- Email: [email protected]
Epinephrine inhibits feeding nonspecifically in the rat
Physiology &Behavior, Vol. 40, pp. 109--115.Copyright©PergamonJourllals Ltd., 1987.Printed in the U.S.A.
0031.9384/87$3.00 + .00
Epinephrine Inhibits Feeding Nonspecifically in the Rat VERONICA HINTON, MANUEL ESGUERRA, NAUDER FARHOODY, JENNIFER GRANGER AND NORI GEARY 1
Department of Psychology, Box 28 Schermerhorn Hall, Columbia University, New York, N Y 10027 R e c e i v e d 5 N o v e m b e r 1986 HINTON, V., M. ESGUERRA, N. FARHOODY, J. GRANGER AND N. GEARY. Epinephrine inhibits feeding nonspecifically in the rat. PHYSIOL BEHAV 40(1) 109-115, 1987.--The hypothesis that epinephrine (EPI) and pancreatic glucagon (laG) inhibit feeding by activating a common physiological satiety mechanism was tested by comparing the two agents' behavioral effects. In several tests of specificity, EPI and laG had functionally different inhibitory actions. Intraperitoneal injection of 6.25-50/zg/kg EPI and 100-400/zg/kg PG elicited overlapping dose-related inhibitions of intake of milk diet in rats maintained ad lib on pelleted chow. Twenty-five to 50/zg/kg EPI also elicited anomalous behaviors that are not normally associated with feeding, including supine postures with limbs extended and crawling with trunk dorsoflexed and abdomen pressed against cage floor. EPI elicited similar anomalous behaviors in rats that either sham fed with open gastric cannulas, drank after water deprivation, or were presented neither food nor water. Fifty to 200/zg/kg EPI also inhibited water intake in the thirsty rats, and 25-50/zg/kg EPI inhibited sham feeding, laG, in contrast, neither elicited anomalous behaviors nor inhibited water intake nor inhibited sham feeding. These data demonstrate that the inhibitory actions of exogenous EPI and PG are functionally dissociable. We conclude that 25-200/zg/kg EPI acts nonspecifically to produce anorexia and adipsia, while PG elicits postprandial satiety. Satiety
Glucagon
Food intake
Water intake
Sham feeding
Liver
Behavior
rather than to elicit postprandial satiety. This dissociates the behavioral actions of EPI and PG and suggests that EPI is not a satiety agent. Lower doses of EPI may, however, inhibit feeding specifically.
P E R I P H E R A L administration of either epinephrine (EPI) or pancreatic glucagon (PG) in the rat stimulates hepatic production of glucose and other energy metabolites and also inhibits food intake. Because of this, Russek and others [22, 24, 29, 31, 35] have proposed that these inhibitory effects of EPI and PG are due to a common hepatic metabolic satiety signal. If PG and EPI do inhibit feeding via a common mechanism, their inhibitory effects should be functionally similar. The inhibitory effect of exogenous PG fulfdls several criteria of behavioral specificity [8, 21, 37]. For example, after intraperitoneal injection of PG in diurnal tests, hungry rats eat smaller, shorter meals and display the normal behavioral sequence of postprandial satiety. Further, PG does not inh/bit water intake in thirsty rats. These fmdings are consistent with the hypothesis that PG specifically inhibits feeding by stimulating a normal neuroendocrine mechanism of postprandial satiety. Although there are several reports that intraperitoneal EPI inhibits feeding [2, 24, 27, 30, 35, 36], the behavioral specificity of this effect has not been as extensively investigated. We conducted these experiments to test the behavioral specificity of EPI's inhibitory effect on feeding in rats and to compare it to that of PG. Doses of EPI comparable or lower to those used in previous research (i.e., 25-50/~g/kg) inhibited feeding potently, but appeared to act nonspecificaUy
EXPERIMENT 1 Rats display a characteristic sequence of behaviors after meals during the light phase. When feeding ends, rats groom and engage in exploratory behaviors such as locomotion and sniffing, before finally resting or sleeping [1, 4, 20, 25, 34]. The postprandial sequence of behaviors biologically defines satiety: if it does not occur, it is unlikely that satiety has occurred [34]. This criterion differentiates satiety from nonspecific inhibitions of feeding such as those elicited by injection of amphetamine [28] or lithium chloride [3]. Injection of PG reduces the size of milk meals in rats without disrupting the satiety sequence [8]. EPI has not been similarly tested. Therefore, we compared the effects of several doses of EPI and PO on meal size and periprandial behavior.
Method Subjects were male rats (Sprague Dawley SPF, Charles River, Wilmington, MA) weighing between 330--600 g. They were individually housed and maintained on pelleted chow (5012 Purina, St Louis, MO) and tap water ad lib, except
1Requests for reprints should be addressed to N. Geary.
109
110
HINTON ET AL. TABLE 1 DIFFERENT EFFECTS OF EPI AND PG ON BEHAVIORS DURING AND AFTER MEALS
Feeding
Frequency Resting
Anomalous
Experiment la Control EPI 6.25 EPI 12.5 EPI 25 EPI 50
9.0 (7.5-12) 10.5 (7-12) 6.5 (5-7)`[ 4.5 (3-6),[ 4.0 (2-5)`[
34.5 (32-39) 36.0 (32-40) 29.0 (19-37) 26.5 (18-33)`[ 23.5 (18-33)`[
0 (0-1) 0 (0-1) 1.0 (0-14) 8.5 (4-18)1' 18.0 (6-23)T
Experiment lb Control EPI 6.25 EPI 9.375 EPI 12.5
11.0 (9-14) 10.5 (9-12) 9.5 (7-13)`[ 8.0 (7-10)`[
14.0 (11.5-18.5) 18.0 (16-22.5) 18.0 (13-20) 16.0 (9-21)
0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0)
10.0 (7-12)$ 9.5 (7--12)~ 8.0 (6--10)`[
17.5 (12-21) 18.0 (16-25)1' 21.5 (15-25)1'
0 (0-0) 0 (0-0) 0 (0-0)
PG 100 PG 200 PG 400
Values are median number of observations of each behavior class, with the first and third quartiles given in parentheses. See text for definition of behaviors. There were 60 observations in Experiment la and 40 in Experiment lb. EPI and PG doses are in t~g/kg. Arrows indicate significant increases (D or decreases (`[) in frequencies, p<0.05, compared to control data, sign tests after significant overall effects determined by median tests. Frequencies of grooming, exploring, and other behaviors were not significantly different across drug groups.
l
-
5c _z :~
4c
3c
~
2o
0 6.25
9.375
12.5 DOSE EF'I ( r n c ~ )
25
50
FIG. 1. Reduction of MS after EPI injection. Data are mean±SE percent inhibition MS as compared to control for 16 rats in Experiment la and 22 rats in Experiment lb. Mean control MS were 13.9+_3.7 ml for Experiment la and 19.3-+1.1 ml in Experiment lb. *p<0.05, **p<0.01 post hoc t-test, after overall ANOVA. ANOVA effects: F(4,60)=17.99, p<0.01, in Experiment la and F(5,105)=3.63, p<0.01, in Experiment lb.
during the test period when pellets were removed. Temperature was maintained between 22° and 26°C. Rats were adapted to handling and intraperitoneal injections of 1 ml/kg 0.9% NaCI near the middle of the light phase of a 14:10 LD cycle. Evaporated milk (Carnation, Los Angeles, CA) was presented in calibrated (_-.1 ml) drinking tubes (Wahmann,
Timonium, MD) immediately after the injections. Milk intake was measured for 60 minutes. An experimenter blind to the injection protocol observed each rat's behavior once a minute during tone-signalled 0.6 sec observation periods for 40 to 60 min [8,11], Behaviors were classified using operational definitions and recorded. F o r example, grooming was defined as biting or licking the coat, paws, genitals, or tail or scratching or stroking the head, face, or vibrassae with one or both paws. Resting was defined as a stationary position in which the abdomen is on the floor of the cage and no other behavior is displayed. Because pilot studies indicated that rats sometimes displayed anomalous behaviors following EPI injections, operational definitions for these were developed. Three anomalous behaviors occurred: crawling--locomotor behavior with spine dorsoflexed, limbs extended, and abdomen pressed against cage floor; s u p i n e - - a stationary position with the rat on its back or side with all four limbs extended; and p r o n e - - a stationary position with abdomen on the cage floor, limbs extended rostrally and caudally, and spine dorsoflexed. These behaviors were rated conservatively; if they appeared simultaneously with any other behavior (such as s ~ ) , the other behavior was given precedence in rating. Experiments began when for at least 80% o f the rats, the standard deviation of the mean milk intake for five consecutive adaptation days was less than 30% of the mean. Experiments were done Tuesdays through Fridays; the rats were readapted Mondays. Two similar experiments were done. In Experiment la, 16 rats were each intraperitoneally injected with 6.25, 12.5, 25 or 50/zg/kg EPI (epinephrine hydrochloride, Parke Davis,
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY Morris Plains, NJ), 50 100, 200 or 400/zg/kg PG (Eli Lilly Research Laboratories, Indianapolis, IN), and 3.0% dimethylsulfoxide-0.9% NaC1 on six days as a control. Crystalline PG was dissolved first in DMSO and then diluted with saline to achieve the desired concentration. EPI was dissolved in premixed DMSO-saline. All injections were 1.0 ml/kg body weight. The order of tests was random for each rat. Because the PG was ineffective in Experiment la, we compared the batch used to a fresh batch and discovered the PG used was no longer active. Therefore, only the EPI data were analyzed. In Experiment lb 22 rats each received 6.25, 9.375 or 12.5 /~g/kg EPI, 100, 200 or 400/~g/kg of new PG, and two 3.0% DMSO-0.9% NaCl controls in random order. Lower doses of EPI were used to obtain effects comparable to those expected to be elicited by PG. Meal size (MS) was defined as the amount of milk ingested during the initial postinjection bout of feeding before the rats displayed the behavioral satiety sequence ending in at least four consecutive observations of resting. This was identical to 30 min milk intake. Rats seldom ate again before the end of the test. Percent inhibition of MS was defined as 100 - [100 (MS after drug injection)/(mean MS of control tests)]. Effects on MS were analyzed with repeated measures ANOVA and post-hoc t-tests [13]. Behavioral observations were grouped into categories of feeding, grooming, exploring (including sniffing, locomotion, rearing, and licking cage), resting, anomalous behaviors, and all other behaviors (such as standing in a stationary position with abdomen off the cage floor, urinating, defecating, yawning, etc.). The number of times behaviors from each category occurred was compiled, and changes in the frequency of occurrence were analyzed with median and sign tests [38]. Results
Rats fed immediately after milk presentation, but ingested less after EPI or PG injection than after control injections. EPI elicited a dose-related inhibition of MS (Fig. 1). 6.25 /~g/kg EPI decreased MS significantly in Experiment lb, but not in Experiment la. 50/xg/kg EPI, the largest dose tested, decreased MS more than 50%. After control injections, rats fed almost continuously for about 10 minutes. Then they displayed mainly grooming and exploratory behaviors before finally resting or sleeping. This behavioral satiety sequence was disrupted by EPI. EPIinjected rats fed less, but failed to rest more (Table 1). Rather, after 25-50 p.g/kg EPI, resting was replaced by dramatic behavioral postures not normally associated with postprandial satiety. These anomalous behaviors accounted for up to 30% of the observations after EPI injection, as opposed to 0% of the observations after control injection. There were no consistent changes in frequencies of postprandial grooming, exploring or other behaviors (for this reason these data are not presented). Rats fed less without increasing time spent resting after 6.25-12.5/zg/kg EPI and significantly decreased time spent resting after 25-50/~g/kg EPI. Despite this, latency to rest, which was quite variable, was not significantly increased. Injection of 100--400 /~g/kg PG decreased MS 13-32% (Fig. 2). In contrast to the EPI results, decreases in MS elicited by PG were accompanied by increases in resting and were not accompanied by anomalous behaviors (Table 1). The amount of resting increased by as much as 19%. Latency to the first rest, however, was not decreased by PG. This
lll 40
< ao
} --7 z
~0
i 100
200
400
DO~ PG(moo~)
FIG. 2. Reduction in MS after PG injection. Data are mean_SE percent inhibition MS as compared to control for 22 rats in Experiment lb. Mean control MS was 19.3---1.1. *p<0.05, **p<0.01, post hoc t-test after overall significant ANOVA. ANOVA effect: F(5,105)=3.63, p<0.01.
appears to result from the variability of latency data and not from any change in the sequence of postprandial behaviors. PG also did not change the frequency of either grooming or exploring. Discussion
These results dissociate the effects of exogenous EPI and PG on feeding and periprandial behavior. As previously [8], PG inhibited MS without altering the normal behavioral sequence of postprandial satiety. The only behavioral changes elicited by PG were that rats ate smaller, shorter meals and increased the time spent resting. This is consistent with the hypothesis that PG inhibits feeding by activating a specific satiety mechanism. In contrast, EPI-injected rats did not display the normal behavioral satiety sequence. Doses of 6.25-12.5/~g/kg EPI decreased MS 13-42% but did not increase the frequency of resting, whereas doses of PG that inhibited meal size 25% or more increased the frequency of resting. These data do not clearly indicate whether doses of 6.25-12.5 EPI act specifically to inhibit feeding. 25-50/~g/kg EPI significantly reduced the incidence of resting and also elicited anomalous behaviors. This suggests that 25-50/~g/kg EPI decreases MS nonspecifically by eliciting behaviors that compete with feeding rather than by signalling postprandial satiety. Thus, because the postprandial satiety sequence may be considered necessary for the demonstration of satiety under these test conditions, we conclude that the inhibitory action of feeding of 25-50/~g/kg EPI is a nonspecific anorectic effect, not a satiety effect. This calls into question the specificity of previously reported inhibitory effects, which with only two exceptions [2,36], involved doses larger than ours [24, 27, 30, 35, 36]. In contrast to EPI, no doses of PG have elicited such behavior in this or similar tests [8,9]. This functional dissociation suggests that PG and EPI act differently to inhibit meal size. EXPERIMENT 2
Hypothesized satiety signals including PG [8,21] and cholecystokinln (CCK) [1] inhibit feeding but do not inhibit water intake in water deprived rats. In order to determine whether EPI's inhibitory action also selectively inhibits feed-
112
HINTON ET AL. TABLE 2 DIFFERENTEFFECTSOF EPI AND PG ON WATERINTAKEANDBEHAVIORSDURINGAND AFTER DRINKING Inhibition of Water Intake
Drinking
Frequency Resting
Anomalous
Control EPI 6.25
9.3 _+ 4.4
9.0 (8--10) 7.5 (9--11)
2.0 (0--5) 4.5 (2-8)
0 0
(0-0) (0-0)
Control EPI 12.5
1.5 +_ 5.1
9.0 (7-10) 9.0 (8-12)
2.0 (2-4) 9.5 (3-17)
0 0
(0-0) (0-0)
Control EPI 25 PG 100
1.3 --_ 17.5 0.7 -+ 20.0
6.0 (4-8) 7.0 (5-8) 7.0 (5-9)
3.0 (1-8) 3.0 (1-9) 2.0 (0-9)
0 (0-1) 0 (0-0) 0 (0-1)
Control EPI 50 PG 200
30.0 _ 8.8* 18.5 ± 20.8
6.5 (4-8) 3.0 (3-6)$ 6.5 (6--7)
1.0 (0-2) 9.5 (3-12)~" 1.5 (0-5)
0 (0-0) 2.0 (0-6)1' 0 (0-1)
Control EPI 100 PG 400
37.7 -+ 8.9* 6.9 ± 9.9
6.0 (4-7) 3.0 (2-5)~ 8.0 (7-10)]'
2.0 (0-9) 9.0 (3-11)1' 3.0 (0-6)
0 (0-1) 3.0 (0-9)1" 0 (0-1)
Control EPI 200 PG 800
76.7 ± 5.3* -5.4+ 11.7
6.0 (6-8) 1.0 (0-2)+ 8.0(6-11)
1.0 (0-5) 11.0 (8--12)'r 8.0(3-11)
0 (0-0) 12.0 (6-17)1, 0 (0-1)
Inhibitions are % of water intake after control tests. Behavior frequencies are median number of 40 total observations, with the first and third quartiles given in parentheses. See text for definitions of behaviors. *Significant inhibition of water intake, post hoc t-test, p<0.01, after significant ANOVA. Arrows indicate significant increases (1') or decreases (~,)in frequencies of behaviors, sign test, p <0.05. Frequencies of grooming, exploring, and other behaviors were not significantly different across drug groups.
Food was always available except during the first hour of the drinking period. Injections were done just before water presentation, and water intake and behaviors were measured as previously. This schedule elicited initial post-deprivation water intakes (10-22 ml) that were similar to the mean control milk meal sizes (14-19 ml) in Experiment 1. Also, under these conditions, rats drank immediately upon water presentation, and the post-deprivation bouts of drinking were similar in length to milk meals. EPI (6.25-200 t~g/kg) and 100-800 /~g/kg PG were prepared as in Experiment 1 and compared to paired control injections in separate experiments.
80 70
,[xx;~
60 50 40 30 20 10 0
I
6.25
12.5
25 50 DOSE EPI (mog,~)
100
Results
200
FIG. 3. Inhibitionsof 30 min water intake in Experiment 2. Data are mean+SE percent inhibition as compared to control for 15 rats in separate saline-paired tests. Mean control intakes were 10.4+ 1.2 to 21.6-+1.2 ml. **p<0.01, post hoc t-test, after significant ANOVA.
ing, we compared the effects of EPI and PG on water intake in thirsty rats. Method Fifteen rats were adapted for one month to 6 hr/day of water access, beginning near the middle of the light phase.
Injection of 50-200/xg/kg EPI inhibited water intake up to 77% (Fig. 3). These inhibitions were accompanied by the same anomalous behaviors that EPI elicited in the feeding tests (Table 2). These EPI doses also signifuzantly increased resting behavior. Resting accounted for 2.5-5% of the observations after control injection and 22.5-28% after 50-200 /,g/kg EPI, p's<0.05. Doses of 6.25-25 t~g/lq~ EPI did not inhibit water intake, although there was a nonsignificant trend for resting to increase. Resting accounted for 5-8% of the observations after control injection and 8--24% after 6.25-25 t~g/kg EPI (p's<0.06). No dose of PG tested affected water intake (inhibitions of 30 minute water intake ranged from - 5 . 4 + 1 1 . 7 to 18.5_+20.8%, (p's>0.05) or the frequency of any behavior.
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY
113
Discussion
The demonstration that 50-200/tg/kg EPI inhibits water intake indicates that EPI's inhibitory effect on ingestive behavior is not specific to feeding. The smallest dose that inhibited water intake (50/zg/kg EPI) was, however, larger than the smallest doses that inhibited feeding (12.5/xg/kg EPI in Experiment la and 6.25 /~g/kg EPI in Experiment lb). Thus, EPI may have a relative specificity of action on feeding in the very low dose range. Our results contrast with Rodriguez et al.'s [27] report that 125-500/zg/kg EPI does not significantly inhibit water intake in 6 hour food and water deprived adult rats. But their test was not compelling. The amount drunk in their control condition was so small, only about 4.4-5.3 ml, that inhibitory effects may have been difficult to observe. Further, this amount of water intake was less than a third of the control milk intake in the feeding test (14.4-17.3 ml), so the drinking test did not closely mimic the feeding test. Rodriguez et al. [27] also demonstrated that EPI did inhibit water intake in 3 to 10 day old rat pups as potently as it inhibited milk intake in a situation in which the control intakes were comparable. Thus, when similar amounts of water and liquid food were ingested, EPI inhibited both feeding and drinking, just as we demonstrated here. As previously [8], PG had no effect on water intake. PG also failed to affect the behaviors associated with water ingestion. Therefore, the effects of EPI and PG on water intake are dissociable. Doses of 50-200/zg/kg EPI inhibit drinking and elicit unusual behavior patterns. PG neither inhibits drinking nor elicits unusual behaviors. This indicates that EPI, unlike PG, is a nonspecific agent that can elicit adipsia as well as anorexia. EXPERIMENT
3
Experiments 1 and 2 demonstrated that EPI elicits similar anomalous behaviors in feeding and drinking tests. To determine whether these behaviors were dependent on ingestive responses, we tested EPI's behavioral effects in the absence of feeding or drinking. Method
Eighteen rats were maintained with ad lib access to food and water except during a 40 min daily test period. Rats were each intraperitoneally injected with either 3% DMSO-0.9% NaCI or 6.25, 12.5, 25 or 50/~g/kg EPI in random order, and behaviors were rated as previously. Results
50 t~g/kg EPI elicited the same anomalous behaviors that occurred during the feeding and drinking tests. Anomalous behaviors did not occur after control injections but accounted for a median frequency (lst-3rd quartile) of 40 (2644)% of the behavioral observations (z=3.76, p<0.01) after 50/zg/kg EPI. Anomalous behaviors accounted for 25 (1750)% of the observations after 25 ttg/kg EPI, but this was not significant (z= 1.00, p>0.05). Discussion
EPI eficited anomalous behaviors in rats that were neither feeding nor drinking. Thus, ingestion of food or water is not necessary for the expression of this behavioral effect of EPI. This is consistent with the hypothesis that EPI inhibits feeding and drinking nonspecifically by eficiting competing be-
70 6O Z
50
10 0 6.25
12.5 25 DOSEEPI(rrog4~)
50
FIG. 4. Reduction of 60 min sham milk intake after EPI injection in Experiment 4. Data are mean__+SE percent inhibition as compared to control for 7 to 9 rats per test. Mean control intakes were 82.6±9.4 to 103___8.7ml. **p<0.01, t-test. haviors that do not normally contribute to the inhibition of ingestion, but interfere with normal ingestive behavior. EXPERIMENT
4
To compare further the inhibitory actions of EPI and PG, we tested EPI's effect on rats sham feeding with open gastric cannulas. PG does not inhibit sham feeding [9,17]. Therefore, if EPI and PG inhibit feeding by the same mechanism, EPI should also fail to inhibit sham feeding. Method
Eleven rats were implanted with chronic gastric cannulas using a previously described method [1,9]. Rats were adapted one month to testing conditions. Pelleted chow was removed from the cages 12 hours prior to injection. The cannulas were opened just before the tests, and each rat's stomach was rinsed with warm 0.9% saline until no food was detected in the drainage. One ml/kg saline was intraperitoneally injected and a plastic collection tube was attached to the cannula. The tubes drained into a collection pan below the cage. After each test, the volume collected was measured. Data from tests in which this volume was less than the rat's intake volume were not used. Doses of 6.25-50/~g/kg EPI were compared to saline control injections according to separate crossover designs. Milk intake and behaviors were observed for 60 rain. Data analysis was done for the 6--9 rats in each test that met the criterion for successful sham feeding on each test day. Sixty min sham intakes were used for the analysis because in control conditions rats often did not stop sham feeding until after 30 min. Results
Doses of 6.25-12.5/~g/kg EPI did not detectably affect sham intake or behavior, but 25 and 50/~g/kg EPI each inhibited sham intake more than 60% (Fig. 4). Anomalous behaviors did not occur after control injections but were emitted by five of the six rats with 25/~g/kg EPI [median frequency (lst-3rd quartile), 48 (14-56)%, z= 1.79, p>0.05] and by six of the eight rat tested with 50/zg/EPI [3 (3-30)%, z=2.04, p<0.05]. As in the real feeding test (Experiment 1), rats fed less after 25-50/~g/kg EPI (75 and 50% decreases,
114
HINTON E T A L .
z's=2.05, 2.44, p's<0.05), but failed to rest more (z's= 1.38, 1.24, p's>0.05). Discussion
In previous studies, sham feeding was not inhibited by doses of up to 2,500/zg/kg PG, despite that this is 16-100 times higher than the doses that inhibit real feeding in similar tests [9,17]. This suggests that PG's satiety effect depends on some postgastric food stimulus that is absent during sham feeding. In contrast, EPI doses only slightly higher than those necessary to inhibit real feeding potently inhibited sham feeding. This suggests that EPI's inhibitory action, unlike PG's, does not require postgastric food stimuli. 100-2,500 /zg/kg PG also failed to elicit anomalous behaviors [9,17], whereas doses of EPI that inhibited sham feeding did elicit such behaviors. These distinctions are further evidence that EPI and PG do not inhibit feeding by the same mechanism. GENERALDISCUSSION Under the conditions of our tests, normal postprandial satiety in rats is associated with a specific sequence of behaviors that includes grooming and exploratory behavior and terminates in rest or sleep [1, 4, 20, 25, 34]. A satiety agent should decrease meal size without disrupting this satiety sequence. Intraperitoneal injection of 25-50 /xg/kg EPI potently decreased meal size, but also disrupted the satiety sequence by eliciting anomalous behaviors that are not associated with normal meals. A selective satiety agent also should not affect other ingestive behavior, but 50--200/zg/kg EPI inhibited drinking as well as feeding. Therefore, we conclude that in this dose range EPI is a nonspecific anorectic and adipsic agent, not a satiety agent. The same anomalous behaviors that were elicited by EPI in feeding tests also occurred when EPI was administered to rats drinking water and to rats that were neither feeding nor drinking. Thus, these behaviors can be elicited independently of EPI's inhibitory action on feeding. This is consistent with the hypothesis that EPI inhibits ingestive behavior nonspecifically by eliciting competing behaviors that interfere with ingestion. The EPI doses that elicited nonspecific effects are 2 to 3 times smaller than those used in most previous investigations of EPI's effects on feeding [24, 27, 30, 35, 36]. Only two of those studies included tests of the hypothesis that EPI has a specific satiety effect. The evidence produced is at best ambiguous. As discussed above, Rodriguez et al. [27] failed to detect an effect of 125-500 tzg/kg EPI on water intake in adult rats, but the amount of water drunk in their tests was much less than the amount of liquid food ingested. And these EPI doses did inhibit water intake in rat pups. Russek and Racotta [31] noted that EPI elicits postprandial decreases in activity in rats tested in jiggle cages and suggested that this might reflect normal postprandial sedation. Our data suggest this decrease in activity might have been due to the appearance of anomalous stationary postures. Finally, because the anomalous behaviors we observed also occur in the absence of feeding, our data suggest they do not reflect "oversatiation" as has been suggested by Russek [29,31] to account for the observation that vomiting sometimes accompanies EPI-anorexia in dogs. Therefore, because inhibition of feeding alone is not sufficient evidence for postprandial satiety,
we conclude that the literature provides no direct support for the hypothesis that EPI elicits satiety. The smallest EPI doses that we tested did inhibit feeding without eliciting nonspecific behavioral effects. Thus, it is possible that EPI may act specifically at smaller doses and nonspecifically only at doses larger than 25/zg/kg. This requires further investigation, however, because there were suggestions of nonspecificity in the lower dose range. First, in Experiment 1, doses of 12.5-25/zg/kg EPI inhibited feeding as much or more than 200-400 ~g/kg PG, but only PG and not EPI increased resting. Higher doses of EPI elicited decreases in the frequency of resting behavior. Second, in the drinking test there was a trend toward an anomalous increase in the frequency of resting after smaller doses of EPI, and larger doses significantly increased the frequency of resting. Control injected rats continued to be active after drinking until they were fed at the end of the test. Since the anomalous behaviors consist largely of stationary postures, it is possible this EPI-induced increase in resting in the drinking test represents a lower intensity of the same response. PG, in contrast, did not increase resting. Thus, whether any EPI treatment elicits a behaviorally specific inhibition of feeding remains unclear. The nonspecific effects of EPI that we observed indicate that EPI's inhibitory action on feeding is functionally dissimilar from PG's. In contrast to EPI, PG did not disrupt the behavioral satiety sequence, elicit anomalous behaviors, or inhibit water intake [5, 8, 16, 37]. Thus, PG fulfills several behavioral criteria for a postprandial satiety agent that EPI does not. The sham feeding test in Experiment 4 further dissociates PG and EPI. EPI inhibited sham feeding whereas PG has consistently failed to [9,17]. It has been hypothesized that the inhibitory actions of PG and EPI are initiated by a single peripheral neural mechanism that is sensitive to their common effect on hepatic carbohydrate metabolism [22, 24, 29, 31, 35, 36]. This hypothesis predicts that the behavioral characteristics of the two agents' inhibitory effects should be similar. Therefore, the behavioral dissociations that we report here suggest that EPI and PG do not activate a single mechanism to inhibit feeding. This complements more direct lines of evidence against this hypothesis. For example, the stimulatory effects of both PG [2, 7-9, 15] and EPI [2,14] on hepatic glycogenolysis and hepatic glucose production have been dissociated from their inhibitory effects on feeding. This fails to support the hypothesis that hepatic carbohydrate metabolism mediates the agents' feeding effects. Further, while hepatic vagotomy [10,19], total liver denervation [18], and intrahepatic injection of alloxan [26] have sometimes blocked PG's satiety effect, none of these treatments affects EPI anorexia [18, 19, 26]. This suggests that PG's satiety effect and EPI's anorectic effect are mediated by different peripheral neural mechanisms.
ACKNOWLEDGEMENTS A preliminary report of these data was given at the 55th Annual Meeting of the Eastern PsychologicalAssociation, Baltimore, MD, March, 1984 [5]. This research was supported by N.I.H. Research Grant AM 32448to N.G. We thank Michael Krumper, Tom Comaechia and Amy Ventry for help with the experiments.
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY
115
REFERENCES 1. Antin, J., J. Gibbs, J. Holt, R. C. Young and (3. P. Smith. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J Comp Physiol Psychol 89: 784-790, 1975. 2. Bellinger, L. L. and F. E. Williams. (31ucngon and epinephrine suppression of food intake in liver-denervated rats. Am J Physiol 251: R349-358, 1986. 3. Crawley, J. N. Divergent effects of cholecystokinin, bombesin, and lithium on rat exploratory behaviors. Peptides 4: 405-410, 1983. 4. Danguir, J., S. Nicolaidis and H. Gerard. Relations between feeding and sleep patterns in the rat. J Comp Physiol Psycho193: 820-830, 1979. 5. Esguerra, M., N. Farhoody, V. Hinton and N. Geary. Epinephrine inhibits feeding nonspecifically in the rat. Proc EPA 55: 121, 1984. 6. (3eary, N., N. Farhoody and A. Gersony. Food deprivation dissociates pancreatic glucagon's effects on satiety and hepatic glucose production. Physiol Behav 39: 507-511, 1987. 7. Gear/, N., W. Langhans and E. Scharrer. Metabolic concomitants of glucagon-induced suppression of feeding in the rat. Am J Physiol 241: E330-E335, 1981. 8. (3eary, N. and G. P. Smith. Pancreatic glucagon and postprandial satiety in the rat. Physiol Behav 28: 313-322, 1982. 9. Geary, N. and G. P. Smith. Pancreatic glucagon fails to inhibit sham feeding in the rat. Peptides 3: 163-166, 1982. 10. Geary, N. and G. P. Smith. Selective hepatic vagotomy blocks pancreatic glucagon's satiety effect. Physiol Behav 31: 391-394, 1983. 11. Gibbs, J., L. Gray, C. F. Martin, W. T. Lhamon and J. A. Stuckey. Quantitative behavioral analysis of neuropeptides which suppress food intake. Soc Neurosci Abstr 6: 530, 1980. 12. Gibbs, J. and (3. P. Smith. The neuroendocrinology of postprandial satiety. In: Frontiers in Neuroendocrinology, vol 8, edited by L. Martini and W. F. Ganong. New York: Raven Press, 1984, pp. 223-245. 13. Hays, W. L. Statistics for Psychologists. New York: Holt, Rinehart and Winston, 1963. 14. Langhans, W., K. Pantel and E. Scharrer. Dissociation of epinephrine's hyperglycemic and anorectic effect. Physiol Behav 34: 457-463, 1985. 15. Langhans, W., E. Scharrer and N. Geary. Pancreatic glucagon's effect on satiety and hepatic glucose production are independently affected by diet composition. Physiol Behav 36: 483487, 1986. 16. Langhans, W., U. Zeigler, E. Scharrer and N. Geary. Stimulation of feeding in rats by intraperitoneal injection of ~mtibodies to glucagon. Science 218: 894-896, 1982. 17. Le Sauter, J. and N. Geary. Pancreatic glucagon corr/bines with cholecystokinin to inhibit sham feeding in the rat. Am J Physiol, submitted, 1986. 18. MacIssac, L. and N. Geary. Hepatic vagotomy or total liver denervation can block pancreatic glucagon's satiety effect. Soc Neurosci Abstr 15: 58, 1985. 19. MacIssac, L. and N. (3eary. Partial liver denervations dissociate the inhibitory effects of pancreatic glucagon and epinephrine on feeding. Physiol Behav 35: 233--237, 1985.
20. Mansbach, R. S. and D. Lorenz. Cholecystokinin (CCK-8) elicits prandial sleep in rats. Physiol Behav 30: 179-183, 1983. 21. Martin, J. R. and D, Novin. Decreased feeding in rats following hepatic-portal infusion of glucagon. Physiol Behav 19: 461-466, 1977. 22. Novin, D., K. Robinson, L. A. Culbreth and M. (3. Tordoff. Is there a role for the liver in the control of food intake?Am J Clin Nutr 42: 1050-1062, 1985. 23. Penick, S. B. and (3. P. Smith. The effects of glucagon on food intake and body weight in man. J Obesity 1: 1-5, 1964. 24. Racotta, R., M. Islas-Chaires, C. Vega, M. Soto-Mora and M. Russek. Glycogenolytic substances, hepatic and systemic lactate, and food intake in rats. Am J Physiol 246: R247-R250, 1984. 25. Richter, C. A behavioristic study of the activity of the rat. Comp Psychol Monogr 1: 1-55, 1922. 26. Ritter, S., S. C. Weatherford and S. L. Stone. Glucagoninduced inhibition of feeding is impaired by hepatic portal alloxan injection. Am J Physiol 250: R682-R690, 1986. 27. Rodriguez-Zendejas, A. M., (3. Chambert, M. C. Lora-Vilchis, A. Epstein and M. Russek. Ontogeny of epinephrine-induced anorexia in rats. Am J Physiol 250: 313-317, 1986. 28. Rosofsky, M. and N. Geary. Specific and nonspecific effects of phenylpropanolamine and amphetamine on feeding in rats. Proc EPA 57: 57, 1986. 29. Russek, M. Current status of the hepatostatic theory of food intake control. Appetite 2: 137-143, 1981. 30. Russek, M., G. J. Mogenson and J. A. F. Stevenson. Calorigenic, hyperglycemic and anorexigenic effects of adrenaline and noradrenaline. Physiol Behav 2: 429-433, 1967. 31. Russek, M. and R. Racotta. A possible role of adrenaline and glucagon in the control of food intake. Front Horm Res 6: 120137, 1980. 32. Russek, M., A. M. Rodriguez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenaline and glucose. Physiol Behav 3: 249-257, 1968. 33. Russek, M. and J. A. F. Stevenson. Correlation between the effects of several substances on food intake and on the hepatic concentration of reducing sugars. Physiol Behav 8: 245-249, 1972. 34. Smith, G. P. and J. Gibbs. Postprandial satiety. In: Progress in Psychobiology and Physiological Psychology, vol 8, edited by J. Sprague and A. E. Epstein. New York: Academic, 1979, pp. 179-242. 35. Tordoff, M. G. and D. Novin. Celiac vagotomy attenuates the ingestive responses to epinephrine and hypertonic saline but not insulin, 2-deoxy-d-glucose, or polyethelene glycol. Physiol Behav 29: 605-613, 1982. 36. Tordoff, M. G., D. Novin and M. Russek. Effects of hepatic denervation on the anorexic response to epinephrine, amphetamine, and lithium chloride: a behavioral identification of glucostatic afferents. J Comp Physiol Psycho196: 361-375, 1982. 37. Weick, B. (3. and S. Ritter. Dose-related suppression of feeding by intraportal glucagon infusions in the rat. Am J Physiol 250: R676-R681, 1986. 38. Winer, B. J. Statistical Principles in Experimental Design, 2nd edition. New York: McGraw-Hill, 1971.
0031.9384/87$3.00 + .00
Epinephrine Inhibits Feeding Nonspecifically in the Rat VERONICA HINTON, MANUEL ESGUERRA, NAUDER FARHOODY, JENNIFER GRANGER AND NORI GEARY 1
Department of Psychology, Box 28 Schermerhorn Hall, Columbia University, New York, N Y 10027 R e c e i v e d 5 N o v e m b e r 1986 HINTON, V., M. ESGUERRA, N. FARHOODY, J. GRANGER AND N. GEARY. Epinephrine inhibits feeding nonspecifically in the rat. PHYSIOL BEHAV 40(1) 109-115, 1987.--The hypothesis that epinephrine (EPI) and pancreatic glucagon (laG) inhibit feeding by activating a common physiological satiety mechanism was tested by comparing the two agents' behavioral effects. In several tests of specificity, EPI and laG had functionally different inhibitory actions. Intraperitoneal injection of 6.25-50/zg/kg EPI and 100-400/zg/kg PG elicited overlapping dose-related inhibitions of intake of milk diet in rats maintained ad lib on pelleted chow. Twenty-five to 50/zg/kg EPI also elicited anomalous behaviors that are not normally associated with feeding, including supine postures with limbs extended and crawling with trunk dorsoflexed and abdomen pressed against cage floor. EPI elicited similar anomalous behaviors in rats that either sham fed with open gastric cannulas, drank after water deprivation, or were presented neither food nor water. Fifty to 200/zg/kg EPI also inhibited water intake in the thirsty rats, and 25-50/zg/kg EPI inhibited sham feeding, laG, in contrast, neither elicited anomalous behaviors nor inhibited water intake nor inhibited sham feeding. These data demonstrate that the inhibitory actions of exogenous EPI and PG are functionally dissociable. We conclude that 25-200/zg/kg EPI acts nonspecifically to produce anorexia and adipsia, while PG elicits postprandial satiety. Satiety
Glucagon
Food intake
Water intake
Sham feeding
Liver
Behavior
rather than to elicit postprandial satiety. This dissociates the behavioral actions of EPI and PG and suggests that EPI is not a satiety agent. Lower doses of EPI may, however, inhibit feeding specifically.
P E R I P H E R A L administration of either epinephrine (EPI) or pancreatic glucagon (PG) in the rat stimulates hepatic production of glucose and other energy metabolites and also inhibits food intake. Because of this, Russek and others [22, 24, 29, 31, 35] have proposed that these inhibitory effects of EPI and PG are due to a common hepatic metabolic satiety signal. If PG and EPI do inhibit feeding via a common mechanism, their inhibitory effects should be functionally similar. The inhibitory effect of exogenous PG fulfdls several criteria of behavioral specificity [8, 21, 37]. For example, after intraperitoneal injection of PG in diurnal tests, hungry rats eat smaller, shorter meals and display the normal behavioral sequence of postprandial satiety. Further, PG does not inh/bit water intake in thirsty rats. These fmdings are consistent with the hypothesis that PG specifically inhibits feeding by stimulating a normal neuroendocrine mechanism of postprandial satiety. Although there are several reports that intraperitoneal EPI inhibits feeding [2, 24, 27, 30, 35, 36], the behavioral specificity of this effect has not been as extensively investigated. We conducted these experiments to test the behavioral specificity of EPI's inhibitory effect on feeding in rats and to compare it to that of PG. Doses of EPI comparable or lower to those used in previous research (i.e., 25-50/~g/kg) inhibited feeding potently, but appeared to act nonspecificaUy
EXPERIMENT 1 Rats display a characteristic sequence of behaviors after meals during the light phase. When feeding ends, rats groom and engage in exploratory behaviors such as locomotion and sniffing, before finally resting or sleeping [1, 4, 20, 25, 34]. The postprandial sequence of behaviors biologically defines satiety: if it does not occur, it is unlikely that satiety has occurred [34]. This criterion differentiates satiety from nonspecific inhibitions of feeding such as those elicited by injection of amphetamine [28] or lithium chloride [3]. Injection of PG reduces the size of milk meals in rats without disrupting the satiety sequence [8]. EPI has not been similarly tested. Therefore, we compared the effects of several doses of EPI and PO on meal size and periprandial behavior.
Method Subjects were male rats (Sprague Dawley SPF, Charles River, Wilmington, MA) weighing between 330--600 g. They were individually housed and maintained on pelleted chow (5012 Purina, St Louis, MO) and tap water ad lib, except
1Requests for reprints should be addressed to N. Geary.
109
110
HINTON ET AL. TABLE 1 DIFFERENT EFFECTS OF EPI AND PG ON BEHAVIORS DURING AND AFTER MEALS
Feeding
Frequency Resting
Anomalous
Experiment la Control EPI 6.25 EPI 12.5 EPI 25 EPI 50
9.0 (7.5-12) 10.5 (7-12) 6.5 (5-7)`[ 4.5 (3-6),[ 4.0 (2-5)`[
34.5 (32-39) 36.0 (32-40) 29.0 (19-37) 26.5 (18-33)`[ 23.5 (18-33)`[
0 (0-1) 0 (0-1) 1.0 (0-14) 8.5 (4-18)1' 18.0 (6-23)T
Experiment lb Control EPI 6.25 EPI 9.375 EPI 12.5
11.0 (9-14) 10.5 (9-12) 9.5 (7-13)`[ 8.0 (7-10)`[
14.0 (11.5-18.5) 18.0 (16-22.5) 18.0 (13-20) 16.0 (9-21)
0 (0-0) 0 (0-0) 0 (0-0) 0 (0-0)
10.0 (7-12)$ 9.5 (7--12)~ 8.0 (6--10)`[
17.5 (12-21) 18.0 (16-25)1' 21.5 (15-25)1'
0 (0-0) 0 (0-0) 0 (0-0)
PG 100 PG 200 PG 400
Values are median number of observations of each behavior class, with the first and third quartiles given in parentheses. See text for definition of behaviors. There were 60 observations in Experiment la and 40 in Experiment lb. EPI and PG doses are in t~g/kg. Arrows indicate significant increases (D or decreases (`[) in frequencies, p<0.05, compared to control data, sign tests after significant overall effects determined by median tests. Frequencies of grooming, exploring, and other behaviors were not significantly different across drug groups.
l
-
5c _z :~
4c
3c
~
2o
0 6.25
9.375
12.5 DOSE EF'I ( r n c ~ )
25
50
FIG. 1. Reduction of MS after EPI injection. Data are mean±SE percent inhibition MS as compared to control for 16 rats in Experiment la and 22 rats in Experiment lb. Mean control MS were 13.9+_3.7 ml for Experiment la and 19.3-+1.1 ml in Experiment lb. *p<0.05, **p<0.01 post hoc t-test, after overall ANOVA. ANOVA effects: F(4,60)=17.99, p<0.01, in Experiment la and F(5,105)=3.63, p<0.01, in Experiment lb.
during the test period when pellets were removed. Temperature was maintained between 22° and 26°C. Rats were adapted to handling and intraperitoneal injections of 1 ml/kg 0.9% NaCI near the middle of the light phase of a 14:10 LD cycle. Evaporated milk (Carnation, Los Angeles, CA) was presented in calibrated (_-.1 ml) drinking tubes (Wahmann,
Timonium, MD) immediately after the injections. Milk intake was measured for 60 minutes. An experimenter blind to the injection protocol observed each rat's behavior once a minute during tone-signalled 0.6 sec observation periods for 40 to 60 min [8,11], Behaviors were classified using operational definitions and recorded. F o r example, grooming was defined as biting or licking the coat, paws, genitals, or tail or scratching or stroking the head, face, or vibrassae with one or both paws. Resting was defined as a stationary position in which the abdomen is on the floor of the cage and no other behavior is displayed. Because pilot studies indicated that rats sometimes displayed anomalous behaviors following EPI injections, operational definitions for these were developed. Three anomalous behaviors occurred: crawling--locomotor behavior with spine dorsoflexed, limbs extended, and abdomen pressed against cage floor; s u p i n e - - a stationary position with the rat on its back or side with all four limbs extended; and p r o n e - - a stationary position with abdomen on the cage floor, limbs extended rostrally and caudally, and spine dorsoflexed. These behaviors were rated conservatively; if they appeared simultaneously with any other behavior (such as s ~ ) , the other behavior was given precedence in rating. Experiments began when for at least 80% o f the rats, the standard deviation of the mean milk intake for five consecutive adaptation days was less than 30% of the mean. Experiments were done Tuesdays through Fridays; the rats were readapted Mondays. Two similar experiments were done. In Experiment la, 16 rats were each intraperitoneally injected with 6.25, 12.5, 25 or 50/zg/kg EPI (epinephrine hydrochloride, Parke Davis,
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY Morris Plains, NJ), 50 100, 200 or 400/zg/kg PG (Eli Lilly Research Laboratories, Indianapolis, IN), and 3.0% dimethylsulfoxide-0.9% NaC1 on six days as a control. Crystalline PG was dissolved first in DMSO and then diluted with saline to achieve the desired concentration. EPI was dissolved in premixed DMSO-saline. All injections were 1.0 ml/kg body weight. The order of tests was random for each rat. Because the PG was ineffective in Experiment la, we compared the batch used to a fresh batch and discovered the PG used was no longer active. Therefore, only the EPI data were analyzed. In Experiment lb 22 rats each received 6.25, 9.375 or 12.5 /~g/kg EPI, 100, 200 or 400/~g/kg of new PG, and two 3.0% DMSO-0.9% NaCl controls in random order. Lower doses of EPI were used to obtain effects comparable to those expected to be elicited by PG. Meal size (MS) was defined as the amount of milk ingested during the initial postinjection bout of feeding before the rats displayed the behavioral satiety sequence ending in at least four consecutive observations of resting. This was identical to 30 min milk intake. Rats seldom ate again before the end of the test. Percent inhibition of MS was defined as 100 - [100 (MS after drug injection)/(mean MS of control tests)]. Effects on MS were analyzed with repeated measures ANOVA and post-hoc t-tests [13]. Behavioral observations were grouped into categories of feeding, grooming, exploring (including sniffing, locomotion, rearing, and licking cage), resting, anomalous behaviors, and all other behaviors (such as standing in a stationary position with abdomen off the cage floor, urinating, defecating, yawning, etc.). The number of times behaviors from each category occurred was compiled, and changes in the frequency of occurrence were analyzed with median and sign tests [38]. Results
Rats fed immediately after milk presentation, but ingested less after EPI or PG injection than after control injections. EPI elicited a dose-related inhibition of MS (Fig. 1). 6.25 /~g/kg EPI decreased MS significantly in Experiment lb, but not in Experiment la. 50/xg/kg EPI, the largest dose tested, decreased MS more than 50%. After control injections, rats fed almost continuously for about 10 minutes. Then they displayed mainly grooming and exploratory behaviors before finally resting or sleeping. This behavioral satiety sequence was disrupted by EPI. EPIinjected rats fed less, but failed to rest more (Table 1). Rather, after 25-50 p.g/kg EPI, resting was replaced by dramatic behavioral postures not normally associated with postprandial satiety. These anomalous behaviors accounted for up to 30% of the observations after EPI injection, as opposed to 0% of the observations after control injection. There were no consistent changes in frequencies of postprandial grooming, exploring or other behaviors (for this reason these data are not presented). Rats fed less without increasing time spent resting after 6.25-12.5/zg/kg EPI and significantly decreased time spent resting after 25-50/~g/kg EPI. Despite this, latency to rest, which was quite variable, was not significantly increased. Injection of 100--400 /~g/kg PG decreased MS 13-32% (Fig. 2). In contrast to the EPI results, decreases in MS elicited by PG were accompanied by increases in resting and were not accompanied by anomalous behaviors (Table 1). The amount of resting increased by as much as 19%. Latency to the first rest, however, was not decreased by PG. This
lll 40
< ao
} --7 z
~0
i 100
200
400
DO~ PG(moo~)
FIG. 2. Reduction in MS after PG injection. Data are mean_SE percent inhibition MS as compared to control for 22 rats in Experiment lb. Mean control MS was 19.3---1.1. *p<0.05, **p<0.01, post hoc t-test after overall significant ANOVA. ANOVA effect: F(5,105)=3.63, p<0.01.
appears to result from the variability of latency data and not from any change in the sequence of postprandial behaviors. PG also did not change the frequency of either grooming or exploring. Discussion
These results dissociate the effects of exogenous EPI and PG on feeding and periprandial behavior. As previously [8], PG inhibited MS without altering the normal behavioral sequence of postprandial satiety. The only behavioral changes elicited by PG were that rats ate smaller, shorter meals and increased the time spent resting. This is consistent with the hypothesis that PG inhibits feeding by activating a specific satiety mechanism. In contrast, EPI-injected rats did not display the normal behavioral satiety sequence. Doses of 6.25-12.5/~g/kg EPI decreased MS 13-42% but did not increase the frequency of resting, whereas doses of PG that inhibited meal size 25% or more increased the frequency of resting. These data do not clearly indicate whether doses of 6.25-12.5 EPI act specifically to inhibit feeding. 25-50/~g/kg EPI significantly reduced the incidence of resting and also elicited anomalous behaviors. This suggests that 25-50/~g/kg EPI decreases MS nonspecifically by eliciting behaviors that compete with feeding rather than by signalling postprandial satiety. Thus, because the postprandial satiety sequence may be considered necessary for the demonstration of satiety under these test conditions, we conclude that the inhibitory action of feeding of 25-50/~g/kg EPI is a nonspecific anorectic effect, not a satiety effect. This calls into question the specificity of previously reported inhibitory effects, which with only two exceptions [2,36], involved doses larger than ours [24, 27, 30, 35, 36]. In contrast to EPI, no doses of PG have elicited such behavior in this or similar tests [8,9]. This functional dissociation suggests that PG and EPI act differently to inhibit meal size. EXPERIMENT 2
Hypothesized satiety signals including PG [8,21] and cholecystokinln (CCK) [1] inhibit feeding but do not inhibit water intake in water deprived rats. In order to determine whether EPI's inhibitory action also selectively inhibits feed-
112
HINTON ET AL. TABLE 2 DIFFERENTEFFECTSOF EPI AND PG ON WATERINTAKEANDBEHAVIORSDURINGAND AFTER DRINKING Inhibition of Water Intake
Drinking
Frequency Resting
Anomalous
Control EPI 6.25
9.3 _+ 4.4
9.0 (8--10) 7.5 (9--11)
2.0 (0--5) 4.5 (2-8)
0 0
(0-0) (0-0)
Control EPI 12.5
1.5 +_ 5.1
9.0 (7-10) 9.0 (8-12)
2.0 (2-4) 9.5 (3-17)
0 0
(0-0) (0-0)
Control EPI 25 PG 100
1.3 --_ 17.5 0.7 -+ 20.0
6.0 (4-8) 7.0 (5-8) 7.0 (5-9)
3.0 (1-8) 3.0 (1-9) 2.0 (0-9)
0 (0-1) 0 (0-0) 0 (0-1)
Control EPI 50 PG 200
30.0 _ 8.8* 18.5 ± 20.8
6.5 (4-8) 3.0 (3-6)$ 6.5 (6--7)
1.0 (0-2) 9.5 (3-12)~" 1.5 (0-5)
0 (0-0) 2.0 (0-6)1' 0 (0-1)
Control EPI 100 PG 400
37.7 -+ 8.9* 6.9 ± 9.9
6.0 (4-7) 3.0 (2-5)~ 8.0 (7-10)]'
2.0 (0-9) 9.0 (3-11)1' 3.0 (0-6)
0 (0-1) 3.0 (0-9)1" 0 (0-1)
Control EPI 200 PG 800
76.7 ± 5.3* -5.4+ 11.7
6.0 (6-8) 1.0 (0-2)+ 8.0(6-11)
1.0 (0-5) 11.0 (8--12)'r 8.0(3-11)
0 (0-0) 12.0 (6-17)1, 0 (0-1)
Inhibitions are % of water intake after control tests. Behavior frequencies are median number of 40 total observations, with the first and third quartiles given in parentheses. See text for definitions of behaviors. *Significant inhibition of water intake, post hoc t-test, p<0.01, after significant ANOVA. Arrows indicate significant increases (1') or decreases (~,)in frequencies of behaviors, sign test, p <0.05. Frequencies of grooming, exploring, and other behaviors were not significantly different across drug groups.
Food was always available except during the first hour of the drinking period. Injections were done just before water presentation, and water intake and behaviors were measured as previously. This schedule elicited initial post-deprivation water intakes (10-22 ml) that were similar to the mean control milk meal sizes (14-19 ml) in Experiment 1. Also, under these conditions, rats drank immediately upon water presentation, and the post-deprivation bouts of drinking were similar in length to milk meals. EPI (6.25-200 t~g/kg) and 100-800 /~g/kg PG were prepared as in Experiment 1 and compared to paired control injections in separate experiments.
80 70
,[xx;~
60 50 40 30 20 10 0
I
6.25
12.5
25 50 DOSE EPI (mog,~)
100
Results
200
FIG. 3. Inhibitionsof 30 min water intake in Experiment 2. Data are mean+SE percent inhibition as compared to control for 15 rats in separate saline-paired tests. Mean control intakes were 10.4+ 1.2 to 21.6-+1.2 ml. **p<0.01, post hoc t-test, after significant ANOVA.
ing, we compared the effects of EPI and PG on water intake in thirsty rats. Method Fifteen rats were adapted for one month to 6 hr/day of water access, beginning near the middle of the light phase.
Injection of 50-200/xg/kg EPI inhibited water intake up to 77% (Fig. 3). These inhibitions were accompanied by the same anomalous behaviors that EPI elicited in the feeding tests (Table 2). These EPI doses also signifuzantly increased resting behavior. Resting accounted for 2.5-5% of the observations after control injection and 22.5-28% after 50-200 /,g/kg EPI, p's<0.05. Doses of 6.25-25 t~g/lq~ EPI did not inhibit water intake, although there was a nonsignificant trend for resting to increase. Resting accounted for 5-8% of the observations after control injection and 8--24% after 6.25-25 t~g/kg EPI (p's<0.06). No dose of PG tested affected water intake (inhibitions of 30 minute water intake ranged from - 5 . 4 + 1 1 . 7 to 18.5_+20.8%, (p's>0.05) or the frequency of any behavior.
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY
113
Discussion
The demonstration that 50-200/tg/kg EPI inhibits water intake indicates that EPI's inhibitory effect on ingestive behavior is not specific to feeding. The smallest dose that inhibited water intake (50/zg/kg EPI) was, however, larger than the smallest doses that inhibited feeding (12.5/xg/kg EPI in Experiment la and 6.25 /~g/kg EPI in Experiment lb). Thus, EPI may have a relative specificity of action on feeding in the very low dose range. Our results contrast with Rodriguez et al.'s [27] report that 125-500/zg/kg EPI does not significantly inhibit water intake in 6 hour food and water deprived adult rats. But their test was not compelling. The amount drunk in their control condition was so small, only about 4.4-5.3 ml, that inhibitory effects may have been difficult to observe. Further, this amount of water intake was less than a third of the control milk intake in the feeding test (14.4-17.3 ml), so the drinking test did not closely mimic the feeding test. Rodriguez et al. [27] also demonstrated that EPI did inhibit water intake in 3 to 10 day old rat pups as potently as it inhibited milk intake in a situation in which the control intakes were comparable. Thus, when similar amounts of water and liquid food were ingested, EPI inhibited both feeding and drinking, just as we demonstrated here. As previously [8], PG had no effect on water intake. PG also failed to affect the behaviors associated with water ingestion. Therefore, the effects of EPI and PG on water intake are dissociable. Doses of 50-200/zg/kg EPI inhibit drinking and elicit unusual behavior patterns. PG neither inhibits drinking nor elicits unusual behaviors. This indicates that EPI, unlike PG, is a nonspecific agent that can elicit adipsia as well as anorexia. EXPERIMENT
3
Experiments 1 and 2 demonstrated that EPI elicits similar anomalous behaviors in feeding and drinking tests. To determine whether these behaviors were dependent on ingestive responses, we tested EPI's behavioral effects in the absence of feeding or drinking. Method
Eighteen rats were maintained with ad lib access to food and water except during a 40 min daily test period. Rats were each intraperitoneally injected with either 3% DMSO-0.9% NaCI or 6.25, 12.5, 25 or 50/~g/kg EPI in random order, and behaviors were rated as previously. Results
50 t~g/kg EPI elicited the same anomalous behaviors that occurred during the feeding and drinking tests. Anomalous behaviors did not occur after control injections but accounted for a median frequency (lst-3rd quartile) of 40 (2644)% of the behavioral observations (z=3.76, p<0.01) after 50/zg/kg EPI. Anomalous behaviors accounted for 25 (1750)% of the observations after 25 ttg/kg EPI, but this was not significant (z= 1.00, p>0.05). Discussion
EPI eficited anomalous behaviors in rats that were neither feeding nor drinking. Thus, ingestion of food or water is not necessary for the expression of this behavioral effect of EPI. This is consistent with the hypothesis that EPI inhibits feeding and drinking nonspecifically by eficiting competing be-
70 6O Z
50
10 0 6.25
12.5 25 DOSEEPI(rrog4~)
50
FIG. 4. Reduction of 60 min sham milk intake after EPI injection in Experiment 4. Data are mean__+SE percent inhibition as compared to control for 7 to 9 rats per test. Mean control intakes were 82.6±9.4 to 103___8.7ml. **p<0.01, t-test. haviors that do not normally contribute to the inhibition of ingestion, but interfere with normal ingestive behavior. EXPERIMENT
4
To compare further the inhibitory actions of EPI and PG, we tested EPI's effect on rats sham feeding with open gastric cannulas. PG does not inhibit sham feeding [9,17]. Therefore, if EPI and PG inhibit feeding by the same mechanism, EPI should also fail to inhibit sham feeding. Method
Eleven rats were implanted with chronic gastric cannulas using a previously described method [1,9]. Rats were adapted one month to testing conditions. Pelleted chow was removed from the cages 12 hours prior to injection. The cannulas were opened just before the tests, and each rat's stomach was rinsed with warm 0.9% saline until no food was detected in the drainage. One ml/kg saline was intraperitoneally injected and a plastic collection tube was attached to the cannula. The tubes drained into a collection pan below the cage. After each test, the volume collected was measured. Data from tests in which this volume was less than the rat's intake volume were not used. Doses of 6.25-50/~g/kg EPI were compared to saline control injections according to separate crossover designs. Milk intake and behaviors were observed for 60 rain. Data analysis was done for the 6--9 rats in each test that met the criterion for successful sham feeding on each test day. Sixty min sham intakes were used for the analysis because in control conditions rats often did not stop sham feeding until after 30 min. Results
Doses of 6.25-12.5/~g/kg EPI did not detectably affect sham intake or behavior, but 25 and 50/~g/kg EPI each inhibited sham intake more than 60% (Fig. 4). Anomalous behaviors did not occur after control injections but were emitted by five of the six rats with 25/~g/kg EPI [median frequency (lst-3rd quartile), 48 (14-56)%, z= 1.79, p>0.05] and by six of the eight rat tested with 50/zg/EPI [3 (3-30)%, z=2.04, p<0.05]. As in the real feeding test (Experiment 1), rats fed less after 25-50/~g/kg EPI (75 and 50% decreases,
114
HINTON E T A L .
z's=2.05, 2.44, p's<0.05), but failed to rest more (z's= 1.38, 1.24, p's>0.05). Discussion
In previous studies, sham feeding was not inhibited by doses of up to 2,500/zg/kg PG, despite that this is 16-100 times higher than the doses that inhibit real feeding in similar tests [9,17]. This suggests that PG's satiety effect depends on some postgastric food stimulus that is absent during sham feeding. In contrast, EPI doses only slightly higher than those necessary to inhibit real feeding potently inhibited sham feeding. This suggests that EPI's inhibitory action, unlike PG's, does not require postgastric food stimuli. 100-2,500 /zg/kg PG also failed to elicit anomalous behaviors [9,17], whereas doses of EPI that inhibited sham feeding did elicit such behaviors. These distinctions are further evidence that EPI and PG do not inhibit feeding by the same mechanism. GENERALDISCUSSION Under the conditions of our tests, normal postprandial satiety in rats is associated with a specific sequence of behaviors that includes grooming and exploratory behavior and terminates in rest or sleep [1, 4, 20, 25, 34]. A satiety agent should decrease meal size without disrupting this satiety sequence. Intraperitoneal injection of 25-50 /xg/kg EPI potently decreased meal size, but also disrupted the satiety sequence by eliciting anomalous behaviors that are not associated with normal meals. A selective satiety agent also should not affect other ingestive behavior, but 50--200/zg/kg EPI inhibited drinking as well as feeding. Therefore, we conclude that in this dose range EPI is a nonspecific anorectic and adipsic agent, not a satiety agent. The same anomalous behaviors that were elicited by EPI in feeding tests also occurred when EPI was administered to rats drinking water and to rats that were neither feeding nor drinking. Thus, these behaviors can be elicited independently of EPI's inhibitory action on feeding. This is consistent with the hypothesis that EPI inhibits ingestive behavior nonspecifically by eliciting competing behaviors that interfere with ingestion. The EPI doses that elicited nonspecific effects are 2 to 3 times smaller than those used in most previous investigations of EPI's effects on feeding [24, 27, 30, 35, 36]. Only two of those studies included tests of the hypothesis that EPI has a specific satiety effect. The evidence produced is at best ambiguous. As discussed above, Rodriguez et al. [27] failed to detect an effect of 125-500 tzg/kg EPI on water intake in adult rats, but the amount of water drunk in their tests was much less than the amount of liquid food ingested. And these EPI doses did inhibit water intake in rat pups. Russek and Racotta [31] noted that EPI elicits postprandial decreases in activity in rats tested in jiggle cages and suggested that this might reflect normal postprandial sedation. Our data suggest this decrease in activity might have been due to the appearance of anomalous stationary postures. Finally, because the anomalous behaviors we observed also occur in the absence of feeding, our data suggest they do not reflect "oversatiation" as has been suggested by Russek [29,31] to account for the observation that vomiting sometimes accompanies EPI-anorexia in dogs. Therefore, because inhibition of feeding alone is not sufficient evidence for postprandial satiety,
we conclude that the literature provides no direct support for the hypothesis that EPI elicits satiety. The smallest EPI doses that we tested did inhibit feeding without eliciting nonspecific behavioral effects. Thus, it is possible that EPI may act specifically at smaller doses and nonspecifically only at doses larger than 25/zg/kg. This requires further investigation, however, because there were suggestions of nonspecificity in the lower dose range. First, in Experiment 1, doses of 12.5-25/zg/kg EPI inhibited feeding as much or more than 200-400 ~g/kg PG, but only PG and not EPI increased resting. Higher doses of EPI elicited decreases in the frequency of resting behavior. Second, in the drinking test there was a trend toward an anomalous increase in the frequency of resting after smaller doses of EPI, and larger doses significantly increased the frequency of resting. Control injected rats continued to be active after drinking until they were fed at the end of the test. Since the anomalous behaviors consist largely of stationary postures, it is possible this EPI-induced increase in resting in the drinking test represents a lower intensity of the same response. PG, in contrast, did not increase resting. Thus, whether any EPI treatment elicits a behaviorally specific inhibition of feeding remains unclear. The nonspecific effects of EPI that we observed indicate that EPI's inhibitory action on feeding is functionally dissimilar from PG's. In contrast to EPI, PG did not disrupt the behavioral satiety sequence, elicit anomalous behaviors, or inhibit water intake [5, 8, 16, 37]. Thus, PG fulfills several behavioral criteria for a postprandial satiety agent that EPI does not. The sham feeding test in Experiment 4 further dissociates PG and EPI. EPI inhibited sham feeding whereas PG has consistently failed to [9,17]. It has been hypothesized that the inhibitory actions of PG and EPI are initiated by a single peripheral neural mechanism that is sensitive to their common effect on hepatic carbohydrate metabolism [22, 24, 29, 31, 35, 36]. This hypothesis predicts that the behavioral characteristics of the two agents' inhibitory effects should be similar. Therefore, the behavioral dissociations that we report here suggest that EPI and PG do not activate a single mechanism to inhibit feeding. This complements more direct lines of evidence against this hypothesis. For example, the stimulatory effects of both PG [2, 7-9, 15] and EPI [2,14] on hepatic glycogenolysis and hepatic glucose production have been dissociated from their inhibitory effects on feeding. This fails to support the hypothesis that hepatic carbohydrate metabolism mediates the agents' feeding effects. Further, while hepatic vagotomy [10,19], total liver denervation [18], and intrahepatic injection of alloxan [26] have sometimes blocked PG's satiety effect, none of these treatments affects EPI anorexia [18, 19, 26]. This suggests that PG's satiety effect and EPI's anorectic effect are mediated by different peripheral neural mechanisms.
ACKNOWLEDGEMENTS A preliminary report of these data was given at the 55th Annual Meeting of the Eastern PsychologicalAssociation, Baltimore, MD, March, 1984 [5]. This research was supported by N.I.H. Research Grant AM 32448to N.G. We thank Michael Krumper, Tom Comaechia and Amy Ventry for help with the experiments.
EPINEPHRINE INHIBITS FEEDING NONSPECIFICALLY
115
REFERENCES 1. Antin, J., J. Gibbs, J. Holt, R. C. Young and (3. P. Smith. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. J Comp Physiol Psychol 89: 784-790, 1975. 2. Bellinger, L. L. and F. E. Williams. (31ucngon and epinephrine suppression of food intake in liver-denervated rats. Am J Physiol 251: R349-358, 1986. 3. Crawley, J. N. Divergent effects of cholecystokinin, bombesin, and lithium on rat exploratory behaviors. Peptides 4: 405-410, 1983. 4. Danguir, J., S. Nicolaidis and H. Gerard. Relations between feeding and sleep patterns in the rat. J Comp Physiol Psycho193: 820-830, 1979. 5. Esguerra, M., N. Farhoody, V. Hinton and N. Geary. Epinephrine inhibits feeding nonspecifically in the rat. Proc EPA 55: 121, 1984. 6. (3eary, N., N. Farhoody and A. Gersony. Food deprivation dissociates pancreatic glucagon's effects on satiety and hepatic glucose production. Physiol Behav 39: 507-511, 1987. 7. Gear/, N., W. Langhans and E. Scharrer. Metabolic concomitants of glucagon-induced suppression of feeding in the rat. Am J Physiol 241: E330-E335, 1981. 8. (3eary, N. and G. P. Smith. Pancreatic glucagon and postprandial satiety in the rat. Physiol Behav 28: 313-322, 1982. 9. Geary, N. and G. P. Smith. Pancreatic glucagon fails to inhibit sham feeding in the rat. Peptides 3: 163-166, 1982. 10. Geary, N. and G. P. Smith. Selective hepatic vagotomy blocks pancreatic glucagon's satiety effect. Physiol Behav 31: 391-394, 1983. 11. Gibbs, J., L. Gray, C. F. Martin, W. T. Lhamon and J. A. Stuckey. Quantitative behavioral analysis of neuropeptides which suppress food intake. Soc Neurosci Abstr 6: 530, 1980. 12. Gibbs, J. and (3. P. Smith. The neuroendocrinology of postprandial satiety. In: Frontiers in Neuroendocrinology, vol 8, edited by L. Martini and W. F. Ganong. New York: Raven Press, 1984, pp. 223-245. 13. Hays, W. L. Statistics for Psychologists. New York: Holt, Rinehart and Winston, 1963. 14. Langhans, W., K. Pantel and E. Scharrer. Dissociation of epinephrine's hyperglycemic and anorectic effect. Physiol Behav 34: 457-463, 1985. 15. Langhans, W., E. Scharrer and N. Geary. Pancreatic glucagon's effect on satiety and hepatic glucose production are independently affected by diet composition. Physiol Behav 36: 483487, 1986. 16. Langhans, W., U. Zeigler, E. Scharrer and N. Geary. Stimulation of feeding in rats by intraperitoneal injection of ~mtibodies to glucagon. Science 218: 894-896, 1982. 17. Le Sauter, J. and N. Geary. Pancreatic glucagon corr/bines with cholecystokinin to inhibit sham feeding in the rat. Am J Physiol, submitted, 1986. 18. MacIssac, L. and N. Geary. Hepatic vagotomy or total liver denervation can block pancreatic glucagon's satiety effect. Soc Neurosci Abstr 15: 58, 1985. 19. MacIssac, L. and N. (3eary. Partial liver denervations dissociate the inhibitory effects of pancreatic glucagon and epinephrine on feeding. Physiol Behav 35: 233--237, 1985.
20. Mansbach, R. S. and D. Lorenz. Cholecystokinin (CCK-8) elicits prandial sleep in rats. Physiol Behav 30: 179-183, 1983. 21. Martin, J. R. and D, Novin. Decreased feeding in rats following hepatic-portal infusion of glucagon. Physiol Behav 19: 461-466, 1977. 22. Novin, D., K. Robinson, L. A. Culbreth and M. (3. Tordoff. Is there a role for the liver in the control of food intake?Am J Clin Nutr 42: 1050-1062, 1985. 23. Penick, S. B. and (3. P. Smith. The effects of glucagon on food intake and body weight in man. J Obesity 1: 1-5, 1964. 24. Racotta, R., M. Islas-Chaires, C. Vega, M. Soto-Mora and M. Russek. Glycogenolytic substances, hepatic and systemic lactate, and food intake in rats. Am J Physiol 246: R247-R250, 1984. 25. Richter, C. A behavioristic study of the activity of the rat. Comp Psychol Monogr 1: 1-55, 1922. 26. Ritter, S., S. C. Weatherford and S. L. Stone. Glucagoninduced inhibition of feeding is impaired by hepatic portal alloxan injection. Am J Physiol 250: R682-R690, 1986. 27. Rodriguez-Zendejas, A. M., (3. Chambert, M. C. Lora-Vilchis, A. Epstein and M. Russek. Ontogeny of epinephrine-induced anorexia in rats. Am J Physiol 250: 313-317, 1986. 28. Rosofsky, M. and N. Geary. Specific and nonspecific effects of phenylpropanolamine and amphetamine on feeding in rats. Proc EPA 57: 57, 1986. 29. Russek, M. Current status of the hepatostatic theory of food intake control. Appetite 2: 137-143, 1981. 30. Russek, M., G. J. Mogenson and J. A. F. Stevenson. Calorigenic, hyperglycemic and anorexigenic effects of adrenaline and noradrenaline. Physiol Behav 2: 429-433, 1967. 31. Russek, M. and R. Racotta. A possible role of adrenaline and glucagon in the control of food intake. Front Horm Res 6: 120137, 1980. 32. Russek, M., A. M. Rodriguez-Zendejas and S. Pina. Hypothetical liver receptors and the anorexia caused by adrenaline and glucose. Physiol Behav 3: 249-257, 1968. 33. Russek, M. and J. A. F. Stevenson. Correlation between the effects of several substances on food intake and on the hepatic concentration of reducing sugars. Physiol Behav 8: 245-249, 1972. 34. Smith, G. P. and J. Gibbs. Postprandial satiety. In: Progress in Psychobiology and Physiological Psychology, vol 8, edited by J. Sprague and A. E. Epstein. New York: Academic, 1979, pp. 179-242. 35. Tordoff, M. G. and D. Novin. Celiac vagotomy attenuates the ingestive responses to epinephrine and hypertonic saline but not insulin, 2-deoxy-d-glucose, or polyethelene glycol. Physiol Behav 29: 605-613, 1982. 36. Tordoff, M. G., D. Novin and M. Russek. Effects of hepatic denervation on the anorexic response to epinephrine, amphetamine, and lithium chloride: a behavioral identification of glucostatic afferents. J Comp Physiol Psycho196: 361-375, 1982. 37. Weick, B. (3. and S. Ritter. Dose-related suppression of feeding by intraportal glucagon infusions in the rat. Am J Physiol 250: R676-R681, 1986. 38. Winer, B. J. Statistical Principles in Experimental Design, 2nd edition. New York: McGraw-Hill, 1971.