effects of pro-opiomelanocortin (pomc) on food …...174 a. p. coll figure 2 hypothalamic...

Clinical Science (2007) 113, 171–182 (Printed in Great Britain) doi:10.1042/CS20070105 171

R E V I E W

Effects of pro-opiomelanocortin (POMC) onfood intake and body weight: mechanisms and

therapeutic potential?

Anthony P. COLLDepartment of Clinical Biochemistry, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, CambridgeCB2 2XY, U.K.

A B S T R A C T

POMC (pro-opiomelanocortin) is a complex polypeptide precursor which is cleaved into smallerbiologically active peptides such as the melanocortins, α-, β- and γ -melanocyte-stimulatinghormone. Data from human genetic and murine studies convincingly show that an intact centralmelanocortin signalling pathway is critical for normal energy homoeostasis. Not only does a lossof normal melanocortin signalling lead to obesity, but there are also data implicating increasedmelanocortin activity in the pathogenesis of cachexia. The study of POMC biology has lead tosome fundamental insights into the mechanisms controlling food intake and body weight. Thisincreased understanding of the physiological roles of the melanocortin system has opened up thepotential for the design and development of rational therapies to treat perturbations in energyhomoeostasis.

INTRODUCTION

The biology of POMC (pro-opiomelanocortin) iscomplex and diverse. Smaller peptide fragments derivedfrom the inert POMC precursor play a crucial role inintegrating vital physiological functions, with POMCextensively processed in a highly tissue-specific mannerto yield a range of peptides involved in a whole range ofprocesses. Historically, the most well-known roles forthese peptides have been in skin pigmentation, adrenalsteroid synthesis and inflammation. However, over thelast decade, a wealth of data have clearly shown thatPOMC-derived peptides, in particular those synthesizedin neurons of the hypothalamus, play a critical role incontrolling food intake and body weight.

Building upon these fundamental insights into theregulation of body weight, there is now the potential forthe development of rational, mechanistic-based therapies

to treat perturbations in energy homoeostasis. In thisreview, I will highlight some of the key findings from ex-tensive murine and human genetic studies that haveled to the central melanocortin system being the best-characterized neuronal pathway involved in the regu-lation of mammalian energy homoeostasis. Furthermore,I will outline current developments in potential therapiesto manipulate this pathway and discuss their potentialclinical uses in treating both obesity and cachexia.

POMC

In humans and mice the POMC gene consists of threeexons. Exon 1 is untranslated, exon 2 codes for a signalpeptide and the N-terminal region, with exon 3 codingfor most of the translated mRNA [1]. Although POMCmRNA can be detected in a number of tissues, the gene is

Key words: body weight, food intake, energy homoeostasis, melanocortin, melanocortin receptor, pro-opiomelanocortin (POMC).Abbreviations: ACTH, adrenocorticotropic hormone; AgRP, agouti-related protein; CART, cocaine and amphetamine-relatedtranscript; GABA, γ -aminobutyric acid; LPS, lipopolysaccharide; MSH, melanocyte-stimulating hormone; MTI, melanotan I;MTII, melanotan II; NPY, neuropeptide Y; PC, prohormone convertase; POMC, pro-opiomelanocortin.Correspondence: Dr Anthony P. Coll (email [email protected]).

C© The Authors Journal compilation C© 2007 Biochemical Society

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Figure 1 POMC processing in humansPOMC is a large precursor peptide processed into smaller biologically activefragments by cleavage at dibasic cleavage sites (solid lines). ACTH, together withα-,β- and γ -MSH are together known as the melanocortins (pale blue). NT, N-ter-minal fragment; JP, joining peptide; β-LPH, β-lipotropin; β-END, β-endorphin.

expressed at physiologically significant levels in a limitedrange of tissues. These include the corticotrophs of theanterior pituitary, neurons originating in the arcuate nuc-leus of the hypothalamus and the brainstem, plus cells inthe dermis and the lymphoid system [1]. The transcribedPOMC pro-peptide itself is functionally inert but, duringtranslocation of the nascent protein through the endo-plasmic reticulum and Golgi apparatus, it is extensivelypost-translationally processed, undergoing a series ofproteolytic cleavages and chemical transformations togenerate a series of smaller biologically active peptides [2].

Human POMC is made up of 241 amino acid residues[3]. It contains eight pairs and one quadruplet of basicamino acids which are cleavage sites for processingenzymes (Figure 1) [1]. The key cleavage enzymeswhich act at these sites are a family of endoproteases,the PCs (prohormone convertases), with the repertoireof POMC products seen in a particular tissue largelydependent on the range of processing enzymes expressedin that tissue. Thus pituitary corticotrophs express PC1(prohormone convertase 1), but not PC2, resulting in theproduction of the N-terminal peptide, joining peptide,ACTH (adrenocorticotropic hormone) and β-lipo-tropin. In contrast, the expression of both PC1 andPC2 within the hypothalamus leads to the productionof smaller peptide fragments such as α-, β- and γ -MSH(melanocyte-stimulating hormone). These three peptides,together with ACTH, are collectively known as themelanocortins.

MELANOCORTIN RECEPTORS

The actions of the melanocortin peptides are mediatedthrough a family of five melanocortin receptors (termedMC1R to MC5R; Table 1) [4]. These receptors show con-siderable homology, all being seven transmembrane do-main G-protein-coupled receptors. MC1Rs are expressedwithin a range of cell types in the skin, including kera-tinocytes, melanocytes and endothelial cells. Signalling atMC1R is a key control point in melanogenesis, causinga switch in production from the red/yellow pigmentphaeomelanin to the brown/black pigment eumelanin.MC2R is the classical adrenocortical ACTH receptor,expressed in the cortex of the adrenal gland [4]. MC3Rsare expressed in the brain, chiefly in the hypothalamus,cortex, thalamus and hippocampus [5,6]. Of note, MC3Rscan be found on some POMC hypothalamic neurons,where they may act as an autoinhibitory receptor.

MC4Rs are widely expressed within the centralnervous system being present in the hypothalamus, thal-amus, hippocampus, limbic system, brainstem and spinalcord [7]. Compelling human genetic and murine data haveestablished that MC4R is a crucial molecular componentof the homoeostatic circuit that regulates energy balance.Finally, MC5Rs are expressed at low levels in numeroustissues, including sebaceous, lachrymal and pheromone-producing exocrine glands [8,9]. Targeted disruption ofMC5R in mice results in animals that have problemswith thermoregulation and repelling water from their coatbecause of decreased production of sebaceous lipids [8].

MELANOCORTINS

Melanocortins derive their name from the ability of thepeptides to stimulate melanogenesis in the melanocyteand/or steroidogenesis in adrenal cortical cells [10]. Thisfamily of peptides possess structural similarity to acharacteristic invariant tetrapeptide sequence (His–Phe–Arg–Trp) at their core being an absolute requirement forbinding and activity at melanocortin receptors [11].

α-MSH is a 13-amino-acid-residue melanocortinagonist. α-MSH has a well-defined role in the skin where,acting via MC1R, it can influence pigmentation [12].It also has anti-inflammatory and immunomodulatory

Table 1 Distribution and function of melanocortin receptors

Receptor Tissue Ligand Function

MC1R Skin α-MSH> β-MSH > γ -MSH > ACTH Pigmentation and immune functionMC2R Adrenal cortex ACTH SteroidogenesisMC3R Hypothalamus, thalamus, γ -MSH = α-MSH > β-MSH Cardiovascular control and sodium and energy homoeostasis

kidney and gutMC4R Brain and spinal cord β-MSH> α-MSH � γ -MSH Food intake and energy expenditureMC5R Exocrine glands, lungs, spleen α-MSH= ACTH> β-MSH >γ -MSH Sebaceous gland secretion

and pancreas


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properties [13]. More recently, α-MSH has been proposedto be the melanocortin with the most important rolein energy balance, acting upon MC4R (and to a lesserextent MC3R) in regions of the brain known to beinvolved in feeding behaviour. However, there remainscontention regarding the physiological hierarchy of themelanocortin peptides and, in particular, whether α-,β- and γ -MSH have unique, overlapping or redundantroles in the processes that control energy balance.

β-MSH is a 22-amino-acid peptide which in vitro has ahigh affinity at MC4Rs [14,15]. Although historically thephysiological role of β-MSH has been poorly defined,there are now data from human studies which reportthat β-MSH may play a critical role in the hypothalamiccontrol of body weight in humans (reviewed below)[16,17].

The role of γ -MSH in energy homoeostasis is less well-defined, although it can bring about a reduction in foodintake when given to a mouse lacking all endogenouslyderived melanocortin peptides [18]. More data suggestthat the primary role of γ -MSH may be in the regulationof the cardiovascular system [19].

ACTH is a 39-amino-acid peptide that is able toactivate all melanocortin receptors. However, ACTH isthe only melanocortin that can act on MC2R [20] tocause the secretion of glucocorticoids and, to a lesserextent, androgenic steroids and mineralocorticoids.

AGOUTI AND AGRP (AGOUTI-RELATEDPROTEIN) MELANOCORTIN ANTAGONISTS

Melanocortin receptors are unique among seven-trans-membrane-domain-receptor families in that, in additionto a range of agonist ligands, there also exist two endo-genously produced ligands which act as antagonists. Thisprovides a mechanism to tightly regulate melanocortinreceptor activity and to integrate opposing signals atthe receptor level.

Agouti is a protein involved in regulating pigmentation[21]. It is secreted within the hair follicles to act in aparacrine fashion, antagonizing the action of α-MSH atMC1R expressed on the surface of melanocytes. Agoutiinduces a switch in pigment production from eumelanin(black/brown) to phaeomelanin (red/yellow). A numberof dominant agouti alleles, such as Ay and Avy, result inwidespread ectopic expression of the agouti protein, giv-ing rise to a phenotype of obesity, hyperphagia and yellowcoat colour [21]. The link between hair colour and anobesity syndrome was made clearer when agouti wasfound to antagonize MC4R as well as MC1R. Additionalinsights came with the identification in the hypothalamusof a peptide named AgRP (due to its significant homologyto agouti) [22]. AgRP acts as an antagonist at hypo-thalamic MC4Rs, with transgenic mice that ubiquitouslyexpress human AGRP developing hyperphagia and an

obesity phenotype indistinguishable from that of theagouti mouse without an effect on pigmentation [23,24].

In a similar manner to POMC, AgRP undergoesprocessing by PC1 to produce a smaller, more biologi-cally active peptide. The C-terminal fragment of AgRP,AgRP83−132, is 6-fold more potent at MC4Rs than full-length AgRP [25]. However, there are still some areasof uncertainty regarding the functions of AgRP and theroles of the N-terminal regions of AgRP have yet to befully determined.

There are also some interesting in vitro data from twoindependent groups proposing that AgRP may be ableto act not simply as an antagonist, but also as an inverseagonist at MC4R [26,27]. This agrees well with anothermodel proposed by Srinivasan and co-workers [28] inwhich the N-terminal domain of MC4R functions as atethered intra-molecular ligand, maintaining constitutiveactivity of the receptor and thereby bringing about tonicinhibition of food intake [28]. Such a model is intriguingnot only because of the unique bi-directionality it wouldconfer upon signalling at MC4Rs, but also because, iftrue, molecules based upon the N-terminal domain of thereceptor may lead to novel therapeutic agents targetingthis receptor.

MELANOCORTIN SIGNALLING IN THEHYPOTHALAMUS

An overriding theme which has come to prominence overthe last decade is the critical role the central nervoussystem plays in co-ordinating metabolic functions inperipheral tissue. In particular, the hypothalamus isrecognized to receive and integrate neural, metabolicand humoral signals from the periphery [29]. Within thehypothalamus, the arcuate nucleus, situated betweenthe third ventricle and the median eminence, is consideredto act as a primary sensor of alterations in energystores to control appetite and body weight. Key to thisrole are two distinct subsets of arcuate neurons (Figure 2).The first population of neurons express POMC,with the majority of these cells also co-expressing theanorectic peptide CART (cocaine and amphetamine-related transcript). The mouse arcuate nucleus containsapproximately 3000 of such POMC-positive cells. Thesecond subset expresses the potent orexigenic peptidesNPY (neuropeptide Y) and AgRP. Both sets of neuronssend out dense projections to other nuclei within thehypothalamus, in particular to regions such as the para-ventricular nucleus and lateral hypothalamus whichexpress MC3R and MC4R. Additionally arcuate POMCneurons have widespread extra-hypothalamic descendingprojections to the brainstem, medulla and spinal cord [30].The hypothalamic melanocortin system can thereforebe defined as the neural circuits that include thesetwo separate populations of neurons within the arcuate


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Figure 2 Hypothalamic melanocortin systemNeurons in the arcuate nucleus express leptin receptors and integrate peripheral signals to maintain energy homoeostasis. Both NPY/AgRP and POMC neurons respondto leptin but do so in an opposite manner; NPY/AgRP neurons are inhibited but POMC neurons are activated. These first-order neurons signal through downstreamsecond-order neurons to bring about changes in feeding behaviour and energy expenditure. 3v, third ventricle; PVN, paraventricular nucleus; LH, lateral hypothalamus.

nucleus and the downstream second-order neuronsexpressing MC3R and MC4R which receive projectionsfrom POMC and/or NPY/AgRP neurons.

These two populations of arcuate neurons express thelong form of the leptin receptor (termed ObRb) [31]and are considered to be one of the key first-orderneurons through which leptin exerts its effects. Leptinregulates these two neuronal populations in a reciprocalmanner inhibiting NPY/AgRP neurons while stimulatingPOMC/CART neurons.

On the basis of these anatomical and functionaldata, a prevalent model of energy homoeostasis hasgained widespread acceptance (Figure 2). In situationsof excess energy, high levels of leptin activate POMCneurons and trigger the release of melanocortins fromPOMC axon terminals. This in turn goes on to activateMC4R thereby leading to suppressed food intake and

increased energy expenditure. Simultaneously, leptinsuppresses the activity of arcuate AgRP/NPY, whichwould otherwise antagonize the effects of α-MSH onMC4R through the release of AgRP. In contrast, in timesof energy depletion when leptin levels are low, thereis reduced anorexigenic POMC neuron activity butincreased orexigenic NPY/AgRP neuron activity. TheNPY/AgRP system also robustly and directly inhibitsPOMC neurons through both NPY and the inhibitoryneurotransmitter GABA (γ -aminobutyric acid) with thisunidirectional interaction providing a tonic inhibition ofPOMC neurons whenever NPY/AgRP cells are active.

However, our knowledge of these pathways continuesto rapidly evolve and, although the importance of thehypothalamic leptin–melanocortin signalling pathway inthe control of energy homoeostasis is still clear, themodel outlined above is undoubtedly an oversimplified


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paradigm. For example, POMC neurons and MC4Rsare also found in the brainstem [32], with melanocortinsignalling in this region probably playing a role inintegrating satiety signals from the gut [33]. Furthermore,the influence of leptin outside of the hypothalamus hasbeen clearly shown by recent data indicating that it can actupon dopaminergic neurons in the ventral tegmental area(a region of the brain involved in reward and motivation)to influence food consumption and ingestive behaviour[34,35].

Finally, in addition to responding to peripheral leptinlevels, arcuate neurons also integrate signals from a widerange of other nutrients and hormones (reviewed in [36]).For example, the effects of insulin on energy balance arelikely to be integrated at the level of POMC neurons [37].

MELANOCORTIN SIGNALLING AND OBESITY

Genetically modified mouse models can be highlyuseful in gaining understanding of complex physiologicalsystems. This is particularly true in the case of POMCbiology and energy homoeostasis, where a powerfulsynergistic relationship between human genetics andmurine modelling has been particularly fruitful indetermining how the melanocortin system brings aboutchanges in food intake and energy expenditure.

Human POMC deficiencyThe first description of humans congenitally lacking thePOMC gene products appeared in 1998. Krude et al. [38]reported two patients, one a compound heterozygotefor two nonsense mutations in exon 3, and a secondhomozygous for a mutation that introduced an additionalout-of-frame start site and interfered with POMC trans-lational initiation [38]. As a consequence of ACTH defi-ciency, both subjects presented with the metabolic con-sequences of hypocortisolaemia in early childhood. Bothwent on to develop severe early-onset obesity associatedwith hyperphagia (due to reduced hypothalamic melano-cortinergic signalling) and both subjects had pale skin andred hair (the result of reduced signalling through MC1Ron melanocytes in skin and hair follicles). Since the firstdescription of loss-of-function mutations in the humanPOMC gene, Gruter and co-workers have gone on toreport three additional children with the same phenotype[39]. Co-workers here in Cambridge have also reporteda patient from a Turkish family who is homozygous for anovel frameshift mutation which causes a stop codon toappear at the N-terminal end of POMC and therefore lossof all POMC-derived peptides [40]. Although affectedby obesity and adrenal insufficiency, this child was thefirst reported patient with POMC deficiency who didnot have red hair. This contrasts with the earlier otherreported cases who were all white European Caucasians.This indicates that different ethnic groups may display avariable dependence on POMC peptides for eumelanin

synthesis. Furthermore, this patient demonstrates thatthe absence of red hair in a non-Caucasian patientwith obesity and adrenal insufficiency does not excludePOMC deficiency as a potential underlying diagnosis.

Humans heterozygous for mutations in POMC havealso been studied. The heterozygous parents of theprobands reported in the initial report of Krude et al. [38]were all found to have high, normal or mildly elevatedbody weight, suggesting a dosage effect of POMC geneproducts on human weight regulation. This was borneout when a number of extended family members of theTurkish patient were also studied [40]. Eleven out of 12subjects heterozygous for the null mutation in POMCwere either overweight or obese compared with only oneout of seven wild-type family members. The finding thateven haploinsufficiency of this gene appears to confer asubstantial obesity risk emphasizes the critical role of thecentral POMC system in the control of human energybalance.

Pomc-null miceThere are now two independent mouse models withdisruption of both alleles of the Pomc gene [41,42]. Thephenotypes seen in both closely match the clinical picturereported in patients congenitally deficient in POMCpeptides and indicate that the melanocortin pathwayswhich regulate energy homoeostasis and adrenal functionare very similar in humans and mice (Figure 3). Usingsuch a model lacking all endogenously derived POMCpeptides (Pomc−/−) our group has demonstrated thatPOMC deficiency results in an increase of both fatand lean tissue mass [41]. Pomc−/− mice also havea significant increase in body length compared withwild-type littermates. Additionally, Pomc−/− mice havea reduction in basal oxygen consumption and plasmathyroxine concentrations, indicating that the obesityphenotype may be the consequence of a reducedmetabolic rate as well as increased food intake. We havealso studied mice heterozygous for a null mutation inthe Pomc allele (Pomc+/−) and, in keeping with thehuman data outlined above, have found they too havedisordered energy homoeostasis. Although on standardchow, food intake and body weight in Pomc+/− mice areindistinguishable from wild-type, when challenged witha high-fat diet, although wild-type mice maintain thesame body weight, Pomc+/− mice become significantlyhyperphagic and develop obesity. This is in contrast withwild-type littermates which maintain the same energyintake and body weight [41].

Disruption of activity at melanocortinreceptorsBy screening the coding region of POMC in 262Caucasian subjects with a history of severe obesity fromchildhood, Challis et al. [43] identified two children whowere heterozygous for a missense mutation (Arg236Gly)


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Figure 3 Pomc-null mouseMice lacking POMC have a clear red/yellow colour on their ventral surface (A), are hyperphagic (B), develop obesity (C) and have small, dysmorphic adrenal glands (D).(B) and (C) reproduced from Challis, B. G., Coll, A. P., Yeo, G. S., Pinnock, S. B., Dickson, S. L., Thresher, R. R., Dixon, J., Zahn, D., Rochford, J. J., White, A., Oliver,R. L., Millington, G., Aparicio, S. A., Colledge, W. H., Russ, A. P., Carlton, M. B. and O’Rahilly, S. (2004) Mice lacking pro-opiomelanocortin are sensitive to high-fat feedingbut respond normally to the acute anorectic effects of peptide-YY(3–36), Proc. Natl. Acad. Sci. U.S.A. 101, 4695–4700 with permission, c© 2004. (D) reproducedfrom Coll, A. P., Challis, B. G., Yeo, G. S., Snell, K., Piper, S. J. Halsall, D., Thresher, R. R. and O’Rahilly, S. (2004) The effects of proopiomelanocortin deficiency onmurine adrenal development and responsiveness to adrenocorticotropin. Endocrinology 145, 4721–4727 with permission, c© 2004, The Endocrinology Society.

that disrupts the dibasic amino acid cleavage site betweenβ-MSH and β-endorphin [43].

The Arg236Gly mutation completely prevented thenormal processing of these two peptides, resulting inan aberrant β-MSH/β-endorphin fusion peptide. Thisfusion peptide had an affinity to MC4R comparable withthat of β-MSH, but the ability to activate the receptoronce bound was much reduced, thereby giving the mutantprotein the capacity to interfere with melanocortinsignalling. Indeed, mutations disrupting the β-MSH/β-endorphin cleavage site were found in 0.9 % of childrenwith severe-onset obesity compared with only 0.2 % ofnormal weight controls, suggesting that mutations at thissite may make an appreciable contribution to the geneticpredisposition to severe childhood obesity.

Two more recent studies have directly addressed theuncertainty regarding the relative importance of partic-ular POMC-derived melanocortin ligands in control of

energy balance. Lee and co-workers [16] screened thecoding regions of the POMC gene for mutations in 538U.K. Caucasian subjects with severe early-onset obesity.They identified five subjects who were heterozygousfor a missense variant in the region encoding β-MSH(Tyr221Cys). The obese children carrying the Tyr221Cysvariant were hyperphagic and had increased lineargrowth, in a similar manner to subjects with MC4R defi-ciency. This variant was found at a significantly higher fre-quency in the obese study population than in unselectedU.K. Caucasian controls (five out of 538 compared withfour out of 5152 respectively) and Tyr221Cys co-seg-regated with obesity in affected family members. Finally,in vitro studies clearly demonstrated that, comparedwith wild-type β-MSH, the variant peptide had reducedbinding and activity at MC4Rs. Interestingly, the samestudy also identified a single proband which was hete-rozygous for a missense mutation in α-MSH, in which


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the highly conserved histidine residue within the classicalHis–Phe-Arg–Trp receptor-binding motif was replacedby a glutamine residue. In keeping with canonicalmelanocortin biology, in vitro studies with a syntheticmutant α-MSH peptide showed this to have a deleteriouseffect on its function and, although the proband wasobese, remarkably the same variant was found in one leanfamily member and one lean unrelated control. Thus thisstudy favoured β-MSH rather than α-MSH as having arole in the control of human energy balance.

Biebermann et al. [17] also reported a missensemutation within the coding region of β-MSH which wasassociated with early-onset human obesity. In addition,this study reported data from postmortem human brainstudies to lend further support to the hypothesis thatβ-MSH plays a role in the hypothalamic control of humanbody weight. These findings have direct relevance to thedevelopment of novel anorexigenic compounds targetingthe melanocortin system as they now provide a rationaleto specifically base pharmacological analogues uponβ-MSH.

Loss of AgRP actionCentral administration of the potent orexigen AgRPhas a profound long-lasting effect upon food intake[44]. The first report of an Agrp-null mouse wastherefore somewhat surprising because, in contrast withthe hypophagic lean animal one might expect with lossof antagonism at MC4R, the food intake and bodycomposition of Agrp−/− mice was indistinguishable fromwild-type littermates [45]. Interestingly, a more recentreport has shown that Agrp−/− mice do actually displaya modest lean phenotype late on in life, although thisis the result of an increase in energy expenditure ratherthan hypophagia [46]. However, when AgRP neuronsare ablated in the postnatal period, the results are verydifferent [47]. In contrast with the very modest impact ofAgrp deletion, loss of AgRP neurons in adult life leads toprofound life-threatening hypophagia, thereby appearingto confirm that AgRP has a critical role in the regulation offood intake. However, deletion of a neuronal populationis a more profound insult than removing a single peptidefrom the same neuronal population. AgRP neurons alsoexpress NPY and GABA and both have been shown toplay a major role in the regulation of feeding. The loss ofthese or other, as yet uncharacterized, neurotransmittersfrom AgRP neurons may play a part in the dramaticresult observed as much as the loss of the AgRPpeptide.

At present, there is no compelling genetic evidencethat abnormal elevation in AGRP expression or AgRPactivity can cause an obesity phenotype in humans.However, there are reports of SNPs (single nucleotidepolymorphisms) in both the promoter and the codingregion that are associated with BMI (body mass index)and fat mass [48–50].

Murine MC4R deficiencyMc4r-deficient mice develop a marked obesity syndromeassociated with hyperphagia, hyperinsulinaemia, hyper-glycaemia and an increase in linear growth comparedwith wild-type [51]. Furthermore, heterozygous mice(Mc4r+/−) have an intermediate phenotype between wild-type and homozygous null mice, indicating a clear genedosage effect. In the intervening decade since Huszaret al. [51] first reported the phenotype of Mc4r deficiency,Mc4r−/− mice have been extensively studied. Measure-ments of metabolic rate and data from pair-feeding studieshave shown that, like Pomc-null mice, the obesity inMc4r-null mice is as a result of defective regulation ofenergy expenditure as well as hyperphagia [52]. High-fatfeeding also causes Mc4r−/− mice to develop obesity atan accelerated rate due to sustained hyperphagia, a muchreduced level of diet-induced thermogenesis and a lack ofincrease in motor activity. Together with the data fromPomc null mice, this is highly suggestive that an intactmelanocortin system is necessary to effect appropriatechanges in homoeostatic mechanisms in response tochanges in the caloric content of the diet [53].

Human MC4R deficiencyIn 1998, two groups reported heterozygous mutations inhumans in MC4R that were associated with dominantlyinherited obesity [54,55]. Since then, mutations in MC4Rhave been reported in obese humans from various ethnicgroups and are responsible for up to 5 % of cases ofsevere childhood obesity and between 0.5 and 2.5 %of adult obesity [56,57]. Furthermore, the finding thatclose to 1 in 1000 of the general population may havean MC4R mutation makes MC4R deficiency one of themost common single gene disorders [58].

As well as the increase in fat mass, MC4R mutantsubjects also have an increase in lean mass that is notseen in other monogenic obesity syndromes such as leptindeficiency [59]. Affected children have increased lineargrowth with a height S.D. score of +2 compared withpopulation standards. MC4R-deficient subjects also havehigher levels of fasting insulin than age, sex and BMISD score-matched children [59]. The accelerated lineargrowth and the disproportionate early hyperinsulinaemiaare consistent with observations in the Mc4r−/− mouse.

Affected subjects are objectively hyperphagic althoughthis appears not to be as severe as that seen with leptindeficiency [59]. However, it is noteworthy that theseverity of receptor dysfunction seen in in vitro assayscan predict the amount of food ingested during a testmeal by the subject harbouring that particular mutation[59].

MC3R deficiencyHomozygous-null Mc3r (Mc3r−/−) mice have an unusualphenotype, in that, although not significantly heavier thanwild-type mice, they have an increased fat mass with a


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reduction in lean mass [60,61]. Mc3r−/− mice also havea reduction in the body length. Our current understand-ing of the role of MC3R is that it influences feedingefficiency and the partitioning of fuel stores into fat.The non-redundant role of MC3R and MC4R is clearlyillustrated by the finding that mice lacking both centralmelancortin receptors become heavier than mice lackingMC4R alone [60].

Direct sequencing of the MC3R gene-coding sequencein populations with Type 2 diabetes mellitus and obesityhas identified a number of sequence variants. However,these have all been detected in unaffected controls withsimilar frequencies or have been absent in family memberswho were also obese, and as yet there is no convincingevidence for a major role of MC3R mutations in causinga severe metabolic phenotype in humans [62,63].

MELANOCORTIN PATHWAYS AND CACHEXIA

As outlined above, disruption of central melanocortinpathways clearly leads to obesity in humans and rodents.It is perhaps not surprising then that overactivity of thisimportant system is also being increasingly implicated inpathological states characterized by energy wasting.

Cachexia is a state of malnutrition characterized bya decrease in appetite and food intake combined withan inappropriate increase in metabolic rate, resultingin a loss of both fat and lean mass [64]. It is not thesame as a voluntary fast when the sensation of hunger isnot dulled and mechanisms entrained by reduced energyintake aim to restore energy supplies by increasing intakeand reducing energy expenditure. Cachexia is commonlya feature of malignant diseases but is also associated withcardiac and renal failure, connective tissue disorders andchronic suppurative conditions. It impacts heavily uponquality of life and tolerance to therapy and has an impacton overall morbidity and mortality.

All of these disease have in common the productionof systemic pro-inflammatory cytokines with the charac-teristic deleterious changes in appetite and metabolismascribed to the direct action of cytokines altering therelease and function of a number of neurotransmitters[65]. In particular, an increasing number of studies ofmelanocortin signalling have demonstrated that POMCneurons may be key transducers of stimuli which drivecachexia. For example, not only are the phenotypicfeatures of MC4R deficiency the opposite of those seenin cachexia, but also activation of central MC4R leadsto reduced food intake, increased energy expenditureand loss of body weight, replicating the response seenwith cytokine administration. Further evidence thatblockade of the central melanocortin signalling pathwaycan ameliorate the effects of cytokine administrationcomes from the findings that Mc4r−/− mice are resistant toLPS (lipopolysaccharide)-induced anorexia [66], whereas

melanocortin antagonists given to wild-type animals cansignificantly ameliorate the reduction in food intake seenfollowing LPS administration [66].

Perturbations in central melanocortin signalling canalso result in differential responses to the induction ofcachexia by tumours [67]. The hypophagia and weightloss induced by sarcoma growth can be reversed andprevented by administration of AgRP. Tumour-bearingMc4r−/− mice are able to continue accumulating bothlean and fat mass in the face of tumour growth, whereaswild-type control animals lose both lean and fat massunder identical conditions [67].

Mc4r−/− mice are also resistant to cachexia associatedwith renal disease. A subtotal nephrectomy in wild-typeanimals causes loss of lean and fat mass with an increase inresting metabolic rate, whereas Mc4r−/− mice undergoingthis procedure continue to increase their lean and fat mass[68].

Intriguingly, Mc3r−/− mice suffer enhanced cachexia,both in response to a challenge with LPS and with tumourgrowth, when they lose more weight than wild-typemice during tumour-induced cachexia [67]. These datalend support to the hypothesis that MC3R may function,in part, as inhibitory autoreceptors on POMC neurons. Inthe absence of this putative braking function, a cachexicstimulus upon POMC neurons can proceed unabatedleading to an overall increase in melanocortin tone anda more marked impact on energy balance [67].

THERAPEUTIC MANIPULATION OF THEMELANOCORTIN PATHWAY

The majority of the evidence presented above is based ona strategy of studying loss of gene function, be it naturallyoccurring or genetically engineered. However, this doesnot conclusively prove that therapeutic strategies directedto increase gene expression levels or activity of the geneproduct will ameliorate the phenotype seen withgene loss. A number of studies have addressed thisissue and investigated the effect of increased melancortinsignalling brought about either by overexpressing Pomcor by administering potent melanocortin analogues.

Genetic manipulationLi et al. [69] transgenically overexpressed neuronalPOMC in genetically obese Zucker rats. There wasa sustained reduction in food intake, a significantattenuation of weight gain and a 24 % decrease in visceraladiposity. There are also data demonstrating that targetedPomc gene therapy in the hypothalamus can reduce bodyweight and visceral adiposity in aged obese rats [70].

Savontaus et al. [71] generated transgenic miceoverexpressing α- and γ -MSH under the control of theCMV (cytomegalovirus) promoter [71]. This resultedin a 2-fold increase in both α-and γ -MSH within the


Effects of POMC 179

Figure 4 Amino acid sequence of melanocortin agonistsThe core melanocortin motif of His-Phe-Arg-Try is highlighted in red.

hypothalamus and reduced weight gain and adiposityin lean wild-type male mice. Transgenic homozygousmice were also crossed with two obese strains of micewith disrupted melanocortin signalling, leptin-receptor-deficient db3J/db3J and yellow agouti Ay/a mice and againobesity was attenuated. The same group used a similarstrategy of transgenic overexpression to determinewhether long-term melanocortinergic activation couldattenuate the metabolic effects of a high-fat diet[72]. Their data showed that long-term melanocortinactivation reduced body weight, adiposity and hepaticfat accumulation in the setting of diet-induced obesity.

Although such genetic manipulations are not in therealm of current available therapy, they neverthelesssuggest that long-term melanocortinergic activationcould serve as a potential strategy for the treatment ofobesity.

Pharmacological manipulationThe fact that central melanocortin receptors are mem-brane-bound G-protein-coupled receptors with knownpeptide ligands, together with the wealth of evidencefrom the studies outlined above, makes the melanocortinsystem a very attractive target for rationally basedtherapies to treat disorders of energy homoeostasis.

However, the endogenous melanocortins are linearpeptides which are rapidly degraded by proteolyticenzymes and therefore hold limited promise as effectivepharmacological agents. Thus many structural and func-tional studies over the last two decades have concentratedon trying to manipulate the basic melanocortin peptidesequence into compounds with improved potencyand resistance to enzymatic degradation [73]. Severalsuch compounds now exist (Figure 4). For example, ifl-phenylalanine at position 7 of α-MSH is replaced byd-phenylalanine, the resultant peptide [NDP-α-MSH,also known as MTI (melanotan I)] is a more potentmelanocortin agonist. Another potent melanocortinagonist is MTII (melanotan II), being a cyclic lactamring derivative of the seven amino acids which makeup α-MSH between positions 4–10. However, if thephenylalanine at position 7 of MTII is replaced by 2-naphthylalanine, this results in a high-affinity antagonistof both MC3R and MC4R called SHU9119.

Both MTII and SHU9119 have been used extensivelyin pharmacological studies of the melanocortin system

[74]. In 1997, Fan et al. [75] demonstrated that centraladministration of MTII significantly decreased foodintake in four different mouse models, including micewith a disrupted leptin–melanocortin system (ob/ob andAy mice) [75]. Furthermore, not only did SHU9119bring about a significant increase in food intake, butco-administration of SHU9119 with MTII significantlyabrogated the anorectic effect of the latter. More recentlyperipheral administration of MTII had been shown tosuppress food intake and cause progressive weight loss ina dose-dependent manner in both lean and diet-inducedobese mice [76,77].

In addition to manipulating the structure of melano-cortin ligands, the concept of ‘priviledged structure’ hasbeen used to develop compounds active at melanocortinreceptors. This approach is based upon the idea that anumber of molecular structural scaffolds are able to bindwith high affinity to multiple receptor families. Suchorganic cores, e.g. piperazine and cyclohexane, are oftenfound in a number of commercially available drugs [73].Using this approach, researchers at Merck have developedan active, small molecule peptide mimetic MC4R agonistnamed THIQ. Studies in mice demonstrate it is able toinhibit feeding in both acute and chronic study paradigmswith no effect being seen in MC4R knockout mice[78].

A number of small molecule MC4R antagonists havealso been successfully used in animal models of cachexia.NBI-12i is a potent MC4R antagonist that has nano-molar affinity and high specificity for MC4R [79].As one might predict, it has no effect in Mc4r−/−

mice, but can significantly increase food intake anddecrease the metabolic rate in wild-type mice whengiven peripherally. Importantly, when used in amurine cancer model (subcutaneous implantation of lungadenocarcinoma), NBI-12i can significantly attenuate theresultant cachexia with the ability to preserve lean body.Another compound, ML00253764, was discovered byhigh-throughput screening of non-peptide benzamidinecompounds with potential activity at MC4Rs. Thismolecule was able to penetrate into the central nervoussystem after peripheral administration and has beenshown to be able to effectively reduce tumour-inducedweight loss in a xenograft mouse model [80].

The melanocortin pathway: therapeuticpromise and therapeutic problems

There are drugs which specifically target melanocortinreceptors that have undergone trials in humans [81].Melanotan, as its name suggests, was developed to mimicthe action of α-MSH on MC1R and induce melanogene-sis in the hope that the resulting skin tan wouldprotect against UV-related skin cancers. The relatedanalogue MTII can also act upon skin melanocytes to


180 A. P. Coll

increase pigmentation but in early clinical trials had theunexpected side effect of producing spontaneous penileerections which were intermittently experienced for 1–5 hafter MTII dosing.

Further studies have shown that MTII also appears toincrease sexual desire but interestingly can also inducenausea, stretching and yawning [82], remininscent inpart of the ‘stretching–yawning syndrome’ seen manydecades ago when POMC peptides were first centrallyadministered to dogs [83]. More recently, small trials ofa nasally administered cyclic heptapeptide melanocortinanalogue, PT-141, have also reported beneficial effects inmen with erectile dysfunction [84].

Inevitably, a drug that can induce a bronzed tan,improve libido and sexual function and which may alsohave the potential to suppress appetite and cause weightloss has given rise to many titillating media commentarieswith MTII attracting the unfortunate moniker of ‘TheBarbie Drug’ [85].

As yet, the published data on melanocortin ligands forthe treatment of obesity or cachexia are not as advanced.A small study looked at the effect of 6 weeks of intranasalACTH4−10 on healthy normal-weight volunteers [86].Compared with placebo, ACTH4−10 did have a significanteffect, reducing body fat by 1.68 kg and body weight by0.79 kg. Gruter and co-workers have reported a 3 monthtrial of treatment with intranasal ACTH4−10 in the twoinitial probands reported with total POMC deficiencybut sadly there was no reduction in body weight or achange in eating behaviour in either [39]. One explanationmay be that, compared with α-MSH, ACTH4−10 has a1000-fold lower affinity at MC4R.

However, there are on-going ‘proof-of-concept’ trialsinvolving MC4R agonists designed specifically for thetreatment of obesity and the results from these are eagerlyawaited.

CONCLUSIONSKnowledge of the physiological roles of the melanocortinsystem has hugely increased in recent years, withPOMC-derived peptides firmly placed at centre stagewhen it comes to our understanding of the molecularmechanisms controlling energy homoeostasis. Muchevidence from human and murine studies gives credenceto the possibility of successfully pharmacologicallymanipulating these pathways. The early evidence fromclinical trials using MTII gives encouragement thatadministration of melanocortin analogues is, in the shortterm at least, safe. However, the unusual side effectsobserved with these agents add a note of caution.Although serendipitously leading to a new treatment forerectile dysfunction, they should provoke investigatorsto move away from a ‘hypothalamo-centric’ view ofthe melanocortin system and be wary of other hithertounexpected effects when manipulating such a crucialcentral-nervous-system signalling pathway. Of course,

unexpected does not always mean unwelcome and asmelanocortin agents move over into the clinical arena wecan anticipate interesting times ahead.

ACKNOWLEDGMENTS

A. P. C. is an MRC (Medical Research Council) ClinicianScientist.

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Received 28 March 2007/8 May 2007; accepted 8 May 2007Published on the Internet 13 July 2007, doi:10.1042/CS20070105


effects of pro-opiomelanocortin (pomc) on food …...174 a. p. coll figure 2 hypothalamic...

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