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Influence of dietary protein concentrations or of duodenal amino acid infusion on cholecystokinin release in goats
Camp.Biochem.Physiol.Vol. Printed in Great Britain
101A. No. 3, pp. 635-638,
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
1992
INFLUENCE OF DIETARY PROTEIN CONCENTRATIONS OR OF DUODENAL AMINO ACID INFUSION ON CHOLECYSTOKININ RELEASE IN GOATS MITSUHIRO FURUSE,* MASAYA KATO, SUNG IK YANG, KOSHIASAKURA Laboratory
and JUNICHIOKUMURA
of Animal Nutrition, School of Agriculture, Nagoya University, Nagoya 464-01, Japan. Telephone: (052)78 1-5111 (Received 10 June 1991)
Abstract-1. Whether dietary protein levels or duodenal infusion of amino acids alters the release of cholecystokinin (CCK) in blood plasma of goats was investigated in three experiments. The CCK determination was done by radioimmunoassay with specific CCK-8 antibody. The male adult Shiba goat, a miniature Japanese native goat, was used. 2. In Experiment 1, four goats were offered a diet containing 4.94 g crude protein (CP)/kg BW“.” for the first 7 days. They were then given a diet containing 5.86g CP/kg BWo75 for 7days and thereafter 6.79 g CP/kg Bw” for the following 7 days. On the last day of each experimental period, blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360min after the feeding of the diet. Plasma CCK levels were not affected by dietary protein levels and time after feeding. 3. Influence of phenylalanine or tryptophan (2 mmol/20 ml) infusion into the duodenum was investigated by a 3 x 3 latin square in Experiment 2. Plasma CCK level was determined at 15 min intervals for 1 hr. Both phenylalanine and tryptophan gradually enhanced plasma CCK concentrations with time after infusion. 4. Branched-chain amino acids such as leucine and isoleucine were supplemented intraduodenally in Experiment 3 as in Experiment 2. No significant change in plasma CCK levels was observed.
INTRODUCTION Cholecystokinin (CCK), one of the major regulators of pancreatic enzyme secretion and an inhibitor of food intake (Baile et al., 1986), is released into circulation after ingestion of food (Meyer, 1975). In species such as rats, where feedback inhibition of pancreatic enzyme secretion occurs, CCK release may be controlled by the level of active intraluminal protease (Louie et al., 1985). The response of CCK to soya-bean trypsin inhibitor (SBTI) administration appears to differ between species: the plasma CCK level was increased in rats (Liddle et al., 1984; Smith et al., 1989) and in chickens (Furuse et al., 1990), whereas it was unchanged in humans (Holm et al., 1988a,b) and in dogs (Sale et al., 1977) when SBTI was added to the diet. Species differences may also exist in CCK response to nutrients such as dietary amino acids: an enhanced CCK release into circulation was found in chickens (Yang et al., 1989; Furuse et al., 1991a), dogs (Chang and Chei, 1983) and in humans (Owyang et al., 1986), while no stimulation of CCK by dietary amino acids was reported in rats (Liddle et al., 1986). Recently, Furuse et al. (1991b) reported that plasma CCK concentrations of dairy cows were almost constant for over 6 hr after feeding, and suggested that this might reflect a continuous flow of digesta into the small intestine. However, which nutrients, such as dietary protein and amino acids, stimulate CCK release in ruminants has not been investigated. The present study was carried out to clarify whether the release of CCK into the circulation was influenced *Author to whom correspondence should be addressed.
by different levels of dietary protein (Experiment 1) or by duodenal infusion of aromatic amino acids (Experiment 2) and branched-chain amino acids (Experiment 3) in goats.
MATERIALSAND METHODS Animals and diets Experiment 1. The male adult Shiba goat, a miniature Japanese native strain, was reared in metabolism cages in a temperature-controlled room (24°C). Four goats, weighing 17-21 kg, were offered a control diet consisting of 20.7 g hay, 2O.Og wheat bran and 3.2g soya-bean oil/kg BW.” once a day at 1000 hr for 7 days. The control diet was mixed to meet the protein and energy requirement recommended by the National Research Council (NRC, 1981). In the second and third weeks, they received additionally, 2.1 or 4.2 a sova-bean meal/ka BW.75. Dailv total crude urotein (CPj iniakes/kg B+75 were 4.94, 5.86 and 6.79 g, respectively. On the last day of each experimental period, about 2.5ml blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360min after the feeding of the diets. Plasma was obtained by centrifugation at 900g at 4°C for lOmin, and stored at -80°C with 3.22 ymol/ml of EDTA (Wake Pure Chemical Industries, Japan) and 500 KIU/ml of aprotinin (Bayer Ltd., Germany) until the CCK assay. Experiment 2. Three male adult Shiba goats, weighing 16-20 kg, were used and the experiment was done using a 3 x 3 latin square. Both goats were attached to duodenal cannulas (2.7 mm outer diameter, I .6 mm inner diameter and 400mm long; Atom, Japan) under anaesthetization with sodium pentobarbital, and given the control diet used in Experiment 1, once a day at 1100 hr. The blood sampling was done at 1000 hr at 2-day intervals and was obtained from the jugular vein at zero (before infusion), 15, 30, 45 and 635
MITSUHIRO FURU~Eet al.
636 6Omin after alanine and dissolved in All solutions cannula.
infusion of 20ml of saline (control), phenyltryptophan solution. The amino acids were saline at a level of 2nunol per 20ml saline. were warmed at 40°C and flushed into the
Experiment 3. Three male adult Shiba goats, weighing 18-20 kg, were used and the procedure was similar to Experiment 2, except for the amino ‘acids. Leucine and isoleucine were used at a level of 2mmol per 20ml saline. Assay of&ma
CCK comentration
One ml of the plasma was mixed with 3 ml of ethanol, incubated at 4°C for 30 min and then centrifuged at 2200 g for 30min. The supematant was obtained by decantation and dried under a nitrogen stream at 45°C followed by dissolving in 1 ml of 0.03 M phosphate buffer, pH 7.6, containing 20 mM EDTA, 0.1% bovine serum albumin (Sigma Chemical Co., U.S.A.), 2OOKIU/mi aprotinin and 0.02% NaN, (Wake Pure Chemical Industries). CCK-8 antibody, izJI CCK-39, normal rabbit serum and goat antirabbit y-globulin antiserum were purchased from Otsuka Pha~a~uti~ls, Japan. Plasma CCK ~n~nt~tions were measured by a ~dio~unoa~ay in which 0.2ml of the speciftc CCK-8 N-terminal antibody, 0.2ml of the plasma sample, 0.2 ml of “‘1 CCK-39 (approximately 10,000 cpm) and 0.2 ml of phosphate buffer were incubated at 4°C for 48 hr. After the incubation, 0.1 ml of diluted normal rabbit serum and 0.1 ml of diluted goat anti-rabbit y-globulin antiserum were added, mixed and incubated at 4°C for 18-24 hr. The antisera specifically reacted with an arninoterminal region of CCK 8 but not with the non-sulphate form of CCK 8, nor with the carboxy-terminal region which shares a cross-reactive determinant among gastrin and CCK related peptides (CCK 4, CCK 8, caerulein, CCK 33 and CCK 39). Details of ~un~herni~l characteristics of the CCK-8 antibody have been described elsewhere (Hashimura et al., 1982). The inter-assay and intra-assay coefficients of variation were 4.1 and 2.6%, respectively (Iateishi et al., 1983). The tubes containing the incubation mixtures were then centrifuged at 175Og at 4’C for 30min. The radioactivity in total and precipitate fractions was counted by an automatic gamma counter (Aloka, Japan). The immunoreactivity of CCK was expressed in terms of the CCK-8 equivalent.
k 0
f 15 Time
30 after
45
a0
intualon (min)
Fig. 2. Effect on time course changes in plasma CCK concentrations of into-du~enal ad~nis~tion of saline (O), phenylalanine (A) or tryptophan (Cl) in Shiba goats. The amino acid (2 nunol) was dissolved in 20 ml saline and infused. Plasma CCK levels are expressed as CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean f SEM. Closed symbols indicate ~~~~t differences from the mean of the binding preinfusion values (P < 0.05). *Significantly different from the control at the same time period at P c: 0.05.
RESULTS
1 shows the time course change in plasma CCK of goats after the commencement of feeding of three different levels of dietary protein. The plasma CCK level was almost constant over the experimental period, except for the value at 30 min, being lower, hut not si~~antly so. No significant difference was detected among diktary treatments. Figure
Srafisrics Analysis of variance was used to evaluate the data statistically, and comparison of means was done by r-test.
a
3
20’
u” 15 -
10’
8 * 0
3aeo
‘
‘ tp
Time after
ma
so
feedlngfmin)
Fig. 1. Effect on time course changes in plasma CCK concentrations of dietary protein levels in Shiba goats. The diet, containing 4.94 g (O), 5.86 g (A) or 6.79 g (Cl) CPjkg Bw0.‘5 was given once a day. Plasma CCK levels are expressedas CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean 1 SEM.
,
I
0
15
Time
after
30
45
80
infuefon (mfn)
Fig. 3. Effect on time course changes in plasma CCK concentrations of intra-duodenal administration of saline (0). leucine (0) or isoleucine (A) in Shiba goats. The amino acid (2 mmol) was dissolved in 20 ml saline and infused. Plasma CCK levels are expressed as CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean f SEM.
CCK release in goats
Time course changes in CCK response when the goat was intra-duodenally infused with saline, phenylalanine or tryptophan are shown in Fig. 2. No significant changes in CCK concentration were observed in the control group, whereas the final values from both the amino acid treatments significantly increased compared to the corresponding initial values. At 45 and 60 min, the values for phenyialanine treatment were significantly higher than those for the control. Influence on time course changes in CCK
concentrations of intra-duodenal administration of saline, leucine or isoleucine in goats is indicated in Fig. 3. There were no significant differences between treatments or times.
whereas the rat is an omnivorous animal. The latter generally eat storage proteins in seed, beans and grains, which contain fewer free amino acids than meat. It is not surprising that CCK release was stimulated by amino acids, because the ruminant is largely dependent on proteins of bacteria and protozoa, which can be considered as animal protein. In the present study, amino acids were infused under the condition in which the digesta from the abomasum continuously stimulated the mucosa of the intestine; consequently, the effect of amino acids on CCK release might be underestimated. Infusion of amino acids into the duodenum after removing the digesta from the intestine would clarify the effect of amino acids on CCK release in ruminants. REFERENCES
DISCUSSION
In single-stomached animals, plasma CCK level rapidly increased after the ingestion of food, although it was almost constant in dairy cows over 6 hr at 1 hr intervals (Furuse et al., 1991b). The observation in the ruminant may reflect a continuous flow of digesta into the small intestine, which seems to be continuously stimulated by the digesta and rumen microbes, because the flow of digesta from the rumen and abomasum is constant (Faichney, 1975). In the present study, plasma CCK concentration of goats was almost constant. However, the value for 30 min after feeding tended to be low. This finding was largely different from the monogastric animals. The pylorus of abomasum could be closed just after feeding by some factors (e.g. neural factors), and duodenal stimulations by the digesta and microbes might be reduced, though exact reasons are not yet known. Recently, Fushiki and Iwai (1989) reviewed two hypotheses on the feedback regulation of pancreatic enzyme secretion, which were mediated by a trypsinsensitive, CCK-releasing peptide. In these hypotheses, affinities of trypsin for dietary protein and trypsin inhibitor are the most important problem. In the present experiment, three dietary protein levels were used, but no difference was observed among treatments. It might be suggested that dietary protein did not stimulate CCK release in goats as well as dogs (Sale, 1977). However, Bunting et al. (1989) reported that abomasum N flows were not changed even if dietary protein levels increased two-fold. Furthermore, even at the lowest level of dietary protein in the present study, CCK release might be fully stimulated. Further experiments need to be carried out to clarify the feedback regulation in ruminants. The highest activity for release of CCK was exhibited by tryptophan and phenylalanine in dogs (Konturek et nl., 1973). The present study clearly
showed that aromatic amino acid had a stimulatory effect on CCK release in goats. In dogs, all essential amino acids except threonine were effective in CCK release (Konturek et al., 1973), although branchedchain amino acids did not stimulate CCK release in the present study. The specificity of amino acids for CCK release might be present in goats. Fushiki and Iwai (1989) speculated as to why amino acids are major stimulants for pancreatic enzyme secretion in the dog but not in the rat. According to them, a dog eats a meat meal that contains many free amino acids,
637
Baile C. A., McLaughlin C. L. and Della-Fera M. A. (1986)
Role of cholecystokinin and onioid pentides in control of food intake. physiol. Rev. 66; 172-234. Buntina L. D.. Boline J. A. and MacKown C. T. (1989) Effect of dietary protein level on nitrogen metabolism in the growing bovine: 1. Nitrogen recycling and intestinal protein supply in calves. J. Anim. Sci. 67, 810-819. Chang T. and Chey W. Y. (1983) Radioimmunoassay of choiecystokinin. big. Dis. &i. &, 456-468. _ Faichnev G. J. (1975) The effect of formaldehvde treatment of a concentrate diet on the passage of soluik and particle markers through the gastrointestinal tract of sheep. Aust. J. Agric. Res. 13, 319-327.
Furuse M., Choi Y. H., Yang S. I., Kita K. and Okumura J. (1991a) Enhanced release ofcholccystokinin in chickens fed diets high in phenylalanine or tyrosine. Comp. Biochem. Physiol. 99A, 449-451. Furusc M., Yang S. I., Choi Y. H., Kawamura N., Takahashi A. and Okumura J. (1991b) A note on plasma cholecystokinin concentration in dairy cows. Amin. Prod. 53, 123-125.
Furuse M., Yang S. I., Muramatsu T. and Okumura J. (1990) Enchanced release of cholecystokinin by soya-bean trypsin inhibitor in chickens. &and. J. Gustroenterol. 25, 1242-1246. Fushiki T. and Iwai K. (1989) Two hypotheses on the feedback regulation on pancreatic enzyme secretion. FASEB J. 3, 121-126. Hashimura E., Shimizu F., Nishino T., Imagawa K., Tateishi K. and Hamaoka T. (1982) Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response. J. Immunol. Merhoak 55, 375-387.
Holm H., Hanssen L. E., Krogdahl A. and Florholmen J. (1988a) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in viuo comparison with bovine serum albumin. J. Nutr. 118, 515-520. Holm H., Krogdahl A. and Hanssen L. E. (1988b) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in vitro comparison of proteinase inhibition. J. N&r. 118, 521-525. Konturek S. J., Radecki T., Thor P. and Dembinski A. (1973) Release of cholecvstokinin bv amino acids. Proc. &c. kxp. Biol. Med. 14j, 305-309. _
Liddle R. A., Goldtine I. D. and Williams J. A. (1984) Bioassay of plasma cholecystokinin in rats: effect of food, trypsin inhibitor and alcohol. Gasboenrerology 87, 542-549. Liddle R. A., Green G. M., Conrad C. K. and Williams J. A. (1986) Protein but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am. J. Physiol. 251, G243-G248.
638
MIIWHIRO FURUSE et
Louie D. S., May D., Miller P. and Owyang C. (1985) Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats. Am. J. Physiol. 250, G252ZG259. Meyer J. H. (1975) Release of secretin and cholecystokinin. In Gastrointestinal Hormones (Edited by Thompson J. C.), pp. 475-489. University of Texas Press, Texas. National Research Council (1981) Nutrient Requirements of Goats. National Academy Press, Washington DC, U.S.A. Owyang C., Louie D. S. and Tatum D. (1986) Feedback regulation of pancreatic enzyme secretion: suppression of cholecystokinin release by trypsin. J. clin. Invest. 77, 2042-2047. Sale J. K., Goldberg D. M., Fawcett A. N. and Wormsley K. G. (1977) Chronic and acute studies indicating absence of exocrine pancreatic feedback inhibition in dogs. Digestion 15, 540-555.
al.
Smith G. P., Greenberg D., Falasco J. D., Avilion A. A. and Gibbs J. (1989) Endogenous cholecystokinin does not decrease food intake or gastric emptying in fasted rats. Am. J. Physiol. 251, R1462-R1466. Tateishi K., Hayashi C., Himeno S., Kanayama S., Tarui S., Hamaoka T., Hashimura E. and Imagawa K. (1983) Establishment of cholecystokinin-specific radioimmunoassay by using non-crossreactive antiserum (in Japanese). In Proceedings of the Fifth Gut Hormone Conference (Edited by Miyoshi A., Abe K., Itoh Z., Kanno T.; Fujita T., Matsuo Y. and Yanaihara N.), pp. 63-71. Igaku Tosyo Syuppan, Tokyo. Yang S. I., Furuse M., Muramatsu T. and Okumura J. (1989) Enhanced release of cholecystokinin by dietary amino acids in chicks. Comp. Biochem. Physiol. 92A, 319-322.
101A. No. 3, pp. 635-638,
0300-9629/92$5.00+ 0.00 0 1992Pergamon Press plc
1992
INFLUENCE OF DIETARY PROTEIN CONCENTRATIONS OR OF DUODENAL AMINO ACID INFUSION ON CHOLECYSTOKININ RELEASE IN GOATS MITSUHIRO FURUSE,* MASAYA KATO, SUNG IK YANG, KOSHIASAKURA Laboratory
and JUNICHIOKUMURA
of Animal Nutrition, School of Agriculture, Nagoya University, Nagoya 464-01, Japan. Telephone: (052)78 1-5111 (Received 10 June 1991)
Abstract-1. Whether dietary protein levels or duodenal infusion of amino acids alters the release of cholecystokinin (CCK) in blood plasma of goats was investigated in three experiments. The CCK determination was done by radioimmunoassay with specific CCK-8 antibody. The male adult Shiba goat, a miniature Japanese native goat, was used. 2. In Experiment 1, four goats were offered a diet containing 4.94 g crude protein (CP)/kg BW“.” for the first 7 days. They were then given a diet containing 5.86g CP/kg BWo75 for 7days and thereafter 6.79 g CP/kg Bw” for the following 7 days. On the last day of each experimental period, blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360min after the feeding of the diet. Plasma CCK levels were not affected by dietary protein levels and time after feeding. 3. Influence of phenylalanine or tryptophan (2 mmol/20 ml) infusion into the duodenum was investigated by a 3 x 3 latin square in Experiment 2. Plasma CCK level was determined at 15 min intervals for 1 hr. Both phenylalanine and tryptophan gradually enhanced plasma CCK concentrations with time after infusion. 4. Branched-chain amino acids such as leucine and isoleucine were supplemented intraduodenally in Experiment 3 as in Experiment 2. No significant change in plasma CCK levels was observed.
INTRODUCTION Cholecystokinin (CCK), one of the major regulators of pancreatic enzyme secretion and an inhibitor of food intake (Baile et al., 1986), is released into circulation after ingestion of food (Meyer, 1975). In species such as rats, where feedback inhibition of pancreatic enzyme secretion occurs, CCK release may be controlled by the level of active intraluminal protease (Louie et al., 1985). The response of CCK to soya-bean trypsin inhibitor (SBTI) administration appears to differ between species: the plasma CCK level was increased in rats (Liddle et al., 1984; Smith et al., 1989) and in chickens (Furuse et al., 1990), whereas it was unchanged in humans (Holm et al., 1988a,b) and in dogs (Sale et al., 1977) when SBTI was added to the diet. Species differences may also exist in CCK response to nutrients such as dietary amino acids: an enhanced CCK release into circulation was found in chickens (Yang et al., 1989; Furuse et al., 1991a), dogs (Chang and Chei, 1983) and in humans (Owyang et al., 1986), while no stimulation of CCK by dietary amino acids was reported in rats (Liddle et al., 1986). Recently, Furuse et al. (1991b) reported that plasma CCK concentrations of dairy cows were almost constant for over 6 hr after feeding, and suggested that this might reflect a continuous flow of digesta into the small intestine. However, which nutrients, such as dietary protein and amino acids, stimulate CCK release in ruminants has not been investigated. The present study was carried out to clarify whether the release of CCK into the circulation was influenced *Author to whom correspondence should be addressed.
by different levels of dietary protein (Experiment 1) or by duodenal infusion of aromatic amino acids (Experiment 2) and branched-chain amino acids (Experiment 3) in goats.
MATERIALSAND METHODS Animals and diets Experiment 1. The male adult Shiba goat, a miniature Japanese native strain, was reared in metabolism cages in a temperature-controlled room (24°C). Four goats, weighing 17-21 kg, were offered a control diet consisting of 20.7 g hay, 2O.Og wheat bran and 3.2g soya-bean oil/kg BW.” once a day at 1000 hr for 7 days. The control diet was mixed to meet the protein and energy requirement recommended by the National Research Council (NRC, 1981). In the second and third weeks, they received additionally, 2.1 or 4.2 a sova-bean meal/ka BW.75. Dailv total crude urotein (CPj iniakes/kg B+75 were 4.94, 5.86 and 6.79 g, respectively. On the last day of each experimental period, about 2.5ml blood samples were taken from the jugular vein at zero (before feeding), 30, 60, 120, 240 or 360min after the feeding of the diets. Plasma was obtained by centrifugation at 900g at 4°C for lOmin, and stored at -80°C with 3.22 ymol/ml of EDTA (Wake Pure Chemical Industries, Japan) and 500 KIU/ml of aprotinin (Bayer Ltd., Germany) until the CCK assay. Experiment 2. Three male adult Shiba goats, weighing 16-20 kg, were used and the experiment was done using a 3 x 3 latin square. Both goats were attached to duodenal cannulas (2.7 mm outer diameter, I .6 mm inner diameter and 400mm long; Atom, Japan) under anaesthetization with sodium pentobarbital, and given the control diet used in Experiment 1, once a day at 1100 hr. The blood sampling was done at 1000 hr at 2-day intervals and was obtained from the jugular vein at zero (before infusion), 15, 30, 45 and 635
MITSUHIRO FURU~Eet al.
636 6Omin after alanine and dissolved in All solutions cannula.
infusion of 20ml of saline (control), phenyltryptophan solution. The amino acids were saline at a level of 2nunol per 20ml saline. were warmed at 40°C and flushed into the
Experiment 3. Three male adult Shiba goats, weighing 18-20 kg, were used and the procedure was similar to Experiment 2, except for the amino ‘acids. Leucine and isoleucine were used at a level of 2mmol per 20ml saline. Assay of&ma
CCK comentration
One ml of the plasma was mixed with 3 ml of ethanol, incubated at 4°C for 30 min and then centrifuged at 2200 g for 30min. The supematant was obtained by decantation and dried under a nitrogen stream at 45°C followed by dissolving in 1 ml of 0.03 M phosphate buffer, pH 7.6, containing 20 mM EDTA, 0.1% bovine serum albumin (Sigma Chemical Co., U.S.A.), 2OOKIU/mi aprotinin and 0.02% NaN, (Wake Pure Chemical Industries). CCK-8 antibody, izJI CCK-39, normal rabbit serum and goat antirabbit y-globulin antiserum were purchased from Otsuka Pha~a~uti~ls, Japan. Plasma CCK ~n~nt~tions were measured by a ~dio~unoa~ay in which 0.2ml of the speciftc CCK-8 N-terminal antibody, 0.2ml of the plasma sample, 0.2 ml of “‘1 CCK-39 (approximately 10,000 cpm) and 0.2 ml of phosphate buffer were incubated at 4°C for 48 hr. After the incubation, 0.1 ml of diluted normal rabbit serum and 0.1 ml of diluted goat anti-rabbit y-globulin antiserum were added, mixed and incubated at 4°C for 18-24 hr. The antisera specifically reacted with an arninoterminal region of CCK 8 but not with the non-sulphate form of CCK 8, nor with the carboxy-terminal region which shares a cross-reactive determinant among gastrin and CCK related peptides (CCK 4, CCK 8, caerulein, CCK 33 and CCK 39). Details of ~un~herni~l characteristics of the CCK-8 antibody have been described elsewhere (Hashimura et al., 1982). The inter-assay and intra-assay coefficients of variation were 4.1 and 2.6%, respectively (Iateishi et al., 1983). The tubes containing the incubation mixtures were then centrifuged at 175Og at 4’C for 30min. The radioactivity in total and precipitate fractions was counted by an automatic gamma counter (Aloka, Japan). The immunoreactivity of CCK was expressed in terms of the CCK-8 equivalent.
k 0
f 15 Time
30 after
45
a0
intualon (min)
Fig. 2. Effect on time course changes in plasma CCK concentrations of into-du~enal ad~nis~tion of saline (O), phenylalanine (A) or tryptophan (Cl) in Shiba goats. The amino acid (2 nunol) was dissolved in 20 ml saline and infused. Plasma CCK levels are expressed as CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean f SEM. Closed symbols indicate ~~~~t differences from the mean of the binding preinfusion values (P < 0.05). *Significantly different from the control at the same time period at P c: 0.05.
RESULTS
1 shows the time course change in plasma CCK of goats after the commencement of feeding of three different levels of dietary protein. The plasma CCK level was almost constant over the experimental period, except for the value at 30 min, being lower, hut not si~~antly so. No significant difference was detected among diktary treatments. Figure
Srafisrics Analysis of variance was used to evaluate the data statistically, and comparison of means was done by r-test.
a
3
20’
u” 15 -
10’
8 * 0
3aeo
‘
‘ tp
Time after
ma
so
feedlngfmin)
Fig. 1. Effect on time course changes in plasma CCK concentrations of dietary protein levels in Shiba goats. The diet, containing 4.94 g (O), 5.86 g (A) or 6.79 g (Cl) CPjkg Bw0.‘5 was given once a day. Plasma CCK levels are expressedas CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean 1 SEM.
,
I
0
15
Time
after
30
45
80
infuefon (mfn)
Fig. 3. Effect on time course changes in plasma CCK concentrations of intra-duodenal administration of saline (0). leucine (0) or isoleucine (A) in Shiba goats. The amino acid (2 mmol) was dissolved in 20 ml saline and infused. Plasma CCK levels are expressed as CCK-8 equivalents determined with the CCK-8 specific antibody. Each point is the mean f SEM.
CCK release in goats
Time course changes in CCK response when the goat was intra-duodenally infused with saline, phenylalanine or tryptophan are shown in Fig. 2. No significant changes in CCK concentration were observed in the control group, whereas the final values from both the amino acid treatments significantly increased compared to the corresponding initial values. At 45 and 60 min, the values for phenyialanine treatment were significantly higher than those for the control. Influence on time course changes in CCK
concentrations of intra-duodenal administration of saline, leucine or isoleucine in goats is indicated in Fig. 3. There were no significant differences between treatments or times.
whereas the rat is an omnivorous animal. The latter generally eat storage proteins in seed, beans and grains, which contain fewer free amino acids than meat. It is not surprising that CCK release was stimulated by amino acids, because the ruminant is largely dependent on proteins of bacteria and protozoa, which can be considered as animal protein. In the present study, amino acids were infused under the condition in which the digesta from the abomasum continuously stimulated the mucosa of the intestine; consequently, the effect of amino acids on CCK release might be underestimated. Infusion of amino acids into the duodenum after removing the digesta from the intestine would clarify the effect of amino acids on CCK release in ruminants. REFERENCES
DISCUSSION
In single-stomached animals, plasma CCK level rapidly increased after the ingestion of food, although it was almost constant in dairy cows over 6 hr at 1 hr intervals (Furuse et al., 1991b). The observation in the ruminant may reflect a continuous flow of digesta into the small intestine, which seems to be continuously stimulated by the digesta and rumen microbes, because the flow of digesta from the rumen and abomasum is constant (Faichney, 1975). In the present study, plasma CCK concentration of goats was almost constant. However, the value for 30 min after feeding tended to be low. This finding was largely different from the monogastric animals. The pylorus of abomasum could be closed just after feeding by some factors (e.g. neural factors), and duodenal stimulations by the digesta and microbes might be reduced, though exact reasons are not yet known. Recently, Fushiki and Iwai (1989) reviewed two hypotheses on the feedback regulation of pancreatic enzyme secretion, which were mediated by a trypsinsensitive, CCK-releasing peptide. In these hypotheses, affinities of trypsin for dietary protein and trypsin inhibitor are the most important problem. In the present experiment, three dietary protein levels were used, but no difference was observed among treatments. It might be suggested that dietary protein did not stimulate CCK release in goats as well as dogs (Sale, 1977). However, Bunting et al. (1989) reported that abomasum N flows were not changed even if dietary protein levels increased two-fold. Furthermore, even at the lowest level of dietary protein in the present study, CCK release might be fully stimulated. Further experiments need to be carried out to clarify the feedback regulation in ruminants. The highest activity for release of CCK was exhibited by tryptophan and phenylalanine in dogs (Konturek et nl., 1973). The present study clearly
showed that aromatic amino acid had a stimulatory effect on CCK release in goats. In dogs, all essential amino acids except threonine were effective in CCK release (Konturek et al., 1973), although branchedchain amino acids did not stimulate CCK release in the present study. The specificity of amino acids for CCK release might be present in goats. Fushiki and Iwai (1989) speculated as to why amino acids are major stimulants for pancreatic enzyme secretion in the dog but not in the rat. According to them, a dog eats a meat meal that contains many free amino acids,
637
Baile C. A., McLaughlin C. L. and Della-Fera M. A. (1986)
Role of cholecystokinin and onioid pentides in control of food intake. physiol. Rev. 66; 172-234. Buntina L. D.. Boline J. A. and MacKown C. T. (1989) Effect of dietary protein level on nitrogen metabolism in the growing bovine: 1. Nitrogen recycling and intestinal protein supply in calves. J. Anim. Sci. 67, 810-819. Chang T. and Chey W. Y. (1983) Radioimmunoassay of choiecystokinin. big. Dis. &i. &, 456-468. _ Faichnev G. J. (1975) The effect of formaldehvde treatment of a concentrate diet on the passage of soluik and particle markers through the gastrointestinal tract of sheep. Aust. J. Agric. Res. 13, 319-327.
Furuse M., Choi Y. H., Yang S. I., Kita K. and Okumura J. (1991a) Enhanced release ofcholccystokinin in chickens fed diets high in phenylalanine or tyrosine. Comp. Biochem. Physiol. 99A, 449-451. Furusc M., Yang S. I., Choi Y. H., Kawamura N., Takahashi A. and Okumura J. (1991b) A note on plasma cholecystokinin concentration in dairy cows. Amin. Prod. 53, 123-125.
Furuse M., Yang S. I., Muramatsu T. and Okumura J. (1990) Enchanced release of cholecystokinin by soya-bean trypsin inhibitor in chickens. &and. J. Gustroenterol. 25, 1242-1246. Fushiki T. and Iwai K. (1989) Two hypotheses on the feedback regulation on pancreatic enzyme secretion. FASEB J. 3, 121-126. Hashimura E., Shimizu F., Nishino T., Imagawa K., Tateishi K. and Hamaoka T. (1982) Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response. J. Immunol. Merhoak 55, 375-387.
Holm H., Hanssen L. E., Krogdahl A. and Florholmen J. (1988a) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in viuo comparison with bovine serum albumin. J. Nutr. 118, 515-520. Holm H., Krogdahl A. and Hanssen L. E. (1988b) High and low inhibitor soybean meals affect human duodenal proteinase activity differently: in vitro comparison of proteinase inhibition. J. N&r. 118, 521-525. Konturek S. J., Radecki T., Thor P. and Dembinski A. (1973) Release of cholecvstokinin bv amino acids. Proc. &c. kxp. Biol. Med. 14j, 305-309. _
Liddle R. A., Goldtine I. D. and Williams J. A. (1984) Bioassay of plasma cholecystokinin in rats: effect of food, trypsin inhibitor and alcohol. Gasboenrerology 87, 542-549. Liddle R. A., Green G. M., Conrad C. K. and Williams J. A. (1986) Protein but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in the rat. Am. J. Physiol. 251, G243-G248.
638
MIIWHIRO FURUSE et
Louie D. S., May D., Miller P. and Owyang C. (1985) Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats. Am. J. Physiol. 250, G252ZG259. Meyer J. H. (1975) Release of secretin and cholecystokinin. In Gastrointestinal Hormones (Edited by Thompson J. C.), pp. 475-489. University of Texas Press, Texas. National Research Council (1981) Nutrient Requirements of Goats. National Academy Press, Washington DC, U.S.A. Owyang C., Louie D. S. and Tatum D. (1986) Feedback regulation of pancreatic enzyme secretion: suppression of cholecystokinin release by trypsin. J. clin. Invest. 77, 2042-2047. Sale J. K., Goldberg D. M., Fawcett A. N. and Wormsley K. G. (1977) Chronic and acute studies indicating absence of exocrine pancreatic feedback inhibition in dogs. Digestion 15, 540-555.
al.
Smith G. P., Greenberg D., Falasco J. D., Avilion A. A. and Gibbs J. (1989) Endogenous cholecystokinin does not decrease food intake or gastric emptying in fasted rats. Am. J. Physiol. 251, R1462-R1466. Tateishi K., Hayashi C., Himeno S., Kanayama S., Tarui S., Hamaoka T., Hashimura E. and Imagawa K. (1983) Establishment of cholecystokinin-specific radioimmunoassay by using non-crossreactive antiserum (in Japanese). In Proceedings of the Fifth Gut Hormone Conference (Edited by Miyoshi A., Abe K., Itoh Z., Kanno T.; Fujita T., Matsuo Y. and Yanaihara N.), pp. 63-71. Igaku Tosyo Syuppan, Tokyo. Yang S. I., Furuse M., Muramatsu T. and Okumura J. (1989) Enhanced release of cholecystokinin by dietary amino acids in chicks. Comp. Biochem. Physiol. 92A, 319-322.