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The effect of cholecystokinin-pancreozymin on hunger and thirst in mice
BEHAVIORALBIOLOGY,7, 441-444 (1972), Abstract No. I33R
BRIEF COMMUNICATION The Effect of Cholecystokinin-Pancreozymin o n H u n g e r a n d Thirst in Mice HENRY S. KOOPMANS, J. A. DEUTSCH and PATRICIA J. BRANSON University o f California, San Diego La Jolla, California 92037 The gastrointestinal hormone, cholecystokinin-pancreozymin,was injected into food or water-deprived C3H mice. The lowest concentration of CCK-PZ that was effective in reducing food intake also reduced water consumption. A lower dose of CCK-PZ was without effect on either eating or drinking. These findings suggest that CCK-PZ has no specific effect on hunger in mice. Many experiments have shown that neural signals from the mouth, throat and stomach are relatively unimportant in reducing hunger. Hull et al. (1951) prevented food from reaching the stomach of a dog by creating an esophogeal fistula. They found that the dog grossly overate and, thus, was unaffected by neural signals from the mouth and throat. Grossman et al. (1947) observed that dogs with denervated stomachs ate normally and responded to insulin injections by increasing their food intake by the same amount as normal dogs. Harris et aL (1947) also noted that stomach-denervated dogs ate normally and showed that amphetamine injections produced similar reductions of food intake in normal and denervated dogs. Recently, Snowdon (1970) found that vagotomized rats ate normal meals when fed food pellets. When given a liquid diet, the rats ate smaller and more frequent meals, probably because of the more rapid dumping of the stomach contents following vagotomy. The failure to find clear-cut satiating effects of neural signals suggests that the signal terminating hunger may be blood-borne. Steffens (1969) has recently shown that blood glucose levels increase and free fatty acid levels fall within a few minutes after a rat has begun a meal of lab chow. However, these blood changes are not necessarily the signal terminating hunger but may simply coexist with hunger satiety. The absorption of food is accompanied by the release of gastrointestinal hormones. Schally et al. (1967) have demonstrated a reduction of food intake by mice as a result of enterogastrone injection. This paper reports the effects of injecting cholecystokinin-pancre441 Copyright © 1972 by Academic Press, Inc.
442
KOOPMANS, DEUTSCH AND BRANSON
ozymin (CCK-PZ), 1 another gastrointestinal hormone, on the satiation of hunger in mice. The release of CCK-PZ into the blood stream causes an increase in flow of digestive enzymes from the pancreas and, in larger mammals, provokes contraction of the gall bladder. It is released when food, particularly fat, is present in the small intestines. Fara et al. (1969) have recently demonstrated that CCK-PZ increases superior mesenteric blood flow and induces drowsiness in cats. It is conceivable that CCK-PZ would have another central effect: that of reducing hunger. Our experiment shows that CCK-PZ prepared from hog mucosa reduces not only hunger but also thirst and, therefore, can not be considered a specific satiety signal. Eighty naive C3H mice weighing approximately 25 g each were deprived of food for 18 hr and assigned to one of five groups. Each group was injected ip with one of four doses of freshly prepared CCK-PZ in 0.1 ml saline or with 0,1 ml saline alone and allowed to rest for 10 rain so that the injection could take effect. The amount of Metrecal liquid diet ingested was measured for each 15-min period following the presentation of food. Figure 1 presents the results for four different doses measured in Ivy Dog Units (IDU) and for the control. An overall F test on the levels of food intake during the first 15 min is significant (F = 65.3, p < 0.01). Brackets in Fig. 1 indicate standard deviations. A multiple comparison Tukey (a) test shows significant differences Co < 0.01) between the intake in the first 15 min by mice injected with any of the three higher doses of CCK-PZ and the intake by the control mice. Mice injected with 0.4 IDU were not significantly different from control mice. The O Sol/he control
1.25
m 0.4 1.0.U./mouse
u 1 I.DU./moase t, 2.5 1.0.U./mouse x 6 I.O.U./mouse
1.00
i ~ o.r5 ~ 0.50
P ~ -~.
....o
..It----/r-x
~__...~.~_~.~--/
0.25
- 7 "
1 0-15
I 15-30
"
I 50-45
I 45-60
"
I 60-75
--X~
~-~... --, I 75-90
I 90-105
I 105-120
Minutes,
Fig. 1. Mice were injected with four doses of CCK-PZ or with saline. The ordinate indicates the amount of Metrecal consumed during each 15-min period following the presentation of food. Brackets indicate standard deviations.
l w e thank Professor J. E. Jorpes and Dr. V. Mutt, Karolinska Institutet, Stockholm, Sweden, for providing us with the hormone preparation.
EFFECT OF CCK-PZ ON HUNGER AND THIRST
443
data from the 0.4 IDU group was collected only during the first 15 min. As a control for nonspecific effects of injection, 30 C3H mice were deprived of water but not of food for 18 hr. They were then injected ip with either 0.4 IDU or 1 IDU of CCK-PZ in .05 ml saline or with .05 ml saline alone and allowed to rest for 10 rain before water was presented. The overall F test was significant (F = 15.3, p < 0.01). Figure 2 shows that, as in the hunger experiment, the 0.4 IDU dose had no significant effect on consumatory behavior. Our lowest dosage effective in reducing food intake in mice, 1 IDU of CCK-PZ, also significantly inhibits drinking during the first 10 min after water presentation. (Tukey (a) test, p < 0.01). Thus, CCK-PZ does not selectively inhibit eating in mice. o Saline control • 0.4 LD.U/mouse a I I.O.U./mouse
0.50
freshly prepare/ 0.40
0.30
0.20
O.lO
0
A
fi~'\- / \\%__.j° 0-10
10-20
20-30
30-40
40-50
50-60
Minules
Fig. 2. Mice were injected with 0.4 or 1 IDU of freshly prepared CCK-PZ, or with 1 IDU of denatured CCK-PZ or with saline. The ordinate indicates the amount of water consumed during each 10-min period following the presentation of water. Brackets indicate standard deviations.
The lowest effective dose in our experiment is seven times greater per kilogram body weight than the amount of endogenous release of CCK-PZ in a dog after his daily meal (Jonson et al., 1967). SchaUy et al. (1967) report a nearly identical ratio in their enterogastrone experiment. Our data suggest that the lowest effective dose of CCK-PZ is nonphysiological in mice. Physiological levels of CCK-PZ would not be expected to inhibit thirst. Normally, drinking follows the end of a meal and the release of CCK-PZ. Our mice were outwardly healthy and normal when injected with the three lower doses of CCK-PZ. When 6 IDU was injected, restricted movement and piloerection was observed. Thus, eating and drinking behavior can be affected before other changes in behavior or appearance can be readily observed. A second control was run to determine the cause of the injection's inhibitory effect on drinking. Ten C3H mice that were water deprived for 18
444
KOOPMANS, DEUTSCH AND BRANSON
hr were injected with 1 IDU of sterile CCK-PZ that had remained at 4 ° for 3 days before the usual injection procedure. A few hours are usually sufficient time for CCK-PZ decomposition (Jorpes and Mutt, personal communication). Figure 2 shows that the group injected with decomposed CCK-PZ was significantly different (Tukey (a) test, p < 0.01) from the group injected with freshly prepared CCK-PZ but was not significantly different from the control group (p > 0.05). Thus, some decomposable ingredient in the 10% pure hormone preparation produced the inhibitory effect on drinking. CCK-PZ is the only identified decomposable ingredient. Our experiments show that CCK-PZ does not have a specific effect on hunger in mice. The lowest dosage that decreases food intake also affects water consumption. Moreover, we have shown that the reduction of water intake is due to a decomposable ingredient of the hormone preparation. The only identified decomposable ingredient is CCK-PZ. The procedure used in the present experiment is similar to that of an earlier experiment using the gastrointestinal hormone, enterogastrone. Schally et al. (1967) showed that enterogastrone reduced food intake in mice without producing stress symptoms or fever. CCK-PZ has also reduced food intake without producing stress symptoms. Comparative hormone levels for both studies are nearly identical (7 times normal dose for dogs/ kg body wt). However, the drinking control in the present study shows that the effect of CCK-PZ on eating is nonspecific and thus CCK-PZ can not be considered a hunger satiety signal. A similar control is necessary for the enterogastrone study before that data can be conclusively interpreted. REFERENCES Fara, J. W., Rubinstein, E. H., and Sonnenschein, R. R. (1969). Visceral and behavioral responses to intraduodenal fat. Science 166,110. Grossman, M. I., Cummings, C. M., and Ivy, A. C. (1947) The effect of insulin on food intake after vagotomy and sympathectomy. Amer. J. Physiol. 149, 100. Harris, S. C., Ivy, A. C., and Searle, L. M. (1947). The mechanism of amphetamine-induced loss of weight. J. Amer. Med. Ass. 134, 1468. Hull, C. L., Livingston, J. R., Rouse, R. O., and Barker, A. N. (1951). True, sham and esophageal feeding as reinforcements. J. Comp. Physiol. Psych. 44, 236. Jonson, G., Svartengren, G., and Thulin, G. (1967). The effect of cholecystokinin-pancreozymin preparations on hepatic bile output in fasting and digesting dogs. Acta Physiol. Seand. 69, 23. Schally, A. V., Redding, T. W., Lucien, H. W. and Meyer, J. (1967). Enterogastrone inhibits eating in fasted mice. Science 157, 211. Snowdon, C. T. (1970). Gastrointestinal sensory and motor control of food intake. J. Comp. Physiol. Psych. 71, 68. Steffens, A. B. (1969). Bloodglucose and FFA levels relation to the meal pattern in the normal rat and the ventromedial hypothalamic lesioned rat. Physiol. Behav. 4, 215.
BRIEF COMMUNICATION The Effect of Cholecystokinin-Pancreozymin o n H u n g e r a n d Thirst in Mice HENRY S. KOOPMANS, J. A. DEUTSCH and PATRICIA J. BRANSON University o f California, San Diego La Jolla, California 92037 The gastrointestinal hormone, cholecystokinin-pancreozymin,was injected into food or water-deprived C3H mice. The lowest concentration of CCK-PZ that was effective in reducing food intake also reduced water consumption. A lower dose of CCK-PZ was without effect on either eating or drinking. These findings suggest that CCK-PZ has no specific effect on hunger in mice. Many experiments have shown that neural signals from the mouth, throat and stomach are relatively unimportant in reducing hunger. Hull et al. (1951) prevented food from reaching the stomach of a dog by creating an esophogeal fistula. They found that the dog grossly overate and, thus, was unaffected by neural signals from the mouth and throat. Grossman et al. (1947) observed that dogs with denervated stomachs ate normally and responded to insulin injections by increasing their food intake by the same amount as normal dogs. Harris et aL (1947) also noted that stomach-denervated dogs ate normally and showed that amphetamine injections produced similar reductions of food intake in normal and denervated dogs. Recently, Snowdon (1970) found that vagotomized rats ate normal meals when fed food pellets. When given a liquid diet, the rats ate smaller and more frequent meals, probably because of the more rapid dumping of the stomach contents following vagotomy. The failure to find clear-cut satiating effects of neural signals suggests that the signal terminating hunger may be blood-borne. Steffens (1969) has recently shown that blood glucose levels increase and free fatty acid levels fall within a few minutes after a rat has begun a meal of lab chow. However, these blood changes are not necessarily the signal terminating hunger but may simply coexist with hunger satiety. The absorption of food is accompanied by the release of gastrointestinal hormones. Schally et al. (1967) have demonstrated a reduction of food intake by mice as a result of enterogastrone injection. This paper reports the effects of injecting cholecystokinin-pancre441 Copyright © 1972 by Academic Press, Inc.
442
KOOPMANS, DEUTSCH AND BRANSON
ozymin (CCK-PZ), 1 another gastrointestinal hormone, on the satiation of hunger in mice. The release of CCK-PZ into the blood stream causes an increase in flow of digestive enzymes from the pancreas and, in larger mammals, provokes contraction of the gall bladder. It is released when food, particularly fat, is present in the small intestines. Fara et al. (1969) have recently demonstrated that CCK-PZ increases superior mesenteric blood flow and induces drowsiness in cats. It is conceivable that CCK-PZ would have another central effect: that of reducing hunger. Our experiment shows that CCK-PZ prepared from hog mucosa reduces not only hunger but also thirst and, therefore, can not be considered a specific satiety signal. Eighty naive C3H mice weighing approximately 25 g each were deprived of food for 18 hr and assigned to one of five groups. Each group was injected ip with one of four doses of freshly prepared CCK-PZ in 0.1 ml saline or with 0,1 ml saline alone and allowed to rest for 10 rain so that the injection could take effect. The amount of Metrecal liquid diet ingested was measured for each 15-min period following the presentation of food. Figure 1 presents the results for four different doses measured in Ivy Dog Units (IDU) and for the control. An overall F test on the levels of food intake during the first 15 min is significant (F = 65.3, p < 0.01). Brackets in Fig. 1 indicate standard deviations. A multiple comparison Tukey (a) test shows significant differences Co < 0.01) between the intake in the first 15 min by mice injected with any of the three higher doses of CCK-PZ and the intake by the control mice. Mice injected with 0.4 IDU were not significantly different from control mice. The O Sol/he control
1.25
m 0.4 1.0.U./mouse
u 1 I.DU./moase t, 2.5 1.0.U./mouse x 6 I.O.U./mouse
1.00
i ~ o.r5 ~ 0.50
P ~ -~.
....o
..It----/r-x
~__...~.~_~.~--/
0.25
- 7 "
1 0-15
I 15-30
"
I 50-45
I 45-60
"
I 60-75
--X~
~-~... --, I 75-90
I 90-105
I 105-120
Minutes,
Fig. 1. Mice were injected with four doses of CCK-PZ or with saline. The ordinate indicates the amount of Metrecal consumed during each 15-min period following the presentation of food. Brackets indicate standard deviations.
l w e thank Professor J. E. Jorpes and Dr. V. Mutt, Karolinska Institutet, Stockholm, Sweden, for providing us with the hormone preparation.
EFFECT OF CCK-PZ ON HUNGER AND THIRST
443
data from the 0.4 IDU group was collected only during the first 15 min. As a control for nonspecific effects of injection, 30 C3H mice were deprived of water but not of food for 18 hr. They were then injected ip with either 0.4 IDU or 1 IDU of CCK-PZ in .05 ml saline or with .05 ml saline alone and allowed to rest for 10 rain before water was presented. The overall F test was significant (F = 15.3, p < 0.01). Figure 2 shows that, as in the hunger experiment, the 0.4 IDU dose had no significant effect on consumatory behavior. Our lowest dosage effective in reducing food intake in mice, 1 IDU of CCK-PZ, also significantly inhibits drinking during the first 10 min after water presentation. (Tukey (a) test, p < 0.01). Thus, CCK-PZ does not selectively inhibit eating in mice. o Saline control • 0.4 LD.U/mouse a I I.O.U./mouse
0.50
freshly prepare/ 0.40
0.30
0.20
O.lO
0
A
fi~'\- / \\%__.j° 0-10
10-20
20-30
30-40
40-50
50-60
Minules
Fig. 2. Mice were injected with 0.4 or 1 IDU of freshly prepared CCK-PZ, or with 1 IDU of denatured CCK-PZ or with saline. The ordinate indicates the amount of water consumed during each 10-min period following the presentation of water. Brackets indicate standard deviations.
The lowest effective dose in our experiment is seven times greater per kilogram body weight than the amount of endogenous release of CCK-PZ in a dog after his daily meal (Jonson et al., 1967). SchaUy et al. (1967) report a nearly identical ratio in their enterogastrone experiment. Our data suggest that the lowest effective dose of CCK-PZ is nonphysiological in mice. Physiological levels of CCK-PZ would not be expected to inhibit thirst. Normally, drinking follows the end of a meal and the release of CCK-PZ. Our mice were outwardly healthy and normal when injected with the three lower doses of CCK-PZ. When 6 IDU was injected, restricted movement and piloerection was observed. Thus, eating and drinking behavior can be affected before other changes in behavior or appearance can be readily observed. A second control was run to determine the cause of the injection's inhibitory effect on drinking. Ten C3H mice that were water deprived for 18
444
KOOPMANS, DEUTSCH AND BRANSON
hr were injected with 1 IDU of sterile CCK-PZ that had remained at 4 ° for 3 days before the usual injection procedure. A few hours are usually sufficient time for CCK-PZ decomposition (Jorpes and Mutt, personal communication). Figure 2 shows that the group injected with decomposed CCK-PZ was significantly different (Tukey (a) test, p < 0.01) from the group injected with freshly prepared CCK-PZ but was not significantly different from the control group (p > 0.05). Thus, some decomposable ingredient in the 10% pure hormone preparation produced the inhibitory effect on drinking. CCK-PZ is the only identified decomposable ingredient. Our experiments show that CCK-PZ does not have a specific effect on hunger in mice. The lowest dosage that decreases food intake also affects water consumption. Moreover, we have shown that the reduction of water intake is due to a decomposable ingredient of the hormone preparation. The only identified decomposable ingredient is CCK-PZ. The procedure used in the present experiment is similar to that of an earlier experiment using the gastrointestinal hormone, enterogastrone. Schally et al. (1967) showed that enterogastrone reduced food intake in mice without producing stress symptoms or fever. CCK-PZ has also reduced food intake without producing stress symptoms. Comparative hormone levels for both studies are nearly identical (7 times normal dose for dogs/ kg body wt). However, the drinking control in the present study shows that the effect of CCK-PZ on eating is nonspecific and thus CCK-PZ can not be considered a hunger satiety signal. A similar control is necessary for the enterogastrone study before that data can be conclusively interpreted. REFERENCES Fara, J. W., Rubinstein, E. H., and Sonnenschein, R. R. (1969). Visceral and behavioral responses to intraduodenal fat. Science 166,110. Grossman, M. I., Cummings, C. M., and Ivy, A. C. (1947) The effect of insulin on food intake after vagotomy and sympathectomy. Amer. J. Physiol. 149, 100. Harris, S. C., Ivy, A. C., and Searle, L. M. (1947). The mechanism of amphetamine-induced loss of weight. J. Amer. Med. Ass. 134, 1468. Hull, C. L., Livingston, J. R., Rouse, R. O., and Barker, A. N. (1951). True, sham and esophageal feeding as reinforcements. J. Comp. Physiol. Psych. 44, 236. Jonson, G., Svartengren, G., and Thulin, G. (1967). The effect of cholecystokinin-pancreozymin preparations on hepatic bile output in fasting and digesting dogs. Acta Physiol. Seand. 69, 23. Schally, A. V., Redding, T. W., Lucien, H. W. and Meyer, J. (1967). Enterogastrone inhibits eating in fasted mice. Science 157, 211. Snowdon, C. T. (1970). Gastrointestinal sensory and motor control of food intake. J. Comp. Physiol. Psych. 71, 68. Steffens, A. B. (1969). Bloodglucose and FFA levels relation to the meal pattern in the normal rat and the ventromedial hypothalamic lesioned rat. Physiol. Behav. 4, 215.