An Alternative Explanation for Apparent Satiating ... - Science Direct

Physiology & Behavior, Vol. 39, pp. 191-202.Copyright©PergamonJournals Ltd., 1987.Printedin the U.S.A.

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An Alternative Explanation for Apparent Satiating Properties of Peripherally Administered Bombesin and Cholecystokinin in Domestic Fowls C. J O H N S A V O R Y Agricultural and F o o d Research Council's Poultry Research Centre Roslin, Midlothian EH25 9PS, Scotland R e c e i v e d 21 O c t o b e r 1985 SAVORY, C. J. An alternative explanation for apparent satiating properties of peripherally administered bombesin and cholecystokinin in domesticfowls. PHYSIOL BEHAV 39(2) 191-202, 1987.--This paper describes a series of experiments with domestic fowls designed to investigate different effects of peripherally administered bombesin (BBS) and cholecystokinin octapeptide (CCK8), with a view to gaining a better understanding of their proposed roles as physiological satiety signals. Following intravenous injections of 1-10 txg/kg BBS or CCK8, short periods of complete inhibition of feeding coincided with periods of abnormal gizzard motility, and longer periods of reduced feeding were associated with periods of abnormal gastrointestinal (GI) motility. Increases in heart rate, measured in a separate experiment, coincided with the periods of altered GI and feeding activities. Effects of the peptides on GI motility and feeding were strongly related to dose with CCK8, but not with BBS, and effects of (2 ~g/kg) BBS and CCK8 on feeding were additive. Evidence from conditioned avoidance tests suggested that consequences of (10/zg/kg) BBS and CCK8 injections may be mildly aversive, perhaps more so with BBS, and in another experiment inhibition of feeding by (8/~g/kg) BBS (but not CCKS) was almost abolished in birds tranquillised with a reserpine derivative drug. CCK8 was more potent than BBS at suppressing feeding only when relatively high doses (8 and 10/xg/kg) were injected. It is suggested that peripherally administered BBS and CCK8 may act on feeding in similar ways, with animals being distracted by possible abdominal discomfort associated with the abnormal GI responses. The results indicate that immediate discomfort may be more severe with BBS, but that discomfort associated with CCK8 may last longer. It is further suggested that satiating properties of the peptides are less apparent in situations where animals are less easily distracted by discomfort, when their arousal is reduced, when their perception of discomfort is reduced and when their motivation to feed is increased. Bombesin Heart rate

Cholecystokinin Food intake Feeding activity Satiety Conditioned avoidance Tranquillisers Domestic fowl

BOMBESIN (BBS) and cholecystokinin (CCK) have received much attention in recent years as putative physiological satiety factors. Both are among the growing number of polypeptides that have a dual distribution in the brain and in neurons and endocrine cells in the alimentary tract. They have a wide variety of biological actions, and can function in endocrine, paracrine, neurotransmitter and neuroendocrine roles [11]. As with various mammals, they have short-term inhibitory effects on feeding when administered centrally (intracerebroventricularly) or peripherally (intravenously) in domestic fowls ([48, 50-52]; Savory and Gentle, unpublished data). The purpose of this paper is to examine in more detail the nature and interpretation of effects of peripheral administration of exogenous BBS and CCK in fowls. In the avian gut, there is high BBS-like immunoreactivity in the proventriculus and gizzard (glandular and muscular stomachs), and high CCK-like immunoreactivity in the intestine from the antrum to the ileum [10, 20, 46, 59]. The

Gastrointestinal motility

assumption is that BBS and CCK released in the gut in response to passage of food may provide preabsorptive signals contributing to termination of meals. However, this implies an intermittent rather than a regular flow of food through the stomach and upper intestine, which in herbivorous birds seems unlikely in view of their brief and frequent meals, and the presence of a crop which allows food to be stored in the oesophageal region [15,47]. With mammals, the claims that (peripheral) BBS and CCK may act as satiety signals are based on evidence that their effects are dose-related, they elicit behaviour characteristic of satiety, treated animals drink normal amounts after water deprivation, do not appear ill and are not hyperthermic [1, 23, 24, 26, 29, 30, 39]. Some authors, however, have reported that BBS and CCK produce conditioned (taste and place) aversions in rats [7, 9, 58], though others have found no such effect [29, 30, 36]. There is also evidence that rats habituate to repeated injections and become tolerant to

191

192

SAVORY TABLE 1 MEAN FOOD INTAKE (g MASH) AFTER NON-INJECTION, SALINE, BBS AND CCK8 TREATMENTS (ALL DOSES COMBINED) Non-

10.00-10.15 10.15-10.30 10.30-11.00

Injection

Saline

BBS

CCK8

SED~

SEDz

9.2* 5.0*+ 7.3*

9.3* 5.0*t 6. It

8. It 4.7* 7.5*

7.8t 5.6t 7.7*

0.6 0.6 0.6

0.4 0.4 0.4

*tin each time period, treatment means having the same superscript do not differ significantly (/9>0.05, by analyses of variance). SED~ is the standard error of the difference between non-injection and any other mean, and SED2 is the standard error of the difference betweeen saline, BBS and CCK8 means (both SEDs have 72 degrees of freedom).

chronic infusion of CCK [5, 41,62]. After injections of CCK, patterns of gastrointestinal (GI) myoelectric activity and duodenal motility are quite unlike those seen during normal satiety [8,63], exogenous CCK may suppress food intake by inhibiting gastric emptying [43], and in man abdominal cramps and nausea have been reported after injections of quite a small dose [57]. Other authors, too, have suggested that the reduced feeding observed following CCK administration is due to aversive consequences and not satiety [21]. Like CCK, exogenous BBS also causes abnormal GI motility in mammals [2, 40, 64], and it is even possible that BBS may stimulate release of CCK [19]. Domestic fowls do not really show behaviour that is characteristic of satiety, and do not appear ill after injections of BBS or CCK. In turkeys and fowls, however, intravenous (IV) injections of 0.5-15 /zg/kg body weight of CCK octapeptide (CCK8) cause gizzard inhibition together with increased activity in the upper intestine, and it has been suggested that this abnormal motility may cause abdominal discomfort sufficient to distract birds from feeding [49]. There is evidence that such discomfort may be less distracting when birds have greater motivation to eat [50]. In order to obtain more information on how BBS and CCK might act on feeding behaviour, this paper describes a series of experiments designed to examine associations between various responses to IV injections of the peptides, and possible aversive properties.

E X P E R I M E N T 1 (DOSE R E S P O N S E TEST) Although it has been established that IV injections of CCK8 inhibit feeding in a dose-related way in fowls [48,50], a similar dose response test has not been done with BBS. The first experiment, therefore, tested feeding responses to three doses of BBS and CCK8 in order to compare dose effects and relative potencies of the two peptides. Procedure

Ten immature medium-hybrid hens (Rhode Island Red x Light Sussex) were housed and tested in individual cages in a room illuminated from 07.00-21.00 hr and maintained at about 20°C. They were given access to a standard layers' mash diet (for composition see [52], 16% protein, 11 MJ/kg metabolisable energy) from 10.00-16.00 hr each day, having

been trained to this schedule for a week before testing started. They were tested for two weeks from 15 to 17 weeks of age, when their mean body weights increased from 1.36_+0.05 (SE) to 1.50_+0.06 kg. Food consumption was measured in the periods 10.00-10.15, 10.15-10.30 and 10.30-11.00 hr every day from Monday to Friday. On these days at 10.00 hr, immediately before receiving its food, each bird was injected by wing vein with either 1, 5 or 10 tzg/kg of BBS (Sigma, London) or CCK8 (Squibb Institute for Medical Research, N J), both dissolved in 0.9% NaCI solution (10 /xg/ml), or equivalent volumes of the 0.9% saline, or was handled as for injection but not injected. Every bird received each of the 10 treatments (three each of BBS, CCK8 and saline, and one non-injection) once, in random order, according to a Latin square arrangement. Results

In the first 15 min access to food, after 18 hr deprivation, IV injections of BBS and CCK8 caused significant reductions in intake compared to that after saline and noninjection treatments (Table 1). There was no significant difference between BBS and CCK8 treatments in the first 15 min, but birds ate more after CCK8 than after BBS in the next 15 min, and less after saline than other treatments in the last 30 min of testing. In Fig. 1, food intakes in the first 15 min, with the three doses of saline, BBS and CCK8, are compared proportionally with the non-injection treatment. The reductions in feeding with BBS and CCK8 increased with dose. The CCK8 response then was strongly related to dose (linear regression coefficient, p0.05, by analyses of variance). SED is the standard error of the difference between means (with 32 degrees of freedom).

weeks old and weighed 1.56---0.06 kg. They were kept in the same conditions as before, with the same 10.00-16.00 hr feeding schedule, and food intake was measured from 10.00-10.15, 10.15-10.30 and 10.30-11.00 hr every day from Monday to Friday. On these days at 10.00 hr, immediately before receiving its food, each bird was injected by wing vein with either 2/zg/kg BBS or 2/xg/kg CCK8, both dissolved in 0.9% saline (5/zg/ml), or 2/xg/kg BBS plus 2/zg/kg CCK8 in an equal mixture of the two peptides ( 10 /zg/ml), or an equivalent volume of saline, or was handled but not injected. On a body weight basis, therefore, all injections were isovolaemic. Every bird received each of the five treatments once, in random order, according to a Latin square arrangement, with two birds receiving each treatment on each day. Results As in Experiment 1, BBS and CCK8 caused significant reductions in food intake in the first 15 min after IV injection, compared with saline and non-injection treatments (Table 3). There were no differences in intake between treatments in the remaining 45 min of testing. In the first 15 rain the combined peptide treatment (2 + 2 /zg/kg) suppressed feeding more than did BBS or CCK8 alone (both 2 /zg/kg). The additivity of BBS and CCK8 was tested by analysis of vari-

ance, with the null hypothesis that there was no interaction between the two peptides when combined, and hence that the reduction in intake (relative to the saline or non-injection controls) with the combined treatment was equal to the sum of the reductions with BBS and CCK8 alone. This null hypothesis was not rejected with either the saline treatment comparison, t(32)=0.35, NS, or the non-injection comparison ( t = - 0 . 1 4 ) , indicating that, as with rats, effects of BBS and CCK8 on feeding (with this dose) are additive in fowls. EXPERIMENT 3 (GI MOTILITY AND F E E D I N G ACTIVITY RESPONSES) The ways in which the avian gut respond to IV injections of CCK have already been described [49], but GI responses to BBS have not. The third experiment investigated the association between feeding and GI responses to the peptides in two ways, by observing the alimentary tract with an X-ray image intensifier and by making simultaneous recordings of GI motility and feeding activity. RADIOGRAPHICVIDEORECORDINGS Procedure Four immature medium-hybrid hens, aged 14 weeks and

194

SAVORY

TABLE 4 NUMBERS OF GIZZARDCONTRACTIONSAND DURATIONS OF ABNORMALINTESTINALMOTILITYON VIDEORECORDINGS OF GI RESPONSES OF FOUR BIRDSTO TWO IV DOSES OF BBS AND CCK8 Time Before and After Injection (min)

2/xg/kg BBS

8 tzg/kg BBS

2/xg/kg CCK8

8/xg/kg CCK8

Numbers of Gizzard Contractions -3 -2

1 1

1 2

3 3

4 3

-I

1

1

3

4

2 2 3 2

13 13 6 4 2

0 1 3 3 3

0f 2 2 3 3

2 3 4 5 6

,

changes in intestinal motility for 7 and 12 min (Table 4). The abnormal activity of the gizzard appeared as 'pulsating,' or incomplete contractions, and that of the intestine as 'rushing' of material through the duodenum and ileum, first in one direction and then the other, without any apparent coordinated propulsive contractions. This rushing was more or less continuous, and was accompanied by occasional nonpropulsive 'segmenting' contractions [28] in the upper ileum. The bird that received 8/zg/kg BBS was noticeably restless in the first minute after injection. Increased GI activity following treatment with BBS has also been found with rats and dogs [2, 40, 64]. IV injections of 2 and 8/xg/kg CCK8 caused reduced gizzard motility for 3-4 min and abnormal intestinal motility for 8 and 14 min. This abnormal motility was similar to that described previously in [49] and appeared mainly as constriction of the duodenum with segmenting contractions in the ileum, and some rushing of material (as with BBS) in the first few minutes after injection (referred to as 'refluxes' in [49]).

Duration of Abnormal Intestinal Motility (rain) 7

12

8

14

*Incomplete contractions--'pulsating.' tRemained in contracted shape.

weighing 1.20-1.48 kg, were each implanted with a permanent indwelling 8 cm cannula in the right jugular vein three days before testing, using the method described in [53]. Food was withdrawn from the birds at least 20 hr before testing. At the start of testing each bird was infused with 20 ml liquid barium sulphate contrast medium into its crop, a polyethylene tubing extension was attached to its jugular cannula for injecting peptides remotely, and it was placed in a narrow cardboard box in which it could not turn round. The box was placed on a platform in front of an X-ray machine (Siemens Triplex Optimatic Generator, energising 125 kV) linked to an image intensifier with a direct recording facility to a video cassette recorder (Panasonic NV-8200). Recordings were made of either lateral or dorso-ventral aspects of the birds' abdominal regions, both aspects being equally good for analysing GI motility. Each bird was injected 10-15 min after being put in the box, and recording continued for 20-30 min after injection. Treatments were allocated to the four birds at random. One bird received 2/zg/kg BBS, one received 8/zg/kg BBS, one 2/zg/kg CCK8 and one 8/zg/kg CCK8. The concentration of both peptide solutions was 10/xg/ml and sufficient saline was injected immediately afterwards to ensure that all the peptide entered the bird. Measurements were made later from the video cassette of numbers of gizzard contractions seen in each of the three mintues before and six minutes after injection, and of durations of abnormal (altered) intestinal motility after injection (this last measurement was somewhat subjective since abnormal motility ceased gradually rather than abruptly). With only one bird per treatment, the aim of this experiment was simply to provide information with which to interpret the subsequent GI motility recordings. Results IV injections of 2 and 8/~g/kg BBS caused increases in gizzard motility in the first 2 and 4 min respectively, and

RECORDINGSOF GI MOTILITYAND FEEDINGACTIVITY Procedure GI motility was recorded by suturing one extraluminal strain gauge transducer (SGT) to the surface of the gizzard and another to the mid-proximal duodenum in each of seven immature medium-hybrid hens, when they were aged between 12 and 14 weeks. One bird was implanted and tested at a time, over a period of many months. Preparation of SGTs (Micro-Measurements, type SA-06-125AC-350), their implantation and use in several bird species have been described in [12,14], and examples of recording traces from turkeys and fowls can be seen in [12, 14, 49]. The birds were also implanted with permanent indwelling 8 cm jugular cannulae (as in [53]) to allow remote injection of peptide solutions. When they were not being tested the birds were kept in the same housing conditions and fed ad lib on the same mash diet as described for Experiment 1. Subjects were deprived of food for 18 hr (overnight) before each recording session, and there was only one such session in a day. At the start of each test the subject's two external leads to its SGTs were linked to a four-channel recorder (Elcomatic) with conditioning amplifiers (Fylde), and an extension was attached to its jugular cannula. Wires and the tubing extension were brought out through the top of the test cage to allow the bird freedom of movement. This cage, which was in a different room, was provided with a water dispenser and had a hole in its side to allow the bird access to a food pan. The pan contained the same mash as before and it rested on a springy metal bar to which was attached another SGT; this in turn was linked to the four-channel recorder. The bird's pecking at food in the pan caused the metal bar to move, and feeding could thus be recorded simultaneously with motility of the gizzard and duodenum. After connecting up the bird the recorder chart was set to run at 5 cm/min, the bird was given 5 min to settle down, it was then given access to the food in the pan and allowed to feed for 10 rain before being injected remotely with the test solution (time of injection being indicated with an event marker), and recording was terminated 30 rain after injection. Doses of BBS and CCK8 injected were 1, 5 and 10/xg/kg (10/xg/ml), saline was injected immediately afterwards to ensure all the peptide entered the bird, and a control saline treatment was

E F F E C T S OF SATIETY PEPTIDES IN FOWLS BBS 5 p g / k g

BBS l p g / k g

0L

L5

I

8 oL ~120[

Saline

lO,nnH OO O

~: c~ 1.01-

~ .~10"0t

BBS 10 pg/kg

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~2"0 f

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0. . 6. .12 . 18 24 - 6 0' 6' 12 ' 18 ' 24' - 6 Time before and after injection (min)

0

6

12 '

18 2 4

FIG. 2. Mean (n=5) times spent feeding and frequencies of gizzard and duodenal contractions, in each 3-min period in the 6 min before and 24 rain after IV injections of three doses of BBS and one dose of saline.

TABLE 5 ASSOCIATIONSBETWEENFEEDINGACTIVITYAND GI RESPONSES, FROMSIMULTANEOUSRECORDINGS,AFTERIV INJECTIONSOF BBS, CCK8 (THREE DOSES) AND SALINE Total Time Feeding in First 15 min

Time to Start of Feeding

Time to First Gizzard Contraction

Time to Normal Gizzard Contractions

Time to First Duodenal Contraction

Time to Normal Duodenal Contractions

2.7* 3.3* 4.6*

3.1" 3.3* 2.9*

0.6*# 0.4*t 0.4*

3.1"¢ 2.9"~t 3.5*

0.4* 0.4* 0.3*

10.9"? 15.6" 9.2"t

2.5* 4.0* 1.3t 5.7* 1.9

1.8" 5.5* 11.9t 1.1" 2.6

1.7"I 2.0t 3.8~ 0.3" 0.7

2.4"~ 5.0*t 7.9t 0.3~ 1.4

1.2t 1.4t 0.9*t 0.7*t 0.3

8.5t 11.8*t 14.8"t 1.2~; 3.0

BBS

1/~g/kg 5/zg/kg 10/zg/kg CCK8 1/zg/kg 5 p.g/kg 10 p.g/kg Saline SED

All mean (n=5) values are in minutes. Other details as in Table 3. The SEDs have 9 degrees of freedom.

equivalent in volume to the largest dose of peptide. Subjects weighed between 1.08 and 1.72 kg at the time of testing, and treatments were applied in random order. Owing to SGTs ceasing to function, however, some of the birds did not receive all seven treatments and data were eventually collected from five birds for each treatment. From the recordings, total times spent feeding and frequencies of gizzard and duodenal contractions were measured in each 3-min period in the six minutes before and 24

min after injections. The times after each injection until the start of the first subsequent feeding bout, the first subsequent gizzard and duodenal contractions, and the returns to 'normal' gizzard and duodenal contractions were also measured. These last two measurements were somewhat subjective, and were based on returns to the form and amplitude of contractions before injection, rather than on returns to preinjection frequencies. Contractions of the proventriculus, gizzard and duodenum are usually highly synchronised [18],

196

SAVORY

CCK8 lpg/kg

CCK8 51ug/kg

I

~ 20 f

CCK8 lOpg/kg

Saline

I

I

Hn l'HHHnnHnn" Hfi'nnnn HH'nnnnnnHH

.~ 1"0I-

~-~ ol

H,~nnnnnOO

nOMn

,

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c 4"0I

nnHHHUH HH HH' onnnnnnHHHHHHHH I

~._N .~_ ~.o

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~ .~'°°f D

8

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nF1

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flnnn i

-6

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I-I I

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6 12 18 24-6

0

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I

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I

I

I

i

6 12 18 24 -6 0 6 12 18 24 -6 Time before and after injection (min)

I

0

I

I

I

I

6 12 18 24

FIG. 3. Details as in Fig. 2., with three doses of CCK8 and one of saline.

280 f

I

Saline 240 t" f l O f l 320f

~os

FIn~n~nnFlnn

I

** • **

320[

~r]fln.

oos

lfl...

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f HiiiiiiiiOnnnno.r,.nH o 'llfinr]nnn

5pg/kg240L

flflfl

240 t.

320f

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320[

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5"pg/kg280f f l f l f l 240t. .

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10JJg/kg280 240 t .

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6

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flflfl

~lHF]~]F-~flflflfl 0

,

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24 30 -6 O Time before and after injection (min)

,

6

,

12

,

18

.

24

.

30

FIG. 4. Mean (n=8) heart rates (beats/min) in each 3-min period in the 9 min before and 30 min after IV injections of two doses of BBS and CCK8 and one dose of saline. Post-injection values were compared with overall mean preinjection values by paired t-test. *p