| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Articles |
INSERM U-45, Hôpital Edouard Herriot, Lyon, France
Address all correspondence and requests for reprints to: Dr. Martine Cordier-Bussat, INSERM U-45, Hôpital Edouard Herriot, Pavillon H Bis, place d’Arsonval, 69437 Lyon Cedex 3, France.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Many physiological factors modulating CCK release in vivo and ex vivo have been described, including nutrients, hormones, and neuropeptides. In the rat, dietary proteins are the major stimulus of CCK release (2, 3). Little, however, is known about the mechanisms regulating CCK secretion. The hypothesis that intact proteins stimulate CCK release by protecting CCK-releasing peptides from proteolytic inactivation by pancreatic enzymes in the intestinal lumen has recently been substantiated by the purification and sequencing of three distinct protease-sensitive CCK-releasing peptides, namely the monitor peptide from rat pancreatic juice (4), the luminal CCK-releasing factor from rat intestinal secretion (5), and finally, a peptide identical to the porcine diazepam binding inhibitor, which was purified and characterized from porcine intestinal mucosa (6). The release of the latter peptide was inhibited by the cholinergic muscarinic receptor antagonist atropine, implying that CCK secretion was under the indirect control of a cholinergic nervous pathway. Noteworthy, a recent in vivo study conducted in rats indeed showed that peptone-induced CCK secretion was dependent on the activation of intestinal submucosal cholinergic neurons (7). Alternatively, peptones induced a marked CCK release in an isolated vascularly perfused rat duodeno-jejunum preparation (8). In this case, the underlying mechanisms did not involve the participation of intramural nerves, as neither the axonal blocker tetrodotoxin nor atropine altered peptone-induced CCK release (8).
Overall, these results suggest that peptones may stimulate CCK release via both indirect and direct mechanisms. The latter hypothesis was tested in the present study. Ideally, these experiments should have been performed with purified intestinal CCK-producing cells. However, the isolation of native intestinal I cells remains a tedious process and produces fairly low yields (9). Hence, the intestinal CCK-producing cell line STC-1 was instead selected, as these cells harbor many features of native intestinal CCK-producing cells (10, 11). We here show that peptones stimulate CCK secretion and gene transcription, and that peptones dose-dependently increase the transcriptional activity of an 800-bp fragment of the CCK gene promoter transfected in STC-1 cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-Amanitin (Sigma) was
dissolved in distilled water. [-32P]Deoxy-CTP,
[-32P]UTP, and the random priming labeling kit were
purchased from Amersham (Les Ulis, France). Agarose, positively charged
nylon membranes, and restriction endonucleases were obtained from
Appligene-Oncor (Illkirch, France). Other chemicals and reagents, of
analytical grade, were purchased from Sigma.
Probes and plasmids
A 575-bp rat CCK complementary DNA (cDNA) fragment (12) and an
800-bp fragment from the 5'-flanking region of the rat CCK gene (13)
were provided by Dr. Dixon (Michigan Medical School, Ann Harbor, MI). A
2-kb human ß-actin cDNA fragment was obtained from Clontech (Ozyme,
Montigny-le-Bretonneux, France). cDNA inserts used as probes were
excised from plasmids by digestion with restriction endonucleases, and
purified from agarose after electrophoresis gel separation. pGL2, pGL3,
pTK-RN plasmids, and luciferase reporter assay systems were obtained
from Promega (Charbonnières, France).
Cell lines and culture conditions
Several CCK-expressing cell lines were used in the present
study. The STC-1 plurihormonal cell line is derived from an endocrine
tumor that developed in the small intestine of a double transgenic
mouse expressing the rat insulin promoter linked to the simian virus 40
large T antigen and to the polyoma virus small t antigen, respectively
(14). It was provided by Dr. Andrew B. Leiter (Department of Medicine,
New England Medicine Center, Boston, MA). The GLUTag cell line is
derived from a large bowel tumor developed in a transgenic mouse
carrying the glucagon/simian virus 40 large T antigen transgene (15)
and was provided by Dr. Daniel J. Drucker (Clinical Biochemistry and
Molecular Genetics, Toronto Hospital, Toronto, Canada). The CA77 cell
line is derived from a rat medullary thyroid carcinoma (16) and was
kindly provided by Dr. A. Kervran (INSERM U-376, Montpellier, France).
The RINm5F cell line has been established from a nude mouse
heterotransplant of a rat radioinduced pancreatic cell tumor described
previously (17). It was provided by Dr. M. Laburthe (INSERM U-410,
Paris, France).
STC-1 and RINm5F cell lines were grown in RPMI 1640 medium supplemented with 5% FCS, 2 mM glutamine, penicillin (100 IU/ml), and streptomycin (50 µM). CA77 cells were grown in medium containing Ham’s F-10 nutrient mixture and DMEM (1:1, vol/vol) with 4.5 g/liter glucose, supplemented with 10% FCS plus penicillin (100 IU/ml), 2 mM glutamine, and streptomycin (50 µM). GLUTag cells were grown in DMEM containing 10% FCS plus 2 mM glutamine, penicillin (100 IU/ml), and streptomycin (50 µM).
Secretion studies
Two days before the experiments, cells were seeded into 24-well
culture plates. When cells reached 85% confluence, the supernatant was
replaced by Krebs-Ringer bicarbonate buffer containing 0.1% (wt/vol)
BSA, with or without the tested agents or peptones. These were directly
prepared at the desired concentrations in the Krebs-Ringer buffer, and
pH was adjusted to 7.2. Cells were then incubated for 2 h at 37 C
before the supernatants were collected and frozen at -20 C for
subsequent RIA of CCK as previously described (18). Briefly, CCK
immunoreactivity was measured using antiserum 39A that cross-reacts at
100% with CCK-33 and CCK-8, at 12% with sulfated gastrin-17, at 5%
with unsulfated gastrin, and at less than 0.1% with unsulfated CCK-8,
unsulfated CCK-7-Gly, and gastrin-34.
RNA isolation and quantitation
A total of 15 x 106 cells were seeded in
100-mm culture plates in the corresponding culture medium, as described
above. After 24 h, the growth medium was replaced by fresh medium
containing the agents to be tested. Peptones were directly dissolved at
the desired concentration in RPMI 1640 medium without FCS, containing
0.3% (wt/vol) BSA, glutamine, penicillin, and streptomycin. The pH was
adjusted to 7.2, and the mixture was filtered before use. Cells were
then incubated at 37 C for 18 h (except in time-course
experiments), then washed once in PBS buffer and harvested in the same
buffer. Dry cell pellets were stored at -70 C before RNA extraction.
RNA was extracted from dry cell pellets using the rapid mini-prep
method described by Wilkinson (19). Briefly, cells were resuspended in
400 µl Tris-saline [25 mM Tris (pH 7.4), 130
mM NaCl, and 5 mM KCl] plus 100 µl ice-cold
NDD buffer, containing 1% Nonidet P-40, 0.5% sodium deoxycholate, and
0.01% dextran sulfate, in Tris-saline. The nuclear pellet was
discarded, and the supernatant, containing RNA, was extracted twice
with phenol-chloroform before ethanol precipitation. Ten micrograms of
RNA per assay were separated on a 1% agarose-formaldehyde denaturing
gel and then blotted onto nylon-positive membrane. Blots were
prehybridized in 1 M NaCl, 1% SDS, and 10% dextran
sulfate at 60 C for 4 h and then hybridized overnight with
106 cpm/ml [-32P]deoxy-CTP-labeled probe
at 60 C in prehybridization solution. Blots were then washed in 2
x SSC (0.3 M NaCl and 0.03 M sodium
citrate)-2% SDS and exposed to Kodak X-Omat AR films (Sigma) for 6–24
h at -70 C with intensifying screens. Quantitative analysis of
autoradiograms was performed using a Bio-Rad scanning densitometer
(Ivry sur Seine, France) coupled with the Bio-Rad PC molecular analyst
program.
Nuclear run-on experiments
Gene transcription rates were determined by quantification of
nascent CCK and ß-actin transcripts in nuclei from control and
peptone-treated cells, as described previously (20). STC-1 cells were
first incubated with peptone (MH; 1%, wt/vol) for 18 h. Nuclei
were isolated by disrupting the cells in Nonidet P-40 buffer and stored
at -70 C until nuclear transcripts were labeled. Each assay was
performed on 2.5 x 107 nuclei in the presence of 100
µCi [-32P]UTP in a classical run-on reaction mixture
for 30 min at 37 C. RNA extraction was then performed. In parallel,
-amanitin was added at a concentration of 5 µg/ml (a concentration
that preferentially inhibits polymerase II) in one set of nuclei. RNA
was hybridized to 10 µg linearized plasmid containing (or not) CCK or
ß-actin cDNA fragments bound to nylon-positive membrane using a
slot-blot apparatus. Filters were hybridized for 48 h at 60 C in
the same hybridization solution as that used for Northern blot
analysis, then washed twice in 2 x SSC-0.1% SDS at 60 C for 15
min, once in 2 x SSC for 5 min at 60 C, once in 2 x SSC
containing 10 µg/ml of ribonuclease A at 37 C for 30 min, and finally
once in 2 x SSC during 30 min at 37 C. Filters were exposed to
Kodak X-Omat AR for 7–10 days at -70 C with intensifying screens.
Quantitative analysis of autoradiograms was performed as described
above.
Transfection experiments and luciferase activity measurement
STC-1 cells were transfected by electroporation. Briefly,
exponentially growing cells were trypsinated and washed twice in PBS
before being electroporated in RPMI 1640 medium without FCS. A total of
15 x 106 cells per assay were submitted to a 250
V/960 µF pulse in a Bio-Rad Gene Pulser in the presence of 20 µg
plasmid pGL3CCK800, 10 µg sonicated salmon sperm DNA, and
2.5 µg plasmid pRL-TK. pGL3CCK800 was obtained by
subcloning an 800-bp KpnI/SacI CCK promoter
fragment into the corresponding restriction sites of PGL3basic vector.
Transfected cells were maintained overnight at 37 C in the presence of
RPMI 1640 medium containing 5% FCS to allow cell attachment. Then, the
medium was replaced by RPMI 1640 without FCS but containing 0.3% BSA
and increasing concentrations of peptones. After 24 h, cells were
collected in PBS. Luciferase activities were measured using the dual
luciferase reporter assay system and a Dynatech luminometer (Elvetec,
Vénissieux, France). Similar to firefly luciferase, the
luminescence reaction catalyzed by Renilla luciferase
provides high sensitivity and a linear range extending over 7 orders of
magnitude of enzyme concentration.
Calculations and statistics
Data in figures are presented as the mean ±
SEM. Statistical significance was assessed using one-way
ANOVA, followed by multiple comparison tests. Differences were
considered significant when P < 0.05%.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, AEH induced a dose-dependent increase in CCK
release, reaching 1420 ± 191% of the control value (n = 16;
P < 0.01) upon treatment with AEH (1%, wt/vol). AEH
treatment did not increase CCK release after 2-h incubation at 4 C,
when exocytosis was inhibited (data not shown).
|
) induced CCK release
that was approximately half (or less) of that obtained with the highest
doses of AEH [647 ± 107% (n = 6) and 349 ± 27%
(n = 8) of the control, respectively; P < 0.05].
In contrast, incubation of STC-1 cells with the amino acid mixture HA25
(1%, wt/vol) or with BSA (1%, wt/vol) had only a modest effect on CCK
release, representing 132 ± 9% (P < 0.05) and
227 ± 17% (P < 0.05) of the control value,
respectively (n = 6 for each; Fig. 1).
Peptones dose-dependently increase CCK steady state RNA level
The effect of peptones on CCK gene expression was examined at the
RNA level by Northern blot analysis. The single 750-bp CCK transcript
and the 2.1-kilobase ß-actin transcript that were previously
described in intestinal extracts (21) are shown in Fig. 2. Incubation of STC-1 cells with various peptones (1%,
wt/vol) induced an increase in the CCK steady state RNA level,
representing 339 ± 54% of the control with AEH, 512 ± 41%
with MH, 334 ± 77% with SH, and 235 ± 62% with CH (n
= 3 for each). The level of ß-actin RNA always remained constant in
all of these experiments. Moreover, incubation of STC-1 cells with HA25
or BSA (1%, wt/vol) did not elicit any detectable increase in CCK RNA
level (data not shown).
|
). The
lowest active concentration was 0.25% (wt/vol) for MH and 1% (wt/vol)
for AEH. The maximal response obtained was 499 ± 49% of the
control value with AEH (n = 6) and 866 ± 136% of the
control value with MH (n = 6; P < 0.01). Because
of its potency, MH was selected for subsequent experiments.
|
, the increase in the CCK RNA level was
significant from 4 h of MH treatment (269 ± 18% of control;
n = 3). A plateau value was then observed between 6 h
(333 ± 53% of control; n = 3) and 48 h (307 ±
17% of control; n = 4).
|
).
|
STC-1 cells were first incubated for 2–24 h with MH (1%, wt/vol) in
the presence or absence of actinomycin D (5 µg/ml), which was added
30 min before MH and maintained during the entire incubation. Figure 6 shows that the time-dependent increase in CCK RNA that
appeared when STC-1 cells were incubated with MH alone was clearly
abolished by actinomycin D. After 24 h, the CCK RNA level was
378 ± 34% of the control value (n = 6) with MH alone and
52 ± 7% of the control value (n = 6) with MH plus
actinomycin D. The observed decrease in ß-actin RNA level upon
incubation with actinomycin D reflected adequate inhibition of
transcription.
|
show
that the CCK hybridization signal obtained with MH-treated nuclei was
clearly increased over the CCK signal obtained with MH-untreated nuclei
(48 ± 6% vs. 15 ± 5%, respectively; n =
2). In comparison, the ß-actin signal remained unchanged. The
addition of 5 µg/ml -amanitin (a concentration that preferentially
inhibits RNA polymerase II) abolished the hybridization signals,
indicating that the observed CCK signal resulted from RNA polymerase II
activity.
|
The results reported in Fig. 8 show that MH
dose-dependently stimulated the transcriptional activity of the 800-bp
CCK promoter fragment from 163,445 ± 10,309 relative light units
(RLU)/OD unit with 0.25% peptone to 507,688 ± 26,825 RLU/OD unit
with 3% MH (n = 3), whereas herpes simplex thymidine kinase
promotor activity was not changed (57,022 ± 2,808 to 58,927
± 8,096 RLU/OD unit, respectively; n = 3).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously showed that CCK release was potently stimulated by AEH in the isolated perfused rat duodeno-jejunum preparation (8). Arterial infusion of tetrodotoxin or atropine had no significant effect on this peptone-induced CCK release, thus excluding any major involvement of intramural nerves in CCK secretion. The present study indicates that incubation of STC-1 cells with the same peptones for 2 h elicited a dose-dependent release of CCK over the concentration range 0.05–3%. Additionally, incubation of STC-1 cells with peptones at 4 C had no effect on CCK secretion. These experiments indicate that the peptone-induced CCK response cannot be related to any toxic effect on STC-1 cells. The profile of the STC-1 cell response to various protein-derived agents was highly consistent with the pattern obtained using the isolated rat duodeno-jejunum preparation. AEH potently stimulated CCK release as well as the other peptones tested, namely meat, casein, and soybean hydrolysates (8). In contrast, a mixture of free amino acids or undigested proteins were weak stimulants of CCK release. These results are in good agreement with those reported in two separate studies that showed a weak stimulatory effect of aromatic amino acids on CCK release from STC-1 cells (11, 24). Overall, it is suggested that the products of protein hydrolysis are capable of directly triggering peptide release from CCK-producing cells. Peptones mainly consist of oligopeptides with a mol wt below 1200 (8). The sequence specificity of CCK-releasing oligopeptide remains to be established. It should be noted that proteins and protein digests did not stimulate CCK release in a perifusion system containing isolated mucosa cells from the rat duodeno-jejunum (25). However, due to the heterogeneity of such cell preparations, a direct stimulatory effect of peptones could not be assessed.
The possible effect of peptones on the regulation of CCK gene transcription had not been documented. It was previously shown that infusion of soybean trypsin inhibitor into the duodenum of anesthetized rats for 12–24 h induced a 3- to 4-fold increase in the CCK RNA level that was the result of an enhanced transcriptional activity of the CCK gene (21). The rationale of the experiment, however, was stimulation of CCK release through interruption of the negative feedback with luminal trypsin, not the effect of peptones. In man, ingestion of mixed nutrients was followed by an increase in the CCK messenger RNA level in extracts of mucosal biopsies (26), but the contribution of protein-derived nutrients was not specifically assessed. We show that various peptones elicit a dose-dependent increase in the CCK RNA level in the STC-1 cell line. This effect appears highly specific, as no parallel change in the ß-actin RNA level was observed. Additionally, RNA levels of two other intestinal peptides secreted by STC-1 cells, namely glucose-dependent insulinotropic peptide and secretin, were not modified upon peptone incubation (our unpublished observations). The peptone-induced increase in the CCK RNA level was remarkably tissue and cell specific. Indeed, no variation in the CCK transcript level was recorded when using three additional cell lines (GLUTag, Ca77, and RINm5F), respectively, of colonic, thyroid (C cell), and pancreatic origin. Among the four CCK-expressing cell lines tested, only the STC-1 cell line, established from an endocrine tumor of the small bowel, displayed a response pattern that would fit the alleged reactivity of intestinal I cells to luminal nutrients. The suppression of the peptone-induced CCK RNA increase by actinomycin D as well as the results of run-on experiments both indicated that transcription of CCK gene was stimulated when STC-1 cells were incubated with peptones. Whether RNA stabilization was additionally involved cannot be ruled out. Further experiments are required to test this hypothesis.
The transcriptional mechanisms regulating CCK gene expression in response to physiological stimuli such as nutrients are unknown. We here obtained evidence that the transcriptional activity of a 800-bp fragment of the CCK gene promoter region (located immediately upstream of the transcriptional start site) was markedly increased in peptone-treated STC-1 cells. Experiments aimed at determining the precise location of the promoter elements that confer peptone inducibility are currently underway.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received October 16, 1996.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  S. Choi, M. Lee, A. L. Shiu, S. J. Yo, G. Hallden, and G. W. Aponte GPR93 activation by protein hydrolysate induces CCK transcription and secretion in STC-1 cells Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1366 - G1375. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Choi, M. Lee, A. L. Shiu, S. J. Yo, and G. W. Aponte Identification of a protein hydrolysate responsive G protein-coupled receptor in enterocytes Am J Physiol Gastrointest Liver Physiol, January 1, 2007; 292(1): G98 - G112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. P. Darcel, A. P. Liou, D. Tome, and H. E. Raybould Activation of Vagal Afferents in the Rat Duodenum by Protein Digests Requires PepT1 J. Nutr., June 1, 2005; 135(6): 1491 - 1495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hira, H. Hara, F. Tomita, and Y. Aoyama Casein Binds to the Cell Membrane and Induces Intracellular Calcium Signals in the Enteroendocrine Cell: A Brief Communication Experimental Biology and Medicine, July 1, 2003; 228(7): 850 - 854. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishi, H. Hara, and F. Tomita Soybean {beta}-Conglycinin Peptone Suppresses Food Intake and Gastric Emptying by Increasing Plasma Cholecystokinin Levels in Rats J. Nutr., February 1, 2003; 133(2): 352 - 357. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Claustre, F. Toumi, A. Trompette, G. Jourdan, H. Guignard, J. A. Chayvialle, and P. Plaisancie Effects of peptides derived from dietary proteins on mucus secretion in rat jejunum Am J Physiol Gastrointest Liver Physiol, September 1, 2002; 283(3): G521 - G528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-C. Gevrey, M. Cordier-Bussat, E. Nemoz-Gaillard, J.-A. Chayvialle, and J. Abello Co-requirement of Cyclic AMP- and Calcium-dependent Protein Kinases for Transcriptional Activation of Cholecystokinin Gene by Protein Hydrolysates J. Biol. Chem., June 14, 2002; 277(25): 22407 - 22413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nishi, H. Hara, T. Hira, and F. Tomita Dietary Protein Peptic Hydrolysates Stimulate Cholecystokinin Release via Direct Sensing by Rat Intestinal Mucosal Cells Experimental Biology and Medicine, December 1, 2001; 226(11): 1031 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bernard, A. Sutter, C. Vinson, C. Ratineau, J.-A. Chayvialle, and M. Cordier-Bussat Peptones Stimulate Intestinal Cholecystokinin Gene Transcription via Cyclic Adenosine Monophosphate Response Element-Binding Factors Endocrinology, February 1, 2001; 142(2): 721 - 729. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Deavall, R. Raychowdhury, G. J. Dockray, and R. Dimaline Control of CCK gene transcription by PACAP in STC-1 cells Am J Physiol Gastrointest Liver Physiol, September 1, 2000; 279(3): G605 - G612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hara, S. Ohyama, and T. Hira Luminal dietary protein, not amino acids, induces pancreatic protease via CCK in pancreaticobiliary-diverted rats Am J Physiol Gastrointest Liver Physiol, June 1, 2000; 278(6): G937 - G945. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Nemoz-Gaillard, C. Bernard, J. Abello, M. Cordier-Bussat, J.-A. Chayvialle, and J.-C. Cuber Regulation of Cholecystokinin Secretion by Peptones and Peptidomimetic Antibiotics in STC-1 Cells Endocrinology, March 1, 1998; 139(3): 932 - 938. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
