Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan
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ABSTRACT |
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An oligopeptide transporter (PEPT1) in
the small intestine plays an important role in the absorption of small
peptides and peptide-like drugs. We examined the effect of thyroid
hormone 3,5,3'-L-triiodothyronine (T3) on the
activity and expression of PEPT1 in human intestinal Caco-2 cells.
Treatment of Caco-2 cells with T3 inhibited
[14C]glycylsarcosine uptake in a time- and dose-dependent
manner. [14C]glycylsarcosine uptake was reduced by
pretreatment of the cells with 100 nM T3 for 4 days (67%
of control value), whereas
methyl--D-[U-14C]glucopyranoside and
[3H]threonine uptake were not decreased. Kinetic analysis
showed that T3 treatment significantly decreased the
maximum uptake (Vmax) value for
[14C]glycylsarcosine uptake but had no effect on the
Km value. Moreover, T3 treatment
caused a significant decrease in the amount of PEPT1 mRNA (25% of the
control). Western blotting indicated that the amount of PEPT1 protein
in the apical membrane was decreased (70% of the control). These
findings indicate that T3 treatment inhibits the uptake of
[14C]glycylsarcosine by decreasing the transcription
and/or stability of PEPT1 mRNA.
intestinal absorption; intestinal oligopeptide transporter; hormonal regulation; gene expression
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INTRODUCTION |
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THE PEPTIDE TRANSPORT
SYSTEM in the small intestine has physiological roles in the
absorption of small peptides to maintain protein nutrition (7,
13, 19, 20). Moreover, the intestinal peptide transport system
mediates the absorption of a broad range of peptide-like drugs, such as
-lactam antibiotics, the anti-cancer agent bestatin, and angiotensin
converting enzyme inhibitors, thereby playing an important
pharmacological role for oral drug delivery (11, 34, 36).
Recently, cDNA encoding an oligopeptide transporter (PEPT1) was
isolated from rabbit (4), human (15), rat
(25), and mouse (5), and its structural and
functional characteristics have been elucidated. In addition, molecular
identification of PEPT1 provides a novel opportunity to determine the
mechanisms of its regulation. The activity of PEPT1 varies
significantly in response to several factors that have been reported to
upregulate the activity of PEPT1, such as its own substrates (32,
35), diet (28), insulin (33),
2-adrenergic agonists (1), and the
-receptor ligand (+)-pentazocine (6). For these
stimuli, the regulation of PEPT1 activity is considered to be at the
level of gene expression, transport function, or protein recruitment to
the plasma membrane. On the other hand, it has been reported that cAMP
(22) and protein kinase C activation (3)
inhibited the activity of PEPT1. These inhibitory effects are likely to be due to posttranscriptional modifications involving
phosphorylation/dephosphorylation of PEPT1. Therefore, the mechanism of
downregulation of PEPT1 at the level of expression has not been elucidated.
Thyroid hormone is secreted from the thyroid to maintain normal growth and development, normal body temperature, and normal energy levels. Most of its effects appear to be mediated by activation of nuclear receptors that lead to increased formation of mRNA and subsequent protein synthesis (24). Thyroid hormone has prominent effects on gastrointestinal development, structure, and function. Absorption of various nutritional substrates from the small intestine was shown to be altered in response to a thyroid state (14). Because 3,5,3'-L-triiodothyronine (T3) is known to increase the metabolism of glucose, the effects of T3 on the hexose transport systems in the small intestine (SGLT1, GLUT5) have been studied extensively (16, 17). On the other hand, no information is available about the effects of T3 on the absorption of oligopeptides, which are also important nutritional substrates.
To elucidate the effects of T3 on the absorption of oligopeptides, we investigated the activity of PEPT1 by measuring [14C]glycylsarcosine uptake by a human intestinal epithelial cell line Caco-2. Caco-2 cells are known to express PEPT1 and have been used to study the regulation of its activity and expression (1, 3, 22, 32, 33, 35). We report here that T3 decreases the transport activity of PEPT1 by inhibition of the transcription and/or the decrease in the stability of PEPT1 mRNA.
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MATERIALS AND METHODS |
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Materials.
[14C]Glycylsarcosine (1.78 GBq/mmol) was
obtained from Daiichi Pure Chemicals (Ibaraki, Japan).
L-[3-3H]threonine (485 GBq/mmol) was from
Amersham Pharmacia Biotech (Little Chalfont, UK).
Methyl--D-[U-14C]glucopyranoside
(
-methyl-D-glucoside; 9.66 GBq/mmol) was from Moravek Biochemical (Brea, CA). T3 was purchased from
Nacalai Tesque (Kyoto, Japan). AG-1-X8 anion exchange resin (chloride form; 200-400 mesh) was obtained from Bio-Rad (Hercules, CA). All
other chemicals were of the highest purity available.
Cell culture. Caco-2 cells at passage 18 obtained from the American Type Culture Collection (ATCC HTB-37) were maintained by serial passage in plastic culture dishes (Falcon; Becton Dickinson, Lincoln Park, NJ) as described previously (12, 18). For uptake studies, 35-mm plastic dishes were inoculated with 2 × 105 cells in 2 ml of complete culture medium. The medium consisted of DMEM (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (Whittaker Bioproducts, Walkersville, MD) and 1% nonessential amino acids (Invitrogen) without antibiotics before T3 treatment. Cells were used for experiments on the 15th day after seeding. In this study, Caco-2 cells were used between passages 36 and 41.
Cell treatment. A stock solution of T3 was prepared as 1 mM solution in 0.1 M NaOH. For T3 treatment, serum was treated by anion exchange resin AG-1-X8 to remove the thyroid hormone according to the method of Samuels et al. (26). T3 concentration in treated serum was below the level of detection (<0.15 ng/ml) by an enzyme immunoassay method (IMx; Dainabot, Tokyo, Japan). To expose the Caco-2 cell monolayers to T3, T3 was added to culture medium containing T3-depleted serum. T3 treatment was applied to postconfluent monolayers. The control cells were incubated with the same concentration of 0.1 M NaOH in each experiment.
Uptake studies by cell monolayers.
The uptake of [14C]glycylsarcosine was measured in cells
grown on 35-mm plastic dishes as described previously
(31). In all experiments, the uptake was measured on
day 15. Radioactivity was determined in 5 ml of ACS
II (Amersham Pharmacia Biotech) by liquid scintillation
counting. The protein contents of cell monolayers solubilized in 1 N
NaOH were determined according to the method of Bradford
(2) using a Bio-Rad protein assay kit with bovine
-globulin as the standard.
Competitive PCR. Competitive PCR was performed according to the method of Siebert and Larrick (29) with some modifications. Briefly, aliquots of 1 µg of total cellular RNA, isolated from Caco-2 cells using the RNeasy Mini Kit (Qiagen, Hilden, Germany), were reverse-transcribed in 20 µl of reaction mixtures and diluted to 200 µl. Aliquots of 5 µl of diluted reaction mixtures, in combination with semilogarithmic serial dilution of mimic competitor DNA from 250 to 1 zmol, were amplified by PCR according to the following method: 5 µM human PEPT1 sense primer (5'-CTGCAAATCCCGCAGTATTTTCTT-3'; corresponding to bases 1803-1826) and human PEPT1 antisense primer (5'-CATCTGTTTCTGTGAATTGGCCCC-3'; corresponding to bases 2157-2186) in 20 µl were incubated according to the following PCR cycle: an initial denaturation step of 95°C for 3 min followed by 34 cycles of 95°C for 1 min, 65°C for 1 min, and 72°C for 1 min, and a final elongation step of 72°C for 10 min. PCR products were then size-fractionated by 1.5% agarose gel electrophoresis. The amplified cellular fragment (target) was 355 bp and the mimic competitor was 538 bp. The amount of competitor DNA yielding equal molar amounts of product gave that of target human PEPT1 mRNA. The densitometric data were normalized for each batch of RNA by correcting the amount of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA as an internal control.
Polyclonal antibodies against human PEPT1 and Western blot analysis. Polyclonal antibodies were raised in rabbits against a synthetic peptide (RFRHRSKAFPKREHWLDW) corresponding to positions 247-264 of human PEPT1 (15). The peptide was synthesized with cysteine at its NH2 terminus and conjugated to keyhole limpet hemocyanin. The apical membrane fraction was purified from Caco-2 cells according to the method of Inui et al. (10) with some modifications. Briefly, Caco-2 cells were homogenized in buffer A consisting of (in mM) 250 sucrose, 10 Tris, 0.5 EGTA, and 0.5 phenylmethylsulfonyl fluoride; pH 7.5. Magnesium chloride was added to a final concentration of 10 mM, and the mixture was allowed to stand for 15 min (step 1). The suspension was centrifuged at 1,000 g for 10 min, and the resulting supernatant was centrifuged at 27,000 g for 30 min (step 2). The pellet from high-speed centrifugation was resuspended in buffer A. Steps 1 and 2 were repeated on this homogenate, and the resulting pellet was resuspended in buffer B (250 mM sucrose, 10 mM Tris, and 0.5 mM phenylmethylsulfonyl fluoride; pH 7.5) by repeated passage through a 27-gauge needle. The protein concentration of the membrane suspension was measured using a Bio-Rad protein assay kit. The membrane fractions were solubilized in lysis buffer (2% SDS, 125 mM Tris, and 20% glycerol). Samples were separated by 8.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon-P; Millipore, Bedford, MA) by semidry electroblotting. Blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris, 137 mM NaCl, pH 7.5) with 0.1% Tween 20 (TBS-T) for 3 h at room temperature. The blots were washed in TBS-T and then incubated with the affinity-purified anti-PEPT1 antibody (1:50 dilution) and left overnight at 4°C. Blots were washed three times with TBS-T, and the bound antibody was detected on X-ray film by enhanced chemiluminescence with a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Amersham Pharmacia Biotech).
Statistical analysis. Data were analyzed statistically by nonpaired t-test or one-way ANOVA followed by Scheffé's test when multiple comparisons were needed. Probability values <5% were considered significant.
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RESULTS |
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Effect of T3 pretreatment concentration and time on
[14C]glycylsarcosine uptake in Caco-2 cells.
To investigate the effect of T3 on
[14C]glycylsarcosine transport in Caco-2 cells, we used
serum treated with anion exchange resin to deplete thyroid hormone.
Supplementation of culture medium with thyroid hormone-depleted serum
for 3 days had no effect on [14C]glycylsarcosine uptake
compared with medium containing untreated serum (data not shown).
Therefore, in the present study we used thyroid hormone-depleted serum
during treatment of Caco-2 cells with T3. Figure
1 shows the effect of various
concentrations of T3 (0.1 nM to 1 µM) on
[14C]glycylsarcosine uptake by Caco-2 cells.
H+-coupled [14C]glycylsarcosine transport was
decreased by T3 pretreatment for 3 days in a
concentration-dependent manner.
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Specificity of T3-induced inhibition of
[14C]glycylsarcosine uptake.
We then examined the effect of T3 pretreatment on
-methyl-D-glucoside and [3H]threonine
uptake by Caco-2 cells to determine whether the effect of
T3 was specific to peptide transport. As shown in Fig.
3,
-methyl-D-glucoside and
[3H]threonine uptake were not diminished by
T3 treatment, indicating that the inhibitory effect of
T3 on [14C]glycylsarcosine uptake appeared to
be specific.
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Kinetic analysis of [14C]glycylsarcosine uptake.
To determine the effect of T3 on the kinetics of
[14C]glycylsarcosine uptake, the concentration dependence
of [14C]glycylsarcosine uptake was examined in Caco-2
cells pretreated with or without T3. Figure
4 shows the initial uptake of
[14C]glycylsarcosine as a function of the substrate
concentration. Specific uptake was calculated by subtracting the
nonspecific uptake, which was estimated in the presence of excess
unlabeled dipeptide, from total uptake. Although the osmolarity of the
incubation medium without excess unlabeled dipeptide was not adjusted,
we confirmed that hyperosmotic solution has little effect on PEPT1 activity using incubation medium including 50 mM mannitol. The [14C]glycylsarcosine uptake values in the presence or
absence of 50 mM mannitol were 197.0 ± 2.0 and 183.9 ± 4.7 pmol · mg protein1 · min
1,
respectively. Kinetic parameters were calculated according to the
Michaelis-Menten equation using nonlinear least-squares regression analysis. The Michaelis constants (Km) in the
cells treated without and with T3 were 0.87 ± 0.01 and 1.02 ± 0.26 mM (mean ± SE of three separate
experiments, P = 0.567), respectively, and the maximum
uptake rates (Vmax) in the cells treated without and with T3 were 9.90 ± 0.84 and 5.22 ± 1.06 nmol · mg protein
1 · min
1
(mean ± SE of three separate experiments, P < 0.05), respectively. Thus treatment of the cells with T3
significantly decreased the maximum velocity value for
[14C]glycylsarcosine uptake, whereas the apparent
Km did not change significantly.
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Human PEPT1 mRNA expression.
Decrease in the Vmax value for
[14C]glycylsarcosine uptake by T3 treatment
suggested decreased expression of the peptide transporter PEPT1 in
Caco-2 cells. Therefore, we then examined the expression of PEPT1 mRNA
by competitive PCR in Caco-2 cells pretreated with T3. In a
preliminary experiment, PEPT1 was amplified from cDNA in the presence
of serial dilutions of the PEPT1 competitor to find the most
appropriate amount of competitor (data not shown). Quantitative
investigation of PEPT1 mRNA was then carried out using total RNA
isolated from Caco-2 cells pretreated with or without T3.
Typical results of competitive PCR analysis are shown in Fig.
5A. Densitometric
quantification indicated that treatment with 100 nM T3 for
4 days led to a decrease in PEPT1 mRNA content (25% of the control,
P < 0.01) (Fig. 5B). Therefore, it was
suggested that the decrease in [14C]glycylsarcosine
transport activity was due to the decreased transcription of PEPT1
mRNA. The amount of GAPDH mRNA used as an internal standard was not
changed significantly (P = 0.752) by this treatment
(data not shown).
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Human PEPT1 protein expression.
To investigate the effect of T3 on the expression of PEPT1
protein, apical membranes were purified from Caco-2 cells and were subjected to immunoblot analysis. A primary band of ~80 kDa was detected using affinity-purified anti-PEPT1 antibodies, which disappeared when the antibody was preabsorbed to the synthetic antigen
peptide (Fig. 6A).
Densitometric quantification indicated that expression of PEPT1 protein
was decreased in cells pretreated with T3 (Fig. 6,
B and C, P < 0.05). An
additional band below PEPT1 was also detected. This band may correspond
to a degradation product of PEPT1 or other proteins detected by
anti-PEPT1 antibodies. Because the identity of the smaller bands are
not known, we did not include the smaller bands in the quantitative
analysis.
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DISCUSSION |
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In the present study, we showed that the
[14C]glycylsarcosine uptake by Caco-2 cells was inhibited
by T3 treatment. The effect of T3 on
[14C]glycylsarcosine uptake appeared to be specific to
PEPT1 and to be different from those on glucose and amino acid
transport, because -methyl-D-glucoside and
[3H]threonine uptake were not decreased by T3
treatment. Previously, Matosin-Matekalo et al. (16) showed
that T3 treatment stimulated
-methyl-D-glucoside uptake via the
Na+-glucose cotransporter (SGLT1) in Caco-2 cells. Their
observations were inconsistent with the present results. Although
reasons for this discrepancy are not clear, differences in the results
of these two studies may have been due to the characteristics of Caco-2
cells and/or experimental conditions used. They used a clone,
Caco-2/TC7, showing markedly higher levels of SGLT1 mRNA expression
than parental Caco-2 cells. In addition, they treated the cells with 1 µM T3 for 20 days, whereas we treated with 0.1 µM
T3 for 4 days. These concentrations were higher than the
physiological concentration of T3 (~2.3 nM). However, we
supposed the condition of hyperthyroidism and the concentration of
T3 used was adequate.
Earlier investigations suggested that the activity of PEPT1 varies in
response to several factors. The regulatory effects of substrate and
dietary factors have been widely investigated (28, 32,
35). It was shown that the presence of dipeptide in the culture
medium stimulated the uptake of dipeptide by Caco-2 cells (32,
35). The mechanism of this stimulation by dipeptide appeared to
be increased levels of PEPT1 protein and mRNA. Recently, Shiraga et al.
(28) identified the promoter region of the rat PEPT1 gene
and showed the presence of an amino acid-responsive element in the
promoter region. They demonstrated that upregulation of dipeptide
transport activity by dietary protein was caused by transcriptional
activation of the PEPT1 gene by selected amino acids and dipeptides in
the diet. Hormonal regulation of PEPT1 in Caco-2 cells, for example by
insulin, has also been studied (33). Insulin stimulated
dipeptide uptake by Caco-2 cells by increasing the amount of PEPT1 in
the plasma membrane. The mechanism of this effect appeared to be
increased translocation of PEPT1 to the plasma membrane from a
preformed cytoplasmic pool. In addition, 2-adrenergic
agonists stimulated oligopeptide transport in Caco-2 cells by
increasing translocation to the apical membrane of preformed cytoplasmic transporter molecules (1). Moreover, peptide
transport in the small intestine was relatively resistant to
starvation, protein-caloric malnutrition, and intestinal damage
(9, 30). It has been reported that the resistance of
peptide transporter to tissue damage may be due to a relative increase
in PEPT1 synthesis (30) and that PEPT1 expression was
enhanced in the rat jejunum under various conditions of malnutrition
(9). In contrast, it has been reported that cAMP
(22) and protein kinase C activation (3)
inhibited the activity of PEPT1 in Caco-2 cells. Because human PEPT1
possesses sites for phosphorylation by protein kinase C, it is likely
that PEPT1 is regulated by posttranslational modifications involving
phosphorylation/dephosphorylation. A change in the phosphorylation state of the transport protein could result in the inhibition of its
activity. In the present study, we demonstrated that T3 treatment inhibited the activity of PEPT1 and decreased the
Vmax value for [14C]glycylsarcosine uptake
without changing the Km value. In addition, the
expression levels of PEPT1 mRNA and protein in Caco-2 cells were
decreased by T3 treatment. It was reported that treatment with thyroid hormone increased the expression of
Na+-K+-ATPase
1-protein and did
not change the expression of SGLT1 protein in Caco-2 cells
(16). In addition, the uptake of
-methyl-D-glucoside and threonine was not decreased by
T3 treatment (Fig. 3), suggesting that the expression of
these transporters was not decreased. Therefore, it seems likely that
T3 treatment does not lead to a general decrease in protein
expression. This is the first study showing the hormonal downregulation
of PEPT1 expression. During the preparation of this manuscript, it was
reported that expression of PEPT1 was decreased by epidermal growth
factor treatment in Caco-2 cells (23).
Thyroid hormone is responsible for optimal growth, development, function, and maintenance of all body tissues. In the small intestine, the processes of enterocyte growth and differentiation are altered by various developmental, dietary, and hormonal factors (8). Thyroid hormone is among the most potent regulators of intestinal epithelial growth and differentiation. For example, T3 regulates the developmental changes in the activity of several brush-border enzymes such as alkaline phosphatase and lactase (8). Developmental expression of PEPT1 in the rat small intestine has also been reported (21, 27). Shen et al. (27) showed that expression levels of PEPT1 mRNA and protein were maximal 3-5 days after birth in the duodenum, jejunum, and ileum, and then declined rapidly. They suggested that this change in PEPT1 expression might be related to an adaptive response to changes in the diet, from high-protein milk to an adult diet containing more carbohydrate than protein. However, it was reported that the serum concentration of thyroid hormone rises from postnatal day 5 to 15 (8) at the same time that expression levels of PEPT1 decline (27). Therefore, it seems likely that thyroid hormone regulates the expression of PEPT1 during development. Further studies are required to determine the precise mechanism of developmental regulation of PEPT1 expression.
Mechanisms of action of thyroid hormone are quite diverse. Although thyroid hormone may exert its effects via a number of cellular loci, its major effect appears to be on transcriptional regulation of target genes. Thyroid hormone enters the cells and proceeds to the nucleus, where it binds to the thyroid hormone receptor. The formation of ligand-bound thyroid hormone receptor complexes specifically interacting with thyroid hormone-responsive elements located in regulatory regions of target genes is presumably a necessary first step for activation or suppression of target genes (37). In the present study, we demonstrated that T3 treatment markedly decreased PEPT1 mRNA levels (Fig. 5). It is likely that the inhibition of [14C]glycylsarcosine transport by T3 is due to the inhibitory effect of T3 on the transcription of PEPT1 mRNA. However, it is not yet known whether the inhibition of transcription of PEPT1 mRNA is due to a direct effect of T3 on PEPT1 mRNA. Recently, promoter analyses of mouse and rat PEPT1 were reported (5, 28). These analyses revealed the putative nucleotide sequence upstream of the translation start site and the corresponding transcription factor. Thyroid hormone-responsive elements were not found in the promoter regions of rat and mouse PEPT1 genes, although these may be differences among rat, mouse, and human homologues. We have examined the effect of thyroid hormone on PEPT1 activity and expression in hyperthyroid rats and found that treatment of thyroid hormone decreased the activity of PEPT1 in rat small intestine (unpublished observations by K. Ashida, T. Katsura, H. Saito, and K. Inui). Although the precise mechanism of the effect of T3 on PEPT1 expression remains to be determined, it is possible that T3 may act indirectly with PEPT1 mRNA in rats and humans.
In conclusion, we demonstrated that the activity of PEPT1 is inhibited by T3 and that the inhibitory effect of T3 is due to inhibition of the transcription and/or the decrease in the stability of PEPT1 mRNA. These results may have important implications for protein nutrition as well as for drug absorption in thyrotoxicosis.
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ACKNOWLEDGEMENTS |
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This work was supported, in part, by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Inui, Dept. of Pharmacy, Kyoto Univ. Hospital, Sakyo-ku, Kyoto 606-8507, Japan (E-mail: inui{at}kuhp.kyoto-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 12, 2001;10.1152/ajpgi.00344.2001
Received 7 August 2001; accepted in final form 7 December 2001.
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