API

Relationship: 2028

Title

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Inhibition, trypsin leads to Increased monitor peptide

Upstream event

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Inhibition, trypsin

Downstream event

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Increased monitor peptide

Key Event Relationship Overview

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AOPs Referencing Relationship

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AOP Name Adjacency Weight of Evidence Quantitative Understanding
Trypsin inhibition leading to pancreatic acinar cell tumors adjacent Moderate Low

Taxonomic Applicability

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Term Scientific Term Evidence Link
Homo sapiens Homo sapiens Low NCBI
Macaca fascicularis Macaca fascicularis Low NCBI
Rattus norvegicus Rattus norvegicus High NCBI
Mus musculus Mus musculus High NCBI

Sex Applicability

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Sex Evidence
Mixed High

Life Stage Applicability

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Term Evidence
All life stages High

Key Event Relationship Description

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Pancreatic acinar cells secrete digestive enzymes including trypsin into the small intestine.

In rats, one of the pancreatic soluble trypsin inhibitors (TIs), monitor peptide (MP), is simultaneously secreted in the pancreatic juice. MP forms complexes with trypsin in the empty intestine, which keeps the intestinal level of free MP low. Once the gastric contents are transported to the small intestine, secretion of the pancreatic proteases including trypsin and MP is induced, where trypsin is used for protein hydrolysis, and the level of free MP is subsequently increased. The increased MP level stimulates CCK release from I cells lining the small intestinal mucosa via MP receptors, and the resulting increase in CCK stimulates exocrine secretion including MP from the pancreas. Increased MP further stimulates CCK secretion via a positive feedback loop as long as duodenal contents remain to consume trypsin for proteolysis.

After trypsin inhibitors are ingested, the intestinal content of free MP increases rapidly, especially in an empty intestine, via positive feedback regulation.

Evidence Supporting this KER

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TBD

Biological Plausibility

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Trypsin is a digestive enzyme secreted by pancreatic acinar cells that cleaves peptide bonds at the carboxyl end of basic amino acids (lysine and arginine). Secretion of pancreatic digestive enzymes including trypsin is regulated mainly by cholecystokinin (CCK) released from enteroendocrine I cells located in the duodenal mucosa of the small intestine [Wang BJ and Cui ZJ, 2007], and CCK release is controlled by multiple mechanisms [Caron J et al, 2017]. These mechanisms involve feedback regulation of trypsin-sensitive CCK-releasing peptides, one being positive feedback regulation of MP and the other negative feedback regulation of luminal CCK-releasing factor (LCRF) [Miyasaka K and Funakoshi A, 1998; Wang BJ and Cui ZJ, 2007; Guan D et al, 1990].

MP is one of the PSTIs in rats, which stimulates CCK release from duodenal enteroendocrine I cells as well as inhibition of trypsin activity. MP consists of 61 amino acids and has a molecular weight of approximately 6000. MP was first purified from rat pancreatic juice, and its amino acid sequence was subsequently determined [Iwai K et al, 1987; Lin YZ et al, 1990].

MP is bound to trypsin in the empty intestine. Once gastric contents are transported into the small intestine, secretion of the pancreatic proteases with MP is increased, where trypsin instead hydrolyzes these proteins, leading to an increase in the free MP level [Iwai K et al, 1988; Liddle RA, 1995; Graf R, 2006]. The increased level of MP stimulates CCK release from I cells, and then pancreatic exocrine secretion is stimulated [Liddle RA et al, 1992; Guan D et al, 1990; Cuber JC et al, 1990]. It was shown that MP binds to the surface of CCK-immunoreactive mucosal cells of the small intestine [Yamanishi R et al, 1993a; Yamanishi R et al, 1993b].

Following the increased secretion of pancreatic enzymes, proteolysis decreases intestinal protein contents, which once again decreases the intestinal level of free MP due to the excess of trypsin and in turn CCK release [Liddle RA, 1995; Miyasaka K and Funakoshi A, 1998; Graf R, 2006].

When raw soya flour (RSF), which contains trypsin inhibitory activity, or TIs such as camostat are ingested, trypsin activity is inhibited to increase the intestinal level of free MP especially in the empty intestine, followed by an increase in the blood level of CCK [Liddle RA, 1995; Miyasaka K and Funakoshi A, 1998]. TI ingestion-induced increases in blood levels of CCK leads to further CCK release due to increased pancreatic secretion of proteins including MP in a positive feedback manner. On the other hand, TIs may elevate the luminal concentration of LCRF to stimulate CCK release; however, this increase might not be as exaggerated as that of MP, because increased blood level of CCK does not induce further secretion of LCRF.

Empirical Evidence

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No study has shown a relationship between the trypsin inhibitor dose or degree of trypsin activity and the luminal concentration of MP. Trypsin inhibitor dosing and CCK levels  are presented here. Considering that MP directly stimulates CCK release from I cells in the small intestine in rodents, increased plasma CCK levels induced by TIs seems to be appropriate as a surrogate of increased luminal MP levels.

The plasma CCK8 level in rats after 18-hour fasting was 0.31 ± 0.05 pM (mean ± SE) and increased to 6.2 ± 1.8 pM 7.5 minutes after feeding and increased to 10.3 ± 1.8 pM 15 minutes after intragastric instillation of a soybean trypsin inhibitor [Liddle RA et al, 1984].

Immediately after oral feeding of camostat at 400 mg/kg in rats, the plasma CCK level increased 10-fold above that in controls, reached a maximum after 90 min, remained elevated for more than 6 h, and then returned to control levels 24 h after administration of camostat [Goke B et al, 1986].

Plasma concentrations of CCK were measured after administration of a single dose of 200 mg/kg camostat by gavage or 2.5 μg/kg CCK8 by subcutaneous administration to rats. The maximum CCK level, 9.6 ± 2.7 pM, was reached at 30 min after administration of CCK8, and that of 4.9 ± 1.2 pM over the time period of 15–240 min per animal with basal CCK concentration of about 2.5 pM [Douglas BR et al, 1989].

In isolated vascularly perfused rat duodenum/jejunum 30-min of infusion of trypsin with ovalbumin hydrolysate reduced CCK release by approximately 60% of that induced by the peptone alone. This effect was reversed by co-infusion of soybean trypsin inhibitor with the trypsin–peptone mixture [Cuber JC et al, 1990].

Eleven healthy volunteers consumed one of two meals: one containing raw soya flour and the other heat-treated soya flour. The two flours contained 34 and 3 mg trypsin/g flour, respectively. The peak CCK response was 16.8 ± 8.1 (mean ± SE) pmol/l for the raw soya flour diet versus 4.9 ± 2.8 pmol/l for the heat-treated soya flour diet (P < 0.05) [Calam J et al, 1987].

Uncertainties and Inconsistencies

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In normal rats, positive regulation of CCK release by MP seems to require some level of pancreatic secretion before to be effective. In the presence of nutritional protein in the duodenum, trypsin is used for digestion of protein and increased levels of MP stimulates CCK release. On the other hand, after most of the protein is digested, increased free MP might be inactivated with excess of trypsin or other proteases, as follows [Foltz M, 2008]:

1) MP is degraded by trypsin and other proteases.

2) MP forms a complex with trypsin as other PSTIs.

3) MP forms a complex with trypsin, thereafter degraded by proteases.

Quantitative Understanding of the Linkage

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TBD

Response-response Relationship

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No study has shown a direct quantitative relationship between MIE and KE1.

Time-scale

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No study has reported the time from trypsin inhibition to alteration of intestinal MP content. However, as mentioned above, treatment with trypsin inhibitors or MP increased the plasma concentration of CCK within 30 min in rats.

Known modulating factors

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Raw soya flour and trypsin inhibitors such as camostat inhibit trypsin activity, leading to an increase in CCK release from the upper intestine into the bloodstream, where the increased CCK released seems to be mediated by increased luminal concentration of MP due to trypsin inhibition [Green GM and Miyasaka K, 1983; Liddle RA et al, 1984; Goke B et al, 1986; Douglas BR et al, 1989; Cuber JC et al, 1990; Playford RJ et al, 1993; Obourn JD et al, 1997; Tashiro M et al, 2004; Komarnytsky S et al, 2011; Calam J et al, 1987] .

Known Feedforward/Feedback loops influencing this KER

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MP stimulates CCK release from intestinal I cells, and the increased CCK level in turn promotes pancreatic acinar cells to secrete pancreatic enzymes including CCK-stimulating MP. Therefore, MP-mediated CCK release is under positive feedback regulation [Liddle RA, 1995; Wang BJ and Cui ZJ, 2007; Chey WY and Chang T, 2001], and the effects of trypsin inhibitors seem robust. As discussed previously, trypsin-sensitive LCRF released from intestinal mucosal cells also stimulate duodenal I cells to release CCK with negative feedback loop.

Domain of Applicability

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Isoforms of trypsin are found in many species, for example, cationic and anionic trypsins (trypsins 1 and 2) and mesotrypsin in humans, cationic and anionic trypsins in cows, and anionic trypsin and P23 in rats [Chen JM and Claude Férec C, 2013; Fukuoka S and Nyaruhucha CM, 2002] . Despite differences among species, the three-dimensional structures of the isoforms are highly conserved among species, and the natural substrates for the enzymes are generally any peptide that contains Lys or Arg [Baird Jr TT, 2017]. The active site of trypsin has a specific catalytic triad structure composed of serine, histidine, and aspartate, and the flanking amino acid sequences are entirely conserved [Baird Jr TT and Craik CS, 2013; Baird Jr TT, 2017]. Therefore, trypsin inhibitors have comparable effects on the enzymatic activity of trypsin isoforms among animal species including humans and rats [Savage GP and Morrison SC, 2003].

MP secreted from rat pancreatic acinar cells into the small intestine stimulates I cells of the small intestinal mucosa to release CCK.

MP-like peptides are also found in rats and other mammalian species [Eddeland A and Ohlsson K, 1976]. Rat soluble trypsin inhibitor [Tsuzuki S et al, 1992; Tsuzuki S et al, 1991], human soluble trypsin inhibitor [Pubols MH et al, 1974; Kikuchi N et al, 1985], and bovine soluble trypsin inhibitor [Greene LJ and Giordano JS Jr, 1969; Guy O et al, 1971] are homologous peptides, all of which show trypsin inhibitory activity but no CCK-stimulatory activity [Miyasaka K et al, 1989a; Miyasaka K et al, 1989b; Marchbank T et al, 1998; Voet D and Voet JG, 1995].

References

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 2.    Baird Jr TT: Trypsin. Elsevier,2017

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 6.    Chey WY, Chang T: Neural hormonal regulation of exocrine pancreatic secretion. Pancreatology 1:320-335,2001

 7.    Cuber JC, Bernard G, Fushiki T, Bernard C, Yamanishi R, Sugimoto E, Chayvialle JA: Luminal CCK-releasing factors in the isolated vascularly perfused rat duodenojejunum. Am J Physiol 259:G191-197,1990

 8.    Douglas BR, Woutersen RA, Jansen JB, de Jong AJ, Rovati LC, Lamers CB: Modulation by CR-1409 (lorglumide), a cholecystokinin receptor antagonist, of trypsin inhibitor-enhanced growth of azaserine-induced putative preneoplastic lesions in rat pancreas. Cancer Res 49:2438-2441,1989

 9.    Eddeland A, Ohlsson K: Purification of canine pancreatic secretory trypsin inhibitor and interaction in vitro with complexes of trypsin-alpha-macroglobulin. Scand J Clin Lab Invest 36:815-820,1976

10.    Foltz M, Ansems P, Schwarz J, Tasker MC, Lourbakos A, Gerhardt CC: Protein hydrolysates induce CCK release from enteroendocrine cells and act as partial agonists of the CCK1 receptor. J Agric Food Chem 56:837-843,2008

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26.    Lin YZ, Isaac DD, Tam JP: Synthesis and properties of cholecystokinin-releasing peptide (monitor peptide), a 61-residue trypsin inhibitor. Int J Pept Protein Res 36:433-439,1990

27.    Marchbank T, Freeman TC, Playford RJ: Human pancreatic secretory trypsin inhibitor. Distribution, actions and possible role in mucosal integrity and repair. Digestion 59:167-174,1998

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37.    Tsuzuki S, Miura Y, Fushiki T, Oomori T, Satoh T, Natori Y, Sugimoto E: Molecular cloning and characterization of genes encoding rat pancreatic cholecystokinin (CCK)-releasing peptide (monitor peptide) and pancreatic secretory trypsin inhibitor (PSTI). Biochim Biophys Acta 1132:199-202,1992

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41.    Yamanishi R, Kotera J, Fushiki T, Soneda T, Saitoh T, Oomori T, Satoh T, Sugimoto E: A specific binding of the cholecystokinin-releasing peptide (monitor peptide) to isolated rat small-intestinal cells.. Biochem J 291 ( Pt 1):57-63,1993b