3.1.1. MC3R: Preclinical Studies on Eating Behavior

The MC3R, compared to the MC4R subtype, exhibits a more limited distribution in the brain, being predominantly found in the hypothalamic nuclei and limbic regions, with dense expression in the ARC, ventromedial hypothalamus, VTA and medial habenula, structures in which it is supposed to regulate energy homeostasis and food seeking behavior [46,50,73–75]. MC3R and MC4R KO mice have been used to investigate the role of each receptor in regulating energy homeostasis, and many studies revealed that MC3Rs and MC4Rs might function independently, playing a complementary but non-redundant role in the regulation of energy balance [65,66,68,76]. Targeted deletion of the MC3R gene in mice promotes a modest obesity syndrome and increased accumulation of fat mass that is not related to hyperphagia, with a normal anorectic response to melanocortin agonists [65,66], suggesting that this receptor could be mostly involved in the regulation of energy homeostasis and metabolic processes, rather than in the control of feeding behavior. However, a study by Zhang et al. showed that MC3Rs and MC4Rs are of approximately equal importance in preventing weight gain during a high-fat chow diet, and that the absence of MC3Rs compromises leptin's ability to decrease food consumption [76], evidencing an altered anorectic response in MC3R null mice. Moreover, male MC3R KO mice, backcrossed onto the C57BL/6J background, showed a mild hyperphagia after exposure to a purified high-fat diet [6]. Sutton et al. demonstrated that obesity associated with MC3R deficiency is dependent on the dietary fat, considering that, if exposed to a low-fat diet, MC3R KO mice exhibited a modest increase in adiposity and a normal body weight, while during a high-fat diet, fat mass was comparable to that of MC4R KO littermates [68]. Additionally, MC3R KO mice were not hyperphagic under a low-fat diet, but showed a modest increase in food consumption under the high-fat diet, an effect that was gender specific, being mainly observed in male mice [68]. A recent experiment in mice with "humanized" MC3Rs further evidenced the role of the MC3R in appetite control: in this mouse model, the murine MC3R was replaced with the Wild Type (WT) human MC3R (MC3RhWT/hWT) or the

double-mutant C17A (Thr6Lys) + G241A (Val81Ile) human MC3R (MC3RhDM/hDM) [77], characterized by a reduced receptor binding, signaling transduction and less protein expression, and associated with a greater risk of childhood obesity in human homozygous carriers [78–80]. Mutant homozygous mice with the double mutation (MC3RhDM/hDM) had an increased adiposity and energy intake, compared to WT human MC3R (MC3RhWT/hWT) littermates and were also hyperphagic [77], highlighting the contribution of MC3R signaling to energy homeostasis, metabolism and feeding behavior.

The behavioral phenotype linked to MC3R-deficiency may be also contextual and dependent on energy balance. In fact, MC3R-deficient mice appear to be less sensitive to the "pain" of hunger, and are not motivated to avoid unpleasant experiences associated with nutrient scarcity [81], as described by investigations using hypocaloric restricting feeding protocols. In this context, MC3Rs seem to be essential for entrainment of anticipatory behavior toward feeding time [81,82]. Food anticipatory behavior, consisting of a progressive rise of activity preceding food presentation, assessed using running wheels and measuring home cage activity, is attenuated in MC3R KO mice, compared to WT, under a restricting feeding protocol [73,82,83]. Moreover, the same mice did not exhibit the increased wakefulness generally coincident with food presentation and normally observed in non-mutant rodents [82]. Hypocaloric feeding protocols are known to promote binge-like eating behavior in WT mice, which reduce meal frequency but increase meal size and duration, with most of the food consumed within the first hour of presentation [21,83,84]. This behavioral phenotype is markedly attenuated in MC3R KO mice, without compensation in the feeding cycle later or changes in the meal structure, a finding that supports the essential role of an intact MC3R signaling in the compulsive eating response, observed after exposure to situations of poor nutrient availability and prolonged negative energy balance [83,84]. Additionally, the motivation to self-administer a food reward is markedly attenuated in MC3R-deficient mice, exposed to a caloric restriction protocol, while being normal if mice are fed in ad libitum conditions, reducing self-administration of chocolate flavored pellets [21]. The abnormal behavioral features associated with the deletion of MC3Rs could be partially explained by the neuroendocrine alterations found in the brain of MC3R-deficient mice, which failed to present the increase in the potent orexigenic neuropeptides AgRP and NPY during fasting and hypocaloric conditions [5,84,85]. Intriguingly, MC3R-deficient mice also exhibit altered responses of the hypothalamic-pituitary-adrenal (HPA) axis during caloric restriction, showing a lack of corticosterone serum increase in response to fasting, which instead is found in WT mice [84,85]. Furthermore, the dysregulation of fasting-induced corticosterone release was accompanied by a defect in the upregulation of hypothalamic corticotropin-releasing hormone (CRH) mRNA in the MC3R KO mice [85], indicating that both hypothalamic and adrenal functions are compromised by the absence of this receptor. Considering both the dysfunctional eating behaviors and the altered activity of the HPA axis observed in MC3R KO mice during fasting, future studies should be conducted to investigate if deletion or antagonism of the MC3R could reduce the compulsive-like eating in a preclinical model of binge eating, where a binge eating episode is elicited by the combination of food restriction and stress, trying to further characterize the role of this receptor in both homeostatic and non-homeostatic eating [86,87].

The information obtained from studies with MC3R KO mice are in accordance with the putative role of the MC3R as an inhibitory autoreceptor on POMC neurons [49,88,89], where α- and γ-MSH, released by POMC nerve terminals within the ARC, are supposed to regulate the activity of POMC neurons through activation of MC3R subtypes, and studies with selective MC3R agonists confirmed this observation. Indeed, Marks et al. found that stimulation of the MC3R, by peripheral administration of the selective MC3R agonist [D-trp8]-γ-MSH, results in the inhibition of POMC neuronal activity, which in turn leads to an increase in food intake in WT mice, while having no effect on feeding in MC3R KO littermates [90]. The suppression of POMC neuronal activity, after injection of [D-trp8]-γ-MSH, was demonstrated to be a consequence of an increased inhibitory synaptic transmission, due to the activation of GABAergic NPY neurons in the ARC, releasing GABA on POMC neurons [88,89]. Considering the highly potent orexigenic activity of NPY [91], this can explain the observed increase in food intake after stimulation of the MC3R [90], as reported in Figure 1.

**Figure 1.** The potential MC3R mechanisms leading to increase food intake and motivation for highly palatable food (HPF) in preclinical studies. ↓: decrease; ↑: increase; ARC: Arcuate nucleus of the hypothalamus; HPF: Highly palatable food; MC3R: Melanocortin-3 receptor; NPY: neuropeptide Y; POMC: Pro-opiomelanocortin; VTA: Ventral Tegmental Area.

However, the study by Marks et al. had the limitation of investigating the effect of the MC3R agonist only by a peripheral administration, not clearly explaining if the results obtained were due to a peripheral or central action. Subsequently, Lee et al., using a rat model, obtained a similar finding, analyzing the effect of the same compound directly injected in the CNS, through intracerebroventricular (i.c.v.) injections, resulting in an increased food intake in treated rats, confirming a central mechanism of action [92]. Interestingly, it was examined if antagonism at MC3R would have the opposite effect, inhibiting feeding, but a strange result was obtained, observing that the MC3R antagonist PG-932, at a low dose, suppressed food intake, while at a higher dose significantly increased food consumption and body weight. These effects could be explained with the possible antagonism profile of PG-932 even at MC4R, when injected at high doses in rats [92].

Taken together, these studies confirm that the MC3Rs, despite their functions are still not completely understood, could represent important targets for the treatment of obesity and could also play a role in the aberrant feeding patterns that characterize eating disorders.
