Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto 606-01, Japan and * Ajinomoto Co., Inc., Life Science Laboratories, Central Research Laboratories, Kawasaki 210, Japan
We investigated pancreatic enzyme secretion in response to soybean trypsin inhibitor (SBTI) in rats fed amino acids as a nitrogen source, from the fetal stage to adulthood. Pregnant rats were divided into two groups 4 d before parturition. During gestation and nursing, one group was fed a 15% protein diet (protein-fed rats) and the other (amino acid-fed rats) a 15% amino acid mixture diet that simulated the composition of the protein diet. Each male offspring was weaned at 4 wk after parturition and fed the same diet as fed to its dam for an additional 6 wk. Pancreatic amylase secretion in response to an intraduodenal infusion of SBTI (10 mg/rat) was observed in the protein-fed rats but not in the amino acid-fed rats. Amylase secretion in response to an intravenous injection of cholecystokinin (CCK) (10 ng/kg rat) was observed in both groups, and the magnitude of the response was significantly higher in the amino acid-fed rats than in the protein-fed rats. An increase in the level of plasma CCK in response to SBTI was observed in the protein-fed rats but not in the amino acid-fed rats. These results suggest that the long-term amino acid diet, because of its ability to inhibit the SBTI-stimulated CCK-releasing process in the small intestine of rats, reduced the pancreatic enzyme secretion response to a trypsin inhibitor. Six rats fed the amino acid mixture until 1 wk after weaning were fed the protein diet for the next 5 wk. These rats showed no pancreatic amylase secretion in response to SBTI, suggesting that dietary components around the weaning stage may affect the development of the ability of small intestinal cells to recognize a trypsin inhibitor.
KEY WORDS:
pancreatic enzyme secretion ·
trypsin inhibitor ·
amino acids ·
cholecystokinin ·
rats
Pancreatic enzyme secretion is one of the most important steps in the biological process promoting the overall digestion and absorption of nutrients. The secretion step includes the exocytosis of pancreatic zymogen granules, which could be termed the "short-term response" in pancreatic enzyme secretion. In rats, proteins and trypsin inhibitors are the most potent stimulants among food components for pancreatic enzyme secretion (Fushiki et al. 1984
, Schneeman et al. 1977). Trypsin inhibitor is proposed to stimulate pancreatic enzyme secretion through the same process as dietary proteins (Green et al. 1973). In rats, an amino acid mixture did not stimulate pancreatic enzyme secretion (Fushiki et al. 1984, Liddle et al. 1986, Schneeman et al. 1977). Except for proteins, several other dietary components tested did not induce pancreatic enzyme secretion (Fushiki et al. 1984, Liddle et al. 1986, Schneeman et al. 1977). This suggests that mainly proteins in the diet regulate the short-term response in pancreatic enzyme secretion in rats.
It is largely unknown how and at what stage of development dietary proteins first act as potent stimulants for pancreatic enzyme secretion in rats. It has been shown that nutritional manipulations influence the maturation of the gastrointestinal tract. For example, the ontogeny of Ia messenger RNA in the small intestinal epithelium of mice is modulated by diet (Sanderson et al. 1993). In rats, the complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, is a developmental event that is strongly influenced by the manipulation of nutritional factors during the weaning period (Lenoir et al. 1995). Thus, dietary manipulations during development might influence the rat recognition system for dietary proteins as stimulants of pancreatic enzyme secretion.
In the present study, we determined the short-term pancreatic enzyme secretion in response to intraduodenal infusion of soybean trypsin inhibitor (SBTI) in rats fed an amino acid diet throughout their lives. The pancreatic enzyme secretion response to the trypsin inhibitor disappeared as a result of the amino acid diet. The absence of pancreatic enzyme secretion response to SBTI seemed to be caused by the consumption of the diet around the weaning stage.
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Table 2.
Composition of the amino acid mixture1
[View Table]
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MATERIALS AND METHODS 
Animals and diets.
Pregnant female Wistar rats (Kitayama, Kyoto, Japan) were divided into two groups 4 d before parturition. One group was fed a 15% protein (whole egg) diet, and the other was fed a 15% amino acid mixture diet that simulated the composition of the whole egg protein diet, until they had weaned their offspring. Each dam was separately maintained in a dark cage, and the amount of daily diet was matched with that fed to a rat in the other group. The compositions of the protein and the amino acid mixture diets are shown in Table 1; composition of the amino acid mixture simulating whole egg is shown in Table 2. Three days after parturition, male offspring were selected and nursed. Litter sizes were five per dam. Each male pup was weaned at the age of 4 wk and was reared separately from its dam, being fed the same diet as that given fed the dam until the age of 10 wk. One week after weaning, six pups of the amino acid diet group were fed the protein diet until the end of the experimental period. Each dietary group of six pups was maintained in a cage, and amounts of diet fed were pair-matched between the dietary groups throughout the experimental period.
Animal operation.
At the end of the experimental period, the rats were deprived of food overnight and then injected with urethane (500 mg/kg) (TCI, Tokyo, Japan) and chloralose (50 mg/kg) (Wako Pure Chemical, Osaka, Japan) every hour. One hour after the first injection of anesthetics, a duodenal cannula was inserted. The main bile-pancreatic duct was then ligated at the Cater papilla, followed by the insertion of a bile-pancreatic cannula. Two hours after the operation, bile-pancreatic juice was collected at 15-min intervals for 2 h, the volume was measured, and then the juice was returned to the intestine, less a 10-µL sample retained for the enzyme assays. Ten milligrams of SBTI (type I-S, Sigma Chemical, St. Louis, MO) in 1 mL of saline, which was sufficient for maximal stimulation of pancreatic enzyme secretion in rats (Fushiki et al. 1987), was infused slowly into the duodenum via the duodenal cannula.
Fig. 1.
Time courses of soybean trypsin inhibitor (SBTI) stimulation on pancreatic amylase secretion in rats fed whole egg protein diet and rats fed the amino acid mixture diet. The SBTI was infused into the rat small intestine via a duodenal cannula. Bile-pancreatic juice was collected via a bile-pancreatic cannula and was returned to the small intestine continuously except for 10 µL for use in the enzyme assay at 15-min intervals. Values are expressed as the percentage of the secretion at time point 0. Values are means ± SEM, n = 7. The P values obtained with repeated measures ANOVA with diet as the variable and time as the repeated measure were 0.04 for the diet effect, 0.03 for the time effect and 0.03 for the diet × time interaction.
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Five rats (protein diet group) or six (amino acid diet group) rats were used to determine the pancreatic responsiveness to cholecystokinin (CCK). After the monitoring of the basal amylase secretion, CCK-8 (10 ng/kg) (obtained from Peptide Institute, Osaka, Japan) was injected intravenously as a bolus into each rat, and then bile-pancreatic juice was collected for 30 min. The content of amylase in the bile pancreatic juice was determined. After the determination of pancreatic amylase secretion, the pancreas, liver, spleen and kidney were removed and weighed.
This study was approved by the Kyoto University Animal Committee (Food Science and Technology Branch), and the animals were maintained in accordance with the guidelines of Kyoto University for the care and use of laboratory animals.
Enzyme assays.
Amylase (EC 3.2.1.1) activity was determined using a Diacolor-Amy kit (Toyobo Co., Osaka, Japan). This assay kit is based on a method using 
-(2,4-dichlorophenyl) maltopentaoside as a substrate. The activity was estimated by measuring the absorbance at 500 nm. Crystalline porcine amylase (type I-A, Sigma Chemical) was used as a standard (Tsuzuki et al. 1991).
Assay of plasma cholecystokinin concentration.
Ten milligrams of the SBTI in 1 mL of saline was infused into the proximal intestine of the rats after the above-mentioned treatments. Before or 30 min after infusion, blood was obtained from the portal vein with a heparinized syringe. The plasma was obtained by centrifugation at 900 × g for 10 min. Duplicate plasma samples were stored at 
60°C in tubes containing 20 mmol/L EDTA and 50 mg/L of aprotinin until the CCK assay. One milliliter of rat plasma was mixed with 3 mL of ethanol. The mixture was incubated for 30 min at 4°C and then centrifuged at 2200 × g for 30 min. The supernatant was collected and then evaporated. The dried material was dissolved in 12 mL of 0.03 mol/L phosphate buffer, pH 7.6, containing 20 mmol/L EDTA, 1 g/L bovine serum albumin and 20 mg/L aprotinin. Plasma CCK concentrations were measured by means of radioimmunoassay using a CCK-8 N-terminal specific antibody, OAL-656 (Iwai et al. 1986). The characteristics of this antibody were described elsewhere (Hashimura et al. 1982).
Statistics.
Results are expressed as means ± SEM. The data in Table 5 and Figures 1, 2 and 3 were analyzed by repeated measures ANOVA. When a significant difference was obtained, the means of individual groups were compared using two-tailed paired or unpaired t test. The data in Tables 3 and 4 were analyzed by the two-tailed unpaired t test. The difference between SBTI-stimulated and basal amylase secretion ratios in Table 5 was analyzed by one-way ANOVA with Bonferroni's test (Miller 1981). Differences with P < 0.05 were considered significant.
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Table 5.
Effect of protein, amino acid, and amino acid/protein diets on rat pancreatic amylase secretion in response to intraduodenal infusion of soybean trypsin inhibitor (SBTI)1
[View Table]
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Fig. 2.
Effects of long-term feeding of whole egg protein diet or amino acid mixture diet on basal- and cholecystokinin (CCK)-stimulated pancreatic amylase secretion. Bile-pancreatic juice was collected via a bile-pancreatic cannula for 30 min, and then CCK (10 ng/kg rat) was intravenously injected followed by the collection of bile pancreatic juice for 30 min. Values are means ± SEM (five rats for the protein diet group, six rats for the amino acid diet group). The P values obtained were 0.01 for the diet effect, <0.0001 for the CCK treatment effect and 0.007 for the diet × CCK interaction. Differences from the corresponding basal values: (*P < 0.05, **P < 0.01, paired t test).
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Fig. 3.
Effect of long-term feeding of whole egg protein diet or amino acid mixture diet on cholecystokinin (CCK) release into plasma in response to intraduodenal infusion of soybean trypsin inhibitor (SBTI). The SBTI was infused into the rat small intestine via a duodenal cannula. Thirty minutes after the infusion of SBTI, the plasma of each rat was collected. The methods for plasma preparation and CCK determination by radioimmunoassay are described in Materials and Methods. Values are means ± SEM (four rats for the protein diet group, six rats for the amino acid diet group). The P values obtained were 0.71 for the diet effect, 0.0005 for the SBTI effect and 0.0008 for the diet × SBTI interaction. Difference from the corresponding basal value: (*P < 0.01, paired t test).
[View Larger Version of this Image (66K GIF file)]
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Table 3.
Effect of diets containing whole egg protein or amino acid mixture on rat final body and organ weights of pancreas, spleen liver and kidney1
[View Table]
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Table 4.
Effect of diets containing whole egg protein or amino acid mixture on rat pancreatic amylase secretion response to soybean trypsin inhibitor (SBTI)1
[View Table]
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RESULTS
As shown in Table 3, the final mean body weights in the protein-fed group and the amino acid-fed group did not differ and were comparable with those of a similar previous study (Itoh et al. 1973). The liver (P < 0.01) and kidney (P < 0.05) weights in the rats fed the amino acid diet were significantly higher than those of rats fed the protein diet. These organ weight increases are probably due to increased nitrogen metabolism. The pancreas and spleen weights were not significantly different between the two groups.
The time courses of the SBTI stimulation on pancreatic amylase secretion are shown in Figure 1. The properties of the pancreatic amylase secretion response to SBTI in both dietary groups are summarized in Table 4. The amylase secretion at the peak time (45 min after SBTI infusion), the peak to basal ratio of the amylase secretion, and the integrated amylase secretion for 75 min after the SBTI infusion were significantly higher in the protein-fed rats than in the amino acid-fed rats (P < 0.05). The amylase secretion at the peak time was significantly higher than the corresponding basal secretion in the protein-fed rats (P < 0.05) but not in the amino acid-fed rats. The basal amylase secretion was not significantly different between the two groups. Taken together, these results indicate that the pancreatic enzyme secretion response to SBTI occurred in the protein-fed rats but not in the amino acid-fed rats.
The amylase secretions in response to the intravenous injection of CCK are shown in Figure 2. The amylase secretion was significantly increased in response to CCK in both groups. The CCK-stimulated to basal amylase secretion ratio was significantly higher in the amino acid-fed rats than in the protein-fed rats (5.32 ± 0.59 for the protein-fed rats, 11.2 ± 0.61 for the amino acid-fed rats; P < 0.0001, unpaired t test).
The intraperitoneal injection of carbachol (80 µg/kg), an analogue of acetylcholine that exerts pancreatic enzyme stimulatory effects via the nervous system, produced maximal pancreatic amylase secretions in both the protein- and the amino acid-fed rats, indicating that there was no difference in the pancreatic enzyme secretory function via the nervous system between the two groups (data not shown).
The CCK release into the blood in response to the intraduodenal infusion of SBTI in the two groups is shown in Figure 3. An elevation in the plasma CCK level in response to SBTI was observed in the protein-fed rats (P < 0.01) but not in the amino acid-fed rats. The SBTI-stimulated to basal CCK release ratio was significantly higher in the protein-fed rats than in the amino acid-fed rats (1.42 ± 0.19 for the protein diet group, 1.05 ± 0.11 for the amino acid diet group; P < 0.01).
The final body weight and pancreas weight in the rats first fed amino acids and then protein were not significantly different from those of the protein- or amino acid-fed rats (data not shown). The amylase secretions for 60 min before or after the intraduodenal infusion of SBTI in the three groups are shown in Table 5. No change in pancreatic amylase secretion in response to SBTI was observed in the rats fed amino acids and then protein (P = 0.07). The SBTI-stimulated to basal amylase secretion ratios were 1.36 ± 0.064 in the protein-fed rats, 0.86 ± 0.051 in the amino acid-fed rats and 0.96 ± 0.010 in the rats fed amino acids and then protein. The ratios for rats fed the protein diet and rats fed amino acids and then protein differed significantly (P < 0.001).
DISCUSSION
In the present study, amylase activity was measured as an index of the short-term response in pancreatic enzyme secretion. The change in amylase activity can be considered representative of the total pancreatic enzyme secretion, because each digestive enzyme is contained in a single zymogen granule in the pancreas, and the ratio between each secreted enzyme level in a short-term response is constant (Fushiki et al. 1984, Palade 1975).
Our results included the following: 1 ) an amino acid diet reduced the pancreatic response to SBTI and increased the pancreatic enzyme secretory response to CCK; 2 ) no apparent elevation of plasma CCK in response to SBTI occurred in the amino acid-fed rats; and 3 ) there was no apparent pancreatic amylase secretion response to SBTI in the rats fed amino acids and then protein. This is the first report of an amino acid mixture diet reducing pancreatic enzyme secretion in response to intraduodenal infusion of SBTI in rats.
The mechanism underlying the absence of the pancreatic enzyme secretion in response to SBTI is not clear. However, at least three possible factors are thought to be involved in the reduction of the pancreatic response: 1 ) pancreatic atrophy; 2 ) desensitization of pancreatic acinar cells to plasma CCK, or a post-CCK receptor defect; and 3 ) a defect of CCK production and/or CCK release in the small intestine. In the present study, the amount of food intake and body weight in each group were equally controlled by pair-feeding. Under the present experimental conditions, no significant loss of pancreas weight was observed in the amino acid-fed rats compared with the protein-fed rats. Thus, pancreatic atrophy does not seem to explain the reduction in the pancreatic response. The level of plasma CCK, which is produced in the small intestine (Schneeman and Lyman 1972), can be increased by intraduodenal infusion of SBTI, which stimulates the pancreas to secrete digestive enzymes (Rosewicz et al. 1989). Thus, there was a possibility that the absence of the pancreatic enzyme secretion response to SBTI in rats fed the amino acid diet was due to a reduction of the pancreatic responsiveness to CCK. However, it is also unlikely that the desensitization of pancreatic acinar cells to plasma CCK or a post-CCK receptor defect explains the reduction of the pancreatic enzyme secretion, because the pancreatic amylase secretion response to intravenous injection of CCK was unexpectedly higher in the amino acid-fed rats than in the protein-fed rats (Fig. 2). Taken together, the findings suggest that serious damage to pancreatic function did not reduce the pancreatic amylase secretion response to SBTI in the amino acid-fed rats. It is also unlikely that any serious defect of the nervous system to the pancreas occurred in the amino acid-fed rats, because carbachol maximally stimulated the pancreatic enzyme secretion in the amino acid-fed rats as well as in the protein-fed rats.
Although it is unclear whether CCK was produced in the small intestinal cells and released into plasma in the rats fed the amino acid diet, the lack of elevation of plasma CCK in response to the intraduodenal infusion of SBTI in the amino acid-fed rats can be attributed to the reduction of the pancreatic enzyme secretion. Intraduodenal infusion of trypsin inhibitors increased the production of CCK in the small intestine (Liddle et al. 1988) and enhances the release of CCK into plasma in rats (Rosewicz et al. 1989). Therefore, it is possible that prolonged amino acid diet intake causes a defect of small intestinal cells in terms of CCK release and/or production in response to the intraduodenal infusion of SBTI.
In the present study, no apparent pancreatic amylase secretion in response to SBTI was shown in the rats fed amino acids and then protein (Table 5). In other words, no pancreatic enzyme secretion in response to the trypsin inhibitor occurred even in the presence of dietary protein during growth after exposure to the amino acid mixture during weaning. This finding implies that the presence of dietary proteins around the weaning stage may be crucial for the normal development of the small intestine in rats for the secretion of pancreatic enzymes in response to dietary proteins. It has recently been reported that dietary manipulations at weaning time strongly influence the maturation of the small intestine; for example, the complete maturation of the brush-border membrane glycoproteins, and particularly their terminal fucosylation, was a developmental event that was strongly influenced by the manipulation of nutritional factors during the weaning period (Lenoir et al. 1995). Pierzynowski et al. (1990) reported that, in pigs, after weaning, the pancreatic juice secretion markedly increased with respect to both basal and postprandial levels and the enzyme composition of the pancreatic juice changed qualitatively during this period. They concluded that there was both an increase in exocrine pancreas function and a qualitative change in the hydrolytic enzyme pattern during porcine postnatal ontogeny, apparently correlated with the changes in diet around weaning. This may be a crucial period in the animal's life, because optimum food digestion and nutrient absorption are essential for its growth and health status. Taken together, these findings and ours indicate that it is possible that an exposure of the small intestinal cells of rats to an amino acid mixture during the weaning period causes some impairment in the rats' ability to recognize dietary proteins. At present, however, the possibility that the absence of dietary proteins around the weaning period can defeat the normal development of the small intestinal cells in rats cannot be excluded.
In conclusion, prolonged consumption of the amino acid diet reduced the SBTI-stimulated CCK-releasing process in the small intestine of rats. An impairment of the ability of small intestinal cells to recognize the trypsin inhibitor may have been caused by the exposure to amino acids and/or to the absence of dietary proteins around weaning. Stimuli (including dietary proteins) around the time of weaning may act on the development of the gastrointestinal tract via the hormonal system or nervous system or both.
Manuscript received 7 October 1996. Initial reviews completed 18 December 1996. Revision accepted 11 March 1997.
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ABSTRACT
