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Preference for palatable food is reduced by the gamma-hydroxybutyrate analogue GET73, in rats
Pharmacological Research 55 (2007) 271–279
Preference for palatable food is reduced by the gamma-hydroxybutyrate analogue GET73, in rats Alessandra Ottani a , Sheila Leone b , Francisca Belen Garcia Vergara c , Raffaella Tacchi e , Antonella Loche d , Alfio Bertolini e,∗ b
a Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, 41100 Modena, Italy Department of Pharmacological Sciences, Section of Pharmacology and Pharmacognosy, University of Chieti “G. D’Annunzio”, 66013 Chieti, Italy c Area ´ de Psicobiolog´ıa, Facultad de Psicolog´ıa, Universidad de M´alaga, 29071 M´alaga, Spain d Laboratorio Farmaceutico CT, 18038 Sanremo, Italy e Department of Diagnostic Services, Section of Clinical Pharmacology, School of Medicine, University of Modena and Reggio Emilia, Largo del Pozzo 71, 41100 Modena, Italy
Accepted 7 December 2006
Abstract Palatability and variety of foods are major reasons for “hedonic” eating, and hence for overeating and obesity. Palatable food and drugs of abuse share a common reward mechanism, and compounds that block the reinforcing effect of drugs of abuse preferentially suppress the intake of palatable foods. This research was aimed at studying the influence of the gamma-hydroxybutyrate analogue N-(4-trifluoromethylbenzyl)-4methoxybutanamide (GET73) – that inhibits alcohol consumption – on consumption and reinforcing effect of palatable food. Adult male rats were used. For place preference conditioning, sweetened corn flakes were used as the reinforcer, and GET73 (50, 100 and 200 mg kg−1 ) or vehicle were orally (p.o.) administered either 30 min before each training session and the test session, or only before the test session. To study the influence on consumption, GET73 was given p.o. at the same doses once daily for 12 days to rats given free access to both palatable and varied food (cafeteria diet) or to standard chow. Both acquisition and expression of palatable food-induced conditioned place preference were inhibited by GET73, either administered throughout the conditioning period or only before the test session. GET73 reduced also the consumption of cafeteria food, while that of standard chow was increased. At these doses, GET73 had no detrimental effect on open-field behaviour. GET73 seems to specifically attenuate the gratification produced by varied and palatable food, without affecting the consumption of not particularly palatable chow. Since, overweight and obesity are mostly due to the overeating of palatable and varied foods, drugs like GET73 could represent a somewhat ideal and rational approach to obesity treatment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Cafeteria diet; Feeding; N-(4-Trifluoromethylbenzyl)-4-methoxybutanamide (GET73); Open-field; Overeating; Palatability; Place preference; Rat
1. Introduction Obesity is a rampant disease, the greatest threat to public health in the developed world [1]; the most important cause being overeating, coupled with inactivity. In the modern world, in fact, overeating is driving a biological system that has been mainly designed to deal with energy depletion but not energy surplus, and the result is steadily increasing prevalence of overweight and obesity [2,3]. In the brain, the control of food intake and energy balance involves brainstem, hypothalamus, mesolimbic and corticolim-
∗
Corresponding author. Tel.: +39 059 4224064; fax: +39 059 4224069. E-mail address: [email protected] (A. Bertolini).
1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.12.002
bic structures. While the former structures are basically involved in the control of “homeostatic”, “energy balance-regulated”, eating [4–7], the latter structures essentially comprise the brain reward system, including the (basolateral) amygdala [8], and are involved in the “non-homeostatic”, “hedonic”, eating [2,3,9,10]. Overeating is stimulated by the abundance of food cues in the modern environment and the ready and easy availability (low physical effort and relatively low cost) of palatable, energy-rich food [2,11–14]. Palatability and pleasantness are mostly responsible for “hedonic”, not “hunger/satiety-controlled”, eating. Indeed, typical of “hedonic”, “reward”, eating is that the driving force is gratification rather than energy deficit [15]. Palatability is probably the single most important non-homeostatic factor determining food intake [2]. Enhancing the taste and flavour of
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food as, for example, in high-fat and sucrose-sweetened diets, can in fact elicit further food intake in satiated subjects, increase energy intake over the long term, and ultimately lead to obesity [16,17]. Moreover, while proteins are the most satiating among macronutrients, fats appear to have a weak satiating capacity [18]. In fact, subjects readily overeat in response to high fat foods [19]. This effect has been described as “the fat paradox”, “high fat hyperphagia”, or “passive overconsumption”. Besides the sensory perception of palatability, the choice of a variety of palatable foods, also known as “cafeteria diet”, is a particularly strong stimulator of food consumption in rats [20,21]. The power of variety to stimulate further food intake in satiated subjects – the so-called “dessert phenomenon” – has also been demonstrated in humans [22,23] (beside being obvious and common knowledge). Long-term overconsumption of palatable food has been compared to drug addiction [9,24,25], and indeed abundant experimental data support the view that palatable food may cause dependence [3,26–28]; moreover, food is more reinforcing for obese people than non-obese people [29–33]. Palatable food activates the same circuitries and pathways that are activated by drugs of abuse. For example, chronic access to a preferred flavour (chocolate-fat solution) produces the same transmitter changes in the nucleus accumbens as chronic morphine or ethanol, suggesting a common reward mechanism for palatable food and conventional drugs of abuse [34]. The prefrontal cortex, which seems to be particularly responsive in conditioning paradigms, receives highly processed sensory inputs from amygdala and hippocampus, and influences motivational pathways through projections to nucleus accumbens and lateral hypothalamus. The potent effect that olfactory, gustatory, or visual stimuli can have on appetite may in part recruit prefrontal cortical circuits that drive downstream appetitive motivational systems [35]. A by now abundant (albeit not always concordant) literature indicates that indeed compounds able to block the reinforcing effect of drugs of abuse are also able to block the reinforcing effect of food. Thus, the compound SR141716A (SR), a selective antagonist at central cannabinoid receptors (CB1) [36], preferentially attenuates the intake of palatable foods, suppresses sucrose eating and drinking, and reduces intake of sweet milk at doses that do not affect normal food or water intake [37–40]. And the opioid receptor antagonists naloxone and naltrexone have been reported to preferentially suppress the intake of foods high in carbohydrate and fat content [41–46]. Moreover, it has been shown that the CB1-antagonist SR also selectively and significantly reduces alcohol ingestion in C57BL/6 mice [37], a strain genetically predisposed to alcohol consumption [47]. Gamma-hydroxybutyric acid (GHB) has long been used for the treatment of alcohol dependence [48–50]. Indeed, treatment with GHB has been reported to effectively decrease alcohol craving and consumption as well as alcohol withdrawal symptoms in alcoholics [49]. Several lines of evidence indicate that GHB exerts its antialcohol effects by a substitution mechanism [50]. The GHB-analogue N(4-trifluoromethylbenzyl)-4-methoxybutanamide (GET73) has been recently synthesized with the aim of prolonging the
duration of action of GHB. And in fact, in a strain of alcoholpreferring rats (Sardinian alcohol-preferring) [51] GET73, in the dose range of 75–200 mg kg−1 , produces a strong inhibition of alcohol intake that is still highly significant after 6 h (60–65% reduction of alcohol intake during the first 6 h after GET73 administration, compared with vehicle-treated rats) (G. Colombo, unpublished data). However, GET73 is not a prodrug of GHB and, contrary to GHB, it has no affinity for GHB and GABAB receptors and a very low affinity for GABAA (30% inhibition of specific binding at 1 × 10−5 M) and 5-HT3 (13% inhibition of specific binding at 1 × 10−5 M) receptors (A. Loche, unpublished data). After oral administration in rats, the bioavailability of GET73 is about 50%, and the drug freely crosses the blood–brain barrier (A. Loche, unpublished data). The effect of GET73 on alcohol intake in Sardinian alcohol-preferring rats is neither associated with behavioural or locomotor deficits nor with reduction of water drinking and standard food consumption, at the doses that almost completely inhibit alcohol intake (100–200 mg/kg) (A. Loche, unpublished data). Thus, we were interested in investigating whether GET73 – likewise other compounds that inhibit alcohol consumption and consumption of palatable food as well – may alike selectively reduce the consumption and the reinforcing effect of palatable food, besides alcohol. 2. Materials and methods 2.1. Animals Adult male rats of an SPF Wistar strain (Harlan, Corezzana, Milano, Italy), weighing 190–210 g upon arrival, were used. They were housed in plexiglas cages (40 cm × 25 cm × 15 cm), two per cage (except for the cafeteria diet preference, where rats were one per cage), in climatized colony rooms (22 ± 1 ◦ C; 60% humidity), on a 12 h/12 h light/dark cycle (light phase: 07:00 h–19:00 h), with tap water and food in pellets (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value, 3.20 kcal/g) (TRM, Harlan, Teklad) freely available (when not otherwise specified). Principles of laboratory animal care were followed. Housing conditions and experimentation procedures were strictly in accordance with the European Community ethical regulations on the care of animals for scientific research (CEE Council 86/609; Italian D.L. 27/01/92 no. 116), and the research protocol was approved by the Animal Experimentation Ethical Committee of the University of Modena and Reggio Emilia. The rats were accustomed to our housing conditions for at least 1 week before being used. Moreover, during this period, rats were handled daily to habituate them to human handling. 2.2. Drug and treatment N-(4-Trifluoromethylbenzyl)-4-methoxybutanamide (GET73) was a kind gift of Laboratorio CT (Sanremo, Italy). It was suspended in vehicle [0.5% solution of methocell (Sigma, Milan, Italy) in distilled water] shortly before adminis-
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tration. Animals were treated by oral gavage with GET73 at the doses of 50, 100 or 200 mg kg−1 (these doses were selected as the most effective ones in reducing alcohol intake); control animals received vehicle (2 ml/kg) by the same route. Each animal was used for only one experiment and only one level of dosage, and at least 10 rats/group or dosage of the drug were used. Doses of GET73 were chosen on the basis of its activity on alcohol intake. 2.3. Open-field test This test was used in order to evaluate any possible effect of GET73 on spontaneous motility and general behaviour. 2.3.1. Procedure The open-field apparatus consisted of a 1 m square wooden box with 30 cm high boundary walls, divided into 25 equal squares by black lines marked on the floor. It was located in a darkened soundproof room, and was lit by a 60 W bulb placed 100 cm above the centre of the arena. The rat was placed in the central square and observed for 5 min. The test was performed between 09:00 h and 14:00 h. After each trial the fecal boluses were removed and the arena was cleaned with dry towels. The observed forms of behaviour were: the outer ambulation [the number of outer squares (squares along the sides of the open-field) crossed], the inner ambulation (the number of inner squares crossed) [the four paws have to enter the square to be recorded as an event], rearing (the frequency of standing on hind limbs), grooming (the time spent in grooming activity) and defecation (the number of fecal boluses). The observer (always the same) was blind to the treatment. 2.3.2. Single treatment In a set of experiments, rats (12 per group) were randomly assigned to one of the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage 30 min before the test. The 30 min latency was chosen on the basis of previous pharmacokinetic experiments (A. Loche, unpublished data). 2.3.3. Repeated treatments In another set of experiments, rats (12 per group) were randomly assigned to one the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage once daily for 7 consecutive days, the last treatment being administered 30 min before the test.
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2.4.1. Procedure Place preference conditioning was conducted in a woodconstructed three-chambered apparatus [55]. The two conditioning chambers were identical in size (30 cm × 15 cm × 15 cm), but were distinguishable both visually and tactually. The interior of one chamber was entirely white, the floor was covered with wood sawdust that was changed prior to each trial; the other chamber was entirely black and had no sawdust on the floor. The conditioning chambers were connected through removable walls to a third compartment (15 cm × 15 cm × 15 cm), painted a neutral grey, with the floor without wood sawdust, which served as the entrance to the conditioning chambers during place preference testing. The white and the grey arms were covered by a wire mesh; the black arm was covered by a black glass. The rats were transported to the testing room daily for 3 consecutive days to habituate them to the experimental environment. The place preference study lasted 11 days. For the first 3 days the animals were allowed to explore freely the whole apparatus for 10 min daily. On day 4, the time spent in each of the three chambers was measured (by means of a chronometer) in a 10 min “pre-conditioning test”. In this pre-conditioning test (on day 4), all rats preferred the black compartment, and the random assignment produced groups whose mean times spent in the black, or in the white compartment, were not significantly different. On days 5–10, the animals received a daily 10 min training trial (preceded by 21 h 50 min food deprivation) in which the animals were confined on alternate days either in the white or in the black chamber. Sweetened corn flakes, used as the reinforcer, were freely available in the white chamber, whereas no food was available in the black chamber. Following this 10 min confinement, the animals were replaced in their home cage with standard food in pellets available for 2 h. Changes in side-preference (with respect to the day 4 “pre-conditioning test”) were measured on day 11 in a 10 min “post-conditioning test”, with both conditioning chambers opened and without reward (sweetened corn flakes) in the white one. Place preference conditioning and testing were performed between 09:00 h and 14:00 h in a sound-proof room, under moderate lighting, by observers unaware of the group to which each animal belonged.
2.4. Place preference test
2.4.2. Single treatment In a set of experiments, after the pre-conditioning test, rats were randomly assigned to four groups and, at the end of the training period, 30 min before the post-conditioning test, were given by oral gavage one of the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage 30 min before the post-conditioning test.
The experimental animal literature has a rich history of using the conditioned place preference paradigm to understand how drugs influence reward, and conditioned place preference has also been produced in response to food reward [52–54]; thus, we used this paradigm in order to see whether GET73 might be able to inhibit/prevent the rewarding effect of palatable food.
2.4.3. Repeated treatments In another set of experiments, after the pre-conditioning test rats were randomly assigned to one the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage once daily for 7 consecutive days, 30 min before each training trial and the post-conditioning test.
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2.5. Cafeteria diet preference test 2.5.1. Procedure The different groups of animals were given three palatable foods for human consumption, in addition to the standard chow (into a separate trough). The three supplementary items were changed at random each day. Palatable food and standard chow were contained in two identical troughs, put side by side in the same place in the cage; each day the positions were reversed in order to avoid the possibility that the location of the food rather than its palatability might influence the rat choice. The list of palatable items included cafeteria-style foods (as usually served with aperitif in Modena caf´es): chips of parmigiano–reggiano cheese, cubes of Bologna sausage, potato chips, roasted hazelnuts, cookies, curls of salt butter and bits of chocolate torrone. The average composition of this diet was: 30% fat, 56% carbohydrate, and 14% protein as percent kcal (3.87 kcal/g). This kind of palatable diet is currently used in our laboratory, in researches of feeding behaviour. The sawdust bedding was replaced with a thick millboard, so as to absorb the urine and to allow the picking up of the spillage. The millboard was changed every 12 h. Animals were treated once daily for 12 days as follows: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 . Food consumption was measured 1, 2 and 3 h after the first treatment, and 24 h after each daily treatment. 2.6. Statistical analysis All data are presented as means ± S.E.M. Data concerning place preference test are presented as percent variation of the time spent in the different chambers during the postconditioning test, with respect to pre-conditioning test, and were analyzed using the t-test for paired data (comparison between pre-conditioning and post-conditioning data in the same group) or ANOVA followed by Student–Newman–Keuls’ test (comparison of values in post-conditioning test among the different groups). Data concerning cafeteria diet preference test are presented as consumption in kcal of the ingested food or as percentage of consumption of the different kind of food with respect to total ingested food, and were analysed by means of ANOVA followed by Student–Newman–Keuls’ test. Data concerning the open-field test were analyzed using ANOVA followed by Student–Newman–Keuls’ test. Results were considered statistically significant when p < 0.05. 3. Results 3.1. Open-field 3.1.1. Acute treatment As shown in Fig. 1, GET73, at the dose of 50 mg kg−1 p.o. 30 min before testing, produced an increase of inner ambulation and of grooming; at the dose of 100 mg kg−1 , it produced an increase of rearing and a decrease of fecal boluses; at the dose of 200 mg kg−1 , it had no significant effect.
Fig. 1. Influence of acute treatment with GET73 in the open-field test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before the test (observation period: 5 min). Data are expressed as the mean ± S.E.M. of: the number (n) of the squares crossed (outer, inner and total), the number (n) of rearing and fecal boluses, the seconds (s) spent in grooming; 12 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
3.1.2. Chronic treatment As shown in Fig. 2, at the dose of 50 mg kg−1 p.o. for 7 consecutive days GET73 produced an increase of grooming; at the dose of 200 mg kg−1 it produced an increase of rearing and outer ambulation; the dose of 100 mg kg−1 had an effect similar to that produced by the dose of 200 mg kg−1 , albeit less pronounced; the number of fecal boluses was significantly reduced at the dose of 200 mg kg−1 . 3.2. Place preference test 3.2.1. Acute treatment As shown in Fig. 3, in the test session (post-conditioning test) (after 6 daily training sessions), rats treated with the vehicle spent significantly more time in the white arm (+29.10%) (t = −5.541, p = 0.000) and significantly less time in the black arm (−38.60%) (t = 4.933, p = 0.000), in comparison with the pre-conditioning test, as expected. The administration of GET73 at the doses of 50 and 200 mg kg−1 p.o., 30 min before the test session, significantly reduced the preference for the white arm (+14% and +14.55%, respectively) in comparison with vehicle-treated rats (F(3,36) = 4.98, p = 0.005), so that the time spent in the black arm was significantly longer than in controls [F(3,36) = 3.22, p = 0.034]. The behaviour of rats treated with the dose of 100 mg kg−1 was not significantly different from controls, with regard to the white harm (+25.15%).
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Fig. 2. Influence of 7-day treatment with GET73 in the open-field test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before the test (observation period: 5 min). Data are expressed as the mean ± S.E.M. of: the number (n) of the squares crossed (outer, inner and total), the number (n) of rearing and fecal boluses, the seconds (s) spent in grooming; 12 animals per group. (*) p < 0.05 with respect to vehicle-treated group and (䊉) p < 0.05 with respect to all the other groups (ANOVA followed by Student–Newman–Keuls’ test).
3.2.2. Chronic treatment As shown in Fig. 4, in the test session (post-conditioning test), rats treated with the vehicle spent significantly less time in the black arm (−42.87%) (t = 7.572, p = 0.000) and
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Fig. 4. Influence of a 7-day treatment with GET73 on the place preference conditioning to a palatable food. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered once daily for 7 days, 30 min before each training trial and the post-conditioning test. Data are expressed as per cent variation (means ± S.E.M.) (with respect to the pre-conditioning test) of the time spent in the different compartments (white, black and grey) during the post-conditioning test; 10 animals per group. (䊉) p < 0.05 with respect to pre-conditioning test in the same group (t-test for paired data); (*) p < 0.05 with respect to vehicle-treated group and () p < 0.05 with respect to all the other groups (ANOVA followed by Student–Newman–Keuls’ test).
significantly more time in the white and in the grey arms (+20.30% and +22.57%, respectively) (t = −8.515, p = 0.000 and t = −4.708, p = 0.001, respectively), in comparison with the preconditioning test. The administration of GET73 at the doses of 50 and 100 mg kg−1 p.o. 30 min before each of the six training sessions, significantly reduced the preference for the white arm (+6.70% and +11.70%, respectively) (F(3,36) = 6.49, p = 0.001), and for the grey arm (+10.70% and +5.37%, respectively) (F(3,36) = 3.85, p = 0.0174), in comparison with vehicle treated rats, so that the time spent in the black arm was significantly longer (−17.4% and −17.07%, respectively) than in controls (F(3,36) = 4.49, p = 0.009). At the highest dose (200 mg kg−1 p.o.) GET73 caused on the contrary a great increase in the preference for the white arm (+38.70%: almost twice the time spent in this arm by controls) (F(3,36) = 6.49, p = 0.001), and a reduction in the time spent in the black arm (−43.47%, similar to that observed in controls) (F(3,36) = 4.49, p = 0.009). The time spent in the grey arm was almost the same spent in this arm in the pre-conditioning test. 3.3. Cafeteria diet preference
Fig. 3. Influence of acute treatment with GET73 on the place preference conditioning to a palatable food. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before post-conditioning test. Data are expressed as per cent variation (means ± S.E.M.) (with respect to the pre-conditioning test) of the time spent in the different compartments (white, black and grey) during the post-conditioning test; 10 animals per group. (䊉) p < 0.05 with respect to pre-conditioning test in the same group (t-test for paired data); (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
As shown in Fig. 5, vehicle-treated rats had a clear preference for the cafeteria diet, that was eaten in significantly larger amounts than the standard diet, throughout the test period. Treatment with GET73 reduced the preference for the cafeteria diet, and this effect was statistically significant throughout the test period with the dose of 100 and 200 mg kg−1 day−1 (the maximum effect being produced by the dose of 100 mg kg−1 day−1 ). With the dose of 50 mg kg−1 the effect was significant on days 1, 2, 10, 11 and 12. The lower intake of cafeteria food was associated with a concurrent larger intake of standard food: this effect
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Fig. 5. Influence of a 12-day treatment with GET73 on the cafeteria diet preference test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered once daily for 12 days. Food was weighed 24 h after each treatment. Data are presented as consumption in kcal of the ingested food (a) or as percentage of consumption of the different kinds of food with respect to total ingested food (b); 10 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
was more evident during the first 6 days of the test period, while it was less pronounced during the last days of the test. The 12 days treatment with GET73 did neither modify body weigh nor body composition (data not presented). The effect of GET73 on cafeteria food intake was already significant 1 h after the first administration for the doses of 50 and 200 mg kg−1 (Fig. 6). 4. Discussion Our present results show that the GHB analogue N(4-trifluoromethylbenzyl)-4-methoxibutanamide (GET73) selectively reduces the rewarding and incentive properties of highly palatable and varied foods, and their consumption, at doses that have no detrimental effect on the gross behaviour and motor activity of rats. Indeed, in the open-field, both single and repeated administrations of GET73 in the dose range of 50–200 mg kg−1 p.o. produced either an increase in ambulation, grooming and rearing, and a reduction of fecal boluses, or no significant effect: a picture that is considered expression of disinhibition, reduction of anxiety and – as far as grooming is concerned – “de-arousal” [the interpretation of grooming is not unambiguous, however (see for a comprehensive scrutiny: Ref. [56])] Both acquisition and expression of a palatable foodinduced conditioned place preference were inhibited by GET73. In fact, such conditioned place preference was reduced either by the administration of GET73 throughout the whole conditioning
period, or by a single administration of GET73 30 min before the post-conditioning test. The bell-shaped dose–effect curve obtained with the acute treatment is not unusual for behaviouraffecting drugs [57–59]. More intriguing is the contradictory effect of the highest dose (200 mg kg−1 day−1 ) of GET73 when administered throughout the whole conditioning period. In the light of the so far available data, this effect of the highest dose of GET73 in the place-preference test might be explained by the fact that this dose produces an increase in the exploratory activity associated with a reduction in anxiety (significant increase of outer and inner ambulation and rearing, and a decrease of the number of fecal boluses, in the open-field test). As far as the consumption is concerned, in the presence of both palatable food (cafeteria food) and standard laboratory chow, with free choice between them, in vehicle-treated rats the daily amount of ingested food was composed by 80–90% of cafeteria food and by 10–20% of standard chow. Treatment with GET73 reduced the consumption of cafeteria food and concurrently increased the consumption of standard chow, so that at the maximum effective dose of 100 mg kg−1 daily−1 , the total daily amount of ingested food was composed by 55–70% of cafeteria food and by 30–45% of standard chow. No tolerance developed to this effect of GET73, at least for the duration of our experiment (12 consecutive days). Since, we measured the total food intake, we do not know whether GET73 did selectively inhibit the consumption of some specific component of the cafeteria food.
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Fig. 6. Influence of acute treatment with GET73 on the cafeteria diet preference test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered, and food was weighed 1, 2, 3 and 24 h after treatment. Data are presented as consumption in kcal of the ingested food (a) or as percentage of consumption of the different kinds of food with respect to total ingested food (b); 10 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
On the whole, our data indicate that GET73 seems to specifically attenuate the gratification produced by varied and palatable food. The data that we have so far obtained concerning the binding and neurochemical properties of GET73 do not allow by now a straightforward explanation of the mechanism underlying this peculiar effect on the consumption of palatable food. Palatability, pleasantness and variety of foods are considered to be the major reasons for overeating, and hence for overweight and obesity (for reviews see, for example [2,3,60]). Palatability cues, particularly taste and smell, are strong modulators of food reward and a number of studies have suggested that food reward and drug reward share common neural substrates of the brain reward system (for a review [9]). Thus, several data indicate that endogenous opioid and cannabinoid systems are involved in the hedonic value of food intake (see, for example [46,61]). And the melanocortin system, which is the main physiological antagonist of the opioid systems [62–64] and plays a central role in the inhibition of feeding [5,65–70], not only contributes to the homeostatic control of feeding, but also to its hedonic aspects via the melanocortin 4 receptors (MC4-R) located in the nucleus accumbens (for a review [60]). In fact, the blockade of and k opioid receptors and of CB1 receptors inhibits consumption of palatable foods and alcohol, whereas the blockade of MC4 receptors in the nucleus accumbens has orexic effects, particularly for palatable foods, that
may persist for up to 6 days [71]. The brain system processing the non-homeostatic, hedonic aspects of ingestive behaviour are thus likely to play a crucial role in the development of obesity [2]. So, promising and rational pharmacological interventions for obesity may be those that interfere with the reinforcing value of food [9]. Our present data suggest that GET73 might be a drug that meets such requirements.
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Preference for palatable food is reduced by the gamma-hydroxybutyrate analogue GET73, in rats Alessandra Ottani a , Sheila Leone b , Francisca Belen Garcia Vergara c , Raffaella Tacchi e , Antonella Loche d , Alfio Bertolini e,∗ b
a Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, 41100 Modena, Italy Department of Pharmacological Sciences, Section of Pharmacology and Pharmacognosy, University of Chieti “G. D’Annunzio”, 66013 Chieti, Italy c Area ´ de Psicobiolog´ıa, Facultad de Psicolog´ıa, Universidad de M´alaga, 29071 M´alaga, Spain d Laboratorio Farmaceutico CT, 18038 Sanremo, Italy e Department of Diagnostic Services, Section of Clinical Pharmacology, School of Medicine, University of Modena and Reggio Emilia, Largo del Pozzo 71, 41100 Modena, Italy
Accepted 7 December 2006
Abstract Palatability and variety of foods are major reasons for “hedonic” eating, and hence for overeating and obesity. Palatable food and drugs of abuse share a common reward mechanism, and compounds that block the reinforcing effect of drugs of abuse preferentially suppress the intake of palatable foods. This research was aimed at studying the influence of the gamma-hydroxybutyrate analogue N-(4-trifluoromethylbenzyl)-4methoxybutanamide (GET73) – that inhibits alcohol consumption – on consumption and reinforcing effect of palatable food. Adult male rats were used. For place preference conditioning, sweetened corn flakes were used as the reinforcer, and GET73 (50, 100 and 200 mg kg−1 ) or vehicle were orally (p.o.) administered either 30 min before each training session and the test session, or only before the test session. To study the influence on consumption, GET73 was given p.o. at the same doses once daily for 12 days to rats given free access to both palatable and varied food (cafeteria diet) or to standard chow. Both acquisition and expression of palatable food-induced conditioned place preference were inhibited by GET73, either administered throughout the conditioning period or only before the test session. GET73 reduced also the consumption of cafeteria food, while that of standard chow was increased. At these doses, GET73 had no detrimental effect on open-field behaviour. GET73 seems to specifically attenuate the gratification produced by varied and palatable food, without affecting the consumption of not particularly palatable chow. Since, overweight and obesity are mostly due to the overeating of palatable and varied foods, drugs like GET73 could represent a somewhat ideal and rational approach to obesity treatment. © 2006 Elsevier Ltd. All rights reserved. Keywords: Cafeteria diet; Feeding; N-(4-Trifluoromethylbenzyl)-4-methoxybutanamide (GET73); Open-field; Overeating; Palatability; Place preference; Rat
1. Introduction Obesity is a rampant disease, the greatest threat to public health in the developed world [1]; the most important cause being overeating, coupled with inactivity. In the modern world, in fact, overeating is driving a biological system that has been mainly designed to deal with energy depletion but not energy surplus, and the result is steadily increasing prevalence of overweight and obesity [2,3]. In the brain, the control of food intake and energy balance involves brainstem, hypothalamus, mesolimbic and corticolim-
∗
Corresponding author. Tel.: +39 059 4224064; fax: +39 059 4224069. E-mail address: [email protected] (A. Bertolini).
1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.12.002
bic structures. While the former structures are basically involved in the control of “homeostatic”, “energy balance-regulated”, eating [4–7], the latter structures essentially comprise the brain reward system, including the (basolateral) amygdala [8], and are involved in the “non-homeostatic”, “hedonic”, eating [2,3,9,10]. Overeating is stimulated by the abundance of food cues in the modern environment and the ready and easy availability (low physical effort and relatively low cost) of palatable, energy-rich food [2,11–14]. Palatability and pleasantness are mostly responsible for “hedonic”, not “hunger/satiety-controlled”, eating. Indeed, typical of “hedonic”, “reward”, eating is that the driving force is gratification rather than energy deficit [15]. Palatability is probably the single most important non-homeostatic factor determining food intake [2]. Enhancing the taste and flavour of
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food as, for example, in high-fat and sucrose-sweetened diets, can in fact elicit further food intake in satiated subjects, increase energy intake over the long term, and ultimately lead to obesity [16,17]. Moreover, while proteins are the most satiating among macronutrients, fats appear to have a weak satiating capacity [18]. In fact, subjects readily overeat in response to high fat foods [19]. This effect has been described as “the fat paradox”, “high fat hyperphagia”, or “passive overconsumption”. Besides the sensory perception of palatability, the choice of a variety of palatable foods, also known as “cafeteria diet”, is a particularly strong stimulator of food consumption in rats [20,21]. The power of variety to stimulate further food intake in satiated subjects – the so-called “dessert phenomenon” – has also been demonstrated in humans [22,23] (beside being obvious and common knowledge). Long-term overconsumption of palatable food has been compared to drug addiction [9,24,25], and indeed abundant experimental data support the view that palatable food may cause dependence [3,26–28]; moreover, food is more reinforcing for obese people than non-obese people [29–33]. Palatable food activates the same circuitries and pathways that are activated by drugs of abuse. For example, chronic access to a preferred flavour (chocolate-fat solution) produces the same transmitter changes in the nucleus accumbens as chronic morphine or ethanol, suggesting a common reward mechanism for palatable food and conventional drugs of abuse [34]. The prefrontal cortex, which seems to be particularly responsive in conditioning paradigms, receives highly processed sensory inputs from amygdala and hippocampus, and influences motivational pathways through projections to nucleus accumbens and lateral hypothalamus. The potent effect that olfactory, gustatory, or visual stimuli can have on appetite may in part recruit prefrontal cortical circuits that drive downstream appetitive motivational systems [35]. A by now abundant (albeit not always concordant) literature indicates that indeed compounds able to block the reinforcing effect of drugs of abuse are also able to block the reinforcing effect of food. Thus, the compound SR141716A (SR), a selective antagonist at central cannabinoid receptors (CB1) [36], preferentially attenuates the intake of palatable foods, suppresses sucrose eating and drinking, and reduces intake of sweet milk at doses that do not affect normal food or water intake [37–40]. And the opioid receptor antagonists naloxone and naltrexone have been reported to preferentially suppress the intake of foods high in carbohydrate and fat content [41–46]. Moreover, it has been shown that the CB1-antagonist SR also selectively and significantly reduces alcohol ingestion in C57BL/6 mice [37], a strain genetically predisposed to alcohol consumption [47]. Gamma-hydroxybutyric acid (GHB) has long been used for the treatment of alcohol dependence [48–50]. Indeed, treatment with GHB has been reported to effectively decrease alcohol craving and consumption as well as alcohol withdrawal symptoms in alcoholics [49]. Several lines of evidence indicate that GHB exerts its antialcohol effects by a substitution mechanism [50]. The GHB-analogue N(4-trifluoromethylbenzyl)-4-methoxybutanamide (GET73) has been recently synthesized with the aim of prolonging the
duration of action of GHB. And in fact, in a strain of alcoholpreferring rats (Sardinian alcohol-preferring) [51] GET73, in the dose range of 75–200 mg kg−1 , produces a strong inhibition of alcohol intake that is still highly significant after 6 h (60–65% reduction of alcohol intake during the first 6 h after GET73 administration, compared with vehicle-treated rats) (G. Colombo, unpublished data). However, GET73 is not a prodrug of GHB and, contrary to GHB, it has no affinity for GHB and GABAB receptors and a very low affinity for GABAA (30% inhibition of specific binding at 1 × 10−5 M) and 5-HT3 (13% inhibition of specific binding at 1 × 10−5 M) receptors (A. Loche, unpublished data). After oral administration in rats, the bioavailability of GET73 is about 50%, and the drug freely crosses the blood–brain barrier (A. Loche, unpublished data). The effect of GET73 on alcohol intake in Sardinian alcohol-preferring rats is neither associated with behavioural or locomotor deficits nor with reduction of water drinking and standard food consumption, at the doses that almost completely inhibit alcohol intake (100–200 mg/kg) (A. Loche, unpublished data). Thus, we were interested in investigating whether GET73 – likewise other compounds that inhibit alcohol consumption and consumption of palatable food as well – may alike selectively reduce the consumption and the reinforcing effect of palatable food, besides alcohol. 2. Materials and methods 2.1. Animals Adult male rats of an SPF Wistar strain (Harlan, Corezzana, Milano, Italy), weighing 190–210 g upon arrival, were used. They were housed in plexiglas cages (40 cm × 25 cm × 15 cm), two per cage (except for the cafeteria diet preference, where rats were one per cage), in climatized colony rooms (22 ± 1 ◦ C; 60% humidity), on a 12 h/12 h light/dark cycle (light phase: 07:00 h–19:00 h), with tap water and food in pellets (3.5% fat, 63% carbohydrate, 14% protein, 19.5% other components without caloric value, 3.20 kcal/g) (TRM, Harlan, Teklad) freely available (when not otherwise specified). Principles of laboratory animal care were followed. Housing conditions and experimentation procedures were strictly in accordance with the European Community ethical regulations on the care of animals for scientific research (CEE Council 86/609; Italian D.L. 27/01/92 no. 116), and the research protocol was approved by the Animal Experimentation Ethical Committee of the University of Modena and Reggio Emilia. The rats were accustomed to our housing conditions for at least 1 week before being used. Moreover, during this period, rats were handled daily to habituate them to human handling. 2.2. Drug and treatment N-(4-Trifluoromethylbenzyl)-4-methoxybutanamide (GET73) was a kind gift of Laboratorio CT (Sanremo, Italy). It was suspended in vehicle [0.5% solution of methocell (Sigma, Milan, Italy) in distilled water] shortly before adminis-
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tration. Animals were treated by oral gavage with GET73 at the doses of 50, 100 or 200 mg kg−1 (these doses were selected as the most effective ones in reducing alcohol intake); control animals received vehicle (2 ml/kg) by the same route. Each animal was used for only one experiment and only one level of dosage, and at least 10 rats/group or dosage of the drug were used. Doses of GET73 were chosen on the basis of its activity on alcohol intake. 2.3. Open-field test This test was used in order to evaluate any possible effect of GET73 on spontaneous motility and general behaviour. 2.3.1. Procedure The open-field apparatus consisted of a 1 m square wooden box with 30 cm high boundary walls, divided into 25 equal squares by black lines marked on the floor. It was located in a darkened soundproof room, and was lit by a 60 W bulb placed 100 cm above the centre of the arena. The rat was placed in the central square and observed for 5 min. The test was performed between 09:00 h and 14:00 h. After each trial the fecal boluses were removed and the arena was cleaned with dry towels. The observed forms of behaviour were: the outer ambulation [the number of outer squares (squares along the sides of the open-field) crossed], the inner ambulation (the number of inner squares crossed) [the four paws have to enter the square to be recorded as an event], rearing (the frequency of standing on hind limbs), grooming (the time spent in grooming activity) and defecation (the number of fecal boluses). The observer (always the same) was blind to the treatment. 2.3.2. Single treatment In a set of experiments, rats (12 per group) were randomly assigned to one of the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage 30 min before the test. The 30 min latency was chosen on the basis of previous pharmacokinetic experiments (A. Loche, unpublished data). 2.3.3. Repeated treatments In another set of experiments, rats (12 per group) were randomly assigned to one the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage once daily for 7 consecutive days, the last treatment being administered 30 min before the test.
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2.4.1. Procedure Place preference conditioning was conducted in a woodconstructed three-chambered apparatus [55]. The two conditioning chambers were identical in size (30 cm × 15 cm × 15 cm), but were distinguishable both visually and tactually. The interior of one chamber was entirely white, the floor was covered with wood sawdust that was changed prior to each trial; the other chamber was entirely black and had no sawdust on the floor. The conditioning chambers were connected through removable walls to a third compartment (15 cm × 15 cm × 15 cm), painted a neutral grey, with the floor without wood sawdust, which served as the entrance to the conditioning chambers during place preference testing. The white and the grey arms were covered by a wire mesh; the black arm was covered by a black glass. The rats were transported to the testing room daily for 3 consecutive days to habituate them to the experimental environment. The place preference study lasted 11 days. For the first 3 days the animals were allowed to explore freely the whole apparatus for 10 min daily. On day 4, the time spent in each of the three chambers was measured (by means of a chronometer) in a 10 min “pre-conditioning test”. In this pre-conditioning test (on day 4), all rats preferred the black compartment, and the random assignment produced groups whose mean times spent in the black, or in the white compartment, were not significantly different. On days 5–10, the animals received a daily 10 min training trial (preceded by 21 h 50 min food deprivation) in which the animals were confined on alternate days either in the white or in the black chamber. Sweetened corn flakes, used as the reinforcer, were freely available in the white chamber, whereas no food was available in the black chamber. Following this 10 min confinement, the animals were replaced in their home cage with standard food in pellets available for 2 h. Changes in side-preference (with respect to the day 4 “pre-conditioning test”) were measured on day 11 in a 10 min “post-conditioning test”, with both conditioning chambers opened and without reward (sweetened corn flakes) in the white one. Place preference conditioning and testing were performed between 09:00 h and 14:00 h in a sound-proof room, under moderate lighting, by observers unaware of the group to which each animal belonged.
2.4. Place preference test
2.4.2. Single treatment In a set of experiments, after the pre-conditioning test, rats were randomly assigned to four groups and, at the end of the training period, 30 min before the post-conditioning test, were given by oral gavage one of the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage 30 min before the post-conditioning test.
The experimental animal literature has a rich history of using the conditioned place preference paradigm to understand how drugs influence reward, and conditioned place preference has also been produced in response to food reward [52–54]; thus, we used this paradigm in order to see whether GET73 might be able to inhibit/prevent the rewarding effect of palatable food.
2.4.3. Repeated treatments In another set of experiments, after the pre-conditioning test rats were randomly assigned to one the following treatments: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 , that were given by oral gavage once daily for 7 consecutive days, 30 min before each training trial and the post-conditioning test.
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2.5. Cafeteria diet preference test 2.5.1. Procedure The different groups of animals were given three palatable foods for human consumption, in addition to the standard chow (into a separate trough). The three supplementary items were changed at random each day. Palatable food and standard chow were contained in two identical troughs, put side by side in the same place in the cage; each day the positions were reversed in order to avoid the possibility that the location of the food rather than its palatability might influence the rat choice. The list of palatable items included cafeteria-style foods (as usually served with aperitif in Modena caf´es): chips of parmigiano–reggiano cheese, cubes of Bologna sausage, potato chips, roasted hazelnuts, cookies, curls of salt butter and bits of chocolate torrone. The average composition of this diet was: 30% fat, 56% carbohydrate, and 14% protein as percent kcal (3.87 kcal/g). This kind of palatable diet is currently used in our laboratory, in researches of feeding behaviour. The sawdust bedding was replaced with a thick millboard, so as to absorb the urine and to allow the picking up of the spillage. The millboard was changed every 12 h. Animals were treated once daily for 12 days as follows: (1) vehicle, 2 ml/kg; (2) GET73, 50 mg kg−1 ; (3) GET73, 100 mg kg−1 ; (4) GET73, 200 mg kg−1 . Food consumption was measured 1, 2 and 3 h after the first treatment, and 24 h after each daily treatment. 2.6. Statistical analysis All data are presented as means ± S.E.M. Data concerning place preference test are presented as percent variation of the time spent in the different chambers during the postconditioning test, with respect to pre-conditioning test, and were analyzed using the t-test for paired data (comparison between pre-conditioning and post-conditioning data in the same group) or ANOVA followed by Student–Newman–Keuls’ test (comparison of values in post-conditioning test among the different groups). Data concerning cafeteria diet preference test are presented as consumption in kcal of the ingested food or as percentage of consumption of the different kind of food with respect to total ingested food, and were analysed by means of ANOVA followed by Student–Newman–Keuls’ test. Data concerning the open-field test were analyzed using ANOVA followed by Student–Newman–Keuls’ test. Results were considered statistically significant when p < 0.05. 3. Results 3.1. Open-field 3.1.1. Acute treatment As shown in Fig. 1, GET73, at the dose of 50 mg kg−1 p.o. 30 min before testing, produced an increase of inner ambulation and of grooming; at the dose of 100 mg kg−1 , it produced an increase of rearing and a decrease of fecal boluses; at the dose of 200 mg kg−1 , it had no significant effect.
Fig. 1. Influence of acute treatment with GET73 in the open-field test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before the test (observation period: 5 min). Data are expressed as the mean ± S.E.M. of: the number (n) of the squares crossed (outer, inner and total), the number (n) of rearing and fecal boluses, the seconds (s) spent in grooming; 12 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
3.1.2. Chronic treatment As shown in Fig. 2, at the dose of 50 mg kg−1 p.o. for 7 consecutive days GET73 produced an increase of grooming; at the dose of 200 mg kg−1 it produced an increase of rearing and outer ambulation; the dose of 100 mg kg−1 had an effect similar to that produced by the dose of 200 mg kg−1 , albeit less pronounced; the number of fecal boluses was significantly reduced at the dose of 200 mg kg−1 . 3.2. Place preference test 3.2.1. Acute treatment As shown in Fig. 3, in the test session (post-conditioning test) (after 6 daily training sessions), rats treated with the vehicle spent significantly more time in the white arm (+29.10%) (t = −5.541, p = 0.000) and significantly less time in the black arm (−38.60%) (t = 4.933, p = 0.000), in comparison with the pre-conditioning test, as expected. The administration of GET73 at the doses of 50 and 200 mg kg−1 p.o., 30 min before the test session, significantly reduced the preference for the white arm (+14% and +14.55%, respectively) in comparison with vehicle-treated rats (F(3,36) = 4.98, p = 0.005), so that the time spent in the black arm was significantly longer than in controls [F(3,36) = 3.22, p = 0.034]. The behaviour of rats treated with the dose of 100 mg kg−1 was not significantly different from controls, with regard to the white harm (+25.15%).
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Fig. 2. Influence of 7-day treatment with GET73 in the open-field test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before the test (observation period: 5 min). Data are expressed as the mean ± S.E.M. of: the number (n) of the squares crossed (outer, inner and total), the number (n) of rearing and fecal boluses, the seconds (s) spent in grooming; 12 animals per group. (*) p < 0.05 with respect to vehicle-treated group and (䊉) p < 0.05 with respect to all the other groups (ANOVA followed by Student–Newman–Keuls’ test).
3.2.2. Chronic treatment As shown in Fig. 4, in the test session (post-conditioning test), rats treated with the vehicle spent significantly less time in the black arm (−42.87%) (t = 7.572, p = 0.000) and
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Fig. 4. Influence of a 7-day treatment with GET73 on the place preference conditioning to a palatable food. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered once daily for 7 days, 30 min before each training trial and the post-conditioning test. Data are expressed as per cent variation (means ± S.E.M.) (with respect to the pre-conditioning test) of the time spent in the different compartments (white, black and grey) during the post-conditioning test; 10 animals per group. (䊉) p < 0.05 with respect to pre-conditioning test in the same group (t-test for paired data); (*) p < 0.05 with respect to vehicle-treated group and () p < 0.05 with respect to all the other groups (ANOVA followed by Student–Newman–Keuls’ test).
significantly more time in the white and in the grey arms (+20.30% and +22.57%, respectively) (t = −8.515, p = 0.000 and t = −4.708, p = 0.001, respectively), in comparison with the preconditioning test. The administration of GET73 at the doses of 50 and 100 mg kg−1 p.o. 30 min before each of the six training sessions, significantly reduced the preference for the white arm (+6.70% and +11.70%, respectively) (F(3,36) = 6.49, p = 0.001), and for the grey arm (+10.70% and +5.37%, respectively) (F(3,36) = 3.85, p = 0.0174), in comparison with vehicle treated rats, so that the time spent in the black arm was significantly longer (−17.4% and −17.07%, respectively) than in controls (F(3,36) = 4.49, p = 0.009). At the highest dose (200 mg kg−1 p.o.) GET73 caused on the contrary a great increase in the preference for the white arm (+38.70%: almost twice the time spent in this arm by controls) (F(3,36) = 6.49, p = 0.001), and a reduction in the time spent in the black arm (−43.47%, similar to that observed in controls) (F(3,36) = 4.49, p = 0.009). The time spent in the grey arm was almost the same spent in this arm in the pre-conditioning test. 3.3. Cafeteria diet preference
Fig. 3. Influence of acute treatment with GET73 on the place preference conditioning to a palatable food. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered 30 min before post-conditioning test. Data are expressed as per cent variation (means ± S.E.M.) (with respect to the pre-conditioning test) of the time spent in the different compartments (white, black and grey) during the post-conditioning test; 10 animals per group. (䊉) p < 0.05 with respect to pre-conditioning test in the same group (t-test for paired data); (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
As shown in Fig. 5, vehicle-treated rats had a clear preference for the cafeteria diet, that was eaten in significantly larger amounts than the standard diet, throughout the test period. Treatment with GET73 reduced the preference for the cafeteria diet, and this effect was statistically significant throughout the test period with the dose of 100 and 200 mg kg−1 day−1 (the maximum effect being produced by the dose of 100 mg kg−1 day−1 ). With the dose of 50 mg kg−1 the effect was significant on days 1, 2, 10, 11 and 12. The lower intake of cafeteria food was associated with a concurrent larger intake of standard food: this effect
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Fig. 5. Influence of a 12-day treatment with GET73 on the cafeteria diet preference test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered once daily for 12 days. Food was weighed 24 h after each treatment. Data are presented as consumption in kcal of the ingested food (a) or as percentage of consumption of the different kinds of food with respect to total ingested food (b); 10 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
was more evident during the first 6 days of the test period, while it was less pronounced during the last days of the test. The 12 days treatment with GET73 did neither modify body weigh nor body composition (data not presented). The effect of GET73 on cafeteria food intake was already significant 1 h after the first administration for the doses of 50 and 200 mg kg−1 (Fig. 6). 4. Discussion Our present results show that the GHB analogue N(4-trifluoromethylbenzyl)-4-methoxibutanamide (GET73) selectively reduces the rewarding and incentive properties of highly palatable and varied foods, and their consumption, at doses that have no detrimental effect on the gross behaviour and motor activity of rats. Indeed, in the open-field, both single and repeated administrations of GET73 in the dose range of 50–200 mg kg−1 p.o. produced either an increase in ambulation, grooming and rearing, and a reduction of fecal boluses, or no significant effect: a picture that is considered expression of disinhibition, reduction of anxiety and – as far as grooming is concerned – “de-arousal” [the interpretation of grooming is not unambiguous, however (see for a comprehensive scrutiny: Ref. [56])] Both acquisition and expression of a palatable foodinduced conditioned place preference were inhibited by GET73. In fact, such conditioned place preference was reduced either by the administration of GET73 throughout the whole conditioning
period, or by a single administration of GET73 30 min before the post-conditioning test. The bell-shaped dose–effect curve obtained with the acute treatment is not unusual for behaviouraffecting drugs [57–59]. More intriguing is the contradictory effect of the highest dose (200 mg kg−1 day−1 ) of GET73 when administered throughout the whole conditioning period. In the light of the so far available data, this effect of the highest dose of GET73 in the place-preference test might be explained by the fact that this dose produces an increase in the exploratory activity associated with a reduction in anxiety (significant increase of outer and inner ambulation and rearing, and a decrease of the number of fecal boluses, in the open-field test). As far as the consumption is concerned, in the presence of both palatable food (cafeteria food) and standard laboratory chow, with free choice between them, in vehicle-treated rats the daily amount of ingested food was composed by 80–90% of cafeteria food and by 10–20% of standard chow. Treatment with GET73 reduced the consumption of cafeteria food and concurrently increased the consumption of standard chow, so that at the maximum effective dose of 100 mg kg−1 daily−1 , the total daily amount of ingested food was composed by 55–70% of cafeteria food and by 30–45% of standard chow. No tolerance developed to this effect of GET73, at least for the duration of our experiment (12 consecutive days). Since, we measured the total food intake, we do not know whether GET73 did selectively inhibit the consumption of some specific component of the cafeteria food.
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Fig. 6. Influence of acute treatment with GET73 on the cafeteria diet preference test. GET73 (50, 100 or 200 mg kg−1 ) or vehicle were orally administered, and food was weighed 1, 2, 3 and 24 h after treatment. Data are presented as consumption in kcal of the ingested food (a) or as percentage of consumption of the different kinds of food with respect to total ingested food (b); 10 animals per group. (*) p < 0.05 with respect to vehicle-treated group (ANOVA followed by Student–Newman–Keuls’ test).
On the whole, our data indicate that GET73 seems to specifically attenuate the gratification produced by varied and palatable food. The data that we have so far obtained concerning the binding and neurochemical properties of GET73 do not allow by now a straightforward explanation of the mechanism underlying this peculiar effect on the consumption of palatable food. Palatability, pleasantness and variety of foods are considered to be the major reasons for overeating, and hence for overweight and obesity (for reviews see, for example [2,3,60]). Palatability cues, particularly taste and smell, are strong modulators of food reward and a number of studies have suggested that food reward and drug reward share common neural substrates of the brain reward system (for a review [9]). Thus, several data indicate that endogenous opioid and cannabinoid systems are involved in the hedonic value of food intake (see, for example [46,61]). And the melanocortin system, which is the main physiological antagonist of the opioid systems [62–64] and plays a central role in the inhibition of feeding [5,65–70], not only contributes to the homeostatic control of feeding, but also to its hedonic aspects via the melanocortin 4 receptors (MC4-R) located in the nucleus accumbens (for a review [60]). In fact, the blockade of and k opioid receptors and of CB1 receptors inhibits consumption of palatable foods and alcohol, whereas the blockade of MC4 receptors in the nucleus accumbens has orexic effects, particularly for palatable foods, that
may persist for up to 6 days [71]. The brain system processing the non-homeostatic, hedonic aspects of ingestive behaviour are thus likely to play a crucial role in the development of obesity [2]. So, promising and rational pharmacological interventions for obesity may be those that interfere with the reinforcing value of food [9]. Our present data suggest that GET73 might be a drug that meets such requirements.
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