Physiology &Behavior. Vol. 49, pp. 439--442. ©PergamonPress pie, 1991. Printedin the U.S.A.
0031-9384/91 $3.00 + .00
Peripheral Administration of Bombesin Increases Metabolism in the Rat P. C. E V E N , Z. DE S A I N T H I L A I R E A N D S. N I C O L A I D I S
Laboratoire de Neurobiologie des R~gulations, C.N.R.S. U.A. 637, Colldge de France 11 Place M. Berthelot, 75231 Paris Cedex 05, France Received 15 June 1990
EVEN, P. C., Z. ve SAINT HILAIRE AND S. NICOLA~DIS. Peripheraladministration ofbombesin increases metabolism in the rat. PHYSIOL BEHAV 49(3) 439--442, 1991.--Bombesin has been shown to decrease food intake and increase the duration of slow wave sleep, as well as to increase blood glucose and glucagon. A suspected mechanism of bombesin's anorexigenic and hypnogenic action could be via its possible effect on the enhancement of metabolism itself. Therefore, the effect of bombesin on rat metabolism was investigated in an open circuit computerized respirometer. This device was specifically conceived to extract the fraction of metabolism which is not due to the energy cost of muscular effort. This fraction of metabolism was designed background metabolism. In the absence of food, peripheral administration of bombesin increased the background metabolism at all doses. The higher dose of bombesin (32 I~g/kg IP) elicited a four-hour enhancement of background metabolism, the magnitude of which was equivalent to that induced by a meal of about 1.5 grams. Therefore, it is suggested that the effect of bombesin on feeding and sleep could be achieved, at least partly, by its effect on the background metabolism, which ultimately may modulate the behavioral responses. Bombesin
Metabolism
Rat
Control of feeding
Sleep
BOMBESIN and mammalian bombesin-like peptides were reported to decrease food intake when administered peripherally in several species including rats and humans (1, 11, 12, 14, 15). More recently, it was shown that the intraperitoneal (IP) administration of mildly anorexigenic doses of bombesin enhances slow wave sleep during about four hours, i.e., as long as bombesin produces satiety (6). These reports are in agreement with the fact that feeding and sleep have been reported to be closely related via the metabolic effect of feeding (4,5). This double anorexigenic and hypnogenic effect of bombesin could be attributed to some common factor affected by the peptide. Such a common factor can be suspected from previous investigations that revealed that bombesin increased blood glucose and glucagon while decreasing insulin (2,3). Therefore, it could be hypothesized that bombesin affects both feeding and sleep responses by acting upon a glucose-mediated increase in metabolic rate itself. More specifically, an increased metabolic rate was previously shown to result in a reduction of feeding and enhancement of sleep (5), precisely what bombesin does (6,11). It could be expected that peripheral administration of bombesin would enhance the metabolic rate and particularly the background metabolic rate previously described as m(tabolisme de fond (7,9) or as "locomotion free metabolic rate" (8,9). The background metabolism was shown to control the onset and offset of feeding (16,17). The present experiment was conducted to test this hypothesis. Does bombesin increase the background metabolism and, if so, is the increase due to an enhancement of endogenous glucose utilization and is the time course of the so-induced hypermetabolism close to the duration of bombesin's anorexigenic and hypnogenic action? These questions can today be dealt with because a new type
of metabolic device allows factoring out the metabolic cost of locomotion. As a result, background metabolic changes become measurable whatever the effect of treatment on locomotor activity. METHOD
Animals and Housing Sixteen male Wistar rats weighing 250-300 grams were used in this experiment. A light/dark cycle with lights on from 0800 to 2000 h and a temperature regulated at 26°C were maintained in the room. Except for during the experimental session in the calorimeter, the rats had continuous free access to standard food and to water.
Experimental Procedure in the Calorimeter Continuous measurement of energy expenditure from which the portion due to locomotor activity can be separately computed at any time was obtained by using an open circuit calorimeter which has been described previously in detail (7-9, 17). Briefly, metabolic rate was measured from oxygen (02) consumption of the rat. Simultaneous measurement of the rat's carbon dioxide (COz) production enables the system to compute the respiratory quotient (RQ). The RQ is defined as: RQ = CO 2 release/O2 consumption. Data acquisition was performed on-line every ten seconds by a HP 9835A computer. The measured parameters were: cage temperature, air flow, intensity of locomotor activity, and percent variation of 0 2 and CO2 content of the air flow through the cage. These figures were electronically integrated during the ten-second interval between two computer scannings.
439
440
Background metabolism is the result of factoring out the part of metabolic rate due to locomotor activity from total metabolic rate and is expressed in watts. The definition of "background metabolism" is different from that of "resting metabolism" since it can be calculated in resting or in active animals. Background metabolism is also different from basal metabolism since it can be calculated in starving or in satiated animals, at any ambient temperature and whatever the locomotor activity level. In contrast to the other parameters, background metabolism is not measured but calculated. Its calculation requires precise and quantitative measurement of the intensity of locomotion (resting piezoelectric accelerometers) and of the corresponding energy expenditure. Energy expenditure due to locomotor activity is calibrated by establishing the relationship between various levels of locomotor activity and the corresponding increases of total metabolic rate. The relation between the instantaneous signal monitored from the accelerometers and the delayed signal monitored from the gas analysers blunted by the volume of the metabolic chamber (metabolic rate) requires an elaborate procedure of synchronization described in detail previously (7) and summarized in several other papers (7-9). Using this device it was previously shown that both the start and finish of a spontaneous meal were preceded by typical changes of background metabolism; in the same way, satiety would occur as long as the background metabolism remains at a high level (17).
Experiment I The purpose of Experiment I was to compare the parameters of energy metabolism (metabolic rate and respiratory quotient) with other endocrine (glucagon and glucose) and behavioral (feeding and sleep) parameters published in the literature. The common dose used in the previously published experiments was that of 32 Fg/kg, and therefore this dose was used in the first experiment. Eight rats were individually housed in the open-circuit calorimeter for 20 hour recording sessions (1100 to 0700 the next day). Food was not available but water remained unrestricted. At 1900, each rat received a single IP injection of 32 Ixg/kg of BBS or 0.15 M NaC1 as vehicle control, then was immediately reintroduced into the metabolic chamber.
Experiment H The purpose of this experiment was to assess the effect of various doses of BBS on the same parameters of energy metabolism as in the Experiment I, The experimental schedule was the same as in Experiment I except that nine rats were injected IP with BBS (2, 4, 8, 16 or 32 IJ,g/kg) or 0.15 M NaC1 as the vehicle control.
EVEN, DE SAINT HILAIRE AND NICOLA|DIS
RO MR (Waits) 35
~l i.p. 8 B S
In Experiment I, changes in background metabolism (expressed in watts) and in RQ induced either by saline or by bombesin treatments or by a 4-g meal were measured in reference to their baseline values computed as the mean level of those values during the hour that immediately preceded the treatments. Differences between saline- and bombesin-treated subjects were statistically analyzed using the Mann-Whitney U-test. In Experiment II, the dose-response effect of bombesin treatment on respiratory quotient (RQ) and background metabolism was analyzed by regression analysis. The significance of the regression was estimated using the Spearrnen rank correlation coefficient.
& (Vcl!sl 2C
9 3.
16 RO
2.5 12
RE/
8
15
18H
19H
2OH
2IN
22H CLOCK TIME
FIG, 1. An example of the computer recording of the effects of bombesin (BBS) treatment (32 ~g/kg) on background metabolism (BgMR), respiratory quotient (RQ), and locomotor activity (ACT or LA). Black bars: intensity of spontaneous activity. Vertically shaded area: metabolic cost of activity. Horizontally shaded area: Increase in BgMR vs. preinjection level.
RESULTS
Experiment I Figures 1 and 2 show examples of recordings of the main parameters explored in this experiment extending from one hour before to four hours after the IP injection of either 32 IJ,g/kg of BBS (Fig. 1) or vehicle (Fig. 2). The main response to BBS was a rapid (10 to 15 min), dramatic (almost equal in shape and duration to a 1.5-g-meal-induced thermogenesis) and sustained (significant during 4 hours) enhancement of background metabolism (Figs. 1 and 3). RQ remained practically unchanged with this dose.
Experiment H All the doses of BBS used in this experiment were followed by an increase in background metabolism (Fig. 4). The response to the dose of 2 Ixg/kg was minimal, and the doses of 4, 8, 16 and 32 t~g/kg induced increases that varied poorly in relation to the dose of bombesin, as if the effect of bombesin on metabolism reached close to its maximal intensity somewhere between 4 and 8 Ixg/kg. As for the time course of metabolic response, the only significant dose effect on metabolism was found when the logarithm of the doses was used, from 60 to 120 min after treatment (Fig. 4).
RQ MR IWotlsl 4(
Statistical Analysis
INJECTION 152 vg/kg)
12
ip
SALINE INJECTION
LA(VoIfsI
35
16
3( 12
IO 9 2.~ 2(
"q,, ,
18H
.,Ihi
19H
l.l.,., 20H
.CT i ,
2(H
, 22H CLocK TIME
FIG. 2. An example of the computer recording of the effects of saline injection on background metabolism (BgMR), respiratory quotient (RQ), and locomotor activity (ACT or LA). Symbols as in Fig. I.
BOMBESIN AND METABOLISM
441
BBS TREATMENT p g / k ~ - - ~
O. 22 120
0.17 - ~00
~0.12 --
go
32
8o
0.07
t~
--
70
z
0.02
6o
--
5o ~o
-0. 03 - 2o! -0, 08
~
A
2
4
INJECTION
6
8
TIME
10
°' -I(]
(Houre)
FIG. 3. Time course of the effect of the IP injectionof bombesin, 32 p,g/ kg, on the backgroundmetabolism(MF). *p
0031-9384/91 $3.00 + .00
Peripheral Administration of Bombesin Increases Metabolism in the Rat P. C. E V E N , Z. DE S A I N T H I L A I R E A N D S. N I C O L A I D I S
Laboratoire de Neurobiologie des R~gulations, C.N.R.S. U.A. 637, Colldge de France 11 Place M. Berthelot, 75231 Paris Cedex 05, France Received 15 June 1990
EVEN, P. C., Z. ve SAINT HILAIRE AND S. NICOLA~DIS. Peripheraladministration ofbombesin increases metabolism in the rat. PHYSIOL BEHAV 49(3) 439--442, 1991.--Bombesin has been shown to decrease food intake and increase the duration of slow wave sleep, as well as to increase blood glucose and glucagon. A suspected mechanism of bombesin's anorexigenic and hypnogenic action could be via its possible effect on the enhancement of metabolism itself. Therefore, the effect of bombesin on rat metabolism was investigated in an open circuit computerized respirometer. This device was specifically conceived to extract the fraction of metabolism which is not due to the energy cost of muscular effort. This fraction of metabolism was designed background metabolism. In the absence of food, peripheral administration of bombesin increased the background metabolism at all doses. The higher dose of bombesin (32 I~g/kg IP) elicited a four-hour enhancement of background metabolism, the magnitude of which was equivalent to that induced by a meal of about 1.5 grams. Therefore, it is suggested that the effect of bombesin on feeding and sleep could be achieved, at least partly, by its effect on the background metabolism, which ultimately may modulate the behavioral responses. Bombesin
Metabolism
Rat
Control of feeding
Sleep
BOMBESIN and mammalian bombesin-like peptides were reported to decrease food intake when administered peripherally in several species including rats and humans (1, 11, 12, 14, 15). More recently, it was shown that the intraperitoneal (IP) administration of mildly anorexigenic doses of bombesin enhances slow wave sleep during about four hours, i.e., as long as bombesin produces satiety (6). These reports are in agreement with the fact that feeding and sleep have been reported to be closely related via the metabolic effect of feeding (4,5). This double anorexigenic and hypnogenic effect of bombesin could be attributed to some common factor affected by the peptide. Such a common factor can be suspected from previous investigations that revealed that bombesin increased blood glucose and glucagon while decreasing insulin (2,3). Therefore, it could be hypothesized that bombesin affects both feeding and sleep responses by acting upon a glucose-mediated increase in metabolic rate itself. More specifically, an increased metabolic rate was previously shown to result in a reduction of feeding and enhancement of sleep (5), precisely what bombesin does (6,11). It could be expected that peripheral administration of bombesin would enhance the metabolic rate and particularly the background metabolic rate previously described as m(tabolisme de fond (7,9) or as "locomotion free metabolic rate" (8,9). The background metabolism was shown to control the onset and offset of feeding (16,17). The present experiment was conducted to test this hypothesis. Does bombesin increase the background metabolism and, if so, is the increase due to an enhancement of endogenous glucose utilization and is the time course of the so-induced hypermetabolism close to the duration of bombesin's anorexigenic and hypnogenic action? These questions can today be dealt with because a new type
of metabolic device allows factoring out the metabolic cost of locomotion. As a result, background metabolic changes become measurable whatever the effect of treatment on locomotor activity. METHOD
Animals and Housing Sixteen male Wistar rats weighing 250-300 grams were used in this experiment. A light/dark cycle with lights on from 0800 to 2000 h and a temperature regulated at 26°C were maintained in the room. Except for during the experimental session in the calorimeter, the rats had continuous free access to standard food and to water.
Experimental Procedure in the Calorimeter Continuous measurement of energy expenditure from which the portion due to locomotor activity can be separately computed at any time was obtained by using an open circuit calorimeter which has been described previously in detail (7-9, 17). Briefly, metabolic rate was measured from oxygen (02) consumption of the rat. Simultaneous measurement of the rat's carbon dioxide (COz) production enables the system to compute the respiratory quotient (RQ). The RQ is defined as: RQ = CO 2 release/O2 consumption. Data acquisition was performed on-line every ten seconds by a HP 9835A computer. The measured parameters were: cage temperature, air flow, intensity of locomotor activity, and percent variation of 0 2 and CO2 content of the air flow through the cage. These figures were electronically integrated during the ten-second interval between two computer scannings.
439
440
Background metabolism is the result of factoring out the part of metabolic rate due to locomotor activity from total metabolic rate and is expressed in watts. The definition of "background metabolism" is different from that of "resting metabolism" since it can be calculated in resting or in active animals. Background metabolism is also different from basal metabolism since it can be calculated in starving or in satiated animals, at any ambient temperature and whatever the locomotor activity level. In contrast to the other parameters, background metabolism is not measured but calculated. Its calculation requires precise and quantitative measurement of the intensity of locomotion (resting piezoelectric accelerometers) and of the corresponding energy expenditure. Energy expenditure due to locomotor activity is calibrated by establishing the relationship between various levels of locomotor activity and the corresponding increases of total metabolic rate. The relation between the instantaneous signal monitored from the accelerometers and the delayed signal monitored from the gas analysers blunted by the volume of the metabolic chamber (metabolic rate) requires an elaborate procedure of synchronization described in detail previously (7) and summarized in several other papers (7-9). Using this device it was previously shown that both the start and finish of a spontaneous meal were preceded by typical changes of background metabolism; in the same way, satiety would occur as long as the background metabolism remains at a high level (17).
Experiment I The purpose of Experiment I was to compare the parameters of energy metabolism (metabolic rate and respiratory quotient) with other endocrine (glucagon and glucose) and behavioral (feeding and sleep) parameters published in the literature. The common dose used in the previously published experiments was that of 32 Fg/kg, and therefore this dose was used in the first experiment. Eight rats were individually housed in the open-circuit calorimeter for 20 hour recording sessions (1100 to 0700 the next day). Food was not available but water remained unrestricted. At 1900, each rat received a single IP injection of 32 Ixg/kg of BBS or 0.15 M NaC1 as vehicle control, then was immediately reintroduced into the metabolic chamber.
Experiment H The purpose of this experiment was to assess the effect of various doses of BBS on the same parameters of energy metabolism as in the Experiment I, The experimental schedule was the same as in Experiment I except that nine rats were injected IP with BBS (2, 4, 8, 16 or 32 IJ,g/kg) or 0.15 M NaC1 as the vehicle control.
EVEN, DE SAINT HILAIRE AND NICOLA|DIS
RO MR (Waits) 35
~l i.p. 8 B S
In Experiment I, changes in background metabolism (expressed in watts) and in RQ induced either by saline or by bombesin treatments or by a 4-g meal were measured in reference to their baseline values computed as the mean level of those values during the hour that immediately preceded the treatments. Differences between saline- and bombesin-treated subjects were statistically analyzed using the Mann-Whitney U-test. In Experiment II, the dose-response effect of bombesin treatment on respiratory quotient (RQ) and background metabolism was analyzed by regression analysis. The significance of the regression was estimated using the Spearrnen rank correlation coefficient.
& (Vcl!sl 2C
9 3.
16 RO
2.5 12
RE/
8
15
18H
19H
2OH
2IN
22H CLOCK TIME
FIG, 1. An example of the computer recording of the effects of bombesin (BBS) treatment (32 ~g/kg) on background metabolism (BgMR), respiratory quotient (RQ), and locomotor activity (ACT or LA). Black bars: intensity of spontaneous activity. Vertically shaded area: metabolic cost of activity. Horizontally shaded area: Increase in BgMR vs. preinjection level.
RESULTS
Experiment I Figures 1 and 2 show examples of recordings of the main parameters explored in this experiment extending from one hour before to four hours after the IP injection of either 32 IJ,g/kg of BBS (Fig. 1) or vehicle (Fig. 2). The main response to BBS was a rapid (10 to 15 min), dramatic (almost equal in shape and duration to a 1.5-g-meal-induced thermogenesis) and sustained (significant during 4 hours) enhancement of background metabolism (Figs. 1 and 3). RQ remained practically unchanged with this dose.
Experiment H All the doses of BBS used in this experiment were followed by an increase in background metabolism (Fig. 4). The response to the dose of 2 Ixg/kg was minimal, and the doses of 4, 8, 16 and 32 t~g/kg induced increases that varied poorly in relation to the dose of bombesin, as if the effect of bombesin on metabolism reached close to its maximal intensity somewhere between 4 and 8 Ixg/kg. As for the time course of metabolic response, the only significant dose effect on metabolism was found when the logarithm of the doses was used, from 60 to 120 min after treatment (Fig. 4).
RQ MR IWotlsl 4(
Statistical Analysis
INJECTION 152 vg/kg)
12
ip
SALINE INJECTION
LA(VoIfsI
35
16
3( 12
IO 9 2.~ 2(
"q,, ,
18H
.,Ihi
19H
l.l.,., 20H
.CT i ,
2(H
, 22H CLocK TIME
FIG. 2. An example of the computer recording of the effects of saline injection on background metabolism (BgMR), respiratory quotient (RQ), and locomotor activity (ACT or LA). Symbols as in Fig. I.
BOMBESIN AND METABOLISM
441
BBS TREATMENT p g / k ~ - - ~
O. 22 120
0.17 - ~00
~0.12 --
go
32
8o
0.07
t~
--
70
z
0.02
6o
--
5o ~o
-0. 03 - 2o! -0, 08
~
A
2
4
INJECTION
6
8
TIME
10
°' -I(]
(Houre)
FIG. 3. Time course of the effect of the IP injectionof bombesin, 32 p,g/ kg, on the backgroundmetabolism(MF). *p
