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Decreased pancreatic CCK receptor binding and CCK-stimulated amylase release in Zucker obese rats
Physiology& Behavior,Vol. 32, pp. 961-965. Copyright ©Pergamon Press Ltd., 1984. Printed in the U.S.A.
0031-9384/84 $3.00 + .00
Decreased Pancreatic CCK Receptor Binding and CCK-Stimulated Amylase Release in Zucker Obese R a t s I C A R O L L. M c L A U G H L I N , * T z S T E V E N R, P E I K I N * A N D C L I F T O N A. B A I L E r 2
*Department o f Medicine, Jefferson Medical College, Thomas Jefferson University Philadelphia, PA 19107 and TDepartment o f Clinical Studies at New Bolton Center, School o f Veterinary Medicine University o f Pennsylvania, Kennett Square, PA 19348 R e c e i v e d 25 J u l y 1983 McLAUGHLIN, C. L., S. R. PEIKIN AND C. A. BAILE. Decreased pancreatic CCK receptor binding and CCKstimulated amylase release in Zucker obese rats. PHYSIOL BEHAV 32(6) 961-965, 1984.--In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations cf CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per/xg DNA basis (19.6_+5.1 vs. 47.4_+ 11.4, p<0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10-1° M), less amylase was secreted in obese rats across the dose range tested (/9<0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p<0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p<0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p<0.001) and by carbachol was only 20% of control (p <0.001). Thus, increased size by increased cell number was associated with decreased response per cell. Results of these experiments support the hypothesis that decreased response of the pancreas of obese compared with lean rats to CCK is due to decreased binding of CCK to receptors. A similar decreased binding of CCK to receptors for satiety in obese animals, if present, may be responsible for increased meal size associated with obesity. CCK-receptor Amylase secretion Zucker obese rats Carbachol Pancreatic acini Trypsin inhibitor DNA
Receptor binding
lz~I-CCK-33
Satiety
weight is the same, thus the second possibility appears to be more likely, assuming the kinetics of absorption of the CCK are similar [13,14]. The location of the receptors for the effect of C C K on satiety is unknown, although there is evidence they may be in the stomach [21]. Receptors for the effect of C C K on pancreatic amylase secretion are on pancreatic acinar cells and their characteristics could be used to evaluate responsiveness of obese animals to the effects of CCK on pancreas exocrine function. CCK stimulates less secretion of amylase from the pancreas in vivo of obese than lean rats [15]. In vitro percent of basal amylase secretion from dispersed pancreatic acini in response to CCK was similar in obese and lean rats [15]. In the in vitro experiment, since no measurement of
C H O L E C Y S T O K I N I N (CCK), one of many peptides released from the gastrointestinal tract by the presence of food, stimulates contraction of the gall-bladder and secretion o f enzymes from the pancreas [1,17]. In addition, increased serum concentrations of C C K have been proposed to signal satiety and result in termination of a meal [20]. In obese rats and mice the increase in meal size which is characteristic may be due to release of less C C K during meals [13,14]; however measurement of serum concentrations of CCK have not been reported in rodents. Another possibility is that receptor number or sensitivity to normal or increased concentrations of C C K is decreased in obese rats. Exogenous administration of C C K decreases food intake less in obese than lean rats and mice; the amount received per body
1This work was supported in part by the Cowperthwaite Fund, Jefferson Medical College, and Research Foundation and Feeding Fund, University of Pennsylvania. 2Present address: Department of Preventive Medicine, Washington University, 4566 Scott Avenue, Box 8113, St. Louis, MO 63110. Requests for reprints should be addressed to C. McLaughlin at her present address.
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McLAUGHLIN, PEIKIN AND BAILE
DNA in the incubation medium was made, response per cell was unknown. It was concluded that since the dose-response curve was not shifted rightward, C C K was equally potent in obese and lean rats [15]. However, evaluation of the efficacy of CCK was not possible. In vitro binding of CCK to its receptor on pancreatic acini has been highly correlated with stimulation of amylase secretion by CCK [9]. Thus, the present experiments were designed to determine if decreased effect of CCK on pancreas growth and in vivo exocrine function in obese rats is a result of decreased binding of CCK to its receptor or decreased efficacy of CCK in obese rats as expressed on a per DNA basis. It is possible that decreased pancreatic exocrine response to CCK in obese compared with lean rats is due solely to decreased pancreas size. To determine if pancreas size is related to function, pancreatic amylase secretion in response to CCK-8 stimulation was measured in rats in which pancreas size was increased by the chronic administration of a trypsin inhibitor. METHOD
those reported here the bioactive fractions were selected by incubating 70,000-100,000 dpm of each fraction with acinar cells from normal rats using the procedure above. They were incubated in the presence or absence of a maximally effective concentration of CCK-8 (16 -6 M) to measure nonspecific and total binding respectively. Only the fractions exhibiting the highest specific binding (total minus nonspecific, usually 1 or 2 fractions) were used in the subsequent binding experiments. D. Measurement o f D N A . The DNA content of the incubation medium was measured in 0.20 ml aliquots to which 0.80 ml of 0.4 N KCI had been added using diphenylamine [7] and calf thymus DNA as a standard. E. Data analysis. Percent binding in the presence or absence of CCK-8 was determined for each rat and dose of CCK-8 and data were subjected to analysis of variance for effects of phenotype, dose of CCK-8 and replicate and interactions of these. Similar analyses were used for percent binding/DNA values calculated. Body weight, pancreas weight and D N A content of obese rats were compared to those of lean rats using paired t-tests.
Experiment 1. CCK Receptor Binding A. Rats. Six obese (307-+27 g) and six lean (188_+11 g) female Zucker rats were used. Prior to experimental days they were housed in a room maintained at 21°C and with a 12-hr light-dark cycle. They were fed (Purina Rat Chow pellets) and watered ad lib. On the experimental day food was removed 4-6 hr before the animals were sacrificed. B. Incubation procedure. On experimental days a rat was sacrificed and the pancreas carefully removed and weighed. Dispersed pancreatic acini were prepared as described by Peikin et al. [17]. Briefly, the pancreas was incubated with purified collagenase, and the dispersed acini obtained were suspended in 7.5 ml incubation solution containing 24.5 mM Hepes (pH=7.4), 98 mM NaC1, 6 mM KCI, 2.5 mM Nail2 PO4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium gluconate, 11.5 mM glucose, 2.0 mM CaCl2, 1.0 mM MgCi2, 2 mM glutamine, 0.2% (wt/vol) albumin, 0.01% (wt/vol) trypsin inhibitor and 1% (vol/vol) essential amino acid mixture. The medium was adjusted to contain 0.5 mM calcium, 5 mM theophylline and 1% (wt/vol) albumin. Of the suspended cells 0.5 ml was removed and frozen for subsequent measurement of D N A content. Six 1.0 ml aliquots were incubated with no unlabelled CCK (total binding) or CCK from l0 -~° to 10-n M (non-specific binding) using the methods described by Jensen et al. [12]. To each was added 10/~l of 70,000-100,000 dpm biologically active iodihated CCKaa (Bolton-Hunter technique [3], obtained from Mr. Thomai, Hazelton Laboratories, Vienna, VA). Biological activity was determined by using the method below (c). The tubes were placed in a Dubnoff incubator at 160 oscillations/min at 37° for 30 min then placed on ice. From each tube two 400 ~l aliquot cell suspensions were centrifuged at 10,000 g for 15 sec in a Beckman microcentrifuge. The supernatant was removed with suction and the cells vortexed and washed with ice-cold incubation medium containing 4% albumin and 0.1% (wt/vol) bacitracin. The cells were washed twice more and counted. From each tube two 50/~l aliquants were counted to determine dpm added. On each treatment day both a lean and obese rat were used, with the sequence being reversed on successive days. C. Selection o f bioactive fractions o f ~2"~I-CCKa3. Each batch of ~25I-CCKaa arrived as 9-10 fractions from a Sephadex G-50 column. For preliminary experiments and
Experiment 2. CCK- and Carbachol-Stimulated Amylase Release in Control Rats and Rats Treated With Trypsin Inhibitor A. Rats. Ten obese (304_+14 g) and 12 lean (193_+1 g) female rats were individually housed and fed as in Experiment 1. Five pairs of obese rats and six pairs of lean rats were formed. One member of each pair was administered 2.0 ml/kg water intragastrically for seven days while the other member of each pair was administered 200 mg/kg trypsin inhibitor [TI, N, N-dimethyl-carbamoyl 4-(4-guanidinobenzlyloxy)-pbenyl acetate methanesulfate, obtained from Oro Pharmaceutical Co., Ltd.] for seven days. Food and water intakes and body weights were measured daily. Twenty-four hours after the last dosing the pancreata of two obese or two lean rats were used to measure amylase secretion. The pancreata from obese and lean rats were incubated on alternate days and the control and TI-treated pancreata were incubated first on alternate days, but with the same solutions made for the day. B. Incubation procedure. Pancreatic acini were prepared as in Experiment 1 with the following exceptions. After the 0.5 ml aliquot of suspended cells had been removed for subsequent DNA analysis, the incubation medium volume was increased to 70 ml. One mi aliquants were incubated with C C K - 8 [10 -11 to 10-6 M] or carbachol [10-8 to 10-a M]. After 30 min two 400 p.l aliquants were spun for 15 sec in a Beckman microfuge, 100 /~! of the supernatant from each was added to 0.5 ml of lysing solution [0.01 M sodium phosphate (pH 7.8), 0.1% (wt/vol) albumin and 0.1% (wt/vol) sodium dodecyl sulfate]. Aliquants of 100 #.1 were assayed for amylase activity by the method of Ceska et al. [4] using the Phadebas reagent. Total amylase activity was determined by assay of 100 /~l of 1.5 ml cell suspension which had been added to 13.5 mi lysing solution. Results were expressed as percent of basal amylase released into the incubation medium in 30 min by each concentration of peptide. C. D N A measurement. As in Experiment I. D. Data analysis. Food intake, weight gain and pancreas weight data were each subjected to analysis of variance to measure effects of treatment and phenotype and interaction of treatment with phenotype. In the in vitro experiments, for each peptide, percent of basal amylase release and percent
DECREASED PANCREATIC CCK RECEPTOR BINDING
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Experiment I. CCK Receptor Binding In Zucker obese rats percent total binding of iodinated CCKaa was one-fourth that in lean rats, F(1,55)=85.57, p<0.001, Fig. 1. In lean rats total binding was 4.4% and non-specific binding was 2.8%, with 1.6% specific binding. In obese rats, however, under the same conditions, on the same day, total binding was only I. 1%, with non-specific binding of 0.8% and 0.3% specific binding. In both obese and lean rats percent binding was dose-dependently inhibited by CCK-8, F(5,55)=2.92, p <0.02. Even though D N A content of the incubation medium was less in obese than lean rats [58-+8 vs. 101-+ 11 /~g/ml, paired-t =5.41, p <0.01], percent binding per D N A was also less in obese than lean rats, F(1,55)=90.84, p <0.001.
Experiment 2. CCK- and Carbachol-Stimulated Amylase Secretion in Obese and Lean Control Rats and Rats Treated With Trypsin Inhibitor During the seven-day treatment period food intake was decreased by TI in obese [19.1 vs. 25.5 g, F(1,14)=4.92, p<0.04] but not lean rats (19.6 vs. 18.8 g). Similarly, weight gain was less in obese rats treated with TI compared with control [ - 0 . 3 vs. 2.6 g, F(1,14)=8.53,p<0.01] but not in lean rats (0.9 vs. 0.1 g). Weight o f the pancreas from obese control rats was not different from that o f lean control rats (0.80 vs. 0.86 g). While administration o f trypsin inhibitor increased pancreas size o f both obese [1.19 vs. 0.80, F(1,12)=12.54, p<0.004] and lean [1.62 vs. 0.86 g, F(1,12)=58.85, p<0.001] rats, the increase was greater in lean than in obese rats, F(1,12)=6.53, p<0.03. Comparison o f values from control obese with control lean rats demonstrated that D N A per ml incubation medium (60 vs. 68/~g/ml), basal percent of total amylase release (3.1 vs. 1.8) and basal percent of total amylase release//xg D N A (0.05 vs. 0.03) were not different (Fig. 2). However, in response to stimulation by C C K percent of basal amylase released//zg D N A was less in obese than lean rats,
CCK
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FIG. 2. Percent of basal amylase release/txg DNA stimulated by CCK-8 from pancreata of Zucker obese and lean rats treated with trypsin inhibitor or control. Each line represents the mean of values from six rats.
F(1,64)=7.93, p<0.006, indicating decreased efficacy. The potency of CCK in obese rats was similar to that in lean rats. The dose-response curve of obese rats to carbachol was shifted to the left indicating similar efficacy but increased potency of carbachol in obese compared with lean rats. Compared with control obese and lean rats trypsininhibitor-treated obese and lean rats had increased DNA/ml incubation medium [96 vs. 64/~g, F(1,20)=9.88, p<0.005] but basal percent of total amylase release (3.4 vs. 2.4) or basal percent of total amylase release per D N A (0.04 vs. 0.04) were not different. However, basal release of amylase per D N A was twice that of control rats, F(1,14)=11.24, p<0.001, even though total amylase was similar. When expressed on a p e r / z g DNA/ml basis CCK-stimulated percent o f basal amylase release from the pancreas of TI-treated rats was only 36% o f that from the pancreas o f control rats, F(1,134) = 179.41, p <0.001. The percent of basal amylase release from obese TI-treated rats compared with obese control rats was greater than lean TI-treated rats compared with lean control rats, F(1,134) = 10.05, p <0.002. The shape of the dose-response curve of TI-treated rats was different from that of the control rats, F(6,134)=2.86, p<0.01, in that there was decreased efficacy. When expressed on a/~g/DNA incubation medium basis percent of basal amylase released in 30 min by carbachol from the pancreas of TI-treatedd rats was, on average, only 20 percent that of control rats, F(1,115)= 119.36, p <0.001
964
M c L A U G H L I N , PEIKIN AND BAILE
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FIG. 3. Percent of basal amylase release//xg DNA stimulated by carbachol from pancreata of Zucker obese and lean rats treated with trypsin inhibitor or control. Each line represents the mean of values from six rats.
(Fig. 3). Although stimulation of amylase release by carbachol in TI-treated rats was much lower than that in control rats, the shapes of the dose-response curves were not different, thus carbachol was less efficacious but not less potent. DISCUSSION
Results of these experiments have shown that CCK is less effective but not less potent in obese rats than in lean rats in stimulating amylase secretion and that percent binding of CCK to pancreatic acini was only one-half that in lean rats. Thus, decreased response may be due to decreased receptor number in obese rats even when values are expressed on a per DNA (cell number) basis. In the first experiment even though a significant amount of iodinated CCK-33 was added to the incubation medium (about 70,000 cpm), binding in lean rats was only 4-5 percent; this is similar to that reported in experiments using the same methods [12] of preparation of I125-CCK-33 and incubation. Inhibition of binding of I125-CCK-33 was maximal with 10 7 M CCK-8, and non-specific binding was greater than 513%of total binding, higher than that reported by some but not all other studies with guinea pigs [9, 11, 12]. At the initiation of the experiments the only method of iodinating CCK was use of the Boiton-Hunter technique for labelling CCK-33 [3]. However, newer methods for producing labelled CCK-33 and CCK-8, which have a higher potency for stimulating amylase release from the pancreas [ 16] have been
developed recently and may prove to be better for evaluating binding of CCK to pancreatic acini. Binding of CCK to pancreatic acini from obese rats was only one-fourth that from lean rats. Even though DNA content of the incubation medium of the pancreas from obese rats was one-half that in lean rats, percent binding of CCK to acini from obese rats per DNA was still half that in the pancreas from lean rats. The results were not due to differences in incubation medium since each day, using the same solution the pancreata from one obese and one lean rat were used; and it was not due to sequence of incubation during a specific day since on alternating days the obese rat was used first, then the lean rat was used first. It is possible that the presence of more fat which was evident in some pancreata from obese rats interfered with binding; however, after incubation of cells with collagenase and washing before incubation with P25-CCK-33 no fat was visible in the medium. These results are supported by those of Praissman and Izzo [17] who have demonstrated that membranes of pancreata from obese rats bound one-half the '2~I-CCK-8 as membranes of pancreata from lean rats. They also reported that the number of binding sites of obese rats calculated from Scatchard plots was one-fourth the number of lean rats but that the sites of obese rats had higher affinity for CCK than those of lean rats. Thus these findings may explain the decreased efficacy of CCK in stimulating amylase release from the obese rat pancreas in the present studies. The decreased response of the obese rat to the trophic and secretory effects of CCK in vivo [15] may also be secondary to decreased CCK binding. In previous experiments with Zucker rats it was demonstrated in vivo that CCK-8-stimulated amylase release and pancreas size were decreased in obese compared with lean rats [15]. Schneeman et al. reported that amylase content/DNA was decreased at 3 months of age and older [19]. While in previous experiments in vitro CCK-8-stimulated amylase release in obese rats was not different from that in lean rats, the DNA content of the incubation medium was not measured. In the present experiments it was demonstrated in vitro that CCK-stimulated amylase secretion was decreased on a per ~g DNA in the incubation medium basis. Thus CCK had equal potency but decreased efficacy in obese compared with lean rats. In contrast, the dose-response curve to carbachol is shifted to the left on obese rats indicating increased potency, but no difference in efficacy. This suggests that the decreased responsiveness of the obese rat pancreas is specific for CCK. As demonstrated previously, maximal amylase secretion stimulated by carbachol is similar to that stimulated by CCK, but carbachol is less potent [17]. Pancreatic acini from rats treated with trypsin inhibitor were much less responsive than those from control rats to stimulation of amylase secretion by either CCK or carbachol. Folsch and Wormsley [6] have shown that in vitro CCK-stimulated amylase secretion in 30 min was increased in rats fed raw soybean flour. However, since pancreas size (not reported) was likely to have been increased 50%, as demonstrated in other studies, the increases in pancreatic exocrine secretion were probably less than the increases in pancreas size, indicative of a decreased response per unit pancreas. Chronic administration of CCK also increased CCK-stimulated amylase output in 3 hr from the pancreas in vivo and this effect was significant when expressed per 1 p.g DNA with one of two experiments with the highest dose tested [5].
DECREASED PANCREATIC CCK RECEPTOR BINDING In contrast, in this latter e x p e r i m e n t and 2 others [2, 5, 10] increases in insulin c o n t e n t o f the pancreas as a result of increased pancreas size were associated with d e c r e a s e d in vivo stimulation of insulin secretion f r o m the pancreas. Thus, increased p a n c r e a s size is not necessarily associated with increased e x o c r i n e o r endocrine function. In the present study the pancreatic e x o c r i n e response to stimulation was d e c r e a s e d in rats with increased pancreas size as a result of trypsin inhibitor administration. The major differences bet w e e n this and o t h e r studies were that an in vitro system was used to e v a l u a t e r e s p o n s e and the time c o u r s e of measurement was relatively short (30 min). Also the responses were m e a s u r e d on a p e r / x g D N A basis, an important consideration w h e n c o m p a r i n g results from several studies. The d e c r e a s e d r e s p o n s e o f trypsin inhibitor-treated rats in the present study o c c u r r e d e v e n though D N A content was about 50% greater in the incubation m e d i u m of pancreas from trypsin inhibitor c o m p a r e d with control rats. While basal amylase release from acini was greater in trypsin inhibitor c o m p a r e d with control rats, C C K - s t i m u l a t e d amylase secretion from trypsin inhibitor-treated rats on average was only 36% o f that from control rats. In the present experiments the r e s p o n s e o f trypsin inhibitor-treated obese rats c o m p a r e d with control obese rats was greater than the re-
965 sponse of trypsin inhibitor-treated lean rats c o m p a r e d with control lean rats. This may be correlated with the finding that pancreas growth was stimulated less in o b e s e than lean rat pancreata by trypsin inhibitor, as shown previously [15]. The response o f pancreata from trypsin inhibitor-treated rats to stimulation o f amylase secretion stimulated by carbachol was only 20% of that in p a n c r e a t a from control rats. The maximally effective d o s e o f carbachol, h o w e v e r , was not different a m o n g t r e a t m e n t groups. The results o f these e x p e r i m e n t s support the hypothesis that pancreatic e x o c r i n e response to C C K is diminished in Z u c k e r obese rats c o m p a r e d with lean rats as a result o f d e c r e a s e d binding of C C K to receptors on pancreatic acini. D e c r e a s e d in vivo pancreatic response to C C K , reported previously [15], was correlated with d e c r e a s e d in vitro pancreatic secretory r e s p o n s e to C C K w h e n data w e r e expressed on a per D N A basis. Binding o f C C K to its r e c e p t o r on pancreatic acini in obese rats was o n e - h a l f that in lean rats and results of future e x p e r i m e n t s may show that binding o f C C K to the r e c e p t o r for satiety m a y be d e c r e a s e d in obese animals as well. D e c r e a s e d binding may be responsible for increased meal size associated with obesity and for decreased sensitivity to one peptide postulated to elicit satiety.
REFERENCES
I. Amer, M. S. Studies with cholecystokinin II. Cholecystokinetic potency of porcine gastrins I and II and related peptides in three systems. Endocrinology 84: 1277-1281, 1969. 2. Arnesjo, B., I. Ihse, I. Lindquist and I. Quist. Effects on exocrine and endocrine rat pancreatic functions of bovine lung trypsin inhibitor administered per orally. Stand J Gastroenterol 8: 545-554, 1973. 3. Bolton, A. E. and W. M. Hunter. The labelling of proteins to high specific radioactivities by conjugation to a tz51-containing acylating agent. Biochem J 133: 52%539, 1973. 4. Ceska, M., K. Birath and B. Brown. A new rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Optimal conditions. Clin Chem Acta 26: 437444, 1969. 5. Folsch, U. R., K. Winckler and K. G. Wormsley. Influence of repeated administration of cholecystokinin and secretion on the pancreas of the rat. Stand J Gastroenterol 13: 663-671, 1978. 6. Folsch, U. R. and K. G. Wormsley. The pancreatic secretion of enzymes in rats treated with soybean diet. Stand J Gastroenterol 9: 67%683, 1974. 7. Giles, K. W. and A. Myers. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206: 93-95, 1965. 8. Green, G. M. and R. L. Lyman. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin-inhibitorinduced hypersecretion in rats. Proc Soc Exp Biol Med 140: 6-12, 1972. 9. Hahne, W. F., R. T. Jensen, G. F. Lemp and J. D. Gardner. Proglumide and benzotript. Members of a different class of cholecystokinin receptor antagonists. Proc Nail Acad Sci USA 78: 6304-6308, 1981. 10. Ihse, I., B. Arnesjo and I. Lindquist. Studies on the reversibility of oral trypsin-inhibitor-induced changes of rat pancreatic exocrine enzyme activity and insulin secretory capacity. Stand J Gastroenterol 10: 321-326, 1975. 11. Innis, R. B. and S. H. Synder. Distinct cholecystokinin receptors in brain and pancreas. Proc Natl Acad Sci USA 77: 69176921, 1980.
12. Jensen, R. T., G. F. Lemp and J. D. Gardner. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Natl Acad Sci USA 77: 207%2083, 1980. 13. McLaughlin, C. L. and C. A. Baile. Decreased sensitivity of Zucker obese rats to the putative satiety agent cholecystokinin. Physiol Behav 25: 543-548, 1980. 14. McLaughlin, C. L. and C. A. Baile. Obese mice and the satiety effects of cholecystokinin, bombesin and pancreatic polypeptide. Physiol Behav 26: 433-437, 1981. 15. McLaughlin, C. L., S. R. Peikin and C. A. Baile. Decreased pancreatic exocrine response to cholecystokinin in Zucker obese rats. Ant J Physiol 242: G612-G619, 1982. 16. Miller, L. J., S. A. Rosenzweig and J. D. Jamieson. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor, N~(Iz~I-desaminotyrosyl)CCK-8, and its interaction with pancreatic acini. J Biol Chem 256: 12417-12423, 1981. 17. Peikin, S. R., A. J. Rottman, S. Batzri and J. D. Gardner. Kinetics of amylase release by dispersed acini prepared from guinea pig pancreas. Am J Physiol 235: E743-E749, 1978. 18. Praissman, M. and R. S. Izzo. Pancreatic CCK receptors in genetically obese rats: diminished numbers of binding sites with increased affinity compared to lean rats. Gastroenterology 84: 1276, 1983. 19. Schneeman, B. O., M. D. Inman and J. S. Stern. Pancreatic enzyme activity in obese and lean Zucker rats: a developmental study. J Nutr 113: 921-925, 1983. 20. Smith, G. P. and J. Gibbs. Brain-gut peptides and the control of food intake. In: Neurosecretion and Brain Peptides, edited by J. B. Martin, R. Reichlin and K. L. Brick. New York: Raven Press, 1981, pp. 38%395. 21. Smith, G. P., C. Jerome, B. J. Cushin, R. Eterno and K. J. Simansky. Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science 213: 1036-1037, 1981.
0031-9384/84 $3.00 + .00
Decreased Pancreatic CCK Receptor Binding and CCK-Stimulated Amylase Release in Zucker Obese R a t s I C A R O L L. M c L A U G H L I N , * T z S T E V E N R, P E I K I N * A N D C L I F T O N A. B A I L E r 2
*Department o f Medicine, Jefferson Medical College, Thomas Jefferson University Philadelphia, PA 19107 and TDepartment o f Clinical Studies at New Bolton Center, School o f Veterinary Medicine University o f Pennsylvania, Kennett Square, PA 19348 R e c e i v e d 25 J u l y 1983 McLAUGHLIN, C. L., S. R. PEIKIN AND C. A. BAILE. Decreased pancreatic CCK receptor binding and CCKstimulated amylase release in Zucker obese rats. PHYSIOL BEHAV 32(6) 961-965, 1984.--In Zucker obese rats the response to the effects of CCK on food intake and pancreatic exocrine function are decreased. However, it is unknown whether the decreased responsiveness is due to decreased receptor number and/or sensitivity or abnormal circulating concentrations cf CCK. In these experiments percent total binding of 125I-CCK-33 to pancreatic acini from obese rats was one-half that in lean rats when data was expressed on a per/xg DNA basis (19.6_+5.1 vs. 47.4_+ 11.4, p<0.01). In a second experiment while the maximally effective dose of CCK for stimulating amylase secretion from dispersed pancreatic acini was similar in obese and lean rats (10-1° M), less amylase was secreted in obese rats across the dose range tested (/9<0.001). In contrast, carbachol had similar potency and efficacy in stimulating amylase release from obese and lean pancreatic acini. The increase of pancreas size by use of a trypsin inhibitor was greater in lean than obese rats (p<0.03). In addition, stimulation of amylase release by CCK from obese trypsin inhibitor-treated compared with control obese rats was greater than that from lean trypsin inhibitor-treated compared with control lean rats (p<0.002). However, overall, stimulation of amylase secretion by CCK was only 36% of control (p<0.001) and by carbachol was only 20% of control (p <0.001). Thus, increased size by increased cell number was associated with decreased response per cell. Results of these experiments support the hypothesis that decreased response of the pancreas of obese compared with lean rats to CCK is due to decreased binding of CCK to receptors. A similar decreased binding of CCK to receptors for satiety in obese animals, if present, may be responsible for increased meal size associated with obesity. CCK-receptor Amylase secretion Zucker obese rats Carbachol Pancreatic acini Trypsin inhibitor DNA
Receptor binding
lz~I-CCK-33
Satiety
weight is the same, thus the second possibility appears to be more likely, assuming the kinetics of absorption of the CCK are similar [13,14]. The location of the receptors for the effect of C C K on satiety is unknown, although there is evidence they may be in the stomach [21]. Receptors for the effect of C C K on pancreatic amylase secretion are on pancreatic acinar cells and their characteristics could be used to evaluate responsiveness of obese animals to the effects of CCK on pancreas exocrine function. CCK stimulates less secretion of amylase from the pancreas in vivo of obese than lean rats [15]. In vitro percent of basal amylase secretion from dispersed pancreatic acini in response to CCK was similar in obese and lean rats [15]. In the in vitro experiment, since no measurement of
C H O L E C Y S T O K I N I N (CCK), one of many peptides released from the gastrointestinal tract by the presence of food, stimulates contraction of the gall-bladder and secretion o f enzymes from the pancreas [1,17]. In addition, increased serum concentrations of C C K have been proposed to signal satiety and result in termination of a meal [20]. In obese rats and mice the increase in meal size which is characteristic may be due to release of less C C K during meals [13,14]; however measurement of serum concentrations of CCK have not been reported in rodents. Another possibility is that receptor number or sensitivity to normal or increased concentrations of C C K is decreased in obese rats. Exogenous administration of C C K decreases food intake less in obese than lean rats and mice; the amount received per body
1This work was supported in part by the Cowperthwaite Fund, Jefferson Medical College, and Research Foundation and Feeding Fund, University of Pennsylvania. 2Present address: Department of Preventive Medicine, Washington University, 4566 Scott Avenue, Box 8113, St. Louis, MO 63110. Requests for reprints should be addressed to C. McLaughlin at her present address.
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McLAUGHLIN, PEIKIN AND BAILE
DNA in the incubation medium was made, response per cell was unknown. It was concluded that since the dose-response curve was not shifted rightward, C C K was equally potent in obese and lean rats [15]. However, evaluation of the efficacy of CCK was not possible. In vitro binding of CCK to its receptor on pancreatic acini has been highly correlated with stimulation of amylase secretion by CCK [9]. Thus, the present experiments were designed to determine if decreased effect of CCK on pancreas growth and in vivo exocrine function in obese rats is a result of decreased binding of CCK to its receptor or decreased efficacy of CCK in obese rats as expressed on a per DNA basis. It is possible that decreased pancreatic exocrine response to CCK in obese compared with lean rats is due solely to decreased pancreas size. To determine if pancreas size is related to function, pancreatic amylase secretion in response to CCK-8 stimulation was measured in rats in which pancreas size was increased by the chronic administration of a trypsin inhibitor. METHOD
those reported here the bioactive fractions were selected by incubating 70,000-100,000 dpm of each fraction with acinar cells from normal rats using the procedure above. They were incubated in the presence or absence of a maximally effective concentration of CCK-8 (16 -6 M) to measure nonspecific and total binding respectively. Only the fractions exhibiting the highest specific binding (total minus nonspecific, usually 1 or 2 fractions) were used in the subsequent binding experiments. D. Measurement o f D N A . The DNA content of the incubation medium was measured in 0.20 ml aliquots to which 0.80 ml of 0.4 N KCI had been added using diphenylamine [7] and calf thymus DNA as a standard. E. Data analysis. Percent binding in the presence or absence of CCK-8 was determined for each rat and dose of CCK-8 and data were subjected to analysis of variance for effects of phenotype, dose of CCK-8 and replicate and interactions of these. Similar analyses were used for percent binding/DNA values calculated. Body weight, pancreas weight and D N A content of obese rats were compared to those of lean rats using paired t-tests.
Experiment 1. CCK Receptor Binding A. Rats. Six obese (307-+27 g) and six lean (188_+11 g) female Zucker rats were used. Prior to experimental days they were housed in a room maintained at 21°C and with a 12-hr light-dark cycle. They were fed (Purina Rat Chow pellets) and watered ad lib. On the experimental day food was removed 4-6 hr before the animals were sacrificed. B. Incubation procedure. On experimental days a rat was sacrificed and the pancreas carefully removed and weighed. Dispersed pancreatic acini were prepared as described by Peikin et al. [17]. Briefly, the pancreas was incubated with purified collagenase, and the dispersed acini obtained were suspended in 7.5 ml incubation solution containing 24.5 mM Hepes (pH=7.4), 98 mM NaC1, 6 mM KCI, 2.5 mM Nail2 PO4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium gluconate, 11.5 mM glucose, 2.0 mM CaCl2, 1.0 mM MgCi2, 2 mM glutamine, 0.2% (wt/vol) albumin, 0.01% (wt/vol) trypsin inhibitor and 1% (vol/vol) essential amino acid mixture. The medium was adjusted to contain 0.5 mM calcium, 5 mM theophylline and 1% (wt/vol) albumin. Of the suspended cells 0.5 ml was removed and frozen for subsequent measurement of D N A content. Six 1.0 ml aliquots were incubated with no unlabelled CCK (total binding) or CCK from l0 -~° to 10-n M (non-specific binding) using the methods described by Jensen et al. [12]. To each was added 10/~l of 70,000-100,000 dpm biologically active iodihated CCKaa (Bolton-Hunter technique [3], obtained from Mr. Thomai, Hazelton Laboratories, Vienna, VA). Biological activity was determined by using the method below (c). The tubes were placed in a Dubnoff incubator at 160 oscillations/min at 37° for 30 min then placed on ice. From each tube two 400 ~l aliquot cell suspensions were centrifuged at 10,000 g for 15 sec in a Beckman microcentrifuge. The supernatant was removed with suction and the cells vortexed and washed with ice-cold incubation medium containing 4% albumin and 0.1% (wt/vol) bacitracin. The cells were washed twice more and counted. From each tube two 50/~l aliquants were counted to determine dpm added. On each treatment day both a lean and obese rat were used, with the sequence being reversed on successive days. C. Selection o f bioactive fractions o f ~2"~I-CCKa3. Each batch of ~25I-CCKaa arrived as 9-10 fractions from a Sephadex G-50 column. For preliminary experiments and
Experiment 2. CCK- and Carbachol-Stimulated Amylase Release in Control Rats and Rats Treated With Trypsin Inhibitor A. Rats. Ten obese (304_+14 g) and 12 lean (193_+1 g) female rats were individually housed and fed as in Experiment 1. Five pairs of obese rats and six pairs of lean rats were formed. One member of each pair was administered 2.0 ml/kg water intragastrically for seven days while the other member of each pair was administered 200 mg/kg trypsin inhibitor [TI, N, N-dimethyl-carbamoyl 4-(4-guanidinobenzlyloxy)-pbenyl acetate methanesulfate, obtained from Oro Pharmaceutical Co., Ltd.] for seven days. Food and water intakes and body weights were measured daily. Twenty-four hours after the last dosing the pancreata of two obese or two lean rats were used to measure amylase secretion. The pancreata from obese and lean rats were incubated on alternate days and the control and TI-treated pancreata were incubated first on alternate days, but with the same solutions made for the day. B. Incubation procedure. Pancreatic acini were prepared as in Experiment 1 with the following exceptions. After the 0.5 ml aliquot of suspended cells had been removed for subsequent DNA analysis, the incubation medium volume was increased to 70 ml. One mi aliquants were incubated with C C K - 8 [10 -11 to 10-6 M] or carbachol [10-8 to 10-a M]. After 30 min two 400 p.l aliquants were spun for 15 sec in a Beckman microfuge, 100 /~! of the supernatant from each was added to 0.5 ml of lysing solution [0.01 M sodium phosphate (pH 7.8), 0.1% (wt/vol) albumin and 0.1% (wt/vol) sodium dodecyl sulfate]. Aliquants of 100 #.1 were assayed for amylase activity by the method of Ceska et al. [4] using the Phadebas reagent. Total amylase activity was determined by assay of 100 /~l of 1.5 ml cell suspension which had been added to 13.5 mi lysing solution. Results were expressed as percent of basal amylase released into the incubation medium in 30 min by each concentration of peptide. C. D N A measurement. As in Experiment I. D. Data analysis. Food intake, weight gain and pancreas weight data were each subjected to analysis of variance to measure effects of treatment and phenotype and interaction of treatment with phenotype. In the in vitro experiments, for each peptide, percent of basal amylase release and percent
DECREASED PANCREATIC CCK RECEPTOR BINDING
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Experiment I. CCK Receptor Binding In Zucker obese rats percent total binding of iodinated CCKaa was one-fourth that in lean rats, F(1,55)=85.57, p<0.001, Fig. 1. In lean rats total binding was 4.4% and non-specific binding was 2.8%, with 1.6% specific binding. In obese rats, however, under the same conditions, on the same day, total binding was only I. 1%, with non-specific binding of 0.8% and 0.3% specific binding. In both obese and lean rats percent binding was dose-dependently inhibited by CCK-8, F(5,55)=2.92, p <0.02. Even though D N A content of the incubation medium was less in obese than lean rats [58-+8 vs. 101-+ 11 /~g/ml, paired-t =5.41, p <0.01], percent binding per D N A was also less in obese than lean rats, F(1,55)=90.84, p <0.001.
Experiment 2. CCK- and Carbachol-Stimulated Amylase Secretion in Obese and Lean Control Rats and Rats Treated With Trypsin Inhibitor During the seven-day treatment period food intake was decreased by TI in obese [19.1 vs. 25.5 g, F(1,14)=4.92, p<0.04] but not lean rats (19.6 vs. 18.8 g). Similarly, weight gain was less in obese rats treated with TI compared with control [ - 0 . 3 vs. 2.6 g, F(1,14)=8.53,p<0.01] but not in lean rats (0.9 vs. 0.1 g). Weight o f the pancreas from obese control rats was not different from that o f lean control rats (0.80 vs. 0.86 g). While administration o f trypsin inhibitor increased pancreas size o f both obese [1.19 vs. 0.80, F(1,12)=12.54, p<0.004] and lean [1.62 vs. 0.86 g, F(1,12)=58.85, p<0.001] rats, the increase was greater in lean than in obese rats, F(1,12)=6.53, p<0.03. Comparison o f values from control obese with control lean rats demonstrated that D N A per ml incubation medium (60 vs. 68/~g/ml), basal percent of total amylase release (3.1 vs. 1.8) and basal percent of total amylase release//xg D N A (0.05 vs. 0.03) were not different (Fig. 2). However, in response to stimulation by C C K percent of basal amylase released//zg D N A was less in obese than lean rats,
CCK
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FIG. 2. Percent of basal amylase release/txg DNA stimulated by CCK-8 from pancreata of Zucker obese and lean rats treated with trypsin inhibitor or control. Each line represents the mean of values from six rats.
F(1,64)=7.93, p<0.006, indicating decreased efficacy. The potency of CCK in obese rats was similar to that in lean rats. The dose-response curve of obese rats to carbachol was shifted to the left indicating similar efficacy but increased potency of carbachol in obese compared with lean rats. Compared with control obese and lean rats trypsininhibitor-treated obese and lean rats had increased DNA/ml incubation medium [96 vs. 64/~g, F(1,20)=9.88, p<0.005] but basal percent of total amylase release (3.4 vs. 2.4) or basal percent of total amylase release per D N A (0.04 vs. 0.04) were not different. However, basal release of amylase per D N A was twice that of control rats, F(1,14)=11.24, p<0.001, even though total amylase was similar. When expressed on a p e r / z g DNA/ml basis CCK-stimulated percent o f basal amylase release from the pancreas of TI-treated rats was only 36% o f that from the pancreas o f control rats, F(1,134) = 179.41, p <0.001. The percent of basal amylase release from obese TI-treated rats compared with obese control rats was greater than lean TI-treated rats compared with lean control rats, F(1,134) = 10.05, p <0.002. The shape of the dose-response curve of TI-treated rats was different from that of the control rats, F(6,134)=2.86, p<0.01, in that there was decreased efficacy. When expressed on a/~g/DNA incubation medium basis percent of basal amylase released in 30 min by carbachol from the pancreas of TI-treatedd rats was, on average, only 20 percent that of control rats, F(1,115)= 119.36, p <0.001
964
M c L A U G H L I N , PEIKIN AND BAILE
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FIG. 3. Percent of basal amylase release//xg DNA stimulated by carbachol from pancreata of Zucker obese and lean rats treated with trypsin inhibitor or control. Each line represents the mean of values from six rats.
(Fig. 3). Although stimulation of amylase release by carbachol in TI-treated rats was much lower than that in control rats, the shapes of the dose-response curves were not different, thus carbachol was less efficacious but not less potent. DISCUSSION
Results of these experiments have shown that CCK is less effective but not less potent in obese rats than in lean rats in stimulating amylase secretion and that percent binding of CCK to pancreatic acini was only one-half that in lean rats. Thus, decreased response may be due to decreased receptor number in obese rats even when values are expressed on a per DNA (cell number) basis. In the first experiment even though a significant amount of iodinated CCK-33 was added to the incubation medium (about 70,000 cpm), binding in lean rats was only 4-5 percent; this is similar to that reported in experiments using the same methods [12] of preparation of I125-CCK-33 and incubation. Inhibition of binding of I125-CCK-33 was maximal with 10 7 M CCK-8, and non-specific binding was greater than 513%of total binding, higher than that reported by some but not all other studies with guinea pigs [9, 11, 12]. At the initiation of the experiments the only method of iodinating CCK was use of the Boiton-Hunter technique for labelling CCK-33 [3]. However, newer methods for producing labelled CCK-33 and CCK-8, which have a higher potency for stimulating amylase release from the pancreas [ 16] have been
developed recently and may prove to be better for evaluating binding of CCK to pancreatic acini. Binding of CCK to pancreatic acini from obese rats was only one-fourth that from lean rats. Even though DNA content of the incubation medium of the pancreas from obese rats was one-half that in lean rats, percent binding of CCK to acini from obese rats per DNA was still half that in the pancreas from lean rats. The results were not due to differences in incubation medium since each day, using the same solution the pancreata from one obese and one lean rat were used; and it was not due to sequence of incubation during a specific day since on alternating days the obese rat was used first, then the lean rat was used first. It is possible that the presence of more fat which was evident in some pancreata from obese rats interfered with binding; however, after incubation of cells with collagenase and washing before incubation with P25-CCK-33 no fat was visible in the medium. These results are supported by those of Praissman and Izzo [17] who have demonstrated that membranes of pancreata from obese rats bound one-half the '2~I-CCK-8 as membranes of pancreata from lean rats. They also reported that the number of binding sites of obese rats calculated from Scatchard plots was one-fourth the number of lean rats but that the sites of obese rats had higher affinity for CCK than those of lean rats. Thus these findings may explain the decreased efficacy of CCK in stimulating amylase release from the obese rat pancreas in the present studies. The decreased response of the obese rat to the trophic and secretory effects of CCK in vivo [15] may also be secondary to decreased CCK binding. In previous experiments with Zucker rats it was demonstrated in vivo that CCK-8-stimulated amylase release and pancreas size were decreased in obese compared with lean rats [15]. Schneeman et al. reported that amylase content/DNA was decreased at 3 months of age and older [19]. While in previous experiments in vitro CCK-8-stimulated amylase release in obese rats was not different from that in lean rats, the DNA content of the incubation medium was not measured. In the present experiments it was demonstrated in vitro that CCK-stimulated amylase secretion was decreased on a per ~g DNA in the incubation medium basis. Thus CCK had equal potency but decreased efficacy in obese compared with lean rats. In contrast, the dose-response curve to carbachol is shifted to the left on obese rats indicating increased potency, but no difference in efficacy. This suggests that the decreased responsiveness of the obese rat pancreas is specific for CCK. As demonstrated previously, maximal amylase secretion stimulated by carbachol is similar to that stimulated by CCK, but carbachol is less potent [17]. Pancreatic acini from rats treated with trypsin inhibitor were much less responsive than those from control rats to stimulation of amylase secretion by either CCK or carbachol. Folsch and Wormsley [6] have shown that in vitro CCK-stimulated amylase secretion in 30 min was increased in rats fed raw soybean flour. However, since pancreas size (not reported) was likely to have been increased 50%, as demonstrated in other studies, the increases in pancreatic exocrine secretion were probably less than the increases in pancreas size, indicative of a decreased response per unit pancreas. Chronic administration of CCK also increased CCK-stimulated amylase output in 3 hr from the pancreas in vivo and this effect was significant when expressed per 1 p.g DNA with one of two experiments with the highest dose tested [5].
DECREASED PANCREATIC CCK RECEPTOR BINDING In contrast, in this latter e x p e r i m e n t and 2 others [2, 5, 10] increases in insulin c o n t e n t o f the pancreas as a result of increased pancreas size were associated with d e c r e a s e d in vivo stimulation of insulin secretion f r o m the pancreas. Thus, increased p a n c r e a s size is not necessarily associated with increased e x o c r i n e o r endocrine function. In the present study the pancreatic e x o c r i n e response to stimulation was d e c r e a s e d in rats with increased pancreas size as a result of trypsin inhibitor administration. The major differences bet w e e n this and o t h e r studies were that an in vitro system was used to e v a l u a t e r e s p o n s e and the time c o u r s e of measurement was relatively short (30 min). Also the responses were m e a s u r e d on a p e r / x g D N A basis, an important consideration w h e n c o m p a r i n g results from several studies. The d e c r e a s e d r e s p o n s e o f trypsin inhibitor-treated rats in the present study o c c u r r e d e v e n though D N A content was about 50% greater in the incubation m e d i u m of pancreas from trypsin inhibitor c o m p a r e d with control rats. While basal amylase release from acini was greater in trypsin inhibitor c o m p a r e d with control rats, C C K - s t i m u l a t e d amylase secretion from trypsin inhibitor-treated rats on average was only 36% o f that from control rats. In the present experiments the r e s p o n s e o f trypsin inhibitor-treated obese rats c o m p a r e d with control obese rats was greater than the re-
965 sponse of trypsin inhibitor-treated lean rats c o m p a r e d with control lean rats. This may be correlated with the finding that pancreas growth was stimulated less in o b e s e than lean rat pancreata by trypsin inhibitor, as shown previously [15]. The response o f pancreata from trypsin inhibitor-treated rats to stimulation o f amylase secretion stimulated by carbachol was only 20% of that in p a n c r e a t a from control rats. The maximally effective d o s e o f carbachol, h o w e v e r , was not different a m o n g t r e a t m e n t groups. The results o f these e x p e r i m e n t s support the hypothesis that pancreatic e x o c r i n e response to C C K is diminished in Z u c k e r obese rats c o m p a r e d with lean rats as a result o f d e c r e a s e d binding of C C K to receptors on pancreatic acini. D e c r e a s e d in vivo pancreatic response to C C K , reported previously [15], was correlated with d e c r e a s e d in vitro pancreatic secretory r e s p o n s e to C C K w h e n data w e r e expressed on a per D N A basis. Binding o f C C K to its r e c e p t o r on pancreatic acini in obese rats was o n e - h a l f that in lean rats and results of future e x p e r i m e n t s may show that binding o f C C K to the r e c e p t o r for satiety m a y be d e c r e a s e d in obese animals as well. D e c r e a s e d binding may be responsible for increased meal size associated with obesity and for decreased sensitivity to one peptide postulated to elicit satiety.
REFERENCES
I. Amer, M. S. Studies with cholecystokinin II. Cholecystokinetic potency of porcine gastrins I and II and related peptides in three systems. Endocrinology 84: 1277-1281, 1969. 2. Arnesjo, B., I. Ihse, I. Lindquist and I. Quist. Effects on exocrine and endocrine rat pancreatic functions of bovine lung trypsin inhibitor administered per orally. Stand J Gastroenterol 8: 545-554, 1973. 3. Bolton, A. E. and W. M. Hunter. The labelling of proteins to high specific radioactivities by conjugation to a tz51-containing acylating agent. Biochem J 133: 52%539, 1973. 4. Ceska, M., K. Birath and B. Brown. A new rapid method for the clinical determination of alpha-amylase activities in human serum and urine. Optimal conditions. Clin Chem Acta 26: 437444, 1969. 5. Folsch, U. R., K. Winckler and K. G. Wormsley. Influence of repeated administration of cholecystokinin and secretion on the pancreas of the rat. Stand J Gastroenterol 13: 663-671, 1978. 6. Folsch, U. R. and K. G. Wormsley. The pancreatic secretion of enzymes in rats treated with soybean diet. Stand J Gastroenterol 9: 67%683, 1974. 7. Giles, K. W. and A. Myers. An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature 206: 93-95, 1965. 8. Green, G. M. and R. L. Lyman. Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin-inhibitorinduced hypersecretion in rats. Proc Soc Exp Biol Med 140: 6-12, 1972. 9. Hahne, W. F., R. T. Jensen, G. F. Lemp and J. D. Gardner. Proglumide and benzotript. Members of a different class of cholecystokinin receptor antagonists. Proc Nail Acad Sci USA 78: 6304-6308, 1981. 10. Ihse, I., B. Arnesjo and I. Lindquist. Studies on the reversibility of oral trypsin-inhibitor-induced changes of rat pancreatic exocrine enzyme activity and insulin secretory capacity. Stand J Gastroenterol 10: 321-326, 1975. 11. Innis, R. B. and S. H. Synder. Distinct cholecystokinin receptors in brain and pancreas. Proc Natl Acad Sci USA 77: 69176921, 1980.
12. Jensen, R. T., G. F. Lemp and J. D. Gardner. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Natl Acad Sci USA 77: 207%2083, 1980. 13. McLaughlin, C. L. and C. A. Baile. Decreased sensitivity of Zucker obese rats to the putative satiety agent cholecystokinin. Physiol Behav 25: 543-548, 1980. 14. McLaughlin, C. L. and C. A. Baile. Obese mice and the satiety effects of cholecystokinin, bombesin and pancreatic polypeptide. Physiol Behav 26: 433-437, 1981. 15. McLaughlin, C. L., S. R. Peikin and C. A. Baile. Decreased pancreatic exocrine response to cholecystokinin in Zucker obese rats. Ant J Physiol 242: G612-G619, 1982. 16. Miller, L. J., S. A. Rosenzweig and J. D. Jamieson. Preparation and characterization of a probe for the cholecystokinin octapeptide receptor, N~(Iz~I-desaminotyrosyl)CCK-8, and its interaction with pancreatic acini. J Biol Chem 256: 12417-12423, 1981. 17. Peikin, S. R., A. J. Rottman, S. Batzri and J. D. Gardner. Kinetics of amylase release by dispersed acini prepared from guinea pig pancreas. Am J Physiol 235: E743-E749, 1978. 18. Praissman, M. and R. S. Izzo. Pancreatic CCK receptors in genetically obese rats: diminished numbers of binding sites with increased affinity compared to lean rats. Gastroenterology 84: 1276, 1983. 19. Schneeman, B. O., M. D. Inman and J. S. Stern. Pancreatic enzyme activity in obese and lean Zucker rats: a developmental study. J Nutr 113: 921-925, 1983. 20. Smith, G. P. and J. Gibbs. Brain-gut peptides and the control of food intake. In: Neurosecretion and Brain Peptides, edited by J. B. Martin, R. Reichlin and K. L. Brick. New York: Raven Press, 1981, pp. 38%395. 21. Smith, G. P., C. Jerome, B. J. Cushin, R. Eterno and K. J. Simansky. Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat. Science 213: 1036-1037, 1981.