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Recent studies suggest that there is a chemical perception of dietary fat in the oral cavity. A much greater response in the cephalic phase of pancreatic secretion is obtained by palatable taste stimuli. To examine the perception of dietary fat in the oral cavity, we determined whether or not oral stimulation with fat alters the cephalic phase of pancreatic enzyme secretion. A cannula was inserted into the common bile duct of rats to collect bile-pancreatic juice, and the animals were esophagostomized to prevent stimulation of the gastrointestinal tract with the test substances. The cephalic phase of pancreatic enzyme secretion was enhanced by oral stimulation with the long chain fatty acids, oleic acid, linoleic acid and linolenic acid, but was unaffected by the middle chain fatty acid, caprylic acid, and long chain fatty acid derivatives, methyl oleate and methyl linoleate. This selectivity is consistent with our previous findings that, in a short term two-bottle preference test, rats prefer long chain fatty acids to triglycerides, middle chain fatty acids or long chain fatty acid derivatives. These results suggest that long chain fatty acids trigger chemical reception in the oral cavity, and the length of the fatty acid chain and the presence or absence of carboxylate groups are related to recognition of fat.

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.

We explored the mechanism(s) by which cholecystokinin (CCK) stimulation of AR42J rat pancreatoma cells results in increased mRNA expression of a CCK-releasing peptide [monitor peptide (MP)]. With the use of a newly established reverse transcription-polymerase chain reaction assay system, CCK was shown to increase the level of MP mRNA by about ninefold. When protein synthesis was blocked by addition of cycloheximide, the MP mRNA level remained unchanged in the presence of CCK. Inhibition of transcription with actinomycin D resulted in a half-life for MP mRNA of approximately 17 h, and this rate remained unchanged after CCK treatment, suggesting that CCK may regulate the MP mRNA level by influencing gene transcription. A-23187, bombesin, substance P, and carbachol increased the MP mRNA level. CoCl(2) abolished actions of both CCK and A-23187 on MP mRNA expression. Dibutyryl-adenosine 3',5'-cyclic monophosphate, forskolin, secretin, and vasoactive intestinal polypeptide had no effect on MP mRNA expression. 12-O-tetradecanoylphorbol-13-acetate and phorbol 12,13-dibutyrate also failed to increase MP mRNA. It was therefore proposed that CCK stimulates MP mRNA expression of AR42J cells in a Ca2+-dependent and protein kinase C-independent manner.

The genes encoding a rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and its structurally related peptide, rat pancreatic secretory trypsin inhibitor (PSTI), have been isolated and sequenced. The two genes share extremely high sequence similarity in the 5' flanking regions, suggesting that these regions may be responsible for the characteristic coordinate expression of the two peptides.

Monitor peptide (CCK-releasing peptide) mRNA increased on the administration of CCK in rat pancreas and the AR4-2J pancreatic cell line. Subcutaneous injection of CCK into rats at 8 h intervals increased the level of monitor peptide mRNA in the pancreas. Concomitant injection of CCK antagonist CR-1409 strongly decreased it. The monitor peptide mRNA was also increased by CCK in AR4-2J cells and was decreased by the antagonist. These findings suggest that the plasma CCK induced by prolonged intake of a high protein diet may be responsible for the adaptative increase in the monitor peptide as well as exocrine proteases in the pancreas.

The adaptation to a high protein diet of the concentration and mRNA level of a trypsin-sensitive, cholecystokinin-releasing peptide (monitor peptide), which was proposed to be the mediator of the cholecystokinin release in response to protein intake, was investigated in the rat pancreas. Adult rats were placed on one of two isocaloric diets. One group was fed a 22% casein diet (control diet) and the other a 64% casein diet (high-protein diet) for 14 days. In order to quantify the monitor peptide separately from pancreatic secretory trypsin inhibitor (PSTI-II), which is highly similar in its amino acid and mRNA nucleotide sequences to the monitor peptide but has less cholecystokinin-releasing activity, we used specific assay methods: HPLC was used for determining the monitor peptide concentration in zymogen granules and a synthetic oligonucleotide probe for determining the mRNA of the monitor peptide in the pancreas. The concentrations in the zymogen granules and the mRNA levels in the pancreas of the two peptides increased in parallel during the adaptation to the high protein diet, indicating that these two peptides were under the same control during the adaptation. The concentration and mRNA level of the monitor peptide, which were measured after 0, 3, and 14 days, increased throughout the experiment period, as did the concentration of trypsin. This suggested that the monitor peptide and trypsin may respond to similar signals during the adaptation to a high protein diet and that this apparent coordination may facilitate the adaptation of the pancreas to the diet.

A new model is proposed for pancreatic enzyme secretion in response to food protein intake in rats. We have found a novel peptide in rat bile-pancreatic juice, which exhibits a trypsin-sensitive, cholecystokinin (CCK)-releasing activity. The amino acid sequence of the peptide purified from rat bile-pancreatic juice is very similar to that of a conservative region in pancreatic secretory trypsin inhibitor (PSTI). The peptide loses its CCK-releasing activity during trypsin digestion, but food protein intake prevents this trypsin digestion. Results of a reconstitution experiment indicate that pancreatic enzyme secretion in response to food protein intake only occurs as a result of interaction between trypsin and our purified peptide. Also, a peptide-specific antibody abolished the response. These findings lead us to hypothesize that this peptide acts as an intraduodenal mediator for CCK release in response to food protein intake.

The rat plasma cholecystokinin (CCK) concentration was measured after intestinal administration of a peptide purified from rat bile-pancreatic juice, which has a stimulatory effect on pancreatic enzyme secretion. The plasma CCK concentration was measured by means of a radioimmunoassay using CCK-8 N-terminal specific antibody, OAL-656. In experimental rats with protease-free intestines, intraduodenal infusion of 10 micrograms of the purified peptide, which stimulates pancreatic enzyme secretion 2.0-2.5 fold, induced a significant increase in the plasma CCK level. Furthermore, after removal of CCK from the plasma by immunoabsorption with an OAL-656-bound Sepharose 4B column, the stimulatory effect of the plasma on pancreatic enzyme secretion was abolished when it was injected intravenously into recipient rats. It was concluded that this peptide stimulates the release of CCK in the intestine and that this is responsible at least in part for the pancreatic enzyme secretion-stimulating activity of the peptide.

We review the mechanisms underlying the feedback regulation of pancreatic enzyme secretion in response to a meal. Pancreatic enzyme secretion in the rat and pig is known to be regulated by a negative feedback mechanism mediated by intestinal trypsin and chymotrypsin. Such a mechanism has recently been noted in humans. The presence of these enzymes in the small intestine suppresses pancreatic enzyme secretion, whereas their removal increases it. Two novel peptides have been proposed to account for the stimulation of pancreatic enzyme secretion in response to feeding trypsin inhibitor. One was assumed to be present in rat pancreatic juice and the other to be spontaneously secreted from the rat small intestine. In either case, trypsin and trypsin inhibitors do not directly interact with the luminal surface of the small intestine, but their actions are mediated by a trypsin-sensitive, cholecystokinin-releasing peptide. This is a novel explanation of the well-recognized stimulation of pancreatic enzyme secretion in response to dietary protein intake.

New evidence has been obtained suggesting that the "monitor" peptide is an essential intraluminal mediator in the stimulation of pancreatic enzyme secretion in response to protein intake in rats. Experiments were performed in vivo using a mixture of 50 mg of ovalbumin, alpha-lactalbumin or casein, 2 micrograms of purified protease-sensitive, cholecystokinin-releasing monitor peptide and 1 mg of porcine trypsin which was infused by cannula into the duodenum of atropine-treated rats. The small intestine had previously been washed with bicarbonate to eliminate proteases and the pancreatic juice was diverted. The amount of trypsin secreted in 2 h was comparable to that of rats in which the pancreatic juice was returned into the duodenum. However, in the presence of a monitor peptide--specific antibody which recognizes the N-terminal region of the peptide, the monitor peptide did not induce any pancreatic response. Therefore, the characteristic pattern of pancreatic enzyme secretion in response to protein intake can be reproduced by infusing only three components--dietary proteins, porcine trypsin and the purified monitor peptide.

The effect of atropine on pancreatic enzyme secretion in response to removal of intraluminal protease was investigated using rats with an indwelling bile-pancreatic cannula. Stimulation of pancreatic enzyme secretion by diversion of bile-pancreatic juice was blocked by intravenous atropine [100 micrograms/(kg X h)] injection. On the other hand, stimulation of pancreatic enzyme secretion by intraluminal infusion of soybean trypsin inhibitor (SBTI) was not blocked by atropine administration. These results suggest that the feedback response of pancreatic enzyme secretion after removal of the intraluminal protease is controlled by at least two different systems, an atropine-sensitive mechanism and an atropine-nonsensitive mechanism. We detected and purified a peptide from rat bile-pancreatic juice that enhanced pancreatic enzyme secretion when the peptide was infused into the proximal intestine. In the atropine-administered rat whose small intestine was deprived of protease after intraluminal washing with saline containing SBTI, intraluminal infusion of the purified peptide stimulated pancreatic enzyme secretion markedly. These findings suggest that the atropine-nonsensitive feedback response of pancreatic enzyme secretion is at least in part mediated by the peptide.

Effects of neural blockages on pancreatic enzyme secretion in response to infusing fatty acid into the lumen were investigated using anesthetized rats, equipped with bile-pancreatic and duodenal cannulae, to evaluate the relative contribution of the neural and the hormonal mediations in the pancreatic response. Oleate (0.2 ml) was injected as a bolus into the rat duodenum, and the trypsin output in bile-pancreatic juice was monitored to determine the pancreatic enzyme secretion response with continuous return of bile-pancreatic juice to the intestine. When anticholinergic agents such as atropine sulfate and scopolamine were administrated, although basal level in pancreatic enzyme secretion fell, the increase of pancreatic enzyme secretion from basal level after stimulation by oleate was not significantly different from that of the control (no blockage). However, the ganglion blocker, hexamethonium, inhibited the pancreatic enzyme secretion in response to oleate by 94%. An adrenergic blocker, guanethidine, also led to as much of a decrease as the ganglion blocker-induced decrease. Adrenergic alpha- and beta-blockers partially, but not completely, inhibited the enzyme secretion. Adrenergic blockage also suppressed the basal level in pancreatic enzyme secretion. On the other hand, a specific CCK antagonist, proglumide, significantly inhibited pancreatic enzyme secretion induced by oleate in the presence of scopolamine. These observations suggest that pancreatic enzyme secretion in response to oleate is primarily mediated by CCK and that adrenergic modulation may play an important role in the CCK-mediated pancreatic enzyme secretion in response to oleate, although interpretation of these results may have some restriction related to anesthesia.

The intracellular localization of pancreatic enzyme secretion-stimulating activity in rat pancreas was investigated. We found and purified a pancreatic enzyme secretion-stimulating peptide from rat bile/pancreatic juice. The peptide is trypsin-sensitive (showing temporary trypsin inhibitory activity), and it is hypothesized that it acts as a trypsin-sensitive mediator in the feedback regulation of diet-induced pancreatic enzyme secretion. The zymogen granule fraction was purified 5-fold by ultracentrifugation by the Percoll density gradient method. The purity of the zymogen granule fraction was determined from the specific amylase activity and electron microscopic morphology. The specific enzyme activities of amylase and trypsin and the trypsin inhibitory activity increased in parallel during the purification, and the pancreatic enzyme secretion-stimulating activity was also localized in the zymogen granule fraction. These results suggest that the pancreatic enzyme secretion-stimulating peptide originates from the acinar cells, and that it is secreted through exocytosis of zymogen granules into the small intestine, its ratio to trypsin thus remaining constant. This idea supports our hypothesis that the stimulating peptide acts as a mediator for the feedback regulation of pancreatic enzyme secretion by trypsin.

We purified a peptide from rat pancreatic juice that enhances pancreatic enzyme secretion in a manner different from that of exogenous trypsin inhibitors, such as soybean trypsin inhibitor, when it is infused into the rat intestine. In this paper, we present evidence for the release of gut hormone(s) into the blood of rats after intestinal administration of the peptide. In the presence of atropine, an anesthetized rat small intestine was washed out with saline containing soybean trypsin inhibitor (Kunitz type) to eliminate proteases. Under these conditions, the rat small intestine was divided into four equal parts by ligation. Administration of the peptide into the first quarter of the small intestine stimulated pancreatic enzyme secretion. However, administration into the more distal parts did not stimulate the enzyme secretion. Moreover, intravenous injection of 1 mL of plasma from rats in which the peptide had been infused into the duodenum caused stimulation of pancreatic enzyme secretion in the recipient rats. It was suggested that the purified peptide acts in the proximal small intestine and that it stimulates the release of gut hormone(s) into the blood to enhance pancreatic enzyme secretion. These findings support the hypothesis that the peptide we purified is responsible in part for the humoral control of pancreatic enzyme secretion in the response to food intake.