European Journal of Pharmacology (v.660, #1)

Foreword by Roger A.H. Adan; Roger D. Cone (1).

Over the past century, prevalent models of energy and glucose homeostasis have been developed from a better understanding of the neural circuits underlying obesity and diabetes. From the early hypothalamic lesion reports to the more recent pharmacological and molecular/genetic studies, the hypothalamic melanocortin system has been shown to play a critical role in the regulation of metabolism. This review attempts to highlight contributions to our current understanding of how numerous neuromodulators (leptin, insulin, and serotonin) integrate with the central melanocortin system to coordinate alterations in energy and glucose balance.
Keywords: Leptin; Insulin; Serotonin; Obesity; Diabetes; Patch-clamp;

Physiological roles of the melanocortin MC3 receptor by Benjamin J. Renquist; Rachel N. Lippert; Julien A. Sebag; Kate L.J. Ellacott; Roger D. Cone (13-20).
The melanocortin MC3 receptor remains the most enigmatic of the melanocortin receptors with regard to its physiological functions. The receptor is expressed both in the CNS and in multiple tissues in the periphery. It appears to be an inhibitory autoreceptor on proopiomelanocortin neurons, yet global deletion of the receptor causes an obesity syndrome. Knockout of the receptor increases adipose mass without a readily measurable increase in food intake or decrease in energy expenditure. And finally, no melanocortin MC3 receptor null humans have been identified and associations between variant alleles of the melanocortin MC3 receptor and diseases remain controversial, so the physiological role of the receptor in humans remains to be determined.
Keywords: Melanocortin-3 receptor; Melanocortin MC3 receptor; Melanocortin; Obesity; γ-MSH; Proopiomelanocortin;

The hypothalamic arcuate nucleus contains two anatomically and functionally distinct populations of neurons—the agouti-related peptide (AgRP)- and pro-opiomelanocortin (POMC)-expressing neurons that integrate various nutritional, hormonal, and neuronal signals to regulate food intake and energy expenditure, and thereby help achieve energy homeostasis. AgRP neurons, also co-release neuropeptide Y (NPY) and γ-aminobutyric acid (GABA) to promote feeding and inhibit metabolism through at least three possible mechanisms: (1) suppression of the melanocortin signaling system through competitive binding of AgRP with the melanocortin 4 receptors; (2) NPY-mediated inhibition of post-synaptic neurons that reside in hypothalamic nuclei; (3) GABAergic inhibition of POMC neurons in their post-synaptic targets including the parabrachial nucleus (PBN), a brainstem structure that relays gustatory and visceral sensory information. Acute ablation of AgRP neurons in adult mice by the action of diphtheria toxin (DT) results in precipitous reduction of food intake, and eventually leads to starvation within 6 days of DT treatment. Chronic delivery of bretazenil, a GABAA receptor partial agonist, into the PBN is sufficient to restore feeding and body weight when AgRP neurons are ablated, whereas chronic blockade of melanocortin 4 receptor signaling is inadequate. This review summarizes the physiological roles of a neural circuitry regulated by AgRP neurons in control of feeding behavior with particular emphasis of the GABA output to the parabrachial nucleus. We also describe a compensatory mechanism that is gradually engaged after ablation of AgRP neurons that allows mice to continue eating without them.
Keywords: Agouti-related protein; Anorexia; Benzodiazepine; Feeding behavior; Fos; Synaptic plasticity;

Neurobiology of overeating and obesity: The role of melanocortins and beyond by Rahul Pandit; Johannes W. de Jong; Louk J.M.J. Vanderschuren; Roger A.H. Adan (28-42).
The alarming increase in the incidence of obesity and obesity-associated disorders makes the etiology of obesity a widely studied topic today. As opposed to ‘homeostatic feeding’, where food intake is restricted to satisfy one's biological needs, the term ‘non-homeostatic’ feeding refers to eating for pleasure or the trend to over-consume (palatable) food. Overconsumption is considered a crucial factor in the development of obesity. Exaggerated consumption of (palatable) food, coupled to a loss of control over food intake despite awareness of its negative consequences, suggests that overeating may be a form of addiction. At a molecular level, insulin and leptin resistance are hallmarks of obesity. In this review, we specifically address the question how leptin resistance contributes to enhanced craving for (palatable) food. Since dopamine is a key player in the motivation for food, the interconnection between dopamine, leptin and neuropeptides related to feeding will be discussed. Understanding the mechanisms by which these neuropeptidergic systems hijack the homeostatic feeding mechanisms, thus leading to overeating and obesity is the primary aim of this review. The melanocortin system, one of the crucial neuropeptidergic systems modulating feeding behavior will be extensively discussed. The inter-relationship between neuronal populations in the arcuate nucleus and other areas regulating energy homeostasis (lateral hypothalamus, paraventricular nucleus, ventromedial hypothalamus etc.) and reward circuitry (the ventral tegmental area and nucleus accumbens) will be evaluated and scrutinized.
Keywords: Obesity; Leptin resistance; Addiction; Craving; Melanocortin;

Cardiovascular effects of melanocortins by Michael H. Humphreys; Xi-Ping Ni; David Pearce (43-52).
Melanocortins (MSH's) are three structurally related peptides derived from proopiomelanocortin. They regulate several physiologic functions including energy metabolism, appetite, and inflammation. Recent work in rodents has also identified important effects of MSH's, particularly γ-MSH, on sodium metabolism and blood pressure regulation. Normal rats and mice respond to a high sodium diet with an increase in the plasma concentration of γ-MSH, and remain normotensive, while those with genetic or pharmacologic γ-MSH deficiency become hypertensive on a high sodium diet. This hypertension is corrected by exogenous administration of the peptide. Mice lacking the γ-MSH receptor (the melanocortin 3 receptor, Mc3r) also become hypertensive on a high sodium diet but remain so when administered γ-MSH, and infusions of physiologic levels of the peptide stimulate urinary sodium excretion in normal rats and mice, but not in mice with deletion of Mc3r. The salt-sensitive hypertension in rodents with impaired γ-MSH signaling appears due to stimulation of noradrenergic activity, since plasma noradrenaline is increased and the hypertension is rapidly corrected with infusion of the α-adrenoceptor antagonist phentolamine. In contrast to the antihypertensive property of physiologic levels of γ-MSH, intravenous or intracerebroventricular injections of high levels of the peptide raise blood pressure. This occurs in mice lacking Mc3r, indicating an interaction with some other central receptor. Finally, the salt-sensitive hypertension in rodents with disruption of γ-MSH signaling is accompanied by insulin resistance, an observation which offers a new window into the study of the association of salt-sensitive hypertension with insulin resistance and type II diabetes.
Keywords: Blood pressure; Salt-sensitive hypertension; γ-melanocyte stimulating hormone; Noradrenergic activity; Insulin resistance;

Fish melanocortin system by José Miguel Cerdá-Reverter; Maria Josep Agulleiro; Raúl Guillot R; Elisa Sánchez; Rosa Ceinos; Josep Rotllant (53-60).
Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC1–MC5) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC5 receptor, while pufferfish have 4 receptors with no melanocortin MC3 receptor and one copy of melanocortin MC5 receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC2 receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC1 receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC1 receptor are responsible for reduced melanization. Both melanocortin MC4 and MC5 receptor are expressed in the hypothalamus, and central melanocortin MC4 receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC2 and MC5 receptors. Both sea bass melanocortin MC1 and MC4 receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively.
Keywords: Melanocortin-stimulating hormone (MSH); Proopiomelanocortin (POMC); Agouti-signalling protein (ASIP); Agouti-related protein (AGRP); Melanocortin receptor; Melanocortin receptor accessory protein (MRAP); Food intake; Pigmentation; Stress;

There are conflicting theories about the evolution of melanocortin MC receptors while only few studies have addressed the evolution of agouti-related peptide (AgRP) and agouti signalling peptide (ASIP), which are antagonists at the melanocortin receptors (MCRs), or the melanocortin MC2 receptor accessory proteins (MRAP1 and MRAP2). Previously we have cloned melanocortin MC receptors (MCa and MCb) genes in river lamprey and here we identify orthologues to these melanocortin MC receptor sequences in the sea lamprey. We investigate the putative presence of the melanocortin MC receptor genes in lancelet (amphioxus; Branchiostoma floridae) but we find it unlikely that such gene exists, due to a sharp drop in sequence similarity beyond sequence clusters of known receptors. We show the presence of AgRP and ASIP in elephant shark, a cartilaginous fish belonging to the subclass of Elasmobranchii. However, we do not find any of these genes in lamprey or lancelet after detailed analysis of both targeted and whole proteome regular expression scans. We found MRAP2, but not MRAP1, to be present in elephant shark and sea lamprey while Fugu (T. rubripes) has both genes. This study shows that the most ancient presence of these melanocortin-related sequences is found in elephant shark and lampreys considering the current available sequence data.
Keywords: ASIP; AgRP; Melanocortin receptor; MRAP; (Elephant shark); (Sea lamprey);

Role of central serotonin and melanocortin systems in the control of energy balance by Oliver J. Marston; Alastair S. Garfield; Lora K. Heisler (70-79).
Body weight homeostasis is critically dependent upon the convergence and integration of multiple central and peripheral signalling systems that collectively function to detect and elicit physiological and behavioural responses to nutritional state. To date, only a minority of these signals have been pharmacologically targeted for the treatment of human obesity. One signal that has been effectively manipulated to reduce body weight is the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT); however, the relevant downstream signalling pathways are incompletely understood. Recently, the melanocortin system, a nexus for multiple modulators of energy balance, has emerged as one key mediator of serotonin's effects on appetite. Here we review the serotonin and melanocortin systems with reference to their roles in energy balance and discuss the evidence that the two systems are functionally linked.
Keywords: Serotonin; Melanocortin; Energy homeostasis; Appetite; Obesity;

Implication of the melanocortin-3 receptor in the regulation of food intake by Boman G. Irani; Zhimin Xiang; Hossein N. Yarandi; Jerry R. Holder; Marcus C. Moore; Rayna M. Bauzo; Bettina Proneth; Amanda M. Shaw; William J. Millard; James B. Chambers; Stephen C. Benoit; Deborah J. Clegg; Carrie Haskell-Luevano (80-87).
The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC3 in the regulation of food intake, JRH322-18 a mixed MC3 partial agonist/antagonist and MC4 agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC4) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC4KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC3 and MC4 knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC3 and MC4 knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC3 and MC4 knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC3 plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.
Keywords: Melanotropin; MC3R; MC4R; Knockout mice; Obesity; Food intake; Feeding; Energy homeostasis; AGRP; SHU9119;

Design of novel melanocortin receptor ligands: Multiple receptors, complex pharmacology, the challenge by Victor J. Hruby; Minying Cai; James Cain; Joel Nyberg; Dev Trivedi (88-93).
The major pharmacophore for the melanocortin 1, 3, 4 and 5 receptors is the sequence -His-Phe-Arg-Trp-. There is a need for potent, biologically stable, receptor selective ligands, both agonists and antagonists, for these receptors. In this report we briefly examine the structural and biophysical approaches we have taken to develop selective agonist and antagonist ligands that can cross (or not) the blood brain barrier. Remaining questions and unmet needs are also discussed.
Keywords: Melanocortin receptor ligand SAR; Selective ligand for MCR; Melanocortin receptor selective ligand; Orthosteric and allosteric ligand; Agonists and antagonists for MCR;

Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein by Patricia M. Hinkle; Madhavika N. Serasinghe; Andrea Jakabowski; Julien A. Sebag; Krista R. Wilson; Carrie Haskell-Luevano (94-102).
The melanocortin 2 (MC2) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC2 receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC2 receptor responsible for these characteristics, we created chimeras between MC2 and MC4 receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC2 but not MC4 receptors. Replacing the first transmembrane segment of the MC2 receptor with the corresponding region from the MC4 receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC4 receptor with the corresponding regions from the MC2 receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC2 receptors with MC4 sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC2 receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC2 receptor as important for MRAP dependent-signal transduction and ligand specificity.
Keywords: Accessory protein; ACTH (adrenocorticotropic hormone); G protein-coupled receptor; Melanocortin receptor; MRAP (melanocortin-2 receptor accessory protein); MSH (melanocyte stimulating hormone);

Melanocortin MC1 receptor in human genetics and model systems by Kimberley A. Beaumont; Shu S. Wong; Stephen A. Ainger; Yan Yan Liu; Mira P. Patel; Glenn L. Millhauser; Jennifer J. Smith; Paul F. Alewood; J. Helen Leonard; Richard A. Sturm (103-110).
The melanocortin MC1 receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC1 receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC1 receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC1 receptor and the molecular mechanisms underlying the association between melanocortin MC1 receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC1 receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC1 receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC1 receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC1 receptor polymorphism.
Keywords: MC1R; Melanoma; Red hair colour; Melanocyte; Tanning; Pigmentation;

In the intervening three decades since Panksepp observed for the first time that centrally administered α-melanocyte stimulating hormone decreased food intake (), a wealth of data have accrued to firmly establish melanocortin signaling as a central regulator of food intake and fat mass. Advances in molecular biology have not only allowed detailed studies of spontaneously occurring obese mice with altered melanocortin signaling to be undertaken but also permitted the generation of a plethora of mouse models with precise perturbations at critical steps in the melanocortin system to finesse further the cellular and molecular architecture of relevant pathways. In this article we focus in upon a number of these mouse models which continue to help us tease apart the complexities of this critical system. Further, we review data on the important interaction between pro-opiomelanocortin derived peptides and the adrenal system and the relationship between agonist and antagonist peptides acting at central melanocortin receptors.
Keywords: Melanocortin; POMC; Obesity; Energy homeostasis; Corticosterone; Adrenal;

Effects of deficiency of the G protein Gsα on energy and glucose homeostasis by Min Chen; Nicholas M. Nemechek; Eralda Mema; Jie Wang; Lee S. Weinstein (119-124).
Gsα is a ubiquitously expressed G protein α-subunit that couples receptors to the generation of intracellular cyclic AMP. The Gsα gene GNAS is a complex gene that undergoes genomic imprinting, an epigenetic phenomenon that leads to differential expression from the two parental alleles. Gsα is imprinted in a tissue-specific manner, being expressed primarily from the maternal allele in a small number of tissues. Albright hereditary osteodystrophy is a monogenic obesity disorder caused by heterozygous Gsα mutations but only when the mutations are maternally inherited. Studies in mice indicate a similar parent-of-origin effect on energy and glucose metabolism, with maternal but not paternal mutations leading to obesity, reduced sympathetic nerve activity and energy expenditure, glucose intolerance and insulin resistance, with no primary effect on food intake. These effects result from Gsα imprinting leading to severe Gsα deficiency in one or more regions of the central nervous system, and are associated with a specific defect in melanocortins to stimulate sympathetic nerve activity and energy expenditure.
Keywords: G protein; Genomic imprinting; Obesity; Insulin resistance; Sympathetic nervous system; Melanocortin;

Melanocortin receptors belong to the seven-transmembrane (TM) domain proteins that are coupled to G-proteins and signaled through intracellular cyclic adenosine monophosphate. Many structural features conserved in other G-protein coupled receptors (GPCRs) are found in the melanocortin receptors. There are five melanocortin receptor subtypes and each of the melanocortin receptor subtypes has a different pattern of tissue expression and has its own profile regarding the relative potency of different melanocortin peptides. α-, β-, and γ-MSH and ACTH are known endogenous agonist ligands for the melanocortin receptors. Agouti and AgRP are the only known naturally occurring antagonists of the melanocortin receptors. We have examined the molecular basis of all five human melanocortin receptors for different ligand binding affinities and potencies using chimeric and mutated receptors. Our studies indicate that human melanocortin MC1 receptor, human melanocortin MC3 receptor, human melanocortin MC4 receptor and human melanocortin MC5 receptor utilize orthosteric sites for non selective agonists, α-MSH and NDP-α-MSH, high affinity binding and utilize allosteric sites for selective agonist or antagonist binding. Furthermore, our results indicate that molecular determinants of human melanocortin MC2 receptor for ACTH binding and signaling are different from that of other melanocortin receptors. Many studies also indicate that agonists can induce different conformation changes of melanocortin receptors, which then lead to the activation of different signaling pathways, even when the expression level of receptor and the strength of stimulus–response coupling are the same. This finding may provide new information for the design of drugs for targeting melanocortin receptors.
Keywords: Orthosteric binding; Allosteric binding; Melanocortin receptor; GPCR;

Meal pattern analysis in neural-specific proopiomelanocortin-deficient mice by Christian D. Richard; Virginie Tolle; Malcolm J. Low (131-138).
The central melanocortin system, consisting of melanocortin peptides, agouti gene related peptide and their receptors plays a critical role in the homeostatic control of energy balance. Loss of function mutations in the genes encoding proopiomelanocortin or melanocortin MC4 receptors cause profound obesity and hyperphagia. However, little is known about the functional relationship of melanocortin neurocircuits to the temporal organization of meal-taking behavior. We used an operant paradigm that combined lever pressing for food pellet deliveries with free water intake monitored by lickometers to quantify meal patterns in mutant mice that selectively lack proopiomelanocortin expression in hypothalamic neurons (nPOMCKO). Compared to wildtype siblings, nPOMCKO mice consumed 50% more food and water daily and exhibited a more stereotyped feeding pattern characterized by reduced inter-meal and inter-mouse variations. Average meals were larger in size but shorter in duration, with no change in meal number. Consequently, intermeal intervals were prolonged in nPOMCKO mice. Similar patterns were observed in pre-obese juvenile and frankly obese adult mice suggesting that neither age nor degree of obesity was responsible for the altered phenotypes. Spontaneous locomotion and wheel running were decreased in nPOMCKO mice, but circadian variations in locomotor and feeding activity were conserved. These data show that hyperphagia in male nPOMCKO mice is due to increased meal size but not meal number, and this pattern is established by age of 5 weeks. The combination of larger, more rapidly consumed meals and prolonged intermeal intervals suggests that proopiomelanocortin peptides are necessary for normal meal termination, but not the maintenance of satiety.
Keywords: Operant behavior; Obesity; Satiety; Hyperphagia; Circadian; Locomotor activity;

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay by Jacques Pantel; Savannah Y. Williams; Dehui Mi; Julien Sebag; Jackie D. Corbin; C. David Weaver; Roger D. Cone (139-147).
The melanocortin MC4 receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC4 receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC4 receptor, we created HEK293 cell lines coexpressing the human melanocortin MC4 receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z′ = 0.50). A pilot screen run on the Microsource Spectrum compound library (n  = 2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β2AR agonists – the β2AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.
Keywords: Melanocortin-4 receptor; MC4R; cAMP assay; Allosteric modulator; High throughput screen;

Both reduction of melanocortin signaling and increase in neuropeptide Y signaling in the brain result in obesity. However, where in the brain reduced melanocortin or increased neuropeptide Y signaling mediate these effects is poorly understood. In separate experiments we have injected recombinant adeno-associated viral vectors that overexpressed agouti-related peptide or neuropeptide Y in specific brain regions namely the paraventricular nucleus and the lateral hypothalamus. In this review we compare the results from these studies and discuss these data with previous data from intracerebroventricular or local brain injections. This review shows that the effects of agouti-related peptide clearly differ from those of neuropeptide Y. In addition, these data suggests complementary roles for these neuropeptides in energy balance.
Keywords: Agouti-related peptide; Neuropeptide Y; Recombinant adeno-associated viral vectors; Hypothalamus;

While rare MC4R mutations are the commonest cause of monogenic forms of extreme, early-onset obesity, growing evidence shows that common MC4R variants contribute to obesity in the general population. Candidate gene studies have focussed on the V103I and I251L MC4R variants that both affect MC4 receptor function in vitro. Individual association studies, which are typically small and underpowered, have found no association between V103I (frequency of 103I-allele: ~ 4%) or I251L (251 L-allele: ~ 2%) and the risk of obesity in the general population. However, large-scale meta-analyses have confirmed that both variants reduce the risk of obesity by − 21% in 103I-allele carriers (P  < 10−4) and by − 50% in 251 L-allele carriers (P  < 10−4). Recently, genome-wide association studies have identified a common variant (minor allele frequency: ~ 27%) at ~ 188 kb downstream of MC4R showing robust association (P  < 5 × 10−8) with BMI and obesity in adults and children. Each additional minor allele increases BMI by 0.20 kg/m2, body weight by 700–1000 g, and obesity risk by 14% in adults. Interestingly, this variant also showed association with increased height, consistent with the phenotype seen for rare MC4R mutations. Although MC4R is the nearest gene and phenotypic associations are consistent with those of MC4R mutations, it has not yet been established whether this variant indeed reflects MC4 receptor function. Taken together, common MC4R variants contribute to variation in BMI and obesity risk in the general population. Of particular interest is the finding from genome-wide association studies that suggests that the region downstream of MC4R contributes to its regulation.
Keywords: MC4R (Melanocortin 4 receptor); Obesity; Body mass index; Genetic epidemiology; Candidate gene study; Genome-wide linkage study; Genome-wide association study;

Successful methylphenidate treatment of early onset extreme obesity in a child with a melanocortin-4 receptor gene mutation and attention deficit/hyperactivity disorder by Özgür Albayrak; Beate Albrecht; Susann Scherag; Nikolaus Barth; Anke Hinney; Johannes Hebebrand (165-170).
We present the case report of a 2 year old boy with early onset extreme obesity (body mass index (BMI) 34.2 kg/m²; body mass index standard deviation score (BMI-SDS) 5.4) who is heterozygous for a non-conservative functionally relevant melanocortin MC 4 receptor mutation (Glu308Lys) and who also showed severe symptoms of attention deficit/hyperactivity disorder (ADHD). Treatment with the stimulant methylphenidate led to a sharp decrease of BMI to 21.8 kg/m² (BMI-SDS 2.8) within 24 months. We discuss potential mechanisms for this unusually large weight loss and suggest a potential link between the melanocortinergic and the dopaminergic systems, and the sympathetic nervous system. The potential benefit of methylphenidate in obese melanocortin MC 4 receptor mutation carriers with and without co-morbid ADHD warrants further studies.
Keywords: Weight reduction; Stimulant; Dopamine; Melanocortin; Brain derived neurotrophic factor; Sympathetic nervous system;

Effects of melanocortins on adrenal gland physiology by Li F. Chan; Louise A. Metherell; Adrian J.L. Clark (171-180).
The melanocortin-2-receptor (MC2 receptor), also known as the ACTH receptor, is a critical component of the hypothalamic–pituitary–adrenal axis. The importance of MC2 receptor in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency, a potentially fatal disease characterised by isolated cortisol deficiency. MC 2 receptor mutations cause ~ 25% of cases. The discovery of a MC2 receptor accessory protein MRAP, mutations of which account for ~ 15%–20% of familial glucocorticoid deficiency, has provided insight into MC2 receptor trafficking and signalling. MRAP is essential for the functional expression of MC2 receptor. MRAP2, a novel homolog of MRAP, can also facilitate MC2 receptor cell surface expression and function. Like MRAP, MRAP2 is a small transmembrane domain glycoprotein capable of homodimerising. In addition, MRAP/MRAP2 can heterodimerise. The presence of MRAP2 adrenal expression suggests a possible role for MRAP2 in adrenal physiology, which has yet to be elucidated. Importantly, new data shows that the MRAPs can interact with all the other melanocortin receptors (MC1,3,4,5 receptor). In contrast to MC2 receptor, this interaction results in reduced melanocortin receptor surface expression and signalling. MRAP2 is predominantly expressed in brain. Hypothalamic expression has been demonstrated for both MRAP and MRAP2. The ability of MRAPs to modulate different members of the melanocortin receptor family in a bidirectional manner is intriguing. Furthermore, central nervous system expression of MRAPs points to a role beyond MC2 receptor mediated adrenal steroidogenesis.
Keywords: Melanocortin receptor; Accessory protein; Adrenal gland;

The estrogen receptor α colocalizes with proopiomelanocortin in hypothalamic neurons and binds to a conserved motif present in the neuron-specific enhancer nPE2 by Flávio S.J. de Souza; Sofia Nasif; Rodrigo López-Leal; Diego H. Levi; Malcolm J. Low; Marcelo Rubinsten (181-187).
The gene encoding the prohormone proopiomelanocortin (POMC) is mainly expressed in two regions in vertebrates, namely corticotrophs and melanotrophs in the pituitary and a small population of neurons in the arcuate nucleus of the hypothalamus. In this latter region, POMC-derived peptides participate in the control of energy balance and sensitivity to pain. Neuronal expression of POMC is conferred by two enhancers, nPE1 and nPE2, which are conserved in most mammals, but no transcription factors are yet known to bind to these enhancers. In this work, by means of a one-hybrid screening, we identify that nPE2 possesses an element recognized by transcription factors of the nuclear receptor superfamily. This element, named NRBE, is conserved in all known nPE2 enhancers and is necessary to confer full enhancer strength to nPE2-driven reporter gene expression in transgenic mice assays, indicating that the phylogenetic conservation of the element is indicative of its functional importance. In a search for candidate nuclear receptors that might control POMC we observed that estrogen receptor alpha (ESR1) – a known regulator of energy balance at the hypothalamic level – can bind to the NRBE element in vitro. In addition we observed by immunofluorescence that ESR1 is coexpressed with POMC in around 25–30% of hypothalamic neurons of males and females during late embryonic stages and adulthood. Thus, our results indicate that hypothalamic expression of POMC is controlled by nuclear receptors and establish ESR1 as a candidate regulator of POMC.
Keywords: Proopiomelanocortin; Melanocortin; Transcriptional regulation; Estrogen receptor; Transcriptional enhancer;

Utilize conjugated melanotropins for the earlier diagnosis and treatment of melanoma by Minying Cai; Zhonglin Liu; Hongchang Qu; Helen Fan; Zhiping Zheng; Victor J. Hruby (188-193).
Peptides serve as effective drugs and contrast agents in the clinic today. However the inherent drawbacks of peptide structures can limit their efficacy as drugs. To overcome this we have been developing new methods to create ‘tailor-made’ peptides and peptide mimetics with improved pharmacological and physical properties. In this work we introduce novel peptide and small molecule conjugated molecules for earlier diagnosis and treatment of melanoma.
Keywords: Melanotropin; Human melanocortin receptors; Melanoma; Selective ligands for MC receptors; MTII; THIQ;

Maternal undernutrition is associated with programming of obesity in offspring. While previous evidence has linked programming to the hypothalamic, pituitary, and adrenal (HPA) axis it could also affect the hypothalamic neuropeptides which regulate food intake and energy balance. Alpha melanocyte stimulating hormone (αMSH), a key regulator of these neuronal pathways, is derived from pro-opiomelanocortin (POMC) which is therefore a prime target for the programming of obesity. Several models of maternal undernutrition have identified changes in POMC in hypothalami from foetuses or offspring at various ages. These models have also shown that the offspring go on to develop obesity and/or glucose intolerance. It is our hypothesis that programming leads to epigenetic changes in hypothalamic neuropeptide genes. Therefore when there is subsequent increased food availability, the epigenetic changes could cause dysfunctional transcriptional regulation of energy balance. We present evidence of epigenetic changes in the POMC gene promoter in foetal hypothalami after peri-conceptional undernutrition. In this model there are also epigenetic changes in the hypothalamic glucocorticoid receptor with consequent up-regulation of the receptor which could lead to alterations in the regulation of POMC and neuropeptide Y (NPY) in the hypothalamus. Thus maternal undernutrition could cause epigenetic changes in the POMC and glucocorticoid receptor genes, in the foetal hypothalamus, which may predispose the offspring to altered regulation of food intake, energy expenditure and glucose homeostasis, later in life.
Keywords: POMC; Hypothalamus; Glucocorticoid receptor; Maternal undernutrition; Programming; Epigenetics;

Melanocortin-5 receptor and sebogenesis by Li Zhang; Wen-Hwa Li; Michael Anthonavage; Apostolos Pappas; Dianne Rossetti; Druie Cavender; Miri Seiberg; Magdalena Eisinger (202-206).
The melanocortins (α-MSH, β-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC5 and MC1 receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC5 receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC5 receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC5 receptor, led to our search for MC5 receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC5 receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.
Keywords: Melanocortin-5 receptor; Antagonist; Sebaceous gland differentiation; Sebum; Lipids;

An overview on how components of the melanocortin system respond to different high energy diets by José K. van den Heuvel; Andréa J. van Rozen; Roger A.H. Adan; Susanne E. la Fleur (207-212).
High energy diets are used to model the obesity epidemic. Moreover, from a variety of genetic studies, it has become clear that the melanocortin system plays an important role in the regulation of energy metabolism. Since most dietary interventions are not standardized, fat/sugar-induced effects on the melanocortin system vary distinctly among different studies. How components of the melanocortin system are affected by high energy diets remains unclear. Therefore, in this review, we first present an overview of the effects of high energy diets on different elements of the melanocortin system in both mice and rats. The effects of a high energy diet are most consistent for agouti related protein levels which were either not affected or decreased after consumption of a high energy diet, whereas for proopiomelanocortin and the melanocortin receptor expression (and binding) it was difficult to define an overall response to a high energy diet. Because of the complexity of the melanocortin system, it is important to measure more than one element of the system. Only a few studies measured both melanocortin peptide and receptor expression and show that a high fat diet consumed for a longer period of time starting at an early age increases melanocortin signaling, whereas in adulthood a very high fat diet decreases melanocortin signaling. Finally, we review our own data on diet-induced changes in peptide expression and melanocortin binding and show that short term exposure to a free-choice high-fat high-sugar diet also decreases melanocortin signaling which supports hyperphagia observed in this model.
Keywords: POMC (pro-opiomelanocortin); AgRP (agouti-related protein); Diet; Obesity; Melanocortin receptor;

Hypothalamic proopiomelanocortin (POMC) neurons play a key role in regulating energy balance and neuroendocrine function. Much attention has been focused on the regulation of POMC gene expression with less emphasis on regulated peptide processing. This is particularly important given the complexity of posttranslational POMC processing which is essential for the generation of biologically active MSH peptides. Mutations that impair POMC sorting and processing are associated with obesity in humans and in animals. Specifically, mutations in the POMC processing enzymes prohormone convertase 1/3 (PC1/3) and in carboxypeptidase E (CPE) and in the α-MSH degrading enzyme, PRCP, are associated with changes in energy balance. There is increasing evidence that POMC processing is regulated with respect to energy balance. Studies have implicated both the leptin and insulin signaling pathways in the regulation of POMC at various steps in the processing pathway. This article will review the role of hypothalamic POMC in regulating energy balance with a focus on POMC processing.
Keywords: Proopiomelanocortin; POMC; α-MSH; Prohormone convertase; Carboxypeptidase E; Obesity;

Association study of POMC variants with body composition measures and nutrient choice by Andrew Ternouth; Marek K. Brandys; Yvonne T. van der Schouw; Judith Hendriks; John-Olov Jansson; David Collier; Roger A. Adan (220-225).
Genome linkage scans and candidate gene studies have implicated the pro-opiomelanocortin (POMC) locus in traits related to food intake, metabolic function, and body mass index. Here we investigate single nucleotide polymorphisms at the POMC locus in order to evaluate the influence of its genetic variance on body fat distribution and diet in a sample of middle-aged men from The Netherlands. 366 Dutch males from the Hamlet cohort were asked detailed questions about food choice, nutrient intake and exercise. Furthermore, their weight and body fat composition were measured. Each cohort member was genotyped for a set of single nucleotide polymorphisms (SNPs) at the POMC locus. Regression analysis, adjusted for several covariates, was used to test for the association between genetic variants and the phenotypes measured. POMC variation was associated with waist:hip ratio, visceral fat and abdominal fat (rs6713532, P = 0.020, 0.019, and 0.021, respectively), and nutrient choice (rs1042571, P = 0.034), but in light of limited power and multiple testing these results should be taken with caution. POMC is a strong candidate for involvement in appetite regulation as supported by animal, physiological, and genetic studies and variation at the POMC locus may affect an individual's energy intake which in turn leads to variation in body composition and body fat.
Keywords: POMC; Pro-opiomelanocortin; Melanocortine; Waist–hip ratio; BMI; Gene-association;

The melanocortin system is implicated in the expression of many phenotypic traits. Activation of the melanocortin MC1 receptor by melanocortin hormones induces the production of brown/black eumelanic pigments, while activation of the four other melanocortin receptors affects other physiological and behavioural functions including stress response, energy homeostasis, anti-inflammatory and sexual activity, aggressiveness and resistance to oxidative stress. We recently proposed the hypothesis that some melanocortin-physiological and -behavioural traits are correlated within individuals. This hypothesis predicts that the degree of eumelanin production may, in some cases, be associated with the regulation of glucocorticoids, immunity, resistance to oxidative stress, energy homeostasis, sexual activity, and aggressiveness. A review of the zoological literature and detailed experimental studies in a free-living population of barn owls (Tyto alba) showed that indeed melanic coloration is often correlated with the predicted physiological and behavioural traits. Support for predictions of the hypothesis that covariations between coloration and other phenotypic traits stem from pleiotropic effects of the melanocortin system raises a number of theoretical and empirical issues from evolutionary and pharmacological point of views.
Keywords: Barn owl; Evolutionary biology; Glucocorticoid; Melanin-based coloration; Melanocortin system;

Melanocortin MC4 receptor expression sites and local function by Jacqueline E. Siljee-Wong (234-240).
The melanocortin MC4 receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC4 receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC4 receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC4 receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC4 receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC4 receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC4 receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC4 receptor at these sites.
Keywords: MC4R; Melanocortin MC4 receptor; Melanocortin; Expression; Brain; Human; α-MSH; Melanotan-II; (Rat); (Mouse);