**4. Melanocortin System and Stress Responses**

Dieting, stress and negative affect are considered potential factors able to trigger binge eating episodes in patients with BED or Bulimia Nervosa [37,166,167]. Indeed, dieting periods are commonly observed in the history of binge eaters, but hunger alone appears to be non-sufficient to induce a compulsive-like eating, if not accompanied by conditions of stress or negative affect [168,169]. Stress has a central role in the etiology of binge eating, considering that obese individuals with BED, compared to those without, show a higher activity of the HPA axis and cortisol/corticosterone plasma level [170–173]. Additionally, higher cortisol levels, induced by stress, are able to promote a greater consumption of sweet foods [174], and are also positively correlated with the severity of binge eating [175].

The melanocortin system, principally via MC4R, has been demonstrated to play a central role in stress response and negative emotional states, including anxiety and depression [176,177], suggesting the MC4R as a possible target to treat these psychiatric conditions. In fact, MC4Rs are expressed in the limbic system, mainly in several nuclei of the amygdala, such as the central and basolateral nuclei, lateral septal nucleus, hippocampus and in the entorhinal cortex [50]; thus, the distribution of the MC4R in the brain indicates an important involvement of this receptor in promoting negative emotional states [176,177]. Moreover, the MC4R, contrary to MC3R, has been highly detected in the PVN of the hypothalamus, where it is supposed to regulate the activity of the HPA axis, via arginine vasopressin (AVP) and corticotropin releasing factor (CRF) neurons [50,176]. Initial evidence linking the MC4R and stress-related responses comes from studies in which the administration of α-MSH and ACTH in rats was able to increase grooming behavior [99–101,178,179], characterized by many activities directed to the animal body surface, such as face washing, body grooming, licking, scratching and genital grooming, and proposed as a rodent behavioral response to stress and novel environments [178,180]. The effect of α-MSH on grooming is principally due to its agonistic activity on MC4Rs, as demonstrated by Adan et al., who found that grooming behavior, induced by MCR agonists, was positively correlated with a greater affinity and potency for MC4R, rather than for MC3R. On the contrary, the antagonist SHU-9119 attenuated grooming induced by both melanocortins and by exposure to a novel environment [178]. This finding is further confirmed by the fact that the MC4R agonist MTII increased grooming in WT, but not in mutant rats deficient in MC4Rs, confirming that this behavior is principally mediated by MC4Rs, and not by MC3R subtypes [181].

Stress has been demonstrated to have profound effects on MC4R expression and activity in the brain. In fact, the exposure to electric foot shock stress in rats increased the expression of POMC and MC4R mRNA in the hypothalamus and in the amygdala [182], region implicated in the modulation of emotional- and fear-related behaviors [183] and binge eating episodes [184,185].

Furthermore, rats exposed to chronic restraint stress had increased MC4R mRNA expression in the ARC of the hypothalamus, compared to control rats, not exposed to stress [186]. The effect of stress on MC4R and on feeding behavior and appetite may be also dependent on the intensity and duration of the stressor, as supported by the study of Chagra et al., in which chronic exposure to a stress induced a significant decrease in c-fos- and MC4R-expressing cells in the ARC, indicating a shift toward more orexigenic behaviors, differently from control and acutely stressed rats [187].

Pharmacological stimulation of the MC4R is able to promote the activity of the HPA axis, as reported by the study of Von Frijtag et al., in which i.c.v. injection of ACTH1-24 (the N-terminal bioactive fragment of ACTH) in rats, significantly increased plasma concentrations of ACTH and corticosterone, an effect inhibited by pretreatment with the non-selective antagonist SHU-9119 and by the selective MC4R antagonist [D-Arg8] ACTH4-10 [188]. The influence of the melanocortin system on HPA axis tone and activity can be explained considering that MC4Rs are highly expressed in the parvocellular division of the PVN [50,189], the region in which CRF neurons are also predominantly localized and where they receive α-MSH neuronal terminals [190,191].

In fact, activation of MC4Rs by i.c.v. injections of α-MSH or MTII increases gene expression of CRF in the PVN [189,192] and enhances corticosterone plasma levels in rats, suggesting a functional interaction between CRF and the melanocortin system [189]. In the same study, the pretreatment with the CRF antagonist α-helical-CRH9–41 was able to prevent MTII-induced suppression of food intake, evidencing that the melanocortin system can alter endogenous CRF levels in order to modulate appetite [189].

A stress procedure that has been demonstrated to promote activation of the melanocortin neurons, and, consequently, of the HPA axis, is the acute restraint stress. Rats exposed to this stress had a robust c-fos mRNA expression in the medial amygdala (MeA) [18,193], a brain region with high levels of MC4Rs [50], and particularly sensitive to psychological stressors, characterized by an emotional component, such as restraint [194,195]. Lesions of the MeA result in a blunted response of the HPA axis to psychogenic stressors [196], conversely to pharmacological stimulation of the MC4R-expressing

neurons in the MeA, which promotes corticosterone release [18]. Moreover, both stress-induced anorexia and corticosterone release, in response to the acute restraint stress, can be prevented by administration of a MC4R antagonist directly in the MeA [18,193]. The interaction between MC4Rs in the MeA and the CRF system is probably mediated by the efferents from the MeA to the Bed Nucleus of the Stria Terminalis (BNST), brain region enriched in CRF neurons [197], and involved in stress-induced emotional responses and activation of the HPA axis [198,199]. The BNST has been demonstrated to play a pivotal role in stress-induced binge eating for HPF, evoked by a combination of frustration stress and food restriction [200,201], and injection of a non-selective CRF receptor antagonist directly into the BNST was able to counteract this compulsive-like eating episode for HPF selectively in rats exposed to both stress and restriction [200]. These findings support the hypothesis that MC4R can also influence the activation of the HPA axis via extrahypothalamic sites, and thus could represent an important factor for the development of aberrant feeding behaviors in response to stress exposure. Consistently with this evidence, acute stress-induced release of ACTH and corticosterone, as well as neuronal activation in the PVN and MeA, were significantly attenuated in male rats with a *MC4R* mutation, producing a less functional receptor, compared to the WT littermates [202,203]. Intriguingly, it was observed that female rats with the same mutation revealed an unexpected and exaggerated acute stress-induced corticosterone release, contrary to mutant males, highlighting a difference in stress reactivity between male and female rats with the MC4R loss of function [202]. The result of this study suggests a sex-dependent responsivity in the basal HPA axis tone and acute stress-induced corticosterone in rodents with MC4R mutation [202]. Considering the heightened stress reactivity found in female rats with deficient MC4R activity [202], and that stress has been associated with EE [37,38] and binge eating behavior [38,204–206], it would be interesting in the future to evaluate the potential involvement of MC4R signaling in a female rat model of binge eating, in which the binge eating episode is elicited by a combination of food restriction plus stress [86,87] and by using HPF, in order to promote the aberrant feeding behavior and to increase the motivation to overconsume food [38,87,207–209].
