Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss)

General and Comparative Endocrinology 160 (2009) 134–138

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Melanocortin peptides affect the motivation to feed in rainbow trout (Oncorhynchus mykiss) Joachim Schjolden a,*, Helgi B. Schiöth b, Dan Larhammar b, Svante Winberg b, Earl T. Larson b a b

Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, P.O. Box 8146 Dep, Ullevålsveien 72, N-0033 Oslo, Norway Department of Neuroscience, Uppsala University, P.O. Box 593, SE-751 24 Uppsala, Sweden

a r t i c l e

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Article history: Received 13 June 2008 Revised 16 October 2008 Accepted 1 November 2008 Available online 8 November 2008 Keywords: Salmonids Neuropeptides Receptors Feeding Appetite HS024 SHU9119 MTII

a b s t r a c t In this study, we investigated the effects of one melanocortin receptor (MCR) agonist and two antagonists on food intake in juvenile rainbow trout. Baseline food intake was established prior to 1 ll intracerebroventricular injection (ICV) of the non-specific agonist MTII, the MC4R antagonist HS024 and the MC3/4R antagonist SHU9119 at concentrations of 0.3, 1 or 3 nM. Saline-injected fish and untreated fish served as controls. Changes in food intake were observed 1 h after the ICV injections. Our results showed that treatment with MTII significantly decreased food intake at 3 nM compared to control, HS024 significantly increased food intake at 3 nM compared to control and saline-treated fish, and SHU9119 significantly increased food intake at 3 nM compared to saline-treated fish. In conclusion, our study provides further evidence, and hence strengthens the hypothesis, that MC4R participates in the control of energy balance in fish in the same manner as in mammals. Our findings that HS024 is more potent than SHU9119 in increasing food intake suggest that the effects of melanocortin on energy balance in rainbow trout are mainly regulated by activation of MC4R. Hence, HS024 seems an excellent tool as a MC4R antagonist in rainbow trout. Ó 2008 Elsevier Inc. All rights reserved.

1. Introduction The neuronal regulation of appetite involves complex interactions between peripheral metabolic status and the brain, where the hypothalamus in particular mediates the regulation of appetite via a range of neuropeptides stimulating (orexigenic) or inhibiting (anorectic) food intake (Kalra et al., 1999; Volkoff et al., 2005; Arora and Anubhuti, 2006). The communication of metabolic status involves neuropeptides and peripheral endocrine peptides with either orexigenic or anorectic properties. The orexigenic neuropeptides released within the hypothalamus include neuropeptide Y (NPY), melanin-concentrating hormone (MCH), agouti-related peptide (AGRP), galanin, orexin A and B and endogenous opioids comprising the three families of b-endorphins, dynorphins and enkephalins, while the anorexigenic neuropeptides released in the hypothalamus include the cocaine and amphetamine regulated transcript (CART), melanocortins, glucagon-like peptides, corticotropin-releasing hormone (CRH) and neurotensin (NT) (Kalra et al., 1999). In this paper, we will focus on the role of the melanocortin system in the regulation of appetite.

* Corresponding author. Fax: +47 22 59 73 09. E-mail address: [email protected] (J. Schjolden). 0016-6480/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2008.11.003

The melanocortins are polypeptides comprising the pituitary hormones adrenocorticotropin (ACTH) and the a-, b-, c-melanocyte-stimulating hormones (MSH), which are derived from the precursor molecule pro-opiomelanocortin (POMC) (Dores and Lecaude, 2005). The actions of these peptides are mediated by five G protein-coupled receptors of the melanocortin receptor (MCR) gene family (Cone, 2000). Neurons in areas of the mammalian brain implicated in the control of feeding express the MC3R (Roselli-Rehfuss et al., 1993; Gantz et al., 1993a) and the MC4R (Gantz et al., 1993b; Mountjoy et al., 1994). For instance, the MC4R mRNA expressing neurons are found in several sites within the paraventricular nucleus (PVN), ventromedial nucleus (VMN), dorsomedial nucleus (DMN) and nuclei that occupy the medial zone of the hypothalamus (Tatro, 1990; Mountjoy et al., 1994). Intracerebroventricular (ICV) injections of a-MSH, which is distributed throughout the hypothalamus in mammals (Jacobowitz and Odonohue, 1978; Joseph et al., 1983), has been shown to suppress food intake (Panksepp et al., 1976; Poggioli et al., 1986). Moreover, ICV injections of the unspecific MC agonist MTII have also been shown to reduce food intake, an effect which is counteracted by the MC3/4R-specific antagonist SHU9119 (Hruby et al., 1995; Fan et al., 1997) while central administration of SHU9119 increased food intake in mice (Fan et al., 1997). More specific MC4R antagonists such as HS104 and HS024 increase feeding in satiated rats, and long-term intraventricular infusion increases food intake and body weight

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(Schiöth et al., 1998; Kask et al., 1998a,b,c; Skuladottir et al., 1999). These findings showed that a tonic restraint on feeding by melanocortins is dependent on a-MSH and mediated by MC4R (Schiöth et al., 1998; Kask et al., 1998a,b,c; Skuladottir et al., 1999). Most studies on the regulation of food intake in fish have focused on either diet composition and digestion (Simpson et al., 1996; Saether and Jobling, 1999), life history strategies (Simpson et al., 1996) or environmental factors such as photoperiod (Bolliet et al., 2001), temperature (Waiwood et al., 1991; Talbot et al., 1999) and stress (Øverli et al., 1998). However, the neuronal regulation of appetite in fish is poorly understood, but there are studies providing some information on the matter. Early on, it was demonstrated that electric stimulation of the hypothalamic inferior lobe induced feeding behaviour in fish (Demski, 1973; Demski, 1977; Roberts and Savage, 1978). Later, a number of appetite regulating peptides homologous to those in mammals have been isolated, and among these are the melanocortins derived from POMC (see Cerdá-Reverter et al., 2003b), which have been characterized by molecular cloning in all major fish lineages (reviewed by Danielson and Dores, 1999; Takahashi et al., 2001). Winberg and Lepage (1998) obtained results suggesting that POMC is involved in stress-related decreases in food intake in rainbow trout (Oncorhynchus mykiss) as both POMC A and B mRNA increased rapidly in non-feeding subordinate individuals. Further, Cerdá-Reverter and co-workers have also implicated this part of the melanocortin system in food intake in fish. ICV injections of the universal melanocortin agonist NDP-a-MSH, inhibited food intake in goldfish (Carassius auratus) in a dose dependent manner (Cerdá-Reverter et al., 2003b) as did the unspecific agonist MTII (Cerdá-Reverter et al., 2003a). Moreover, Cerdá-Reverter et al. (2003a) also showed that the specific MC4R antagonist HS024 stimulated food intake, suggesting that melanocortins exert a tonic inhibitory effect through central MC4R signalling. These findings together with a conserved expression pattern of MCR genes in the brain, similar to what has been observed in mammals, suggest that the melanocortin system is important in the regulation of energy homeostasis in most vertebrates (reviewed by Schiöth et al., 2005). The aim of the present study was to study the food intake regulation in fish and thus extend the work that Cerdá-Reverter et al. (2003a,b) performed in goldfish on the role of the melanocortin system in control of food intake. In this study we have chosen to investigate the effect of MC3R and MCR4 agonists and antagonists on feeding behaviour in the distantly related rainbow trout. The relation is considered distant because both goldfish and rainbow trout have undergone a fourth independent tetraploidization of the genome after the teleost 3R tetraploidization (Larhammar and Risinger, 1994; Otto and Whitton, 2000; Conlon and Larhammar, 2005), which could mean that the melanocortin system in these two species do not share the same characteristics. Moreover, Cerdá-Reverter et al. (2003a,b) submitted a feeding regime that allowed the fish to eat at individually chosen periods during a stretch of 2 or 4 h 15 min subsequent to ICV injections of the MCR agonist or antagonist. Thus, food pellets first discarded after the fish had become satiated could be eaten later when appetite was regained. An increased food intake in these studies is therefore an excellent measure of increased appetite. However, in this study we aim to implicate a feeding regime that measures the effects of MC3/4R agonists and antagonists on the motivation to feed in rainbow trout. 2. Materials and methods

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with aerated Uppsala tap water at 1 l/min and the temperature varied between 8 and 10 °C. The light/dark regime was automatically controlled and adjusted to conditions at latitude 51°N. While the fish were in the holding tank they were manually fed with commercial trout pellets (Ewos ST40) at 1–2% of the body mass per day. The experimental fish, randomly selected from the holding tank, weighed between 25 and 50 g (mean ± SD = 43.5 ± 6.0 g, n = 142). 2.2. Experimental conditions The experimental set up consisted of eight glass aquaria (250 l) continuously supplied with aerated Uppsala tap water (0.8 l/min, 8–10 °C). Each aquarium was divided into four equal compartments (65 l) using opaque PVC walls. Light was provided by 30 W Lumilux daylight fluorescent tubes placed 10 cm above the water surface of each aquarium. 2.3. Experimental protocol These experiments were conducted according to the guidelines provided by the guide for the care and use of laboratory animals (NRC, 1996) and approved by the Uppsala Animal Research Ethical Committee. For quantification of feed intake, individual fish were fed with one pellet at the time until the fish rejected three pellets in a row. Using these criteria, the feeding was terminated for each fish at a maximum of 15–20 min. This feeding regime allows us to directly monitor the feeding behaviour of the fish. As the fish has to keep eating to receive more food the total number of pellets consumed is a measure of the motivation to feed, as opposed to having food available in abundance within its territory to feed upon at will. The experiment started when fish were individually transferred to the compartments within the glass aquaria. The fish were fed until satiation with commercial feed (EWOS ST40) once a day for 5 days to allow the fish to acclimate. Initially during this period the feed intake increased and then stabilized towards day 5. The number of pellets consumed on day 6–8 laid the foundation for the basal feed intake for each individual fish. After determining basal feed intake the fish were removed from the compartments of the glass aquaria one at a time and lightly anaesthetized in 500 mg/l ethyl-m-aminobenzoate methanesulphonate. Subsequently, random fish were given an intracerebroventricular (ICV) injection (1 ll) of either the non-specific melanocortin receptor (MC) agonist MT II, the MC4R antagonist HS024 or MC3/4R antagonist SHU9119. The different drugs were diluted in Fish Ringer and given at concentrations of 0.3, 1 or 3 nM. Fish given only Fish Ringer served as injection controls (saline) and unmanipulated fish served as an overall control. After the injections fish were returned to their compartments and allowed to recuperate. After 2 h of recuperation the fish were once more fed to satiation and individual food intake was quantified as described above. We chose a recuperation period of 2 h to allow for a maximum recovery after surgery. Previous results on goldfish (Cerdá-Reverter et al., 2003a,b) have shown that the MCR antagonists and agonist we have used in this study have an effect on food intake for at least 2 h after injection, while the effect starts to wear off after 4 h. Food intake was measured again one day after injection to make sure that the fish returned to their basal food intake. Fish that did not resume their basal food intake were removed from the experiment to ensure that the effects we measured on food intake were not affected by the administration method in itself.

2.1. Experimental animals 2.4. Data treatment and statistics Prior to the experiment, juvenile rainbow trout were kept in a 1 m3 indoor holding tank at a density of approximately 0.02 kg/l for approximately 1 month. The tank was continuously supplied

The mean (±SEM) food intake were calculated for each treatment group and presented as percent of the mean basal food intake

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3. Results In the experiment where fish were treated with the unspecific MCR agonist MTII, feed intake was significantly (one-way ANOVA, F4, 47 = 4.328, P = 0.0068) different between the treatment groups (Fig. 1). Feed intake decreased with increasing concentration of MTII, and fish given 3.0 nM of the MCR agonist decreased their feed intake to 37 ± 12% (n = 9) of baseline, which was significantly (Tukey–Kramer post hoc test, P < 0.05) lower compared to control fish (105 ± 10%, n = 10). However, there were no significant (P > 0.05) differences between any of the other groups. In the experiment where fish were treated with the MC4R antagonist HS024 feed intake was also significantly (F4, 38 = 5.219, P = 0.0019) different between the treatment groups (Fig. 2). Feed intake increased with increasing concentration of HS024, and fish given 3.0 nM of this MCR antagonist increased their feed intake to 272 ± 95% (n = 9) of baseline, which was significantly (Tukey– Kramer post hoc test, P < 0.05) higher compared to the control group (105 ± 12%, n = 9) and the group given saline (77 ± 12%, n = 9). There were no significant (P > 0.05) differences between any of the other groups. In the experiment where fish were treated with the MC3R/ MC4R antagonist SHU9119 feed intake was significantly (F4, 42 = 2.680, P = 0.0442) different between the treatment groups (Fig. 3). Feed intake increased with increasing concentration of SHU9119, and fish given 3.0 nM of this MCR antagonist increased their feed intake to 158 ± 32% (n = 10) of baseline, which was significantly (P < 0.05) higher compared to the fish given saline injections (72 ± 16%, n = 9). However, there were no significant differences (P > 0.05) between any of the other groups. 4. Discussion The results from the present study show that the MCR agonist (MTII) decreases, while the MCR antagonists (HS024 and

MTII Food intake (% of baseline)

HS024 b

320 280 240

ab

200 160 120

a

ab a

80 40 control

saline

0.3 nM

1.0 nM

3.0 nM

Treatment Fig. 2. Food intake in juvenile rainbow trout calculated as percent of basal food intake obtained on day 6–8 after transfer to social isolation. The fish were given ICV injections of 0.3, 1.0 and 3.0 nM of the melanocortin receptor 4 specific antagonist HS024. Fish injected with saline and untreated fish served as control. Different letters are assigned to bars which are significantly different (one-way ANOVA, Tukey–Kramer post hoc test, P < 0.05).

SHU9119

240 200

b

160 120

ab ab

ab a

80 40

control

saline

0.3 nM

1.0 nM

3.0 nM

Treatment Fig. 3. Food intake in juvenile rainbow trout calculated as percent of basal food intake obtained on day 6–8 after transfer to social isolation. The fish were given ICV injections of 0.3, 1.0 and 3.0 nM of the melanocortin receptor 3/4 antagonist SHU9119. Fish injected with saline and untreated fish served as control. Different letters are assigned to bars which are significantly different (one-way ANOVA, Tukey–Kramer post hoc test, P < 0.05).

140 120

360

Food intake (% of baseline)

in each group. A one-way ANOVA with Tukey–Kramer post hoc test was applied to test for significant differences between the different treatment groups. Data on feed intake from the fish treated with HS024 were log transformed as values did not follow a normal distribution.

Food intake (% of baseline)

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a ab

100

ab 80

ab

b

1.0 nM

3.0 nM

60 40 20

control

saline

0.3 nM

Treatment Fig. 1. Food intake in juvenile rainbow trout calculated as percent of basal food intake obtained on day 6–8 after transfer to social isolation. The fish were given ICV injections of 0.3, 1.0 and 3.0 nM of the non-specific melanocortin receptor agonist MTII. Fish injected with saline and untreated fish served as control. Different letters are assigned to bars which are significantly different (one-way ANOVA, Tukey– Kramer post hoc test, P < 0.05).

SHU9119) increase food intake in rainbow trout. These findings are in agreement with the results obtained by Cerdà-Reverter et al. (2003a, 2003b) on goldfish. Thus, our study provides further evidence of the participation of MC4R in the control of energy balance in fish. MTII is an unspecific agonist binding to all MC receptors. Although the MC3R has been shown to be involved in the regulation of energy balance in mammals (Chen et al., 2000), the effects of melanocortins on food intake seem to be solely conveyed through the MC4R as food intake in MC4R knockout mice is not affected by ICV injections of MTII (Marsh et al., 1999). Moreover, ICV injections of MTII have also been shown to reduce food intake in mammals, an effect which is counteracted by the MC3/4-specific antagonist SHU9119 (Hruby et al., 1995; Fan et al., 1997). The MC3R has yet to be identified in rainbow trout and it been shown to be absent in Fugu (Takifugu rubripes) (Metz et al., 2006). Both

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spiny dogfish (Squalus acanthias) (Ringholm et al., 2003) and zebrafish have functional copy of the MC3R suggesting that the MC3R has been lost in certain species, while it was present in the ancestors to teleosts. It is not known if the MC3R is present in rainbow trout, but if it is not, there is no evidence that a loss of this gene would affect the physiology or pharmacology of the MC4R. Our results also show that ICV-injections of the MC4R antagonist HS024 results in a more pronounced increase in food intake compared to the unspecific antagonist SHU9119. In fact 3.0 nM of SHU9119 had approximately the same effect on feed intake as 1.0 nM of HS024 (Figs. 2 and 3). Previous studies have shown that SHU9119 had higher affinity to the MC4R in trout compared to HS024 (Haitina et al., 2004). The MC3R is not important for regulating food intake in mammals (Butler and Cone, 2002) but is important for energy storage. It is possible that HS024 simply is functionally more potent than SHU9119 in the trout despite the lower binding affinity of HS024 compared to SHU9119. The pronounced increase in food intake brought on by HS024 in both trout and goldfish suggests that this substance is a very valuable tool to study the melanocortin effects in fish while we can not say for sure if the effect is only due to the MC4R or if the MC3R could also play a role for this effect (Haitina et al., 2004). There are some differences in the effect of HS024 on food intake in our study compared to the study on goldfish (Cerdá-Reverter et al., 2003a) as follows. First, Cerdá-Reverter et al. (2003a) found that food intake did not continue to increase as the concentration of HS024 was raised from 1.2 to 3.2 nM as we did in our study. The reason for this could be a species-specific difference in the expression of the MC4R and/or how their data were calculated. Cerdá-Reverter et al. (2003a) did not relate the food intake in treated fish to food intake before treatment, meaning that the fish injected with 3.2 nM of HS024 could have a lower basal food intake, which could account for the lack of further increase in food intake compared to the 1.2 nM group. Our results therefore suggest that HS024 affects food intake in a dose dependent manner beyond 1.2 nM in fish. However, we cannot rule out the possibility of rainbow trout having a greater potential for increased appetite due to a higher baseline expression of the MC4R. Second, the fish in our study treated with 3.0 nM of HS024 increased their food intake almost three-fold during a period of 15– 20 min compared to a two-fold increase during a period of 4 h in the study by Cerdá-Reverter et al. (2003a). These results show that HS024 has a much larger effect in rainbow trout compared to goldfish. Again this could be accounted for by a greater potential for increased appetite in rainbow trout compared to goldfish. But it could also be explained by the difference in methods used when feeding the fish. In our study, fish were fed with one pellet at the time until the fish rejected three pellets in a row, which represents more scarce food availability as opposed to having food available in abundance within its territory to feed upon at will, as is the case in the study of Cerdá-Reverter et al. (2003a). As the fish in our study had to keep eating to receive more food the total number of pellets consumed is a measurement which implicates the motivation to feed to a larger extent compared to the study on goldfish. In the study by Cerdá-Reverter et al. (2003a), the fish had food available over a period of 4 h in an environment where there was no foraging competition, which might lead to a lower motivation to feed. On the other hand, the motivation to feed in our study might be strengthened by both the scarcity of food availability and by the movement of food through the water column in comparison to food lying in abundance on the bottom of the tank. In our study the fish were allowed to feed for only 15–20 min, which might also contribute to the difference in the amount of food eaten compared to fish allowed to eat for 4 h. Afferent signals from stretch-receptors in the GI tract and chemoreceptors in the liver travel via vagus nerve fi-

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bres to higher control centres where they contribute to satiety in the post-prandial period (Mei, 1985). These signals contribute to the short-term regulation of feeding, and therefore have a lower influence on food intake in our study compared to Cerdá-Reverter et al. (2003a). Hence, motivation to feed is a more direct measurement of behavioural changes that occur as a result of MCR antagonist treatment compared to appetite measured by the number of pellets digested. 5. Conclusions Our study has provided further evidence, and hence strengthened the hypothesis that MC4R participates in control of the energy balance in fish. Our results showing that HS024 is more potent than SHU9119 in increasing food intake, suggest that the effects of melanocortin on energy balance in rainbow trout are regulated by activation of MC4R to a larger degree compared to activation via MC3R. Our results also indicated that HS024 has a larger effect on food intake in rainbow trout compared to goldfish, an effect which can be accounted for by a greater potential for increased appetite in rainbow trout compared to goldfish, but also by the difference in methods used feeding the fish. Our results have shown that HS024 seems an excellent tool as a MC4R antagonist studying the regulation of energy balance and feeding behaviour in rainbow trout. Moreover, we believe that motivation to feed is a far better tool examining the behavioural effects mediated by the MC4R compared to appetite. Acknowledgements The methodology used in this study was approved by the Uppsala Animal Research Ethical Committee. Financially, this study was mainly supported by the Swedish Research Council for Environment, Agriculture Sciences and Spatial Planning (FORMAS), and partly supported by the Swedish Research Council (VR). References Arora, S., Anubhuti, 2006. Role of neuropeptides in appetite regulation and obesity— A review. Neuropeptides 40, 375–401. Bolliet, V., Aranda, A., Boujard, T., 2001. Demand feeding rhythm in rainbow trout and European catfish—Synchronisation by photoperiod and food availability. Physiol. Behav. 73, 625–633. Butler, A.A., Cone, R.D., 2002. The melanocortin receptors: lessons from knockout models. Neuropeptides 36, 77–84. Cerdá-Reverter, J.M., Cerda-Reverter, J.M., Ringholm, A., Schiöth, H.B., Peter, R.E., 2003a. Molecular cloning, pharmacological characterization, and brain mapping of the melanocortin 4 receptor in the goldfish: involvement in the control of food intake. Endocrinology 144, 2336–2349. Cerdá-Reverter, J.M., Schiöth, H.B., Peter, R.E., 2003b. The central melanocortin system regulates food intake in goldfish. Regul. Pept. 115, 101–113. Chen, A.S., Marsh, D.J., Trumbauer, M.E., Frazier, E.G., Guan, X.M., Yu, H., Rosenblum, C.I., Vongs, A., Feng, Y., Cao, L.H., Metzger, J.M., Strack, A.M., Camacho, R.E., Mellin, T.N., Nunes, C.N., Min, W., Fisher, J., Gopal-Truter, S., MacIntyre, D.E., Chen, H.Y., Van der Ploeg, L.H.T., 2000. Inactivation of the mouse melanocortin3 receptor results in increased fat mass and reduced lean body mass. Nat. Genet. 26, 97–102. Cone, R.D., 2000. The Melanocortin Receptors. Humana Press, New Jersey. Conlon, J.M., Larhammar, D., 2005. The evolution of neuroendocrine peptides. Gen. Comp. Endocrinol. 142, 53–59. Danielson, P.B., Dores, R.M., 1999. Molecular evolution of the opioid/orphanin gene family. Gen. Comp. Endocrinol. 113, 169–186. Demski, L.S., 1973. Feeding and aggressive behavior evoked by hypothalamic stimulation in a cichlid fish. Comp. Biochem. Physiol. 44, 685–692. Demski, L.S., 1977. Electrical stimulation of shark brain. Am. Zool. 17, 487–500. Dores, R.M., Lecaude, S., 2005. Trends in the evolution of the proopiomelanocortin gene. Gen. Comp. Endocrinol. 142, 81–93. Fan, W., Boston, B.A., Kesterson, R.A., Hruby, V.J., Cone, R.D., 1997. Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385, 165–168. Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S.J., Delvalle, J., Yamada, T., 1993a. Molecular cloning of a novel melanocortin receptor. J. Biol. Chem. 268, 8246–8250.

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