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Current status of cholecystokinin as a short-term satiety hormone
Neuroscience & Biobehavioral Reviews, Vol. 2, pp. 7 9 - 8 7 , 1978. Printed in the U.S.A.
Current Status of Cholecystokinin as a Short-Term Satiety Hormone K A T H Y R N E M U E L L E R AND SIGMUND HSIAO 1
Department o f Psychology, University o f Arizona, Tucson, A Z 85721 (Received 25 August 1977) MUELLER, K. AND S. HSIAO. Currentstatus of cholecystokinin as a short-term satiety hormone. NEUROSCI. BIOBEHAV. REV. 2(2) 79-87, 1978. - Cholecystokinin (CCK) is a putative short-term satiety hormone which may regulate meal size and the intermeal interval. A series of criteria are proposed to determine whether CCK can be regarded as a short-term satiety hormone. The available data indicate that the CCK-induced inhibition of food intake may not be chemicaUy specific (to CCK) but is behaviorally specific (to ingestive responses). The physiological nature of the necessary doses is unclear. The effects of CCK seem to simulate natural satiety, and these effects appear to be consistent across wide ranges of nutritional deficit. The mechanism of the effect may involve the feeding-associated areas of the brain. The effect has been shown in rats and monkeys, but data from humans have been inconsistent. CCK appears to be a promising factor which may participate in natural satiety along with many other factors. Cholecystokinin
Satiety
Specificity
Dose levels
IN order to maintain an adequate nutritional and energy balance, an organism must regulate energy outflow, energy inflow and potential energy. Energy inflow should match energy outflow; potential energy serves to gap the time differential between the discontinuous inflow and the continuous (obligatory) and episodic outflow of energy. Regulation of energy inflow is accomplished by consuming discrete meals via adjusting the initiation of a meal, duration of a meal, termination of a meal and intermeal intervals. The events surrounding the initiation of a meal are generally referred to as "hunger-related" or as "hunger variables" and those surrounding the termination of a meal are referred to as "satiety-related" or as "satiety variables." This paper will review the experimental data of the satiety-related effects of an intestinal hormone, cholecystokinin (CCK). CCK is a polypeptide hormone existing in two molecular forms, one that contains 33 and another that contains 39 amino acid residues [62]. Its release is stimulated by certain properties of food chyme: hydrogen ions, protein (and amino acids), fat, and magnesium and calcium ions [33, 40, 62]. CCK-secreting cells have recently been identified in the duodenum, jejunum, and ileum but the secretory activity decreases caudally [15,60]. CCK has been classically known to stimulate contraction of the gallbladder and secretion of pancreatic enzymes [40]. It also elicits bicarbonate and insulin release from the pancreas, stimulates intestinal motility, contracts the quiet stomach and pyloric sphincter, stimulates secretion of Brunner's glands and blood flow in the superior mesenteric artery, but decreases systemic arterial pressure. It stimulates
Hypothalamus
pancreatic growth and inhibits contraction of the lower esophagial sphincter, the sphincter of Oddi, absorption of fluid, sodium, potassium, and chloride from the jejunum and ileum, and motility of the active stomach [40,61]. To this list of effects we hope to add satiety induction, but mere demonstration of inhibition of feeding induced by CCK is not sufficient evidence to do so. For some time blood-borne factors have been known to mediate the termination of eating: The food intake of a hungry rat was reduced 50% after its blood had been thoroughly mixed with the blood of a rat which had just finished a meal [ 11 ]. The blood-borne factor disappeared as time elapsed between the end of a meal and the blood transfusion [10]. When a fasted rat received blood from a sated rat, its cortical EEG pattern was altered to that of a sated rat [65]. The blood-borne factor formed in relation to food intake was assumed to promote satiety and when it fell below a critical level the animal initiated its next meal [101. An early study indicated a gut " h o r m o n e , " enterogastrone, inhibited eating in fasted mice; two other digestive hormones, secretin and pancreatic glucagon, did not inhibit eating [66]. The gastrointestinal source of the satiety factor was further suggested by two observations: Duodenal infusions of liquid diet or osmotically potent substances decreased food intake during normal meals and during sham feeding (in which contents of the stomach were allowed to drain through a fistula) [47,74] ; thus the presence of food in the small intestine, in absence of stomach accumulation of food, was sufficient to inhibit feeding in rats [73]. Administration of CCK during sham
1Address reprint requests to S. Hsiao, Department of Psychology, University of Arizona, Tucson, AZ 85721, U.S.A. This publication is supported by NIH Institutional Grant of the University of Arizona. C o p y r i g h t © 1978 A N K H O
I n t e r n a t i o n a l Inc.--0149-7634/78/0202-0079501.05/0
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MUELLFR ANI) HSIAO
feeding mimicked the effects of duodenal infusions of food; sham feeding was inhibited and several responses characteristic of satiety were displayed [25 ]. A "CCK satiety hypothesis" began to be advanced: As food chyme leaves the stomach some of its chemical properties stimulate the CCK-secreting cells in the small intestines and blood-borne CCK interacts with the ventromedial hypothalamus to induce satiety. Presumably, once the concentration of the blood-borne CCK falls below a critical level, eating is resumed. To determine the validity of the CCK hypothesis, several criteria are proposed and the related experimental evidence is discussed. THE CRITERIA TO ADOPT THE CCK HYPOTHESIS 1. The CCK-induced inhibition of food intake should be chemically specific: (a) CCK should suppress food intake in a dose-related manner; (b) Molecules sharing the bioactive component of CCK should also inhibit food intake. Similar concentrations of molecules lacking the bioactive component should be ineffective in suppressing food intake; (c) Events stimulating or blocking endogenous CCK secretion should also affect food intake by either quickening or delaying the termination of a meal. 2. The CCK-induced inhibition of food intake should be behaviorally specific; only the occurrence of ingestive responses should be altered. 3. Experimental doses of CCK which inhibit food intake should be within the physiological range of a species. 4. The CCK-induced inhibition of food intake should be similar to satiety occurring after a free feeding meal: (a) Behaviors following the administration of CCK and the consumption of a free feeding meal should be similar; (b) Physiological responses following the termination of a free feeding meal should occur following the administration of exogenous CCK; (c)CCK, like satiety, should induce a state of affairs which an organism does not avoid and which an organism may even perform behaviors to attain or prolong. 5. The CCK-induced inhibition of food intake should interact in a consistent and adaptive manner with the nutritional deficit of an organism 6. The mechanism of the CCK-induced inhibition of food intake should be experimentally verifiable. 7. The CCK-induced inhibition of food intake should be consistent among those species possessing similar dietary habits and gastrointestinal systems. SUPPORTING AND CONFLICTING EVIDENCE CCK and Dose Dependence 1A. CCK should suppress food intake in a dose-related manner. The degree of suppression of intake should be directly related to the amount of CCK administered or secreted. Assuming the absence of competing behavioral effects of the hormone, the shape of the dose-response relationship has implications for the mechanism of the CCK-suppressive effect. A U-shaped curve may indicate the existence of low affinity inhibitory receptor sites [49] ; a linear dose-response relationship may indicate a neural component; a hyperbolic curve may indicate the direct action of CCK on a limited number of receptors. Most studies in rats have revealed a consistent doseresponse relationship. A positively increasing dose-related suppression of food intake has been consistently reported from 5 to 80 Ivy dog units/kg body weight (the Ivy dog
unit is defined as that amount of dry material which when dissolved in normal saline solution and injected intravenously, during 10 to 15 sec into an anesthetized (tog weighing about 15 kg, results in a more or less immediate ( 1 to 3 rain) rise in intragallbladder pressure of 1 cm of bile [40]), [23, 24, 25, 45, 54, 55, 731. However, 100 Ivy dog units/kg (U/kg) has been reported ineffective in suppressing intake [45]. Thus the dose response curve of CCK in suppressing food intake may be U-shaped as il is in its effect on gastric secretion [7811. In other studies doses of 100 U/kg and above have been reported as more effective than 40 U/kg [73]. However, these data were reported for rats with open gastric fistulas which require doses approximately four times higher than the doses necessary t~ produce equivalent responses in intact rats [251. Therefore, the concept of a U-shaped dose-response cnrve has no[ necessarily been contradicted. Research with humans has failed lo reveal a consistent dose-response relationship. Although rapid intravenous injection of 0.5 U/kg reduces food intake [81], stow intravenous infusions of 3 U/kg have been reported to be ineffective [29] or to increase intake [81 ]. Either the route of administration or the dose may be important in increasing intake -- whether the nature of endogenous hormone release resembles a slow infusion or rapid injection is not known [49]. Dose-related suppressions of food intake have been reported only for relatively high, nonphysiological doses. At lower doses, the relationship between the amount of CCK administered and the degree of suppression of intake is unclear. Evidence for this criterion is therefore inconclusive. Molecular Bioactivity - Structural Specff)cit.v lB. Molecules sharing the bioactive component o f CCK should inhibit food intake. Similar concentrations o f molecules lacking the bioactive component should be ineffective in suppresing food intake. The biologically active portion of the CCK molecule is the C-terminal octapeptide. The sulfonated tyrosine-7 is essential for biological activity; the nonsulfonated molecule is relatively ineffective in contracting the gallbladder [42,58]. A synthetic octapeptide (Sincalide, Squibb) has been shown to be extremely effective in suppressing food intake [22, 24, 45]. Caerulein, a decapeptide isolated from the skin of an Australian frog (Hyla caerulea) shares the common C-terminal octapeptide with CCK, except for an exchange of threonine in caerulein for methionine in CCK. Caerulein is also effective in reducing food intake [79]. On the other hand secretin and pancreatic glucagon [51] which are gastrointestinal hormones but do not share the common C-terminal octapeptide with CCK are not effective in altering food intake [66,73]. Although pentagastrin can inhibit food intake, the doses required are 80 times the dose required for eliciting maximal gastric acid output in the rat and therefore may be the result of a toxic effect. Doses of CCK required to inhibit food intake are within the range for eliciting maximal gastric acid output and maximal pancreatic secretion [73]. It should be mentioned, however, that under certain conditions glucagon can decrease food intake [50] but the mechanism appears to be different than that of CCK. Glucagon fails to suppress feeding when hepatic glycogen has been depleted by moderate fasting [50,66] but CCK is equally effective in suppressing food intake during fairly severe fasting [541.
CHOLECYSTOKININ AND SHORT-TERM SATIETY The suppression of food intake induced by glucagon is probably a result of its various metabolic effects, rather than a result of any direct satiety-inducing properties [50]. The duration of the suppressive effect of CCK and its analogs has not been investigated, but the duration may be shorter for the octapeptide and caerulein than for the parent CCK molecule. The half-life of the parent molecule has been estimated at between 2.5 and 3 min [59, 61, 64, 83], 5 to 7 min [30] with radioimmunoassay or at 1 0 - 1 5 m i n [5] with bioassay in humans or cats. The biological effects of the parent molecule should last longer than those of its shorter analogs since as long as the C-terminal octapeptide is intact the fragment should remain active. Several of the CCK peptides and caerulein are far more potent on a molar basis than the parent molecule [40]. This probably is a function of molecular size, shape and resulting hormone receptor interactions. While many studies could be done to further clarify hormone receptor interactions for satiety induction and the half-life of the CCK analogs, this criterion of the effect of the C-terminal octapeptide of CCK on food intake can be regarded as fulfilled. Thus, CCK receptors for the inhibition of food intake are similar to those in gallbladder.
Stimulation and Blockade o f CCK 1C. Events stimulating or blocking endogenous CCK secretion should also affect food intake by either quickening or delaying the termination o f a meal. Events stimulating the release of CCK should decrease food intake by quickening the termination of a meal. As indicated above, the release of CCK is stimulated by hydrogen ions, fatty acids, peptides or certain amino acids, magnesium and calcium ions. Very large loads of glucose may also stimulate the secretion of CCK but not ordinary amounts [49,88]. However, apart from any particular chemical composition or nutritive value of the ingestia, its osmotic potency has often been referred to as a short-term regulator of food intake [67,70] and therefore tends to confound the CCK hypothesis. Availability of liquid and the osmolality of liquid drank are known to influence amount of food consumed or intake-related responses [31, 34, 36] and blood osmolality can determine the amount and the time of food intake [41,69]. However, the amount of NaC1 in food has been shown not to influence the intake amount even when no liquid is present [34]. Under free feeding conditions, with or without water available, the contents of the stomach after consumption of dry food are diluted by absorption of body fluids [46] such that chyme entering the intestines tends toward isotonicity. Thus, under free feeding conditions, though the osmotic property of the ingesta influences gastric emptying [38], an intestinal osmotic factor per se should not be important in the short-term regulation of meal size in the intact animal [16]. Studies employing direct duodenal infusions should be carefully examined for the possible introduction of "unnatural" osmotic effects. Another factor which may be important in the shortterm regulation of food intake is the presence of (nonnutritive) bulk in the gastrointestinal system. Isotonic bulk has been reported to inhibit food intake in rats [16, 70, 71 ]. However, stomach clearance of the bulk was not determined, and the results may have been due to artificial stomach distention resulting from abnormal clearance of the bulk.
81 Keeping in mind the possible effects of osmolality and bulk, the effects of substances stimulating the release of CCK can be evaluated. Intragastric loads of liquid diet [74] and wet mash [70] inhibit food intake in rats. Liquid diets have been reported to enter the intestines rapidly [3] to stimulate CCK-secreting cells and we have confirmed these observations in our lab. However, this rapid stomach clearance again raises the possibility that at least the initial inhibition of food intake may be due to osmotic, rather than chemical properties of the ingestia. The relative importance of the intestinal factor, the general osmotic factor and the bulk effect on regulation of food intake is not clear, however, these effects may occur in a certain time sequence. Intragastric infusions of phenylalanine (an amino acid) and casein (a phosphoprotein) limit meal size in monkeys [22,51]. Isovolumetric infusions of NaCI or the D-isomer of phenylalanine (which is generally without any biological effectiveness) fail to produce any changes in intake patterns [22]. Chemical properties rather than volume, therefore, appear important in the inhibition of food intake. Pharmacological agents affecting the release of CCK have not been examined for their effects on food intake. Administration of veratrine, which has been reported to potentiate the effects of CCK [5], should suppress food intake to an even greater degree than the sole introduction of those substances which can stimulate the release of CCK. The secretion of CCK is blocked by hexamethonium [5], oxethazaine [ 19], and other local anesthetics [ 19], but the effects of these agents on food intake have not been reported. Delaying termination of a meal by blocking the secretion of CCK is likely to be a difficult task. In addition to being hyperosmotic, most foods contain sugars, which would be expected to activate the neurally mediated glucose mechanism of satiety [57]. This glucose mechanism may operate independently of the CCK mechanism. Therefore one may expect a normal meal duration even in the absence of CCK. Because food intake can be suppressed by osmotic properties or nonnutritive bulk, and because the effects of the appropriate pharmacological interventions have not been reported, this criterion has not been supported.
Behavioral Specificity 2. The CCK-induced inhibition o f food intake shouM be behaviorally specific; only the occurrence o f ingestive responses should be altered. CCK should suppress behaviors selectively, i.e., it should affect food intake directly without activating a diffuse behavioral system. This is a particularly important criterion in view of the tranquilizing effect of CCK (see discussion of Criterion 4). Most studies addressing this criterion have employed rats drinking in response to water deprivation. The assumption is that if the CCK-induced suppression in intake were due to a general tranquilizing effect, water intake would be suppressed by CCK as well. Although a suppression in water intake was reported in mice [44], in rats exogenous CCK seems to be without effect or to increase water intake very slightly [24,33]. These studies, however, are not strictly comparable to those reporting the effects of CCK on food intake since rats tested for water intake were generally deprived of water far longer than those tested for food intake. Recently exogenous CCK was reported to reduce
82 liquid diet in 23.5 hr water-deprived rats without affecting water intake under the same conditions [55]. Sweetness in ingesta is thought to be a food-cue [52], thus apparently when ingesting water the brain interprets CCK as an irrelevant signal, so that changes in intake do not occur. Unpublished data [ 1 ] indicates that open field behavior is not affected by CCK. Since drinking water in response to water deprivation is so behaviorally similar to drinking liquid diet in response to food deprivation and since the former is not suppressed by CCK while the latter is, the likelihood of other less similar behaviors which may have many controlling factors being suppressed by CCK is very low. The effects of CCK appear to fulfill this criterion. Physiological vs Pharmacological Dose 3. Experimental doses o f CCK which inhibit food intake should be within the physiological range o f a species. The amount of exogenous CCK necessary to suppress food intake should be approximately equal to the a m o u n t of endogenous CCK released in response to a meal. Amounts of CCK circulating in the blood have been estimated by radioimmunoassay at 1.4 ng/ml in fasting hogs [ 1 7 ] ; at 0.9 ng/ml [63], 0.7 ng/ml [61], and 0.025 ng/ml [30] in fasting humans; at 119 ng/ml in dogs [63]. Amounts of endogenous CCK circulating postprandially have been estimated at 12 ng/ml [17] and 3 ng/ml [28] in hogs; at 0 . 9 1 2 n g / m l [61], 9 . 1 n g / m l [63] and up to 300 ng/ml [88] in humans. In adult humans after ingestion of one pint of milk serum CCK is raised from a basal level of 25.8 _+4.3 (SEM) pg/ml (range 5 to 80) to about 16 ng/ml within 30 min postprandially [30]. In cats perfusing the intestinal lumen with weak HC1, emulsified fat, long and short chain fatty acids, or 10% peptone results in the release of CCK of up to 5 to 10 milli Ivy dog units/ml serum within 5 to 10 rain as determined by bioassay [5]. The variability of the estimates may be due to different techniques employed as well as to different substances ingested, as certain stimuli are known to be more effective than others in stimulating the secretion of CCK [40]. The effects of CCK on the food intake of dogs, hogs, or cats have not been reported. In humans only one instance of CCK-induced satiety has been reported -~ at 0.5 Ivy dog units/kg [81 ] or approximately 13 ng/ml. If one assumes that rats and monkeys secrete proportional amounts of CCK in response to a meal, amounts of exogenous CCK necessary to induce satiety appear to be within physiological levels. To date the lowest dose of exogenous CCK which has been reported to induce satiety in rats is 2.5 U/kg [24] or approximately 65 ng/ml, and in monkeys is 5 U/kg [22] or approximately 130 ng/ml. However, before drawing conclusions, a cautionary note is advised. In most feeding studies cited above, exogenous CCK was allowed to mix with endogenous CCK (stimulated by passage of food chyme into the intestines) so that the total amount of blood-borne CCK may have surpassed physiological levels. In addition, estimates of circulating CCK reviewed above are limited in indicating the amount of biologically active portions of the hormone in circulation [27]. Any fragment of the molecule containing an intact octapeptide sequence retains its biological activity and such fragments may not be identified by radioimmunoassay techniques. In the same manner these techniques may identify as CCK those molecular fragments with a distorted
MUELLt:R AND HSIA(~ octapeptide sequence which actually posses~ no biologicai activity. Since little is known about the catabolism of CCK. these difficulties may lead either to intlatcd ,)~ reduced estimates of circulating biologically active C('K. Most studies in rats have employed rapid intraperitoneal injections of CCK, therefore actual blood levels of CCK induced experimentally are unknown. Other studies in humans and monkeys have employed either slow or rapid intravenous infusion. In this case although initial plasma levels are known, one must take into account rates of catabolism and tissue uptake of CCK to accurately estimate amounts of circulating CCK [28]. Although the evidence is suggestive, firm conclusions of the effectiveness of physiological levels of CCK in suppressing food intake cannot be drawn. Behavioral Similarity 4A. Behaviors fi)llowing the administration oJ CCK and the consumption o f a free feeding meal should be similar. Relatively little information is available about satietyrelated behaviors. Rats maintained in semi-natural conditions engage in exploratory and sampling behaviors at the close of a meal [4]. In more confined conditions grooming and resting are associated with termination of a meal [1,6]; however, under these conditions very few behavioral alternatives are available. Thus, only the occurrence of sleep or resting behaviorally distinguished between the cessation of feeding due to satiety and that due to quinine adulteration of the diet [ I ]. In cats, sedation also appears to be satiety-related [20]. Administration of exogenous CCK during feeding not only reduced the amount of food eaten, but was followed by grooming and resting in rats [1]. Sedation has been noticed in cats following administration of exogenous CCK or intraduodenal milk and corn oil which elicit the release of endogenous CCK [20]. Further work is clearly necessary to establish satiety-related behaviors in other species. Physiological Response Patterns 4B. Physiological responses following termination oj a free .feeding meal shouM occur following the administration o f exogenous CCK. The cortical EEG pattern appears to be a particularly good indicator of satiety. Satiety is accompanied by consistent changes in the EEG [20, 65, 82] and prolonged EEG changes occur only when both oral and gastric receptors are stimulated [82] just as "intestinal satiety" depends upon both oral and gastrointestinal stimulation [2]. Changes in cortical EEG, appearance of the eye movement and increase in superior mesenteric blood flow have been observed following a meal [20,68] as well as following the administration of exogenous CCK [20]. Appearance of loose stools is also reported following CCK administration in man [81]. We also observed an increased occurrence of defecation in rats following CCK injections in our lab. Whether the increased bowel movement is a part of natural satiety or it represents a sign of supra-physiological dose level of CCK is not clear. Natural Satiety and CCK 4C. CCK, like satiety, should induce a sate o] ~affairs which an organism does not avoid, and which an organism may even perform behaviors to attain or prolong. People often associate satiety with warmth or satisfaction [53]
CHOLECYSTOKININ AND SHORT-TERM SATIETY and we are tempted to speculate that satiety (or CCK) may function as a powerful reinforcer in an operant learning situation. Although the reinforcing properties of CCK have not been investigated, we can conclude that CCK does not function as an aversive stimulus. Attempts to produce a conditioned taste aversion with CCK or its octapeptide have been unsuccessful (but see note) [1, 24, 32]. No toxic effects whatsoever have been observed in rats or mice after eight months of CCK injections two or three times weekly [73] or after single administration of doses up to 2000 U/kg [48]. Some side effects have been noted in humans at 1 U/kg but they were not severe and did not appear to be responsible for changes in food consumption [81]. Administration of exogenous CCK does not change the pattern of food intake in rats, but only reduces the amount consumed [54]. Aversion can therefore be eliminated as the cause of the CCK-suppressive effect.
Generality over Deprivation Levels 5. The CCK-induced inhibition o f food intake should interact in a consistent and adaptive manner with the nutritional deficit o f an organism. CCK should interact in a consistent and adaptive manner with the state of nutritional deficit of the organism with the nature of the interaction indicating something about the nature of the CCKsuppressive effect. The effects of CCK on food intake have been examined at varying levels of deprivation - 5 hr [54], 5.5 hr [23, 24, 45], 17 hr [1, 24, 25, 73], 8 hr [44], 19 hr [54], 4 8 h r [54], and 9 2 h r [72] - and suppressions in intake have occurred at each level except 92 hr. The CCK-suppressive effect appears to remain constant across wide variations in food deprivation levels. For example, 40 U/kg suppresses intake by approximately 50% at 5, 19 and 48 hr deprivation [54]. This result is suggestive of some interaction with nutritional deficit; if no interaction were present, intake would be expected to cease at a given time or given volume rather than at a given percent of control intake. A simple chemical trigger reflex model of the CCK effect on food intake would therefore seem inappropriate. The failure of CCK to suppress food intake at 92 hr deprivation suggests the existence of a second satiety mechanism, a "fail-safe" mechanism. Such an arrangement would be clearly adaptive. It would inhibit excessive caloric intake during moderate deprivation but facilitate larger caloric loads during life-threatening deprivation. Such a shift in locus of control would require a physiological change occuring between 48 and 92 hr deprivation which could trigger a fail-safe mechanism. Transitions from utilization of blood glucose in the free feeding animal to the utilization of body fat and finally body protein during severe deprivation [7] may provide the necessary physiological signal. A shift in locus of control of meal size is not without precedent. A neural short-term satiety mechanism has recently been demonstrated which is activated by glucose and mediated by the vagus nerve. This mechanism is duodenally based under free feeding conditions but hepatic-portally based after 22 hr deprivation [57]. Similarly, during severe deprivation the CCK mechanism may be replaced by some other satiety factor - a "fail-safe" factor. The known effects of CCK fulfill this criterion.
83
Mechanisms o f CCK-Satiety Effect 6. The mechanisms o f the CCK-induced inhibition o f food intake should be experimentally verifiable. CCK may suppress food intake as a result of its classical physiological roles. For example, stomach emptying may be inhibited by CCK to the degree that ingested food accumulates in the stomach causing stomach distention and a suppression of feeding. However, as indicated previously, CCK can induce satiety in the absence of gastric distention. CCK may suppress food intake because of its sedative properties. But, as previously noted, sedation seems to be a part of satiety rather than a cause. In addition a recent series of e x p e r i m e n t s suggested a relationship between the traditional hypothalamic feeding and satiety areas and the CCK satiety effect. Changes in evoked responses to auditory stimuli were recorded from rats following administration of exogenous CCK. These changes were found only after injection of CCK and specifically from the ventromedial hypothalamic (VMH) area. In addition, the time course of these changes appeared similar to the time course of satiety [9]. Lesions in the VMH produced a reduced sensitivity to caerulein (1 ug/kg), but lesions in the lateral hypothalamus (LH) heightened sensitivity. Microinjection of caerulein into the VMH, but not into the LH, limited feeding. Tritiated caerulein was selectively bound to tissue in the VMH [79]. These results indicate possible receptor sites of CCK in the brain and place CCK in a well-known Stellar schema of the control of feeding [76,77] as a chemical signal for regulating the activity of the hypothalamic feeding areas. However, in another recent study lesions in the VMH were reported to have no effect at all on the sensitivity to the CCK-induced suppression of food intake [45]. The similarity between this CCK mechanism of satiety and the angiotensin-related mechanism of thirst is striking. Angiotensin II has been shown clearly to be a signal of thirst which appears to have the subfornical organ as its receptor site [18,35]. How a polypeptide, like Angiotensin II, could enter the brain to signal thirst has been a problem, but renin has been found within the brain itself [21]. Similarly CCK-like peptides have also recently been found in the brain [ 14]. The functions and sources of these brain-originated compounds are not understood at this time. Species Generality 7. The CCK-induced inhibition o f food intake should be consistent among those species possessing similar dietary habits and gastrointestinal systems. CCK should be important in regulating meal size in several mammalian species. Although the satiety-like effects of enterogastrone were first discovered in mice [66], the only report of the effects of exogenous CCK on the food intake of mice is inconclusive [44]. Most studies employing exogenous CCK have been limited to rats. The single failure to find a CCK-induced suppression of intake in rats [26] was probably due to the low dose employed or to the slow rate of administration. Although the effects of exogenous CCK on the meal size of cats have not been reported, available evidence suggests that endogenous CCK seems to evoke satiety-related responses [20, 68, 82]. As indicated earlier, however, this evidence cannot be accepted as conclusive. A single study indicates that exogenous CCK reduces
84
M U E L L E R AND tlSIA() TABLE 1 CURRENT STATUS OF CCK AS A SHORT-TERM SATIETY HORMONE
Criterion
Supporting Evidence
Conflicting Evidence
U-shaped or hyperbolic dose-response relationship at 5 U/kg and above [23, 24, 25, 54, 55, 73].
Lack of clear dose-response relationship at lower doses [29,81].
B. Bioactive component of CCK should suppress food intake.
Suppressions in food intake by octapeptide and caerulein [22, 23, 24, 25, 45, 55, 73].
None
C. Events stimulating or blocking CCK secretion should affect meal size.
Suppressions in food intake by certain substances stimulating the release of CCK [22, 47, 51, 73].
A. Lack of osmotic and non-nutritive bulk controls: Suppression in food intake by bulk, osmotic pressure, and substances not releasing CCK [16, 51, 67, 74].
1. Chemical Specificity A. Dose-related suppression of intake.
B. Lack of appropriate pharmacological interventions. 2. Behavioral Specificity
CCK does not affect water intake or open field behavior in rats [1, 23, 24, 55].
CCK suppresses water intake in mice [441 .
3. Physiological doses of CCK
Suppressions in food intake by exogenous CCK in amounts likely to be secreted during a meal [22, 24, 61, 63, 73, 88].
A. Lack of adequate data about circulating biologically active CCK [27]. B.
4. Natural Satiety A. Behavioral similarity
Mixing of endogenous and exogenous CCK.
Occurrence of grooming, resting and sedation is satiety-related and CCK-related [I, 5, 6, 20, 55].
N one
B. Physiological similarity
Cortical EEG changes and mesenteric vasodilation are satiety-related and CCK-related [20, 65, 68, 82].
None
C. Lack of aversive propertie s
CCK does not produce a conditioned taste aversion [1, 24, 32]. No toxic effects observed after administration of large amounts of CCK [48,73]. CCK does not change pattern of food intake in rats [54].
None (see Addendum)
5. Interaction with deprivation
CCK suppressed food intake in consistent manner to 48 hr deprivation [23, 24, 25, 44, 54, 731.
CCK fails to suppress food intake after 92 hr deprivation [72].
6. Mechanism of suppression
CCK induced changes in evoked responses to auditory stimuli in feeding associated areas [9]. Possible receptors for CCK in the brain [79]. Evidence of CCK-like peptides in the brain [14].
VMH lesions do not alter susceptibility to CCK [451.
7. Generality of CCK effect
CCK reduces food intake in rats [23, 24, 25, 44, 54, 73]. CCK reduces food intake in monkeys [22]. CCK reduces food intake in humans [81]. Elevated CCK levels due to clinical disorders are associated with depressed appetites [30, 61, 84].
CCK has nonspecific effects on intake in mice [44]. CCK does not reduce food intake in rats [26]. CCK can increase food intake or have no effect on food intake in humans [29, 81]. Most studies of food intake in humans suggest regulation based on volume [74, 75,
851.
CHOLECYSTOKININ AND SHORT-TERM SATIETY intake in rhesus monkeys [22] and this report is supported by earlier reports of suppressions of intake induced by substances stimulating the release of endogenous CCK. In humans exogenous CCK has been reported to either be without effect, to increase or to decrease food intake [29,811. However, certain anomalies in appetite associated with clinical disorders suggest that CCK may be important in controlling food intake in humans. Patients experiencing deficiencies of pancreatic exocrine secretion have very high levels of circulating CCK [30]. Apparently, a high threshold of CCK is required to break the inhibition to pancreatic secretion [87]. Very high levels of circulating CCK have also been found in the duodenal ulcer patient [61] and in association with chronic consumption of alcohol [84]. The CCK hypothesis offers a simple explanation of the suppressed appetites accompanying these disorders. Experimental studies exploring the regulation of food intake in humans, however, have generally been inconsistent with the CCK hypothesis - pointing instead to regulation based upon intragastric volume [77]. On a short-term basis humans are unable to compensate for severe dilution of the diet during intragastric feeding [39,75] and instead they accurately compensate for volume. On the long-term basis only about half of the subjects can learn adequate compensation [75]. Although preloads suppress food intake, the suppression is insufficient for proper regulation. Furthermore, no stomach emptying was found to occur during the suppressive effect [85]. The CCK hypothesis would have predicted rapid stomach clearance with the subsequent release of CCK in amounts proportional to the number of CCK receptors filled. A highly diluted diet, therefore, would not be expected to stimulate the release of sufficient CCK to terminate the meal. In addition, protein has been reported to be the most effective stimulus in inducing satiety in humans [8] while fat is believed to be the most effective releasor of CCK [40,88]. The possibility remains that other satiety mechanisms - an osmotic or neural-glucose mechanism - may mask the CCK effect in humans. In this case, however, CCK may be relegated to a small role in the control of food intake in humans. CONCLUSION The status of CCK with respect to the proposed criteria is summarized in Table 1. Although the evidence is suggestive it is far from conclusive. Particularly important are the lack of adequate data about the physiological nature of the doses required to inhibit food intake, the failure to establish the chemical specificity of the effect, and the contradictory results in humans. The proposed criteria suggest that satiety can be precisely defined as a complex series of events including: the deceleration and eventual termination of ingestive
85 responses; the regular occurrence of particular noningestive responses (such as resting and grooming in rats) which may be species-specific or culture-specific; particular physiological responses (such as cortical EEG changes and mesenteric vasodilation); and hedonic events or events which may possess reinforcing properties (such as feelings of warmth and cheerfulness reported by humans). We are encouraged that CCK can induce several of these responses. Although we have concentrated on the possible satietyinducing effects of a single gastrointestinal hormone, CCK, such a complex behavior as satiety is unlikely to be the function of a single stimulus. Several other stimuli which may also be important in the physiological aspects of satiety induction have already been mentioned: the possibility of a glucose sensitive mechanism mediated by the vagus nerve, and the role of bulk and osmotic pressure. In addition fat-related substances in blood [43,86], some nutrient-sensitive cells in the stomach [ 13], caloric sensitive mechanisms in the circulation [56], and even the sweet taste itself [37] may serve to regulate food intake. Once the satiety-inducing stimuli have been identified, their interaction must be determined. Satiety has been suggested to be a conditioned response with CCK and possibly glucose and bulk functioning as internal discriminative stimuli [70,80]. We agree that CCK may play a primary role, particularly in view of the recent research relating CCK and the hypothalamus. To date research on the behavioral effects of CCK have shed some light on its mechanism. The receptors are apparently of the same type as those involved in pancreatic secretion and contraction of the gallbladder. Its actions are apparently more complicated than a simple reflex; the mode of action is apparently hormonal with no neural links. CCK-like peptides have been found in the brain. The similarities of the hunger and thirst systems are striking; both have two basic mechanisms operating. Two mechanisms in the hunger system are: a neuronal mechanism involving duodenal sugar and the vagus nerve [57] and a hormonal mechanism involving duodenal fat, protein, and CCK. In the thirst system, a neuronal mechanism with osmoreceptors located in the lateral preoptic area responds to the hyperosmotic state of the extracellular body fluid to initiate activity in the water intake system, and the hormonal mechanism involving renin-angiotensin secretion which was mentioned previously [18]. These two mechanisms operate independently, as may the two mechanisms in food intake. ADDENDUM After preparation of this manuscrpit, we became aware of a recent report of bait shyness produced by 40 U/kg of CCK. The experimental animals reduced intake from 10.3 ml to 9.6 ml after treatment while the control animals increased intake from 10.3 ml to 14 ml after treatment [12]. Apparently this issue of the toxic-aversive effect of CCK has to be further investigated in terms of CCK doses.
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Current Status of Cholecystokinin as a Short-Term Satiety Hormone K A T H Y R N E M U E L L E R AND SIGMUND HSIAO 1
Department o f Psychology, University o f Arizona, Tucson, A Z 85721 (Received 25 August 1977) MUELLER, K. AND S. HSIAO. Currentstatus of cholecystokinin as a short-term satiety hormone. NEUROSCI. BIOBEHAV. REV. 2(2) 79-87, 1978. - Cholecystokinin (CCK) is a putative short-term satiety hormone which may regulate meal size and the intermeal interval. A series of criteria are proposed to determine whether CCK can be regarded as a short-term satiety hormone. The available data indicate that the CCK-induced inhibition of food intake may not be chemicaUy specific (to CCK) but is behaviorally specific (to ingestive responses). The physiological nature of the necessary doses is unclear. The effects of CCK seem to simulate natural satiety, and these effects appear to be consistent across wide ranges of nutritional deficit. The mechanism of the effect may involve the feeding-associated areas of the brain. The effect has been shown in rats and monkeys, but data from humans have been inconsistent. CCK appears to be a promising factor which may participate in natural satiety along with many other factors. Cholecystokinin
Satiety
Specificity
Dose levels
IN order to maintain an adequate nutritional and energy balance, an organism must regulate energy outflow, energy inflow and potential energy. Energy inflow should match energy outflow; potential energy serves to gap the time differential between the discontinuous inflow and the continuous (obligatory) and episodic outflow of energy. Regulation of energy inflow is accomplished by consuming discrete meals via adjusting the initiation of a meal, duration of a meal, termination of a meal and intermeal intervals. The events surrounding the initiation of a meal are generally referred to as "hunger-related" or as "hunger variables" and those surrounding the termination of a meal are referred to as "satiety-related" or as "satiety variables." This paper will review the experimental data of the satiety-related effects of an intestinal hormone, cholecystokinin (CCK). CCK is a polypeptide hormone existing in two molecular forms, one that contains 33 and another that contains 39 amino acid residues [62]. Its release is stimulated by certain properties of food chyme: hydrogen ions, protein (and amino acids), fat, and magnesium and calcium ions [33, 40, 62]. CCK-secreting cells have recently been identified in the duodenum, jejunum, and ileum but the secretory activity decreases caudally [15,60]. CCK has been classically known to stimulate contraction of the gallbladder and secretion of pancreatic enzymes [40]. It also elicits bicarbonate and insulin release from the pancreas, stimulates intestinal motility, contracts the quiet stomach and pyloric sphincter, stimulates secretion of Brunner's glands and blood flow in the superior mesenteric artery, but decreases systemic arterial pressure. It stimulates
Hypothalamus
pancreatic growth and inhibits contraction of the lower esophagial sphincter, the sphincter of Oddi, absorption of fluid, sodium, potassium, and chloride from the jejunum and ileum, and motility of the active stomach [40,61]. To this list of effects we hope to add satiety induction, but mere demonstration of inhibition of feeding induced by CCK is not sufficient evidence to do so. For some time blood-borne factors have been known to mediate the termination of eating: The food intake of a hungry rat was reduced 50% after its blood had been thoroughly mixed with the blood of a rat which had just finished a meal [ 11 ]. The blood-borne factor disappeared as time elapsed between the end of a meal and the blood transfusion [10]. When a fasted rat received blood from a sated rat, its cortical EEG pattern was altered to that of a sated rat [65]. The blood-borne factor formed in relation to food intake was assumed to promote satiety and when it fell below a critical level the animal initiated its next meal [101. An early study indicated a gut " h o r m o n e , " enterogastrone, inhibited eating in fasted mice; two other digestive hormones, secretin and pancreatic glucagon, did not inhibit eating [66]. The gastrointestinal source of the satiety factor was further suggested by two observations: Duodenal infusions of liquid diet or osmotically potent substances decreased food intake during normal meals and during sham feeding (in which contents of the stomach were allowed to drain through a fistula) [47,74] ; thus the presence of food in the small intestine, in absence of stomach accumulation of food, was sufficient to inhibit feeding in rats [73]. Administration of CCK during sham
1Address reprint requests to S. Hsiao, Department of Psychology, University of Arizona, Tucson, AZ 85721, U.S.A. This publication is supported by NIH Institutional Grant of the University of Arizona. C o p y r i g h t © 1978 A N K H O
I n t e r n a t i o n a l Inc.--0149-7634/78/0202-0079501.05/0
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MUELLFR ANI) HSIAO
feeding mimicked the effects of duodenal infusions of food; sham feeding was inhibited and several responses characteristic of satiety were displayed [25 ]. A "CCK satiety hypothesis" began to be advanced: As food chyme leaves the stomach some of its chemical properties stimulate the CCK-secreting cells in the small intestines and blood-borne CCK interacts with the ventromedial hypothalamus to induce satiety. Presumably, once the concentration of the blood-borne CCK falls below a critical level, eating is resumed. To determine the validity of the CCK hypothesis, several criteria are proposed and the related experimental evidence is discussed. THE CRITERIA TO ADOPT THE CCK HYPOTHESIS 1. The CCK-induced inhibition of food intake should be chemically specific: (a) CCK should suppress food intake in a dose-related manner; (b) Molecules sharing the bioactive component of CCK should also inhibit food intake. Similar concentrations of molecules lacking the bioactive component should be ineffective in suppressing food intake; (c) Events stimulating or blocking endogenous CCK secretion should also affect food intake by either quickening or delaying the termination of a meal. 2. The CCK-induced inhibition of food intake should be behaviorally specific; only the occurrence of ingestive responses should be altered. 3. Experimental doses of CCK which inhibit food intake should be within the physiological range of a species. 4. The CCK-induced inhibition of food intake should be similar to satiety occurring after a free feeding meal: (a) Behaviors following the administration of CCK and the consumption of a free feeding meal should be similar; (b) Physiological responses following the termination of a free feeding meal should occur following the administration of exogenous CCK; (c)CCK, like satiety, should induce a state of affairs which an organism does not avoid and which an organism may even perform behaviors to attain or prolong. 5. The CCK-induced inhibition of food intake should interact in a consistent and adaptive manner with the nutritional deficit of an organism 6. The mechanism of the CCK-induced inhibition of food intake should be experimentally verifiable. 7. The CCK-induced inhibition of food intake should be consistent among those species possessing similar dietary habits and gastrointestinal systems. SUPPORTING AND CONFLICTING EVIDENCE CCK and Dose Dependence 1A. CCK should suppress food intake in a dose-related manner. The degree of suppression of intake should be directly related to the amount of CCK administered or secreted. Assuming the absence of competing behavioral effects of the hormone, the shape of the dose-response relationship has implications for the mechanism of the CCK-suppressive effect. A U-shaped curve may indicate the existence of low affinity inhibitory receptor sites [49] ; a linear dose-response relationship may indicate a neural component; a hyperbolic curve may indicate the direct action of CCK on a limited number of receptors. Most studies in rats have revealed a consistent doseresponse relationship. A positively increasing dose-related suppression of food intake has been consistently reported from 5 to 80 Ivy dog units/kg body weight (the Ivy dog
unit is defined as that amount of dry material which when dissolved in normal saline solution and injected intravenously, during 10 to 15 sec into an anesthetized (tog weighing about 15 kg, results in a more or less immediate ( 1 to 3 rain) rise in intragallbladder pressure of 1 cm of bile [40]), [23, 24, 25, 45, 54, 55, 731. However, 100 Ivy dog units/kg (U/kg) has been reported ineffective in suppressing intake [45]. Thus the dose response curve of CCK in suppressing food intake may be U-shaped as il is in its effect on gastric secretion [7811. In other studies doses of 100 U/kg and above have been reported as more effective than 40 U/kg [73]. However, these data were reported for rats with open gastric fistulas which require doses approximately four times higher than the doses necessary t~ produce equivalent responses in intact rats [251. Therefore, the concept of a U-shaped dose-response cnrve has no[ necessarily been contradicted. Research with humans has failed lo reveal a consistent dose-response relationship. Although rapid intravenous injection of 0.5 U/kg reduces food intake [81], stow intravenous infusions of 3 U/kg have been reported to be ineffective [29] or to increase intake [81 ]. Either the route of administration or the dose may be important in increasing intake -- whether the nature of endogenous hormone release resembles a slow infusion or rapid injection is not known [49]. Dose-related suppressions of food intake have been reported only for relatively high, nonphysiological doses. At lower doses, the relationship between the amount of CCK administered and the degree of suppression of intake is unclear. Evidence for this criterion is therefore inconclusive. Molecular Bioactivity - Structural Specff)cit.v lB. Molecules sharing the bioactive component o f CCK should inhibit food intake. Similar concentrations o f molecules lacking the bioactive component should be ineffective in suppresing food intake. The biologically active portion of the CCK molecule is the C-terminal octapeptide. The sulfonated tyrosine-7 is essential for biological activity; the nonsulfonated molecule is relatively ineffective in contracting the gallbladder [42,58]. A synthetic octapeptide (Sincalide, Squibb) has been shown to be extremely effective in suppressing food intake [22, 24, 45]. Caerulein, a decapeptide isolated from the skin of an Australian frog (Hyla caerulea) shares the common C-terminal octapeptide with CCK, except for an exchange of threonine in caerulein for methionine in CCK. Caerulein is also effective in reducing food intake [79]. On the other hand secretin and pancreatic glucagon [51] which are gastrointestinal hormones but do not share the common C-terminal octapeptide with CCK are not effective in altering food intake [66,73]. Although pentagastrin can inhibit food intake, the doses required are 80 times the dose required for eliciting maximal gastric acid output in the rat and therefore may be the result of a toxic effect. Doses of CCK required to inhibit food intake are within the range for eliciting maximal gastric acid output and maximal pancreatic secretion [73]. It should be mentioned, however, that under certain conditions glucagon can decrease food intake [50] but the mechanism appears to be different than that of CCK. Glucagon fails to suppress feeding when hepatic glycogen has been depleted by moderate fasting [50,66] but CCK is equally effective in suppressing food intake during fairly severe fasting [541.
CHOLECYSTOKININ AND SHORT-TERM SATIETY The suppression of food intake induced by glucagon is probably a result of its various metabolic effects, rather than a result of any direct satiety-inducing properties [50]. The duration of the suppressive effect of CCK and its analogs has not been investigated, but the duration may be shorter for the octapeptide and caerulein than for the parent CCK molecule. The half-life of the parent molecule has been estimated at between 2.5 and 3 min [59, 61, 64, 83], 5 to 7 min [30] with radioimmunoassay or at 1 0 - 1 5 m i n [5] with bioassay in humans or cats. The biological effects of the parent molecule should last longer than those of its shorter analogs since as long as the C-terminal octapeptide is intact the fragment should remain active. Several of the CCK peptides and caerulein are far more potent on a molar basis than the parent molecule [40]. This probably is a function of molecular size, shape and resulting hormone receptor interactions. While many studies could be done to further clarify hormone receptor interactions for satiety induction and the half-life of the CCK analogs, this criterion of the effect of the C-terminal octapeptide of CCK on food intake can be regarded as fulfilled. Thus, CCK receptors for the inhibition of food intake are similar to those in gallbladder.
Stimulation and Blockade o f CCK 1C. Events stimulating or blocking endogenous CCK secretion should also affect food intake by either quickening or delaying the termination o f a meal. Events stimulating the release of CCK should decrease food intake by quickening the termination of a meal. As indicated above, the release of CCK is stimulated by hydrogen ions, fatty acids, peptides or certain amino acids, magnesium and calcium ions. Very large loads of glucose may also stimulate the secretion of CCK but not ordinary amounts [49,88]. However, apart from any particular chemical composition or nutritive value of the ingestia, its osmotic potency has often been referred to as a short-term regulator of food intake [67,70] and therefore tends to confound the CCK hypothesis. Availability of liquid and the osmolality of liquid drank are known to influence amount of food consumed or intake-related responses [31, 34, 36] and blood osmolality can determine the amount and the time of food intake [41,69]. However, the amount of NaC1 in food has been shown not to influence the intake amount even when no liquid is present [34]. Under free feeding conditions, with or without water available, the contents of the stomach after consumption of dry food are diluted by absorption of body fluids [46] such that chyme entering the intestines tends toward isotonicity. Thus, under free feeding conditions, though the osmotic property of the ingesta influences gastric emptying [38], an intestinal osmotic factor per se should not be important in the short-term regulation of meal size in the intact animal [16]. Studies employing direct duodenal infusions should be carefully examined for the possible introduction of "unnatural" osmotic effects. Another factor which may be important in the shortterm regulation of food intake is the presence of (nonnutritive) bulk in the gastrointestinal system. Isotonic bulk has been reported to inhibit food intake in rats [16, 70, 71 ]. However, stomach clearance of the bulk was not determined, and the results may have been due to artificial stomach distention resulting from abnormal clearance of the bulk.
81 Keeping in mind the possible effects of osmolality and bulk, the effects of substances stimulating the release of CCK can be evaluated. Intragastric loads of liquid diet [74] and wet mash [70] inhibit food intake in rats. Liquid diets have been reported to enter the intestines rapidly [3] to stimulate CCK-secreting cells and we have confirmed these observations in our lab. However, this rapid stomach clearance again raises the possibility that at least the initial inhibition of food intake may be due to osmotic, rather than chemical properties of the ingestia. The relative importance of the intestinal factor, the general osmotic factor and the bulk effect on regulation of food intake is not clear, however, these effects may occur in a certain time sequence. Intragastric infusions of phenylalanine (an amino acid) and casein (a phosphoprotein) limit meal size in monkeys [22,51]. Isovolumetric infusions of NaCI or the D-isomer of phenylalanine (which is generally without any biological effectiveness) fail to produce any changes in intake patterns [22]. Chemical properties rather than volume, therefore, appear important in the inhibition of food intake. Pharmacological agents affecting the release of CCK have not been examined for their effects on food intake. Administration of veratrine, which has been reported to potentiate the effects of CCK [5], should suppress food intake to an even greater degree than the sole introduction of those substances which can stimulate the release of CCK. The secretion of CCK is blocked by hexamethonium [5], oxethazaine [ 19], and other local anesthetics [ 19], but the effects of these agents on food intake have not been reported. Delaying termination of a meal by blocking the secretion of CCK is likely to be a difficult task. In addition to being hyperosmotic, most foods contain sugars, which would be expected to activate the neurally mediated glucose mechanism of satiety [57]. This glucose mechanism may operate independently of the CCK mechanism. Therefore one may expect a normal meal duration even in the absence of CCK. Because food intake can be suppressed by osmotic properties or nonnutritive bulk, and because the effects of the appropriate pharmacological interventions have not been reported, this criterion has not been supported.
Behavioral Specificity 2. The CCK-induced inhibition o f food intake shouM be behaviorally specific; only the occurrence o f ingestive responses should be altered. CCK should suppress behaviors selectively, i.e., it should affect food intake directly without activating a diffuse behavioral system. This is a particularly important criterion in view of the tranquilizing effect of CCK (see discussion of Criterion 4). Most studies addressing this criterion have employed rats drinking in response to water deprivation. The assumption is that if the CCK-induced suppression in intake were due to a general tranquilizing effect, water intake would be suppressed by CCK as well. Although a suppression in water intake was reported in mice [44], in rats exogenous CCK seems to be without effect or to increase water intake very slightly [24,33]. These studies, however, are not strictly comparable to those reporting the effects of CCK on food intake since rats tested for water intake were generally deprived of water far longer than those tested for food intake. Recently exogenous CCK was reported to reduce
82 liquid diet in 23.5 hr water-deprived rats without affecting water intake under the same conditions [55]. Sweetness in ingesta is thought to be a food-cue [52], thus apparently when ingesting water the brain interprets CCK as an irrelevant signal, so that changes in intake do not occur. Unpublished data [ 1 ] indicates that open field behavior is not affected by CCK. Since drinking water in response to water deprivation is so behaviorally similar to drinking liquid diet in response to food deprivation and since the former is not suppressed by CCK while the latter is, the likelihood of other less similar behaviors which may have many controlling factors being suppressed by CCK is very low. The effects of CCK appear to fulfill this criterion. Physiological vs Pharmacological Dose 3. Experimental doses o f CCK which inhibit food intake should be within the physiological range o f a species. The amount of exogenous CCK necessary to suppress food intake should be approximately equal to the a m o u n t of endogenous CCK released in response to a meal. Amounts of CCK circulating in the blood have been estimated by radioimmunoassay at 1.4 ng/ml in fasting hogs [ 1 7 ] ; at 0.9 ng/ml [63], 0.7 ng/ml [61], and 0.025 ng/ml [30] in fasting humans; at 119 ng/ml in dogs [63]. Amounts of endogenous CCK circulating postprandially have been estimated at 12 ng/ml [17] and 3 ng/ml [28] in hogs; at 0 . 9 1 2 n g / m l [61], 9 . 1 n g / m l [63] and up to 300 ng/ml [88] in humans. In adult humans after ingestion of one pint of milk serum CCK is raised from a basal level of 25.8 _+4.3 (SEM) pg/ml (range 5 to 80) to about 16 ng/ml within 30 min postprandially [30]. In cats perfusing the intestinal lumen with weak HC1, emulsified fat, long and short chain fatty acids, or 10% peptone results in the release of CCK of up to 5 to 10 milli Ivy dog units/ml serum within 5 to 10 rain as determined by bioassay [5]. The variability of the estimates may be due to different techniques employed as well as to different substances ingested, as certain stimuli are known to be more effective than others in stimulating the secretion of CCK [40]. The effects of CCK on the food intake of dogs, hogs, or cats have not been reported. In humans only one instance of CCK-induced satiety has been reported -~ at 0.5 Ivy dog units/kg [81 ] or approximately 13 ng/ml. If one assumes that rats and monkeys secrete proportional amounts of CCK in response to a meal, amounts of exogenous CCK necessary to induce satiety appear to be within physiological levels. To date the lowest dose of exogenous CCK which has been reported to induce satiety in rats is 2.5 U/kg [24] or approximately 65 ng/ml, and in monkeys is 5 U/kg [22] or approximately 130 ng/ml. However, before drawing conclusions, a cautionary note is advised. In most feeding studies cited above, exogenous CCK was allowed to mix with endogenous CCK (stimulated by passage of food chyme into the intestines) so that the total amount of blood-borne CCK may have surpassed physiological levels. In addition, estimates of circulating CCK reviewed above are limited in indicating the amount of biologically active portions of the hormone in circulation [27]. Any fragment of the molecule containing an intact octapeptide sequence retains its biological activity and such fragments may not be identified by radioimmunoassay techniques. In the same manner these techniques may identify as CCK those molecular fragments with a distorted
MUELLt:R AND HSIA(~ octapeptide sequence which actually posses~ no biologicai activity. Since little is known about the catabolism of CCK. these difficulties may lead either to intlatcd ,)~ reduced estimates of circulating biologically active C('K. Most studies in rats have employed rapid intraperitoneal injections of CCK, therefore actual blood levels of CCK induced experimentally are unknown. Other studies in humans and monkeys have employed either slow or rapid intravenous infusion. In this case although initial plasma levels are known, one must take into account rates of catabolism and tissue uptake of CCK to accurately estimate amounts of circulating CCK [28]. Although the evidence is suggestive, firm conclusions of the effectiveness of physiological levels of CCK in suppressing food intake cannot be drawn. Behavioral Similarity 4A. Behaviors fi)llowing the administration oJ CCK and the consumption o f a free feeding meal should be similar. Relatively little information is available about satietyrelated behaviors. Rats maintained in semi-natural conditions engage in exploratory and sampling behaviors at the close of a meal [4]. In more confined conditions grooming and resting are associated with termination of a meal [1,6]; however, under these conditions very few behavioral alternatives are available. Thus, only the occurrence of sleep or resting behaviorally distinguished between the cessation of feeding due to satiety and that due to quinine adulteration of the diet [ I ]. In cats, sedation also appears to be satiety-related [20]. Administration of exogenous CCK during feeding not only reduced the amount of food eaten, but was followed by grooming and resting in rats [1]. Sedation has been noticed in cats following administration of exogenous CCK or intraduodenal milk and corn oil which elicit the release of endogenous CCK [20]. Further work is clearly necessary to establish satiety-related behaviors in other species. Physiological Response Patterns 4B. Physiological responses following termination oj a free .feeding meal shouM occur following the administration o f exogenous CCK. The cortical EEG pattern appears to be a particularly good indicator of satiety. Satiety is accompanied by consistent changes in the EEG [20, 65, 82] and prolonged EEG changes occur only when both oral and gastric receptors are stimulated [82] just as "intestinal satiety" depends upon both oral and gastrointestinal stimulation [2]. Changes in cortical EEG, appearance of the eye movement and increase in superior mesenteric blood flow have been observed following a meal [20,68] as well as following the administration of exogenous CCK [20]. Appearance of loose stools is also reported following CCK administration in man [81]. We also observed an increased occurrence of defecation in rats following CCK injections in our lab. Whether the increased bowel movement is a part of natural satiety or it represents a sign of supra-physiological dose level of CCK is not clear. Natural Satiety and CCK 4C. CCK, like satiety, should induce a sate o] ~affairs which an organism does not avoid, and which an organism may even perform behaviors to attain or prolong. People often associate satiety with warmth or satisfaction [53]
CHOLECYSTOKININ AND SHORT-TERM SATIETY and we are tempted to speculate that satiety (or CCK) may function as a powerful reinforcer in an operant learning situation. Although the reinforcing properties of CCK have not been investigated, we can conclude that CCK does not function as an aversive stimulus. Attempts to produce a conditioned taste aversion with CCK or its octapeptide have been unsuccessful (but see note) [1, 24, 32]. No toxic effects whatsoever have been observed in rats or mice after eight months of CCK injections two or three times weekly [73] or after single administration of doses up to 2000 U/kg [48]. Some side effects have been noted in humans at 1 U/kg but they were not severe and did not appear to be responsible for changes in food consumption [81]. Administration of exogenous CCK does not change the pattern of food intake in rats, but only reduces the amount consumed [54]. Aversion can therefore be eliminated as the cause of the CCK-suppressive effect.
Generality over Deprivation Levels 5. The CCK-induced inhibition o f food intake should interact in a consistent and adaptive manner with the nutritional deficit o f an organism. CCK should interact in a consistent and adaptive manner with the state of nutritional deficit of the organism with the nature of the interaction indicating something about the nature of the CCKsuppressive effect. The effects of CCK on food intake have been examined at varying levels of deprivation - 5 hr [54], 5.5 hr [23, 24, 45], 17 hr [1, 24, 25, 73], 8 hr [44], 19 hr [54], 4 8 h r [54], and 9 2 h r [72] - and suppressions in intake have occurred at each level except 92 hr. The CCK-suppressive effect appears to remain constant across wide variations in food deprivation levels. For example, 40 U/kg suppresses intake by approximately 50% at 5, 19 and 48 hr deprivation [54]. This result is suggestive of some interaction with nutritional deficit; if no interaction were present, intake would be expected to cease at a given time or given volume rather than at a given percent of control intake. A simple chemical trigger reflex model of the CCK effect on food intake would therefore seem inappropriate. The failure of CCK to suppress food intake at 92 hr deprivation suggests the existence of a second satiety mechanism, a "fail-safe" mechanism. Such an arrangement would be clearly adaptive. It would inhibit excessive caloric intake during moderate deprivation but facilitate larger caloric loads during life-threatening deprivation. Such a shift in locus of control would require a physiological change occuring between 48 and 92 hr deprivation which could trigger a fail-safe mechanism. Transitions from utilization of blood glucose in the free feeding animal to the utilization of body fat and finally body protein during severe deprivation [7] may provide the necessary physiological signal. A shift in locus of control of meal size is not without precedent. A neural short-term satiety mechanism has recently been demonstrated which is activated by glucose and mediated by the vagus nerve. This mechanism is duodenally based under free feeding conditions but hepatic-portally based after 22 hr deprivation [57]. Similarly, during severe deprivation the CCK mechanism may be replaced by some other satiety factor - a "fail-safe" factor. The known effects of CCK fulfill this criterion.
83
Mechanisms o f CCK-Satiety Effect 6. The mechanisms o f the CCK-induced inhibition o f food intake should be experimentally verifiable. CCK may suppress food intake as a result of its classical physiological roles. For example, stomach emptying may be inhibited by CCK to the degree that ingested food accumulates in the stomach causing stomach distention and a suppression of feeding. However, as indicated previously, CCK can induce satiety in the absence of gastric distention. CCK may suppress food intake because of its sedative properties. But, as previously noted, sedation seems to be a part of satiety rather than a cause. In addition a recent series of e x p e r i m e n t s suggested a relationship between the traditional hypothalamic feeding and satiety areas and the CCK satiety effect. Changes in evoked responses to auditory stimuli were recorded from rats following administration of exogenous CCK. These changes were found only after injection of CCK and specifically from the ventromedial hypothalamic (VMH) area. In addition, the time course of these changes appeared similar to the time course of satiety [9]. Lesions in the VMH produced a reduced sensitivity to caerulein (1 ug/kg), but lesions in the lateral hypothalamus (LH) heightened sensitivity. Microinjection of caerulein into the VMH, but not into the LH, limited feeding. Tritiated caerulein was selectively bound to tissue in the VMH [79]. These results indicate possible receptor sites of CCK in the brain and place CCK in a well-known Stellar schema of the control of feeding [76,77] as a chemical signal for regulating the activity of the hypothalamic feeding areas. However, in another recent study lesions in the VMH were reported to have no effect at all on the sensitivity to the CCK-induced suppression of food intake [45]. The similarity between this CCK mechanism of satiety and the angiotensin-related mechanism of thirst is striking. Angiotensin II has been shown clearly to be a signal of thirst which appears to have the subfornical organ as its receptor site [18,35]. How a polypeptide, like Angiotensin II, could enter the brain to signal thirst has been a problem, but renin has been found within the brain itself [21]. Similarly CCK-like peptides have also recently been found in the brain [ 14]. The functions and sources of these brain-originated compounds are not understood at this time. Species Generality 7. The CCK-induced inhibition o f food intake should be consistent among those species possessing similar dietary habits and gastrointestinal systems. CCK should be important in regulating meal size in several mammalian species. Although the satiety-like effects of enterogastrone were first discovered in mice [66], the only report of the effects of exogenous CCK on the food intake of mice is inconclusive [44]. Most studies employing exogenous CCK have been limited to rats. The single failure to find a CCK-induced suppression of intake in rats [26] was probably due to the low dose employed or to the slow rate of administration. Although the effects of exogenous CCK on the meal size of cats have not been reported, available evidence suggests that endogenous CCK seems to evoke satiety-related responses [20, 68, 82]. As indicated earlier, however, this evidence cannot be accepted as conclusive. A single study indicates that exogenous CCK reduces
84
M U E L L E R AND tlSIA() TABLE 1 CURRENT STATUS OF CCK AS A SHORT-TERM SATIETY HORMONE
Criterion
Supporting Evidence
Conflicting Evidence
U-shaped or hyperbolic dose-response relationship at 5 U/kg and above [23, 24, 25, 54, 55, 73].
Lack of clear dose-response relationship at lower doses [29,81].
B. Bioactive component of CCK should suppress food intake.
Suppressions in food intake by octapeptide and caerulein [22, 23, 24, 25, 45, 55, 73].
None
C. Events stimulating or blocking CCK secretion should affect meal size.
Suppressions in food intake by certain substances stimulating the release of CCK [22, 47, 51, 73].
A. Lack of osmotic and non-nutritive bulk controls: Suppression in food intake by bulk, osmotic pressure, and substances not releasing CCK [16, 51, 67, 74].
1. Chemical Specificity A. Dose-related suppression of intake.
B. Lack of appropriate pharmacological interventions. 2. Behavioral Specificity
CCK does not affect water intake or open field behavior in rats [1, 23, 24, 55].
CCK suppresses water intake in mice [441 .
3. Physiological doses of CCK
Suppressions in food intake by exogenous CCK in amounts likely to be secreted during a meal [22, 24, 61, 63, 73, 88].
A. Lack of adequate data about circulating biologically active CCK [27]. B.
4. Natural Satiety A. Behavioral similarity
Mixing of endogenous and exogenous CCK.
Occurrence of grooming, resting and sedation is satiety-related and CCK-related [I, 5, 6, 20, 55].
N one
B. Physiological similarity
Cortical EEG changes and mesenteric vasodilation are satiety-related and CCK-related [20, 65, 68, 82].
None
C. Lack of aversive propertie s
CCK does not produce a conditioned taste aversion [1, 24, 32]. No toxic effects observed after administration of large amounts of CCK [48,73]. CCK does not change pattern of food intake in rats [54].
None (see Addendum)
5. Interaction with deprivation
CCK suppressed food intake in consistent manner to 48 hr deprivation [23, 24, 25, 44, 54, 731.
CCK fails to suppress food intake after 92 hr deprivation [72].
6. Mechanism of suppression
CCK induced changes in evoked responses to auditory stimuli in feeding associated areas [9]. Possible receptors for CCK in the brain [79]. Evidence of CCK-like peptides in the brain [14].
VMH lesions do not alter susceptibility to CCK [451.
7. Generality of CCK effect
CCK reduces food intake in rats [23, 24, 25, 44, 54, 73]. CCK reduces food intake in monkeys [22]. CCK reduces food intake in humans [81]. Elevated CCK levels due to clinical disorders are associated with depressed appetites [30, 61, 84].
CCK has nonspecific effects on intake in mice [44]. CCK does not reduce food intake in rats [26]. CCK can increase food intake or have no effect on food intake in humans [29, 81]. Most studies of food intake in humans suggest regulation based on volume [74, 75,
851.
CHOLECYSTOKININ AND SHORT-TERM SATIETY intake in rhesus monkeys [22] and this report is supported by earlier reports of suppressions of intake induced by substances stimulating the release of endogenous CCK. In humans exogenous CCK has been reported to either be without effect, to increase or to decrease food intake [29,811. However, certain anomalies in appetite associated with clinical disorders suggest that CCK may be important in controlling food intake in humans. Patients experiencing deficiencies of pancreatic exocrine secretion have very high levels of circulating CCK [30]. Apparently, a high threshold of CCK is required to break the inhibition to pancreatic secretion [87]. Very high levels of circulating CCK have also been found in the duodenal ulcer patient [61] and in association with chronic consumption of alcohol [84]. The CCK hypothesis offers a simple explanation of the suppressed appetites accompanying these disorders. Experimental studies exploring the regulation of food intake in humans, however, have generally been inconsistent with the CCK hypothesis - pointing instead to regulation based upon intragastric volume [77]. On a short-term basis humans are unable to compensate for severe dilution of the diet during intragastric feeding [39,75] and instead they accurately compensate for volume. On the long-term basis only about half of the subjects can learn adequate compensation [75]. Although preloads suppress food intake, the suppression is insufficient for proper regulation. Furthermore, no stomach emptying was found to occur during the suppressive effect [85]. The CCK hypothesis would have predicted rapid stomach clearance with the subsequent release of CCK in amounts proportional to the number of CCK receptors filled. A highly diluted diet, therefore, would not be expected to stimulate the release of sufficient CCK to terminate the meal. In addition, protein has been reported to be the most effective stimulus in inducing satiety in humans [8] while fat is believed to be the most effective releasor of CCK [40,88]. The possibility remains that other satiety mechanisms - an osmotic or neural-glucose mechanism - may mask the CCK effect in humans. In this case, however, CCK may be relegated to a small role in the control of food intake in humans. CONCLUSION The status of CCK with respect to the proposed criteria is summarized in Table 1. Although the evidence is suggestive it is far from conclusive. Particularly important are the lack of adequate data about the physiological nature of the doses required to inhibit food intake, the failure to establish the chemical specificity of the effect, and the contradictory results in humans. The proposed criteria suggest that satiety can be precisely defined as a complex series of events including: the deceleration and eventual termination of ingestive
85 responses; the regular occurrence of particular noningestive responses (such as resting and grooming in rats) which may be species-specific or culture-specific; particular physiological responses (such as cortical EEG changes and mesenteric vasodilation); and hedonic events or events which may possess reinforcing properties (such as feelings of warmth and cheerfulness reported by humans). We are encouraged that CCK can induce several of these responses. Although we have concentrated on the possible satietyinducing effects of a single gastrointestinal hormone, CCK, such a complex behavior as satiety is unlikely to be the function of a single stimulus. Several other stimuli which may also be important in the physiological aspects of satiety induction have already been mentioned: the possibility of a glucose sensitive mechanism mediated by the vagus nerve, and the role of bulk and osmotic pressure. In addition fat-related substances in blood [43,86], some nutrient-sensitive cells in the stomach [ 13], caloric sensitive mechanisms in the circulation [56], and even the sweet taste itself [37] may serve to regulate food intake. Once the satiety-inducing stimuli have been identified, their interaction must be determined. Satiety has been suggested to be a conditioned response with CCK and possibly glucose and bulk functioning as internal discriminative stimuli [70,80]. We agree that CCK may play a primary role, particularly in view of the recent research relating CCK and the hypothalamus. To date research on the behavioral effects of CCK have shed some light on its mechanism. The receptors are apparently of the same type as those involved in pancreatic secretion and contraction of the gallbladder. Its actions are apparently more complicated than a simple reflex; the mode of action is apparently hormonal with no neural links. CCK-like peptides have been found in the brain. The similarities of the hunger and thirst systems are striking; both have two basic mechanisms operating. Two mechanisms in the hunger system are: a neuronal mechanism involving duodenal sugar and the vagus nerve [57] and a hormonal mechanism involving duodenal fat, protein, and CCK. In the thirst system, a neuronal mechanism with osmoreceptors located in the lateral preoptic area responds to the hyperosmotic state of the extracellular body fluid to initiate activity in the water intake system, and the hormonal mechanism involving renin-angiotensin secretion which was mentioned previously [18]. These two mechanisms operate independently, as may the two mechanisms in food intake. ADDENDUM After preparation of this manuscrpit, we became aware of a recent report of bait shyness produced by 40 U/kg of CCK. The experimental animals reduced intake from 10.3 ml to 9.6 ml after treatment while the control animals increased intake from 10.3 ml to 14 ml after treatment [12]. Apparently this issue of the toxic-aversive effect of CCK has to be further investigated in terms of CCK doses.
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