![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
,2
* Northern Advancement Center for Science and Technology, Colabo-Hokkaido, Sapporo 001-0021, Japan and
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2To whom correspondence should be addressed. E-mail: hara{at}chem.agr.hokudai.ac.jp.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
and ' subunits ( 212–224 and ' 230–240) had less binding ability than did ß 51–63. Model peptides constructed with arginine (R) and glycine (G), such as GRGRGRG, had strong binding affinity, but peptides containing a single R or RR did not. These results indicate that the ß-conglycinin ß 51–63 fragment is the bioactive appetite suppressant in ß-conglycinin, and multiple arginine residues in the fragment may be involved in this effect.
KEY WORDS: • ß-conglycinin • cholecystokinin • food intake • arginine • brush border membrane
Cholecystokinin (CCK) is an important physiologic endocrine factor in appetite control (1). CCK is produced in the brain and enteroendocrine cells, and food intake suppression by CCK in the central nervous system is well established (2). Peripheral CCK is also involved in food intake suppression because abdominal trunkal vagotomy has been shown to abolish anorexia caused by intraperitoneal injection of CCK octapeptide (CCK-8) (3).
Plasma CCK concentration increases after eating. Dietary protein and fatty acids stimulate CCK release (4,5). Endogenous CCK has an important role in inducting anorexia by intraduodenal infusion of peptone and oleic acid (6–8). In rats, it is thought that dietary protein-induced CCK release from the intestine is regulated by negative feedback control in luminal trypsin activity (4). However, we and other researchers recently found that some kinds of dietary protein hydrolysates react directly on the small intestine and CCK-producing culture cells to release CCK (9–12). Our further study demonstrated that a peptic hydrolysate of soybean ß-conglycinin is a potent stimulator of CCK release from intestinal mucosal cells and inhibits food intake and gastric emptying through this CCK release (13).
The purpose of the present study was to define and characterize the active site in ß-conglycinin concerned with CCK release and food intake suppression. ß-Conglycinin is formed by 3 types of subunits (, ' and ß); the amino acid sequence of each subunit is already known (14,15). We previously showed that the arginine residue in protein structures was responsible for CCK release through direct action on the intestinal cells (11,12). Therefore, to focus on the arginine residues in the ß-conglycinin, we used peptone and tryptone digestion to identify whether internal or C-terminal basic amino acid residues were essential for brush border membrane binding. Then, we synthesized peptides of ß-conglycinin fragments with an arginine-concentrated structure and examined their abilities to bind to the intestinal cell component as well as their effect on CCK release and appetite suppression. Furthermore, we also assessed the binding activities of synthetic model peptides containing multiple arginine residues to examine the role of arginine in peptide structure on binding to the brush border membrane.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Male Sprague-Dawley rats, weighing 
250–350 g (Japan SLC, Hamamatsu, Japan), were kept in individual stainless steel cages in a room maintained at 23 ± 2°C with a 12-h light:dark cycle; rats were fed a casein-based basal stock diet (Table 1) during the acclimation period. The Hokkaido University Animal Committee approved the study, and animals were maintained in accordance with the guidelines for the care and use of laboratory animals of Hokkaido University.
|
|
|
|
Binding of synthetic peptides to rat intestinal brush border membranes.
The binding of ß-conglycinin peptone (peptic hydrolysate), ß-conglycinin tryptone (tryptic hydrolysate), synthetic ß-conglycinin fragments and model peptides to rat intestinal brush border membranes was estimated using a BIACORE 3000 system (BIACORE AB, Uppsala, Sweden) as described (13). Briefly, the soluble components of rat jejunal brush border membrane were prepared by solubilization with detergent Triton X-100; they were immobilized on a flow cell surface of the sensor chip of BIACORE by an amine-coupling procedure (16). Ethanolamine was immobilized on another flow cell of the same tip to serve as a reference flow. Test hydrolysates or peptides in HEPES buffer containing 150 mmol/L NaCl and 3 mmol/L EDTA, pH 7.4, were injected over both the test flow cell and reference flow cell at a rate of 10 µL/min for 2 min. The amount of the fragment bound to the soluble brush border membrane was represented in resonance units (RU) and was calculated from the maximum response value in the immobilized soluble brush border membrane cell subtracted from the increase in the reference flow cell. A representative binding sensorgram of the fragment to the brush border membrane components is shown in Fig. 1A.
|
Rats were implanted with a duodenal cannula (Silascone No. 00, i.d. 0.5 mm, o.d. 1.0 mm; Dow Corning, Kanagawa, Japan) 1 cm distal from the pyloric sphincter. Another cannula for intravenous injection of devazepide was implanted into the jugular vein and was filled with heparinized saline (104 U/L). These cannulae were inserted subcutaneously behind the neck and covered with a protector made of vinyl tubing. The jugular vein cannula was flushed with heparinized saline every other day to maintain patency in the postsurgical period. Rats were allowed to recover for 7 d with a basal diet and then were deprived of food overnight before the experiment.
The duodenal cannula was connected to polyethylene tubing (SP 28, i.d. 0.4 mm, o.d. 0.8 mm; Natsume Seisakusyo, Tokyo, Japan) that was passed through a protective steel pipe, and the test substance in distilled water was infused into the duodenum through the tube for 5 min at a flow rate of 0.5 mL/min. Ten minutes after the end of infusion, the rats were given 15 g of basal diet for 1 h. The weight of the ingested diet was calculated by subtracting the weight of the uneaten portion during the feeding period from the weight of the total diet given. In the devazepide study, rats were administered a bolus intravenous injection of 500 µg/kg devazepide or its vehicle (5% dimethylsulfoxide:5% Tween 80:90% saline) 10 min before intraduodenal infusion of the test substance solution. Devazepide, which is a selective CCK-A receptor antagonist, was kindly donated by ML Laboratories PLC (Liverpool, UK). Each rat was randomly assigned to all groups in a study at 3-d intervals.
Plasma CCK levels after infusion of the ß-conglycinin fragment.
The portal blood for measurement of CCK concentration in the plasma was collected under anesthesia 45 min after the start of infusion of test solution. The plasma separated from the portal blood by centrifugation (2000 x g, 20 min, 4°C) was stored at -40°C until CCK measurement. Plasma CCK was extracted by a Sep-pak C18 cartridge (Waters, Milford, MA), and its concentration was measured by bioassay using rat acini (17). Synthetic CCK-octapeptide sulfate (CCK-8; Peptide Institute, Osaka, Japan) was used as a standard, and CCK concentration in plasma was estimated as a CCK-8 equivalent.
Statistical analyses.
All results are expressed as means ± SEM. Data were analyzed by one- or two-way ANOVA and significant differences between groups were determined by Duncan’s multiple range test (P < 0.05). Comparison of the binding response of each fragment to ß-conglycinin peptone was performed by Student’s t test (P < 0.05).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Both ß-conglycinin peptone and tryptone bound dose dependently to the solubilized rat brush border membrane. The amount of ß-conglycinin peptone bound to the membrane was 200% greater than that of its tryptone (Fig. 1B).
A fragment of the ß-conglycinin ß 51–63 subunit (ß 51–63) had the strongest binding to the rat intestinal brush border membrane. Its binding activity was greater than that of the ß-conglycinin peptone. Other synthetic ß-conglycinin fragments did not have binding activity as high as that of ß 51–63 (Table 2). The binding abilities of ß 51–59, ß 53–63 and ß 53–59, which are shortened peptides of ß 51–63, to the rat intestinal cell components were lower than that of ß 51–63. The binding abilities of 212–224 and ' 230–240, peptides very similar to ß 51–63, to the intestinal cell components were also lower than that of ß 51–63 (Table 3).
Two types of synthetic model peptides containing one arginine (GGGRGGG and GGGGGGR) did not bind to the components of rat intestinal brush border membranes. Among the synthetic peptides containing two arginine residues, GGGRRGG also did not bind to the brush border membrane; however, peptides GGRGRGG, GRGGRGG and GRGGGRG did bind to it. A synthetic peptide containing three arginine residues (GRGRGRG) had greater binding ability than did the peptides containing two arginine residues (Table 4).
Food intake study.
Food intake for 1 h after overnight food deprivation was markedly suppressed by the intraduodenal infusion of 5 mg (2.5 mL of 2 g/L) ß-conglycinin peptone compared with water infusion. The infusion of 50 µg (20 mg/L = 12 µmol/L) of ß 51–63 had an effect similar to that of 5 mg ß-conglycinin peptone (Fig. 2). Note that the concentration of ß 51–63 corresponds to that in 2 g/L ß-conglycinin peptone.
|
|
Food intake in the vehicle-injected group infused with 3 µmol/L of ß 51–63 was lower than in the water-infused vehicle-injected group (Fig. 4A). This decrease did not occur in the group infused with 3 µmol/L of ß 51–63 but injected with devazepide. The portal plasma CCK concentration 45 min after the infusion of 3 µmol/L of ß 51–63 was about threefold greater than after the water infusion. (Fig. 4B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ß-Conglycinin peptone had high binding activity to components of the rat intestinal brush border membrane; however, the binding activity of ß-conglycinin tryptone was markedly lower. Trypsin hydrolyzes protein at basic amino acids (arginine and lysine) in the amino acid sequence of protein, which suggests that internal, but not C-terminal basic amino acid residues in ß-conglycinin are responsible for the binding to the brush border membrane. We demonstrated previously that a peptide structure rich in arginine is involved in the stimulation of CCK release from intestinal mucosal cells (11,12). From these results, we selected peptide fragments containing two or more arginine residues from three subunits of soybean ß-conglycinin and examined their binding ability and suppressive effects on food intake.
The fragment ß 51–63 (VRIRLLQRFNKRS) had the greatest binding ability to components of the rat intestinal brush border membrane of all the ß-conglycinin fragments that had multiple nonadjacent arginine residues. The binding ability of ß 51–63 was also significantly greater than that of ß-conglycinin peptone. We determined previously that the threshold for the suppression of food intake by ß-conglycinin peptone was 2 g/L (13). The duodenal infusion of 20 mg/L (12 µmol/L) ß 51–63 decreased food intake to the same degree as 2 g/L ß-conglycinin peptone (Fig. 2), the respective dose of ß 51–63 (20 mg/L). However, the dose-response experiment showed that the threshold of ß 51–63 was 3 µmol/L (Fig. 3), which is much less than the effective dose of ß-conglycinin peptone noted above. These results suggest that there is a pepsin cleavage site in ß 51–63, and ß-conglycinin peptone may not include the intact active peptide. The active fragment in ß-conglycinin for suppression of food intake, ß 51–63, may be partially degraded by pepsin hydrolysis because the binding ability of ß-conglycinin peptone was much less than that of ß 51–63.
Portal CCK concentration increased markedly with the intraduodenal infusion of 3 µmol/L ß 51–63, and the effect of ß 51–63 on food intake suppression was completely abolished by the intravenous injection of devazepide (Fig. 4). These findings indicate that luminal ß 51–63 stimulates CCK release from the small intestine and the endogenous CCK mediates the reduction of food intake via peripheral CCK receptors. In rats, three types of endogenous CCK-releasing peptides exist in the lumen (18–20). These peptides react directly with intestinal CCK cells to stimulate CCK release from the cells. The peptides are very sensitive to trypsin and contain basic amino acids in the active site. However, their amino acid sequences (19–21) have no homology with those of ß 51–63. Bombesin acts on CCK cells from the basolateral side of the membrane and prompts the release of CCK (22). The amino acid sequence of this peptide hormone (23) is unlike that of ß 51–63. As an exogenous CCK-releasing substance, caseinomacropeptide has CCK-releasing activity via direct reaction with the small intestine (9). However, this peptide also does not resemble ß 51–63, which has five basic amino acids in its amino acid sequence. This peptide likely acts as a substrate of trypsin in the lumen. Duodenal ß 51–63 possibly masks luminal trypsin activity and releases CCK via negative feedback regulation. However, the dose of ß 51–63 used in our food intake study did not mask trypsin (data not shown). We found that ß 51–63 binds strongly to the brush border membrane. ß 51–63 may be a novel dietary protein–derived active peptide, which releases intestinal CCK as a luminal factor. As a nonluminal suppressor, Albutensin A, which originates from serum albumin, is a bioactive peptide that inhibits food intake via intracerebroventricular or intraperitoneal administration (24). However, the amino acid sequence of this peptide also has no homology with ß 51–63. The effects of parenteral administration of ß 51–63 are not known and should be examined in future studies.
To examine the role of arginine in peptides on binding to the brush border membrane, the binding activities of several synthetic model peptides containing arginine were measured. Peptide structures having multiple nonadjacent arginine residues are a necessary condition for binding to the brush border membrane (Table 4). The peptide GRGRGRG had the greatest binding activity. The binding activities of the smaller fragments of ß 51–63 (ß 51–59, ß 53–63 and ß 53–59) to the brush border membrane components were lower than that of intact ß 51–63. The results suggest that the number of arginine residues in the peptide is a factor related to binding to the cell membrane. However, except for ß 51–63, ß-conglycinin fragments did not have high binding activity; in particular, the fragment in which glycine in the GRGRGRG was replaced by proline (PRPRPRP) had only 17% of the binding activity of GRGRGRG (Table 2). Additionally, two fragments similar to ß 51–63 ( 212–224 and ' 230–240) had considerable binding abilities although less than that of ß 51–63 (Table 3). These results suggest that amino acids in addition to arginine also affect the binding activity of the peptide to the brush border membrane.
The present study shows that ß 51–63 suppresses food intake through CCK-A receptors. However, it is not known whether another mediator is involved in the suppression of food intake by ß 51–63. Some reports demonstrated that other endogenous mediators act synergistically with peripheral CCK to suppress food intake (25,26). The role of these mediators in the effect of ß 51–63 on the suppression of food intake also requires clarification.
In conclusion, we found that the ß-conglycinin ß 51–63 fragment is a bioactive peptide that suppresses appetite. It is possible that this peptide in the lumen interacts directly with the intestinal mucosal cells to stimulate CCK release as an exogenous CCK-releasing peptide, and the released CCK suppresses food intake via CCK-A receptors.
|
FOOTNOTES |
|---|
3 CCK, cholecystokinin; CCK-8, CCK-octapeptide sulfate; RU, resonance unit.
Manuscript received 28 January 2003. Initial review completed 4 March 2003. Revision accepted 2 June 2003.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Reidelberger, R. D. (1994) Cholecystokinin and control of food intake. J. Nutr. 124:1327S-1333S.
2. Baile, C. A., McLaughlin, C. L. & Della-Fera, M. A. (1986) Role of cholecystokinin and opioid peptides in control of food intake. Physiol. Rev. 66:172-234.
3. Smith, G. P., Jerome, C., Cushin, B. J., Eterno, R. & Simansky, K. J. (1981) Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science (Washington, DC) 213:1036-1037.
4. Liddle, R. A., Green, G. M., Conrad, C. K. & Williams, J. A. (1986) Proteins, but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am. J. Physiol. 251:G243-G248.
5. Lewis, L. D. & Williams, J. A. (1990) Regulation of cholecystokinin secretion by food, hormones, and neural pathways in the rat. Am. J. Physiol. 258:G512-G518.[Medline]
6. Trigazis, L., Orttmann, A. & Anderson, G. H. (1997) Effect of a cholecystokinin-A receptor blocker on protein-induced food intake suppression in rats. Am. J. Physiol. 272:R1826-R1833.
7. Woltman, T. & Reidelberger, R. (1999) Role of cholecystokinin in the anorexia produced by duodenal delivery of peptone in rats. Am. J. Physiol. 276:R1701-R1709.[Medline]
8. Woltman, T., Castellanos, T. D. & Reidelberger, R. (1995) Role of cholecystokinin in the anorexia produced by duodenal delivery of oleic acid in rats. Am. J. Physiol. 269:R1420-R1433.
9. Beucher, S., Levenez, F., Yvon, M. & Corring, T. (1994) Effects of gastric digestive products from casein on CCK release by intestinal cells in rat. J. Nutr. Biochem. 5:578-584.
10. Némoz-Gaillard, E., Bernard, C., Abello, J., Cordier-Bussat, M., Chayvialle, J. A. & Cuber, J. C. (1998) Regulation of cholecystokinin secretion by peptones and peptidomimetic antibiotics in STC-1 cells. Endocrinology 139:932-938.
11. Nishi, T., Hara, H. & Kasai, T. (1998) Guanidinated casein hydrolysate stimulates pancreatic secretagogue release by direct action to the intestine in rats. Proc. Soc. Exp. Biol. Med. 218:357-364.[Abstract]
12. Nishi, T., Hara, H., Hira, T. & Tomita, F. (2001) Dietary protein peptic hydrolysates stimulate cholecystokinin release via direct sensing by rat intestinal mucosal cells. Exp. Biol. Med. 226:1031-1036.
13. Nishi, T., Hara, H. & Tomita, F. (2003) Soybean ß-conglycinin peptone suppresses food intake and gastric emptying by increasing plasma cholecystokinin levels in rats. J. Nutr. 133:352-357.
14. Sebastiani, F. L., Ferrel, L. B., Schuler, M. A. & Beachy, R. N. (1990) Complete sequence of a cDNA of subunit of soybean ß-conglycinin. Plant Mol. Biol. 15:197-201.[Medline]
15. Doyle, J. J., Schuler, M. A., Godette, W. D., Zenger, V., Beachy, R. N. & Slightom, J. L. (1986) The glycosylated seed storage proteins of Glycine max and Phaseolus vulgaris. Structural homologies of genes and proteins. J. Biol. Chem. 261:9228-9238.
16. Hira, T., Hara, H. & Tomita, F. (2001) Characterization of binding between the rat small intestinal brush-border membrane and dietary proteins in the sensory mechanism of luminal dietary proteins. Biosci. Biotechnol. Biochem. 65:1007-1015.[Medline]
17. Riddle, R. A., Goldfine, I. D. & Williams, J. A. (1984) Bioassay of plasma cholecystokinin in rats: effects of food, trypsin inhibitor, and alcohol. Gastroenterology 87:542-549.[Medline]
18. Iwai, K., Fukuoka, S., Fushiki, T., Kodaira, T. & Ikei, N. (1986) Elevation of plasma CCK concentration after intestinal administration of a pancreatic enzyme secretion-stimulating peptide purified from rat bile-pancreatic juice. Biochem. Biophys. Res. Commun. 136:701-706.[Medline]
19. Spannagel, A. W., Green, G. M., Guan, D., Liddle, R. A., Faull, K. & Reeve Jr, J. R. (1996) Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion. Proc. Natl. Acad. Sci. U.S.A. 93:4415-4420.
20. Herzig, K. -H., Schön, I., Tatemoto, K., Ohe, Y., Li, Y., Fölsch, U. R. & Owyang, C. (1996) Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine. Proc. Natl. Acad. Sci. U.S.A. 93:7927-7932.
21. Iwai, K., Fushiki, T. & Fukuoka, S. (1988) Pancreatic enzyme secretion mediated by novel peptide: monitor peptide hypothesis. Pancreas 3:720-728.[Medline]
22. Liddle, R. A. (1995) Regulation of cholecystokinin secretion by intraluminal releasing factors. Am. J. Physiol. 269:G319-G327.[Medline]
23. Anastasi, A., Erspamer, V. & Bucci, M. (1972) Isolation and amino acid sequences of alytestin and bombesin, two analogous active tetradecapeptides from the skin of European discoglossid frogs. Arch. Biochem. Biophys. 148:443-446.[Medline]
24. Ohinata, K., Inui, A., Asakawa, A., Wada, K., Wada, E. & Yoshikawa, M. (2002) Albutensin A and complement C3a decrease food intake in mice. Peptides 23:127-133.[Medline]
25. Wang, L., Barachina, M. D., Martínez, V., Wei, J. Y. & Taché, Y. (2000) Synergistic interaction between CCK and leptin to regulate food intake. Regul. Pept. 92:79-85.[Medline]
26. Bhavsar, S., Watkins, J. & Young, A. (1998) Synergy between amylin and cholecystokinin for inhibition of food intake in mice. Physiol. Behav. 64:557-561.[Medline]
27. Reeves, P. G. (1989) AIN-76 diet: should we change the formulation?. J. Nutr. 119:1081-1082.
28. American Institute of Nutrition (1977) Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 107:1340-1348.
29. American Institute of Nutrition (1980) Second report of the ad hoc Committee on Standards for Nutritional Studies. J. Nutr. 110:1726.
30. Harper, A. E. (1959) Amino acid balance and imbalance. 1. Dietary level of protein and amino acid imbalance. J. Nutr. 68:405-418.
This article has been cited by other articles:
|
|
 |
|
  A. Aziz, G. H. Anderson, A. Giacca, and F. Cho Hyperglycemia after protein ingestion concurrent with injection of a GLP-1 receptor agonist in rats: a possible role for dietary peptides Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2005; 289(3): R688 - R694. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
