Cholecystokinin suppresses sham feeding in the rhesus monkey

Physiology& Behavior,Vol. 23, pp. 887-890. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.

Cholecystokinin Suppresses Sham Feeding in the Rhesus Monkey I J. D. F A L A S C O , 2 G. P. S M I T H a A N D J. G I B B S 4

Department o f Psychiatry, Cornell University Medical Center and E. W. Bourne Behavioral Research Laboratory, The New York Hospital White Plains, N Y 10605 R e c e i v e d 28 M a r c h 1979 FALASCO, J. D., G. P. SMITH AND J. GIBBS. Cholecystokinin suppresses sham feeding in the rhesus monkey. PHYSIOL. BEHAV. 23(5) 887-890, 1979.--The satiety effect of slow intravenous infusions of impure cholecystokinin (CCK) was investigated in 5 rhesus monkeys during sham feeding. CCK suppressed sham feeding. The dose for 50% inhibition of sham feeding was about 10 U/kg-hr; 20 U/kg-hr abolished sham feeding. No dose produced retching, vomiting, diarrhea or other behavioral signs of toxicity. These results demonstrate the potency of CCK for inhibiting feeding in the monkey when gastric, intestinal and postabsorptive mechanisms are not activated by ingested food. Cholecystokinin Gastric cannula Rhesus monkey Physiological effect Pharmacological effect

C H O L E C Y S T O K I N I N (CCK) inhibits food intake in rats [1,8], lean and obese mice [20], rabbits [121, sheep [11] and rhesus monkeys [7]. In one study using humans [21], a rapid intravenous injection of CCK decreased feeding, while a slow intravenous infusion increased food intake. In another study [13], a slow intravenous infusion of a low dose reduced food intake. In both studies, the inhibitions of food intake occurred without side effects. When CCK is tested in animals that are feeding normally, it can interact with numerous satiety mechanisms activated by ingested food. In the rat these include unidentified preabsorptive mechanisms in mouth and esophagus [14], stomach [4,15] and small intestine [16,19], as well as postabsorptive mechanisms [2,18]. All of these mechanisms except those from pregastric sites can be eliminated by sham feeding. Thus, the demonstration [9,17] that impure CCK and its synthetic C-terminal octapeptide inhibited sham feeding in rats in a dose-related manner provided strong evidence of the potency of CCK to inhibit feeding when most endogenous satiety mechanisms were not operating. We recently reported [6] that rhesus monkeys will sham feed a liquid diet almost continuously for 90 min when the ingested diet drains from a chronic gastric cannula. We used this preparation to test the potency of CCK for inhibiting feeding when CCK was administered in the absence of gastric, intestinal and postabsorptive satiety mechanisms. We report here that slow intravenous infusion of CCK (20% pure) inhibited sham feeding in 5 rhesus monkeys in a doserelated manner without producing any signs of toxicity.

Satiety

Sham feeding

Satiety behavior

METHOD Five male rhesus monkeys, 5.9-8.3 kg, were surgically equipped with a chronic stainless steel Thomas cannula (10.2 I D x 14.6 mm OD) sutured into the most dependent portion of the stomach under intravenous sodium pentobarbital anesthesia (Nembutal, 50 mg initially, supplemented as necessary; monkeys were first subdued with an intramuscular injection of 0.1 mg/kg ketamine HC1--Vetalar, Parke-Davis). The cannula was brought out through a stab wound in the abdominal musculature and closed with a removable screw cap. When this cap was removed, ingested liquid food drained completely from the stomach. This permits the animal to sham feed. A detailed account of the preparation, maintenance, testing and validation of sham feeding in rhesus monkeys has been published [6]. A chronic intravenous Silastic catheter (Dow Coming, 1.02 ID×2.16 mm OD) was also implanted at surgery. The catheter was inserted into the femoral or jugular vein so that its tip lay in the inferior or superior vena cava, respectively. Monkeys were adapted to chronic restraint in primate chairs (BRS Foringer), housed individually in vented and lighted wooden booths and maintained on an 0700-1900 hr light cycle. They were accustomed to a schedule of 16.5 hr food deprivation (1730-1000 hr). Water was always available for drinking. The following testing schedule was employed: at 0900 hr an intravenous infusion (12.5 ml/hr, Harvard Apparatus Pump No. 975) was begun. At 1000 hr the screw cap to the gastric cannula was removed and a collecting tube

'This research was supported by NIMH Research Grant MH-15455 and NIH Research Grant AM-17240. '-'Requests for reprints should be sent to: John D. Falasco, E. W. Bourne Behavioral Research Laboratory, The New York Hospital, 21 BIoomingdale Road, White Plains, NY 10605. :~Supported by NIMH Research Scientist Development Award MH-00149. 4Supported by NIMH Research Scientist Development Award MH-70874.

C o p y r i g h t © 1979 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/79/110887-04502.00/0

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(Silastic, Dow Corning, 9.5 IDx 12.7 mm OD) was attached to the cannula. Liquid food (chocolate Nutrament, Drackett, 1.02 Kcal/ml) was offered and the monkey was allowed to sham feed for 60 min. At 1100 hr, the liquid food was removed, the screw cap was replaced on the cannula and the intravenous infusion was stopped. Solid food was then made available until 1730 hr when the food was removed to begin the deprivation period for the test on the following day. Monkeys were tested on this schedule for five days per week; solid food and water were available ad lib from 1100 hr Friday to 1730 hr Sunday. On the first day of a two-day test sequence, 0.15 M NaCl was infused intravenously for the 2 hr test period. On the second day, CCK (20% pure CCK from AB Kabi Diagnostica, Fac, S-611 01 Nykroping, Sweden) in doses of 0.63, 1.25, 2.5, 5.0, 10.0 and 20.0 Ivy units/kg-hr was dissolved in 0.15 M NaCI and infused intravenously. All solutions for intravenous infusions were pre-warmed to 37°C. Doses were not given in sequential order; at least one saline test separated each hormone test; only one test was run on a single day. Thirteen of the hormone tests were repeated, with a minimum period of 5 days separating duplicate tests. Individual monkeys received at least one test at each dose; one monkey received 4 tests at each of 2 doses. Results from duplicate tests on individual monkeys were averaged before being used in statistical analyses. During the second hour of each infusion, each monkey sham fed liquid food from a 2000 ml cylinder graduated at 20 ml intervals. Gastric contents were collected in a 2000 ml cylinder graduated at 20 ml intervals. Sham intake and gastric drainage were recorded every 3 min and were judged to the nearest 10 ml (food intake and gastric drainage) or 5 ml (water intake). The behavior of each monkey was observed regularly for 3 sec every 3 min through a one-way mirror in the door of each booth, and the incidence of the following behaviors was recorded: (a) feeding: sucking or licking food spout; (b) drinking: sucking or licking water spout; (c) non-ingestive activity: any movement of the body, arms, hands, head or eyes not related to feeding or drinking; (d) resting: no observable movement, and eyes closed or half closed. Results of sham intake were analyzed using matched-pair t test, one-tailed (Hewlett-Packard program ST 1-15B). RESULTS

Intravenous infusions of cholecystokinin during sham feeding produced a large, dose related suppression of sham intake (Fig. 1). The minimal effective dose was l0 U/kg-hr: this dose inhibited sham intake 59 _+ 12% (mean _+ SEM). A dose of 20 U/kg-hr virtually abolished sham feeding (96.6 +- 3.3% suppression). Tachyphylaxis was not observed despite repeated testing. The pattern of this decreased sham intake is shown in Fig. 2. The effect of CCK infusion was a dose-related reduction in the rate of sham feeding at the beginning of the test (rain 3-6: saline= 129 --- 35 ml; 10 U/kg-hr=70 +_ 27 mi, p<0.05) which was sustained throughout the 60 rain test period. Doses of 5 U/kg-hr or less had no effect on the rate of sham feeding, whereas 20 U/kg-hr completely inhibited sham feeding in 3 monkeys, and greatly reduced the rate of sham feeding in the other 2 (rain 3-6: saline, 144 --- 43 ml; 20 U/kg-hr, 10 - 8.7 ml, p<0.025). We previously reported that sham feeding in rhesus monkeys produced a marked reduction in the incidence of

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FIG. 2. Effects of slow intravenous infusions of cholecystokinin (CCK) on sham feeding of liquid food in 5 rhesus monkeys. Solid line denotes sham intakes during successive 3 rain intervals on a day when monkeys received a slow intravenous infusion of 0.15 M NaCI: dotted lines denote sham intakes on 3 days when monkeys received slow intravenous infusions of 3 different doses of 20% pure CCK.

postprandial behaviors that occur after normal feeding. We now report that infusions of cholecystokinin during sham feeding tend to normalize the incidence of these behaviors. A variety of non-parametric tests (binomial, sign, Mann-Whitney U) demonstrated that the only statistically significant change in behavior was the increased incidence of resting behavior with the 20 U/kg-hr dose (saline: median 0, range 0; 20 U CCK: median 6, range 1-20; p<0.05, MannWhitney U test). Clear but statistically non-significanttrends towards increased resting and increased non-ingestive activity were seen in 4 of 5 monkeys with the 10 U/kg-hr dose. Non-ingestive activity was not increased by 20 U/kg hr, presumably because of the increase in resting behavior.

CCK SUPPRESSES SHAM F E E D I N G IN MONKEYS The inhibitory effect of cholecystokinin at these doses was specific for food. Cholecystokinin had no effect on water intake. Cholecystokinin did not inhibit sham feeding by making monkeys sick because retching, vomiting, diarrhea or other noticeable signs of discomfort were not observed during or after any of the tests. DISCUSSION The results demonstrate the potency of impure CCK for inhibiting feeding in the rhesus monkey when gastric, intestinal and postabsorptive satiety mechanisms are not working. The fact that sham feeding could be suppressed in a dose-response fashion, and even abolished without the appearance of retching, vomiting or diarrhea is strong evidence for a specific satiety effect produced by CCK. This finding extends similar observations in the rat [9]. The dose of CCK required for 50% inhibition of sham feeding (10 U/kg-hr) is about 25 times the D50 for pancreatic enzyme secretion in rhesus monkeys under very similar circumstances [51. Grossman [3,10] has argued that when the D50 for a new action of a gut hormone exceeds the D50 for a known visceral action of the same hormone, then the new effect is probably pharmacological, i.e., it is not likely to occur with the quantity of endogenous hormone released in response to an average meal. By this criterion, the satiety effect of CCK is pharmacological in the monkey. However, it is important to note that the conditions of the present experiment are relatively insensitive for the satiety effect for three reasons. First, the animals were sham feeding, and therefore no endogenous neural or humoral signals beyond pregastric sites were likely to be activated by the accumulation and passage of ingested food. Under these conditions, it was necessary for exogenous CCK to act alone in producing satiety, while during normal feeding endogenous CCK will act with other satiety mechanisms.

889 Second, due to the lack of a reliable radioimmunoassay, the pattern of release and the forms of endogenous CCK released during a meal are not known. These factors may be important for its actions. The slow intravenous administration used in the present experiment is a common pattern of infusion employed to establish dose-response curves for physiological functions of exogenous hormones. While a slow infusion is a useful pattern to establish stable baselines so that reliable dose-response curves can be constructed for biological functions, it is very unlikely that gut hormones such as CCK are released in this manner. It is more likely that circulating CCK, like gastrin, rises rapidly and peaks in response to the arrival of food in the gut lumen. The sensitivities of the different systems---pancreas, gall bladder, and satiety--may be different when the techniques are available to mimic, by exogenous infusion, the endogenous release of the various forms of CCK. Third, it is possible that the satiety effect of CCK is produced by a local release of CCK in the wall of the small intestine (paracrine effect) instead of by circulating CCK (endocrine effect). If this were the case, large doses of exogenous CCK would have to be infused in order to achieve the high concentration of locally released CCK. Thus, a criterion which employs comparative bioassays is not a decisive test of the physiological role of CCK in postprandial satiety. We believe this question will not be" settled until the postprandial circulating forms and pattern of CCK release can be measured reliably and until the extent of synergism between CCK and other satiety signals from preabsorptive and postabsorptive sites has been determined. Until this information becomes available, CCK remains a putative physiological satiety signal. Whatever its role in normal postprandial satiety proves to be, these results demonstrate that CCK has a potent satiety effect in rhesus monkeys without any sign of toxicity.

REFERENCES

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10. Grossman, M. 1. The role of hormones in regulation of gastrointestinal motility. In: Proceedings of the Fourth International Symposium on Gastrointestinal Motility. Vancouver: Mitchell Press, 1974, pp. 21%220. 11. Grovum, W. L. Factors that decrease food intake in sheep. Program, 6th International Conference Physiol. of Food and FluM Intake, (abstract), 1977. 12. Houpt, T. R., S. M. Anika and N. C. Wolff. Satiety effects of cholecystokinin and caerulein in rabbits. Am. J. Physiol. 235: R23--R28, 1978. 13. Kissileff, H. R., F. X. Pi-Sunyer, J. Thornton and G. P. Smith. Cholecystokinin octapeptide (CCK-8) decreases food intake in man. Am. J. clin. Nutr. 32: 939, 1979. 14. Kraly, F. S., W. J. Catty and G. P. Smith. Effect of pregastric food stimuli on meal size and intermeal interval in the rat. Physiol. Behav. 20: 77%784, 1978. 15. Kraly, F. S. and G. P. Smith. Combined pregastric and gastric stimulation by food is sufficient for normal meal size. Physiol. Behav. 21: 405-408, 1978. 16. Liebling, D. S., J. D. Eisner, J. Gibbs and G. P. Smith. Intestinal satiety in rats. J. comp. physiol. Psychol. 89: 955-965, 1975. 17. Lorenz, D., J. Gibbs and G. P. Smith. Effect of cholecystokinin and other gut hormones on sham feeding in the rat. In: Gut Hormones. edited by S. R. Bloom. London: Churchill Livingstone, 1978, pp. 224-226.

890 18. Novin, D. Visceral mechanisms in the control of food intake. In: Hunger: Basic Mechanisms and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976, pp. 357-367. 19. Novin, D. and D. A. Vanderweele. Visceral involvement in feeding: there is more to regulation than the hypothalamus. In: Progress in Psychobiology and Physiological Psychology, Vol. 7, edited by J. M. Sprague and A. N. Epstein. New York: Academic Press, 1977, pp. 193-241.

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