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Effects of intravenous infusions of cholecystokinin (CCK)-8 on exocrine and endocrine pancreatic secretion in conscious sheep
Camp. Biochem.Physiol. Vol. lIIA,No. I, pp. 133-138. 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0300-9629/95 $9.50+ 0.00
Pergamon 0300-%29(94)00192-8
Effects of intravenous infusions of cholecystokinin (CCK)-8 on exocrine and endocrine pancreatic secretion in conscious sheep H. Mineo,* N. Iwaki,* K. Kogishi,* R. Zabielski,? T. Onaga* and S. Kate* *Department of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido Japan; and TDepartment of Animal Physiology, Faculty of Veterinary Medicine, Warsaw Agricultural University, 02-766 Warsaw, Poland
069,
The effects of cholecystokinin (CCK)3 on both exocrine and endocrine pancreatic functions were examined simultaneously in five conscious sheep. Intravenous infusions of CCK-8 (0, 5, 10, 20, 30, 60, 120 and 240 pmol/kg/min for 40 min) induced dosedependent increases in flow rate, and in protein and amylase outputs in pancreatic juice. The same CCK-8 infusions induced dosedependent increases in plasma insulin, but no change in plasma glucagon concentrations. The threshold dose (l&30 pmol/kg/min) of CCK-8 infusion for stimulating insulin secretion was similar to that for stimulating amylase output. In conclusion, using amylase output as an indicator of physiological action, CCK is one of the potential candidates as a physiological regulator of insulin, but not glucagon secretion in sheep. Key words: CCK; Exocrine
pancreas; Endocrine
pancreas; Pancreatic juice; Amylase; Insulin;
Glucagon; Sheep; Ruminants. Comp. Biochem. Physiol. 11IA, 133-138, 199.5.
Introduction In non-ruminant animals, a variety of gastrointestinal hormones is known to affect endocrine pancreatic secretions (Rushakoff et al., 1990). In addition to its action on exocrine pancreatic secretion, in vivo and in vitro studies have shown that cholecystokinin (CCK) stimulates pancreatic hormone secretion from the endocrine cells of the pancreas (Karlsson and Ahren, 1992). A variety of evidence supports the hypothesis that CCK is one of the candidates for functioning as a physiological regulator of pancreatic hormone secretion in non-ruminant species (Okabayashi et al., 1989; Rushakoff et al., 1990). On the other hand, there have been Correspondence lo: Dr H. Mineo, Department of Medical Physiology C, Panum Institute, Blegdamsvej 3, DK-2200, Copenhagen N, Denmark. Tel. 45-3532-7516; Fax 45-3532-7537. Received 9 May 1994; revised 8 November 1994; accepted 14 November 1994.
few reports of the effects of CCK on endocrine pancreatic responses in ruminants (Baile et al., 1969; Godden et al., 1981; Takahashi et al., 1983). In these reports, pharmacological doses of CCK were used to induce pancreatic hormone secretion. In a recent report from our laboratory, intravenous infusions of CCK-8 at low doses induced insulin, but not glucagon secretion in sheep (Mine0 et al., 1994). However, it was not known whether the doses of CCK-8 used in that experiment were within the physiological range. One of the physiological actions of CCK is well known to be a stimulating effect on enzyme secretion from the acinar cells of the pancreas. Thus, the aim of the present study was to simultaneously examine the effect of intravenous infusions of CCK-8 on the exocrine and endocrine pancreatic responses, and to compare the dose-response relationships of 133
H. Mineo et al.
134
CCK-8 and its effects on both exocrine and endocrine function of the pancreas in conscious sheep.
Materials and Methods Animals and diets
Five Suffolk cross-bred wethers, aged l-2 years and weighing 3842 kg, were used. They had their left common carotid artery chronically placed in a loop of skin by surgical operation under general anesthesia with pentobarbitone sodium (25 mg/kg) more than 2 months before experiments began. In addition, under general anesthesia introduced by pentobarbital sodium and maintained by halothane and oxygen. each animal was chronically catheterized with silastic tubing (2 mm I.D., 4 mm O.D.) inserted into the common bile duct at two places: one close to the duodenum for collection of pancreatic juice and the other close to the gall-bladder for collection of bile. The common bile duct was ligated immediately distal to both catheter insertions. A third catheter (2 mm I.D., 4mm O.D.) was inserted into the duodenum close to the opening of the common bile duct to return the bile and pancreatic juice. Sheep were housed in individual cages and were fed lucerne pellets (1000 g) and orchard grass hay (100 g) once daily at 19.00. Water was freely available. Pancreatic juice and bile were returned to the duodenum of each sheep at approximately the rate of secretion, using a peristaltic infusion pump. Sheep were accustomed to the experimental environment for at least 10 days after surgery. A polyethylene catheter for infusion of solutions was inserted into the right jugular vein at least 3 days before experiments. The catheter was maintained with a sterilized solution of 3.8% (wt/vol) trisodium citrate. E.yperimental
procedure
On the day of the experiment. a catheter was inserted into the carotid artery at least 3 hr before blood sampling. CCK-8 (Peptide Institute Inc., Osaka, Japan) was dissolved and diluted with 0.9% (wt/vol) NaCl solution to the appropriate concentrations immediately before infusion. CCK-8 solutions were infused at rates of 5, 10, 20, 30, 60, 120 and 240 pmol/kg/min for 40min through the jugular vein catheter. Sterile 0.9% NaCl solution was infused for 40 min as a control. The infusion volume o.f each solution was 0.5 ml/min delivered by using an infusion pump (SJ-1211, ATTO, Tokyo, Japan). In each experiment, CCK-8 infusion was started 30min after initiating pancreatic juice sampling. The flow rate of pancreatic juice was measured every 10 min for 90 min, and
20 ~1 of each sample was taken for analysis. Sampling of blood was undertaken at -20, - 10, 0, 2, 5, 10, 20, 30, 40 and 60 min relative to the starting time of the CCK-8 infusion. Each blood sample (4 ml) was taken by syringe and immediately transferred into a polyethylene test-tube containing EDTA (I .2 mg/ml blood, Kanto Chemical Co., Tokyo, Japan) and aprotinin (500 KIU/ml blood, Hoechst Japan, Tokyo, Japan). Each experiment was started at 10.00. Tests using the same animal were separated by at least 2 days.
The protein concentration of pancreatic juice was determined by the method of Lowry et al. ( 1951) using bovine serum albumin as a standard. Amylase activity was assayed by the method of Kanno (1975). A unit of activity was defined as the amount of enzyme which produced 1 mg maltose/min from starch (Merck, Germany). Plasma was separated by centrifugation (SOOOg for 15 min) at 4°C and stored at -20°C until used for hormone assay. The plasma insulin and glucagon concentrations were determined by radioimmunoassay procedures, using previously described methods for insulin (Sasaki and Takahashi, 1980) and glucagon (Sasaki et cd., 1982). Guinea-pig anti-bovine insulin serum (ICN, Israel) and ““I-labeled porcine insulin (NEN, Boston, U.S.A.) were purchased for insulin assay. Bovine insulin (Sigma, St. Louis, U.S.A.) was used as a standard. Intraand inter-assay coefficients of variation for insulin determination were 5.2% (n = 6) and 8.7% (n = 5), respectively. The antiserum used for the glucagon assay (rabbit antiserum to bovine pancreatic glucagon, Calbiochem Co., California. U.S.A.) was specific for pancreatic glucagon. Porcine glucagon was used as a standard (Eli-Lilly Co., Indianapolis, U.S.A.) and as an iodinated hormone (NEN, Boston, U.S.A.). Intra- and inter-assay coefficients of variation for glucagon determination were 6.5% (n = 6) and 10.5% (n = 5), respectively. The remaining plasma was used for the determination of plasma glucose concentration by the glucoseoxidase method (Kabasakalian et al., 1974). Statistical
analysis
All results are given as means f SE for five sheep. The changes in pancreatic juice flow. protein and amylase outputs, plasma hormones and glucose were expressed as the difference between the peak and basal values (mean value of -20, - 10 and 0 min samples). Statistical analyses were performed using a paired r-test (P < 0.05).
135
CCK-8 on pancreatic function in sheep
Results Figure 1 shows the time course changes in flow rate, and in protein and amylase outputs in pancreatic juice after CCK-8 infusion at doses of 10, 30 and 120 pmol/kg/min. The basal values in flow rate, and in protein and amylase outputs in pancreatic juice were I .78 f 0.79 ml, 81.5 f 42.1 mg and 8.24 + 5.11 kU per lOmin, respectively (n = 5 and 8). Physiological saline (control solution) and 5 pmol/kg/min CCK-8 did not affect exocrine pancreatic secretion (data not shown). Dose-dependent increases in juice flow and protein output were observed at doses between 10 and 120 pmol/kg/min CCK-8. The maximal juice flow response occurred at 120 pmol/kg/min CCK-8 infusion. The juice flow response to 240 pmol/kg/min was the same as that to 120 pmol/kg/min infusion (data not shown). Dose-dependent increases in protein and amylase outputs were observed up to doses of CCK-8 at 120 and 240 pmol/kg/min, respectively. Figure 2 shows the time course changes in plasma insulin, glucagon and glucose concentrations after CCK-8 infusions at doses of 10, 30 and 120 pmol/kg/min. The basal values in
!i q’orb
60 Time (min)
Fig. 2. The time course of changes in plasma insulin, glucagon and glucose concentrations during and after CCK-8 infusion. The values are means k SE for five sheep. The doses are 10 (a), 30 (A), and 120 (m) pmol/kg/min. The horizontal line represents the infusions period (40 min). Open symbols indicate significant differences (P < 0.05) from the pre-infusion value (means of time -20, - IO and 0).
;1
+o-
60 Time (mln)
Fig. 1. The time course of changes in pancreatic juice flow, and in protein and amylase outputs during and after CCK-8 infusion. The values are means f SE for five sheep. The doses are 10 (O), 30 (A), and 120 (m) pmol/kg/min. The horizontal line represents the infusions period (40 min). Open symbols indicate significant differences (P < 0.05) from the pre-infusion value (means of time -20, - 10 and 0).
plasma insulin, glucagon and glucose concentrations were 12.3 + 5.7 pU/ml, 184 i 26 pg/ml and 67.2 f 7.5 mg/lOO ml, respectively (n = 5 and 8). Physiological saline (control solution) and 5 pmol/kg/min CCK-8 did not affect plasma concentrations of pancreatic hormones or glucose (data not shown). Plasma insulin after the CCK-8 increased immediately infusion, with a similar time course of responses to all doses of 10 pmol/kg/min and above. Dosedependent insulin responses were obtained with doses from 10 to 240 pmol/kg/min of CCK-8 infusion (data not shown). Plasma glucagon was not affected by any dose of CCK-8 infusion used in this experiment. Plasma glucose was decreased in step with increases in the dose of CCK-8. Figure 3 shows the dose-response relationships between the infusion rate of CCK-8, and peak increments in amylase output and insulin concentration relative to basal values. Dose-dependent amylase output was observed at a dose of between 10 and 120 pmol/kg/min CCK-8 infusion. The amylase output response
H. Mineo Ed ul
136
y
I,
I
0
10 Dose
of
30
CCK-B
100
’
300
(pmol/kg/mln)
Fig. 3. Dose-response relationships between rates of mfusion of CCK-8 and increments in amylase output and plasma insulin concentrations, The values are means + SE for five sheep. The increments represent the difference between the maximal output or amylase (0) or peak plasma insulin concentration (0) and the pre-infusion value (means of time -20, - IO and 0). Asterisks indicate a significant difference (P < 0.05) from the control (physiological saline Infusion) value.
obtained with 240 pmol/kg/min of CCK-8 was almost the same as that obtained with 120 pmol/kg/min infusion. By contrast, the insulin increment increased in step with the dose of CCK-8, but no maximal response was observed even with the highest dose of CCK-8. The threshold dose of CCK-8 for stimulating both amylase output and insulin secretion was in the range of l&30 pmol/kg/min of CCK-8 infusion.
Discussion Previous reports concerning the effects of CCK on the ruminant endocrine pancreas are limited (Baile et al., 1969; Godden et al., 1981; Takahashi et al., 1983). In those experiments, the doses of CCK were very high compared with physiological levels, and administration was done by a single injection, rather than by continuous infusion. A recent report from our laboratory demonstrated that intravenous infusion of CCK-8 at low doses (3-1000 pmol/ kg/min for 40 min) induced insulin secretion, but did not affect glucagon secretion in sheep (Mine0 et al., 1994). However, it was not known whether the doses of CCK-8 infused intravenously were really within the physiological range. In this experiment, it was possible to determine physiological doses of CCK-8, since the pancreatic exocrine response to exogenously infused CCK-8 was used as an indicator of the physiological effects of the peptide. The present results demonstrate that the intravenous infusion of CCK-8 stimulates exocrine pancreatic secretion in a dose-dependent manner, and, at the same time, increases plasma insulin, but not glucagon concentrations in
conscious sheep. In addition, analysis of the dose-response relationships showed that the threshold dose of CCK-8 for stimulating insulin secretion is similar to that for stimulation of amylase output ( 1O-30 pmol/kg/min). These results suggest that CCK may stimulate insulin secretion within the physiological range of doses which elicit the well-known biological action of CCK-8 on exocrine pancreatic secretion. The simultaneous effects of exogenous CCK on both exocrine and endocrine pancreatic secretions have not been studied previously in order to evaluate the physiological role of CCK on pancreatic hormone secretion in vivo in any there are many reports species. By contrast, which compare the effects of CCK or its related peptides on both the exocrine and endocrine pancreas in vitro, using perfused pancreatic preparations from non-ruminant animals (Otsuki et al., 1979; Jensen et al., 1981; Sakamoto et al., 1982; Okabayashi et al.. 1983). A supraphysiological dose of CCK-8 was needed to stimulate insulin release at low or normal glucose concentrations in the perfusing solution in rats (Otsuki er al., 1979; Sakamoto et al.. 1982; Okabayashi el al., 1983) and pigs (Jensen ef ul., 1981). However, in these experiments, physiological doses of CCK stimulated insulin secretion at high glucose concentrations in the infused medium. An enhancing effect of glucose on CCK-induced insulin secretion was demonstrated using perfused pancreas or isolated pancreatic islets in rats (Sakamoto et al., 1982: Verspohl et al.. 1986; Zawalich et al.. 1986). The effect of CCK on insulin secretion in vitro at normal glucose levels was thus very weak in non-ruminant species. Another approach is to measure plasma concentrations of both pancreatic hormones and CCK, when the latter has been exogenously infused (Rushakoff et al., 1987; Liddle et al.. 1988; Hildebrand et al., 1990; Schmidt et ul., 1991), or endogenously released by normal or test meals (Rushakoff et al., 1987; Liddle et al.. 1988; Hildebrand et al., 1990; Schmidt et al.. 1991). and thereby determine the role of CCK as a physiological regulator of pancreatic hormone secretion. With this approach, although the stimulating effect of CCK on insulin secretion was weak, amino acids enhanced CCK-induced insulin secretion in humans (Rushakoff et al., 1987). It has been reported that pancreatic hormone secretion induced by a pharmacological dose of exogenous CCK was enhanced after a meal (Ahren et al., 1991). Both in vivo and in vitro studies indicate that the effect of CCK in stimulating pancreatic hormone secretion was weak, and that nutrients in the blood or in the perfusing solution enhance CCK-induced secretion of insulin or other
CCK-8 on pancreatic
pancreatic hormones in non-ruminant species (Sakamoto et al., 1982; Verspohl et al., 1986; Zawalich et al., 1986; Rushakoff et al., 1987). At least in sheep, a single CCK-8 infusion could evoke insulin secretion with the same threshold as for amylase output, even though plasma glucose levels are lower than in non-ruminant animals. In this experiment, although the infusion period of CCK was 40 min, the insulin response was obtained only over the first 2 or 5 min after the beginning of the infusion. Although it has not yet been studied, the enhancement effect of nutrients may also play an important role in CCK-induced insulin secretion after feeding in ruminants. In non-ruminant animals, CCK-8 stimulated insulin and glucagon secretion in uivo in dogs (Frame et al., 1975; Williams and Champagne, 1979), mice (Ah& et al., 1986) and pigs (Ahren et al., 1988). On the other hand, in human beings, CCK-8 stimulated insulin, somatostatin and pancreatic polypeptide secretion, but not glucagon (Ah&n et al., 199 1). The form of CCK also affects its activity for pancreatic hormone secretion. In the mouse in viuo, CCK-39 had a greater potential than CCK-33 or CCK-8 for insulin secretion, and CCK-4 did not affect insulin levels (Ah&n et al., 1986). In contrast, CCK-8 and CCK-4 stimulated insulin, glucagon and somatostatin secretion in pigs in z&o, and the potency of the two peptides for pancreatic hormone secretions was the same (Ah& et al., 1988). In vitro experiments, using rats (Okabayashi et al., 1983; Verspohl et al., 1986; Sandberg et al., 1988) dogs (Hermansen, 1984) or pigs (Jensen et al., 1981), also showed that the effect of CCK on pancreatic hormone secretion depended on the species and the form of peptides. Thus, the activity of CCK on pancreatic hormone secretion depends on the molecular form of CCK, and differs between species both in vivo and in vitro. It has been reported that CCK-immunoreactive cells are found in the duodenum and the jejunum of sheep, as in non-ruminants (Calingasan et al., 1984). An increase in plasma CCK levels was observed in the calf after milk ingestion (Toullec et al., 1992; De PassillC et al., 1993). In addition, the postprandial variations of protein output in pancreatic juice were correlated to the changes in plasma CCK concentrations in the milk-fed calf (Le Huerou-Luron et al., 1994). At present, there is no information about determining the changes in exocrine pancreatic secretion and plasma CCK levels simultaneously after feeding in adult ruminants. A postprandial increase in plasma CCK concentration was also reported in rats (Green et al., 1989) and humans (Liddle et al., 1985, 1988; Schmidt et al., 1991). However, plasma CCK
function
in sheep
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levels were not changed after feeding in dairy cows (Furuse et al., 1991) and goats (Furuse et al., 1992). These results might reflect the continuous flow of digesta from the rumen into the duodenum in adult ruminants. Even in non-ruminant species, a physiological role of CCK in regulating pancreatic hormone secretion after a meal has only been established in rats, and was questionable in humans (Karlsson and Ahren, 1992). In conclusion, CCK appears to be one of the candidates for physiological regulation of insulin secretion, but not for glucagon in sheep, using amylase output induced by exogenously infused CCK-8 as an indicator of physiological relevance. However, the detailed mechanism by which CCK-8 induces insulin secretion was not determined in this experiment. Further experiments, such as measuring plasma CCK levels or using CCK antagonists, are needed to clarify the physiological role and the mechanism of CCK-induced insulin secretion in ruminants. Acknowledgement-The authors wish to thank Dr T. E. C. Weekes, University of Newcastle upon Tyne, U.K., for his help in preparing this manuscript,
References Ahren B., Hender P. and Lundquist I. (1986) Effects of six cholecystokinin (CCK) fragments on insulin secretion in the mouse. Acta Pharmac. Toxic. 58, 115-120. Ahrtn B., Martensson H. and Nobin A. (1988) Cholecystokinin (CCK)-4 and CCK-8 stimulate islet hormone secretion in uivo in the pig. Pancreas 3, 279-284. Ahren B., Pettersson M., Uvnis-Moberg K., Gutniak M. and Efendic S. (1991) Effects of cholecystokinin (CCK)-8, CCK-33 and gastric inhibitory polypeptide (GIP) on basal and meal-stimulated pancreatic hormone secretion in man. Diabetes Res. clin. Pratt. 13, 153-162. Baile C. A., Glick Z. and Mayer J. (1969) Effects of secretin and cholecystokinin-pancreozymin on pancreatic juice and insulin secretion of goats. J. Dairy Sci. 52, 513.-5 17. Calingasan N. Y., Kitamura N., Yamada J., Oomori Y. and Yamashita T. (1984) Immunocytochemical study of the gastroenteropancreatic endocrine cells of the sheep. Acfa Anat. 118, 171-180. De Passille A. M. B., Christopherson R. and Rushen J. (1993) Nonnutritive sucking by the calf and postprandial secretion of insulin, CCK, and gastrin. Physiol. Behar. 54, 1069-1073. Frame C. M., Davidson M. B. and Sturdevant R. A. L. (1975) Effects of the octapeptide on cholecystokinin on insulin and glucagon secretion in the dog. Endocrinology 97, 549-553. Furuse M., Kato M., Yang S. I., Asakura K. and Okumura J. (1992) Influence of dietary protein concentrations or of duodenal amino acid infusion on cholecystokinin release in goats. Comp. Biochem. Physiol. lOlA, 635-638. Furuse M., Yang S. I., Choi Y. H., Kawamura N., Takahashi A. and Okumura J. (1991) A note on plasma cholecystokinin concentration in dairy cows. Anim. Prod. 53, 123-125. Godden P. M. M., Ward J. B. and Weekes T. E. C. (1981) The effects of glucagon, tolbutamide, and cholecystokinin octapeptide on plasma insulin and glucose concentrations in sheep. Gen. romp. Endocr. 43, 405408.
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Green G. M., Taguchi S.. Friestman J.. Chey W. Y. and Liddle R. A. (1989) Plasma secretin, CCK, and pancreatic secretion in response to dietary fat in the rat. Am. J. Physiol. 256, G10161021. Hermansen K. (1984) Effects of cholecystokinin (CCK)-4. non-sulfated CCK-8, and sulfated CCK-8 on pancreatic somatostatin, insulin, and glucagon secretion in the dog: studies in vitro. Endocrinology 114, 1770-l 775. Hildebrand P., Ensinck J. W., Mossi S.. Delco F.. Gry K. and Beglinger C. (1990) Cholecystokinin (CCK) is not an incretin in humans. Digestion 46, 1433144. Jensen S. L.. Retheld J. F., Hoist J. J., Nielsen 0. V.. Fahrenkrug J. and Schaffalitzky de Muckadell 0. 8. (1981) Secretory effects of cholecystokinins on the isolated perfused porcine pancreas. Acta Physiol. &and. 111, 225-231. Kabasakalian P.. Kalliney S. and Westcott A. (1974) Enzymatic blood glucose determination by calorimetry of N,N-diethylaniline-4-amonoantipyrine. Clin. Chem. 20, 606607. Kanno T. (1975) The electrogenic sodium pump in the hyperpolarizing and secretory effects of pancreozymin in the pancreatic acinar cell. J. Physiol. 245, 599416. Karlsson S. and Ahren B. (1992) Cholecystokinin and the regulation of insulin secretion. &and. J. Gastroenterol. 27, 161-165. Le Hdrou-Luron I.. Le Drean G., Chayvialle J. A.. Bernard C., Rome V., Toullec R. and Guilloteau P. (1994) Are secretin and cholecystokinin (CCK) involved in the regulation of postprandial pancreatic juice secretion in preruminant calves? Preliminary results concerning juice flow and protein concentration and plasma regulatory peptides levels. Proc. Sot. Nutr. Physiol. 3, 298. Liddle R. A., Goldfine 1. D.. Rosen M. S.. Taplitz R. A. and Williams J. A. (1985) Cholecystokinin bioactivity in human plasma. J. clin. Invest. 75, 1144-1152. Liddle R. A., Rushakoff R. J.. Morita E. T., Beccaria L.. Carter J. D. and Goldfine I. D. (1988) Physiological role for cholecystokinin in reducing postprandial hyperglycemia in humans. J. clin. Invest. 81, 1675-1681. Lowry 0. H.. Rosebrough N. J.. Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mineo H., Iwaki N., Onaga T. and Kato S. (1994) Effects of intravenous infusions of cholecystokinin-8 and pentagastrin on plasma concentrations of insulin and glucagon in sheep. Res. Vet. Sci. 56, 298-302. Okabayashi Y., Otsuki M. and Baba S. (1989) Cholecystokinin in the entero-insular axis. Diabetes Res. Clin. Pracf. 7, S79-85. Okabayashi Y., Otsuki M., Ohki A., Sakamoto C. and Baba S. (1983) Effects of C-terminal fragments of cholecystokinin on exocrine and endocrine secretion from isolated perfused rat pancreas. Endocrinology 113, 2210-2215. Otsuki M., Sakamoto C., Yuu H.. Maeda M., Moita S..
Ohki A., Kobayashi N., Terashi K., Okano K. and Baba S. (1979) Discrepancies between the doses of cholecystokinin- or caerulein-stimulating exocrine and endocrine responses in perfused isolated rat pancreas. J. clin. Invest. 63, 478-484. Rushakoff R. J., Goldfine I. D., Carter J. D. and Liddle R. A. (1987) Physiological concentrations of cholecystokinin stimulate amino-acid induced insulin release in humans. J. clin. Endocr. Metab. 65, 395401. Rushakoff R. J.. Liddle R. A.. Williams J. A. and Goldfine I. D. (1990) The role of cholecystokinin and other gut peptides on regulation of postprandial glucose and insulin levels, In Insulin. Handbook of Experimental Pharmacoiog_v 92 (Edited by Cautrecasas P. and Jacobs S), pp. 125-149. Springer, Berlin. Sakamoto C., Otsuki M., Ohki A.. Yuu H., Maeda M., Yamasaki T. and Baba S. (1982) Glucose-dependent insulinotropic action of cholecystokinin and caerulein in the isolated perfused rat pancreas. Endocrinolog_v 110, 398402. Sandberg E.. Ahren B.. Tendler D. and Efendic S. (1988) Cholecystokinin (CCK)-33 stimulates insulin secretion from the perfused rat pancreas: studies on the structureactivity relationship. Pharmac. Toxic. 63, 4245. Sasaki Y. and Takahashi H. (1980) Insulin secretion in sheep exposed to cold. J. Physiol. 306, 323-335. Sasaki Y.. Takahashi H.. Aso H.. Ohneda A. and Weekes T. E. C. (1982) Effects of cold exposure on insulin and glucagon secretion in sheep. Endocrinology 111, 2070-2076. Schmidt W. E., Creutzfeldt W.. Hacker M.. Nustede R.. Choudhury A. R., Schleser A.. Rovati L. C. and Folsch U. R. (1991) Cholecystokinin receptor antagonist loxiglumide modulates plasma levels of gastro-entero-pancreatic hormones in man. Eur. J. cfin. Invest. 21, 501-505. Takahashi H.. Ohtani F.. Aso H.. Sasaki Y. and Tsuda T. (1983) Insulin response to gastrointestinal hormones in sheep. h Proceedings. The fifth world conference on animal production 2, pp. 319-320. Japanese Society of Zootechnical Science, Tokyo. _ Toullec R.. Chavvialle J. A.. Guilloteau P. and Bernard C. (1992) Early-life patterns of plasma gut regulatory peptide levels in calves.. Effects of age, weaning and- fcehing. Coma. Biochem. Phvsiol. 102A. 203-209. Verspohl E. J., Amman H. P. T., Williams J. A. and Goldfine I. D. (1986) Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. Diabetes 35, 3843. Williams R. H. and Champagne J. (1979) Effects of cholecystokinin, secretin, and pancreatic polypeptide on secretion of gastric inhibitory polypeptide, insulin, and glucagon. Life Sci. 25, 947-956. Zawalich W., Cote S. B. and Diaz V. A. (1986) Influence of cholecystokinin on insulin output from isolated perfused pancreatic islets. Endocrinology 119, 616-621.
Pergamon 0300-%29(94)00192-8
Effects of intravenous infusions of cholecystokinin (CCK)-8 on exocrine and endocrine pancreatic secretion in conscious sheep H. Mineo,* N. Iwaki,* K. Kogishi,* R. Zabielski,? T. Onaga* and S. Kate* *Department of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido Japan; and TDepartment of Animal Physiology, Faculty of Veterinary Medicine, Warsaw Agricultural University, 02-766 Warsaw, Poland
069,
The effects of cholecystokinin (CCK)3 on both exocrine and endocrine pancreatic functions were examined simultaneously in five conscious sheep. Intravenous infusions of CCK-8 (0, 5, 10, 20, 30, 60, 120 and 240 pmol/kg/min for 40 min) induced dosedependent increases in flow rate, and in protein and amylase outputs in pancreatic juice. The same CCK-8 infusions induced dosedependent increases in plasma insulin, but no change in plasma glucagon concentrations. The threshold dose (l&30 pmol/kg/min) of CCK-8 infusion for stimulating insulin secretion was similar to that for stimulating amylase output. In conclusion, using amylase output as an indicator of physiological action, CCK is one of the potential candidates as a physiological regulator of insulin, but not glucagon secretion in sheep. Key words: CCK; Exocrine
pancreas; Endocrine
pancreas; Pancreatic juice; Amylase; Insulin;
Glucagon; Sheep; Ruminants. Comp. Biochem. Physiol. 11IA, 133-138, 199.5.
Introduction In non-ruminant animals, a variety of gastrointestinal hormones is known to affect endocrine pancreatic secretions (Rushakoff et al., 1990). In addition to its action on exocrine pancreatic secretion, in vivo and in vitro studies have shown that cholecystokinin (CCK) stimulates pancreatic hormone secretion from the endocrine cells of the pancreas (Karlsson and Ahren, 1992). A variety of evidence supports the hypothesis that CCK is one of the candidates for functioning as a physiological regulator of pancreatic hormone secretion in non-ruminant species (Okabayashi et al., 1989; Rushakoff et al., 1990). On the other hand, there have been Correspondence lo: Dr H. Mineo, Department of Medical Physiology C, Panum Institute, Blegdamsvej 3, DK-2200, Copenhagen N, Denmark. Tel. 45-3532-7516; Fax 45-3532-7537. Received 9 May 1994; revised 8 November 1994; accepted 14 November 1994.
few reports of the effects of CCK on endocrine pancreatic responses in ruminants (Baile et al., 1969; Godden et al., 1981; Takahashi et al., 1983). In these reports, pharmacological doses of CCK were used to induce pancreatic hormone secretion. In a recent report from our laboratory, intravenous infusions of CCK-8 at low doses induced insulin, but not glucagon secretion in sheep (Mine0 et al., 1994). However, it was not known whether the doses of CCK-8 used in that experiment were within the physiological range. One of the physiological actions of CCK is well known to be a stimulating effect on enzyme secretion from the acinar cells of the pancreas. Thus, the aim of the present study was to simultaneously examine the effect of intravenous infusions of CCK-8 on the exocrine and endocrine pancreatic responses, and to compare the dose-response relationships of 133
H. Mineo et al.
134
CCK-8 and its effects on both exocrine and endocrine function of the pancreas in conscious sheep.
Materials and Methods Animals and diets
Five Suffolk cross-bred wethers, aged l-2 years and weighing 3842 kg, were used. They had their left common carotid artery chronically placed in a loop of skin by surgical operation under general anesthesia with pentobarbitone sodium (25 mg/kg) more than 2 months before experiments began. In addition, under general anesthesia introduced by pentobarbital sodium and maintained by halothane and oxygen. each animal was chronically catheterized with silastic tubing (2 mm I.D., 4 mm O.D.) inserted into the common bile duct at two places: one close to the duodenum for collection of pancreatic juice and the other close to the gall-bladder for collection of bile. The common bile duct was ligated immediately distal to both catheter insertions. A third catheter (2 mm I.D., 4mm O.D.) was inserted into the duodenum close to the opening of the common bile duct to return the bile and pancreatic juice. Sheep were housed in individual cages and were fed lucerne pellets (1000 g) and orchard grass hay (100 g) once daily at 19.00. Water was freely available. Pancreatic juice and bile were returned to the duodenum of each sheep at approximately the rate of secretion, using a peristaltic infusion pump. Sheep were accustomed to the experimental environment for at least 10 days after surgery. A polyethylene catheter for infusion of solutions was inserted into the right jugular vein at least 3 days before experiments. The catheter was maintained with a sterilized solution of 3.8% (wt/vol) trisodium citrate. E.yperimental
procedure
On the day of the experiment. a catheter was inserted into the carotid artery at least 3 hr before blood sampling. CCK-8 (Peptide Institute Inc., Osaka, Japan) was dissolved and diluted with 0.9% (wt/vol) NaCl solution to the appropriate concentrations immediately before infusion. CCK-8 solutions were infused at rates of 5, 10, 20, 30, 60, 120 and 240 pmol/kg/min for 40min through the jugular vein catheter. Sterile 0.9% NaCl solution was infused for 40 min as a control. The infusion volume o.f each solution was 0.5 ml/min delivered by using an infusion pump (SJ-1211, ATTO, Tokyo, Japan). In each experiment, CCK-8 infusion was started 30min after initiating pancreatic juice sampling. The flow rate of pancreatic juice was measured every 10 min for 90 min, and
20 ~1 of each sample was taken for analysis. Sampling of blood was undertaken at -20, - 10, 0, 2, 5, 10, 20, 30, 40 and 60 min relative to the starting time of the CCK-8 infusion. Each blood sample (4 ml) was taken by syringe and immediately transferred into a polyethylene test-tube containing EDTA (I .2 mg/ml blood, Kanto Chemical Co., Tokyo, Japan) and aprotinin (500 KIU/ml blood, Hoechst Japan, Tokyo, Japan). Each experiment was started at 10.00. Tests using the same animal were separated by at least 2 days.
The protein concentration of pancreatic juice was determined by the method of Lowry et al. ( 1951) using bovine serum albumin as a standard. Amylase activity was assayed by the method of Kanno (1975). A unit of activity was defined as the amount of enzyme which produced 1 mg maltose/min from starch (Merck, Germany). Plasma was separated by centrifugation (SOOOg for 15 min) at 4°C and stored at -20°C until used for hormone assay. The plasma insulin and glucagon concentrations were determined by radioimmunoassay procedures, using previously described methods for insulin (Sasaki and Takahashi, 1980) and glucagon (Sasaki et cd., 1982). Guinea-pig anti-bovine insulin serum (ICN, Israel) and ““I-labeled porcine insulin (NEN, Boston, U.S.A.) were purchased for insulin assay. Bovine insulin (Sigma, St. Louis, U.S.A.) was used as a standard. Intraand inter-assay coefficients of variation for insulin determination were 5.2% (n = 6) and 8.7% (n = 5), respectively. The antiserum used for the glucagon assay (rabbit antiserum to bovine pancreatic glucagon, Calbiochem Co., California. U.S.A.) was specific for pancreatic glucagon. Porcine glucagon was used as a standard (Eli-Lilly Co., Indianapolis, U.S.A.) and as an iodinated hormone (NEN, Boston, U.S.A.). Intra- and inter-assay coefficients of variation for glucagon determination were 6.5% (n = 6) and 10.5% (n = 5), respectively. The remaining plasma was used for the determination of plasma glucose concentration by the glucoseoxidase method (Kabasakalian et al., 1974). Statistical
analysis
All results are given as means f SE for five sheep. The changes in pancreatic juice flow. protein and amylase outputs, plasma hormones and glucose were expressed as the difference between the peak and basal values (mean value of -20, - 10 and 0 min samples). Statistical analyses were performed using a paired r-test (P < 0.05).
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CCK-8 on pancreatic function in sheep
Results Figure 1 shows the time course changes in flow rate, and in protein and amylase outputs in pancreatic juice after CCK-8 infusion at doses of 10, 30 and 120 pmol/kg/min. The basal values in flow rate, and in protein and amylase outputs in pancreatic juice were I .78 f 0.79 ml, 81.5 f 42.1 mg and 8.24 + 5.11 kU per lOmin, respectively (n = 5 and 8). Physiological saline (control solution) and 5 pmol/kg/min CCK-8 did not affect exocrine pancreatic secretion (data not shown). Dose-dependent increases in juice flow and protein output were observed at doses between 10 and 120 pmol/kg/min CCK-8. The maximal juice flow response occurred at 120 pmol/kg/min CCK-8 infusion. The juice flow response to 240 pmol/kg/min was the same as that to 120 pmol/kg/min infusion (data not shown). Dose-dependent increases in protein and amylase outputs were observed up to doses of CCK-8 at 120 and 240 pmol/kg/min, respectively. Figure 2 shows the time course changes in plasma insulin, glucagon and glucose concentrations after CCK-8 infusions at doses of 10, 30 and 120 pmol/kg/min. The basal values in
!i q’orb
60 Time (min)
Fig. 2. The time course of changes in plasma insulin, glucagon and glucose concentrations during and after CCK-8 infusion. The values are means k SE for five sheep. The doses are 10 (a), 30 (A), and 120 (m) pmol/kg/min. The horizontal line represents the infusions period (40 min). Open symbols indicate significant differences (P < 0.05) from the pre-infusion value (means of time -20, - IO and 0).
;1
+o-
60 Time (mln)
Fig. 1. The time course of changes in pancreatic juice flow, and in protein and amylase outputs during and after CCK-8 infusion. The values are means f SE for five sheep. The doses are 10 (O), 30 (A), and 120 (m) pmol/kg/min. The horizontal line represents the infusions period (40 min). Open symbols indicate significant differences (P < 0.05) from the pre-infusion value (means of time -20, - 10 and 0).
plasma insulin, glucagon and glucose concentrations were 12.3 + 5.7 pU/ml, 184 i 26 pg/ml and 67.2 f 7.5 mg/lOO ml, respectively (n = 5 and 8). Physiological saline (control solution) and 5 pmol/kg/min CCK-8 did not affect plasma concentrations of pancreatic hormones or glucose (data not shown). Plasma insulin after the CCK-8 increased immediately infusion, with a similar time course of responses to all doses of 10 pmol/kg/min and above. Dosedependent insulin responses were obtained with doses from 10 to 240 pmol/kg/min of CCK-8 infusion (data not shown). Plasma glucagon was not affected by any dose of CCK-8 infusion used in this experiment. Plasma glucose was decreased in step with increases in the dose of CCK-8. Figure 3 shows the dose-response relationships between the infusion rate of CCK-8, and peak increments in amylase output and insulin concentration relative to basal values. Dose-dependent amylase output was observed at a dose of between 10 and 120 pmol/kg/min CCK-8 infusion. The amylase output response
H. Mineo Ed ul
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y
I,
I
0
10 Dose
of
30
CCK-B
100
’
300
(pmol/kg/mln)
Fig. 3. Dose-response relationships between rates of mfusion of CCK-8 and increments in amylase output and plasma insulin concentrations, The values are means + SE for five sheep. The increments represent the difference between the maximal output or amylase (0) or peak plasma insulin concentration (0) and the pre-infusion value (means of time -20, - IO and 0). Asterisks indicate a significant difference (P < 0.05) from the control (physiological saline Infusion) value.
obtained with 240 pmol/kg/min of CCK-8 was almost the same as that obtained with 120 pmol/kg/min infusion. By contrast, the insulin increment increased in step with the dose of CCK-8, but no maximal response was observed even with the highest dose of CCK-8. The threshold dose of CCK-8 for stimulating both amylase output and insulin secretion was in the range of l&30 pmol/kg/min of CCK-8 infusion.
Discussion Previous reports concerning the effects of CCK on the ruminant endocrine pancreas are limited (Baile et al., 1969; Godden et al., 1981; Takahashi et al., 1983). In those experiments, the doses of CCK were very high compared with physiological levels, and administration was done by a single injection, rather than by continuous infusion. A recent report from our laboratory demonstrated that intravenous infusion of CCK-8 at low doses (3-1000 pmol/ kg/min for 40 min) induced insulin secretion, but did not affect glucagon secretion in sheep (Mine0 et al., 1994). However, it was not known whether the doses of CCK-8 infused intravenously were really within the physiological range. In this experiment, it was possible to determine physiological doses of CCK-8, since the pancreatic exocrine response to exogenously infused CCK-8 was used as an indicator of the physiological effects of the peptide. The present results demonstrate that the intravenous infusion of CCK-8 stimulates exocrine pancreatic secretion in a dose-dependent manner, and, at the same time, increases plasma insulin, but not glucagon concentrations in
conscious sheep. In addition, analysis of the dose-response relationships showed that the threshold dose of CCK-8 for stimulating insulin secretion is similar to that for stimulation of amylase output ( 1O-30 pmol/kg/min). These results suggest that CCK may stimulate insulin secretion within the physiological range of doses which elicit the well-known biological action of CCK-8 on exocrine pancreatic secretion. The simultaneous effects of exogenous CCK on both exocrine and endocrine pancreatic secretions have not been studied previously in order to evaluate the physiological role of CCK on pancreatic hormone secretion in vivo in any there are many reports species. By contrast, which compare the effects of CCK or its related peptides on both the exocrine and endocrine pancreas in vitro, using perfused pancreatic preparations from non-ruminant animals (Otsuki et al., 1979; Jensen et al., 1981; Sakamoto et al., 1982; Okabayashi et al.. 1983). A supraphysiological dose of CCK-8 was needed to stimulate insulin release at low or normal glucose concentrations in the perfusing solution in rats (Otsuki er al., 1979; Sakamoto et al.. 1982; Okabayashi el al., 1983) and pigs (Jensen ef ul., 1981). However, in these experiments, physiological doses of CCK stimulated insulin secretion at high glucose concentrations in the infused medium. An enhancing effect of glucose on CCK-induced insulin secretion was demonstrated using perfused pancreas or isolated pancreatic islets in rats (Sakamoto et al., 1982: Verspohl et al.. 1986; Zawalich et al.. 1986). The effect of CCK on insulin secretion in vitro at normal glucose levels was thus very weak in non-ruminant species. Another approach is to measure plasma concentrations of both pancreatic hormones and CCK, when the latter has been exogenously infused (Rushakoff et al., 1987; Liddle et al.. 1988; Hildebrand et al., 1990; Schmidt et ul., 1991), or endogenously released by normal or test meals (Rushakoff et al., 1987; Liddle et al.. 1988; Hildebrand et al., 1990; Schmidt et al.. 1991). and thereby determine the role of CCK as a physiological regulator of pancreatic hormone secretion. With this approach, although the stimulating effect of CCK on insulin secretion was weak, amino acids enhanced CCK-induced insulin secretion in humans (Rushakoff et al., 1987). It has been reported that pancreatic hormone secretion induced by a pharmacological dose of exogenous CCK was enhanced after a meal (Ahren et al., 1991). Both in vivo and in vitro studies indicate that the effect of CCK in stimulating pancreatic hormone secretion was weak, and that nutrients in the blood or in the perfusing solution enhance CCK-induced secretion of insulin or other
CCK-8 on pancreatic
pancreatic hormones in non-ruminant species (Sakamoto et al., 1982; Verspohl et al., 1986; Zawalich et al., 1986; Rushakoff et al., 1987). At least in sheep, a single CCK-8 infusion could evoke insulin secretion with the same threshold as for amylase output, even though plasma glucose levels are lower than in non-ruminant animals. In this experiment, although the infusion period of CCK was 40 min, the insulin response was obtained only over the first 2 or 5 min after the beginning of the infusion. Although it has not yet been studied, the enhancement effect of nutrients may also play an important role in CCK-induced insulin secretion after feeding in ruminants. In non-ruminant animals, CCK-8 stimulated insulin and glucagon secretion in uivo in dogs (Frame et al., 1975; Williams and Champagne, 1979), mice (Ah& et al., 1986) and pigs (Ahren et al., 1988). On the other hand, in human beings, CCK-8 stimulated insulin, somatostatin and pancreatic polypeptide secretion, but not glucagon (Ah&n et al., 199 1). The form of CCK also affects its activity for pancreatic hormone secretion. In the mouse in viuo, CCK-39 had a greater potential than CCK-33 or CCK-8 for insulin secretion, and CCK-4 did not affect insulin levels (Ah&n et al., 1986). In contrast, CCK-8 and CCK-4 stimulated insulin, glucagon and somatostatin secretion in pigs in z&o, and the potency of the two peptides for pancreatic hormone secretions was the same (Ah& et al., 1988). In vitro experiments, using rats (Okabayashi et al., 1983; Verspohl et al., 1986; Sandberg et al., 1988) dogs (Hermansen, 1984) or pigs (Jensen et al., 1981), also showed that the effect of CCK on pancreatic hormone secretion depended on the species and the form of peptides. Thus, the activity of CCK on pancreatic hormone secretion depends on the molecular form of CCK, and differs between species both in vivo and in vitro. It has been reported that CCK-immunoreactive cells are found in the duodenum and the jejunum of sheep, as in non-ruminants (Calingasan et al., 1984). An increase in plasma CCK levels was observed in the calf after milk ingestion (Toullec et al., 1992; De PassillC et al., 1993). In addition, the postprandial variations of protein output in pancreatic juice were correlated to the changes in plasma CCK concentrations in the milk-fed calf (Le Huerou-Luron et al., 1994). At present, there is no information about determining the changes in exocrine pancreatic secretion and plasma CCK levels simultaneously after feeding in adult ruminants. A postprandial increase in plasma CCK concentration was also reported in rats (Green et al., 1989) and humans (Liddle et al., 1985, 1988; Schmidt et al., 1991). However, plasma CCK
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levels were not changed after feeding in dairy cows (Furuse et al., 1991) and goats (Furuse et al., 1992). These results might reflect the continuous flow of digesta from the rumen into the duodenum in adult ruminants. Even in non-ruminant species, a physiological role of CCK in regulating pancreatic hormone secretion after a meal has only been established in rats, and was questionable in humans (Karlsson and Ahren, 1992). In conclusion, CCK appears to be one of the candidates for physiological regulation of insulin secretion, but not for glucagon in sheep, using amylase output induced by exogenously infused CCK-8 as an indicator of physiological relevance. However, the detailed mechanism by which CCK-8 induces insulin secretion was not determined in this experiment. Further experiments, such as measuring plasma CCK levels or using CCK antagonists, are needed to clarify the physiological role and the mechanism of CCK-induced insulin secretion in ruminants. Acknowledgement-The authors wish to thank Dr T. E. C. Weekes, University of Newcastle upon Tyne, U.K., for his help in preparing this manuscript,
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