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Physiology & Behavior, Vol. 63, No. 4, pp. 723–728, 1998 © 1998 Elsevier Science Inc. All rights reserved. Printed in the U.S.A. 0031-9384/98 $19.00 1 .00
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A b-3 Adrenergic Agonist (BRL-37,344) Decreases Food Intake SATORU TSUJII*1 AND GEORGE A. BRAY† *Pennington Biomedical Research Center, Baton Rouge, LA 70808, and †Department of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 18 July 1997; Accepted 5 November 1997 TSUJII, S. AND G. A. BRAY. A b-3 adrenergic agonist (BRL-37,344) decreases food intake. PHYSIOL BEHAV 63(4) 723–728, 1998.—This study evaluated the effect of peripheral injections of a b3 adrenergic agonist, BRL-37,344 on food intake and whether this inhibition could be blocked by a nonspecific b-adrenergic antagonist, propranolol, given peripherally or into the central nervous system. When BRL-37,344 was injected intraperitoneally (i.p.) into lean and obese Zucker rats, food intake was decreased. The reduction of food intake by BRL-37,344 was attenuated when propranolol was administered i.p. prior to giving the b adrenergic agonist. When propranolol was administered into the third cerebral ventricle, it increased food intake in lean rats, but not the fatty rats. Propranolol administered into the third cerebral ventricle attenuated the effect on food intake of i.p. injection of BRL-37,344. These studies support the hypothesis that there are peripheral b-3 adrenergic receptors that can reduce food intake and that there are central b2 or b3 adrenergic receptors that mediate the peripheral effect of the b3 agonist. © 1998 Elsevier Science Inc. Zucker rats
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tissue (2,3,8,25,45). Several b3 adrenergic drugs have been developed (2,45), and some of them have been tested clinically (2,11,45). Using a cloned b3 adrenergic receptor transfected into Chinese hamster ovarian cells, Emorine et al. (13) showed that BRL-37,344 [4-(2-((2-hydroxy-2-(3-chlorophenyl)ethyl)amino)propyl)-phenoxy-acetate] was among the most specific agonists for this receptor. The present studies, initially presented in abstract form (39), were designed to establish whether there are peripheral b3 adrenergic receptors, the activation of which will reduce food intake and whether this is blocked by a nonspecific b-adrenergic blocking drug, propranolol, given either peripherally or into the CNS.
MORE than 30 years ago, Grossman (20) demonstrated that norepinephrine injected into the hypothalamus of rats stimulated feeding. This seminal observation was followed by data from Leibowitz (27) showing that there were a-adrenergic receptors located in the paraventricular nucleus (PVN) that stimulated feeding and a-adrenergic receptors located in the perifornical area that produced satiety when norepinephrine was selectively injected into these regions. Subsequent work from her laboratory (27) and elsewhere (42) showed that there are both a1(43) and a2 adrenergic receptors in the PVN that modulate feeding (19,28,29,41,43). These observations have been extended in several laboratories that have found that clonidine, an a2 adrenergic agonist, can stimulate feeding whether injected into the central nervous system (CNS) or injected peripherally (4,34,41). These effects occur in normal as well as genetically obese animals (12,41). Injection of a-1 agonists into the PVN, on the other hand, decreases food intake (43). Data in the fatty rat have suggested that hyperphagia in these animals may reflect an altered balance between the a2 and b-adrenergic control of feeding (43). Moreover, we have shown that a selective b3 adrenergic agonist produces a dose-dependent reduction in food intake when administered into the third ventricle (43) of fatty and lean rats, even though b3 receptors have not been found in the CNS. This effect is more potent in the fatty rat than in the corresponding lean animals. Conversely, clonidine injected into the third ventricle stimulated food intake more in lean rats than in Zucker fatty rats (43). b3 adrenergic agonists are lipolytic and thermogenic. They are lipolytic in white adipose tissue and thermogenic in brown adipose 1
MATERIALS AND METHODS
Animals The 34 female Zucker fatty rats weighing 290 –390 g and 29 lean female litter mates weighing 190 –240 g used in these experiments were purchased from the Vassar College Colony (Poughkeepsie, NY). Animals had access to commercial laboratory chow (Wayne Lablox, Chicago, IL) and were adapted to eating a powdered chow prior to the experiments. Tap water was available ad libitum, and lights were illuminated from 0600 to 1800 h. The vivarium was maintained at 22 6 2°C. Surgical Procedures Guide cannulas, made of 23 gauge stainless steel, were placed in the third ventricle of lean and Zucker fatty rats, which were
To whom requests for reprints should be addressed, at Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808.
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724 anesthetized with pentobarbital (45 mg/kg) and mounted in a Kopf stereotaxic frame (Tujunga, CA). The tips of the cannulas were placed 2.8 mm posterior to the bregma and 7.8 – 8.3 mm below the cortical surface at the midline sagittal sinus (32). The cannulas were anchored in dental cement held in place by stainless steel screws in the calvarium. A stylet was placed inside of the guide cannula and lightly sealed. Animals were allowed to recover for 7 to 10 days prior to experimentation. At the end of the experiments, animals were injected with methylene blue dye and their brains removed and fixed in 10% formalin prior to the preparation of frozen sections to verify the location of the cannulas. An infusion volume of 5 mL was given into the third ventricle over 10 min. Solutions for injection were prepared in buffered saline. The BRL-37,344 was kindly provided by SmithKline Beecham Laboratories (London, UK). All other reagents were reagent grade. Protocols Rats were deprived of food overnight prior to all experiments. Studies began in the morning between 0900 and 1000 h. Animals were thoroughly accustomed to handling prior to experimentation. Peripheral injections were given into the peritoneum (BRL-37,344 or propranolol). Following injections, animals were returned to their home cage and provided with fresh food. No behavioral changes were noted in either fa/fa or lean rats after the intraperitoneal (i.p.) injections, but when they stopped eating, grooming behavior increased. The food eaten was measured at 0.5, 1, 2, 3, and 6 h when animals were also observed for behavioral changes. Spillage on paper towels beneath the food cup was subtracted to give cumulative food intake. Three experiments were conducted. In each experiment, rats were used in a counter-balanced design so that each rat received all treatments with an interval of 3–5 days between injections. Experiment 1 examined whether peripheral injection of the BRL-37,344 would reduce food intake in animals fasted for 24 h. Saline and doses of 30 and 300 nmol of BRL-37,344 were injected i.p. in 14 fatty and 11 lean animals. These doses were picked to be similar to the doses given by i.c.v. injection in a previous experiment (41). Although we do not know the distribution of this drug in our studies if it is diluted in body water, the concentration would approximate ;1 nmol/mL, which is less than 1% of the concentration that would be anticipated had the 300 nmol given i.c.v. dissolved only in brain water (approximately 2 mL). Experiment 2 examined the interaction between i.p. injections of propranolol at two doses and BRL-37,344 at one dose (300 nmol). Eleven Zucker fatty rats and 10 lean litter mates received i.p. injections of saline or of propranolol at 300 nmol (the molar equivalent of BRL) or at a much higher does of 5 mg/kg (approximately 5000 nmol). These are doses that will block b1 and b2 adrenergic receptors. Injections of propranolol were followed 15 min later by an i.p. injection of saline or BRL-37,344 (300 nmol). Each animal received all treatments in a counter-balanced design with 3 to 5 days between injections. Experiment 3 tested whether propranolol injected into the third ventricle (i.c.v.) influenced the response to peripherally administered BRL-37,344. Nine Zucker fatty rats and 8 lean litter mates with cannulas in the third ventricle were injected i.c.v. with saline or propranolol (300 nmol/5 mL), and 15 min later with saline or BRL-37,344 (300 nmol) i.p. Equimolar doses were selected for this study. Each animal received all treatments in a counterbalanced design.
TSUJII AND BRAY Statistical Analysis Data for Experiment 1 was analyzed by a univariate ANOVA using genotype as a grouping factor and treatment and time as repeated measures variables. Experiments 2 and 3 were analyzed by a univariate ANOVA using genotype as a grouping factor with Treatment 1, Treatment 2, and time as repeated measures variables. In addition, data were evaluated by a one- or two-way ANOVA for each phenotype at each time point where appropriate. If the ANOVA indicated, further comparisons between different doses were performed using the least significant difference test as a post-hoc test. Data are expressed as mean 6 SEM. RESULTS
Experiment 1. Food intake after peripheral injection of BRL-37,344 Figure 1 shows the food intake of lean and obese Zucker rats for 6 h following i.p. injection of BRL 37,344. Food intake was significantly reduced by treatment with BRL-37,344 in both Zucker fatty rats and lean rats [F(2, 46) 5 34.8; p , 0.0001; three-way ANOVA]. For the fatty rats, food intake was decreased compared to saline at 30 min [F(2, 26) 5 21.8; p , 0.01], at 1 h [F(2, 26) 5 20.95; p , 0.01], at 2 h [F(2, 26) 5 21.97; p , 0.01], at 3 h [F(2, 26) 5 21.99; p , 0.01], and at 6 h [F(2, 26) 5 24.75; p , 0.01]. For the lean animals, food intake was also significantly decreased at 30 min [F(2, 20) 5 15.48; p , 0.01], at 1 h [F(2, 20) 5 11.17; p , 0.01], at 2 h [F(2, 20) 5 6.34; p , 0.01], and at 3 h [F(2, 20) 5 9.32; p , 0.01], but not at 6 h. Food intake was decreased by 22% with the low dose and 33% by the high dose at 30 minutes. The fatty (fa/fa) genotype showed a greater percentage reduction than the lean animals at all subsequent times. Experiment 2. Effect of i.p. propranolol on the response to BRL-37,344. Figure 2 shows the data on fatty rats (left) and lean rats (right). Using a four-way ANOVA, there were significant main effects of BRL-37,344 [F(1, 19) 5 90.0099; p , 0.0000] time [F(1, 19) 5 451.4062; p , 0.0000) and an effect of propranolol [F(2, 19) 5 4.6903; p 5 0.0129], but no main effect of genotype. Treatment with BRL-37,344 decreased food intake confirming the previous experiment. There was a significant interactions between propranolol and BRL-37,344 [F(2, 38) 5 11.5088; p , 0.000124]. Propranolol, partially, but not completely, reversed the effect of BRL37,344 (left panel). The effects are most easily separable using the data on the fatty rats. Using the two-way ANOVA, these effects were significant at 0.5 h, 1 h, 2 h, and 3 h in the lean rats and at 1 h, 2 h, and 3 h in the fatty rats. These data suggest that the inhibitory effects of BRL-37,344 on food intake can be partially blocked by the injection of propranolol, a nonspecific b-adrenergic blocking drug. The larger dose of propranolol, 5 mg/kg i.e., ;5000 nmol, by itself reduced food intake in both phenotypes of animals 2 and 3 h after injection. Experiment 3. Effect of propranolol injected in the third ventricle on food intake following peripheral injection of BRL-37,344. Intraperitoneal injection of BRL-37,344 significantly decreased food intake [F(1, 15) 5 61.4176; p , 0.0000; (Fig. 3)] in this experiment as in the previous two. This was more obvious in the fatty rats (left panel) than in the lean rats (right panel), a finding supported by the interaction in the four-way ANOVA [F(1, 15) 5 13.6596; p 5 0.0021]. Propranolol, injected into the third ventricle
BRL-37,344 b-AGONISTS AND FOOD INTAKE
FIG. 1. Effect of BRL-37,344 on food intake of lean and Zucker fatty rats. Vehicle and doses of 30 and 300 nmol of BRL-37,344 were injected i.p. into overnight-starved lean and fatty rats with 3–5 days between injections. Food intake was measured for vehicle and drug-treated animals at 0.5, 1, 2, 3, and 6 h after injection. Data are expressed as mean 6 SEM for 14 fatty and 11 lean animals at each treatment level. *p , 0.01 compared to vehicle treated controls.
FIG. 2. Effect of propranolol on the response to BRL-37,344 in lean and Zucker fatty rats. Each animal received vehicle or propranolol by two routes: i.p. injections of propranolol at 5 mg/kg, and at a different time, intracerebroventricular injections of propranolol (300 nmol) into the third ventricle or they received comparable volumes of vehicle by each route, saline followed 15 min later by BRL-37,344 (300 nmol). Food intake was measured at 0.5, 1, 2, 3, and 6 h after treatment. Data are expressed as mean 6 SEM for 11 fatty and 10 lean rats.
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FIG. 3. Effect of intracerebroventricular injections of propranolol on the response to i.p. BRL-37,344 in lean and Zucker fatty rats. Propranolol (300 nmol) was injected into the third vertebral ventricle 15 min before i.p. injection of BRL- 37,344 (300 nmol). Food intake was measured at 0.5, 1, 2, 3, and 6 h after treatment. Data are expressed as mean 6 SEM. There was no significant attenuation of the response to BRL-37,344 by injecting propranolol i.c.v.
by itself had a marginal effect of increasing food intake in the lean rats [F(1, 15) 5 5.093; p 5 0.039] but it had no effect on food intake in the fatty rats. Propranolol significantly increased food intake at 1 h [F(1, 7) 5 6.48; p , 0.05] and 3 h [F(1, 7) 5 8.15; p , 0.05] in the lean rats. There was an interaction between propranolol and BRL-37,344 [F(1, 15) 5 7.47; p 5 0.015] but no interaction by genotype. These data suggest that there are peripheral b-adrenergic receptors which can reduce food intake and that blockade of CNS b-adrenergic receptors with propranolol may attenuate this effect. ANOVA showed no main effect of genotype in any of the three experiments. Time, a repeated measures variable (or within subject variable), was highly significant in all experiments. DISCUSSION
b-adrenergic receptors have been known to mediate satiety since the work of Grossman (20,21) and Leibowitz (27,28). They showed that injection of norepinephrine into the medial hypothalamic area increased food intake. Leibowitz (27) also showed that propranolol would block the reduction of food intake following perifornical injection of norepinephrine (NE) indicating that NE was probably acting through either b1 or b2 adrenergic receptors. After Arch et al. (3) showed that a non- b1 -non- b2 adrenoceptor, now known to be the b3 adrenoceptor mediated the thermogenic response to NE, a number of agonists were developed as potential thermogenic compounds to treat obesity (2,3,45). Treatment with these drugs reduced body fat in experimental animals without affecting food intake (8). Reduction of body fat to a comparable degree by calorie restriction would be followed by marked hyperphagia when food became available ad libitum, suggesting that hyperphagia does not occur in animals chronically treated with b3 agonists because food in-
take is being simultaneously suppressed. In a previous study, we were the first to test the effects on food intake of a b3 agonist, BRL-37,344 injected into the ventricular system (41). We found that it acutely reduced food intake in fatty rats more than in lean rats (41). We subsequently reported in abstract form that BRL 37,344 also reduced food intake when given peripherally (39). The present study extends these observations by showing that i.p. injection of BRL-37,344 reduces food intake more in Zucker fatty rats than in lean rats. In addition, the response to BRL-37,344 was attenuated by an i.p. injection of propranolol as well as by propranolol injected into the third ventricle. Because 300 nmol of b3 agonist given peripherally in this experiment or centrally in the previous experiment (41) produced a reduction in food intake, it suggests that b-adrenergic receptors responsive to BRL-37,344 are located peripherally and may also be present in the CNS. Although we do not know the pharmacokinetics of BRL-37,344 in these animals that differ markedly in body fat, it is clear that fatty rats showed a greater reduction in food intake, by either route, despite their greater weight and greater quantity of body fat. Several studies using different b3 agonists have shown a decrease in food intake in mice and rats when the drugs are given peripherally (22,26,38,41) or in the diet (10). When given in the diet, the effects on body fat were found to vary with the diet and strain of mice (10). The drug used in the present study, BRL37,344, has a high affinity for b3 adrenergic receptors (13) but will also interact with b2 adrenoceptors at a much lower affinity. Propranolol injected peripherally attenuated, but did not block, the reduction in food intake produced by BRL-37,344. Propranolol is a weak antagonist of b3 adrenoceptors but is an effective antagonist of both b1 and b2 adrenoceptors. There are, as yet, no potent b3 receptor antagonists known to us. In other
BRL-37,344 b-AGONISTS AND FOOD INTAKE studies, however, we have found that a b2 agonist, clenbuterol (44), given peripherally or salbutamol given centrally (41) are both potent inhibitors of food intake. The ED50 for suppressing food intake by the clenbuterol was 0.04 mmol/kg compared to . 1 mmol/kg for a selective b3 agonist, ICI D-7114 (44). The relative potency of these two drugs for thermogenesis was reversed (44), leading to the question of the location and receptor type through which the anorectic effect of b3 adrenoceptor agonists is occurring. There are several possible locations of the b3 adrenoceptors that suppress food intake. Himms–Hagen (25) has recently proposed that the initiation and inhibition of food intake may be related to the effects of b3 agonists on brown adipose tissue, which is a rich source of b3 adrenoceptors because damage to this tissue in transgenic animals leads to weight gain and susceptibility to high fat-induced obesity (23). Grujic et al. (22) have recently provided a critical test of this idea. They used transgenic mice expressing b3 adrenoceptors in brown adipose tissue alone, or in brown and white adipose tissue. The reduction of food intake in the animals expressing the b3 adrenoceptors in both brown and white adipose tissue was much greater than the effect in the animals expressing it only in brown adipose tissue, suggesting that it is the b3 adrenoceptors in white adipose tissue that are involved in reducing food intake. This effect in white fat cells cannot be the result of leptin because it occurs rapidly and because activation of b3 adrenoceptors suppresses leptin mRNA expression (30). The nature of the signal from white adipose tissue following treatment with a b3 agonist that could reduce food intake is unknown. One possibility would be fatty acids. Modifying fatty acid oxidation is known to affect food intake (9,15–17,24,35), and this involves vagal afferents (33). Changes in blood metabolites are associated with the hypophagia following insulin (7,18,31). b-hydroxybutyrate, a metabolite of long chain fatty acids, reduces food intake and is another possible product of metabolism of fatty acids from adipose tissue that could modulate feeding (1). Finally, the fat cell may release TNF-a, which is a cytokine that could suppress feeding. Whether any of these are actually involved requires additional experimental work. A second possible explanation is that the b3 agonists could be acting on b3 receptors in the brain. Although not originally identified in brain, recent studies using RT-PCR by Summers et al. (37) have demonstrated the presence of b3 adrenoceptor mRNA in cerebellum, hippocampus, and striatum. This is consistent with observations that an atypical b-adrenoceptor agonist (SR 58611A) has an antidepressant profile (36). Similarly, the demonstration that the b3 agonist, BRL-37,344 given into the third ventricle reduces food intake is consistent with b3 adrenoceptors in the brain. A third possible site for action on b3 adrenoceptors is in the gut. b3 adrenoceptors have been identified in the fundus of the stomach, in the longitudinal and circular smooth muscle of both colon and ileum, and in the colonic submucosa (14). Afferent signals from the gut to the brain are an important part of the satiety system (33,35). Stimulation of b3 receptors in the gut are involved in gastric acid secretion (6) and may be involved in modulating food intake. Propranolol given into the third cerebral ventricle attenuated the reduction in food intake when BRL-37,344 was given peripherally. This suggests that NE is involved in the central anorectic system activated by peripheral b3 adrenoceptor stimulation. This central adrenoceptor could be either the b2 adrenoceptor, which responds to salbutamol (41) or the b3 adrenoceptor, which has been demonstrated by Summers et al. (37). Differentiation of these possibilities will await the development of selective b3 antagonists. In either case, it suggests that one of the central relays from the peripheral adrenergic receptor system is another adrenergic receptor in the brain.
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FIG. 4. Diagram of peripheral and central circuitry that could account for the effects of b-adrenergic agonists on food intake when injected peripherally or centrally. See text for details. POA, preoptic area; PFA, perifornical area; MPG, motor pattern generator; VMH, ventromedial hypothalamus; BAT, brown adipose tissue.
A proposed model for the central and peripheral effects of b-adrenergic receptors in food intake is shown in Fig. 4. Peripheral injection of BRL-37,344 may stimulate b3 adrenoceptors in white adipose tissue with the production of heat. Increased heat is known to reduce food intake probably through effects mediated by the pre-optic area of the hypothalamus. Himms–Hagen has recently proposed a model of food intake in which the brown adipose tissue plays a central role (25). In this model, stimulation of brown adipose tissue by the systemic nervous system increases glucose utilization and produces the “glucose dip” that is associated with 60% or more of spontaneous meals. Alternatively, the b3 agonist may activate b3 adrenoceptors in the gut with satiety information relayed to the brain over the vagus or splanchnic nerves. The heat signal acting through the pre-optic area or afferent neural signal acting in the hind brain or hypothalamus or elsewhere could reduce food intake through effects on satiety mediated through the perifornical area. The perifornical area acts to inhibit a motor pattern generator, which controls the food seeking activities. Central injection of a b-adrenergic agonist could act directly on the b-adrenergic receptors in the perifornical area that have been identified by Leibowitz (27,28). The satiety that is produced by a meal might also involve the pathways described here as shown by the nutrient or vagal signals that are generated by ingestion of food and transmitted to the ventromedial hypothalamus, where they activate the pathways involved in stimulation of brown adipose tissue. The differences in the fatty Zucker rat and the lean animals may reside in the different relative levels of tonic activity that serves to modulate hypothalamic receptor activity (40 – 42), supporting the hypothesis of a reciprocal relationship between food intake and the sympathetic nervous system (5). In conclusion, there are both peripheral and central b3-adrenergic receptors involved in producing satiety. ACKNOWLEDGEMENTS
Supported by National Institute of Health (NIDDK) Grant 2 R01 DK31988.
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enhances susceptibility to diet-induced obesity, diabetes, and hyperlipidemia. Endocrinology. 137:21–29; 1996. Harris, R. B. S.; Martin, R. J. Lipostatic theory of energy balance: Concepts and signals. Nutr. Behav. 1:253–275; 1984. Himms-Hagen, J. Role of brown adipose tissue thermogenesis in control of thermoregulatory feeding in rats: A new hypothesis that links thermostatic and glucostatic hypotheses for control of foodintake. Proc. Soc. Exp. Biol. Med. 208:159 –169; 1995. Himms–Hagen, J.; Cui, J.; Danforth E., Jr.; Taatjes, D. J.; Lang S. S.; Waters, B. L.; Claus, T. H. Effect of CL-316,243, a thermogenic b-3 agonist, on energy balance and brown and white adipose tissues in rats. Am. J. Physiol. 266:R1371–R1382; 1994. Leibowitz, S. F. Reciprocal hunger-regulating circuits involving alphaand beta-adrenergic receptors located, respectively, in the ventromedial and lateral hypothalamus. Proc. Natl. Acad. Sci. USA. 67:1063– 1070; 1970. Leibowitz, S. F. Brain monoamines and peptides: Role in the control of eating behavior. Fed. Proc. 45:1396 –1403; 1986. Leibowitz, S. F.; Jhanwar-Uniyal, M.; Dvorkin, B.; Makman, M. H. Distribution of a-adrenergic, b-adrenergic and dopaminergic receptors in discrete hypothalamic areas of rat. Brain Res. 233:97–114; 1982. Mantzoros, C. S.; Qu, D.; Frederich, R. C.; Susulic, V. S.; Lowell, B. B.; Maratos-Flier, E.; Flier, J. S. Activation of b3-adrenergic receptors suppresses leptin expression and mediates a leptin-independent inhibition of food intake in mice. Diabetes. 45:909 –914; 1996. Mayer, J. Regulation of energy intake and the body weight: The glucostatic theory and the lipostatic hypothesis. Ann. NY Acad. Sci. 63:15– 43; 1955. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney: Academic Press; 1982. Ritter, S.; Taylor, J. S. Vagal sensory neurons are required for lipoprivic but not glucoprivic feeding in rats. Am. J. Physiol. 258: R1395–R1401; 1990. Ritter, S.; Wise, C. D.; Stein, L. Neurochemical regulation of feeding in the rat: Facilitation by a-noradrenergic, but not dopaminergic, receptor stimulants. J. Comp. Physiol. Psychol. 88:778 –784; 1975. Scharrer, E.; Langhans, W. Control of food intake by fatty acid oxidation. Am. J. Physiol. 250:R1003–R1006; 1986. Simiand, J.; Keane, P. E.; Guitard, J.; Langlois, X.; Gonalons, N.; Martin, P.; Bianchetti, A.; Le Fur, G.; Soubrie, P. Antidepressant profile in rodents of SR 58611A, a new selective agonist for atypical b-adrenoceptors. Eur. J. Pharmacol. 219:193–201; 1992. Summers, R. J.; Papaioannou, M.; Harris, S.; Evans, B. A. Expression of b3-adrenoceptor mRNA in rat brain. Br. J. Pharmacol. 116:2547– 2548; 1995. Susulic, V. S.; Frederich, R. C.; Lawitts, J.; Tozzo, E.; Kahn, B. B.; Harper, M.; Himms-Hagen, J.; Flier, J. S.; Lowell, B. B. Targeted disruption of the b3-adrenergic receptor gene. J. Biol. Chem. 270: 29483–29492; 1995. Tsujii, S.; Bray, G. BRL-37344 (b3-adrenoceptor agonists) and food intake in genetically obese rats. Int. J. Obes. 14:50; 1990. Tsujii, S.; Bray, G. A. Effects of glucose, 2-deoxyglucose, phlorizin, and insulin on food intake of lean and fatty rats. Am. J. Physiol. 258:E476 – 481; 1990. Tsujii, S.; Bray, G. A. Food intake of lean and obese Zucker rats following ventricular infusions of adrenergic agonists. Brain Res. 587:226 –232; 1992. Tsujii, S.; Bray, G. A. GABA-related feeding control in genetically obese rats. Brain Res. 540:48 –54; 1991. Wellman, P. J.; Davies, B. T.; Morien, A.; McMahon, L. Modulation of feeding by hypothalamic paraventricular nucleus a1 and a2-adrenergic receptors. Life Sci. 53:669 – 679; 1993. Yamashita, J.; Onai, T.; York, D. A.; Bray, G. A. Relationship between food intake and metabolic rate in rats treated with b-adrenoceptor agonists. Int. J. Obes. 18:429 – 433; 1994. Yen, T. T. Antiobesity and antidiabetic b-agonists: Lessons learned and questions to be answered. Obes. Res. 2:472– 480; 1994.
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A b-3 Adrenergic Agonist (BRL-37,344) Decreases Food Intake SATORU TSUJII*1 AND GEORGE A. BRAY† *Pennington Biomedical Research Center, Baton Rouge, LA 70808, and †Department of Medicine, University of Southern California, Los Angeles, CA 90033, USA Received 18 July 1997; Accepted 5 November 1997 TSUJII, S. AND G. A. BRAY. A b-3 adrenergic agonist (BRL-37,344) decreases food intake. PHYSIOL BEHAV 63(4) 723–728, 1998.—This study evaluated the effect of peripheral injections of a b3 adrenergic agonist, BRL-37,344 on food intake and whether this inhibition could be blocked by a nonspecific b-adrenergic antagonist, propranolol, given peripherally or into the central nervous system. When BRL-37,344 was injected intraperitoneally (i.p.) into lean and obese Zucker rats, food intake was decreased. The reduction of food intake by BRL-37,344 was attenuated when propranolol was administered i.p. prior to giving the b adrenergic agonist. When propranolol was administered into the third cerebral ventricle, it increased food intake in lean rats, but not the fatty rats. Propranolol administered into the third cerebral ventricle attenuated the effect on food intake of i.p. injection of BRL-37,344. These studies support the hypothesis that there are peripheral b-3 adrenergic receptors that can reduce food intake and that there are central b2 or b3 adrenergic receptors that mediate the peripheral effect of the b3 agonist. © 1998 Elsevier Science Inc. Zucker rats
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tissue (2,3,8,25,45). Several b3 adrenergic drugs have been developed (2,45), and some of them have been tested clinically (2,11,45). Using a cloned b3 adrenergic receptor transfected into Chinese hamster ovarian cells, Emorine et al. (13) showed that BRL-37,344 [4-(2-((2-hydroxy-2-(3-chlorophenyl)ethyl)amino)propyl)-phenoxy-acetate] was among the most specific agonists for this receptor. The present studies, initially presented in abstract form (39), were designed to establish whether there are peripheral b3 adrenergic receptors, the activation of which will reduce food intake and whether this is blocked by a nonspecific b-adrenergic blocking drug, propranolol, given either peripherally or into the CNS.
MORE than 30 years ago, Grossman (20) demonstrated that norepinephrine injected into the hypothalamus of rats stimulated feeding. This seminal observation was followed by data from Leibowitz (27) showing that there were a-adrenergic receptors located in the paraventricular nucleus (PVN) that stimulated feeding and a-adrenergic receptors located in the perifornical area that produced satiety when norepinephrine was selectively injected into these regions. Subsequent work from her laboratory (27) and elsewhere (42) showed that there are both a1(43) and a2 adrenergic receptors in the PVN that modulate feeding (19,28,29,41,43). These observations have been extended in several laboratories that have found that clonidine, an a2 adrenergic agonist, can stimulate feeding whether injected into the central nervous system (CNS) or injected peripherally (4,34,41). These effects occur in normal as well as genetically obese animals (12,41). Injection of a-1 agonists into the PVN, on the other hand, decreases food intake (43). Data in the fatty rat have suggested that hyperphagia in these animals may reflect an altered balance between the a2 and b-adrenergic control of feeding (43). Moreover, we have shown that a selective b3 adrenergic agonist produces a dose-dependent reduction in food intake when administered into the third ventricle (43) of fatty and lean rats, even though b3 receptors have not been found in the CNS. This effect is more potent in the fatty rat than in the corresponding lean animals. Conversely, clonidine injected into the third ventricle stimulated food intake more in lean rats than in Zucker fatty rats (43). b3 adrenergic agonists are lipolytic and thermogenic. They are lipolytic in white adipose tissue and thermogenic in brown adipose 1
MATERIALS AND METHODS
Animals The 34 female Zucker fatty rats weighing 290 –390 g and 29 lean female litter mates weighing 190 –240 g used in these experiments were purchased from the Vassar College Colony (Poughkeepsie, NY). Animals had access to commercial laboratory chow (Wayne Lablox, Chicago, IL) and were adapted to eating a powdered chow prior to the experiments. Tap water was available ad libitum, and lights were illuminated from 0600 to 1800 h. The vivarium was maintained at 22 6 2°C. Surgical Procedures Guide cannulas, made of 23 gauge stainless steel, were placed in the third ventricle of lean and Zucker fatty rats, which were
To whom requests for reprints should be addressed, at Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, LA 70808.
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724 anesthetized with pentobarbital (45 mg/kg) and mounted in a Kopf stereotaxic frame (Tujunga, CA). The tips of the cannulas were placed 2.8 mm posterior to the bregma and 7.8 – 8.3 mm below the cortical surface at the midline sagittal sinus (32). The cannulas were anchored in dental cement held in place by stainless steel screws in the calvarium. A stylet was placed inside of the guide cannula and lightly sealed. Animals were allowed to recover for 7 to 10 days prior to experimentation. At the end of the experiments, animals were injected with methylene blue dye and their brains removed and fixed in 10% formalin prior to the preparation of frozen sections to verify the location of the cannulas. An infusion volume of 5 mL was given into the third ventricle over 10 min. Solutions for injection were prepared in buffered saline. The BRL-37,344 was kindly provided by SmithKline Beecham Laboratories (London, UK). All other reagents were reagent grade. Protocols Rats were deprived of food overnight prior to all experiments. Studies began in the morning between 0900 and 1000 h. Animals were thoroughly accustomed to handling prior to experimentation. Peripheral injections were given into the peritoneum (BRL-37,344 or propranolol). Following injections, animals were returned to their home cage and provided with fresh food. No behavioral changes were noted in either fa/fa or lean rats after the intraperitoneal (i.p.) injections, but when they stopped eating, grooming behavior increased. The food eaten was measured at 0.5, 1, 2, 3, and 6 h when animals were also observed for behavioral changes. Spillage on paper towels beneath the food cup was subtracted to give cumulative food intake. Three experiments were conducted. In each experiment, rats were used in a counter-balanced design so that each rat received all treatments with an interval of 3–5 days between injections. Experiment 1 examined whether peripheral injection of the BRL-37,344 would reduce food intake in animals fasted for 24 h. Saline and doses of 30 and 300 nmol of BRL-37,344 were injected i.p. in 14 fatty and 11 lean animals. These doses were picked to be similar to the doses given by i.c.v. injection in a previous experiment (41). Although we do not know the distribution of this drug in our studies if it is diluted in body water, the concentration would approximate ;1 nmol/mL, which is less than 1% of the concentration that would be anticipated had the 300 nmol given i.c.v. dissolved only in brain water (approximately 2 mL). Experiment 2 examined the interaction between i.p. injections of propranolol at two doses and BRL-37,344 at one dose (300 nmol). Eleven Zucker fatty rats and 10 lean litter mates received i.p. injections of saline or of propranolol at 300 nmol (the molar equivalent of BRL) or at a much higher does of 5 mg/kg (approximately 5000 nmol). These are doses that will block b1 and b2 adrenergic receptors. Injections of propranolol were followed 15 min later by an i.p. injection of saline or BRL-37,344 (300 nmol). Each animal received all treatments in a counter-balanced design with 3 to 5 days between injections. Experiment 3 tested whether propranolol injected into the third ventricle (i.c.v.) influenced the response to peripherally administered BRL-37,344. Nine Zucker fatty rats and 8 lean litter mates with cannulas in the third ventricle were injected i.c.v. with saline or propranolol (300 nmol/5 mL), and 15 min later with saline or BRL-37,344 (300 nmol) i.p. Equimolar doses were selected for this study. Each animal received all treatments in a counterbalanced design.
TSUJII AND BRAY Statistical Analysis Data for Experiment 1 was analyzed by a univariate ANOVA using genotype as a grouping factor and treatment and time as repeated measures variables. Experiments 2 and 3 were analyzed by a univariate ANOVA using genotype as a grouping factor with Treatment 1, Treatment 2, and time as repeated measures variables. In addition, data were evaluated by a one- or two-way ANOVA for each phenotype at each time point where appropriate. If the ANOVA indicated, further comparisons between different doses were performed using the least significant difference test as a post-hoc test. Data are expressed as mean 6 SEM. RESULTS
Experiment 1. Food intake after peripheral injection of BRL-37,344 Figure 1 shows the food intake of lean and obese Zucker rats for 6 h following i.p. injection of BRL 37,344. Food intake was significantly reduced by treatment with BRL-37,344 in both Zucker fatty rats and lean rats [F(2, 46) 5 34.8; p , 0.0001; three-way ANOVA]. For the fatty rats, food intake was decreased compared to saline at 30 min [F(2, 26) 5 21.8; p , 0.01], at 1 h [F(2, 26) 5 20.95; p , 0.01], at 2 h [F(2, 26) 5 21.97; p , 0.01], at 3 h [F(2, 26) 5 21.99; p , 0.01], and at 6 h [F(2, 26) 5 24.75; p , 0.01]. For the lean animals, food intake was also significantly decreased at 30 min [F(2, 20) 5 15.48; p , 0.01], at 1 h [F(2, 20) 5 11.17; p , 0.01], at 2 h [F(2, 20) 5 6.34; p , 0.01], and at 3 h [F(2, 20) 5 9.32; p , 0.01], but not at 6 h. Food intake was decreased by 22% with the low dose and 33% by the high dose at 30 minutes. The fatty (fa/fa) genotype showed a greater percentage reduction than the lean animals at all subsequent times. Experiment 2. Effect of i.p. propranolol on the response to BRL-37,344. Figure 2 shows the data on fatty rats (left) and lean rats (right). Using a four-way ANOVA, there were significant main effects of BRL-37,344 [F(1, 19) 5 90.0099; p , 0.0000] time [F(1, 19) 5 451.4062; p , 0.0000) and an effect of propranolol [F(2, 19) 5 4.6903; p 5 0.0129], but no main effect of genotype. Treatment with BRL-37,344 decreased food intake confirming the previous experiment. There was a significant interactions between propranolol and BRL-37,344 [F(2, 38) 5 11.5088; p , 0.000124]. Propranolol, partially, but not completely, reversed the effect of BRL37,344 (left panel). The effects are most easily separable using the data on the fatty rats. Using the two-way ANOVA, these effects were significant at 0.5 h, 1 h, 2 h, and 3 h in the lean rats and at 1 h, 2 h, and 3 h in the fatty rats. These data suggest that the inhibitory effects of BRL-37,344 on food intake can be partially blocked by the injection of propranolol, a nonspecific b-adrenergic blocking drug. The larger dose of propranolol, 5 mg/kg i.e., ;5000 nmol, by itself reduced food intake in both phenotypes of animals 2 and 3 h after injection. Experiment 3. Effect of propranolol injected in the third ventricle on food intake following peripheral injection of BRL-37,344. Intraperitoneal injection of BRL-37,344 significantly decreased food intake [F(1, 15) 5 61.4176; p , 0.0000; (Fig. 3)] in this experiment as in the previous two. This was more obvious in the fatty rats (left panel) than in the lean rats (right panel), a finding supported by the interaction in the four-way ANOVA [F(1, 15) 5 13.6596; p 5 0.0021]. Propranolol, injected into the third ventricle
BRL-37,344 b-AGONISTS AND FOOD INTAKE
FIG. 1. Effect of BRL-37,344 on food intake of lean and Zucker fatty rats. Vehicle and doses of 30 and 300 nmol of BRL-37,344 were injected i.p. into overnight-starved lean and fatty rats with 3–5 days between injections. Food intake was measured for vehicle and drug-treated animals at 0.5, 1, 2, 3, and 6 h after injection. Data are expressed as mean 6 SEM for 14 fatty and 11 lean animals at each treatment level. *p , 0.01 compared to vehicle treated controls.
FIG. 2. Effect of propranolol on the response to BRL-37,344 in lean and Zucker fatty rats. Each animal received vehicle or propranolol by two routes: i.p. injections of propranolol at 5 mg/kg, and at a different time, intracerebroventricular injections of propranolol (300 nmol) into the third ventricle or they received comparable volumes of vehicle by each route, saline followed 15 min later by BRL-37,344 (300 nmol). Food intake was measured at 0.5, 1, 2, 3, and 6 h after treatment. Data are expressed as mean 6 SEM for 11 fatty and 10 lean rats.
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FIG. 3. Effect of intracerebroventricular injections of propranolol on the response to i.p. BRL-37,344 in lean and Zucker fatty rats. Propranolol (300 nmol) was injected into the third vertebral ventricle 15 min before i.p. injection of BRL- 37,344 (300 nmol). Food intake was measured at 0.5, 1, 2, 3, and 6 h after treatment. Data are expressed as mean 6 SEM. There was no significant attenuation of the response to BRL-37,344 by injecting propranolol i.c.v.
by itself had a marginal effect of increasing food intake in the lean rats [F(1, 15) 5 5.093; p 5 0.039] but it had no effect on food intake in the fatty rats. Propranolol significantly increased food intake at 1 h [F(1, 7) 5 6.48; p , 0.05] and 3 h [F(1, 7) 5 8.15; p , 0.05] in the lean rats. There was an interaction between propranolol and BRL-37,344 [F(1, 15) 5 7.47; p 5 0.015] but no interaction by genotype. These data suggest that there are peripheral b-adrenergic receptors which can reduce food intake and that blockade of CNS b-adrenergic receptors with propranolol may attenuate this effect. ANOVA showed no main effect of genotype in any of the three experiments. Time, a repeated measures variable (or within subject variable), was highly significant in all experiments. DISCUSSION
b-adrenergic receptors have been known to mediate satiety since the work of Grossman (20,21) and Leibowitz (27,28). They showed that injection of norepinephrine into the medial hypothalamic area increased food intake. Leibowitz (27) also showed that propranolol would block the reduction of food intake following perifornical injection of norepinephrine (NE) indicating that NE was probably acting through either b1 or b2 adrenergic receptors. After Arch et al. (3) showed that a non- b1 -non- b2 adrenoceptor, now known to be the b3 adrenoceptor mediated the thermogenic response to NE, a number of agonists were developed as potential thermogenic compounds to treat obesity (2,3,45). Treatment with these drugs reduced body fat in experimental animals without affecting food intake (8). Reduction of body fat to a comparable degree by calorie restriction would be followed by marked hyperphagia when food became available ad libitum, suggesting that hyperphagia does not occur in animals chronically treated with b3 agonists because food in-
take is being simultaneously suppressed. In a previous study, we were the first to test the effects on food intake of a b3 agonist, BRL-37,344 injected into the ventricular system (41). We found that it acutely reduced food intake in fatty rats more than in lean rats (41). We subsequently reported in abstract form that BRL 37,344 also reduced food intake when given peripherally (39). The present study extends these observations by showing that i.p. injection of BRL-37,344 reduces food intake more in Zucker fatty rats than in lean rats. In addition, the response to BRL-37,344 was attenuated by an i.p. injection of propranolol as well as by propranolol injected into the third ventricle. Because 300 nmol of b3 agonist given peripherally in this experiment or centrally in the previous experiment (41) produced a reduction in food intake, it suggests that b-adrenergic receptors responsive to BRL-37,344 are located peripherally and may also be present in the CNS. Although we do not know the pharmacokinetics of BRL-37,344 in these animals that differ markedly in body fat, it is clear that fatty rats showed a greater reduction in food intake, by either route, despite their greater weight and greater quantity of body fat. Several studies using different b3 agonists have shown a decrease in food intake in mice and rats when the drugs are given peripherally (22,26,38,41) or in the diet (10). When given in the diet, the effects on body fat were found to vary with the diet and strain of mice (10). The drug used in the present study, BRL37,344, has a high affinity for b3 adrenergic receptors (13) but will also interact with b2 adrenoceptors at a much lower affinity. Propranolol injected peripherally attenuated, but did not block, the reduction in food intake produced by BRL-37,344. Propranolol is a weak antagonist of b3 adrenoceptors but is an effective antagonist of both b1 and b2 adrenoceptors. There are, as yet, no potent b3 receptor antagonists known to us. In other
BRL-37,344 b-AGONISTS AND FOOD INTAKE studies, however, we have found that a b2 agonist, clenbuterol (44), given peripherally or salbutamol given centrally (41) are both potent inhibitors of food intake. The ED50 for suppressing food intake by the clenbuterol was 0.04 mmol/kg compared to . 1 mmol/kg for a selective b3 agonist, ICI D-7114 (44). The relative potency of these two drugs for thermogenesis was reversed (44), leading to the question of the location and receptor type through which the anorectic effect of b3 adrenoceptor agonists is occurring. There are several possible locations of the b3 adrenoceptors that suppress food intake. Himms–Hagen (25) has recently proposed that the initiation and inhibition of food intake may be related to the effects of b3 agonists on brown adipose tissue, which is a rich source of b3 adrenoceptors because damage to this tissue in transgenic animals leads to weight gain and susceptibility to high fat-induced obesity (23). Grujic et al. (22) have recently provided a critical test of this idea. They used transgenic mice expressing b3 adrenoceptors in brown adipose tissue alone, or in brown and white adipose tissue. The reduction of food intake in the animals expressing the b3 adrenoceptors in both brown and white adipose tissue was much greater than the effect in the animals expressing it only in brown adipose tissue, suggesting that it is the b3 adrenoceptors in white adipose tissue that are involved in reducing food intake. This effect in white fat cells cannot be the result of leptin because it occurs rapidly and because activation of b3 adrenoceptors suppresses leptin mRNA expression (30). The nature of the signal from white adipose tissue following treatment with a b3 agonist that could reduce food intake is unknown. One possibility would be fatty acids. Modifying fatty acid oxidation is known to affect food intake (9,15–17,24,35), and this involves vagal afferents (33). Changes in blood metabolites are associated with the hypophagia following insulin (7,18,31). b-hydroxybutyrate, a metabolite of long chain fatty acids, reduces food intake and is another possible product of metabolism of fatty acids from adipose tissue that could modulate feeding (1). Finally, the fat cell may release TNF-a, which is a cytokine that could suppress feeding. Whether any of these are actually involved requires additional experimental work. A second possible explanation is that the b3 agonists could be acting on b3 receptors in the brain. Although not originally identified in brain, recent studies using RT-PCR by Summers et al. (37) have demonstrated the presence of b3 adrenoceptor mRNA in cerebellum, hippocampus, and striatum. This is consistent with observations that an atypical b-adrenoceptor agonist (SR 58611A) has an antidepressant profile (36). Similarly, the demonstration that the b3 agonist, BRL-37,344 given into the third ventricle reduces food intake is consistent with b3 adrenoceptors in the brain. A third possible site for action on b3 adrenoceptors is in the gut. b3 adrenoceptors have been identified in the fundus of the stomach, in the longitudinal and circular smooth muscle of both colon and ileum, and in the colonic submucosa (14). Afferent signals from the gut to the brain are an important part of the satiety system (33,35). Stimulation of b3 receptors in the gut are involved in gastric acid secretion (6) and may be involved in modulating food intake. Propranolol given into the third cerebral ventricle attenuated the reduction in food intake when BRL-37,344 was given peripherally. This suggests that NE is involved in the central anorectic system activated by peripheral b3 adrenoceptor stimulation. This central adrenoceptor could be either the b2 adrenoceptor, which responds to salbutamol (41) or the b3 adrenoceptor, which has been demonstrated by Summers et al. (37). Differentiation of these possibilities will await the development of selective b3 antagonists. In either case, it suggests that one of the central relays from the peripheral adrenergic receptor system is another adrenergic receptor in the brain.
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FIG. 4. Diagram of peripheral and central circuitry that could account for the effects of b-adrenergic agonists on food intake when injected peripherally or centrally. See text for details. POA, preoptic area; PFA, perifornical area; MPG, motor pattern generator; VMH, ventromedial hypothalamus; BAT, brown adipose tissue.
A proposed model for the central and peripheral effects of b-adrenergic receptors in food intake is shown in Fig. 4. Peripheral injection of BRL-37,344 may stimulate b3 adrenoceptors in white adipose tissue with the production of heat. Increased heat is known to reduce food intake probably through effects mediated by the pre-optic area of the hypothalamus. Himms–Hagen has recently proposed a model of food intake in which the brown adipose tissue plays a central role (25). In this model, stimulation of brown adipose tissue by the systemic nervous system increases glucose utilization and produces the “glucose dip” that is associated with 60% or more of spontaneous meals. Alternatively, the b3 agonist may activate b3 adrenoceptors in the gut with satiety information relayed to the brain over the vagus or splanchnic nerves. The heat signal acting through the pre-optic area or afferent neural signal acting in the hind brain or hypothalamus or elsewhere could reduce food intake through effects on satiety mediated through the perifornical area. The perifornical area acts to inhibit a motor pattern generator, which controls the food seeking activities. Central injection of a b-adrenergic agonist could act directly on the b-adrenergic receptors in the perifornical area that have been identified by Leibowitz (27,28). The satiety that is produced by a meal might also involve the pathways described here as shown by the nutrient or vagal signals that are generated by ingestion of food and transmitted to the ventromedial hypothalamus, where they activate the pathways involved in stimulation of brown adipose tissue. The differences in the fatty Zucker rat and the lean animals may reside in the different relative levels of tonic activity that serves to modulate hypothalamic receptor activity (40 – 42), supporting the hypothesis of a reciprocal relationship between food intake and the sympathetic nervous system (5). In conclusion, there are both peripheral and central b3-adrenergic receptors involved in producing satiety. ACKNOWLEDGEMENTS
Supported by National Institute of Health (NIDDK) Grant 2 R01 DK31988.
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