Physiological responses to melanocortin ligands are mediated by five receptors (MC1R-MC5R) (Cortés et al., 2014). Melanocortin receptor pharmacology is complex, with two antagonists/inverse agonists (AgRP and agouti signaling peptide) and MSH ligands that exhibit varying degrees of receptor specificity (Figure 1B; Cone et al., 1996). Other ligands and cell-surface proteins have been identified that regulate melanocortin signaling (e.g., melanocortin receptor accessory proteins 1 and 2, mahogany, mahoganoid, attractin-like protein, syndecans, ion channels and defensins) (Kaelin et al., 2008; Nix et al., 2013, 2015; Anderson et al., 2016).

Melanocortin regulation of energy balance is mediated by two receptors expressed in the central nervous system. Mc3r and Mc4r mRNA are expressed in overlapping and distinct brain regions linked to appetite and metabolic control (Roselli-Rehfuss et al., 1993; Mountjoy et al., 1994; Kishi et al., 2003; Liu et al., 2003; Lippert et al., 2014; Mavrikaki et al., 2016). Mc3r expression is concentrated in hypothalamic and limbic structures, with dense expression in the ARC, ventromedial hypothalamus (VMH), ventral tegmental area (VTA), and medial habenula (MHb) (Roselli-Rehfuss et al., 1993; Cone, 2005; Lippert et al., 2014; Mavrikaki et al., 2016). Initial observations of expression of both receptors in areas of the rodent brain linked to appetite control (Roselli-Rehfuss et al., 1993; Mountjoy et al., 1994), and stimulation of feeding by melanocortin antagonists administered centrally (Fan et al., 1997), were crucial early steps in revealing the physiological significance of the central nervous melanocortin system. Chronic intracerebroventricular infusion of AgRP, an MC3R/MC4R antagonist/inverse agonist (Ollmann et al., 1997; Shutter et al., 1997), causes a hyperphagic obesity syndrome (Small et al., 2001). The central nervous melanocortin system is thus viewed as a promising target for developing obesity therapies. The first trials of melanocortin agonists for treating obesity failed due to cardiovascular responses (Greenfield, 2011). However, a recent trial investigating RM-493, a small peptide MC3R/MC4R agonist shown to have MC4R-dependent effects on food intake and body weight (Kumar et al., 2009), produced promising outcomes. In humans, RM-493 increased resting energy expenditure and reduced the respiratory quotient (RQ), suggesting enhanced fat oxidation (Chen et al., 2015). In obese non-human primates, administration of RM-493 resulted in weight loss with a transient suppression of food intake, increased total energy expenditure and improvements in insulin resistance and cardiovascular function (Kievit et al., 2013). Importantly, adverse cardiovascular responses that led to the discontinuation of earlier compounds were not evident.

In the absence of selective melanocortin receptor ligands, targeted deletion of the melanocortin receptors provided important information concerning the functional specificity of neural melanocortin receptors. MC3Rs are not required for suppression of food intake in response to MSH analogs (Marsh et al., 1999; Chen et al., 2000a,b; Kumar et al., 2009), and for appetite control during exposure to palatable high-fat/high sucrose diets (Butler et al., 2000, 2001; Albarado et al., 2004; Sutton et al., 2006; Srisai et al., 2011). Unlike MC4Rs, MC3Rs are not required for appetitive and metabolic responses to serotoninergic compounds (Heisler et al., 2002, 2006; Zhou et al., 2007). Deletion of the gene encoding either MC3R or MC4R causes obesity in mice, with both affecting partitioning of nutrients between adipose and non-adipose tissues (Huszar et al., 1997; Butler et al., 2000; Chen et al., 2000a). The two receptors were originally considered to function independently, as Mc3r;Mc4r double knockouts exhibit an additive obese phenotype (Chen et al., 2000a). As discussed later in this review, our data suggest MC3Rs may regulate MC4R activity by altering the response of “1st order” neurons releasing the endogenous ligands to signals of metabolic state.

Genetic screens of obese populations confirmed the importance of normal melanocortin receptor function in the defense of body weight from early childhood. Missense mutations in the POMC and MC4R genes are associated with severe hyperphagic obesity syndromes that manifests within the first 1–2 years of life (Farooqi and O'Rahilly, 2008). The central nervous melanocortin system responds to environmental cues through epigenetic modifications that have long-lasting effects on expression of genes promoting lean phenotypes (Benite-Ribeiro et al., 2016; Kühnen et al., 2016). Methylation in a variably methylated region (VMR) of the POMC gene allele is associated with altered body mass in humans (Kühnen et al., 2016). Methylation of this region is sensitive to metabolic conditions in utero, and to paternal methylation patterns. Altered POMC expression as a consequence of developmental conditions could therefore contribute to obesity later in life. While the evidence for direct causality is less clear, MC3R haploinsufficiency is linked to increased risk of childhood obesity (Feng et al., 2005; Tao, 2010; Lee, 2012; Lee et al., 2016).

Overall, the lack of conclusive evidence supporting a role for MC3Rs in appetite control in ad libitum fed situations, combined with comparatively modest changes in body mass (Butler et al., 2000, 2001; Chen et al., 2000a), explains why many laboratories overlooked neural MC3Rs. Evaluating behavioral and/or metabolic responses of mice to environmental challenges can be informative when investigating the functions of genes involved in behavior and metabolism. For example, cold stress is often used to assess mobilization of energy reserves and futile cycles to maintain body temperature (Kozak and Anunciado-Koza, 2008). Another example is transitioning between chows and obesogenic diets to assess behavioral and metabolic control (Collins et al., 2004). Mc3r-deficient mice tolerate cold and control appetite when challenged with palatable diets (Butler et al., 2000, 2001; Chen et al., 2000a; Sutton et al., 2006; Ellacott et al., 2007). However, a behavioral phenotype is observed in Mc3r−/− mice subjected to hypocaloric restricted feeding protocols to assess motivational responses anticipating food presentation (Sutton et al., 2008; Begriche et al., 2011a,b, 2012; Girardet et al., 2014a, 2017). These outcomes suggest that MC3Rs play a role in mediating appetite responses to situations of nutrient scarcity.

Mice provided unrestricted access to a running wheel exhibit food anticipatory activity (FAA) when subjected to a hypocaloric diet (70–75% of habitual intake) presented at 24 h intervals (Mistlberger, 2011). FAA involves a progressive rise in activity preceding food access, and has been suggested to involve a circadian oscillator (“food-entrainable oscillator,” or FEO) that is independent of the light-entrained master clock. FAA is attenuated in Mc3r−/− mice housed in a 12 h light:dark setting (Sutton et al., 2008); the same study reported that Mc3r−/− mice failed to increase wakefulness in anticipation of food presentation. Entrainment to food presentation is also attenuated, but not completely inhibited, when FAA is assessed in constant dark (Begriche et al., 2011b). Based on the weakened anticipatory responses observed during restricted feeding, MC3Rs may act as a modulator of the inputs (or outputs) of FEOs (Mistlberger, 2011). Entrainment to food availability is thought to involve coordinated responses of FEO distributed throught the body (Mohawk et al., 2012). However, it is no clear how MC3Rs exert regulatory control over rhythms in FEO activity.

A recent paper from Roger Cone's laboratory suggested another interpretation of the FAA phenotype associated with loss of MC3R. Renquist et al. reported that the fasting responses of NAG neurons are not observed in Mc3r−/− mice (Renquist et al., 2012). We subsequently reported increased hypothalamic AgRP and Npy expression in the hypocaloric conditions used to induce FAA is also not observed in Mc3r-deficient mice (Girardet et al., 2014a, 2017). Collectively, these results suggest activation of NAG neurons by signals of negative energy balance contributes to the expression of FAA. Adult mice lacking NAG neurons adapt poorly to a hypocaloric feeding protocol used to induce FAA (Tan et al., 2014). FAA involves increased food seeking and motivational responses to seek food (Aponte et al., 2011; Krashes et al., 2011). Similar responses occur upon activation of NAG neurons (Aponte et al., 2011; Krashes et al., 2011), although another interpretation is that activation of NAG neurons delivers a “negative valence” signal (Betley et al., 2015) causing avoidance of situations associated with a painful experience (hunger).

Mc3r-deficient mice also exhibit attenuated appetitive responses to hypocaloric conditioning. Wild-type mice subjected to hypocaloric feeding protocols exhibit binge-feeding behavior, reducing meal frequency and increasing meal size to consume most of the food within 1 h of presentation (Bruss et al., 2010; Begriche et al., 2012; Girardet et al., 2017). This behavioral adaptation is attenuated in Mc3r−/− mice: food intake in the 1 h following presentation is markedly reduced with no compensation later in the feeding cycle and changes in meal structure (fewer, larger meals) are also attenuated (Begriche et al., 2012; Girardet et al., 2017). Motivation to self-administer food-rewards during hypocaloric conditions is also attenuated in Mc3r-deficient mice (Mavrikaki et al., 2016). However, self-administration is normal in Mc3r-deficient mice in ad libitum feeding conditions and increased motivation to self-administer more palatable sucrose diets is retained (Mavrikaki et al., 2016). The behavioral phenotype associated with MC3R-deficiency is therefore contextual and dependent on energy balance. Mc3r-deficient mice may not experience the “pain” of hunger, and are not be motivated to avoid unpleasant experiences associated with nutrient insufficiency.

These observations also suggest a new and perhaps simpler interpretation of the phenomenon observed in Mc3r−/− mice during restricted feeding. In the absence of MC3Rs, NAG neurons are desensitized to internal cues of metabolic state provided by hormones and metabolites, the release of which follows patterns that are sensitive to food consumption (Tschop et al., 2006). This model also explains why the release of other neuropeptides and neurotransmitters from NAG neurons does not compensate for the absence of MC3Rs. The rapid stimulation of feeding behavior following activation of NAG neurons requires the release of GABA or NPY from NAG neurons, while release of AgRP elicits a delayed yet prolonged increase in feeding behavior that is dependent on MC4Rs (Krashes et al., 2013).

We developed the LoxTBMc3r mouse, allowing us to reactivate of Mc3r transcription using Cre transgenics, inserting a “lox-stop-lox” sequence in the 5′UTR (Begriche et al., 2011a). The response of NAG neurons to hypocaloric conditioning is restored in LoxTBMc3r mice in which hypothalamic expression was rescued using Nkx2.1-Cre (Girardet et al., 2017). This study also observed that restoring FAA in LoxTBMc3r mice is independent of improvements in adiposity. These results suggest that actions involving NKX2.1(+ve);MC3R(+ve) neurons in the hypothalamus are sufficient to restore “normal” activity of NAG neurons. This could indicate a developmental role in which NAG neurons fail to develop normal responses to altered signals of metabolic state in the absence of MC3Rs. Alternatively, MC3Rs in the mature hypothalamus may exert an active “gating” function; determining whether rescuing MC3Rs in the adult mouse restores responses of NAG neurons to metabolic cues could address this question.

MC3Rs expressed in the limbic system may regulate feeding-related motivational responses. MC3Rs are expressed in dopamine transporter (DAT) (+ve) and (−ve) neurons in the VTA, with female Mc3r-deficient mice exhibiting lower dopamine and altered sucrose consumption and taste preferences (Lippert et al., 2014). Operant conditioning experiments suggest increased food-related motivational responses associated with hypocaloric diets are attenuated in Mc3r-deficient mice. Rescuing Mc3r transcription in DAT(+ve) neurons in the VTA improved motivational responses (Mavrikaki et al., 2016) without restoring binge-feeding observed following the prolonged inter-meal interval. Compulsive behavioral responses to consume large meals in situations of negative energy balance may thus require MC3R activation in additional brain areas, and not only in the limbic system. A caveat to interpreting these studies is that they only used male mice; sex differences in the functions of MC3Rs in regulating feeding-related reward pathways exist (Lippert et al., 2014). Further studies using LoxTBMC3R mice to investigate the role of MC3Rs expressed in the VTA of females in regulate sucrose consumption and taste preferences are clearly needed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.