Appetite regulation: The central role of melatonin in Danio rerio

Hormones and Behavior 58 (2010) 780–785

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Hormones and Behavior j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y h b e h

Appetite regulation: The central role of melatonin in Danio rerio Chiara Carla Piccinetti a, Beatrice Migliarini a, Ike Olivotto a, Giuliana Coletti a, Adolfo Amici b, Oliana Carnevali a,c,⁎ a b c

Dipartimento di Scienze del Mare, Università Politecnica delle Marche, Via Brecce Bianche 60131, Ancona, Italy Istituto di Biotecnologie Biochimiche, Università Politecnica delle Marche, Via Brecce Bianche 60131, Ancona, Italy Istituto Nazionale Biostrutture e Biosistemi, viale Medaglie d'Oro 305,00136 Roma, Italy

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Article history: Received 15 March 2010 Revised 27 July 2010 Accepted 28 July 2010 Available online 5 August 2010 Keywords: Food intake Melatonin Leptin Ghrelin MC4R NPY CB1 Fish

a b s t r a c t Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure. The aim of this study was to evaluate the role of melatonin in appetite central control analyzing the involvement of this hormone in the regulation of feeding behavior in the zebrafish Danio rerio. For this purpose, the effect of two different melatonin doses (100 nM and 1 μM) administered for 10 days, via water, to zebrafish adults was evaluated at both physiological and molecular level and the effect of melatonin was considered in relation to the most prominent systems involved in appetite regulation. For the first time, in fact, melatonin control of food intake by the modulation of leptin, MC4R, ghrelin, NPY and CB1 gene expression was evaluated. The results obtained indicate that melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition, such as leptin and MC4R, and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 are showed here. Taken together these results support the idea that melatonin falls fully into the complex network of signals that regulate food intake thus playing a key role in central appetite regulation. © 2010 Elsevier Inc. All rights reserved.

Introduction Central regulation of energy balance in animals is dependent on a complex neuro-anatomical network between the central nervous system and the peripheral districts that integrates information about the status of energy reserves and prevailing climatic conditions (Helwig et al., 2009). Among different environmental synchronizers, the alternation of light and dark (circadian rhythm) is probably the main factor controlling animal behavior. Melatonin, which is mainly produced in the pineal organ and in the retina, is the key signal in the vertebrate circadian clock, and it is related to many functions which have in common a rhythmic expression as well as locomotor activity, thermal preference, osmoregulation, migration, food intake, reproduction and growth (Reiter, 1991; Falcón et al., 2007). Since the finding of melatonin production in the gastrointestinal tract (GIT) by enterochromaffin cells of several vertebrate species (Bubenik, 2002), including fish (Lepage et al., 2005), a growing number of studies have been involved in the relationship between this hormone and food intake processes. Moreover, the high diver⁎ Corresponding author. Department of Marine Science, Università Politecnica delle Marche, Via Brecce Bianche 60131, Ancona, Italy. Fax: +39 071 2204650. E-mail address: [email protected] (O. Carnevali). 0018-506X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2010.07.013

sity in the distribution of melatonin binding sites in the gastrointestinal tract has suggested a variety of possible functions of this hormone in the digestive system and gastric mucosal cell proliferation (Fernández-Durán et al., 2007). Evidence of an involvement of melatonin in appetite regulation has been reported by many authors; melatonin regulates food intake in rat (Huether, 1994), mouse (Bubenik and Pang, 1994), pig (Bubenik et al., 1996), hamster (Bartness and Wade, 1985) and goldfish (Pinillos et al., 2001). Furthermore, although several studies have been carried out on melatonin relationship with appetite and body weight, the results obtained are often contradictory depending on the animal's daily living habits. Melatonin decreases food intake in many diurnal species such as domestic fowl and mouse (Bermúdez et al., 1983; Bubenik and Pang, 1994) while in nocturnal ones, such as rat, the responses are conflicting including reduction, increase or no effect on food consumption (Shaji and Kulkarni, 1998; Wolden-Hanson et al., 2000). Moreover, it is well-known that the physiological control of appetite in animals is regulated by a complex interplay between hormones, neurotransmitters and neuropeptides that interact reciprocally both at central and peripheral level to stimulate or inhibit feeding behavior (York, 1999). Among the molecules that have an orexigenic effect, the neuropeptide Y (NPY) is the most powerful central enhancer of appetite (Kalra et al., 1998; Pu et al., 1999). In fact,

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the central injection of NPY stimulates feeding behavior, food intake, and body weight gain in mammals (Clark et al., 1984; Stanley and Leibowitz, 1985) as well as in fish (López-Patiño et al., 1999). Related to NPY (Kohno et al., 2003), ghrelin is a newly discovered peptide that is released from the stomach and from neurons in the hypothalamic arcuate nucleus (ARC) and potently stimulates growth hormone release and food intake in vertebrates such as mammals (Korbonits et al., 2004) and fish (Unniappan and Peter, 2005). On the other hand, considering anorexigenic signals, leptin plays a key role as a satiety molecule. Both in mammals and fish, leptin is secreted by a variety of tissues such as adipocytes, stomach, muscle, placenta and brain (Ahima et al., 1996; Ahima et al., 2000; Volkoff et al., 2003; Morash et al., 1999). Leptin reduces food intake by upregulating anorexigeic peptides and downregulating orexigenic signals. In particular, leptin acts stimulating melanocyte-stimulating hormone and MC4R, the key receptor in appetite regulation of the melanocortin system (Schwartz et al., 1997; Seeley et al., 1997) and inhibiting the neuropeptide Y as observed in mice (Stephens et al., 1995) and fish (Volkoff et al., 2003). Recently, in mammals, interactions have been described between melatonin, leptin and ghrelin in body weight regulation (Mustonen et al., 2001; Mustonen et al., 2002; Nieminen et al., 2002) demonstrating a new role of melatonin in appetite control. In addition, De Pedro and collaborators (2008) demonstrated in goldfish the effect of melatonin on metabolic resources, in particular on carbohydrates and lipids metabolism, but no relationship was found between this hormone and feeding regulators such as leptin and NPY. Another element which is strongly involved in the regulation of food intake, body weight and energy balance in all vertebrates is the endocannabinoid system. The endocannabinoid system is comprised of cannabinoid receptors (CBrs), their endogenous ligands, among which anandamide and 2-arachidonoylglycerol, and enzymes for their biosynthesis and degradation (Matias and Di Marzo, 2006). The orexigenic effects of cannabinoids are exerted through the cannabinoid type 1 receptor (CB1), mainly expressed in the brain (Devane et al., 1988; Matsuda et al., 1990; Di Marzo et al., 1998) and to a lesser extent in the periphery. Moreover, a correlation between this system and NPY (Gamber et al., 2005; Cottone et al., 2009; Piccinetti et al., 2010), leptin (Di Marzo et al., 2001), ghrelin and MC4R (Di Marzo and Matias, 2005) has been demonstrated in mammals and fish, although possible relationships with melatonin have not yet been studied. This study was aimed at investigating the effect of melatonin treatment on food intake and its possible involvement in the complex network of molecules that control appetite in the brain. For this purpose, food intake and changes in the gene expression of the major molecules involved in feeding regulation were studied in the zebrafish Danio rerio. This experimental model, over the past twenty years, has attracted considerable attention as an excellent vertebrate model system for studying genetics and development (Fishman, 2001), and more recently, for human disease understanding and for the screening of therapeutic drug, so that zebrafish is recognized as a powerful pharmacological tool (Penberthy et al., 2002; Goldsmith, 2004; Sumanas and Lin, 2004; Keller et al., 2006; Shiels et al., 2009). Materials and Methods Animal and maintenance The experiments were carried out in zebrafish (Danio rerio) obtained from a commercial supplier, Acquario di Bologna, Bo (Italy). Fish were maintained in 100-L aquaria, with a constant flow of filtered fresh water. Photoperiod was 14 L:12D (lights on at 07:00) and water temperature was 27 ± 1 °C. Fish were daily fed with a 2% body weight (bw) ration of floating pellets (Sera Vipagran, Heinsberg, Germany). Animals were acclimated to these conditions for at least

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15 days prior to the assay, showing normal feeding and activity patterns during this acclimation period. Care and the use of the animals were in accordance with the Guidelines on the handling and Training of Laboratory Animals by the Universities Federation for Animal Welfare (UFAW) and with the Italian animal welfare legislation (D.L. 116/92). Experimental design Melatonin treatment Two groups of zebrafish (n = 10 fish/group) maintained with the photoperiod previously described, were exposed via water to two different doses of melatonin (Sigma Aldrich, Germany), 100 nM and 1 μM, following Zhdanova and co workers (2008). Treatment was 10 days long, melatonin doses were added daily at 11.00 a.m. and the concentrations were maintained constant throughout the experiment by renewing water every 24 h in each tank. A control group, (n = 10) was kept at the same rearing conditions but without melatonin administration. For the control group and for each group exposed to two melatonin doses, the treatment was performed in three different tanks. Food intake analysis During the experiment, the animals received pre-weighed food in excess (5% bw) every day, 30’ after melatonin treatment. Food intake (FI) was measured at the end of the experiment, during the next 5 h post melatonin administration. FI was calculated as follows: FI = Wi − (Wf × F), where Wi = initial dry food weight, Wf = remaining dry food weight and F = correction factor. F was previously calculated in the absence of fish to determine the effect of water dissolution on food pellets during the feeding time, and represents the reduction in food weight after food remains 5 h into the aquaria (F = 0.856 ± 0.0054) following López-Patiño et al. (1999). At the end of the experiment, zebrafish were anesthetized (0.1 mg/ l MS222, Sigma) and the brains were sampled and stored at −80 °C until analysis. Animals were sacrificed in accordance with the Guidelines on the handling and Training of Laboratory Animals by the Universities Federation for Animal Welfare (UFAW) and with the Italian animal welfare legislation (D.L. 116/92). High-performance liquid chromatography At the end of 10 days treatment zebrafish were sacrificed and brains were collected. Brain samples were sonicated in methanol 60% solution and centrifuged at 12000 g for 10 minutes. Melatonin was extracted from supernatant by reversed-phase chromatography using a C18 Bakerbond SPE cartridge 3 ml (pore size 60 A, particle diameter 40 μm). The columns were conditioned according to the manufacturer's instructions with two portions of methanol followed by two portions of HPLC-grade water. Melatonin was determined by high-performance liquid chromatography (HPLC-FL) with fluorescence detection. HPLC assay was performed with a Varian modular system (Varian Instruments, Inc. USA) with spectrofluorometric detector Varian ProStar 360 (Varian Instruments, Inc. USA). Chromatographic separations were carried out on an Ultrasphere C18 column (i.d., 250 × 4–6 mm; particle diameter, 5 μm; pore size, 80 Å) from Varian Instruments. Excitation and emission wavelengths were set at 286 and 352 nm, respectively. An isocratic elution system was prepared. The mobile phase was 60 % HPLC-grade methanol (Kulczykowska, 1998). RNA extraction and cDNA synthesis Total RNA was extracted from brain samples with RNeasy Mini Kit (Qiagen). Final RNA concentrations were determined by optical density measurement at 260 nm, and the RNA integrity was verified by ethidium bromide staining of 28 S and 18 S ribosomal RNA bands

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on 1% agarose gel. First strand cDNA synthesis was performed as already described in Carnevali and Maradonna (2003). Real time PCR A relative quantification of CB1, NPY, leptin, ghrelin, and MC4R gene expression was carried out by using β-actin, a validated housekeeping gene (McCurley and Callard 2008), as internal standard. The following primers were used at final concentration of 10 pmol/μL: β-actin: For 5'-GGTACCCATCTCCTGCTCCAA-3', Rev 5'-GAGCGT GGCTACTCCTTCACC-3'; Leptin: For 5'-AGCTCTCCGCTCAACCTGTA-3', Rev 5'-CAGCGGGAATCTCTGGATAA-3'; Ghrelin: For 5'-AATACGGT CCCGTGCTTCAG-3', Rev 5'-TGCACCCACTTTGCTACAGA-3'; NPY: For 5'-GTCTGCTTGGGGACTCTCAC-3', Rev 5'-CGGGACTCTGTTTCACCAAT3'; MC4R: For 5'-ATCTCCACGGAGGTCTTCCT-3' Rev 5'-CGAAGCATTGGAGACACTCA-3'. CB1: For 5'-TCTGTGGGAAGCCTGTTTC-3', Rev 5'-ACCGAGTTGAG CCGTTTG-3'. Primers were designed using PCR designer software PRIMER3 (http://frodo.wi.mit.edu/primer3/) starting from zebrafish sequences available in GenBank. PCRs were performed with SYBR green method in a iQ5 Multicolor Real-Time PCR Detection system (BioRad). Triplicate PCR reactions were carried out for each sample analyzed. The reactions were set on a 96-well plate by mixing, for each sample, 1 μl of diluted (1/10) cDNA, 5 μl of 2× concentrated SYBR Green PCR Master Mix (BioRad), containing SYBR Green as fluorescent intercalating agent, 0,3 μM forward primer and 0,3 μM of reverse primer. The thermal profile, for all reactions, was: 3 min 95 °C and then 45 cycles of 10 s at 95 °C, 20 s at 60 °C and 20 s at 72 °C. The fluorescence monitoring occurred at the end of each cycle. Additional dissociation curve analysis was performed and showed in all cases one single peak. Data obtained were treated by iQ5 optical system software version 2.0. Western Blot analysis To study the presence of the CB1 receptor protein, protein concentrations of brain samples were measured by the Coomassie Brilliant Blue method (Bradford, 1976) using BSA as a standard. Samples were then treated as described by Tsou et al. (1998) and proteins were then separated using 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter (Amersham Pharmacia Biotech) at 250 mA for 2 h at 4 °C in order to evaluate CB1 immunoreactivity. Filters were treated with blocking solution (2% BSA, 0.25%, Tween-20 in Tris-buffered saline, TBS, pH 7.6) over night at 4 °C to prevent nonspecific binding and then incubated with the antibody [anti-cannabinoid receptor CB1, Rat (Rabbit) Calbiochem] diluted 1:1000 in 2% blocking solution. As a control protein, β-tubulin was used. For the β-tubulin assay, the primary antibody (GTX15568), diluted 1:1000 was purchased from Gene Tex, Inc.

Fig. 1. Food intake control by melatonin treatment (100 nM and 1 μM) in zebrafish adults. Data are expressed as mean food intake (mg) per body wt (g). Results are expressed as mean ± SEM. Different letters indicate significant differences evaluated by one-way ANOVA with the Tukey post-test (p b 0.05).

found in fish exposed to 100 nM and 40.54 ± 2.4 pg/mg in fish exposed to 1 μM while 30.32 ± 1.2 pg/mg were found in the control (see Table 1). Real time PCR analysis, conducted on key genes in appetite regulation, clearly showed a central role of melatonin in food intake control. Fig. 2 shows a modulation in leptin gene expression that was dose-related. Both melatonin doses caused a significant increase in mRNA levels with respect to the control, with the strongest effect being observed with the higher dose (1 μM). MC4R was also affected by melatonin treatment: a significant increase in mRNA levels in the zebrafish brain for the higher dose tested was observed, while the lower dose did not induce any effect on MC4R gene expression with respect to the control (Fig. 3). The two different doses of melatonin here used also affected orexigenic signals in the brain: ghrelin gene expression showed a significant decrease (more than 2-fold) in mRNA levels with respect to the control for both doses tested (Fig. 4). Similarly, NPY gene expression showed a significant reduction in mRNA levels that was about 4 times lower than the control (Fig. 5). Moreover, melatonin treatment affected the endocannabinoid system. The two melatonin doses (100 nM and 1 μM) showed a modulation of CB1 mRNA levels in the brain that was not dose-dependent. Both the higher and the lower dose of melatonin significantly decreased CB1 mRNA with respect to the control (Fig. 6A). The effect of melatonin observed on CB1 mRNA levels was accompanied by lower levels of CB1-like protein, as assessed by Western Blot analysis, which showed an immunoreactive band for the CB1-like protein of about 63 kDa as previously found by Migliarini and Carnevali (2009) and Piccinetti et al. (2010). The levels of CB1-like protein were affected by melatonin with a trend comparable to that observed in CB1 mRNA levels (Fig. 6B and C). Discussion

Statistical analysis The data obtained were examined by one-way ANOVA followed by the Tukey post test, using a statistical software package, Graph Pad Prism5 (Graph Pad Software Inc., San Diego, CA, USA) with significance set at p b 0.05. Results The effect of melatonin on food intake in zebrafish adults is shown in Fig. 1. The two different doses (100 nM and 1 μM) of the indoleamine, administered via water, significantly reduced food intake with respect to the control group. The strongest decrease in food intake was observed with the higher dose (about 2-fold) (Fig. 1). The melatonin levels in the brain significantly increased in fish exposed to different doses of the hormone: 36.7 ± 1.3 pg/mg were

In this study the effect of melatonin on food intake in fish was evaluated and related with the major signals involved in appetite control. For the first time, the gene modulation of leptin, ghrelin, NPY, MC4R and CB1 by melatonin is described.

Table 1 Levels of melatonin in zebrafish brain after fish hormonal exposure. Asterisks indicate significant differences, respect to control, evaluated by one-way ANOVA with the Tukey post-test (p b 0.05). Results are expressed as mean ± SEM. Melatonin (pg/mg tissue) CTRL 100 nM 1 μm

30.32 ± 1.2 36.70 ± 1.3* 40.54 ± 2.4*

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Relative Leptin mRNA abundance

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Fig. 2. Leptin gene expression in zebrafish brain after exposure to melatonin (100 nM and 1 μM). Results are expressed as mean ± SEM. Different letters indicate significant differences on the basis of the Tukey post-test (p b 0.05).

Fig. 4. Ghrelin gene expression in zebrafish brain after exposure to melatonin (100 nM and 1 μM). Results are expressed as mean ± SEM. Different letters indicate significant differences on the basis of the Tukey post-test (p b 0.05).

Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure (López-Olmeda et al., 2006; Barrenetxe et al., 2004; Falcón et al., 2007). Considering the conflicting data obtained about the involvement of melatonin in food intake and appetite regulation (Pinillos et al., 2001; Wolden-Hanson et al., 2000; De pedro et al., 2008; Bojková et al., 2006; López-Olmeda et al., 2006), the aim of this study was to clarify the possible links between melatonin and the main regulators of appetite in Danio rerio. First of all, the results here obtained showed a clear decrease in food intake after chronic melatonin administration via water. These data are in agreement with previous studies on European sea bass (Rubio et al., 2004) where melatonin was provided to fish by oral gelatin capsules. In addition, the increase of brain melatonin content here obtained, after 10 days of exposure to this hormone, could indicate its involvement on central control of food intake reduction. However, Pinillos and collaborators (2001) showed, on goldfish, that only intraperitoneal injections of melatonin and not intracerebroventricular ones induced a decrease in appetite suggesting a possible indirect/peripheral effect of melatonin on brain food intake regulation. Additional studies are necessary to better elucidate if in zebrafish melatonin can exert a direct as well as an indirect effect. At molecular level data obtained on signals involved in food intake control support the idea that melatonin falls fully into the complex network signal that regulates appetite and its effect is not only due, as some authors have suggested, to a sedative action on locomotor activity (Bermúdez et al., 1983; Murakami et al., 2001; Zhdanova et al., 2002). Data available on the relationship between leptin and circadian/ultradian rhythmicities in the food intake control suggest a possible interaction with melatonin (Kalra et al., 2003; Pu et al.,

2000) although studies conducted so far are somewhat contradictory. In different experimental models melatonin was found to decreases leptin serum levels (Canpolat et al., 2001; Gündüz, 2002; Ambid et al., 1998; Mustonen et al., 2000) while, De Pedro and collaborators (2008) found no correlation between melatonin administration and plasma leptin levels in goldfish. In this study, chronic administration of melatonin positively influences leptin mRNA levels in the zebrafish brain. This raise is in agreement with the increased levels of melatonin in the brain and with the decrease of food intake, moreover an enhancement of MC4R mRNA levels is detectable after high dose of melatonin treatment. These data fit properly with the well-known link between leptin and the melanocortin system (Schwartz et al., 1997; Seeley et al., 1997). The relationship between melatonin and ghrelin, which is recognized as a major physiological appetite stimulator, has recently been studied. It seems clear that indoleamine treatment decreases ghrelin levels in the rat and the results obtained reinforce the hypothesis that ghrelin is antagonistic to leptin (Mustonen et al., 2001). Our findings are in agreement with the results obtained in mammals by Mustonen and collaborators (2001), confirming the negative effect of melatonin on ghrelin and extend the question to another important regulator of feeding: the neuropeptide Y. Both in mammals and fish it is well known that the orexigenic effect of ghrelin is exerted through the upregulation of NPY considering that there is a co-expression of both neurons in the hypothalamic arcuate nucleus (Kalra et al., 2003; Miura et al., 2006) thus indicating that the two signals work in synergy. The suppression of ghrelin and NPY here obtained in response to melatonin administration attested the inhibitory role in feeding behavior played by this hormone. Finally, in this study the endocannabinoid system was investigated. The results achieved here on CB1 gene and CB1-like protein levels

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Fig. 3. MC4R gene expression in zebrafish brain after exposure to melatonin (100 nM and 1 μM). Results are expressed as mean ± SEM. Different letters indicate significant differences on the basis of the Tukey post-test (p b 0.05).

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Fig. 5. NPY gene expression in zebrafish brain after exposure to melatonin (100 nM and 1 μM). Results are expressed as mean ± SEM. Different letters indicate significant differences on the basis of the Tukey post-test (p b 0.05).

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Relative CB1 mRNA abundance

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B 63 kDa - CB1-like protein Fig. 7. Summary diagram of the effect of melatonin on the control of food intake.

Acknowledgments This work has been supported by COFIN 2005 and 2008 awarded to Prof. Oliana Carnevali.

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References

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Fig. 6. CB1 gene expression in zebrafish brain after exposure to melatonin (100 nM and 1 μM) (A). Western blot analysis of CB1-like protein in the presence of melatonin (100 nM and 1 μM). The CB1-like protein band corresponds to 63 kDa and b-tubulin corresponds to 55 kDa (B and C). Results are expressed as mean ± SEM. Different letters indicate significant differences on the basis of the Tukey post-test (p b 0.05).

show for the first time that melatonin is able to influence the endocannabinoid system. Nevertheless, it is not possible to assert whether this effect is direct or due to the interactions with the other feeding signals such as NPY, ghrelin, leptin, and MC4R modulated by melatonin treatment. These results together with the previous, confirm that melatonin is deeply integrated into the network of signals that regulate appetite in the zebrafish. In particular, these findings place melatonin at the center of the dense system of signals that governs feeding and energy balance since it is able to exert its effect by stimulating the anorexigenic and inhibiting the orexigenic signals as suggested in scheme reported in Fig. 7. Furthermore, data obtained in the present study clearly evidenced a physiological effect on food intake and on the expression of genes involved on appetite control by the lower dose (100 mM) of melatonin. The slight differences observed with 10 times higher melatonin concentration indicate the importance to investigate the effect of even lower doses on appetite control.

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