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Further evidence of the inhibitory role of perifornical hypothalamic β-adrenergic receptors in the feeding behaviour of hungry rats
Life Sciences, Vol. 38, pp. 259-266 Printed in the U.S.A.
Pergamon Press
FURTHER EVIDENCE OF THE INHIBITORY ROLE OF PERIFORNICAL HYPOTHALAMIC $-ADRENERGIC RECEPTORS IN THE FEEDING BEHAVlOUR OF HUNGRY RATS C. Bendotti, M. Villa and R. Samanin Istituto di Ricerche Farmacologiche "Mario Negri" via Eritrea, 62 -20157 Milan, Italy (Received in final form October 30, 1985) Summary The reduction of food intake in hungry rats induced by salbutamol (10 mg/kg/i.p.) was prevented by IPS 339 (5 mg/kg, i.p.) a selective B 2 adrenergic antagonist, but not by metoprolol (10 mg/kgi.p.), a blocker of 61 adrenergic receptors. Similarly, bilateral injections of IPS 339 (32 ~g/]~l) but not metoprolol (80 ~g/l pl) in the perifornical hypothalamic area completely antagonized the anorectic effect of intraperitoneal salbutamol, suggesting an involvement of ~2 adrenergic receptors in this brain area. Clenbuterol, a ~adrenergic agonist which readily crosses the blood-brain barrier, was 10-100 times more potent than salbutamol in inhibiting feeding consumption of deprived rats when injected intraperitoneally and this effect was also selectively antagonized by pretreatment with IPS 339. Neither IPS 339 nor metoprolol injected in the perifornical hypothalamus significantly modified the anorectic effect of diethylpropion (5 mg/kg i.p.) whereas it was partially prevented byintraperifornical injection of l-propranolol (52 ~g/2~l), a non-selective 6 antagonist, suggesting that both B I and 62 adrenergic receptors in the hypothalamus contribute to the mechanism by which diethylpropion causes anorexia. There is evidence that brain noradrenaline and adrenaline play an important role in the control of feeding behaviour. Specifically, it has been suggested that the activation of 6-adrenergic sites in the perifornical hypothalamic area produces anorexia in food deprived rats (1,2,3). This hypothesis rises from the finding that injection of adrenaline, noradrenaline or other adrenergic stimulants in this brain area caused a dose-dependent reduction of food intake which was prevented by propranolol, a 6-adrenergic receptor blocker (2,4). Recently it has been observed that systemic injection of salbutamol, an agonist of 62-adrenergic receptors (5), produced a dose-dependent reduction of food intake of starved rats (6). This effect was completely antagonized by intracerebroventricular injections of propranolol and alprenolol, two non-selective 6-adrenergic receptor blockers, suggesting the involvement of central B-adrenergic 0024-3205/86.$3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
260
~-adrenergic
Receptors and Food Intake
Vol.
38, No. 3, 1986
sites (6). In agreement with the findings of Leibowitz and Rossakis (2), it was suggested that the activation of $-adrenergic sites, probably of the $2 type in the perifornical hypothalamus was responsible for the anorectic action of salbutamol injected systemically. To verify this hypothesis, the present study examined the effect of selective $I and ~2 adrenergic blockers, injected systematically or directly into the perifornical hypothalamic area, on the anorectic action of peripherally administered salbutamol. IPS 339 ((t-butyl-amino-3-~-2-propyl)amino-9fluorene HCI), a selective antagonist of $2 adrenergic receptors (7), which has been found to be effective in antagonizing the central action of $-adrenergic stimu]ants (8) and metoprolol, a selective $I adrenergic blocker (9) which easily passes the blood-brain barrier (10) were used for these experiments. To obtain additional information on the role of central $2 adrenergic sites in controlling feeding, the effect on food intake of hungry rats of c]enbuterol, a $2 adrenergic agonist (11) more lipophilic than salbutamol, which could be assumed to pass readily into the brain, was studied and its selectivity of action on ~2 adrenergic receptors assessed. Previous studies have shown that the integrity of the ventral noradrenergic-adrenergic bundles is necessary for the anorectic effect of diethylpropion (12), an appetite suppressant widely used in the treatment of clinical obesity (13,14), but there is no informationonwbich adrenerg~c receptors are involved in its effect. It was therefore of interest to establish the extent to which $2 or $I adrenergic receptors in the hypothalamus are involved in diethylpropion' s ability to reduce food intake in hungry rats.
Methods ~lale CD-COBS Sprague Dawley rats (Charles River, Italy) weighing about 2 5 0 g w e r e trained over 2-3 weeks ot eat their food (Altromin MT for rats, Rieper, Italy) during a daily 4 h period (11-15 h). On the 8th-10th day of this training four groups of 30 rats were stereotaxically imp]anted with bilateral chronic cannulae according to the methods described below. On the day of testing, day 15-20, the rats were placed singly in cages containing a weighed amount of food, and intake during I h was measured to the nearest 0.1 g. The amount of food eaten was corrected for spillage. Cannulae implantation Rats were stereotaxically implanted under ethyl ether anesthesia with chronic bilateral guide cannulae, made of 23 gauge stainless steel tubing in a plexiglass holder. The cannulae were placed with their tip 2 mm above the perifornical hypothalamic area. Stainless steel stylets, 30 gauge, as long as the guide, kept the guide patent until the animals were given intracerebral injections 7-10 days later. The rats were accustomed to handling before testing and on the day of the test the stylets were withdrawn and replaced by bilateral injectionunits (30 gauge, stainless steel tubing) terminating 2 mm below the tips of the guides. The following stereotaxic coordinates from KSnig and Klippel rat brain atlas (15) were used: A = 4890, L = $ 1.2, V = +3.
Vol. 38, No. 3, 1986
B-adrenergic Receptors and Food Intake
261
Drugs and sources Clenbuterol HCI (dr. Karl Thomae GmbH, Biberach an der Riss, Germany), diethylpr6pion HCI (Merrel, Cincinnati, USA), metoprolol tartrate (Ciba-Geigy, Origgio, Italy), salbutamol sulphate (Glaxo, Verona, Italy) were dissolved in saline. 1-Propranolol HCI (ICI Pharma, Macclesfield, Cheshire, England) was dissolved in I% ascorbic acid buffered with NaOH I N. IPS 339 HCI soluble in saline was kindly supplied by dr. Leclerc of University "Louis Pasteur" 9 Strasbourg, France. The doses used for each drug, calculated as base, are reported in the tables. The injection volume in the perifornical area of hypothalamus was I ~1 each side for all drug solutions except for the relatively insoluble compound l-propranolol, which had to be injected in 2~I. Fresh solutions of each drug were prepared immediately before the start of each test. Histological examinations After completion of the experiments, animals were decapitated, brains were removed and immediately frozen in dry ice and 40 ~ sections cut in a cryostat. In these conditions the trace of the cannulae was clearly seen and served to locate the site of injection. Only data from rats in which the eannulae were exactly located bilaterally in the perifornical hypothalamic area were included in the results. Statistics Data were analyzed by Dunnett's test (two-tailed) for multiple comparisons or by factorial analysis of variance followed by Tukey's test.
Results As shown in Table I, the anorectic effect of salbutamol was prevented by a systemically injected IPS 339, a selective antagonist of B2 adrenergic receptors, whereas metoprolol - a blocker of ~ adrenergic receptors - failed to modify salbutamol-induced anorexia.
Table I Effect of IPS 339 and metoprolol on the anorectic action of salbutamol
Food intake Treatment
Vehicle IPS 339 Metoprolol
Dose (m~/kg i.p.) 5.0 10.0
Saline 7.2 + 0.4 6.0 + 0.6 7.2 + 0.5
(g/rat/lh) Mean + S.E. Salbutamol 3.2 + 0.3** 5.9 + 0.9 °o 4.3 + 0.5
IPS 339 and metoprolol were injected 15 min before salbutamol. Salbutamol 10 mg/kg i.p. was administered 15 min before access to food Each group consisted of 6 rats. ANOVA 2x2; IPS 339 treatment: F interaction 10.4 df 1/17 p
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Vol. 38, No. 3, 1986
Similarly, IPS 339, but not metoprolol, injected bilaterally in the perifornical hypothalamic area, completely antagonized the anorectic effect of salbutamol given intraperitoneally (Table 2). That the dose of metoprolol used in this experiment effectively blocked $2 adrenergic receptors was demonstrated by the fact that it significantly antagonized the reduction of food intake induced by subcutaneous injection of isoproterenol (ISO), 0. I mg/kg, a typical non-selective adrenergic agonist which has been suggested to produce anorexia by activating the perifornical hypothalamic ~ adrenergic sites (3) (vehicle + saline = 12.6 g + 1.4, vehicle + ISO = 5.0 g + 1.0, metoprolol + saline = I0.8 g + I.I, metoprolol + ISO = 8.3 g + 1.2; ANOVA 2x2: F interaction 4.35 df 1/15 p
Table 2 Effect of IPS 339 and metoprolol injected bilaterally in the hypothalamic perifornical area on the anorectic action of salbutamol.
Treatment
Dose (pg/l ~I)
Food intake (g/rat/1 h) Mean +S.E. Saline
Salbutamol
Vehicle IPS 339
32
7.0 + 0.3 7.4 + 1.1
2.7 + 0.4** 7.0 + 1.0 °
Vehicle Metoprolol
80
9.8 + 1.3 8.3 + 1.1
2.4 + 0.3** 3.1 + 0.4**
IPS 339 and metoprolol were injected just before salbutamol. Salbutamol 10 mg/kg i.p. was injected 15 min before rats had access to food. Each group consisted of 5-7 rats. ANOVA 2x2; IPS 339 treatment: F interaction 7.7 df 1/19 p
Intraperitoneal injections of clenbuterol at doses 10-100 times lower than salbutamol produced a dose-dependent decrease of food intake in rats (Table 3). No changes in behaviour were apparent. The effect of I mg/kg was prevented by intraperitoneal IPS 339 but not metoprolol (Table 4). Table 5 shows the effect of IPS 339 and metoprolol injected bilaterally into the perifornical hypothalamus on the anorectic effect of intraperitoneal diethylpropion. The fact that saline-treated animals ate more food in these experiments than in earlier ones could be due to their longer training (4-5 weeks) to eat during a daily 4-h period. Neither IPS 339 normetoprolol modified ~eeffect of diethy~ropio~ To examine whether stimulation of both ~I and ~2 adrenergic receptors was responsible for diethylpropion's anorectic activity, the effect of this drug was measured after bilateral injection in the perifornical hypothalamus of lpropranolol, a non-selective adrenergic receptor blocker. Table 5 shows that 52 ~g of l-propranolol, reported to reduce anorexia induced by hypotbalamic injection of adrenaline (2), partially prevented the effect of diethylpropion (p
Vol. 38, No. 3, 1986
Effect
of
~-adrenergic Receptors and Food Intake
clenbuterol
on
Table 3 food intake
Dose Treatment
Saline Clenbuterol Clenbuterol Clenbuterol
of rats.
Food intake
(mg/kg i.p.)
263
(g/rat/1 h )
Mean + S.E.
O.1 0.5 1.0
6.9 4.6 2.9 2.7
+ + + +
0.6 0.8* 0.5** 0.5**
Clenbuterol was injected 15 min before rats had access to food. Each group consisted of 6 rats. Dunnett's
test;
*p
**p
Table 4 Effect of IPS339 and metoprolol on the anorectic action of clenbuterol.
Treatment
Vehicle IPS 339 Metoprolol
Dose
Food intake (g/rat/1 h) Mean + S.E.
(mg/kg i.p.)
Saline
Clenbuterol
5.0 10.0
6.4 + 0.4 6.2 + 0.5 6.4 + 0.4
2.8 + 0.6 ~* 5.0 + 0.3 ° 2.9 + 0.5**
IPS 339 and metoprolol were injected 15 min before clenbuterol. Clenbuterol I mg/kg i.p. was administered 15 min before access to food. Each group consisted of 6 rats. ANOVA 2x2; IPS 339 treatment: F interaction 6.3 df 1/19 p
Discussion The fact that IPS 339, a specific 82 adrenergic receptor blocker, but not metoprolol, which is known to block only 81 adrenergic receptors, prevented the anorectic effect of salbutamol, suggests that ~2 adrenergic sites are selectively involved. Injection of IPS 339 in the perifornieal hypothalamic area completely antagonized the anorexia induced by salbutamol suggesting that 82 adrenergic receptors in this area mediate the suppression of feeding induced by systemic salbutamol.
264
B-adrenergic
Receptors and Food intake
Vol.
38, No. 3, 198@
Table 5 Effect of IPS 339, metoprolol and l-propranolol injected bilaterally into the hypothalamic perifornical area on the anorectic action of diethylpropion.
Treatment
Dose (~g/1~l)
Food intake Saline
(g/rat/lh) Mean + S.E. Diethy]propion
Vehicle IPS 339
32
13.6 + 1.0 15.8 + 1.0
7.0 + 1.3"* 6.5 + 1.1"*
Vehicle Metoprolol
80
11.2 + 1.6 9.3 + 0.8
3.7 + 0.6** 4.9 + 0.4**
11.1 + 0.6 13.2 + 0.9
4.9 + 0 . 6 * * 9.8 + 1.1"*
Vehicle l-Propranolol
(~g/2 91) 52
IPS 339, metoprolol and l-propranolol were injected just before diethylpropion. Diethylpropion 5 mg/kg i.p. was injected 15 min before rats had access to food. Each group consisted of 4-7 rats. ANOVA 2x2; IPS 339 treatment: F interaction 1.4 df 1/19 N.S. Metoprolol treatment: F interaction 3.4 df 1/21N.S. l-Propranolol treatment: F interaction 3.0 df 1/16 N.S. Tukey's test; *p
The involvement of $2 adrenergic sites in the perifornical area in the mechanism of anorexia was previously suggested by Leibowitz and Rossakis (2) who observed that the reduction of food consumption induced by adrenaline injected in this brain area was preferentially blocked by ~2 adrenergic antagonists. The present study shows that systemically administered $2 adrenergic stimulants can effectively activate these central sites involved in the control of feeding behaviour. Metoprolol injected in the perifornical hypothalamus failed to modify the anorectic action of salbutamol, but significantly reduced the feeding suppression induced by subcutaneous isoproterenol, a B I ~2 adrenergic agonist, suggesting that $I adrenergic receptors involved in mediating feeding inhibition in hungry rats are also present in the perifornical hypothalamus. Clenbuterol, another ~2 adrenergic agonist which crosses the blood brain barrier more readily than salbutamol, was 10-100 times more effective than the latter in reducing food intake in deprived rats. Frances et al. (16) reported a similar potency ratio between these two drugs for the reduction of motor activity and the loss of interest of fasting mice in food pellets whereas these drugs showed comparable peripheral activity (11). Selective inw)ivement of ~2 adrenergic receptors in the anorectic effect of clenbuterolwas demonstrated by the fact that it was prevented by pretreatment with IPS 339 but not with metoprolol. The easier access to central ~2 adrenergic sites involved in feeding inhibition and the specificity of action of clenbutero] make it a potentially useful drug for the treatment of obesity.
Vol. 38, No. 3, 1986
B-adrenergic Receptors and Food Intake
265
The anorectic effect of diethylpropion, which was previously shown to be completely antagonized by lesion of the ventral noradrenergic bundles (12), was not modified by intraperifornical injections of IPS 339 or metoprolol, suggesting that different mechanisms are involved in the anorectic effect of salbutamol and diethylpropion. Unlike salbutamol which directly stimulates central B 2 adrenergic receptors (17), diethylpropion releases noradrenaline from nerve terminals (18) and may therefore have a more general effect on ~-adrenergic receptors involved in feeding control. The selective block of a single receptorial population of the perifornical hypothalamus could be insufficient to modify diethylpropion's effect on food intake. This suggestion is supported by the finding that l-propranolol, which block both $I and ~2 adrenergic receptors, partially prevented the effect of diethylpropion when injected in the perifornical hypothalamic area. A problem with this interpretation is that l-propranolol, because of its poor solubility, was injected in the hypothalamus in a larger volume than the other adrenergic antagonists and it may therefore have reached adrenergic sites outside the perifornical area. The fact that diethylpropion-induced anorexia was only partially reduced by inhibiting perifornical adrenergic receptors whereas complete antagonism was observed after the lesion of the ventral bundle of noradrenaline-containing fibers (12) suggests that other noradrenergicadrenergic sites in the hypothalamus or other brain regions innervated by these noradrenergic-adrenergic pathways play a role in the action of diethylpropion. In conclusion, the present study shows that systemically administered salbutamol causes anorexia in hungry rats through the selective activation of ~2adrenerg~ receptors in the perifornical hypothalamic area. Clenbuterol, which easily enters in the brain, is 70-100 times more potent than salbutamol in inhibiting feeding consumption of deprived rats and this effect is selectively mediated by B 2 adrenergic receptors. Finally, it has been shown that B I and B 2 adrenergic receptors in the hypothalamus partially contribute to the mechanism by which diethylpropion causes anorexia in rats. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
S.F. LEIBOWITZ, Nature 226 963-964 (1970). S.F. LEIBOWITZ and C. ROSSAKIS, Neuropharmacology 1 7 691-702 (1978). H.W. GOLD,JAN, D. LEHR and E. FRIEDMAN, Nature 231 453-455 (1971). D. LEHR and W. GOLDMAN, Eur. J. Pharmac. 23 197-210 (1973). V.A. CULLUM, J.B. FARMER, D. JACK and G.P. LEVY, Br. J. Pharmac. 35 14]-151 (1969). F. BORSINI, C. BENDOTTI, P. THURBLY and R. SAMANIN, Life Sci. 30 905-911 (1982). J.L. IMBS, F. MIESCH, J. SCHWARTZ, J. VELLY, G. LECLERC, A. MANN and C.G. VERMUTH, Br. J. Pharmac. 60 357-362 (1977). S.B. ROSS, Acta pharmac, tox. 47 347-350 (1980). B. ABLAD, K.O. BORG, E. CARLSSON, L. EK, G. JOHNSSON, T. MALFORS and C-G. REGARDH, Acta pharmac.tox. 36 suppl.V, 7-23 (1975). J.A. STREET, B.A. HEMSWORTH, A.G. ROACH and M.D. DAY, Archs. int.. Pharmacodyn. Ther. 237 180-190 (~979).
266
11. 12. 13. 14.
15.
$-adrenergic
Receptors
and Food Intake
Vol. 38, No. 3, 1986
V.G. ENGELHARDT, Arzneimittel-Forsch. 26 1404-1420 (1976). F. BORSINI, C. BENDOTTI, M. CARLI, E. POGGESI and R. SAMANIN, Res. Commun. chem. Pathol. Pharmac. 26 3-11 (1979). J.F. MUNRO and M.J. FORD, i n Drugs and Appetite (ED. T. SILVERSTONE), p. 125-158, Academic Press, London (1982). M.T. HOEKENGA, R.H. DILLON and H.M. LEYLAND, irL Central Mechanisms of Anorectic Drugs (ED. S. GARATTINI and R. SAMANIN), p. 391-404, Raven Bess, New York (1978). J.F.R. KONIG and R.A. KLIPPEL, The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams & Wilkins, Baltimore
(1963). 16. 17. 18.
H. FRANCES, A.J. PUECH, S. DANTI and P. SIMON, Eur. J. Pharmac. 92 223-230 (1983). D.B. BYLUND and 8.H. SNYDER, Mol. Pharmac. 12 568-580 (1976). J. OFFERMEIER and H.G. du PREEZ, in Central Mechanisms of Anorectic Drugs (ED. S. GARATTINI and R. SAMANIN), p. 217-231, Raven Press, New York (1978).
Pergamon Press
FURTHER EVIDENCE OF THE INHIBITORY ROLE OF PERIFORNICAL HYPOTHALAMIC $-ADRENERGIC RECEPTORS IN THE FEEDING BEHAVlOUR OF HUNGRY RATS C. Bendotti, M. Villa and R. Samanin Istituto di Ricerche Farmacologiche "Mario Negri" via Eritrea, 62 -20157 Milan, Italy (Received in final form October 30, 1985) Summary The reduction of food intake in hungry rats induced by salbutamol (10 mg/kg/i.p.) was prevented by IPS 339 (5 mg/kg, i.p.) a selective B 2 adrenergic antagonist, but not by metoprolol (10 mg/kgi.p.), a blocker of 61 adrenergic receptors. Similarly, bilateral injections of IPS 339 (32 ~g/]~l) but not metoprolol (80 ~g/l pl) in the perifornical hypothalamic area completely antagonized the anorectic effect of intraperitoneal salbutamol, suggesting an involvement of ~2 adrenergic receptors in this brain area. Clenbuterol, a ~adrenergic agonist which readily crosses the blood-brain barrier, was 10-100 times more potent than salbutamol in inhibiting feeding consumption of deprived rats when injected intraperitoneally and this effect was also selectively antagonized by pretreatment with IPS 339. Neither IPS 339 nor metoprolol injected in the perifornical hypothalamus significantly modified the anorectic effect of diethylpropion (5 mg/kg i.p.) whereas it was partially prevented byintraperifornical injection of l-propranolol (52 ~g/2~l), a non-selective 6 antagonist, suggesting that both B I and 62 adrenergic receptors in the hypothalamus contribute to the mechanism by which diethylpropion causes anorexia. There is evidence that brain noradrenaline and adrenaline play an important role in the control of feeding behaviour. Specifically, it has been suggested that the activation of 6-adrenergic sites in the perifornical hypothalamic area produces anorexia in food deprived rats (1,2,3). This hypothesis rises from the finding that injection of adrenaline, noradrenaline or other adrenergic stimulants in this brain area caused a dose-dependent reduction of food intake which was prevented by propranolol, a 6-adrenergic receptor blocker (2,4). Recently it has been observed that systemic injection of salbutamol, an agonist of 62-adrenergic receptors (5), produced a dose-dependent reduction of food intake of starved rats (6). This effect was completely antagonized by intracerebroventricular injections of propranolol and alprenolol, two non-selective 6-adrenergic receptor blockers, suggesting the involvement of central B-adrenergic 0024-3205/86.$3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
260
~-adrenergic
Receptors and Food Intake
Vol.
38, No. 3, 1986
sites (6). In agreement with the findings of Leibowitz and Rossakis (2), it was suggested that the activation of $-adrenergic sites, probably of the $2 type in the perifornical hypothalamus was responsible for the anorectic action of salbutamol injected systemically. To verify this hypothesis, the present study examined the effect of selective $I and ~2 adrenergic blockers, injected systematically or directly into the perifornical hypothalamic area, on the anorectic action of peripherally administered salbutamol. IPS 339 ((t-butyl-amino-3-~-2-propyl)amino-9fluorene HCI), a selective antagonist of $2 adrenergic receptors (7), which has been found to be effective in antagonizing the central action of $-adrenergic stimu]ants (8) and metoprolol, a selective $I adrenergic blocker (9) which easily passes the blood-brain barrier (10) were used for these experiments. To obtain additional information on the role of central $2 adrenergic sites in controlling feeding, the effect on food intake of hungry rats of c]enbuterol, a $2 adrenergic agonist (11) more lipophilic than salbutamol, which could be assumed to pass readily into the brain, was studied and its selectivity of action on ~2 adrenergic receptors assessed. Previous studies have shown that the integrity of the ventral noradrenergic-adrenergic bundles is necessary for the anorectic effect of diethylpropion (12), an appetite suppressant widely used in the treatment of clinical obesity (13,14), but there is no informationonwbich adrenerg~c receptors are involved in its effect. It was therefore of interest to establish the extent to which $2 or $I adrenergic receptors in the hypothalamus are involved in diethylpropion' s ability to reduce food intake in hungry rats.
Methods ~lale CD-COBS Sprague Dawley rats (Charles River, Italy) weighing about 2 5 0 g w e r e trained over 2-3 weeks ot eat their food (Altromin MT for rats, Rieper, Italy) during a daily 4 h period (11-15 h). On the 8th-10th day of this training four groups of 30 rats were stereotaxically imp]anted with bilateral chronic cannulae according to the methods described below. On the day of testing, day 15-20, the rats were placed singly in cages containing a weighed amount of food, and intake during I h was measured to the nearest 0.1 g. The amount of food eaten was corrected for spillage. Cannulae implantation Rats were stereotaxically implanted under ethyl ether anesthesia with chronic bilateral guide cannulae, made of 23 gauge stainless steel tubing in a plexiglass holder. The cannulae were placed with their tip 2 mm above the perifornical hypothalamic area. Stainless steel stylets, 30 gauge, as long as the guide, kept the guide patent until the animals were given intracerebral injections 7-10 days later. The rats were accustomed to handling before testing and on the day of the test the stylets were withdrawn and replaced by bilateral injectionunits (30 gauge, stainless steel tubing) terminating 2 mm below the tips of the guides. The following stereotaxic coordinates from KSnig and Klippel rat brain atlas (15) were used: A = 4890, L = $ 1.2, V = +3.
Vol. 38, No. 3, 1986
B-adrenergic Receptors and Food Intake
261
Drugs and sources Clenbuterol HCI (dr. Karl Thomae GmbH, Biberach an der Riss, Germany), diethylpr6pion HCI (Merrel, Cincinnati, USA), metoprolol tartrate (Ciba-Geigy, Origgio, Italy), salbutamol sulphate (Glaxo, Verona, Italy) were dissolved in saline. 1-Propranolol HCI (ICI Pharma, Macclesfield, Cheshire, England) was dissolved in I% ascorbic acid buffered with NaOH I N. IPS 339 HCI soluble in saline was kindly supplied by dr. Leclerc of University "Louis Pasteur" 9 Strasbourg, France. The doses used for each drug, calculated as base, are reported in the tables. The injection volume in the perifornical area of hypothalamus was I ~1 each side for all drug solutions except for the relatively insoluble compound l-propranolol, which had to be injected in 2~I. Fresh solutions of each drug were prepared immediately before the start of each test. Histological examinations After completion of the experiments, animals were decapitated, brains were removed and immediately frozen in dry ice and 40 ~ sections cut in a cryostat. In these conditions the trace of the cannulae was clearly seen and served to locate the site of injection. Only data from rats in which the eannulae were exactly located bilaterally in the perifornical hypothalamic area were included in the results. Statistics Data were analyzed by Dunnett's test (two-tailed) for multiple comparisons or by factorial analysis of variance followed by Tukey's test.
Results As shown in Table I, the anorectic effect of salbutamol was prevented by a systemically injected IPS 339, a selective antagonist of B2 adrenergic receptors, whereas metoprolol - a blocker of ~ adrenergic receptors - failed to modify salbutamol-induced anorexia.
Table I Effect of IPS 339 and metoprolol on the anorectic action of salbutamol
Food intake Treatment
Vehicle IPS 339 Metoprolol
Dose (m~/kg i.p.) 5.0 10.0
Saline 7.2 + 0.4 6.0 + 0.6 7.2 + 0.5
(g/rat/lh) Mean + S.E. Salbutamol 3.2 + 0.3** 5.9 + 0.9 °o 4.3 + 0.5
IPS 339 and metoprolol were injected 15 min before salbutamol. Salbutamol 10 mg/kg i.p. was administered 15 min before access to food Each group consisted of 6 rats. ANOVA 2x2; IPS 339 treatment: F interaction 10.4 df 1/17 p
262
~-adrenergic
Receptors and Food Intake
Vol. 38, No. 3, 1986
Similarly, IPS 339, but not metoprolol, injected bilaterally in the perifornical hypothalamic area, completely antagonized the anorectic effect of salbutamol given intraperitoneally (Table 2). That the dose of metoprolol used in this experiment effectively blocked $2 adrenergic receptors was demonstrated by the fact that it significantly antagonized the reduction of food intake induced by subcutaneous injection of isoproterenol (ISO), 0. I mg/kg, a typical non-selective adrenergic agonist which has been suggested to produce anorexia by activating the perifornical hypothalamic ~ adrenergic sites (3) (vehicle + saline = 12.6 g + 1.4, vehicle + ISO = 5.0 g + 1.0, metoprolol + saline = I0.8 g + I.I, metoprolol + ISO = 8.3 g + 1.2; ANOVA 2x2: F interaction 4.35 df 1/15 p
Table 2 Effect of IPS 339 and metoprolol injected bilaterally in the hypothalamic perifornical area on the anorectic action of salbutamol.
Treatment
Dose (pg/l ~I)
Food intake (g/rat/1 h) Mean +S.E. Saline
Salbutamol
Vehicle IPS 339
32
7.0 + 0.3 7.4 + 1.1
2.7 + 0.4** 7.0 + 1.0 °
Vehicle Metoprolol
80
9.8 + 1.3 8.3 + 1.1
2.4 + 0.3** 3.1 + 0.4**
IPS 339 and metoprolol were injected just before salbutamol. Salbutamol 10 mg/kg i.p. was injected 15 min before rats had access to food. Each group consisted of 5-7 rats. ANOVA 2x2; IPS 339 treatment: F interaction 7.7 df 1/19 p
Intraperitoneal injections of clenbuterol at doses 10-100 times lower than salbutamol produced a dose-dependent decrease of food intake in rats (Table 3). No changes in behaviour were apparent. The effect of I mg/kg was prevented by intraperitoneal IPS 339 but not metoprolol (Table 4). Table 5 shows the effect of IPS 339 and metoprolol injected bilaterally into the perifornical hypothalamus on the anorectic effect of intraperitoneal diethylpropion. The fact that saline-treated animals ate more food in these experiments than in earlier ones could be due to their longer training (4-5 weeks) to eat during a daily 4-h period. Neither IPS 339 normetoprolol modified ~eeffect of diethy~ropio~ To examine whether stimulation of both ~I and ~2 adrenergic receptors was responsible for diethylpropion's anorectic activity, the effect of this drug was measured after bilateral injection in the perifornical hypothalamus of lpropranolol, a non-selective adrenergic receptor blocker. Table 5 shows that 52 ~g of l-propranolol, reported to reduce anorexia induced by hypotbalamic injection of adrenaline (2), partially prevented the effect of diethylpropion (p
Vol. 38, No. 3, 1986
Effect
of
~-adrenergic Receptors and Food Intake
clenbuterol
on
Table 3 food intake
Dose Treatment
Saline Clenbuterol Clenbuterol Clenbuterol
of rats.
Food intake
(mg/kg i.p.)
263
(g/rat/1 h )
Mean + S.E.
O.1 0.5 1.0
6.9 4.6 2.9 2.7
+ + + +
0.6 0.8* 0.5** 0.5**
Clenbuterol was injected 15 min before rats had access to food. Each group consisted of 6 rats. Dunnett's
test;
*p
**p
Table 4 Effect of IPS339 and metoprolol on the anorectic action of clenbuterol.
Treatment
Vehicle IPS 339 Metoprolol
Dose
Food intake (g/rat/1 h) Mean + S.E.
(mg/kg i.p.)
Saline
Clenbuterol
5.0 10.0
6.4 + 0.4 6.2 + 0.5 6.4 + 0.4
2.8 + 0.6 ~* 5.0 + 0.3 ° 2.9 + 0.5**
IPS 339 and metoprolol were injected 15 min before clenbuterol. Clenbuterol I mg/kg i.p. was administered 15 min before access to food. Each group consisted of 6 rats. ANOVA 2x2; IPS 339 treatment: F interaction 6.3 df 1/19 p
Discussion The fact that IPS 339, a specific 82 adrenergic receptor blocker, but not metoprolol, which is known to block only 81 adrenergic receptors, prevented the anorectic effect of salbutamol, suggests that ~2 adrenergic sites are selectively involved. Injection of IPS 339 in the perifornieal hypothalamic area completely antagonized the anorexia induced by salbutamol suggesting that 82 adrenergic receptors in this area mediate the suppression of feeding induced by systemic salbutamol.
264
B-adrenergic
Receptors and Food intake
Vol.
38, No. 3, 198@
Table 5 Effect of IPS 339, metoprolol and l-propranolol injected bilaterally into the hypothalamic perifornical area on the anorectic action of diethylpropion.
Treatment
Dose (~g/1~l)
Food intake Saline
(g/rat/lh) Mean + S.E. Diethy]propion
Vehicle IPS 339
32
13.6 + 1.0 15.8 + 1.0
7.0 + 1.3"* 6.5 + 1.1"*
Vehicle Metoprolol
80
11.2 + 1.6 9.3 + 0.8
3.7 + 0.6** 4.9 + 0.4**
11.1 + 0.6 13.2 + 0.9
4.9 + 0 . 6 * * 9.8 + 1.1"*
Vehicle l-Propranolol
(~g/2 91) 52
IPS 339, metoprolol and l-propranolol were injected just before diethylpropion. Diethylpropion 5 mg/kg i.p. was injected 15 min before rats had access to food. Each group consisted of 4-7 rats. ANOVA 2x2; IPS 339 treatment: F interaction 1.4 df 1/19 N.S. Metoprolol treatment: F interaction 3.4 df 1/21N.S. l-Propranolol treatment: F interaction 3.0 df 1/16 N.S. Tukey's test; *p
The involvement of $2 adrenergic sites in the perifornical area in the mechanism of anorexia was previously suggested by Leibowitz and Rossakis (2) who observed that the reduction of food consumption induced by adrenaline injected in this brain area was preferentially blocked by ~2 adrenergic antagonists. The present study shows that systemically administered $2 adrenergic stimulants can effectively activate these central sites involved in the control of feeding behaviour. Metoprolol injected in the perifornical hypothalamus failed to modify the anorectic action of salbutamol, but significantly reduced the feeding suppression induced by subcutaneous isoproterenol, a B I ~2 adrenergic agonist, suggesting that $I adrenergic receptors involved in mediating feeding inhibition in hungry rats are also present in the perifornical hypothalamus. Clenbuterol, another ~2 adrenergic agonist which crosses the blood brain barrier more readily than salbutamol, was 10-100 times more effective than the latter in reducing food intake in deprived rats. Frances et al. (16) reported a similar potency ratio between these two drugs for the reduction of motor activity and the loss of interest of fasting mice in food pellets whereas these drugs showed comparable peripheral activity (11). Selective inw)ivement of ~2 adrenergic receptors in the anorectic effect of clenbuterolwas demonstrated by the fact that it was prevented by pretreatment with IPS 339 but not with metoprolol. The easier access to central ~2 adrenergic sites involved in feeding inhibition and the specificity of action of clenbutero] make it a potentially useful drug for the treatment of obesity.
Vol. 38, No. 3, 1986
B-adrenergic Receptors and Food Intake
265
The anorectic effect of diethylpropion, which was previously shown to be completely antagonized by lesion of the ventral noradrenergic bundles (12), was not modified by intraperifornical injections of IPS 339 or metoprolol, suggesting that different mechanisms are involved in the anorectic effect of salbutamol and diethylpropion. Unlike salbutamol which directly stimulates central B 2 adrenergic receptors (17), diethylpropion releases noradrenaline from nerve terminals (18) and may therefore have a more general effect on ~-adrenergic receptors involved in feeding control. The selective block of a single receptorial population of the perifornical hypothalamus could be insufficient to modify diethylpropion's effect on food intake. This suggestion is supported by the finding that l-propranolol, which block both $I and ~2 adrenergic receptors, partially prevented the effect of diethylpropion when injected in the perifornical hypothalamic area. A problem with this interpretation is that l-propranolol, because of its poor solubility, was injected in the hypothalamus in a larger volume than the other adrenergic antagonists and it may therefore have reached adrenergic sites outside the perifornical area. The fact that diethylpropion-induced anorexia was only partially reduced by inhibiting perifornical adrenergic receptors whereas complete antagonism was observed after the lesion of the ventral bundle of noradrenaline-containing fibers (12) suggests that other noradrenergicadrenergic sites in the hypothalamus or other brain regions innervated by these noradrenergic-adrenergic pathways play a role in the action of diethylpropion. In conclusion, the present study shows that systemically administered salbutamol causes anorexia in hungry rats through the selective activation of ~2adrenerg~ receptors in the perifornical hypothalamic area. Clenbuterol, which easily enters in the brain, is 70-100 times more potent than salbutamol in inhibiting feeding consumption of deprived rats and this effect is selectively mediated by B 2 adrenergic receptors. Finally, it has been shown that B I and B 2 adrenergic receptors in the hypothalamus partially contribute to the mechanism by which diethylpropion causes anorexia in rats. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10.
S.F. LEIBOWITZ, Nature 226 963-964 (1970). S.F. LEIBOWITZ and C. ROSSAKIS, Neuropharmacology 1 7 691-702 (1978). H.W. GOLD,JAN, D. LEHR and E. FRIEDMAN, Nature 231 453-455 (1971). D. LEHR and W. GOLDMAN, Eur. J. Pharmac. 23 197-210 (1973). V.A. CULLUM, J.B. FARMER, D. JACK and G.P. LEVY, Br. J. Pharmac. 35 14]-151 (1969). F. BORSINI, C. BENDOTTI, P. THURBLY and R. SAMANIN, Life Sci. 30 905-911 (1982). J.L. IMBS, F. MIESCH, J. SCHWARTZ, J. VELLY, G. LECLERC, A. MANN and C.G. VERMUTH, Br. J. Pharmac. 60 357-362 (1977). S.B. ROSS, Acta pharmac, tox. 47 347-350 (1980). B. ABLAD, K.O. BORG, E. CARLSSON, L. EK, G. JOHNSSON, T. MALFORS and C-G. REGARDH, Acta pharmac.tox. 36 suppl.V, 7-23 (1975). J.A. STREET, B.A. HEMSWORTH, A.G. ROACH and M.D. DAY, Archs. int.. Pharmacodyn. Ther. 237 180-190 (~979).
266
11. 12. 13. 14.
15.
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Receptors
and Food Intake
Vol. 38, No. 3, 1986
V.G. ENGELHARDT, Arzneimittel-Forsch. 26 1404-1420 (1976). F. BORSINI, C. BENDOTTI, M. CARLI, E. POGGESI and R. SAMANIN, Res. Commun. chem. Pathol. Pharmac. 26 3-11 (1979). J.F. MUNRO and M.J. FORD, i n Drugs and Appetite (ED. T. SILVERSTONE), p. 125-158, Academic Press, London (1982). M.T. HOEKENGA, R.H. DILLON and H.M. LEYLAND, irL Central Mechanisms of Anorectic Drugs (ED. S. GARATTINI and R. SAMANIN), p. 391-404, Raven Bess, New York (1978). J.F.R. KONIG and R.A. KLIPPEL, The Rat Brain: A Stereotaxic Atlas of the Forebrain and Lower Parts of the Brain Stem, Williams & Wilkins, Baltimore
(1963). 16. 17. 18.
H. FRANCES, A.J. PUECH, S. DANTI and P. SIMON, Eur. J. Pharmac. 92 223-230 (1983). D.B. BYLUND and 8.H. SNYDER, Mol. Pharmac. 12 568-580 (1976). J. OFFERMEIER and H.G. du PREEZ, in Central Mechanisms of Anorectic Drugs (ED. S. GARATTINI and R. SAMANIN), p. 217-231, Raven Press, New York (1978).