Luminal dietary protein, not amino acids, induces pancreatic
protease via CCK in pancreaticobiliary-diverted rats
Hiroshi
Hara,
Sumika
Ohyama, and
Tohru
Hira
Division of Applied Bioscience, Graduate School of
Agriculture, Hokkaido University, Sapporo 060-8589, Japan
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ABSTRACT |
We determined whether pancreatic
adaptation to a high-protein diet depends on ingested protein in the
intestinal lumen and whether such adaptation depends on a CCK or
capsaicin-sensitive vagal afferent pathway in
pancreaticobiliary-diverted (PBD) rats. Feeding a high-casein (60%)
diet but not a high-amino acid diet to PBD rats increased pancreatic
trypsin and chymotrypsin activities compared with those after feeding a
25% casein diet. In contrast, feeding both the high-nitrogen diets
induced pancreatic hypertrophy in PBD rats. These pancreatic changes by
the diets were abolished by treatment with devazepide, a CCK-A receptor
antagonist. Protease zymogen mRNA abundance in the PBD rat was not
increased by feeding the high-casein diet and was decreased by
devazepide. Perivagal capsaicin treatment did not influence the values
of any pancreatic variables in PBD rats fed the normal or high-casein
diet. We concluded that luminal protein or peptides were responsible
for the bile pancreatic juice-independent induction of pancreatic
proteases on feeding a high-protein diet. The induction was found to be dependent on the direct action of CCK on the pancreas. Pancreatic growth induced by high-protein feeding in PBD rats may depend at least
partly on absorbed amino acids.
pancreatic serine protease; pancreatic hypertrophy; messenger RNA; vagal afferent pathway
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INTRODUCTION |
THE EXOCRINE PANCREAS IS DOUBLY controlled by
endogenous and exogenous factors. Bile acids and pancreatic proteases
are endogenous factors in the intestinal lumen (11, 13, 30, 36), and protein as a dietary component is an exogenous stimulator of the exocrine pancreas (6, 10, 12, 39). The relationship between these
control mechanisms for pancreatic adaptation is not fully understood.
Recently, it has been reported that the secretion level of CCK, a
potent stimulator of the exocrine pancreas, is increased by dietary
protein in a manner independent of bile pancreatic juice (BPJ) (3, 5,
27, 28). However, the regulatory action of food components is in
concert with the endogenous factors, bile acids and pancreatic
proteases, in normal rats, and it is difficult to examine the action of
dietary protein itself for regulation of the exocrine pancreas in
normal rats. Rats with chronic BPJ diversion from the proximal small
intestine [pancreaticobiliary-diverted (PBD) rats] are
suitable models for examining the effects of dietary protein
independent of BPJ, and we have previously shown that feeding a
high-protein diet induces pancreatic growth and protease production in
PBD rats (15). On the other hand, we have also shown that feeding a
high-amino acid level diet induces pancreatic growth and results in an
increase in the protease content of the pancreas and that these effects
are not dependent on CCK secretion in normal rats (14). Feeding a
high-protein diet results in increases in the amino acid concentrations
in the intestinal contents and in the blood, and these increments are
possibly involved in pancreatic adaptation to a high-protein diet in
PBD rats.
The aims of the present study were to determine whether or not the
induction of pancreatic growth and protease production observed on
feeding a high-protein diet requires luminal protein and to determine
whether this induction depends on CCK or afferent vagal nerve signals.
In our previous study, BPJ was diverted from the proximal small
intestine through a bile pancreatic duct catheter (15). However, the
catheterized rats could not be used for long-term experiments (>10
days) because of catheter problems. First, we confirmed the
BPJ-independent pancreatic growth and protease induction under
conditions of chronic BPJ diversion established by transposition of the
intestinal segment, including the ampulla of Vater, to the upper ileum
(26, 40).
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MATERIALS AND METHODS |
Animals and diets.
This study was performed as four separate experiments. Male
Sprague-Dawley rats (Japan SLC, Hamamatsu, Japan), weighing ~220 g,
were fed a semipurified sucrose-casein-based diet for 5-7 days for
acclimation. After a 24-h fast, surgery was performed to divert the BPJ
to the ileum by transposition of a duodenal segment, including the
ampulla of Vater, under anesthesia by intraperitoneal injection of
pentobarbital sodium (40 mg/kg body wt; Abbott Laboratories, North
Chicago, IL). The acclimated rats were divided into two groups in
experiments 1 and 2. Rats of one group in
experiments 1 and 2 and all rats in experiments 3 and 4 were operated on to establish chronic
pancreaticobiliary diversion (26). Briefly, a 2- to 3-cm segment of the
duodenum containing the ampulla of Vater was cut off after ligation of
the proximal end of the segment. End-to-side anastomosis was carried
out between the cut edge of the anal side of the segment and the
lateral opening on the upper ileum (45 cm distal to the ligament of
Treitz), and both cut edges of the duodenum were end-to-end anastomosed
(PBD rat). The rats of another group in experiments 1 and 2 were subjected to a sham operation; that is, a 1-cm segment distal
to the ampulla of Vater was transected and end-to-end anastomosed (sham
rat). After the operation, the rats were fed a semipurified
sucrose-based diet containing casein (250 g casein/kg diet) for a
recovery period of 7-14 days and then fed the assigned test diet.
Rats in all groups had free access to the assigned diet and water. The
experiments were performed in a room controlled at 23 ± 2°C, with
a 12:12-h light-dark cycle (8:00-20:00 light period). The study
was approved by the Hokkaido University Animal Committee, and the
animals were maintained in accordance with the guidelines for the care
and use of laboratory animals of Hokkaido University.
On the last day of the experiments, a segment of pancreatic tissue
(~50 mg) from the dorsal area (experiments 2, 3, and
4) and a 10-cm jejunum segment immediately distal to the
ligament of Treitz (experiment 3) were removed from the rats
under pentobarbital anesthesia for extraction of RNA. The mucosa was
lightly scraped from the jejunal segment using a glass slide. The
pancreatic tissue and mucosa were immediately homogenized in Isogen RNA
extraction mixture (Nippon Gene, Tokyo, Japan) using a Polytron
homogenizer (Kinematica, Amlehnhalde, Switzerland). The concentration
of extracted RNA was measured spectrophotometrically (absorbance at 260 nm). The residual pancreatic tissue was removed and frozen in liquid nitrogen.
In experiment 1, sham and PBD rats after recovery from
operative damage were divided into two subgroups. The rats of one
subgroup of group were fed a 25% casein, fat-free test diet, and the
rats of the other subgroups were fed a 60% casein, fat-free test diet (Table 1; Refs. 1, 32) for 3 days to get
reproducibility of the results of the previous study. The 3-day feeding
period of test diets was adopted in the previous work using PBD rats with bile pancreatic duct cannulation.
In experiment 2, the sham rats were fed the 25% casein,
fat-free test diet for 14 days to clarify the role of luminal protein in the pancreatic adaptation and to evaluate a long-term adaptation. The PBD rats were divided into three subgroups. The rats of one subgroup were fed the same diet as the sham group. The PBD rats of the
second subgroup were fed the 60% casein, fat-free diet, and the PBD
rats of the last subgroup were fed a 25% casein, fat-free diet
containing a 35% amino acid mixture simulating casein, prepared by
replacement of sucrose (Table 1; Refs. 1, 32), in which the nitrogen
level was the same as that of the 60% casein, fat-free diet, for 14 days.
In experiment 3, the PBD rats of two groups were treated with
devazepide, a potent CCK-A receptor antagonist, and the PBD rats of the
other two groups were treated with the vehicle of the devazepide
solution. The devazepide solution and the vehicle solution were
injected subcutaneously three times a day (6 mg · kg
body
wt
1 · day
1; 2 mg/kg body wt at 8:00, 18:00, and 1:00) for 5 days. Devazepide, which
was kindly provided by ML Laboratories (London, UK), was dissolved in a
mixture of dimethylsulfoxide and Tween 80 (1:1, 5 g/l) as a stock
solution. The stock solution was diluted with saline to 1 g
devazepide/l for use. The PBD rats treated with devazepide or vehicle
were divided into two subgroups, and the rats of two subgroups were
each fed the 25% casein, fat-free diet and those of the other two
subgroups were fed the 60% casein, fat-free diet during the 5-day
devazepide or vehicle treatment period.
In experiment 4, the PBD rats of two groups were treated with
capsaicin (20), and the PBD rats of the other two groups were treated
with the vehicle of the capsaicin solution. A small piece of gauze
soaked with the capsaicin solution (0.2 ml, 10 g/l olive oil; Sigma
Chemical, St. Louis, MO) was applied to the abdominal vagal trunk for
>30 min during the transposition operation. The capsaicin- or
vehicle-treated PBD rats after a 14-day period of recovery from the
operation were divided into two subgroups. The rats of two subgroups
were each fed the 25% casein, fat-free diet and those of the other two
subgroups were fed the 60% casein, fat-free diet for 5 days.
Analyses.
Enzyme mRNA in the total pancreatic RNA and CCK and
-actin mRNA in
polyadenylated [poly(A)+] RNA from the upper
(10 cm) jejunal mucosa were quantified by the Northern blotting method
by digoxigenin (DIG)-labeled cDNA hybridization (21, 37).
Poly(A)+ RNA was extracted from total RNA of the jejunal
mucosa using an oligo(dT) latex polymer (Oligotex-dT30 Super; Takara
Suzo, Tokyo, Japan). Extracted total or poly(A)+ RNA was
electrophoresed on a 1% agarose gel, and the RNA was transferred from
the agarose gel to a nylon membrane (Hybond-N; Amersham International,
Little Chalfont, UK). The Northern blot was hybridized with DIG-labeled
trypsinogen I or III, chymotrypsinogen B, CCK, or
-actin cDNA as
DIG-labeled hybridization probes and visualized using a DIG luminescent
detection kit (Boehringer Mannheim, Mannheim, Germany). The intensity
of each mRNA band was quantified by exposing the blots to X-ray film
and subsequent scanning densitometry (Flying-Spot scanner; Shimadzu,
Kyoto, Japan).
The cDNAs for trypsinogen I and III and chymotrypsinogen B were the
RT-PCR products from total RNA of the pancreas, and for
-actin they
were the RT-PCR products from total RNA of the jejunal mucosa. CCK cDNA
was prepared by RT-PCR from poly(A)+ RNA of the jejunal
mucosa. Trypsinogen I and III and CCK probes were labeled by DIG-PCR
(21) from the RT-PCR products using Taq polymerase (Gene Taq,
Nippon Gene), specific primers, and DIG DNA labeling mixture
(Boehringer Mannheim). Other probes were labeled by means of a DIG DNA
labeling kit (Boehringer Mannheim). Primers for CCK were those
described previously (25). Pancreatic enzymes and
-actin cDNAs were
prepared using a sense primer (position 46-68) and an antisense
primer (position 461-486) for rat trypsinogen I (24), a sense
primer (position 213-232) and an antisense primer (position
794-813) for rat trypsinogen III (23), a sense primer (position
378-401) and an antisense primer (position 906-929) for rat
chymotrypsinogen B (2), and a sense primer (position 31-51) and an
antisense primer (position 1000-1020) for rat
-actin (29).
Pancreatic ribosomal RNA, blotted onto membranes, was stained with
methylene blue solution (0.04%) and quantified by densitometry. Trypsinogen and chymotrypsinogen in freeze-dried pancreas were activated by enterokinase (Sigma Chemical) at 30°C for 20 min in 15 mmol/l Tris buffer (pH 8.1). Trypsin and chymotrypsin activity levels
were estimated photometrically using synthetic substrates, TAME (33),
and N-benzoyl-L-tyrosine ethyl ester (34),
respectively. Protein was measured by a modified version of Lowry's
method (22, 38). The concentration of total RNA was determined
colorimetrically by the orcinol method (18) following extraction as
described by Fleck and Munro (8).
Calculations.
The content of protein and RNA in the pancreas and the activities of
the pancreatic enzymes were measured in the freeze-dried residual
pancreas, and these were expressed as units per pancreas in the whole
pancreas (the residual pancreas was >90% of the whole pancreas based
on wet weight, and the sum of the wet weight of the pancreas segment
used for RNA extraction plus that of the residual pancreas was equal to
the weight of the whole pancreas). One unit of trypsin or chymotrypsin
activity was defined as the amount of activity hydrolyzing 1 µmol
substrate/min at 30°C. CCK mRNA abundance was expressed as a ratio
of
-actin mRNA. Pancreatic enzyme mRNA was expressed as a ratio of
18S ribosomal RNA. The data were analyzed by ANOVA, and the
significance of the difference between groups was determined by
Duncan's multiple-range test (P < 0.05; SAS version 6.07, SAS Institute, Cary, NC).
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RESULTS |
In Table 2, body weight gain and food
intakes in all experiments are summarized. Food intakes were lower in
the 60% casein groups than in the 25% casein groups in experiment
1 but not in other experiments. Devazepide and capsaicin treatments
did not influence body weight gain and food intakes.
Effects of feeding a high-protein diet on pancreatic growth and
protease in PBD rats (experiment 1).
Pancreatic protein and RNA content were higher in the PBD rats than in
the sham rats. In the PBD groups, the protein and RNA content were
further increased in rats fed the 60% casein diet compared with the
levels in rats fed the 25% casein diet (Table 3). The
trypsin and chymotrypsin activities in the pancreas were higher in the
PBD groups than in the sham groups and were further increased as a
result of feeding a high-protein diet in the PBD groups (Fig.
1). The increase in the levels
of these proteases on feeding the 60% casein diet compared with the
25% casein diet in PBD rats was similar to that in sham rats.
High-protein feeding for 3 days induced pancreatic growth and protease
in both normal and PBD rats.
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Table 3.
Pancreatic growth indicator values after feeding a diet with a normal
or high protein level to sham-operated and chronic PBD rats for 3 days
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Fig. 1.
Trypsin (A) and chymotrypsin (B) activity levels in
pancreas of sham and chronic pancreaticobiliary-diverted (PBD) rats
after feeding a diet with a normal protein level (250 g casein/kg diet)
or a high protein level (600 g casein/kg diet) for 3 days. Values are
means ± SE [8 rats per group, except for PBD rats fed
high-casein diet (7 rats)]. P values estimated by 2-way
ANOVA for PBD vs. sham rats (D), normal vs. high protein level (P), and
D × P were 0.020, 0.021, and 0.790, respectively, for trypsin,
and 0.010, 0.010, and 0.984 for chymotrypsin. Mean values not sharing a
lower case letter are significantly different between groups (P < 0.05).
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Comparison between the effects of high-protein and high-amino acid
feeding on the pancreatic adaptation (experiment 2).
The pancreatic protein and RNA content was higher in the PBD group than
in the sham group of rats fed the 25% casein diet (Table
4). In the PBD rats, the values of these
two growth variables were further increased by feeding the 60% casein
diet or the 25% casein + 35% amino acid diet. Trypsin and
chymotrypsin activity levels in the pancreas were increased as a result
of chronic BPJ diversion, and the levels were further increased by
feeding the 60% casein diet but not by feeding the 25% casein + 35%
amino acid diet in the case of the PBD rats (Fig.
2).
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Table 4.
Pancreatic growth indicator values after feeding a diet with a normal
protein level to sham rats and after feeding a diet with a normal
protein level, a high protein level or a high amino acid level
to chronic PBD rats for 14 days
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Fig. 2.
Trypsin (A) and chymotrypsin (B) activity levels in the
pancreas after feeding a diet with a normal protein level (25C), a high
protein level (60C), or a high amino acid level (250 g casein + 350 g
amino acid mixture/kg diet; 25C+35A) to chronic PBD rats for 14 days
compared with those in sham rats fed a normal casein diet. Details on
the amino acid mixture are provided in MATERIALS AND
METHODS. Values are means ± SE (8 rats for the 25% casein-fed
groups and 9 rats for the 60% casein-fed groups in the case of both
sham and PBD rats). P values estimated by 1-way ANOVA were
<0.001 for trypsin and chymotrypsin. Mean values not sharing a lower
case letter are significantly different between groups (P
<0.05).
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The abundance of trypsinogen I and III mRNA in the PBD groups was
higher than that in the sham group, and switching to the high-protein
or the high amino acid level diet did not affect the abundance (Fig.
3). The changes in abundance of
chymotrypsinogen B mRNA tended to be similar to those in the case of
trypsinogen mRNA, but there was no significant difference between
groups in chymotrypsinogen B mRNA. Dietary protein, but not amino
acids, increased pancreatic proteases without any changes in protease mRNA levels; however, both nitrogen sources induced pancreatic growth
in PBD rats.

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Fig. 3.
Pancreatic trypsinogen I (A), trypsinogen III (B), and
chymotrypsinogen B (C) mRNA abundance after feeding a diet with
a normal protein level, a high protein level, or a high amino acid
level (250 g casein + 350 g amino acid mixture/kg diet) to chronic PBD
rats for 14 days compared with those in sham rats fed a normal-casein
diet. At top, representative Northern blots of each enzyme mRNA
are shown with 18S ribosomal RNA (18S rRNA) as a reference. Values are
means ± SE (8 rats for the 25% casein-fed groups and 9 rats for the
60% casein-fed groups in the case of both sham and PBD rats).
P values estimated by 1-way ANOVA were 0.035 for trypsinogen I
mRNA, 0.004 for trypsinogen III mRNA, and 0.152 for chymotrypsinogen B
mRNA. Mean values not sharing a lower case letter are significantly
different between diet groups (P <0.05).
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Effects of devazepide treatment on pancreatic growth and protease
induced by dietary protein in PBD rats (experiment 3).
The pancreatic protein content in the vehicle-treated PBD rats was
increased by feeding the 60% casein diet (Table
5), similar to the case of the PBD rats in
experiment 1. Treatment with the CCK-A receptor antagonist,
devazepide, resulted in a decrease in the values of both growth
variables, and feeding a high-protein diet did not influence the values
of these variables in the case of devazepide-treated PBD rats. Trypsin
and chymotrypsin activity levels were increased by feeding the 60%
casein diet in the case of the vehicle-treated PBD rats (Fig.
4). These activity levels were decreased by
devazepide treatment in rats fed either of the test diets, and the
levels were not increased by feeding the high-protein diet in the case
of the devazepide-treated PBD rats.
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Table 5.
Pancreatic growth indicator values after feeding a diet with a normal
or high protein level to chronic PBD rats treated with the CCK-A
receptor antagonist devazepide or vehicle for 5 days
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Fig. 4.
Trypsin (A) and chymotrypsin (B) activity levels in the
pancreas after feeding a diet with a normal protein level or a high
protein level to chronic PBD rats treated with devazepide (6 mg · kg body
wt 1 · day 1) or
vehicle for 5 days. Values are means ± SE (9 rats for vehicle-treated
groups and 6 or 7 rats for devazepide-treated groups fed the 25% or
60% casein diet, respectively). P values estimated by 2-way
ANOVA for devazepide vs. vehicle (D), normal vs. high protein level
(P), and D × P were <0.001, <0.001, and 0.020, respectively,
for trypsin and <0.001, 0.007, and 0.079 for chymotrypsin. Mean
values not sharing a lower case letter are significantly different
between groups (P <0.05).
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The abundance of trypsinogen III mRNA and chymotrypsinogen B mRNA was
lower in the devazepide-treated groups than in the vehicle-treated groups. Also, the abundance of the mRNA of all three protease zymogens
was significantly influenced by devazepide treatment, as indicated by
the results of two-way ANOVA (Fig. 5). The
mRNA abundance was not influenced by diet in the PBD rats.

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Fig. 5.
Pancreatic trypsinogen I (A), trypsinogen III (B), and
chymotrypsinogen B (C) mRNA abundance after feeding a diet with
a normal protein level or a high protein level to chronic PBD rats
treated with devazepide (6 mg · kg body
wt 1 · day 1) or
vehicle for 5 days. At top, representative Northern blots of
each enzyme mRNA are shown with 18S rRNA as a reference. Values are
means ± SE (9 rats for vehicle-treated groups and 6 or 7 rats for
devazepide-treated groups fed the 25% or 60% casein diet,
respectively). P values estimated by 2-way ANOVA for
devazepide vs. vehicle (D), normal vs. high protein level (P), and D × P were 0.031, 0.248, and 0.847, respectively, for trypsinogen I
mRNA, <0.001, 0.426, and 0.751 for trypsinogen III mRNA, and
<0.001, 0.762, and 0.531 for chymotrypsinogen B mRNA. Mean values not
sharing a lower case letter are significantly different between groups
(P <0.05).
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CCK mRNA abundance in the jejunal mucosa was increased as a result of
feeding a high-protein diet in the case of vehicle-treated PBD rats but
not in the case of devazepide-treated rats (Fig. 6). Devazepide treatment increased the
abundance of CCK mRNA in rats fed the 25% casein diet. Devazepide
treatment abolished both the pancreatic growth and protease induction
evoked by feeding a high-protein diet in PBD rats.

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Fig. 6.
Jejunal CCK mRNA abundance after feeding a diet with a normal protein
level or a high protein level to chronic PBD rats treated with
devazepide (6 mg · kg body
wt 1 · day 1) or
vehicle for 5 days. Top, representative Northern blot with
-actin mRNA as a reference. Values are means ± SE (9 rats for
vehicle-treated groups and 6 or 7 rats for devazepide-treated groups
fed the 25% or 60% casein diet, respectively). P values
estimated by 2-way ANOVA were 0.025, 0.060, and 0.075 for devazepide
vs. vehicle (D), normal vs. high protein level (P), and D × P. Mean values not sharing a lower case letter are significantly different
between groups (P < 0.05).
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Effects of capsaicin treatment on pancreatic growth and protease
induced by dietary protein in PBD rats (experiment 4).
Pancreatic protein and RNA content, trypsin, and chymotrypsin activity
levels in the pancreas in the case of the vehicle-treated PBD rats were
increased by feeding the 60% casein diet (Table 6 and Fig. 7),
which was similar to the case of the PBD rats in experiment 1.
Denervation by perivagal capsaicin treatment did not influence the
values of any variables concerning the pancreas. Also, capsaicin
treatment did not influence abundance of mRNA of pancreatic protease
zymogens measured in experiments 2 and 3 (data not
shown).
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Table 6.
Pancreatic growth indicator values after 5-day feeding of a normal or
high protein level diet to chronic PBD rats with perivagal
treatment of capsaicin or vehicle
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Fig. 7.
Trypsin (A) and chymotrypsin (B) activity levels in the
pancreas after feeding a diet with a normal protein level or a high
protein level for 5 days to chronic PBD rats given perivagal treatment
with capsaicin or vehicle. Details are provided in MATERIALS AND
METHODS. Values are means ± SE (7 or 8 rats for the 25%
casein-fed groups treated with capsaicin or vehicle-treated rats,
respectively, and 6 rats for the 60% casein-fed groups with either
treatment). P values estimated by 2-way ANOVA for capsaicin vs.
vehicle (C), normal vs. high protein level (P), and C × P were
0.092, <0.001, and 0.512, respectively, for trypsin and 0.413, 0.001, and 0.806 for chymotrypsin. Mean values not sharing a lower case letter
are significantly different between groups (P <0.05).
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DISCUSSION |
We have previously shown that exocrine pancreatic growth and pancreatic
protease production were induced by dietary protein independent of BPJ
in PBD rats (15). In our previous study, BPJ was diverted from the
proximal small intestine via a bile pancreatic duct catheter, which was
a simple technique to divert BPJ with minimum operative damage to the
small intestine. However, the catheter became broken or occluded after
>10 days when the rats were allowed free access to the diet and free
movement. In the present study, we adopted a procedure involving
transposition of an intestinal segment including the ampulla of Vater
to the ileum to divert the BPJ, a procedure that is used in some
studies to prepare PBD rats (36, 40). With this transposition
procedure, the effects of the operation on the small intestine are
greater than those in the case of the cannulation procedure; however, it is suitable to achieve long-term BPJ diversion. First, we showed that induction of pancreatic growth and induction of protease production occur on feeding a high-protein diet to rats with BPJ diversion established by the transposition procedure, as in the case of
PBD rats with bile pancreatic duct cannulation (experiment 1,
Table 1, and Fig. 1). This transposition model is suitable for studying
the mechanisms of adaptation of the exocrine pancreas to dietary
protein without any effects of the endogenous factor, BPJ.
We examined whether protein or peptides derived from the diet in the
small intestinal lumen are responsible for induction of BPJ-independent
pancreatic growth and induction of protease production on feeding a
high-protein diet (experiment 2). Luminal dietary protein or
peptides, but not amino acids, are involved in the induction of
pancreatic serine proteases in PBD rats (Fig. 2). The result indicates
that induction of protease production in PBD rats on feeding a
high-protein diet is not due to an increase in nitrogen flow into the
body, but it may be due to an increase in the concentration of peptides
derived from dietary protein in the intestinal lumen. In normal rats,
we demonstrated that dietary amino acids induced pancreatic protease
(14). In the PBD rats, the effects of amino acids in induction of
protease production may be masked by the strong and sustained
stimulation provided by chronic BPJ diversion. In contrast, the values
of the pancreatic growth variables, protein and RNA content, were increased in PBD rats fed a high-amino acid diet similar to those observed in the case of PBD rats fed a high-protein diet (Table 4).
This result suggests that amino acids largely absorbed under conditions
of high-nitrogen feeding are involved in the trophic effects of dietary
protein on the pancreas. The finding also suggests that the pancreatic
growth associated with high-protein feeding is regulated differently
from the induction of pancreatic protease.
Treatment with devazepide, a potent CCK-A receptor antagonist,
abolished pancreatic growth and the increase in levels of serine proteases observed on feeding a high-protein diet in the case of PBD
rats. The result indicates that the induction of pancreatic protease
production by dietary protein in the PBD rats depends on CCK. It is
reported that dietary protein or protein hydrolysate directly
stimulates CCK secretion by isolated mucosal cells of the rat small
intestine (28) or the neuroendocrine cell line STC-1 (5). The result of
the present study supports these previous findings. Recently, Lacourse
et al. (19) reported that the pancreatic protease levels in genetically
CCK-deficient mice changed as a result of pancreatic adaptation to
dietary protein levels. Our finding that induction of pancreatic
protease production by dietary protein in PBD rats totally depends on
CCK does not agree with their result. The reason for the discrepancy
between the results of the Lacouse report and ours is not known.
The results of an experiment using devazepide also showed that CCK was
involved in pancreatic growth induced by feeding the high-protein diet.
We suggest that absorbed amino acids play a role in the pancreatic
growth in rats fed a high-protein diet with the results of
experiment 2 as described above. However, amino acids may not
influence CCK release. We speculate that absorbed amino acids in rats
fed a high-protein diet do not stimulate CCK output, but the amino acid
may need CCK action to induce the pancreatic growth in PBD rats.
CCK mRNA abundance was increased as a result of feeding a high-protein
diet in the case of PBD rats (Fig. 5). This finding suggests that CCK
is involved in the BPJ-independent induction of pancreatic growth and
the induction of protease production by dietary protein. Also, this is
consistent with the finding that blocking of CCK action abolishes the
increases in the values of pancreatic growth variables and protease
activity levels, as shown in Table 5 and Fig. 4. The finding also
strengthens the view that pancreatic adaptation to a high-protein diet
does not depend on luminal BPJ in the ileum in the case of PBD rats
because enteroendocrine cells secreting CCK are mainly distributed in the proximal small intestine (16) and BPJ is absent from this segment
of the intestine in PBD rats. We confirmed that CCK mRNA is not
detectable in the ileum mucosa by poly(A)+ RNA Northern
blot analyses (unpublished data). Also, it is reported that
BPJ-dependent stimulation of pancreatic secretion does not work in the
ileum of normal rats (36) or PBD rats (17). Devazepide injection
increased the abundance of CCK mRNA in rats fed the 25% casein diet
(Fig. 5). CCK-A receptor antagonist treatment induces hypersecretion of
CCK and an increase in CCK mRNA levels in the intestinal tissue of
normal rats (9, 31). The results of the present study show that CCK-A
receptor blockage increases the abundance of intestinal CCK mRNA in PBD
rats, similar to that in normal rats.
Denervation of capsaicin-sensitive afferent nerves did not affect the
induction of pancreatic growth or the induction of pancreatic protease
production on feeding a high-protein diet in the case of PBD rats
(Table 6 and Fig. 7). It is reported that CCK released from the
proximal small intestine stimulates pancreatic secretion via the vagal
afferent pathway (7, 20). However, the present study using PBD rats
showed that feeding a high-protein diet induces pancreatic growth and
protease production independent of the capsaicin-sensitive vagal
afferent pathway. The increased level of CCK in PBD rats fed a
high-protein diet may act directly on the pancreas.
Increase in trypsinogen and chymotrypsinogen content in the pancreas by
feeding a high-protein diet was not associated with the increase in
mRNA abundance in PBD rats (see PBD rats in Fig. 3 and vehicle groups
in Fig. 5). This finding is consistent with the results of a previous
study using cannulated PBD rats (15), indicating that increases in
pancreatic protease in PBD rats are induced at the translation stage or
later. In contrast, the abundance of pancreatic serine protease mRNA
was increased by BPJ diversion (Fig. 3). The protease mRNA abundance in
PBD rats was markedly decreased as a result of devazepide treatment
(Fig. 5). These results indicate that the increases in these pancreatic
protease mRNA levels occurring as a result of BPJ diversion largely
depend on CCK action. It is reported that increases in pancreatic
protease mRNA levels were induced in the case of
hypercholecystokininemia (4, 35). The findings in the present study
show that induction of pancreatic protease with BPJ diversion depends
on mRNA levels; however, BPJ-independent induction of the pancreatic
protease by dietary protein is not associated with mRNA levels.
In conclusion, dietary protein was found to induce pancreatic growth
and pancreatic protease production in a manner independent of luminal
BPJ. Luminal protein or peptides derived from dietary protein are
responsible for the induction of pancreatic protease production but not
for the induction of pancreatic growth. The induction of pancreatic
growth and protease production in PBD rats is dependent on the direct
action of CCK on the pancreas.
 |
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: H. Hara,
Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido Univ., Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
(E-mail: hara{at}chem.agr.hokudai.ac.jp).
Received 23 August 1999; accepted in final form 7 January 2000.
 |
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