To eat or not to eat; regulation by the melanocortin system

Physiology & Behavior 89 (2006) 97 – 102

To eat or not to eat; regulation by the melanocortin system Jacquelien J.G. Hillebrand a,b , Martien J.H. Kas a , Roger A.H. Adan a,⁎ a

Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, Utrecht, The Netherlands, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands b Altrecht–Rintveld Eating Disorders, Zeist, The Netherlands Received 29 November 2005; accepted 10 January 2006

Abstract The central melanocortin (MC) system is one of the best-characterized neuropeptidergic systems involved in the regulation of energy balance. This short review describes the role of the central MC system in feeding behavior. Pharmacological, anatomical and genetic studies show that activation of the MC system reduces meal size, whereas de-activation of the MC system increases meal size. Several brain regions, including distinct hypothalamic nuclei and the hindbrain, are involved in this process. Further dissection of MC pathways in feeding behavior is the subject of recent and probably future studies. As the MC system is involved in animal models of obesity and (possibly) anorexia, it appears that this is a target system for development of drugs for the treatment of disturbed human eating behavior. © 2006 Elsevier Inc. All rights reserved. Keywords: Melanocortins; Feeding behavior; Adiposity; Obesity; Anorexia nervosa

1. The MC system: an introduction During food scarcity animals increase physical activity to search for food. In periods of overfeeding, animals store energy in body fat for physical activity and thermogenesis as well as growth and reproduction in periods of restriction. The three parameters of energy homeostasis, sc. feeding, physical activity and thermogenesis, are tightly regulated, especially for defending the lower limit of body weight. The central melanocortin (MC) system is one of the best characterized neuropeptidergic systems implicated in energy homeostasis with effects on feeding and thermogenesis, while it is also involved in other, possibly related, biological processes (e.g. pigmentation, adrenal steroidogenesis, modulation of blood pressure and heart rate) [1–5]. In this short review, we focus on the involvement of MCs in feeding behavior and describe which neuroanatomical areas are involved. MCs are processed from pro-opiomelanocortin (POMC), which is expressed in the arcuate nucleus (ARC), nucleus of the solitary tract (NTS), pituitary gland and other peripheral tissue

⁎ Corresponding author. Tel.: +31 302538517; fax: +31 302539032. E-mail address: [email protected] (R.A.H. Adan). 0031-9384/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2006.01.034

[6,7]. Central MCs are downstream mediators of leptin, an adiposity hormone that acts in the brain to reduce food intake and body weight [8]. Absence of leptin or functional leptin receptors causes morbid obesity [9]. The MC system consists of an endogenous agonist, α-melanocyte-stimulating hormone (α-MSH, derived from POMC) as well as an endogenous antagonist, Agouti-related protein (AgRP), which both act on central MC receptors (MC-Rs). This unique characteristic implicates a tight regulation of MC function in energy homeostasis. Arcuate POMC neurons express leptin receptors [10]. Leptin increases the activity of POMC neurons, resulting in hypophagia and increased sympathetic activity [11,12]. Leptin levels are related to the amount of adipose tissue. Fasting (low leptin) results in a diminished activation of POMC neurons, whereas overfeeding (high leptin) results in a stimulation of POMC neurons [13,14]. AgRP shows great similarity with Agouti, a paracrine factor involved in pigmentation [15]. AgRP is centrally expressed in distinct ARC neurons that also express leptin receptors [16]. AgRP neurons are inhibited in the overfed state and stimulated during fasting [14,17]. While often considered a competitive antagonist of the MC system, AgRP appears in fact an inverse agonist of the constitutively active MC3 / 4-Rs [18,19].

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The biological effects of MCs are mediated by five different MC-Rs [20–25]. The effects on energy homeostasis are mediated by the central MC3-R and MC4-R. Both receptors are expressed in several regions of the brain, including the hypothalamus. The MC4-R has high affinity for α-MSH and is evidently implicated in feeding behavior (see below) [26,27]. The MC3-R has highest affinity for γ-MSH (which does not influence feeding behavior [2]) and appears to be mainly involved in regulation of adiposity rather than food intake (see below). Interestingly, MC3-Rs are expressed on ARC POMC and AgRP neurons suggesting auto-regulation of POMC and AgRP release [28]. Indeed, it was recently shown that peripheral administration of a MC3-R agonist increases food intake in rats [29]. 2. The MC system: genetic variants The role of central MC-Rs and endogenous ligands in feeding behavior became more evident from genetic studies in rodents and humans. Targeted disruption of the MC4-R in mice leads to increased longitudinal growth and obesity as a result of hyperphagia and reduced energy expenditure [30]. MC3-R deficient mice have an increased adipose mass, but are hypophagic (with increased feed efficiency) and hypoactive [31,32]. POMC deficient mice are obese and hyperphagic and also show impaired pigmentation (via MC1-R) and adrenal development (via MC2-R) [33]. In contrast to the above, AgRP deficient mice do not have an obvious (anorexic) phenotype. AgRP deficient mice exhibit normal feeding behavior, body weight, and locomotor activity [34]. However, it was recently demonstrated that ablation of AgRP neurons in adult mice (but not in neonatal mice) does result in hypophagia and reduced body weight. This suggests that when AgRP is absent during development (as in AgRP knockout mice), compensating mechanisms exist to secure normal energy balance [35]. Indeed, reducing AgRP gene expression by RNA interference results in lower body weight gain due to an increased metabolic rate [36]. On the other hand, over-expression of AgRP leads to a hyperphagic obese phenotype, similar to Agouti yellow (Ay) mice that ubiquitously express Agouti [16,37]. Natural variations in human MC genes also result in specific phenotypes. Variations in the POMC gene are rare, but result in severe early onset obesity, red hair pigmentation and adrenal insufficiency [38]. MC4-R haplo-insufficiency is the most common monogenic cause of severe obesity [39,40]. In addition, MC4-R SNPs are also associated with adiposity and physical activity [41,42]. Branson et al. recently showed that all obese carriers of MC4-R mutations in their population met binge eating disorder (BED) criteria, whereas other clinical characteristics (fat mass, energy expenditure) were similar to obese people without MC4-R mutations [43]. This finding was, however, not confirmed in another population [44]. SNPs in the MC3-R gene are common but are not associated with obesity [45,46]. We screened the human AgRP gene and found two SNPs in linkage disequilibrium of which one, G760A, leads to nonconservative amino acid substitution (Ala67Thr). This SNP was

significantly enriched in anorexia nervosa patients versus healthy controls [47]. Homozygosity of the same allele (Thr67) was associated with a low body mass index and fat mass in another white population [48] and was protective against late onset obesity in a third white population [49]. Up to now no differences have been found in cellular distribution, processing and secretion of the AgRP variant [48,50]. Moreover, in vitro antagonism of the MC4-R in reporter gene assays and in vivo stimulation of food intake appears similar to the normal protein [50]. 3. The MC system: food intake and selection Intracerebroventricular (icv) or local infusion of α-MSH or its synthetic analogue melanotan-II (MTII) decreases food intake, increases energy expenditure and stimulates HPA axis activity in rats, but not in MC4-R deficient mice [51–55]. The efficacy of α-MSH and MTII to reduce food intake depends on the experimental paradigm. Chronic injections appear less effective than acute injections [56] and efficacy appears attenuated during scheduled feeding, when rats have learned to consume large amounts of food in a short period of time [57]. Central injections of SHU9119, a synthetic competitive antagonist of MC3 / 4-Rs, antagonize α-MSH/MTII-induced hypophagia [51]. Chronic injections of SHU9119 in the third ventricle (i3vt) enhances body weight gain by increasing food intake and decreasing energy expenditure by thermogenesis [58]. AgRP is a potent stimulant of food intake and is implicated in the maintenance of feeding rather than the initiation of feeding [59]. Therefore, AgRP(83–132) is most effective when injected in the natural feeding period or following food deprivation. A single icv or local injection in the paraventricular nucleus (PVN) increases food intake for at least 24 h (up to 7 days) and blocks α-MSH/MTII-induced hypophagia [59–61]. The mechanisms underlying the long-lasting hyperphagia are unclear but seem to be independent of persistent MC4-R blockade or crosstalk with the opioid system [60,62]. Chronic icv AgRP(83–132) treatment increases body weight gain by increasing food intake and decreasing energy expenditure [63]. Although genetic and pharmacological studies support an effect of the MC system in feeding behavior, exactly how this is brought about is unknown. The MC-system may e.g. affect meal initiation, meal size or meal choice. Meal pattern analysis studies in rats show that MTII, similar to leptin, decreases food intake by reducing meal size and meal duration rather than meal frequency or inter-meal interval [64]. Oppositely, SHU9119 increases food intake by selectively increasing meal size [65]. MCs are also involved in food selection. Peripheral MTII injections in diet-induced obese (DIO) wildtype mice reduce fat intake (but not protein or carbohydrates) when these mice were offered a three-choice macronutrient diet [66]. Likewise, central AgRP(83–132) enhances the intake of specifically high fat diets in rats [67]. Obese Ay mice also have enhanced preference for fat intake when compared to wildtype littermates [68]. The preference for an energy-dense diet might be mediated by crosstalk between the MC and the opioid system since

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opioidergic signaling is implicated in food intake as well as food selection [69]. The opioid antagonist naltrexone suppresses intake of specifically high fat diets and reduces AgRP(83–132)induced feeding when administered simultaneously with AgRP(83–132) [67,70]. Interestingly, AgRP(83–132) does not increase consumption of a sucrose solution, which is also considered a preferred diet. In fact, AgRP(83–132) (i3vt) specifically increased chow intake in rats when a choice of chow or sucrose was given [67]. Thus, it appears that AgRP(83–132) drives ingestion of energy dense foods and does not increase motivation for preferred foods perse. Another group, however, showed that local injection of AgRP(83–132) in the dorsal medial hypothalamus (DMH) results in enhanced intake of a sucrose diet compared to a corn starch diet in a choice paradigm, suggesting that MC signaling in the DMH is involved in rewarding aspects of food intake [71]. Hence, local injection or expression of AgRP in distinct brain regions/ventricles might result in different phenotypical outcomes, which will be further described below. Two polymorphisms in the human AgRP gene also appear to be associated with macronutrient intake. The Thrallele of the aforementioned Ala67Thr SNP is associated with reduced energy intake from (saturated and monounsaturated) fat and increased energy intake from carbohydrates in white subjects [72]. Another SNP, − 38 C/T, in the promoter region of the gene is associated with protein intake in black subjects. Homozygotes of the mutant T allele show reduced protein intake versus carriers of the C allele. The TT genotype is associated with a low promoter activity and low affinity for transcription factors and was previously found to be protective of obesity [73]. The macronutrient content of a diet also influences the sensitivity to MC ligands. Clegg, Benoit and co-workers demonstrated that rats fed on a high fat diet showed an attenuated response to MTII [57,74]. Others, however, found that MTII-induced hypophagia and body weight loss were enhanced in rodents that were fed a palatable high fat diet, whereas the response to specific MC4-R antagonist HS014 was not changed suggesting that feeding on a high fat palatable diet results in a lower endogenous MC tone [66,75]. Furthermore αMSH-induced hypophagia appeared enhanced in DIO rats [76]. Discrepancies in these findings might be explained by differences in experimental procedures, like duration and constituents of the diet, dose of MTII and body adiposity. It was recently shown that chronic MTII treatment reduces food intake in rats fed a high fat diet as well as in food-deprived rats. MTII was, however, less effective in reducing food intake and body weight in rats with a lower body fat mass. This indicates that activity of the MC system is tied to the defended level of body adiposity, suggesting that the main function of the MC system is regulating body adiposity rather than food intake [77]. 4. The MC system: site of action Arcuate POMC and AgRP neurons influence feeding behavior and energy expenditure through their effects on MC3 / 4-Rs in adjacent brain regions, including the hypothalamic PVN, DMH and lateral hypothalamus (LHA) [26,78]. Local injections of α-MSH/MTII in these regions decrease food

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intake, whereas local injections of AgRP or SHU9919 increase food intake [59,71,79]. From these nuclei, neurons project to areas in the hindbrain and corticolimbic regions. Whereas studies on MC activity in the hypothalamus dominate the literature, the brainstem appears particularly important in regulation of feeding behavior. The brainstem can execute several aspects of feeding behavior by interaction with gastrointestinal satiety signals, independent of the forebrain. Experiments with decerebrate rats, however, showed that the brainstem is not capable of managing long-term energy homeostasis [80]. POMC and MC4-Rs are expressed in the brainstem [26]. Injections of MTII in the fourth ventricle (i4vt) result in hypophagia whereas i4vt injections of SHU9119 result in enhanced food intake [65]. Several new techniques now make it possible to dissect MC pathways thoroughly to establish which neuronal populations are critical in modulation of food intake and energy expenditure. By using transgenic POMC-EGFP it was discovered that POMC cells from the NTS are activated by peripheral cholecystokinin (CCK) treatment as well as food-induced satiety [81]. CCK-8induced hypophagia was observed in wildtype and MC3-R deficient mice, but not in MC4-R deficient mice. SHU9119 treatment prevents CCK-8-induced hypophagia when injected i4vt, but only partly when injected i3vt, indicating that hindbrain MC4-Rs are important regulators of food intake via peripheral signals [81]. Retrograde tracer studies revealed that a small population of ARC POMC neurons projects directly to the hindbrain [65]. Hence, in the hindbrain integration might take place between POMC neurons regulating acute and chronic responses to energy homeostasis [65]. Future studies should focus on specific characteristics of both types of POMC neurons. Chronic local antagonism of MC4-Rs results in brain-region specific forms of obesity [82]. Rats that were bilaterally injected with an adeno-associated viral (AAV)-Agouti vector in the PVN showed hyperphagia and increased body weight starting one week after injection and continuing during the 6-week experiment. Interestingly, we found that injection of AAVAgouti in the DMH resulted in late-onset hyperphagia and obesity (day 21), whereas injections in the LHA at first sight did not result in obese rats. Nevertheless, these LHA-Agouti rats gained significantly more weight than LHA-EGFP control rats on a high fat diet by insufficient reduction of intake of this high caloric diet. This implicates that MC4-Rs in the LHA might protect rodents against DIO. Balthasar et al. recently described the use of the Cre-LoxP system to manipulate MC4-R gene expression in a specific population of neurons [83]. LoxP MC4-R mice are obese (similar to MC4-R null mice), and total or neuronal reactivation of MC4-Rs by Cre recombinase resulted in a phenotype identical to wildtype mice. Uni-lateral reactivation of MC4-Rs in the PVN by AAV-Cre injections decreased body weight and tended to reduce food intake in LoxTB MC4-R mice, although LoxTB MC4-R mice were still heavier than wildtype mice. Next, a transgene-driven Cre expression approach was taken to investigate the reactivation of MC4-R in distinct brain areas. Reactivation of MC4-R in Sim1 (a transcription factor expressed in the PVN and amygdala, but not in the hindbrain) containing brain regions resulted in a

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partial reduction of obesity and body fat mass, whereas food intake and longitudinal length were similar to wildtype mice. MC4-Rs in the PVN and amygdala are thus involved in food intake, while MC4-Rs in other brain regions, e.g. the dorsal vagal complex are involved in energy expenditure and body composition [83]. The data from Kas and Balthasar together with previous pharmacological data suggest that the PVN is a major site for effects of MCs on food intake [59,62,82,83].

endorphin is implicated in rewarding aspects of food intake, which can (normally) be overridden by homeostatic systems [98]. Furthermore, it was recently discovered that reduced βendorphin signaling (in μ-opioid receptor deficient mice) attenuates anticipatory activity during scheduled food access [99]. Future studies should further explore the (possible) involvement of the MC system (specific receptors and brain regions) and crosstalk with the opioid system in development of the anorexic phenotype.

5. The MC system: involvement in anorexia nervosa? 6. Perspectives Whereas reduced activity of the MC system is evidently implicated in obesity, there is only limited information on the putative involvement of this system in a human disorder on the other end of the body weight spectrum: anorexia nervosa (AN). AN is a psychiatric disorder that is often characterized by extreme hypophagia, low body weight, obsessive fears of being fat and hyperactivity, symptoms which assume activation of the MC system [84,85]. As already described above, we found that the Ala67Thr SNP was enriched in AN patients, but no other SNP in the MC system have been associated with AN yet. Plasma levels of leptin and central levels of POMC are reduced in ill AN patients [86,87], while antibodies against α-MSH have been found in ill AN patients which were associated with psycho-behavioral abnormalities [88]. In absence of genetic mouse models of anorexia (except for the anx/anx mice [89]), we used a behavioral animal model of anorexia to gain knowledge on central mechanisms involved in anorexia. The activity-based anorexia model is based on scheduled feeding (1 h/day) in combination with running wheel access (24 h/day). Rats exposed to this model show (food-anticipatory) hyperactivity and reduced food intake, resulting in a rapid decline of body weight as well as activation of the HPA axis and hypothermia [90]. We showed that MC binding sites in the ventromedial hypothalamus (VMH) are increased in ABA rats [91]. Chronic treatment of ABA rats with AgRP(83–132) prevented selfstarvation by increasing food intake and influencing body temperature [91]. We also found that chronic icv administration of α-MSH enhanced the development of ABA by reducing food intake and stimulating food-anticipatory activity (in contrast to Ref. [57]) [92]. Next we recently described transient upregulation of POMC in the ARC during early exposure to ABA, followed by a significant down-regulation after one week [91,93]. These data indicate hyperactivity of the MC system in ABA rats. Further studies should, however, be performed since icv SHU9119 treatment did not rescue rats from self-starvation, opposite to AgRP [93]. This suggests that inverse agonism of MC-Rs is important in development/rescue from ABA. In addition, other peptides derived from the (up-regulated) POMC gene might be involved in ABA. β-Endorphin is processed from POMC and seems to stimulate food intake as well as rewarding processes and nociception [94–96]. Male β-endorphin deficient mice are however obese and hyperphagic, indicating an anorexic potency of β-endorphin [97]. These mice showed reduced operant responding towards food when ad libitum fed, but not when food-restricted, suggesting that β-

The MC system is clearly involved in energy homeostasis. Gene expression of POMC and AgRP is regulated by energy balance, POMC and AgRP neurons express leptin receptors and administration of natural or synthetic MC-R ligands influence food intake and body fat mass, implying that the MC system is a target for treatment of disturbed eating behavior in humans. Further dissection of the MC pathway by studying the phenotype of MC-R deficient mice, taught us that the MC4-R is probably mainly involved in feeding behavior, whereas the MC3-R is probably mostly involved in regulating adiposity. It was recently argued that the MC system (MTII) is specifically involved in defending body adiposity rather than regulating food intake. This theory needs to be further investigated in future studies. Specific ligands acting on either MC3-R or MC4-R are therefore necessary. Nevertheless feeding behavior is not solely regulated by homeostatic processes. Future studies should also focus on mechanisms of non-homeostatic feeding by e.g. investigating crosstalk between MCs, opioids and mesolimbic dopamine in several regions of the brain to investigate the involvement of MCs in liking and wanting of food in relation to human expression of disturbed eating behavior. References [1] Cone RD. The central melanocortin system and energy homeostasis. Trends Endocrinol Metab 1999;10:211–6. [2] Nijsen MJ, de Ruiter GJ, Kasbergen CM, Hoogerhout P, de Wildt DJ. Relevance of the C-terminal Arg-Phe sequence in gamma(2)-melanocytestimulating hormone (gamma(2)-MSH) for inducing cardiovascular effects in conscious rats. Br J Pharmacol 2000;131:1468–74. [3] Tatro JB, Sinha PS. The central melanocortin system and fever. Ann N Y Acad Sci 2003;994:246–57. [4] Von Frijtag JC, Croiset G, Gispen WH, Adan RA, Wiegant VM. The role of central melanocortin receptors in the activation of the hypothalamus– pituitary–adrenal-axis and the induction of excessive grooming. Br J Pharmacol 1998;123:1503–8. [5] Thody AJ. Alpha-MSH and the regulation of melanocyte function. Ann N Y Acad Sci 1999;885:217–29. [6] Jacobowitz DM, O'Donohue TL. Alpha-melanocyte stimulating hormone: immunohistochemical identification and mapping in neurons of rat brain. Proc Natl Acad Sci U S A 1978;75:6300–4. [7] Gee CE, Chen CL, Roberts JL, Thompson R, Watson SJ. Identification of proopiomelanocortin neurones in rat hypothalamus by in situ cDNAmRNA hybridization. Nature 1983;306:374–6. [8] Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994;372:425–32.

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