Effect of insulin on cephalexin uptake and transepithelial transport in the human intestinal cell line Caco-2

European Journal of Pharmaceutical Sciences 21 (2004) 87–95

Effect of insulin on cephalexin uptake and transepithelial transport in the human intestinal cell line Caco-2 Kazuhiro Watanabe∗ , Kazuaya Terada, Toshiya Jinriki, Juichi Sato Hokkaido College of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido 047-0264, Japan Received 25 March 2003; received in revised form 21 July 2003; accepted 6 October 2003

Abstract We investigated whether cephalexin transport in Caco-2 cells is regulated by insulin. After the insulin pretreatment, cephalexin uptake, and transport as well as PEPT1 mRNA and protein expression in the cells were measured. Cephalexin uptake was significantly increased by the insulin pretreatment. Insulin significantly increased cephalexin saturable uptake, but had no significant effect on the non-saturable one. PEPT1 protein expression on the apical membrane, but not PEPT1 mRNA expression, was increased by the insulin pretreatment. The enhancement of cephalexin uptake by the insulin pretreatment was inhibited by genistein, a tyrosine kinase inhibitor, and colchicine, an agent that disrupts protein translocation. Apical-to-basolateral transport of cephalexin has increased by the insulin pretreatment at the apical side and long-term insulin pretreatment at the basolateral side. It is considered that insulin mainly binds to its receptor on the apical and basolateral membranes, thereby promoting PEPT1 translocation from the intracellular pool to the apical membrane surface; consequently, PEPT1 protein expression on the apical membrane is increased. © 2003 Elsevier B.V. All rights reserved. Keywords: PEPT1; Cephalexin; Insulin; Caco-2 cells; Uptake; Transport

1. Introduction Intestinal drug transporters play an important role in the absorption of orally administered drugs. PEPT1, an H+ /oligopeptide transporter, is localized not only in the apical membrane but also in the lysosomal membrane (Ogihara et al., 1996; Lee, 2000). PEPT1, which has wide substrate specificity, mediates the cellular transport of small peptides (Leibach and Ganapathy, 1996) and such drugs as ␤-lactam antibiotics (Terada et al., 1998) and angiotensin-converting enzyme inhibitors (Boll et al., 1994). Terada et al. (1999) found that another peptide transporter was expressed in the basolateral membrane. The basolateral peptide transporter and PEPT1 cooperate in the efficient transepithelial transport of small peptides and peptide-like drugs from the cells to the circulating blood. Recently, there is some available information on the regulation of PEPT1 activity and PEPT1 mRNA expression; for instance, triiodothyronine (Ashida et al., 2002) and epidermal growth factor (EGF, Nielsen et al., 2001) inhibited dipeptide transport activity and PEPT1 mRNA expression, ∗

Corresponding author. Tel.: +/fax: +81-134-62-1849. E-mail address: [email protected] (K. Watanabe).

0928-0987/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2003.10.002

on the other hand, leptin (Buyse et al., 2001) and ␴-receptor ligand, pentazocine (Fujita et al., 1999), enhanced dipeptide transport activity and PEPT1 protein expression. However, little is known about the relationship between PEPT1 activity and disorders. In our previous study (Watanabe et al., 2003), we investigated the intestinal absorption and pharmacokinetics of cephalexin, as well as the PEPT1 mRNA and protein expression in diabetic rats. Cephalexin disappearances from the duodenum loop and plasma cephalexin concentrations after oral administration were higher in hyperinsulinemic type 2 diabetic GK and Zucker fatty rats than in streptozotocin-induced (STZ) type 1 diabetic rats and control rats. Moreover, the intestinal brush-border membrane vesicle PEPT1 protein expression levels were higher in GK and Zucker fatty rats than in STZ type 1 diabetic and control rats. Thamotharan et al. (1999) reported that insulin stimulated glycylglutamine uptake in Caco-2 cells by increasing the membrane population of PEPT1 while not changing PEPT1 gene expression. The human intestinal cell line Caco-2 constitutively expresses such drug transporters as peptide transporter, P-glycoprotein, organic cation transporter and organic anion transporter (Tsuji et al., 1994). Therefore, this cell line has been widely used as an excellent model system for

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investigating intestinal drug absorption. In addition, Caco-2 cells show several functional properties of the small intestine, such as receptor function and various enzyme activities. Insulin receptors are also present on the apical and basolateral membranes of Caco-2 cells (MacDonald et al., 1993) and intestinal mucosal cells (Gallo-Payet and Hugon, 1984). In the present study, we investigated whether cephalexin transport and PEPT1 expression in Caco-2 cells are regulated by insulin, as reported previously (Thamotharan et al., 1999). As a result, cephalexin uptake and PEPT1 protein expression on the apical membrane of Caco-2 cells were increased by insulin pretreatment; however, PEPT1 mRNA expression was not changed by the pretreatment. We also investigated the effects of single and long-term pretreatment with insulin at the apical and/or basolateral sides on the apical-to-basolateral transport and the basolateral-to-apical transport of cephalexin in Caco-2 cells, with consideration of the circulating insulin. 2. Materials and methods 2.1. Materials and high-performance liquid chromatography (HPLC) N-2-Hydroxyethylpiperazine-N -2-ethanesulfonic acid (HEPES), 2-(N-morpholino) ethanesulfonic acid (MES) and colchicine were purchased from ICN Biomedicals Inc. (Costa Mesa, CA, USA). Dulbecco’s modified Eagle medium (DMEM), non-essential amino acid, fetal bovine serum and cephradine were purchased from Sigma (St. Louis, MO, USA). Cephalexin, genistein, brefeldin, and insulin (human recombinant) were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Precision protein standard (prestained; broad range) and 100 bp DNA ladder were purchased from Bio-Rad (Richmond, CA, USA) and New England Biolabs (Beverly, MA, USA), respectively. All other chemicals used in the experiments were of the highest purity commercially available. HPLC (Watanabe et al., 2003) was carried out using a model LC-10AS pump (Shimadzu, Kyoto, Japan) equipped with an SPD-10A detector (262 nm, Shimadzu, Kyoto, Japan). A reversed-phase column packed with LiChrospher 100 RP-18(e) (LiChroCART 250-4, 5 ␮m, 250 mm × 4 mm i.d., Kanto Chemical, Tokyo, Japan) was used at 50 ◦ C. The mobile phase used a mixture of 30 mM phosphate buffer (pH 7.0) and methanol (72/28 (v/v)), and the flow rate was 1.0 ml/min. Calibration curves were obtained by injecting cephalexin (2–4000 ng) containing a quantitative amount (200 ng) of cephradine as an internal standard into the HPLC system. 2.2. Cell culture and insulin treatment Caco-2 cells at passage 41 were obtained from RIKEN GeneBank (Tsukuba, Japan) and cultured as described previously (Watanabe et al., 2002a,b). The cells from passages

45–55 were used. For the uptake study, Caco-2 cells were seeded at a density of 8 × 105 cells per dish on 60 mm plastic culture dishes coated with rattail collagen type I (Becton Dickinson, Franklin Lakes, NJ, USA). Caco-2 cell monolayers on the fifth day of culture were used for the experiments. For the transport study, Caco-2 cells were seeded at a density of 3.5 × 105 cells per permeable membrane (cell culture insert, 0.4 ␮m, 4.3 cm2 growth area, Becton Dickinson) in the culture medium. Each permeable membrane was replaced into in a Multiwell plate (6-well, Becton Dickinson) with 2.2 ml of the basolateral side medium and 1.5 ml of the apical side medium. The medium was replaced every 3–4 days after inoculation. Caco-2 cell monolayers cultured for 14–16 days were used in the experiments. The quality of the monolayers grown on the permeable membrane was assessed by measuring the transepithelial electrical resistances using Millicell-ERS (Millipore, Bedford, MA, USA). The resistances were higher than 390  cm2 . The culture medium was removed, and the Caco-2 cell monolayers were washed twice with preincubation medium consisting of HEPES buffer (145 mM NaCl, 3 mM KCl, 1 mM CaCl2 , 0.5 mM MgCl2 , 5 mM d-glucose, 5 mM HEPES (pH 7.4)). After the wash, the monolayers were preincubated with 3 ml of the preincubation medium for 10 min at 37 ◦ C. The medium was removed after the preincubation, and the monolayers were incubated with 3 ml of fresh preincubation medium containing insulin (5 nM) for the indicated time at 37 ◦ C. Insulin was dissolved in HEPES buffer. Furthermore, the effects of treatment with genistein, brefeldin, and colchicine were examined on insulin-stimulated cephalexin uptake in Caco-2 cell monolayers. In the long-term insulin treatment experiment, Caco-2 cell monolayers were subcultured with insulin (0, 5 or 50 nM) in the culture medium for 15 days, post confluence (14–16 days). The subculture medium containing insulin (0, 5 or 50 nM) was exchanged three times (09:00, 14:00, and 19:00) per day. After the subculture, the monolayers were washed and treated with insulin as described above. 2.3. Uptake study The preincubation medium was removed, and the monolayers were washed twice with incubation medium consisting of MES buffer (145 mM NaCl, 3 mM KCl, 1 mM CaCl2 , 0.5 mM MgCl2 , 5 mM d-glucose, 5 mM MES (pH 6.0)). After the wash, the monolayers were preincubated with 3 ml of the incubation medium for 10 min at 37 ◦ C. The incubation medium was removed after the preincubation, and the monolayers were incubated with 3 ml of fresh incubation medium containing cephalexin for the indicated time at 37 ◦ C. Cephalexin was dissolved in MES buffer, and other compounds were dissolved in DMSO or H2 O. The final concentration of DMSO in the incubation medium was 1%. The uptake was terminated by aspiration of the medium, and the monolayers were washed twice with ice-cold incubation medium. To measure the amount of cephalexin, an internal

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standard (cephradine, 2.0 ␮g/10 ␮l) was added to the monolayers that were scraped using a Cell Scraper (Becton Dickinson) with 0.5 ml of 30 mM phosphate buffer (pH 7.0), and the mixture was homogenized at 23,000 rpm for 30 s using an Omni type ␮H homogenizer (St. Louis, MO, USA). To an aliquot (0.3 ml) of the homogenate was added 0.3 ml of methanol, and then the mixture was filtered through a Millipore filter (Millex-LG, Millipore). The filtrates were subjected to HPLC. The protein concentrations of cells solubilized in 30 mM phosphate buffer (pH 7.0) were measured by using a Bio-Rad Protein Assay Kit with bovine serum albumin (BSA) as reference. 2.4. Transport study The culture medium of both sides was removed by aspiration, and the Caco-2 cell monolayers were washed twice with incubation medium consisting of HEPES buffer (basolateral side) or MES buffer (apical side). After washing, the monolayers were preincubated for 10 min at 37 ◦ C with 1.5 ml (MES buffer) and 2.2 ml (HEPES buffer) of the incubation medium in the apical and basolateral sides, respectively. After the preincubation, the medium was removed immediately and the incubation medium containing cephalexin (1.0 mM) was added to either the apical side (1.5 ml) or the basolateral side (2.2 ml), and the incubation medium (without cephalexin) was added to the opposite side (basolateral, 2.2 ml; apical, 1.5 ml). The monolayers were incubated for the indicated time at 37 ◦ C. Cephalexin was dissolved in MES or HEPES buffer, and other compounds were dissolved in DMSO or H2 O. The final concentration of DMSO in the incubation medium was 1%. After the incubation, the medium in the opposite side was transferred to a sample tube and filtered through a Millipore filter (Millex-LG), and the concentration of cephalexin in the filtrate was determined by the HPLC method as described above. For the determination of the intracellular accumulation of cephalexin, the membrane filters were detached from the insert well after incubation, and then the cells on the membrane filters were extracted with 0.5 ml of 30 mM phosphate buffer (pH 7.0)/methanol (50/50 (v/v)) for 1 h. The extract was centrifuged at 6000 rpm for 20 min, and the supernatant filtered through a Millipore filter. The filtrate was subjected to HPLC. 2.5. Kinetics of cephalexin uptake To estimate the kinetic parameters of the saturable uptake by Caco-2 cell monolayers, the uptake rate was fitted to the following equation that consists of both saturable and non-saturable linear terms, using a non-linear regression analysis program MULTI (Yamaoka et al., 1981): Jmax × [S] J= + Kd × [S] (1) Km + [S] where J is the rate of cephalexin uptake, Jmax the maximal carrier-mediated uptake rate, Km the Michaelis

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constant, Kd the rate constant for non-saturable uptake and [S] is cephalexin concentration. 2.6. Preparation of membrane protein Apical membrane vesicles (AMV) from Caco-2 cells were prepared according to the method of Minami et al. (1992). Protein concentration in the membrane suspension was measured by using a Bio-Rad Protein Assay Kit with BSA as reference. Membrane purity was determined by assaying for the activities of alkaline phosphatase as the apical membrane marker enzyme (Forstner et al., 1968) and Na+ /K+ -adenosine triphosphatase as the basolateral membrane marker enzyme (Del Catillo and Robinson, 1982). 2.7. Western blot analysis The polyclonal antibody was prepared by Nitsuka Techno Service Co. (Hitachi-shi, Ibaraki, Japan). Briefly, for the preparation of the antibody, a synthetic multiple antigen peptide (MAP) corresponding to the 15 carboxy-terminal amino acids (SNPYFMSGANS QKQM) of human PEPT1 was used as the epitope. Stepwise buildup of the peptide was done by the Fmoc chemistry solid-phase method. The crude MAP peptide was purified by passing through a Sephadex G-50 column with 0.1% acetic acid. Rabbit was immunized with 0.2 mg of MAP per immunization. The MAP was administered with Freund’s complete adjuvant in the first injection and with Freund’s incomplete adjuvant in booster injections. Polyclonal antibody response was examined by ELISA. AMV proteins (100 ␮g) were suspended in SDS buffer (4% SDS, 0.125 M Tris–HCl (pH 6.8), 20% glycerol, 0.125% dithiothreitol). Samples were subjected to 4–20% SDS-PAGE in a Laemmli system (Laemmli, 1970). Resolved proteins were transferred to PVDF membranes (Bio-Rad) and blocked with 1% gelatin–2% BSA in PBS buffer (pH 7.4) for 18 h. After washing with PBS buffer, the membranes were incubated with polyclonal antibody (1:1000) raised against PEPT1 protein at 25 ◦ C for 1 h. After the incubation with polyclonal antibody, the PVDF membranes were washed with PBS buffer and incubated with the second antibody (alkali phosphatase (AP) conjugated goat anti-rabbit IgG, 1:3000, Bio-Rad). After the incubation with the second antibody, the PVDF membranes were washed with PBS buffer and incubated with the AP conjugated substrate kit (Bio-Rad). Band intensity was quantified using Luminous Imager Ver. 2.0 (Aisin Cosmos R&D Co., Aichi, Japan). 2.8. RT-PCR Total RNA was isolated from Caco-2 cells using an RNeasy mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. For RT-PCR, the upstream primer, 5 -GCAGTCACCTCAGTAAGCT-3 ,

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corresponded to nucleotide positions 342–360, and the downstream primer, 5 -CAAACAAGGCCCAGAACATT-3 , corresponded to nucleotide positions 929–948 of human PEPT1 cDNA, were used. First-strand cDNA synthesis from 1.0 ␮g of total RNA and PCR reaction were performed using RT-PCR high Plus (Toyobo, Osaka, Japan). PCR reaction was performed in a GeneAmp PCR system 9600 (Applied Biosystems, Norwalk, CT, USA) and the amplification program was 50 ◦ C for 30 min and 94 ◦ C for 2 min, followed by 94 ◦ C for 1 min and 54 ◦ C for 1.5 min for 30 cycles, and 54 ◦ C for 7 min in the last cycle. The RT-PCR products were subjected to 2.0% agarose gel electrophoresis and stained with ethidium bromide. For semiquantitative purposes, the expression of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) mRNA as an internal control was measured using the primers, yielding a 450 bp fragment, appended to the RT-PCR kit reagent. Fluorescence intensity was measured by Luminous Imager Ver. 2.0, and was normalized to that of internal control G3PDH. 2.9. Statistical analysis The results are expressed as mean ± S.D. Differences between groups were evaluated using the Student’s t-test or Welch’s t-test after initial analysis by the F-test. Three or more groups were evaluated using Bonferroni/Dunn’s multiple comparison test or Scheffe’s multiple comparison test after analysis by the Bartlett test and one-way analysis of variance (ANOVA) test. P values less than 0.05 were considered significant.

Table 1 Kinetic parameters for cephalexin uptake in Caco-2 cell monolayers Parameter

Insulin (−)

Insulin (+)

Vmax (nmol/mg protein per 15 min) Km (mM) Kd (␮l/mg protein per 15 min)

14.83 ± 1.80 5.16 ± 0.43 0.89 ± 0.12

32.28 ± 3.91∗ 5.37 ± 0.33 0.96 ± 0.09

Each value represents the mean ± S.D. of three different passages. ∗ : Significantly different between the two groups (P < 0.05).

uptake by Caco-2 cells. Therefore, we pretreated Caco-2 cells with 5 nM insulin for 2 h in cephalexin uptake study. To determine the effect of insulin pretreatment on the kinetic parameters of cephalexin uptake, we investigated the effect of insulin pretreatment on concentration-dependent cephalexin uptake in Caco-2 cell monolayers. As shown in Fig. 1, cephalexin uptake was higher in insulin-treated Caco-2 cell monolayers than in control. The uptake components were analyzed according to Eq. (1) in Section 2. The dashed line represents cephalexin uptake for the saturable component calculated from the kinetic parameters, and the dotted line represents that for the non-saturable component calculated also from the kinetic parameters. The kinetic parameters of cephalexin uptake are shown in Table 1. The Jmax of cephalexin uptake with insulin pretreatment was significantly increased compared to that without insulin pretreatment. The stimulation of cephalexin uptake by insulin pretreatment was due to an increase of Jmax , and not Km and Kd . 3.2. Effect of insulin pretreatment on PEPT1 mRNA and PEPT1 protein expression

3. Results 3.1. Effect of insulin pretreatment on cephalexin uptake Thamotharan et al. (1999) demonstrated that insulin treatment (5 nM, 1–2 h) markedly stimulated glycylglutamine

The results of RT-PCR analysis of PEPT1 mRNA in Caco-2 cells with or without insulin pretreatment are shown in Fig. 2. The expression of PEPT1 mRNA was not affected by the insulin pretreatment. We performed Western blot analysis to determine PEPT1 protein expression in the

60

Insulin ( +)

4

V2

40

0 0

30

60

V/S

20 0

0

5

10

15

Cephalexin (mM)

20

25

Cephalexin (nmol/mg protein/15 min)

Cephalexin (nmol/mg protein/15 min)

Insulin ( _ ) 60

6

V3

40

0 0

50

100

V/S

20 0

0

5

10

15

20

25

Cephalexin (mM)

Fig. 1. Effects of insulin pretreatment on the kinetic parameters of cephalexin uptake in Caco-2 cell monolayers. Inset: Eadie–Hofstee plots of cephalexin uptake after correction for non-saturable component. Caco-2 cell monolayers were pretreated with or without insulin (5 nM) at 37 ◦ C for 2 h. After the pretreatment, the monolayers were incubated with cephalexin (0.1–25 mM) at 37 ◦ C for 15 min. Each value represents the mean ± S.D. of three different passages. Dashed and dotted lines represent cephalexin uptake for saturable and non-saturable components calculated from the kinetic parameters, respectively.

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Table 2 Effects of genistein, brefeldin, and colchicines on the stimulation of cephalexin uptake by insulin pretreatment in Caco-2 cell monolayers Compound

Uptake (nmol/mg protein per 15 min)

Insulin (−) Insulin (+) Genistein Genistein + insulin Brefeldin Brefeldin + insulin Colchicine Colchicine + insulin

2.53 4.28 2.32 2.56 2.70 4.86 2.58 2.46

± ± ± ± ± ± ± ±

0.08 0.15∗ 0.34 0.41# 0.45 0.65∗ 0.12 0.11#

Caco-2 cell monolayers were treated with or without genistein (80 ␮M, for 24 h), brefeldin (5 ␮M, for 20 min) and colchicine (10 ␮M, for 20 min). The monolayers were preincubated with or without insulin (5 nM) at 37 ◦ C for 2 h. After the preincubation, the monolayers were incubated with cephalexin (1 mM) at 37 ◦ C for 15 min. Each value represents the mean ±S.D. of three different passages. ∗ : Significantly different from the insulin (−) group (P < 0.05). # : Significantly different from the insulin (+) group (P < 0.05).

Fig. 2. RT-PCR analysis of PEPT1 mRNA in Caco-2 cells. Caco-2 cell monolayers were pretreated with or without insulin (5 nM) at 37 ◦ C for 2 h. After the pretreatment, total RNA was isolated from Caco-2 cell monolayers. The RT-PCR products were subjected to 2.0% agarose gel electrophoresis and stained with ethidium bromide. Each value represents the mean±S.D. of three different passages. NS: not significantly different.

AMVs in Caco-2 cells. As shown in Fig. 3, insulin-pretreated Caco-2 cells showed a significant increase in PEPT1 protein expression on the apical membrane compared with insulin non-pretreated Caco-2 cells.

3.3. Mechanism of cephalexin uptake stimulation by insulin pretreatment To clarify the mechanism of cephalexin uptake stimulation by insulin pretreatment, we investigated the effects of a tyrosine kinase inhibitor, genistein (Milovic et al., 1995), a selective destruction agent of the Golgi apparatus, brefeldin (Thamotharan et al., 1998), and a disruption agent of protein translocation, colchicine (Achler et al., 1989). As shown in Table 2, genistein, brefeldin, and colchicine alone had no effect on cephalexin uptake. However, genistein and colchicine completely inhibited the stimulatory effect of insulin pretreatment on cephalexin uptake, whereas brefeldin had no effect on the stimulatory effect of insulin pretreatment. 3.4. Effect of insulin pretreatment on cephalexin transepithelial transport

Fig. 3. Western blot analysis of PEPT1 protein on the apical membrane in Caco-2 cells. Caco-2 cells monolayers were pretreated with or without insulin (5 nM) at 37 ◦ C for 2 h. After the pretreatment, AMVs were prepared according to Section 2. AMVs proteins (100 ␮g) were subjected to Western blot analysis. For each analysis, AMVs were prepared from Caco-2 cells in 3–5 culture tubes (250 cm2 ). Each value represents the mean ± S.D. of three different passages. ∗ Significantly different between two groups (P < 0.05).

Next, we investigated the effect of insulin pretreatment on cephalexin transepithelial transport using Caco-2 cell monolayers cultured on a permeable membrane. Fig. 4A and B show the effect of insulin pretreatment on apical-to-basolateral transport and intracellular accumulation of cephalexin in Caco-2 cell monolayers. When insulin was added to the apical side, and to both apical and basolateral sides, apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin in Caco-2 cell monolayers were significantly increased compared with those of control. However, the addition of insulin to the basolateral side did not affect apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin. On the other hand, basolateral-to-apical transport and intracellular accumulation from the basolateral side of cephalexin in the monolayers were not affected by the addition of insulin to the apical and/or basolateral sides (Fig. 4C and D).

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Fig. 4. Effects of insulin pretreatment on transport (A, C) and intracellular accumulation (B, D) of cephalexin in Caco-2 cell monolayers. Caco-2 cell monolayers were pretreated with or without insulin (5 nM, apical and/or basolateral) at 37 ◦ C for 2 h. After the pretreatment, the monolayers were incubated with cephalexin (1 mM, apical or basolateral) at 37 ◦ C for 15 min. Each value represents the mean ± S.D. of three different passages. ∗ : Significantly different from the insulin (–) group (P < 0.05).

3.5. Effect of long-term insulin pretreatment on cephalexin transepithelial transport We finally investigated the effects of long-term insulin pretreatment for 15 days on cephalexin transepithelial transport in Caco-2 cell monolayers. As shown in Table 3, apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin were significantly increased compared with those of control, when insulin pretreatment (5, 50 nM) was carried out on the apical side and the basolateral side (except accumulation from apical side at 5 nM). However, long-term insulin pretreatment of the apical or basolateral side did not affect basolateral-to-apical transport and intracellular accumulation from the basolateral side.

4. Discussion Thamotharan et al. (1999) demonstrated that the addition of insulin at physiological concentrations (5 nM) to the incubation medium markedly stimulated glycylglutamine

uptake by Caco-2 cells. This stimulation was blocked when genistein, an inhibitor of tyrosine kinase, was added to the incubation medium. Also, there was no significant change in the Michaelis–Menten constant for glycylglutamine transport, but there was a nearly two-fold increase in its maximal velocity. They demonstrated that insulin might stimulate the translocation of PEPT1 to the cell surface in a microtubule-dependent manner. Finally, with insulin pretreatment, there was no change in PEPT1 mRNA expression, but the expression of PEPT1 protein in the apical membrane was increased. The mechanism appears to involve increased translocation of the transporter from a preformed cytoplasmic pool. Diabetes mellitus is a disorder related to insulin (Sacks, 1997; Withers and White, 2000). There is little useful information on the intestinal absorption of dipeptides in diabetes mellitus. Intestinal absorption of dipeptide was the same in control and STZ-1 type diabetic model rats (Schedl et al., 1978). In our previous report, we indicated that the intestinal absorption of drugs mediated by intestinal PEPT1 is affected by plasma insulin concentration (Watanabe et al., 2003). In the present study, we used cephalexin as a model

K. Watanabe et al. / European Journal of Pharmaceutical Sciences 21 (2004) 87–95 Table 3 Effects of long-term (15 days) insulin pretreatment on transepithelial transport and intracellular accumulation of cephalexin in Caco-2 cell monolayers Insulin Treatment side

Concentration (␮M)

Transport (nmol/cm2 per 60 min)

Accumulation (nmol/cm2 per 60 min)

(A) Apical-to-basolateral + Control – Apical 5 Apical 50 Basolateral 5 Basolateral 50

5.06 11.50 12.31 6.41 7.75

± ± ± ± ±

0.44 1.10∗ 1.83∗ 0.52∗ 0.45∗

1.66 2.73 2.93 1.72 2.33

± ± ± ± ±

0.27 0.28∗ 0.24∗ 0.06 0.27∗

(B) Basolateral-to-apical + Control – Apical 5 Apical 50 Basolateral 5 Basolateral 50

2.98 2.68 2.76 2.73 2.68

± ± ± ± ±

0.44 0.36 0.25 0.32 0.23

1.23 1.15 1.12 1.20 1.11

± ± ± ± ±

0.04 0.06 0.12 0.30 0.18

Caco-2 cell monolayers were pretreated with or without insulin (5, 50 nM; apical or basolateral) for 15 days. After the subculture, the monolayers were pretreated with or without insulin (apical or basolateral) at 37 ◦ C for 2 h, and were incubated with cephalexin (1 mM, apical or basolateral) at 37 ◦ C for 15 min. Each value represents the mean ± S.D. of three different passages. ∗ : Significantly different from the insulin (−) group (P < 0.05).

drug to investigate the effect of insulin pretreatment on its uptake and transport mediated by PEPT1 in Caco-2 cells. Cephalexin uptake consists of two components, the saturable and the non-saturable one. Eadie–Hofstee plots of the saturable component showed the presence of a single transport system in both control and insulin-pretreated cells. From the kinetic analysis, insulin pretreatment significantly increased Jmax , but had no significant effect on Km . From these findings, it was considered that the affinity of cephalexin for PEPT1 was not altered but the expression of PEPT1 protein on the apical membrane was increased in insulin-pretreated Caco-2 cells. To clarify the mechanism underlying the increase in PEPT1 protein expression on the apical membrane, we investigated the expression of PEPT1 mRNA and PEPT1 protein on the apical membrane in insulin-pretreated Caco-2 cells. PEPT1 protein expression on the apical membrane was increased in insulin-pretreated Caco-2 cells; however, there was no change in PEPT1 mRNA expression. When insulin binds to its receptor, tyrosine kinase in the ␤-subunit of the receptor is activated by autophosphorylation. We were interested in whether the stimulation of cephalexin uptake by insulin pretreatment requires the binding of insulin to its receptor. To investigate the mechanism underlying the stimulation of cephalexin uptake by insulin pretreatment, we studied the effect of various drugs on cephalexin uptake. The enhancement of cephalexin uptake by insulin pretreatment was inhibited by the addition of a tyrosine kinase inhibitor, genistein, and a translocation inhibitor, colchicine. From these findings, it is considered that insulin binds to its receptor on the apical membrane and biological signals are transmitted. Following the transmission, the translocation

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of PEPT1 from the intracellular PEPT1 pool to the apical membrane surface is promoted, and consequently, PEPT1 protein expression on the apical membrane is increased. To summarize, the increase in PEPT1 protein expression on the apical membrane is due to the enhancement of translocation of PEPT1 from the intracellular PEPT1 pool. The peptide transport system in the intestinal epithelial cell consists of PEPT1 on the apical membrane and the basolateral peptide transporter on the basolateral membrane (Inui et al., 1992; Terada et al., 1999). In the transport study using Caco-2 cell monolayers cultured on a permeable membrane, apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin were increased only when insulin was added to the apical side. Insulin addition to the basolateral side did not affect apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin. We attempted to clarify the results. Insulin concentration at the apical (lumen) side is low compared with that at the basolateral (blood) side. Most of the insulin receptors are concentrated on the basolateral membrane rather than on the brush-border membrane of mucosal cells (Gallo-Payet and Hugon, 1984). Furthermore, we investigated the effects of long-term insulin pretreatment on cephalexin transport in Caco-2 cells. Apical-to-basolateral transport and intracellular accumulation from the apical side of cephalexin were significantly increased by long-term insulin pretreatment at the basolateral side. However, long-term insulin pretreatment at the apical or basolateral side did not affect basolateral-to-apical transport and intracellular accumulation from the basolateral side. These results suggest that insulin does not affect the basolateral peptide transporter. PEPT1 mRNA expression was not changed by long-term insulin pretreatment (15 days, 50 nM) at the basolateral side (data not shown). It is considered that the long-term insulin pretreatment at the basolateral side in Caco-2 cell monolayers may increase PEPT1 protein expression on the apical membrane. The effect of the short-term insulin treatment from basolateral side was not observed for the apical-to-basolateral transport of cephalexin. There might be a difference between the effect of insulin from basolateral side and that from apical side though permeable membrane filters. More recently, Nielsen et al. (2003) showed that short-term treatment with EGF or insulin at the basolateral side, but not at the apical side, in Caco-2 cell monolayers grown for 26–28 days caused an increase in apical uptake of glycylsarcosine. Thamotharan et al. (1999) demonstrated that the addition of insulin at physiological concentrations (5 nM) to the incubation medium markedly stimulated glycylglutamine uptake by Caco-2 cells grown on plastic supports for 14 days. In the present paper, we showed that single treatment at apical side, and long-term treatment at both apical and basolateral sides with insulin in Caco-2 cell monolayers grown on collagen-coated plastic support and permeable membrane caused an increase in apical uptake and apical-to-basolateral transport of cephalexin. The cause of these discrepancies is

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unclear, but it might depend on the different culture conditions such as the culture period and the supports used, as Nielsen et al. (2003) proposed. Apical/basolateral ratios of insulin receptors depend on the differentiation of Caco-2 cells (MacDonald et al., 1993; Garrouste et al., 1997). Nielsen et al. (2003) suggested that not fully differentiated (less than 20 days of culture) Caco-2 cell monolayers can be stimulated by apical application of insulin, and fully differentiated Caco-2 monolayers respond to basolateral insulin stimulation. However, in the present paper, fully differentiated Caco-2 cell monolayers (29–31 days) respond to both apical and basolateral insulin stimulation. Caco-2 cells after 4 days in culture demonstrated the high affinity, low capacity receptors possessed a Kd = 2.23 × 10−10 M and R0 = 330 receptors per cell, and the low affinity, high capacity receptors possessed a Kd = 7.52 × 10−8 M and R0 = 21, 000 receptors per cell (MacDonald et al., 1993). The high affinity, low capacity receptors/low affinity, high capacity receptors ratio might change according to the various culture conditions of Caco-2 cells. From our previous paper and the present findings, the enhancement of intestinal cephalexin absorption is thought to be due to the accelerated PEPT1 protein translocation and the subsequent increase in PEPT1 protein expression on the apical membrane by insulin pretreatment. Recently, it has been reported that leptin, an ob gene product (Schmitz et al., 1997), increases the intestinal absorption of oligopeptide and cephalexin through PEPT1 (Buyse et al., 2001). The mechanism seems to be similar to that of insulin. It is considered that not only insulin but also leptin participates in intestinal cephalexin absorption.

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