Cholecystokinin: Induced suppression of feeding in fed, fasting and hypothalamic island rats

Brain ResearchBulletin, Vol. 21, pp. 225-231. ©PergamonPress plc, 1988. Printedin the U.S.A.

0361-9230/88$3.00 + .00

Cholecystokinin: Induced Suppression of Feeding in Fed, Fasting and Hypothalamic Island Rats N. D A F N Y , M. A. G I L L M A N * A N D F. J. L I C H T I G F E L D *

University of Texas Medical School, P.O. Box 20708, Department of Neurobiology and Anatomy Houston, TX 77225 and *South African Brain Research Institute, Johannesburg, South Africa R e c e i v e d 5 A u g u s t 1987 DAFNY, N., M. A. GILLMAN AND F. J. LICHTIGFELD. Cholecystokinin: Induced suppression offi'eding in fed. fitsting and hypothalamic island rats. BRAIN RES BULL 21(2) 225-231, 1988.--Experiments were undertaken to examine the involvement of CCK in the regulation of feeding behavior. The results indicate that 1) intact normal animals exhibited rhythmic circadian feeding behavior, with onset of feedings starting about two hr prior to dark period and continuinguntil 1 to 2 hr prior to the light period; 2) food deprivation and surgical isolation of the hypothalamus alter this pattern of feeding behavior, to the point that animals spend more time eating with no identifiable pattern; 3) administration of CCK to either fed, fasted or hypothalamic island animals has a profound dose related effect on suppressing feeding behavior and in modifying the eating pattern over 24 hr. A possible involvement of CCK with the opiate system in controlling feeding behavior is discussed. Cholecystokinin

Suppression of feeding

Fed

Fasting

THE regulation of postprandial satiety is believed to be in part mediated by gastrointestinal hormones that are released by ingested food. Evidence has been obtained that cholecystokinin (CCK), a classical gut hormone, can reduce food intake in various species (19,32) of animals including rats and humans, and has thus been proposed to act as a satiety regulator (33). There is now considerable evidence that exogenous cholecystokinin (CCK) can produce feeding satiety although it is not clear whether this is a physiological or pharmacological effect. Despite the large volume of work in this area little has been done regarding the behavioral effects of CCK in fed, starved and hypothalamic deafferented obese animals, although there are reports of changes in CCK receptor binding after fasting (12,30). We therefore decided to investigate the effects of CCK on feeding behavior in intact fed and fasted rats (deprived of food for 24 and 48 hours) as well as in rats with complete hypothalamic deafferentation which included mainly the ventro- and dorsomedial hypothalamus.

Hypothalamic island

The average weight of each rat at the beginning of the experiments ranged from 170-200 grams. The rats were divided into 15 experimental groups (each N=8); control, chow ad lib, plus saline injection (group 1); group 2; control plus 1.0 I.U. CCK (Squibb, synthetic C-terminal octapeptide of cholecystokinin; 1/xcg= 1.0 I. U.); control plus 2.5 I.U. CCK (group 3); fasted for 24 hours plus saline (group 4); fasted for 24 hours plus 1.0 I.U. CCK (group 5); fasted for 24 hours 2.5 I.U. CCK (group 6); fasted for 48 hours plus saline (group 7); fasted for 48 hours plus 1.0 I.U. CCK (group 8); and fasted for 48 hours plus 2.5 I.U. CCK (group 9). Hypothalamic deafferentation was performed in additional six groups of animals (each N--8) according to the method of Halasz (17) and Halasz and Pupp (18) with minor modification (11). These groups (10 to 15) were identical to the intact groups 1 to 6 respectively except for deafferentation. All injections were in a volume of one ml and given intraperitoneally. In order to control for differences in the powder and normal chow 2 groups of 8 rats each were weighed each after being exposed to either powder or routine chow. A feeding dish containing the powdered chow with a depth of 4 cm was positioned so that the rat's head would interfere with a light source attached to a photo-electric cell (10 sec response time) in such a manner that feeding would be impossible without interfacing with the light source. This system was connected to a recorder that noted both time and duration of feeding. During all experiments access to pine

METHOD Male albino rats of the Hebrew University strain were fed with standard rat chow that had been ground to a fine powder for one week prior to the commencement of the experiments. They were exposed to light for 14 hours (0600-2200) per day with 10 hours darkness and fed ad lib while becoming acclimatized to the powdered food, each in a separate cage.

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DAFNY, GILLMAN AND LICHTIGFELD

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w o o d sticks w a s allowed to p r o t e c t the integrity o f d e n t a l structures which may have been damaged because of the powdered chow. E x p e r i m e n t a l s e s s i o n s s t a r t e d at 0900 e a c h d a y a f t e r a 24 h o u r s p e r i o d o f a d a p t a t i o n to the e x p e r i m e n t a l cages, treatm e n t (saline or C C K ) w a s g i v e n 15 min b e f o r e e x p e r i m e n t a l s e s s i o n b e g a n . D u r i n g food d e p r i v a t i o n , rats w e r e g i v e n w a t e r ad lib. All d a t a was s u b j e c t e d to a M a n n - W h i t n e y U - W i l c o x o n r a n k s u m W - t e s t as well as a n a n a l y s i s o f v a r i a n c e . T h e d a t a c o n s i d e r e d w a s ; time to first feed in m i n u t e s ; total d u r a t i o n o f e a t i n g in first 6 h o u r s ; total d u r a t i o n of eating d u r i n g entire 24 h o u r s t e s t i n g period; f r e q u e n c y o f eating in first 6 h o u r s ; f r e q u e n c y o f eating o v e r e n t i r e 24 hours.

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FIG. 2. Histograms showing the time at which feeding started (latency) in control and experimental animals. The rats were divided into 15 experimental groups (each N=8); control chow ad lib (fed), treated with saline injection (group 1); fed animals injected with 1.0 I.U. CCK (group 2); fed animals treated with 2.5 I. U. CCK (group 3); food deprived for 24 hours then injected with saline (group 4); food deprived for 24 hours then treated with 1.0 I.U. CCK (group 5); food deprived for 24 hours then treated with 2.5 I.U. CCK (group 6); food deprived for 48 hours then treated with saline (group 7); food deprived for 48 hours then treated with 1.0 CCK (group 8); food deprived for 48 hours then injected with 2.5 I.U. CCK (group 9). Hypothalamic deafferentation was performed in additional six groups of animals (N=8). These groups (I0 to 15) were identical to the intact groups 1 to 6 respectively except for deafferentation. All injections were in a volume of one ml and given intraperitoneally. The Mann-Whitney test was used to identify differences among groups compared to control group.

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FIG. 3. Six hours frequency histograms of eating pattern in two fed (control) animals and four deprived animals (24 and 48 hr) before and after CCK (1.0 or 2.5 I.U.) treatments respectively.

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RESULTS

Powdered chow feeding made no statistically significant difference to the weight of those rats that received it when compared with rats fed normal chow (6_+4 g/day to 5-+3 g/day respectively). Intact animals exhibit some characteristic patterns, i.e., the number of feeding bouts increased between 5 p.m. two hr before the light went offand persisted until about 3 to 4 a.m. one to two hr before the light went on (Fig. 1). Food deprivation and hypothalamic deafferentation

(Fig. 1) as well as CCK injection modifies this eating pattern. Hypothalamic deafferentation dramatically increased the daily weight gain of these animals over at least two weeks period from 5±3 g/day to 18±5 g/day. It can be seen that the latency to feeding decreased as the length of fast increased. This was also seen as a result of hypothalamic deafferentation (Figs. I and 2 groups 4, 7, 11 and 14), and all these differences were significant (p <0.001--using the analysis of variance). In preliminary experiments fasted animals were injected

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FIG. 5. Histograms summarizing the amount of time in min/6 hr (in A) and min/24 hr (in B) animals were spending in eating for each of the 15 groups respectively (abbreviation and groups see Fig. 2).

with 1.0 or 2.5 I.U. CCK and were immediately placed in the testing cage. These animals started to eat immediately and exhibited identical feeding behavior as uninjected animals. When animals were put into the testing cage 15 rain after C C K injection different observations were obtained. Therefore, in this experiment animals were tested 15 rain postinjection. The latency (in rain) to the first feed when compared to intact animals following 1.0 I.U. CCK or 2.5 I.U. CCK was delayed by 743% and 81 I% respectively (Fig. 2), those fasted for 24 hr by 3170% and 2670% (groups 5 and 6 compared to 4), those fasted for 48 hr 3915% and 7000% (groups 8 and 9 compared to 7). In deafferented fed animals CCK (1.0 I.U. and 2.5 CCK respectively) delayed the first fed by 4140% and 6186% (groups 12 and 13 compared to 11) and 13100% and 29700% (groups 16 and 15 compared to group 14) in deafferented 24 hr fasted animals respectively (Fig. 2) namely dose response relationships were obtained (p<0.01). Figures 3 and 4 show representative feeding behaviors of the animals during the initial 6 hr for intact animals and for hypothalamic deafferentation respectively and Fig. 5A summarizes that data obtained for the initial 6 hour period. It is clear that CCK in nondeprived animals decreased the amount of eating significantly but this was not dose related. In Fig. 5B which illustrates total feeding time in the 24 hour period 1.0 I.U. CCK (group 2) decreased the total feeding time by 5% and 2.5 I.U. CCK (group 3) by 34%. F o r animals fasted for 24 hours 1.0 I.U. unit and 2.5 I.U. reduced total feeding over 24 hours by 23% and 22% respectively (Fig. 3B; groups 5 and 6) whilst in 48 hour fasted animals the reduction in eating over the 24 hour period was 35% and 45% for 1.0 I.U. and 2.5 I.U. respecitvely (Fig. 5B; groups 8 and 9). In hypothalamic deafferented animals dose related characteristics were observed (20% and 45% reduction in feeding

time) in fed animals (groups 11 and 12 compared to 10 Fig. 5B) and 55% and 71% in 24 hr fasted animals following 1.0 and 2.5 I.U. CCK respectively (groups 14 and 15 compared to 13)(Fig. 5B). Figure 6A summarizes the frequency of eating over the initial 6 hours period. In fed animals the low dose of CCK reduced the frequency of eating by 66% while the higher dose reduced it by 83% (Fig. 6A; groups 2 and 3 compared to 1). The 24 hour fasted animals had their frequency of eating reduced by 46% and 61% by the low and high doses of CCK (Fig. 6A; groups 5 and 6 compared to 4). Forty-eight hours of fasting resulted in a decrease in frequency of 46% and 54% respectively (Fig. 6A; groups 8 and 9 compared to group 7). Similar observations were obtained in deafferented animals 50% and 67%; and 45% and 82% in fed and 24 hr starved rats following 1.0 and 2.5 I.U. CCK respectively (Fig. 6A). In fed animals CCK had no effect on frequency of eating over 24 hours (Fig. 6B; groups 2 and 3 compared to 1). In animals fasted for 24 hours 1.0 I.U. CCK had no effect over 24 hr whilst 2.5 I.U. reduced frequency of eating over 24 hours by 27% (Fig. 6B; groups 5 and 6). In the animals fasted for 48 hours the lower dose of CCK reduced feeding frequency by 31% whilst the higher dose reduced it by 23% (Fig. 6B; groups 8 and 9 compared to 7). Similar observations were obtained in hypothalamic deafferented animals when over a 24 hr period a single injection of CCK 1.0 or 2.5 I.U. was able to significantly reduce the frequency of eating in fed and fasted animals (Fig. 6B). DISCUSSION

It has been known for some time that there exists a blood-borne factor associated with satiety. When blood from a sated rat is mixed with the blood of a hungry rat, the hungry rat eats only about 50% of what it eats when it re-

CHOLECYSTOKININ AND FEEDING

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FIG. 6. Histograms summarizing the frequency (number of times) of eating in the initial 6 hr (A) and during 24 hr (B) for each of the 15 groups respectively (see Fig. 2).

TABLE 1 S U M M A R Y OF T H E F V A L U E S (F) AND T H E S I G N I F I C A N C E L E V E L S (/)) B E T W E E N G R O U P S AND T R E A T M E N T FOR T H E INITIAL 6, 20 AND 24 HOURS, R E S P E C T I V E L Y

Time at which feeding started Frequency of feeding Time spent in eating

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F 4.07p<0.001

F 12.39p<0.000

F 6.59p<0.000

F 5.73p<0.000

ceived blood from another hungry rat (19). Teleologically GI hormones are excellent candidates to be considered as participants in the regulation of food intake. They are secreted in response to quality and quantity of the ingesta entering the GI tract, and through the blood stream could transfer this information to a central regulating mechanism (16). Satiety after feeding must be due to rapid physiological consequences of eating (5, 19, 33). It has been long known that single daily meals offered to animals can drastically affect behavioral and physiological rhythms. Daily food intake in the rat occurs in a series of discrete meals separated by internal intervals (29). Several researchers have found that both the size and frequency of meals increased during the dark segment of a LD cycle (29); similar observations were obtained in our study. It has also been known for some time that lesioning the VMH could disrupt feeding patterns (28). Hence, it seems evident that the VMH is involved in the mechanism underly-

ing the development of circadian rhythmicity associated with periodic feeding (21). Our hypothalamic deafferented animals produced a similar picture of disrupted feeding. It is well accepted that the major regulating areas for appetite are though to be in the hypothalamus (6,7). Two centers have been described: one situated in the VMH responsible for producing the sensation of fullness, i.e., the satiety center, and one situated in the LH that appears to initiate feeding (24,31). These areas are enriched with both opioids (2) and CCK (9). Knife cuts placed between the lateral and medial hypothalamus produce obesity and hyperphagia suggesting that a medial lateral connection carries inhibiting influences from the VMH to the L H (1). Histochemical localization has shown that the most dense collections of CCK cells occurred in the periaqueductal gray and in the dorsomedial hypothalamus (20). Strauss and Yallow (34) showed that ob/ob mice who manifested hyperphagia have strikingly lower CCK content than their lean

230 littermates and other normal mice, suggesting that the lower amount of CCK in the brain may be causally related to the unrestrained appetite of these mice. It is clear from these results that a single CCK injection has a profound effect on suppressing feeding behavior and in modifying the eating pattern for the initial 6 hr in all experimental animals and in some groups over 24 hr. Our observations are in line with those observed by Hsiao et al. (19) that a synthetic solution containing only CCK-OP can delay onset of eating substantially in food deprived rats. This provides additional support for the role of CCK as a satiety hormone. This effect was reported to be overridden in rats after some days of repetitive CCK injections at mealtimes and resulted in an increase in the frequency of smaller meals eaten by these animals (35). However, in our work where the animals were studied for only 24 hours such compensation did not occur. The effect of fast length on the response to CCK has been measured in few studies. In rats 1 ~g/kg CCK decreased food intake by 70% of control after a 12 hr fast but had not effect after a 48 hr fast; 10/~g/kg was required to produce a similar reduction in 48 hr fasted rats (3,4). After a 23 hr fast, partially purified CCK decreased the rate of eating in both obese mice and their nonobese littermates (3,27). In Zucker obese rats, injection of low doses of CCK-8 just before meals decreased food intake less than in lean rats (23), while in our experiment CCK was effective in hypothalamic deafferented as well as in intact animals which is in disagreement with the above observation. It would appear from our experiments that deafferentation produces a striking increase in the animal's sensitivity to the satiety effects of CCK after food deprivation. The mechanism underlying this finding could be related to deafferentation hypersensitivity of CCK receptors within the disconnected area of the hypothalamus. This would apply particularly to fibers from the parabrachial nucleus to the VMH which may have been severed (13). In addition, even if the deafferentation occurred distal to the VMH, hypersensitivity could have been produced by transneuronal degeneration. It is not possible from our present work to exclude the effects of deafferentation hypersensitivity on either the opioid or CCK systems and either of which might have a significant bearing on our observations. Notwithstanding the foregoing the enhanced activity of CCK in deafferented animals may well have occurred as a result of a disturbance of the balance between the CCK and opioids that may be critical in the homeostatic control of feeding. It is therefore of interest to note that opioid upregulation supersensitivity has been demonstrated in rats where the VMH nuclei were ablated (22). These workers showed that such lesions produced suppression of the symptoms of morphine abstinence and dependence, thereby supporting the view that addiction and feeding may have a common representation at the hypothalamic level (22). We have suggested a link between feeding and addiction through interactions between CCK and the endogenous opioids (15), and it is possible that this interaction could be mediated at this level. Other evidence suggests that animals which are hyperphagic with resultant obesity are less sensitive to the effects of CCK on satiety (3). This illustrates the complexity of the homeostatic mechanisms involved in appetitive behaviors and that in regard to feeding many systems must be involved (25,26), one of which is opioid (8). Because of the opposite effects that opiate and CCK peptides have in various physiological systems, including feeding behavior, it is tempting to suggest that these two peptide systems interact

DAFNY, GILLMAN AND LICHTIGFELD at some level to modify the effects of one another (3). The finding that CCK was less effective in delaying the time to feeding in fasted animals further indicates the possible activation of the opioid system in such animals (8). Moreover, our results also indicate that the frequency of feeding in the 2 groups of fasted animals was similar, while the time spent eating was significantly greater for the 48 hour fasted animals than was the case for the 24 hours fasted animals. Notwithstanding the fact that the time to first eating was diminished in the longer food deprived animals it might be that the actual amount of food consumed by these animals may well have been greater even though less time was taken to consume this greater volume of food. In addition, this would also seem to apply to anorexia nervosa subjects, who respond to offered food by vomiting and ingesting small amounts of food (5,15); anorexia nervosa is related to excessive opioid stimulation (15). Thus the longer the fast, the more likely it is that excessive opioid production would result in some disorder causing an opioid-CCK synergism instead of the more usual antagonism (15). In addition, the balance between the opioid and antiopioid systems which we have postulated to be involved in other physiological processes as well as in appetitive drives (15) may be disturbed in prolonged fasting. This, therefore, could explain the paradoxical opioid-CCK synergism noted above. It would, therefore, appear that the investigation of various fasting states might further elucidate the relationships existing between the antagonistic opioid systems as well as their relationship to CCK. Furthermore, it has been postulated that CCK is a natural anti-opioid substance (10,14). Moreover, Faris has shown the presence of a CCK network in the hypothalamic paraventricular nucleus, which is a critical area involved in opioid stimulated food intake (10). In addition, the vagal afferents mediating the actions of peripherally administered CCK synapse in the nucleus of the solitary tract, which is itself well endowed with opioid receptors (33). In view of the work showing that receptor binding of brain CCK is altered in fasted animals (12,30), it is possible also to infer that the effect of CCK in such rats is mediated by central as well as peripheral mechanisms. Baile et al. (3) provide evidence from various sources to support the above concept of the interaction between the opioids and CCK in the control of feeding behavior, in which the opioids stimulated and the CCK peptides inhibited feeding. However, they indicate that the evidence for these interactions are suggestive and not conclusive, since neither the site nor mechanism of this interaction are clear. Our present work provides evidence for the postulate that it is a disturbed balance between the opioid and CCK systems that underlies eating disorders in human subjects (15) and that hypothalamic connections are important in this regard. It would seem that further clinical work in feeding disorders using CCK and opioid agonists and antagonists may elucidate this disturbed balance. ACKNOWLEDGEMENTS Supported by grant NS 18372 and a grant from Anglo-American and De Beers (S.A.) Ltd. The authors gratefully acknowledge Dr. S. Feldman and N. Conforti from the Laboratory of Neurophysiology, Department of Neurology, Hadassah University Hospital and Hebrew University-Hadassah Medical School, Jerusalem where this work was performed. The statistical analyses were done by Drs. Y. Wax and I. Einot from the Statistical Consultant Unit, Department of Statistics, Social Sciences, Hebrew University, Mount Scopus, Jerusalem. The secretarial assistance of D. Parker is appreciated.

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