the hypothalamus and obesity

Current Drug Targets, 2005, 6, 225-240 225

1389-4501/05 $50.00+.00 © 2005 Bentham Science Publishers Ltd.

The Hypothalamus and Obesity

Peter J. King*

Johnson & Johnson Pharmaceutical Research and Development, A Division of Janssen Pharmaceutica N.V., MetabolicDisorders, Turnhoutseweg 30, B-2340 Beerse, Belgium

Abstract: Obesity, a condition already at epidemic proportions in the developed world, is largely attributable to anindulgent lifestyle. Biologically we feel hunger more acutely than feeling ‘full-up’ (satiety). The discovery over a decadeago of leptin, an adiposity signal, revolutionised our understanding of hypothalamic mechanisms underpinning the centralcontrol of ingestive behaviour. The structure and function of many hypothalamic peptides (Neuropeptide Y (NPY),Melanocortins, Agouti related peptide (AGRP), Cocaine and amphetamine regulated transcript (CART), Melaninconcentrating hormone (MCH), Orexins and endocannabinoids) have been characterised in rodent models. Thepharmacological potential of several endogenous peripheral peptides released prior to, during and/or after feeding arebeing explored. Short-term signal hormones including Cholecystokinin (CCK), Ghrelin, Peptide YY (PYY3-36) andGlucagon-like peptide 1 (GLP-1) control meal size via pathways converging on the hypothalamus. Long-term regulationis provided by the main circulating hormones leptin and insulin. These systems among others, implicated in hypothalamicappetite regulation all provide potential “drugable” targets by which to treat obesity.

Key Words: Food intake, Obesity, Energy homeostasis, Hypothalamus, Anti-obesity drugs.

INTRODUCTION

Obesity across the major markets is at epidemicproportions (the prevalent population across the USA,Europe and Japanese markets reached 114 million in 2003)and is forecast to grow approximately 20 % by 2013(reaching 140 million) [1]. Paralleling such growth will bethe commercial opportunities this offers, with obesityresearch set to rise from $500 million in 2003 to $2.3 billionin 2013 [1]. It has been proposed that centrally acting anti-obesity agents will make up approximately three quarters ofthe future obesity market, despite the lingering safety con-cerns following withdrawal of dexfenfluramine (Adifax®/Redux®) and fenfluramine due to unwanted cardiovascularside effects [2, 3] in the late 1990’s and sibutramine’stemporary withdrawal from the Italian markets in 2002.

Even though there have been many scientific break-throughs in the understanding of the regulation of foodintake and energy disposal throughout the last few decades,new anti-obesity drugs have not reached the marketplace.The current marketed drugs available for the treatment ofobesity are insufficient to cope with the expanding obesitypopulations now seen in both western and developingcountries. Not only are they limited in number, but also intheir efficacy at producing sustained weight loss beyond 10% (Table 1). This is partly due to the complex neuronalcircuitry in the central nervous system (CNS) and peripherythat regulate energy deposition and expenditure, and thedifficulty of extrapolating findings in experimental animals(mostly rodents) to humans. This plus contributing geneticand environmental factors, plus the need for potent and safe

*Address correspondence to this author at the Johnson & Johnson Pharma-ceutical Research and Development, A Division of Janssen PharmaceuticaN.V., Metabolic Disorders, Turnhoutseweg 30, B-2340 Beerse, Belgium;Tel: +32(0)14/60.31.06; Fax: +32(0)14/60.54.03;E-mail: [email protected]

pharmacological interventions, the discovery of noveldisease targets has been tardy and unfruitful. Despite thesehurdles, abdominal obesity clearly represents a major contri-buting factor and also initiator of the metabolic syndrome(defined by the World Health Organization (WHO) asinsulin resistance plus two of the five additional criteria ofhypertension, high triglycerides, low HDL cholesterol, highbody mass index (BMI), and high urinary albumin excre-tion), treating obesity is a heavily pursued area for academicand industrial researchers and several new chemical entitiesare currently being investigated for their treatment of obesity(Fig. 1). Many excellent reviews have recently beenpublished outlining the potential therapeutic targets for thetreatment of obesity [4-10].

This article will highlight some of the newer prospective“drugable” targets for treating obesity, particularly pathwaysconverging on the hypothalamus or some of the manypeptides and neurotransmitters expressed in this small brainregion, that influence feeding and energy homeostasis.

REGULATION OF ENERGY BALANCE

Obesity results from an energy imbalance, where theenergy intake exceeds energy expenditure. The likelihood ofobesity varies considerably between individuals of any givenpopulation, with certain individuals being able to consumealmost any food without ‘putting on an ounce of weight’ soto speak, in comparison to those who easily ‘put on thepounds’. Such paradigms are paralleled in rodents withdistinct high and low weight gain subset populations emerg-ing from a genetically uniform single population of rats fed ahighly palatable diet [11]. It is not clear whether thisdifference is attributable to metabolic adaptations limitingexcessive weight gain, but effects of lifestyle can obviouslybe ruled out in the case of such a rodent model, which mostclosely resembles human obesity.

226 Current Drug Targets, 2005, Vol. 6, No. 2 Peter J. King

Naturally occurring mutations, as well as ablative lesions,have shown that the brain regulates both aspects of energybalance and that abnormalities in energy expenditure contri-butes to obesity. Indeed, early evidence pointing towards amodel for hypothalamic control of energy balance camefrom specific brain lesion experiments, either through physi-cal or chemical destruction of neurones. In particular lesionsof the ventromedial hypothalamic nucleus (VMH), resultedin obesity, whilst lesions of the lateral hypothalamic area(LHA) lead to anorexia and weight loss [12]. From this firstconception, it is now understood that sensory informationfrom the upper gastrointestinal (GI) tract, abdominal visceraand taste information from the oral cavity [13] are allintegrated in the nucleus of the tractus solitarius (NTS), anarea in the caudal brainstem. Satiety-inducing signals,initiated by mechanical or chemical stimulation of thestomach and small intestine, neural-inputs related to energymetabolism in the liver [14] and humoral signals alsoconverge on the NTS via ascending vagal fibres from thespinal cord [15]. Afferent fibres then carry the signals to thehypothalamus and other forebrain regions (Fig. 2).

PERIPHERAL SIGNALS AFFECTING REGULATIONOF METABOLISM

Leptin

For much of the past two or so decades, the attention ofobesity and diabetes research has been directed towardsleptin. However, this circulating hormone is neither the mostpotent nor the most physiologically important regulator ofthe hypothalamic neurones, or indeed of energy balance.Leptin, the 146-amino acid residue product of the ob gene, is

synthesised mainly in white adipose tissue (WAT) [16] andalso in brown adipose tissue (BAT) [17], stomach, skeletalmuscle, the placenta and mammary tissues, albeit at muchlower levels [18]. It is now generally accepted that leptin actsas a signal that indicates the size of the adipose tissue massto the CNS centres that regulate feeding behaviour [19].Considerable support for the involvement of leptin in thecentral regulation of feeding has been provided by studiesusing three genetic models of obesity, ob/ob and db/db miceand the fa/fa Zucker rat [20-22]. All of these models havedifferent single-gene mutations that consequently result insevere obesity due to hyperphagia.

Leptin injected into the bloodstream of experimentalanimals readily enters the mediobasal hypothalamus andarcuate nucleus (ARC), where the blood-brain barrier isspecifically modified to allow the passage of large molecules[23]. It is also the site where an extended functional variantof the leptin receptor (Ob-Rb) encoded by the db gene [24] isexpressed on various ARC neuron populations [25-29].Although leptin is a potent inhibitor of feeding, thehyperphagia and obesity in both animals and humans seemsto go hand in hand with hyperleptinaemia related to over-secretion of this hormone (and also insulin) [30]. Thecounter-regulatory effects of leptin (that in normal weightmammals decreases food intake through the hypothalamus),is evidently lacking in obese mammals, with such mech-anisms being more apparent in individuals or animals thatare predisposed to weight gain and diabetes [31]. Impairedtransport of leptin into the CNS is hypothesized to beresponsible for the proposed for the ‘leptin resistance’ [32].This insensitivity or “resistance” to leptin may explain the

Table 1. Current Drug Therapies for the Treatment of Obesity

Agent Company/Brand Advantages Disadvantages

Pancreatic lipase inhibitor

Orlistat Roche’s Xenical

Trials show that 5-10 gk or 5-10 % body weight is lost over 6months to 1 year. Up to 30 % of patients maintain their weightloss over two years therapy. Patients have reduced serum lipid

levels and improvements in risk factors for developingcardiovascular disease and diabetes

Gastoinstestinal side effects include fecalincontinence, flatulence and oily

spotting. Malabsorption of lipid-solublevitamins can occur in some patients

taking orlistat.

Serotonin and norepinephrine reuptake inhibitors

SibtramineAbbott’s Meridia &

Reductil

Trials show that 3-6 kg or 5-10 % of body weight is lost over 6months to 1 year. Up to 30 % of patients maintain their weightloss over two years therapy. A study demonstrated that obese

patients with concurrent binge-eating disorder exhibitedimproved control of eating behaviour while taking sibutramine

Common side effects include elevationin blood pressure, increased heart rate,

dry mouth, anorexia, insomnia andnausea. Sibutramine is contraindicated in

patients taking antidepressants or whohave uncontrolled hypertension.

Noradrenergic anorectic agents

PhentemineGlaxoSmithKline’s Fastin,UCB’s lonamin, generics

MazindolWyeth-Ayerst’s Mazanor,

Novartis’s Sanorex

Trials demonstrate a weight loss of 0.5 kg/week or less than 5% of body weight lost over two months of therapy

Side effects include overstimulation ofthe CNS, restlessness, dizzness,

insomnia, euphoria, dysphoria, tremorand headache. Drugs in this class are

approved for short-term use only.

Adapted from Metabolic Disorders Study # 11, Obesity, August 2004 with permission from Decision Resources, Inc., Waltham, MA [1].

The Hypothalamus and Obesity Current Drug Targets, 2005, Vol. 6, No. 2 227

Fig. (1). Emerging neural and hormonal targets in development for the treatment of obesity. Adapted from Metabolic Disorders Study # 11,Obesity, August 2004 with permission from Decision Resources, Inc., Waltham, MA.


Fig. (2). Simplified schematic overview of the hypothalamic structures (based on rat data) involved in food intake regulation and energyexpenditure. Coronal section showing the relative positions of the arcuate (ARC), ventromedial (VMH), dorsomedial (DMH), paraventricular(PVN) nuclei and lateral hypothalamic area (LHA) with respect to each other through the brain. Circuits allowing communications betweenthese neuronal populations are indicated.

disappointing trials of peripherally administered recombinantleptin or leptin analogues [31, 33, 34]. Despite these frustrat-ing results other agonists of leptin are being developed.Cambridge Biotechnology is investigating a series of leptinmimetics, including CBT-001452 for the treatment ofobesity. Preclinical data showed centrally administered CBT-001452 reduced food intake and body weight with effectsbeing evident in lean rats when administering the compoundeither i.p. or p.o. This reduced food intake was not seen inthe Zucker fa/fa rats, proof that CBT-001452 works via theleptin receptor1. Abbott Laboratories in collaboration withMillenmium Pharmaceuticals Inc. (recently terminated)developed antisense oligonucleotides against the product ofgene 46a (involved in the leptin breakdown) that cause areduction in 24 h food intake in ob/ob mice to a greaterextent than leptin treatment [35].

Insulin

Insulin has similar central catabolic effects to leptin onenergy homeostasis, in that it inhibits feeding, stimulates

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1 Manuef, Y.; Higginbottom, M.; Pritchard, M.; Ashford, M.; Lione, L.; Ho, M.; Payne,K.; Littlefield, S.; Peck, E.; Chapman, E.; Horton, J.; Guyen, V-A.; Simpson, I.;Stygall, J.; Tyzack, C.; Sedgwick, T. and Richardson, P. (2004) Diabetes, 53, Abs 64-LB.

thermogenesis and induces weight loss. Insulin injectedcentrally inhibits feeding and stimulates thermogenesis [36],while injection of neutralizing antibodies targeted againstinsulin specifically into the VMH stimulates feeding [37,38]. Contrary systemic injection of insulin at pharmacologi-cal levels increases body weight in patients [39]. Generallycirculating insulin levels parallel body fat mass and, likeleptin, is a plausible hormonal signal of adiposity, withplasma insulin concentrations being decreased in all states ofenergy deficiency, such as starvation, insulin-deficientdiabetes (IDDM), lactation and physical exercise, which isconsistent with data showing insulin to act centrally anddirectly to inhibit both hypothalamic neuropeptide Y (NPY)-and galanin producing neurones under normal feedingconditions [40, 41]

Despite wide distribution throughout the brain, insulinreceptors are especially rich in the ARC and paraventricularnucleus (PVN) [42], with circulating insulin entering themedian eminence (ME) and ARC through specializedregions of the blood brain barrier. Confirming insulin’s rolein the regulation of food intake and body weight, selectiveknockout of insulin receptors in the brain leads to hyper-phagia and obesity [38]. The use of small molecule insulinmimetics, have previously been shown to stop the progressof obesity in diet-induced obese mice by reducing foodintake and body weight gain, together with lessening adipo-


sity and insulin resistance [43], suggesting their potential usein the treatment of obesity.

Cholecystokinin (CCK)

The gut peptide, CCK, was one of the first satiety factorsto be discovered. CCK is released upon nutrient stimulationof neuroendocrine secreting cells lining the intestinal lumen[44]. It dose-dependently decreases meal size in rodents [45]without affecting body weight. CCK’s proposed pathway ofaction via afferent vagal fibres to the NTS, may account forthe observation that CCK can decrease hypothalamic andhippocampal NPY levels in the rodent brain withoutaffecting levels elsewhere in the brain [46, 47]. This resultsuggests a negative relationship between NPY and CCK-peptides, which is not surprising given their opposite roles inthe control of feeding.

The physiological effects of CCK are mediated throughtwo G-protein coupled receptor (GPCR)’s, CCK1 (formerlyCCK-A) and CCK2 (formerly gastrin/CCK-B). Severalstudies have confirmed that exogenous CCK acts viaperipheral CCK1 to elicit satiety in cats and rodents [48-52].However, the receptor site of action for endogenous CCK inthe periphery remains unclear. Dourish and colleagues [53]demonstrated that endogenous CCK acts predominantly atcentral CCK2 expressed in the VMH and PVN to promotesatiety. Several reports state that antagonism of CCK1increases food intake in contrast to CCK2 antagonism [51,52, 54, 55]. Meal termination induced by satiety signals canbe demonstrated when all neuronal connections between theforebrain and hindbrain are severed [56] implying that thebrain areas involved are independent of hypothalamicinfluence. Both leptin [57] and insulin [58] enhance thesatiating effects of CCK. Evidence shows that leptin poten-tiates activation of NTS neurones by CCK [59, 60], thusdemonstrating that signals involved in energy homeostasismodulate the response of NTS neurones to input related tosatiety. Indeed melanocortin (MC) 4 receptors [61], leptinreceptors [62, 63], NPY receptors [64] and pro-opiomelano-cortin (POMC) neurones are present in the NTS [65]. Morerecently, Fan and colleagues have demonstrated brainstemPOMC neurones to be activated by CCK and feedinginduced satiety, and that activation of the MC4-receptor isrequired for CCK induced suppression of feeding [66].

Human studies [67, 68] demonstrated that CCK-8 (anoctapeptide form of CCK released from the small intestineafter eating) reduced food intake in normal subjects byaltering the size of individual meals, but that repeatedadministration did not alter body weight [69]. In the late1990’s GSK reported on orally active CCK1 selective benzo-diazepine derivatives (GI-181771), which was followed threeyears later by Sanofi-Sythelabo publishing on SR 146131, athiazole derivative, a potent, selective and orally activeCCK1 agonist that entered phase II clinical trials for obesity.However, out of these two promising drugs only GI-181771has progressed into advanced clinical trials, according toCurrent Drugs © IDdb3, accessed Aug 2004.

Ghrelin

Ghrelin, a recently discovered 28 amino acid gastricpeptide [70-72] with orexigenic and adipogenic properties, is

the only known endogenous ligand identified for the growthhormone secretagogue receptor (GHSR)-1 to date [73, 74]. Aunique feature of ghrelin is that specific post-translationalmodification of serine-3 to be n-octanoylated is fundamentalfor its bioactivity. [75-77]. Ghrelin is primarily expressed (asmRNA) in the stomach [70, 78, 79], with concentrationsdecreasing distally along the intestinal tract (antrum >duodenum > ileum > colon). Ghrelin expression is alsoobserved in the pancreas, kidney, liver, hypothalamus(mainly ARC), pituitary gland, immune cells (B and T cells,neutrophils) and placenta [70, 80-83].

Despite the robust effect on growth hormone (GH) secre-tion and stimulation of feeding/weight gain upon repeatedadministration [84, 85], ghrelin’s lesser physiological acti-vities in rodents include enhancing gastric motility, gastricacid secretion, gastric and insulin release [86, 87]. Theorexigenic activity of exogenous ghrelin, is thought to bemediated, at least in part, by brain NPY and agouti-relatedprotein (AGRP) [88, 98] as administration of either neutrali-zing antibodies or antagonists to these neuropeptides abol-ishes ghrelins augmentation of feeding in animals [90, 91].In humans, as in rodents, Ghrelin 1) stimulates GH secretionand increases food intake and appetite [92, 93]; 2) secretionlevels are increased in states of negative energy balance,such as fasting and 3) levels rise between meals and fallrapidly after eating [85, 94-96]. While obese human subjectshave much reduced ghrelin levels [85], the post-prandialsuppression of ghrelin after a meal appears to be absent [97]that may contribute to the development of obesity. Indeed,significantly increased ghrelin levels are present in morbidlyobese patients with Prader-Willi syndrome and may be afactor to their extreme hyperphagia [94, 98]

Despite ghrelin knockout only exhibiting a preference forfat utilization [99, 100], the selective knockout of the GHSR-1 in the ARC nucleus of transgenic animals show a leanerphenotype that is characterised by reduced epididymal andmesenteric fat masses in addition to decreased food intakeand body weight [101]. Taken together with the limitedavailability of ghrelin antagonists, such as [D-Lys3]GHRP-6that reduces ghrelin-stimulated food intake in mice [102],and administration (i.c.v.) of polyclonal anti-ghrelin anti-bodies decreases food intake in both fasted and normal darkphase feeding rats [89], emphasize a role of ghrelin in feed-ing. Two recently published papers highlight new interest inghrelin. Helmling and colleagues described the use stableRNA-based compounds, named “spiegelmers” (spiegelmeaning mirror in German) to bind active n-octanoyl ghrelinwith nanomolar affinities, inhibiting ghrelin mediated GHSRactivation in-vitro and growth hormone release in-vivo whenadministered i.v. in rats [88]. The second discloses novelanalogs to human ghrelin with enhanced stability, increasedaffinity for the GHSR, and ability to stimulate weight gain[103]. This second paper also corroborates previous researchindicating that another receptor other than the GHSRmediates the feeding activity of ghrelin [91, 104-107].

All these observations point to the presence of aregulatory axis between the gastrointestinal system and thehypothalamus-pituitary unit, with ghrelin as the intermediaryand makes antagonising the ghrelin GHSR system in theCNS, a viable pharmacological approach to reduce foodintake, and ultimately reduced body weight.


Peptide YY (PYY3-36)

Professor Bloom and colleagues first described PeptideYY3-36’s (PYY3-36) effect on satiety and eating behaviour[108]. Belonging to the same family as NPY, PYY3-36 isreleased postprandially by the endocrine L-cells in the distalileum and colon in proportion to the nutrient content ingestedfrom a meal [109, 110]. Believed to act through the NPY-Y2

autoreceptors, PYY3-36 reduces the activities of NPY contain-ing neurones, which in turn removes the tonic inhibitory tonenormally suppressing the anorexigenic POMC neurones, thusoverall inhibiting food intake [108, 111]. Like CCK, PYY3-36

transiently inhibits food intake, with its actions only lasting3-4 hours post injection [108, 112, 113], though many havefound these results hard to reproducibly replicate [114, 115].Unlike CCK, PYY3-36 does not cause its inhibition on foodintake via neuronal activation in the brainstem, but directlyin hypothalamic neurones that express POMC [112], inparticular those neurones in the ARC [108]. In a one study[108] PYY3-36 given i.p. was able to suppress fasted-inducedfeeding in rats and mice via the NPY-Y2 receptors.

Even though it has been shown that fasting and post-prandial levels of PYY3-36 are significantly lower in obesesubjects [116], variants in the genes encoding for PYY andthe NPY-Y2 receptor are not commonly found in humanswith severe early-onset obesity [117]. Despite this, PYY3-36

is a credible target for human obesity, as it has also beenshown to inhibit appetite and energy intake acutely in bothrodent and humans [35, 108, 116]. Indeed, there is consider-able interest in the role of this gut hormone for suppressionof appetite, according to information in Current Drugs ©IDdb3 (accessed Aug 2004). Amylin Pharmaceuticals Inc., iscurrently developing the human gut PYY3-36 for the potentialtreatment of obesity and diabetes. In January 2004, theysubmitted patents on PYY3-36 for the treatment of obesitywith clinical trials planned. Nastech Pharmaceutical Co. Inc.,has reported positive results from phase I studies with a nasalPYY spray, showing that a mean reduction in food intake forall doses of PYY was 8.2 % in the 9 of 11 responders, 5 ofwhich had greater than a 24 % reduction in food intake. PYYwas also well tolerated with the moderate side effects(nausea, headache and dizziness) being resolved withouttreatment. Merck are now set to co-develop PYY3-36 drugwith Nastech, with Merck assuming primary responsibilityfor clinical and non-clinical studies and regulatory affairs,with Nastech responsible for manufacturing.

BRAIN SIGNALS AFFECTING REGULATION OFMETABOLISM

Neuropeptide Y (NPY)

NPY, a 36 amino acid neurotransmitter [118] is one ofthe most abundant and widely distributed neurotransmittersin the mammalian brain [119] and is an important regulatorypeptide in both the central and peripheral nervous system[120, 121]. Anatomical mapping of the sites involved inNPY-mediated changes in energy homeostasis has localizedthe effects to the PVN, dorsomedial hypothalamic nuclei(DMH), ARC and (LHA), with short projections that termi-nate within the ARC itself [122], suggesting that NPY rele-ased within the ARC acts as a short negative feedback loop

involving NPY autoreceptors. Indeed this inhibition is thefocus of PYY3-36 effects on feeding as discussed previously.

The effects of NPY on feeding are robust and elicited bynanomolar doses injected into the lateral ventricles or hypo-thalamic structures (PVN or LHA) [123, 124], by increasingboth carbohydrate consumption [125, 126] and the size andduration of the first meal, rather than by affecting mealnumber [127]. Chronic administration of NPY into the PVN,with the resulting 3-10 fold increase in hyperphagia demons-trates that NPY is capable of overriding powerful short- andlong-term mechanisms of satiety and body weight regulation[128]. Such effects of NPY are also attributed to NPY’sinsulin secretatogue actions, as insulin receptors located inthe hypothalamus [25, 129] are in near proximity to thosecontaining NPY, and anti-thermogenic properties, which aremediated through the autonomic nervous system.

Inappropriate and unrepressed over-activity of the ARCNPY neurones leads to obesity, as seen in rodent models ofobesity that is due to interruption of the leptin signalingsystem, which is expected in view of leptins inhibitory effecton the NPY neurones. Also, obesity develops through thecombination of decreased BAT activity, thermogenesis andhyperphagia. Raised NPY mRNA levels are present in all ofthese models, with increased NPY levels in the ARC, PVNand DMH and enhanced NPY secretion in the PVN in fattyZucker rat [130]. Moreover, down-regulation of NPYreceptors, specifically of non-NPY-Y1 receptors (putativelyNPY-Y5 receptors) sites in the perifornical LHA, was shownin the fatty Zucker rat [131]. All these observations strengthenthe hypothesis that overactivity of the ARC-PVN projectionplays a role in the hyperphagia and reduced energy expendi-ture that leads to obesity in these models. By contrast,dietary-induced obesity leads to a fall in hypothalamic NPYmRNA levels and an up-regulation of the non-NPY-Y1

receptors in the LHA [132]. This suggests that the hypo-thalamic NPY-ergic activity, particularly of the ARC-PVNneurones, does not drive the hyperphagia associated withexposure to a palatable diet [132], indeed these sameneurones appear to be inhibited, perhaps in an attempt tolimit hyperphagia and weight gain.

It is proposed that multiple NPY receptor subtypeslocated in the brain, are involved in the regulation of foodintake (six characterised and cloned to date), with the notionthat NPY-Y1 receptors mediate the stimulatory effect of NPYon carbohydrate intake and meal size, while NPY-Y2

receptors have the opposite effect of suppressing carbo-hydrate intake [125, 127], with much recent attention comingfrom PYY3-36. Nonetheless, much attention has focused onthe NPY-Y5 receptor subtype as being the ‘feeding’ receptor[133]. However, the fact that the initial highly selectiveantagonists developed as anti-obesity drugs did not havemajor effects on normal feeding or body weight in rats [134],and anecdotal evidence from Phase-2a trials with humanNPY-Y5 receptor antagonists not being successful, havefueled the debate as to the involvement of each of NPY’sreceptors in the regulation of food intake

Melanocortins & Agouti Related Protein (AGRP)

The melanocortin (MC) system has come under intenseacademic and pharmaceutical scrutiny for it’s potential role


in several physiological processes including pigmentation,sexual function, inflammation, cardiac function, memory andother neuronal processes and critically, energy homeostasis.The latter being highlighted as there are several reportslinking alterations in POMC or MC4-receptor genes withobesity in humans [135-137]. For a more detailed role of themelanocortin system in obesity and the current peptide, non-peptide small molecule ligands under development the readeris referred to the recent review [138] and references therein.

MC4-R and, to a lesser extent, MC3-R are the keyreceptor sub-types implicated for pivotal roles in the controlof food intake. MC-receptors are activated by one of severalpeptides derived from the POMC pituitary precursor (includ-ing ATCH, α-, β-, γ-melanocortin stimulating hormone(MSH), β-endorphin [139, 140]. Agouti and AGRP arecompetitive antagonists at the MC4-R and their uniqueendogenous existence confers an extra level of control on thesystem. Evidence for this came from early studies on themutant yellow obese agouti (Ay/a) mouse. In this modelexpression of agouti (a 131 amino acid peptide) in the fairfollicles antagonises the action of α-MSH at the MC-1receptor switching production of eumelanin (brown-black)induced by α-MSH, to phaeomelanin (yellow-red), resultingin the yellow fur pigmentation that is characteristic in suchmice [141, 142]. The obesity in the agouti mouse is aconsequence of agouti’s ectopic expression antagonising thehypothalamic MC-3 and MC-4 receptors in the brain [142],to which α-MSH binds to with high affinity [143, 144].Furthermore, injection of either α-MSH, or its stableanalogue MTII (i.c.v.), inhibits normal feeding, with theextent of α-MSH’s inhibition of feeding being extended tofour mice models of hyperphagia, including fasted obesityprone C57BL/6J, ob/ob, (Ay/a) mice and NPY-inducedhyperphagic mice [145]. By contrast, the selective MC-4peptide receptor antagonists, HS024, HS028 and SHU9119,increased feeding in both normal and satiated rodents [145-147]. Huszar and colleagues (1997) observed that null micefor the MC4-R are hyperphagic and exhibit an obesephenotype [142].

A vast number of agonistic compounds have beenscreened but to date none have been clinically evaluated.MC4-R antagonists block anorexia in rodents induced bycentral administration of both endogenous agonist ligand, α-MSH and other synthetic peptide agonists [148]. Chiron havereported on more encouraging potent and selective non-peptidic molecules [149], with GSK under license fromChiron Corp2, Proctor & Gamble Co. [150-152], Amgen Inc.[153], Novartis, Neurocrine Biosciences Inc., MillenniumPharmaceuticals Inc, Eli Lily & Co [154], and their subsid-iary company Novasite Pharmaceuticals Inc., Merck & CoInc., and the University of California [155], to highlight justa few are all investigating MC receptor ligands for the poten-tial treatment of obesity, cachexia and erectile dysfunction.

Clearly the ability to separate anti-obesity activity fromspontaneous erectile activity will be a significant factor indetermining whether any MC4-R agonist, peptide or not, willbe developed as a weight loss agent. With the high level of

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2 Chiron Corp (2003) Press Release., Chiron and GSK to codevelop MC-4R

validation for the involvement of the MC4-R in energyhomeostasis, it is to be expected that selective small mole-cule MC4-R agonists will eventually be clinically evaluatedfor the treatment of obesity.

Cocaine and Amphetamine Regulated Transcript(CART)

In 1998, the peptide cocaine- and amphetamine-regulatedtranscript (CART) was added to the list of neuropeptidesidentified for affecting feeding behaviour (Fig. 3). CARTmRNA was initially identified using a PCR differentialdisplay technique whose levels in the brain were specificallyinduced by psychomotor stimulants, cocaine and amphet-amine [156]. In the brain regions sensitive to the actions ofthe psycho-stimulants, such as the striatum, CART wasshown to be induced 4 - 5 fold [157]. Further investigationrevealed that CART mRNA was found enriched in thehypothalamus [158], with low levels also present in the eye,adrenal and pituitary, with no other signals detectedelsewhere in the rat [157, 159, 160].

Food restriction decreased CART levels in the ARC[161, 162] and obese animals were shown to have minimalor no expression of CART, fueling speculation that CART isinvolved in the regulation of feeding behaviour.Considerable evidence now exists to suggest that CART isan endogenous satiety factor modulating the actions of NPYand leptin. Studies showed that administration of recombi-nant CART (i.c.v.) inhibited both normal and starvation-induced feeding [162-164] and completely inhibited NPY-induced feeding in rodents [162], whereas central infusion ofanti-CART antibodies resulted in higher food consumption[162-164]. Furthermore, peripheral administration of leptinin obese animals induced the expression of CART in theARC [162].

Unfortunately, the CART peptide fragments, CART (82-103) and CART (55-102), have been shown to onlytransiently decrease the feeding response induced by NPY,with central intra-hypothalamic administration of CARTshown to actually increase feeding [165]. In addition, CARTknockout mice only become obese when fed a high-caloriediet [166]. However, CART, and in particular its putativereceptor (not yet identified) represent another possibletherapeutic target to control food intake and obesity, with thehypothesis that a proportional reduction in NPY and anincrease in CART, control the leptin-mediated suppressionof food intake [164].

Melanin Concenrtating Hormone (MCH)

A role for MCH in feeding was initially demonstrated bystudies showing that its expression is higher in thehypothalamus of both ob/ob and fasted mice and that acutei.c.v. injection of MCH to rats and mice stimulate feeding[167-171]. MCH is a cyclic neuropeptide with rat, mouseand human sharing a high degree of homology with salmonMCH, from which MCH was initially isolated [172-174]. Inrodents and humans prepro MCH is expressed predominantlywith the LH and zona incerta of the CNS [175, 176].

These MCH expressing neurones project widelythroughout the CNS (including pituitary) suggesting MCH tobe involved in many neuronal functions [177], including


Fig. (3). Neural and hormonal mediators of hunger and satiety. Adapted from Metabolic Disorders Study # 11, Obesity, August 2004 withpermission from Decision Resources, Inc., Waltham, MA.


regulating the release of lutenising hormone [178, 179] andadrenocorticotropic factor (ACTH) [180, 181]. In the rat andhuman approximately 70-% of the MCH neurones in the LHand nearly 95 % in the ZI co-express the anorexigenic pep-tide CART [182, 183], whereas in the rat, such populationsof neurones are also in close proximity to orexin cells in theLHA [184]. Two MCH receptors (both GPCR’s) have beenidentified. The MCH-1R (as known as SLC-1 or GPCR 24)[185, 186] is found in rodents and higher mammalian species[187] and is distributed widely throughout the brainincluding the nucleus accumbens, VMH, DMH and ARC ofthe hypothalamus [185, 188, 189], a pattern consistent withthe terminal fields of MCH neurones. The second, MCH-2Rhas only a 37 % homology with MCH-1R [190, 191], butunlike MCH-1R it is not expressed in rodents but is presentin ferrets, dogs, rhesus monkeys and humans [187, 192]. Dueto MCH-2R’s lower and more restricted expression, plus itsdiffering species-species expression pattern it is currentlyunclear as to its role in food intake and energy expenditure.

The most intensively study role for MCH is in the regul-ation of feeding behaviour and energy homeostasis. MCH-expressing neurones in the LH receive afferent projectionsfrom NPY/AGRP and POMC expressing neurones in theARC that respond to circulating leptin and insulin. Speci-fically, fibres for NPY and AGRP, and the anorexigenicpeptide α-MSH innervate MCH neurones, with this innerva-tion most intense in the perifornical area [193]. Chroniccentral infusions of MCH to mice on a high fat diet producepersistent hyperphagia together with increased adiposity,hyperinsulinaemia and hyperleptinaemia [194, 195] whereasi.c.v. administration of a potent MCH-1R agonist to ratsproduced similar effects [196]. Intriguingly, no sustainedhyperphagia is produced following repeated injections [197].Transgenic mice overexpressing MCH are hyperphagic,obese and insulin-resistant [198]. Conversely, MCH knoc-kout mice have a lean phenotype characterised by hypo-phagia and increased energy expenditure [199]. Interestingly,mice lacking both MCH and leptin, despite consumingsimilar amounts of food, are leaner compared to ob/ob micedue to their increased energy expenditure and improvedglucose tolerance [200]. This hypermetabolic phenotype wasalso observed in the MCH-1R null mice, despite beinghyperphagic, they are lean and have decreased leptin andinsulin levels that are similar to the MCH deficient mice[197, 201]. Such hyperphagia is not explained by alterationsin the expression of NPY, AGRP, orexin or CART andPOMC, nor in the tone of endogenous orexigenic signals asevidenced by a normal response to exogenously administeredNPY or AGRP [197, 201].

The promising pharmacology of the MCH-1R has madethis an attractive target for the development of small-molecule antagonists by several companies. A recent reviewdetails the advances in MCH-1R antagonists [202]. High-lighting just a few of the in-vivo effects, Merck have des-cribed the activity of a peptide MCH-1R antagonist [196],while Takeda [203] and Synaptic [204] have small molecularweight antagonists. All reverse the orexigenic effects ofMCH-1R agonists in rats. Arena Pharmaceuticals Inc., havealso reported an orally bioavailable MCH receptor modulatorable to reduce body weight equivalent to that of sibutramine3.On a cautionary note, increased heart rate has been observed

in MCH-1R null mice, and as with NPY receptors, the widedistribution of MCH-1R in the CNS indicates the likelihoodof multiple roles for MCH-1R signaling [189].

Glucagon-Like Peptide 1 (GLP-1)

Extra hypothalamic targets may include the neurones ofthe medulla that express the gut peptide, GLP-1, an alter-native cleavage product of the preproglucagon gene. Pre-proglucagon mRNA and GLP-1 like immunoreactivity havebeen shown in cell bodies of the nucleus of the solitary tract,that project to the PVN, one of the main feeding controlcentres in the hypothalamus [205]. Specific GLP-1 receptorsare also located in the PVN and amygdala [206] and GLP-1has been shown to reduce food intake and body weight wheninjected centrally in rats [207]. More recently both centraland peripheral administration of exendin-4 (a small peptideagonist of GLP-1 that is isolated from salivary venom of theGila monster) reduced food intake in rats4.

GLP-1 is convincingly implicated as a short actingendogenous, meal related satiety peptide [208], as it has beenshown to interact with other regulators of food intake, as itcompletely inhibits the effects of NPY on food intake [209].The role of leptin in GLP-1’s actions is mixed. Even thoughprolonged treatment in the diabetic db/db mice with exendin-4 had no effect on body weight [210], contrary data hasshown that both lean and obese (fa/fa) Zucker rats havereduced food intake and body weight after chronic treatmentwith exendin-4 [211, 212]. GLP-1 has also been shown toactivate the hypothalamo-pituitary adrenocortical axisthrough stimulation of CRH neurones, and this activationmay also be responsible for the inhibition of feeding beha-viour [206].

However, the actions of GLP-1 on feeding are too short[207] to play a central role in the long-term regulation offeeding (possible due to the very short half-life of GLP-1 inthe circulation system) and are thought to mediate post-prandial satiety. Despite this, GLP-1 angonism throughincreased stability and biological activity, or inhibition ofdipeptidy peptidase IV (DPP IV) that rapidly inactivatesGLP-1 may prove promising therapies for the treatment ofobesity and also diabetes. Amylin and Eli Lily [213]presented data at various meetings5 showing subjects treatedwith Exendin-4 (Exenatide and Exenatide-LAR), as havingsignificant weight loss6, with such data reflected in C57Bl/6Jmice fed a high fat diet7. Several competitors including NovoNordisk’s Liraglutide and again Amylin Pharmaceutical withPramlintide may offer effective prospects for anti-obesitytreatment.

____________________________

3 Bjenning, C.A.; Whelan, K.; Creehan, K.; Gonzalez, L.; Al-Shammal, H.; Thomsen,W.J.; Tran, T.; Semple, G.; Funakoshi, T.; Nishiguchi, M.; Kanuma, K.; Sekiguchi, Y.and Chaki, S. (2004) Soc. Neurosci. Abst., 34, (San Diego) Abs. 629.8.4 Al-Barazanjika, K.A.; Buckingham, R.E.; Tadayyon, M. and Arch, J.R.S. (1998) Int.J. Obesity, 22, suppl 23, S73.5 64th ADA scientific meeting in Orlando, USA; 40th EASD Annual meeting inMunich, Germany; SMi’s annual Diabetes meeting in London, UK.6 Mack, C.M.; Wilson, J.K.; Young, A.A. and Parkes, D.G. (2004) Diabetes, 53, Abs1717-P.7 Moore, C.X.; Jodka, C.M.; Hoyt, J.A.; Young, A.A. and Sams-Dodd, F. (2003)Diabetes, 52, suppl 6, Abs 1691-P.


Orexins/Hypocretins

Several hypothalamic neuropeptides function in thecentral control of food intake, but until the discovery oforexins (also known as hypocretins) only MCH was speci-fically produced in the LHA [214]. Even though centraladministration of either peptide stimulates food intake 215,216], orexins and MCH are not co-localised in the lateralhypothalamus (LH) [187]. Orexin-A and orexin-B (33 & 28amino-acids, respectively) are found to be abundant in the rathypothalamus, medulla-pons, and midbrain-thalamus, withmoderate expression found in the cerebral cortex, with theirmRNA being restricted to cell bodies present in the LH [187,216]. However, levels of orexin-B were found to be muchgreater than that of orexin-A in the LH, as well in a lesserextent in other brain regions [217, 218]. Immunohistochem-ical mapping of orexins have demonstrated that orexin-containing fibres project to selective nuclei in the hypo-thalamus, central gray, dorsal raphe nucleus of the midbrain,as well as the locus coeruleus of the medulla [219]. Thiswould explain the high orexin contents of the midbrain-thalamus, medulla-pons and cortical mantle that receiveabundant projections from the LH [219]. After 48 hourfasting, both orexin-A and -B exhibited a trend to increase inthe LH, with prepro-orexin mRNA being up-regulated 2.4fold after [216]. There are contradictory data with respect toeither of the orexins’ involvement in feeding. Both orexin-Aand orexin-B equally and dose-dependently stimulated feed-ing in rats when injected in the cerebral ventricles [216].However, single injections of orexin-A into the thirdventricle of C57BL/6J mice 3 hours into the light phaseincreased metabolic rate but only slightly stimulated feedingand orexin-B had no effect [220]. Despite the speciesdifferences these data suggest that orexins are more likely tobe involved in control of energy metabolism rather than foodintake. Further to this notion, Cai and colleagues (1999)observed an increase in hypothalamic prepro-orexin mRNAonly in rats fasted for 48 hours, or when rats were madeacutely hypoglycaemic but only in the absence of food.Intriguingly, chronic 6-day food restriction, streptozotocin-induced diabetes, hypoglycaemia in which rats were allowedto feed, neuroglycopenia provoked by 2-deoxy-D-glucose,and dietinduced obesity all failed to bring about changes inorexin mRNA levels. Moreover, no obvious relationship oforexin expression was observed with respect to insulin,leptin or body fat mass observed [221].

The roles of the orexins remain unclear, but the abovedata suggests that orexin peptides may be linked to nutri-tional status of the circadian cycle together with the state ofhunger rather than to satiety. Orexins also are stronglyinvolved in the sleep-wake patterns, being documented ascausing narcolepsy in animal models when its actions areblocked, therefore, shedding doubt as to its direct effects onfeeding [222, 223].

Serotonin (5HT)

Although the brain 5-HT system has been known sincethe 1970’s to be intrinsically linked to feeding, it is onlyrecently that we have been able to define the receptor sub-types responsible for and understand 5HT-mediated hypo-phagia.

It is believed that fourteen 5-HT receptor subtypes existand these are conveniently divided into 7 classes, 5HT1 to5HT7. Of these, research into potential treatments for obesityin terms of food intake has centred upon 5HT1A, 5HT1B,

5HT2A and 5HT2C with the latter currently the best candidate.

5HT1A is an autoreceptor that reduces 5HT release ontopost-synaptic 5HT receptors, activation of which, in rats,results in hyperphagia [224, 225]. The precise role of 5HT1B

in the control of feeding is still up for discussion. Althoughactivation of 5HT1B receptors has been shown to attenuatefeeding in animal models [226-228] and transgenic 1Breceptor knockout mice gain more weight than their WTlittermates [229], these results are in no way universal [230].Hypophagia associated with activation of 5-HT2A receptors isthought to be as a result of non-specific behavioural disrup-tion, namely sedation and nausea [231].

More importantly, there is considerable evidence that 5-HT2C specifically mediates hypophagia in both animals andman, and appears to be a consequence of increased satiety.Recent studies have concentrated on the 5-HT2C receptors.The presence of 5-HT2C receptor mRNA in the choroidplexus, various cortical areas, hippocampus, amygdala,nucleus accumbens, substantia nigra and brainstem nuclei inthe rat, monkey and humans [232, 233] has been confirmedby immunohistochemical analysis and in situ hybridizationexperiments. Moreover, 5-HT2C receptor expression wasnoted in the VMH in humans [234]. Immunohistochemicalstudies, rather than autoradiographical studies of the rat brainhave reported 5-HT2C receptor expression in the ARC,DMH, PVN and LH nuclei [235-237]. There is some evi-dence also to suggest a role for 5-HT2C receptor mediatedhypophagia in hypothalamic control of feeding. It has beenhypothesised that 5-HT2C receptor activation of ARC POMCneurones results in α-MSH activation of MC3 and MC4receptors to produce the hypophagic effect [238]. Support forthis came in another study that revealed that d-fenfluramine-mediated hypophagia is prevented by prior treatment ofanimals with the MC receptor antagonist, SHU9119 [238]. Ithas also been reported that sibutramine indirectly causesMC3/4 receptor down-regulation in certain hypothalamicnuclei8 thereby adding further evidence for downstream acti-vation of the ARC melanocortin system by 5-HT2C receptors.

Equally efficacious as both sibutramine and dexfenflur-amine is AR-10A, an orally active 5-HT2C receptor agonistpresented by Arena Pharmaceuticals Inc., acute adminis-tration of which dose-dependently reduced food consump-tion in free-fed SD rats, an effect reversed by the 5-HT2C

antagonist, SB-242084, but not by a 5-HT2A antagonist,according to information in Current Drugs © IDdb3(accessed August 2004).

There are a number of compounds which while notspecific act more selectively as 5-HT2C receptor agonists.Agonists tri-fluoromethylphenylpiperazine (TFMPP) and m-chlorophenylpiperazine (mCPP) [239-243], have been shownto increase the latency to feed, reduce the size of the firstmeal following treatment and slow the rate of feeding [226,

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8 King, P.J. and Berwaer, M. (2003) NAASO meeting, Ft Lauderdale, FL, USA.Obesity Research, 11, Abs. 124-OR


227, 244]. A recent review by Bickerdike, 2003 [245]describes in more details MK-212, Org 37684, Ro 60-0175,PNU-22394 and VER 3323, all of which acutely attenuatefood intake by satiety processes in animal models [246-2529,

10]. However, this does not mean that a prolonged drop infood intake and/or loss of body weight results from chronictreatment with these compounds. The possibility of receptordesensitization in-vitro [253, 254] as well as in-vivo has beenwell documented [255-259], even though chronic adminis-tration doesn’t appear to promote tolerance in drug-inducedhypophagia [260, 261].

Cillary Neurotrophic Factor (CNTF)

An alternative approach to finding small moleculeagonists for the leptin receptor is to mimics leptin’s actions.This approach has been adopted by Regeneron, using agenetically engineered version of human CNTF (Axokine)for the treatment of obesity (Fig. 1).

CNTF is a naturally occurring protein that functions as aregulator of cell differentiation and survival, and is expressedin hypothalamic regions of the brain that effect energybalance. Although the mechanism by which CNTF adminis-tration leads to weight loss in humans and animals [262-264]is still unclear, it is believed to work through leptin-likeindependent mechanisms. CNTF receptors are present in theARC of the hypothalamus and upon activation, signal tosatiety centres in the brain [265]. Studies in ob/ob, db/db andDIO mice showed that CNTF and CNTFAx15 (Axokine, asecond generation analog) can suppress food intake withoutincreasing NPY levels and causing muscle wasting [263-265], indicating that CNTF acts, at least in part, downstreamof the leptin receptor, bypassing the leptin resistance. Onestudy showed that CNTF affects adipocyte signaling inrodents and induces insulin action in vitro [266] suggestingthat the ability of CNTF to induce weight loss is not solelymediated by the CNS.

However, due to the fact that approximately 70 % ofusers in clinical trials formed neutralizing antibodies duringtreatment, according to information in Current Drugs ©IDdb3 (accessed August 2004), it will make this drugsmarket more selective.

CONCLUDING REMARKS

One of the problems to unraveling the underlyingmechanisms associated with obesity, is the extrapolation ofexperimental data from genetically and dietary-inducedobese rodents to humans. The recognition that invasivehypothalamic tumours can cause disruption of appetite andthermoregulation, just as lesions of the VMH or LHA resultin rodents becoming obese or anorectic, respectively,demonstrates that the hypothalamus plays an important roleof controlling food intake within humans.

____________________________

9 McCall, R.B.; Franklin, S.R.; Hyslop, D.K.; Knauer, C.S.; Chio, C.L.; Haber, C.L.and Fitzgerald, L.W. (2001) Soc. Neurosci. Abstr., 27, 309.2.10 Vickers, S.P.; Bass, C.; Bickerdike, M.J.; Kennett, G.A. and Dourish, C.T. (2002)5th IUPHAR Satellite Meeting on Serotonin, Acapulco.

Keeping the brain’s ‘feeding’ centres in mind, muchattention now focuses on the myriad of interacting systemscontrolling both the short-term (meal-to-meal) and long-term(energy balance equilibrium) signals that converge on thehypothalamus, working in partnership to control feedingbehaviour, as depicted in Fig. (3). However, due to numerouspathways involved in weight regulation, many of which stillremain to be fully explored, up and coming drug targets arelikely be effective for only certain patient groups.

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