subsequently, the supernatant was discarded, and the pellet was resuspended in final mannitol buffer (50 mM mannitol, 50 mM Tris-HCl, pH 7.4, and 1 mM PMSF, with 5 µg/ml pepstatin, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). The membrane proteins were contained in the final mannitol buffer, and they were quantified as described for total protein.
Western Blot Analysis. Proteins (20 µg) were separated by electrophoresis on 8% SDS-polyacrylamide gel electrophoresis gels. Proteins were then transferred to nitrocellulose membranes, and they were subjected to immunoblot analysis. The blots were blocked for 1 h with 10% nonfat dry milk in 1x Tween 20 Tris-buffered saline [20 mM Tris, 200 mM NaCl, and 0.1 and 20% (v/v) Tween 20, pH 7.5]. After washing in 1x Tween 20 Tris-buffered saline, membranes were incubated overnight at 4°C with either a 1:1000 dilution of rabbit anti-rat/mouse-PepT1 antibody serum (gift from Dr. L. Barbot and Pr. N. Kapel) or a 1:5000 dilution of mouse anti--actin antibody serum (clone AC74; Sigma-Aldrich). The membranes were washed and incubated for 1 h at room temperature with anti-rabbit (for PepT1) or anti-mouse (for actin) peroxidase-conjugated antibodies (DakoCytomation Denmark A/S, Glostrup, Denmark) diluted to 1:10,000. The membranes were washed and then probed using the enhanced chemiluminescence system (Perkin Elmer Life and Analytical Sciences, Waltham, MA). The membranes were exposed to Kodak film (Sigma-Aldrich). The intensity of the bands was quantified using Scion Image (Scion Corporation, Frederick, MD). The level of PepT1 protein for each lane was normalized to the abundance of -actin protein.
Real-Time PCR Analysis. Small intestine samples from 18-h-fasted rats and mice were collected in the same way as for protein experiments. Total RNA was isolated using the guanidine thiocyanate method with RNAble (Eurobio, Les Ulis, France) according to the manufacturer's instructions, and its concentration and purity were verified by an optical density260/optical density280 absorption ratio greater than 1.7. Integrity of RNA was verified by visualization of the 18S and 28S rRNA bands after agarose (1%) electrophoresis and ethidium bromide staining.
First strand cDNA was synthesized by reverse transcription using 5 µg of total RNA and the SuperScript II reverse transcriptase (Invitrogen, Cergy Pontoise, France). Reaction was incubated for 45 min at 37°C.
For PCR amplification of cDNA, 5 µl of the 1:20 diluted reverse transcription products were added to 5 µl of the solution containing 0.3 µM forward and reverse primers each, 3 mM MgCl2, and 1x PCR buffer (Roche Diagnostics) (containing FastStart TaqDNA polymerase, dNTP, and SYBR Green I dye). Two or three housekeeping genes (-actin, GAPDH, and ubiquitin C) were used according to the GeNorm strategy (Vandesompele et al., 2002). Primers were designed with the Primer3 software (Rozen and Skaletsky, 2000) (Table 1).
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Amplification occurred in two-step procedures: denaturation at 95°C for 8 min and 45 cycles, which consisted of denaturation (5 s at 95°C), annealing (5 s at designated temperatures), and extension (72°C for designated times) (Table 1). PCR products were separated by electrophoresis through a 2% agarose gel, followed by ethidium bromide staining to ensure the amplification of an appropriate size product.
Cell Culture
Caco-2 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 20% fetal bovine serum (Invitrogen), 1% nonessential amino acids, and 1% penicillin/streptomycin in 5% CO2, 95% humidity at 37°C. Cells were seeded on Costar Transwell membrane inserts with 0.4-µm pores (Corning Glassworks, Corning, NY) at a density of 5 x 104 cells/cm2. The medium was changed on the 3rd day after seeding and every day thereafter. Confluence was reached after 10 days of culture, and cell monolayer were subsequently treated with 0.2 nM leptin, which corresponds to normoleptinemia in human (R&D Systems) or without leptin (control) for 7 days. Leptin was used on apical (mimicking gastric leptin) or basolateral (mimicking systemic leptin) side. Leptin concentration was measured in the medium by RIA using human leptin RIA kit following the manufacturer's instructions (Linco Research Inc.).
Transport of Cephalexin in Caco-2 Cells. On the day of the experiment, the medium culture was removed, and apical and basolateral compartments were washed three times with a Krebs' modified buffer at pH 6 at 37°C or with a Krebs' modified buffer at pH 7.4 at 37°C, respectively, and transepithelial electrical resistance was measured in each well using an EVOM epithelial tissue voltohmmeter (WPI, Saratosa, FL). No monolayer was used if transepithelial electrical resistance values were below 150 
/cm2. Cells were then incubated for 15 min with soft circular shaking at 37°C. After incubation, the apical buffer was removed, and it was replaced with a Krebs' modified buffer at pH 6 containing 1 mM cephalexin (Sigma-Aldrich), and 50-µl aliquots were taken on the basolateral compartment at t = 5, 10, 15, 20, 25, and 30 min under soft circular shaking at 37°C. Cephalexin concentration was determined by high-performance liquid chromatography (HPLC), and the apparent permeability coefficients were estimated by the following equation: Papp = (1/A)(dQ/dt), where A is the surface area of the Transwell membrane and dQ/dt is the flux across the membrane insert.
HPLC Determination of Cephalexin Concentration. The HPLC apparatus was composed of a 717plus autosampler (Waters, Guyancourt, France), a CR-5A detector (Shimadzu, Champs-sur-Marne, France), and a LC-6A integrator (Shimadzu). Cephalexine concentration was determined on a C18 Supelcosil 250 x 4 mm, 4-µm particle column (Sigma-Aldrich) at 
= 260 nm. The mobile phase was composed of 0.01 M sodium acetate buffer, pH 5.2 (VWR), and acetonitrile (VWR) (91.5:8.5, v/v). The flow rate was 1 ml/min, and the volume injected was 20 µl. The detection limit was 0.2 mg/l.
Cell Protein Extraction and Western Blot Analysis on Caco-2 Cells. At the end of the treatment period, culture medium was removed, and the monolayers were washed three times with ice-cold PBS. Cells were harvested in 1 ml of ice-cold PBS. Cell suspension was centrifuged 5 min at 3000g, and cells were contained in the pellet. Cell total protein extraction was realized on the pellets as exposed for animals. All of the procedures were identical to those exposed for Western blot analysis in animals apart from the first antibody, which was a 1:1000 dilution of a rabbit anti-human-PepT1 antibody (gift from Dr. D. Merlin, Emory University, Atlanta, GA).
Statistical Analysis
All values were expressed as mean ± S.E.M. For each variable, the Mann-Whitney test was performed to compare two means, and a Kruskal-Wallis test was performed to compare more than two means. For the analysis of body weight, food intake over time, and transport kinetic, statistical comparisons between the control and treated groups were performed by two-way analysis of variance (time x treatment) to test for overall significance with treatment and time as variables, and Bonferroni post test for individual significant differences. A Spearman correlation test was performed to test the correlation between leptinemia and PepT1 expression and activity. Statistical analyses were conducted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). The level of significance was set at P < 0.05 for all analyses.
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Results |
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Induction of acute hyperleptinemia by bolus injection of leptin (0.5 mg/kg) produced a rapid and 12-fold rise in plasma leptin levels (32.00 ± 7.2 in leptin-treated rats versus 2.62 ± 0.75 ng/ml in control rats) (P < 0.001 versus control), and it produced no significant change in plasma insulin levels (data not shown). This acute hyperleptinemia resulted in a transient increase in Gly-Sar transport. As shown in Fig. 3, this increase was rapid, significant as soon as 5 min, and reached a 25% increase at 10 min (P < 0.05 versus control). The calculated AUC0–30 was significantly increased by 39% (P < 0.05 versus control), but it returned to baseline at 60 min.
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), immunoblotting of extracts from different portions of the small intestine revealed one PepT1 immunoreactive band in the small intestine with higher expression found in the jejunum. No PepT1 immunoreactive band could be detected in the large intestine (Fig. 4A). Next, we focused our analysis on PepT1 expression in the jejunum.
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Leptin-Deficiency Is Associated with Reduced PepT1 Activity and Expression in the Jejunum. To determine the physiological significance of data compiled from rats, PepT1 function was studied in ob/ob mice.
Under the conditions of active leptin replacement, hyperphagia was reduced in ob/ob mice with daily food intake being significantly reduced by 34% (P < 0.05 versus untreated ob/ob mice) (Table 3). This effect was associated with a significant 10% weight loss (P < 0.05 versus untreated ob/ob mice), as early as day 2. This weight loss then stabilized, and it was maintained throughout the period of the experiment (Fig. 5). In addition, hyperinsulinemia occurring in ob/ob mice was significantly reduced by 80% in leptintreated ob/ob mice, but plasma insulin remained at higher levels than that in control mice. Furthermore, circulating glucose levels that were 2.2-fold higher in untreated ob/ob mice returned to values similar to those found in control mice upon challenge with leptin (Table 3).
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Gly-Sar apparent diffusion coefficient across the jejunum was 0.26 ± 0.04% in wild-type mice. As shown in Fig. 6, this apparent diffusion was 80% blocked by an excess of Gly-Pro or Gly-Gly, two substrates for PepT1, indicating that Gly-Sar transport is PepT1-specific. Chronic deficiency in leptin was associated with a significant 50% (P < 0.05) decrease in Gly-Sar transport in the jejunum of ob/ob mice in comparison with their lean littermates (Fig. 6). It is noteworthy that residual transport of Gly-Sar measured after the addition of Gly-Pro and Gly-Gly was identical in ob/ob and wild-type mice, and no significant modification was observed in paracellular permeability among the studied groups (data not shown), suggesting that leptin only affects the PepT1-mediated transport of Gly-Sar. This reduced PepT1 activity was accompanied by a 40% reduction (P < 0.05) in the abundance of PepT1 membrane protein and by a significant 2-fold decrease in PepT1 mRNA levels in ob/ob mice compared with their lean control littermates (Fig. 7, A and B).
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Apical or Basolateral Leptin Directly Activates PepT1 Function in Caco-2 Cells in Vitro. To determine whether the in vivo effects of leptin could be direct, the human intestinal enterocyte-like Caco-2 cells were used.
The determination of leptin immunoreactivity in the culture medium after apical or basolateral addition of leptin to Caco-2 monolayer indicated that leptin concentration in the contralateral compartment was no different from that in the control nontreated wells. (Table 4). This suggested that confluent Caco-2 monolayer prevented the flux of leptin from the apical to the basolateral compartment and vice versa.
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As shown in Fig. 9A, challenge of the Caco-2 cells with physiological concentration of leptin (0.2 nM) for 7 days induced a significant increase in cephalexin flux across Caco-2 monolayer. Indeed, we observed a 2.7- and 1.8-fold augmentation when treatment was applied on the apical and basolateral side, respectively. The enhanced PepT1 activity was accompanied by up-regulation of PepT1 protein expression (2.1- and 2.5-fold, respectively; P < 0.05 versus control) (Fig. 9B). These data suggest that leptin enhances the PepT1 function through direct activation of the apical or basolateral sides of the Caco-2 monolayer.
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Discussion |
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Consistent with previous reports (Pelleymounter et al., 1995), chronic peripheral leptin has a potent weight loss property. Indeed, despite a similar amount of food intake, leptin-treated rats exhibited a smaller weight gain than pairfed controls. Furthermore, the hyperphagia characteristic of ob/ob mice was reversed by leptin replacement, and the weight gain of leptin-treated ob/ob mice was reduced compared with ob/ob nontreated mice.
One of the most important findings in this report is that hyperleptinemia induced by leptin infusion in nonobese animals results in a 6-fold increase in the levels of PepT1 mRNA in the jejunum, associated with an increase in the amount of PepT1 protein. These effects may explain the enhancement of PepT1 transport activity from the jejunal mucosa. Moreover, leptin-deficient mice displayed a dramatic reduction in PepT1 expression and activity, whereas leptin replacement reverses this PepT1 phenotype together with metabolic abnormalities seen in leptin-deficient mice.
The hormonal regulation of PepT1 transcription has been the subject of few studies. Within this context, it was reported previously that intestinal absorption of drugs mediated by PepT1 was up-regulated in fa/fa rats exhibiting hyperinsulinemia and hyperleptinemia (Watanabe et al., 2003). Because leptin signal transduction is reduced in these rats (Yamashita et al., 1997), it is reasonable to think that hyperleptinemia alone cannot account for such a regulation of the PepT1 transporter in this model of Zucker rats, making insulin as a putative mediator. However, in our study, the fact that plasma insulin levels did not change upon leptin challenge in rats indicates that increased PepT1 expression is unlikely to involve insulin. Moreover, although ob/ob mice display high plasma insulin levels, we showed notable reduction in PepT1 expression and activity compared with their wild-type littermates. However, this does not exclude the possibility that desensitization of insulin receptors could explain the lack of insulin action on PepT1 in this model (Um et al., 2004).
Alternatively, reports show that growth hormone and sympathetic nervous tone can up-regulate PepT1 expression and activity (Berlioz et al., 2000; Alteheld et al., 2005) and that these factors can be modulated by leptin (Cocchi et al., 1999; Watanobe and Habu, 2002). Thus, one can speculate that changes in growth hormone levels and in sympathetic nervous tone could be indirect regulators of PepT1 induced by leptin in our animals; however, this has yet to be demonstrated.
Nduati et al. (2007) reported that leptin can directly regulate PepT1 expression on enterocyte in an in vitro model. However, leptin concentrations used in this study were supraphysiological, and only a short-term effect was investigated. In our current study, we demonstrated that chronic physiological levels of leptin (0.2 nM corresponding to 3.2 ng/ml for 7 days) are capable of directly enhancing PepT1 expression, regardless of the side of the treatment (apical versus basolateral) in Caco-2 model of enterocytes. This direct effect of leptin is likely to be mediated through leptin receptors Ob-R.
Indeed, we and others have reported previously that intestinal epithelial cells expressed leptin receptors (Morton et al., 1998; Buyse et al., 2001; Barrenetxe et al., 2002). These leptin receptors were reported to signal through different signaling pathways notably involving Janus tyrosine kinase-signal transducer and activator of transcription (JAK-STAT) and mitogen-activated protein kinase (MAPK). These transcription factors then translocate into the nucleus and activate the transcription of a number of target genes (Morton et al., 1998), among which the PepT1 gene could be a candidate. The promoter regions of the rat, mouse and human PepT1 gene (Shiraga et al., 1999; Fei et al., 2000; Shimakura et al., 2005) have been characterized, and they contain a number of potential binding sites for regulatory factors of gene transcription that can be targeted by leptin signaling pathways. Likewise, Nduati et al. (2007) showed that cAMP response element-binding protein (CREB) and caudal-related homeobox 2 (Cdx2) are involved in PepT1 regulation by leptin in vitro. Future studies are needed to unravel the molecular mechanisms of activation of intestinal peptide transporter PepT1 expression by leptin in vivo.
Unexpectedly, we found that induction of hyperleptinemia is associated with high leptin levels in the jejunal juice. Such a finding is consistent with our previous report showing that leptin (free and bound to macromolecules) could be detected in duodenal fluid (Guilmeau et al., 2003). Thus, we cannot exclude that in vivo leptin operating from the apical side could also contribute to the modulation of PepT1 expression shown. This intriguing finding raises the question of transfer of leptin from blood to intestinal lumen. Further investigations are required to determine direct implication of systemic versus luminal leptin in the long-term regulation of PepT1 upon sustained hyperleptinemia.
Chronic hyperleptinemia did not induce any change in PepT1 expression in kidneys, despite the presence of leptin receptors in this organ (Serradeil-Le Gal et al., 1997), indicating a tissue-specific regulation of the transporter by leptin and that renal reabsorption of excreted drugs via PepT1 is not likely to be modulated by leptin.
In this study, we also showed that acute hyperleptinemia achieved by a bolus of leptin precociously and transiently increased PepT1 activity, presumably because of the very short half-life of leptin, which is 9 min in the rat (Zeng et al., 1997). Likewise, we reported previously a rapid and short-term effect of apical leptin in Caco-2 cells involving enhanced translocation of the transporter (Buyse et al., 2001). However, we were unable to show in that study any increase of dipeptide uptake by leptin acting from the basolateral side of Caco-2 cells. This discrepancy might be explained by different experimental conditions (leptin concentrations and in vivo versus in vitro experiments), together with asymmetric distribution of leptin receptors with much lower density of receptors at basolateral than apical side of the enterocyte (Barrenetxe et al., 2002). This early effect was not seen in the chronic hyperleptinemic rats where leptin-induced PepT1 activity up-regulation was observed after a delayed time.
Taken together, we conclude that leptin exerts a dual effect on PepT1 function: 1) a short-term effect consisting in increasing translocation of PepT1 molecules from a preformed cytoplasmic pool to apical membrane consistent with our previous findings; and 2) a long-term effect consisting of activation of the transcription of PepT1 gene and/or enhanced of PepT1 mRNA stability to reconstitute cytoplasmic pool of PepT1 transporter. This dual effect of leptin is similar to that reported for insulin regulation of PepT1 with a cytoplasmic pool translocation after a short-term challenge that is followed by increased mRNA expression after a longer time exposure (Gangopadhyay et al., 2002). Moreover, Benomar et al. (2006) recently showed the same pattern of regulation of another intestinal transporter, glucose transporter-4 (Glut4), where both insulin and leptin could increase translocation of the preformed pool of glucose transporter glucose transporter-4 in vitro after short term treatment (15 min), whereas a longer treatment length (16 h) had transcriptional implications. Furthermore, it is worth mentioning that leptin has been shown to be a key regulator of the transport of the satiety peptide urocortin into the brain barrier (Kastin et al., 2000).
In summary, we showed, for the first time, in vivo that leptin is the key regulator of PepT1 expression and activity. The molecular mechanisms involved remain to be elucidated. Hyperleptinemia is associated with enhanced PepT1 function, and it may have physiological relevance in peptide absorption and drug bioavailability.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PepT1, H+-coupled peptide cotransporter 1; PBS, phosphate-buffered saline; RIA, radioimmunoassay; Gly-Sar, glycylsarcosine; AUC, area under the curve; MES, 2-(N-morpholino)ethanesulfonic acid; PMSF, phenylmethanesulfonyl fluoride; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPLC, high-performance liquid chromatography.
Address correspondence to: Dr. Patrick Hindlet, Department of Clinical Pharmacy (Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 2706), Faculty of Pharmaceutical Sciences Paris XI, 5, rue Jean Baptiste Clément, 92296 Châtenay-Malabry, France. E-mail: patrick.hindlet{at}u-psud.fr
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