Hormonal regulation of oligopeptide transporter Pept-1 in a
human intestinal cell line
Manikkavasagar
Thamotharan,
Shahab Zare
Bawani,
Xiaodong
Zhou, and
Siamak A.
Adibi
Clinical Nutrition Research Unit, Department of Medicine,
University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
15260
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ABSTRACT |
The intestinal oligopeptide transporter (cloned as Pept-1) has
major roles in protein nutrition and drug therapy. A key unstudied question is whether expression of Pept-1 is hormonally regulated. In
this experiment, we investigated whether insulin has such a role. We
used a human intestinal cell monolayer (Caco-2) as the in vitro model
of human small intestine and glycylglutamine (Gly-Gln) as the model
substrate for Pept-1. Results showed that addition of insulin at a
physiological concentration (5 nM) to incubation medium greatly
stimulates Gly-Gln uptake by Caco-2 cells. This stimulation was blocked
when genistein, an inhibitor of tyrosine kinase, was added to
incubation medium. Studies of the mechanism of insulin stimulation
showed the following. 1) Stimulation
occurred promptly (30-60 min) after exposure to insulin.
2) There was no significant change
in the Michaelis-Menten constant of Gly-Gln transport, but there was a
nearly twofold increase in its maximal velocity.
3) Insulin effect persisted even
when Golgi apparatus, which is involved in trafficking of newly
synthesized Pept-1, was dismantled.
4) However, there was complete
elimination of insulin effect by disruption of microtubules involved in
trafficking of preformed Pept-1. 5)
Finally, with insulin treatment, there was no change in Pept-1 gene
expression, but the amount of Pept-1 protein in the apical membrane was
increased. In conclusion, the results show that insulin, when it binds
to its receptor, stimulates Gly-Gln uptake by Caco-2 cells by
increasing the membrane population of Pept-1. The mechanism appears to
be increased translocation of this transporter from a preformed
cytoplasmic pool.
Caco-2 cells; gene expression; glycylglutamine; insulin; microtubules
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INTRODUCTION |
STUDIES IN HUMAN SMALL intestine have established the
presence of an oligopeptide transporter that has major roles in
assimilation of dietary proteins and absorption of peptidomimetic drugs
such as
-lactam antibiotics (2). This transporter was recently cloned and designated Pept-1 (9). This cloning provides a novel opportunity to investigate whether the expression of the intestinal oligopeptide transporter is metabolically regulated and, if so, to
determine the mechanism of its regulation.
In metabolic regulation, insulin is usually the key hormone serving as
a mediator. Therefore, the question becomes whether insulin has any
effect on expression of Pept-1 and, if so, what cellular and/or
molecular mechanisms insulin uses to cause the effect. In this
experiment, we investigated these questions in a human intestinal cell
line (Caco-2 cells) so that we could investigate dipeptide transport in
the absence and presence of insulin as the only hormone.
We used glycylglutamine (Gly-Gln) as a model dipeptide for transport by
the apical membrane of Caco-2 cells. Gly-Gln currently serves as a
stable source of glutamine for cells in culture and for patients
needing nutritional support (15, 21). Our previous studies have
validated the use of the Caco-2 cell monolayer as the in vitro model
for studies of Gly-Gln transport in human intestine (2, 19). For
example, these studies showed that Gly-Gln uptake by Caco-2 cells is
largely as intact dipeptide and that the uptake is mediated by an
oligopeptide transporter with functional and biological similarities to
human Pept-1 (2, 19).
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MATERIALS AND METHODS |
Materials.
The cell line Caco-2 was purchased from the American Type Culture
Collection (Manassas, VA). Custom-synthesized
[Gln-3,4-3H]Gly-Gln
(49 Ci/mmol) was obtained from DuPont-NEN (Boston, MA). Cloned cDNA
encoding Pept-1 was provided by Dr. Matthias A. Hediger (Brigham and
Women's Hospital, Harvard Medical School, Boston, MA).
Other chemicals were purchased from Sigma Chemical (St. Louis, MO) or
Bachem Bioscience (Philadelphia, PA).
Cell culture.
At approximately passage
30, Caco-2 cells were seeded in 200-ml
flasks and passaged in DMEM supplemented with 1% nonessential amino
acids, 10% FCS, 1,000 U/l penicillin, and 1 mg/l streptomycin (complete DMEM). Monolayers were detached with trypsin, resuspended in
medium, and split 3:1. When the cells reached
passages
50-70, they were plated onto 12-well cluster trays at a density of 63,000 cells/cm2. Cells were cultured in
complete DMEM; the medium was replaced every 2-3 days. Monolayers
were kept at 37°C, 5% CO2,
and 90% relative humidity and were used for experiments 4 days
postconfluency, which is equivalent to 12-15 days after seeding.
Serum was withdrawn from 4-day-postconfluent monolayers for 24 h before
each experiment.
Uptake studies.
The culture medium was removed, and the monolayers were washed twice
with an Earle's balanced salt solution (equilibration buffer)
containing 2 mM bicarbonate, 5 mM glucose, and 10 mM HEPES (pH 7.5).
After the wash, the cells were incubated with the equilibration buffer
for 15 min at 23°C. After this interval, the equilibration buffer
was removed and 500 µl of transport buffer containing
[Gln-3,4-3H]Gly-Gln
were added per well. The transport buffer was the same as the
equilibration buffer except that 10 mM HEPES was replaced with 10 mM
MES (pH 6.0). The final concentration of Gly-Gln was 0.1 mM except in
kinetic studies. The incubation time was 5 min. During this incubation,
the plates were circularly and continuously shaken (25 rpm). Uptake
into cells was terminated by gentle suction of the uptake medium,
followed by two washes of the monolayer with ice-cold transport buffer
(pH 6.0). In all experiments, uptake at 0°C was used to determine
the nonspecific binding of Gly-Gln. This value was subtracted from all
uptake values. Isotope was extracted from each well by solubilizing the
monolayers in 500 µl 0.2% SDS in 0.2 N NaOH. Radioactivity was
quantitated using aliquots of the resulting solution. Each value is
expressed as the mean ± SE of three to six wells, and each
experiment was repeated at least twice.
Transport kinetics.
To examine the kinetics of peptide transport by Caco-2 cells, the
initial rates of uptake were measured as a function of dipeptide concentration in the transport buffer. Uptake at 0°C was used to
estimate the nonspecific binding. After correction of the total rate of
uptake for the nonspecific binding, kinetic constants [Michaelis-Menten constant
(Km) and
maximal velocity
(Vmax)]
were derived by a nonlinear regression method with the Michaelis-Menten kinetic equation using GRAFIT (Sigma). To determine the number of
systems involved in the uptake of Gly-Gln, the uptake rates were
transformed according to the Eadie-Hofstee method.
Western blot analyses.
Apical membrane vesicles from Caco-2 cells were prepared as described
previously for preparation of brush-border membrane vesicles from human
enterocytes (12). In brief, Caco-2 cells were homogenized in buffer
[in mM: 60 mannitol, 0.1 phenylmethylsulfonyl fluoride, 10 EDTA,
and 12 Tris (pH 7.4)] in a blender for 2 min. 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 3,000 g for 15 min, and the
resulting supernatant was centrifuged at 27,000 g for 30 min
(step
2). The pellet from the high-speed
spin was resuspended in 35 ml of the above buffer using a
Potter-Elvehjem homogenizer. Steps
1 and 2 were repeated on this homogenate,
and the resulting pellet was resuspended in the above buffer by
repeated passage through an 18-gauge needle. The protein concentration
of the membrane suspension was measured using the Bio-Rad protein assay
(Bio-Rad Laboratories, Hercules, CA).
Identical amounts of apical membrane proteins (100 µg) from control
and insulin-treated Caco-2 cells were suspended in SDS buffer
[4% wt/vol SDS, 0.125 M Tris · HCl (pH 6.8),
20% vol/vol glycerol, and 0.125 wt/vol
DL-dithiothreitol].
Samples were subjected to 10% SDS-PAGE in a Laemmli system (8).
Resolved proteins were transferred to nitrocellulose membranes and
subjected to immunoblot analyses. The membranes were incubated with
polyclonal antibody (1:1,000) raised against Pept-1 protein. For
preparation of antibody, a synthetic peptide
(Glu-Asn-Pro-Tyr-Ser-Ser-Leu-Glu-Pro-Val-Ser-Gln-Thr-Asn-Met) corresponding to the 15 carboxy-terminal amino acids of Pept-1 was used
as epitope. Antibody was generated by immunization of rabbits with the
epitope, and specificity of the antibody was confirmed by Western blot
analysis with antibody that had been preabsorbed with epitope. We (19)
previously validated the use of this antibody for the Western analysis
of Pept-1. After incubation with Pept-1 antibody, the membranes were
washed and incubated with the second antibody (peroxidase-conjugated
goat anti-rabbit IgG, 1:2,000) as previously described (4, 13). Pept-1
protein in Caco-2 apical membrane was detected with an enhanced
chemiluminescence Western blotting system (ECL Plus, Amersham Life
Science, Arlington Heights, IL). The intensity of bands was quantified
using Image PC (Scion, Frederick, MD). Preliminary studies showed
linearity of the Western blot assay from 25 to 200 µg of Caco-2
apical membrane protein. The correlation coefficient between the amount
of protein and ECL image intensity was 0.967.
Northern blot analyses.
For Northern blot analyses, 5 µg of
poly(A)+ RNA (isolated by using a
miniribocep isolation kit from Collaborative Research, Becton
Dickinson, Bedford, MA) from Caco-2 cells were resolved by
electrophoresis in 0.9% agarose gels containing formaldehyde and
transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH) by
capillary action. After transfer, mRNA was immobilized by radiation
with ultraviolet light (UV cross-linker, Stratagene, La Jolla, CA).
Then the membranes were prehybridized overnight at 42°C in
prehybridization solution [50% deionized formamide, 0.25 mM
Na2HPO4
(pH 7.2), 0.25 mM NaCl, 1 mM EDTA, 100 µg/ml heat-denatured herring
sperm DNA, 7% SDS, and 0.1% sodium pyrophosphate]. Specific 32P-labeled cDNA probes (Pept-1 or
-actin) were made by random primer technique using an oligolabeling
kit (Amersham Pharmacia Biotech, Piscataway, NJ) and were added to
fresh aliquots of the prehybridization solution. Hybridization was
performed at 42°C for 24-72 h. To remove the unbound probe,
membranes were washed twice for 20 min in each of the following
buffers: 1) 2× SSC (sodium chloride-sodium citrate buffer) and 0.1% SDS at 42°C,
2) 25 mM Na2HPO4
(pH 7.2), 1 mM EDTA, and 0.1% SDS at 42°C, and
3) 25 mM Na2HPO4
(pH 7.2), 1 mM EDTA, and 1% SDS at 42°C. Hybridization signals
were visualized by autoradiography with Biomax MS film (Kodak,
Rochester, NY) for 48-72 h at
70°C. Densitometric
analyses of the autoradiographs were carried out using Image PC.
Membranes initially hybridized to Pept-1 were subsequently hybridized
to
-actin to normalize for differences in mRNA loading between
wells. Preliminary experiments showed that the abundance of
-actin
mRNA was not affected by the addition of insulin to the culture medium.
Statistics.
Each rate of uptake is the mean ± SE of determinations in three to
six monolayers. Preliminary studies showed that the SD of the
radiotracer method is ~10% of the mean of three replicates. Significance was tested with paired and unpaired tests and ANOVA, as appropriate.
 |
RESULTS |
Effect of insulin.
To measure Gly-Gln transport, Caco-2 cells were incubated for 5 min
with 0.1 mM Gly-Gln at pH 6.0, which is the optimum pH for uptake of
Gly-Gln by Caco-2 cells (2). The incubation conditions were based on
the results of preliminary experiments that showed that the rate of
uptake was linear with regard to time of incubation and concentration
of Gly-Gln.
To investigate whether insulin has any effect on Gly-Gln transport,
Caco-2 cells were preincubated for 30-120 min with 5 nM insulin.
An approximately twofold stimulation of Gly-Gln uptake occurred after
60 min of preincubation with insulin (Fig.
1). Longer preincubation (90-120 min) or
an increase in the insulin concentration did not further
increase Gly-Gln transport. Therefore, for the following studies,
Caco-2 cells were preincubated for 60-120 min with 5 nM insulin.

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Fig. 1.
Transport of glycylglutamine (Gly-Gln) as a function of time of
exposure to insulin. Insulin (5 nM) was added to culture medium of
Caco-2 cell monolayers for 30-120 min. Before uptake measurements,
culture medium was removed, and control and insulin-treated monolayers
were washed twice with equilibration buffer (pH 7.5) and incubated for
10-15 min with same buffer at 23°C. pH of medium was 6.0, concentration of Gly-Gln in medium was 0.1 mM, temperature was
23°C, and incubation time was 5 min. Each rate of uptake is mean ± SE of determinations in 3-6 monolayers.
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The following experiment was performed to investigate whether the
stimulation in dipeptide transport requires binding of insulin to its
receptor, which has been shown to be present in the membrane of Caco-2
cells (3, 10). Insulin action is mediated through the insulin receptor,
a transmembrane glycoprotein with intrinsic protein tyrosine kinase
activity (17). When insulin binds to its receptor, the tyrosine kinase
becomes activated. This activation can be blocked by genistein (11).
Therefore, we added genistein before adding insulin to determine
whether this inhibitor would block the stimulatory effect of insulin on
Gly-Gln transport. The results showed that genistein had no effect on
Gly-Gln transport by Caco-2 cells but completely blocked the
stimulatory effect of insulin on this transport (Fig.
2).

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Fig. 2.
Effect of genistein on Gly-Gln transport. For control experiment,
culture medium was removed, and Caco-2 cell monolayers were washed
twice with equilibration buffer (pH 7.5) and incubated with same buffer
for 10-15 min at 23°C. For uptake measurement, pH of medium
was 6.0, concentration of Gly-Gln in medium was 0.1 mM, temperature was
23°C, and incubation time was 5 min. For study of effect of
genistein, above procedure was followed except for addition of
genistein (80 µM) to culture medium overnight before cells were
washed with equilibration buffer. For study of combined effect of
genistein and insulin, genistein (80 µM) was added to culture medium
overnight, followed by insulin (5 nM) addition to culture medium for
120 min, and then cells were washed with equilibration buffer. Each
rate of uptake is mean ± SE of determinations in 3-6
monolayers.
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Mechanism of insulin effect.
Insulin might increase Gly-Gln uptake by increasing its affinity for
the oligopeptide transporter or by activating a dormant transporter. To
investigate these possibilities, we determined the effect of insulin on
the kinetics of Gly-Gln uptake by Caco-2 cells (Fig.
3). Eadie-Hofstee plots of the data showed
the presence of a single transport system in both control and
insulin-treated cells. Furthermore, kinetic analysis of this system
showed that insulin significantly (P < 0.01) increased the
Vmax (3.53 ± 0.61 vs. 6.31 ± 0.5 nmol · mg
protein
1 · 5 min
1) but had no
significant effect on the
Km (1.49 ± 0.55 vs. 1.36 ± 0.33 mM). These data eliminated the
possibilities of involvement of more than one system in dipeptide
transport by the insulin-treated cells and a change in the affinity of
Gly-Gln for the oligopeptide transporter.

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Fig. 3.
Effect of insulin on kinetics of Gly-Gln transport. Insulin (5 nM) was
added to culture medium for 120 min. Culture medium was then removed,
and Caco-2 cell monolayers were washed twice with equilibration buffer
(pH 7.5) and incubated for 10-15 min with same buffer at 23°C.
For uptake measurement, pH of medium was 6.0, temperature was 23°C,
and incubation time was 5 min. Each rate of uptake is mean ± SE of
determinations in 3-6 monolayers.
Inset: Eadie-Hofstee plots
(V vs.
V/S) of Gly-Gln uptake, where
V is rate of uptake
(nmol · mg
protein 1 · 5 min 1) and S is substrate
concentration (mM).
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The increase in
Vmax suggested
that insulin increased the population of the peptide transporter in the
apical membrane of Caco-2 cells. To investigate this suggestion, we
used Western analyses to determine the amount of Pept-1 protein in the
apical membrane of control and insulin-treated cells. The results are shown in Fig. 4. As previously found by us
(19) and others (5), the observed molecular weight of Pept-1 in Caco-2
cells was higher than its predicted value (9). This difference has been
attributed to glycosylation of Pept-1 (5), which has several sites for this posttranscriptional modification. As is apparent from Fig. 4A, insulin treatment increased the
amount of Pept-1 protein in the apical membrane of Caco-2 cells. This
was confirmed by densitometric analyses (Fig.
4B) of immunoblots
(P < 0.01).

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Fig. 4.
Effect of insulin on amount of Pept-1 protein in apical membrane.
Culture medium of treated cells was enriched with insulin (5 nM) 2 h
before protein measurement. Apical membrane proteins (100 µg) of
control and insulin-treated Caco-2 cells were subjected to Western blot
analyses. For each analysis, apical membranes were prepared from cells
grown in 3-5 culture bottles, each with an area of 175 cm2.
A:
lane
1, control cells;
lane
2, insulin-treated cells.
B: quantitative densitometric analyses
of Western blots for Pept-1 protein. Values are means ± SE of 6 analyses, expressed as percent control. KD, kilodaltons.
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To investigate whether the mechanism of increased Pept-1 protein was
pretranslational, we determined the gene expression of Pept-1 in
control and insulin-treated cells. Quantitative and qualitative
analyses of Northern blots showed that abundance of Pept-1 mRNA was not
affected by insulin treatment (Fig. 5).

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Fig. 5.
Effect of insulin on abundance of mRNA encoding Pept-1. Culture medium
of treated cells was enriched with insulin (5 nM) 2 h before mRNA
measurement. A: mRNA was extracted
from control and insulin-treated Caco-2 cells and was subjected to
Northern blot analysis using
32P-labeled cDNAs encoding Pept-1
and -actin. Lane
1, control cells;
lane
2, insulin-treated cells.
B: densitometric analyses of Northern
blots. Level of mRNA in each sample was normalized to abundance of
-actin mRNA, which was not affected by insulin treatment. Values are
means ± SE of 4 analyses, expressed as percent control.
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Another possible mechanism is that insulin increases the membrane
population of Pept-1 by a direct effect on its translation. To
investigate this possibility, we determined whether insulin stimulates
Gly-Gln transport in Caco-2 cells treated with brefeldin. Stimulation
in transport was used as evidence of increased membrane population of
Pept-1. In a previous study (19), we showed that the brefeldin
treatment of Caco-2 cells selectively dismantles their Golgi apparatus,
which is required for the processing of newly synthesized Pept-1
protein for insertion into plasma membrane. As shown in Fig.
6, brefeldin treatment did not affect
Gly-Gln transport either in the control or in insulin-treated Caco-2
cells.

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Fig. 6.
Effect of brefeldin on Gly-Gln transport. For control experiment,
culture medium was removed, and Caco-2 cell monolayers were washed
twice with equilibration buffer (pH 7.5) and incubated with same buffer
for 10-15 min at 23°C. For uptake measurement, pH of medium
was 6.0, concentration of Gly-Gln in medium was 0.1 mM, temperature was
23°C, and incubation time was 5 min. For study of effect of
brefeldin, above procedure was followed except for addition of
brefeldin (5 µM) to culture medium for 140 min before cells were
washed with equilibration buffer. For study of combined effect of
brefeldin and insulin, above procedure was followed except for addition
of brefeldin (5 µM) to culture medium; after 20 min, insulin (5 nM)
was added to culture medium for 120 min, and then cells were washed
with equilibration buffer. Each rate of uptake is mean ± SE of
determinations in 6 monolayers.
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Finally, we investigated whether the mechanism of increased membrane
population of Pept-1 is an increase in their translocation from a
preformed cytoplasmic pool. To investigate this possibility, we
determined whether insulin stimulates Gly-Gln transport of Caco-2 cells
treated with colchicine. Previous studies have shown that treatment of
cells (including Caco-2 cells) with colchicine results in
depolymerization of microtubules, which disrupts the translocation of
proteins targeted for membrane insertion (1, 14, 22). As shown in Fig.
7, colchicine had no effect on the uptake
of Gly-Gln by the control Caco-2 cell monolayers. However, when
colchicine was added 20 min before the addition of insulin, it
completely abolished the stimulatory effect of insulin on Gly-Gln uptake.

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Fig. 7.
Effect of colchicine on Gly-Gln transport. For control experiment,
culture medium was removed, and Caco-2 cell monolayers were washed
twice with equilibration buffer (pH 7.5) and incubated with same buffer
for 10-15 min at 23°C. For uptake measurement, pH of medium
was 6.0, concentration of Gly-Gln in medium was 0.1 mM, temperature was
23°C, and incubation time was 5 min. For study of effect of
colchicine alone, above procedure was followed except for addition of
colchicine (10 µM) to culture medium for 140 min before cells were
washed with equilibration buffer. For study of effect of insulin alone,
above procedure was followed except for addition of insulin (5 nM) to
culture medium for 120 min before cells were washed with equilibration
buffer. For study of combined effect of colchicine and insulin, above
procedure was followed except for addition of colchicine (10 µM) to
culture medium; after 20 min, insulin (5 nM) was added to culture
medium for 120 min, and then cells were washed with equilibration
buffer. Each rate of uptake is mean ± SE of determinations in
3-6 monolayers.
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 |
DISCUSSION |
The results of the present study bring a new dimension to the field of
peptide transport by providing the first evidence for its stimulation
by a hormone. The stimulation was shown in Caco-2 cells treated with a
physiological concentration of insulin. This observation raises a
question about the specificity of the insulin effect. In other words,
is the transport of other nutrients also increased by insulin treatment
of Caco-2 cells? Among the various nutrients, glucose would be the most
likely candidate for this effect. However, a previous study (10) showed
that insulin treatment does not affect glucose transport by Caco-2
cells. Therefore, the effect of insulin on dipeptide transport appears
to be specific.
The present study also provides novel information about the mechanism
of a regulatory action of insulin regarding a nutrient transport by an
intestinal cell. It shows that insulin increases the membrane
population of oligopeptide transporter by increasing its translocation
from a preformed cytoplasmic pool. The evidence for this is based on
1) increased
Vmax of dipeptide
uptake by the apical membrane of insulin-treated Caco-2 cells and
2) increased amount of Pept-1 in the
apical membrane fraction isolated from these cells.
Our previous studies in Caco-2 cells have suggested that the alteration
in the gene expression may be a mechanism of regulation of Pept-1 (19).
Our present study suggests the existence of another mechanism for this
regulation, namely, intracellular trafficking of Pept-1. The evidence
for this is based on 1) lack of
alteration in the gene expression in insulin-treated cells and
2) abolition of insulin stimulation
of dipeptide transport by disruption of microtubules that are involved
in membrane insertion of proteins from cytoplasmic pools.
Previous studies (16, 20) showed the presence of an oligopeptide
transporter on the basolateral membrane of Caco-2 cells that appears to
be different from Pept-1. Apparently, the function of this transporter
is to provide a mechanism for the exit of dipeptides from the Caco-2
cells. Because we were studying cellular accumulation of Gly-Gln as a
function of Pept-1 activity, the Caco-2 cells were plated on plastic
for determination of uptake. This method does not allow exposure of the
basolateral membrane to the incubation medium and, therefore, prevents
the exit of transported Gly-Gln from the cells. Use of this
experimental design led to the following two questions. Are insulin
receptors present on the apical membrane of Caco-2 cells? If so,
are they involved in the action of insulin on dipeptide transport? The
answer to the first question is provided by previous studies (3, 10) that showed the presence of insulin receptors on the apical membrane of
Caco-2 cells. To answer the second question, we found that, indeed,
blocking the insulin receptors also blocks the action of insulin on
dipeptide transport (Fig. 2).
Finally, questions may be raised about the physiological relevance of
the present results. All we can say at this time is that insulin
receptors are present on both the basolateral and brush-border
membranes of intestinal mucosal cells (6, 7). However, most of the
insulin receptors are concentrated on the basolateral membrane rather
than on the brush-border membrane of mucosal cells (6, 7). Because
there is no reason to believe that there is any difference between
insulin receptors located on the basolateral and apical membranes of
mucosal cells, it could be envisioned that an in vivo action of insulin
is to regulate dipeptide transport in the intestine. However, a
previous study from our laboratory, performed over two decades ago,
found that diabetes has no effect on dipeptide absorption (18).
Although this study needs to be repeated with the tools currently
available, it is possible that other factors besides insulin may
regulate dipeptide transport in the intestine of diabetic rats.
Clearly, the present study needs to be followed by further studies on
physiology and biology of hormonal regulation of dipeptide transport.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. A. Adibi,
UPMC Health System, Montefiore University Hospital, 200 Lothrop St.,
Pittsburgh, PA 15213-2536 (E-mail:
adibi{at}med1.dept-med.pitt.edu).
Received 27 July 1998; accepted in final form 29 December 1998.
 |
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