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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 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 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.

                              
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Table 1.   Composition of test diets

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 beta -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 beta -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 beta -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 beta -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 beta -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 beta -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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

                              
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Table 2.   Body weight gain and food intake in four separate experiments

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).

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).

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).

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).

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).

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 beta -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).

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 (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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 278(6):G937-G945
0193-1857/00 $5.00 Copyright © 2000 the American Physiological Society




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