Leptin signaling in the hypothalamus: emphasis on energy … · 2017-07-14 · Leptin signaling in...

Leptin signaling in the hypothalamus: emphasis onenergy homeostasis and leptin resistance

Abhiram Sahu*

Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S829 Scaife Hall, 3550 Terrace Street,Pittsburgh, PA 15261, USA

Abstract

Leptin, the long-sought satiety factor of adipocytes origin, has emerged as one of the major signals that relay the status of fatstores to the hypothalamus and plays a significant role in energy homeostasis. Understanding the mechanisms of leptin signaling inthe hypothalamus during normal and pathological conditions, such as obesity, has been the subject of intensive research during thelast decade. It is now established that leptin action in the hypothalamus in regulation of food intake and body weight is mediated bya neural circuitry comprising of orexigenic and anorectic signals, including NPY, MCH, galanin, orexin, GALP, a-MSH, NT, andCRH. In addition to the conventional JAK2–STAT3 pathway, it has become evident that PI3K–PDE3B–cAMP pathway plays acritical role in leptin signaling in the hypothalamus. It is now established that central leptin resistance contributes to the devel-opment of diet-induced obesity and ageing associated obesity. Central leptin resistance also occurs due to hyperleptinimia producedby exogenous leptin infusion. A defective nutritional regulation of leptin receptor gene expression and reduced STAT3 signalingmay be involved in the development of leptin resistance in DIO. However, leptin resistance in the hypothalamic neurons may occurdespite an intact JAK2–STAT3 pathway of leptin signaling. Thus, in addition to defective JAK2–STAT3 pathway, defects in otherleptin signaling pathways may be involved in leptin resistance. We hypothesize that defective regulation of PI3K–PDE3B–cAMPpathway may be one of the mechanisms behind the development of central leptin resistance seen in obesity.! 2003 Elsevier Inc. All rights reserved.

1. Introduction

1.1. Historical background

Obesity is one of the major health hazards in humans,particularly in western society. Remarkably in mosthumans body weight is maintained in stable condition.Positive energy balance as a result of less energy ex-penditure as compared to energy intake leads to thestorage of energy in the form of fat. Although cumula-tive evidence gathered mostly over the last two decadessuggest that body weight is regulated by a complex cir-cuitry involving both central and peripheral factorsworking primarily in the brain, particularly in the hy-pothalamus; the idea that some factors originating in theperiphery relay the status of body fat stores to the brainhas originated from the days of Kennedy, almost 50

years ago [160]. In 1953, Kennedy hypothesized that thehypothalamus senses some peripheral factors that pro-vide the information about the body fat stores, and thehypothalamus would then transduce this information tochange food intake to compensate for changes in bodyfat content. Subsequent studies using parabiosis exper-iments in rats, Hervey showed that when one of theparabiotic partner made obese by a lesion in the ven-tromedial hypothalamus, the intact partner became an-orexic and lean [132]. These results suggested that someblood-borne factor produced by the increased fat massacted to induce satiety in the intact partner. Further-more, its lack of effect in the lesioned animals alsosuggested that the action of this factor(s) in the hypo-thalamus is essential for the maintenance of normalbody weight.

In the 1970s, Douglas Coleman!s finding that reces-sive mutations in the mouse ob and db genes resulted inobesity and diabetes [60], provided a critical clue aboutthis peripheral factor that regulates body weight. Usingparabiosis experiments with ob/ob and db/db mice,

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Frontiers in Neuroendocrinology 24 (2004) 225–253

Frontiers inNeuroendocrinology

Coleman concluded that the blood-borne factor wasencoded in the ob gene and the receptor for this factorwas encoded in the db gene [60]. However, the productof the ob gene was not discovered until 1994, whenJeffrey Friedman!s team, using positional cloning,identified and characterized the ob gene and its product,leptin (from the Greek leptos¼ thin). They identifiedleptin as a 16 kDa protein produced primarily in whiteadipose tissue [367]. Although subsequent studies havedemonstrated that leptin is produced in small amount inother tissues such as the placenta [207], stomach [10],pituitary [148,251] and the hypothalamus [221], the roleof this extra adipose tissue-derived leptin is not clearlyunderstood. After the discovery of leptin receptor byTartaglia!s group in 1995 [327], physiological role ofleptin has been the subject of intensive investigation andhas been appreciated not only in regulation of bodyweight, but also in a variety of physiological functionssuch as reproduction, bone formation, and cardiovas-cular systems, etc.

1.2. Leptin physiology

As expected, cumulative evidence suggests that leptinsignals nutritional status to key regulatory centers in thehypothalamus [92,153,294,357] and it has emerged as animportant signal regulating body weight homeostasisand energy balance [42,102,124,243,347]. Mutations thatresult in leptin deficiency are associated with massiveobesity in humans as well as rodents [102,220]. Centralor peripheral administration of leptin decreases foodintake and body weight in a variety of animals, includ-ing rats, mice, and monkeys [102,267,326]. In normalmice, leptin administration reduces weight and correctsdiet-induced obesity [42,124,243]. Leptin treatment hasbeen shown to normalize feeding, reduce body weightand initiate puberty in a leptin deficient girl [99]. It iswell established that leptin plays an important role inthe long-term maintenance of body weight. In addition,the evidence that leptin mRNA levels are decreasedfollowing food deprivation and return to normal afterrefeeding [200,277]; and a rapid decrease of plasmaleptin levels after a short-term fast followed by a rapidrecovery after refeeding in man [34,65,165,166]; the ex-istence of diurnal rhythm of plasma leptin entrained tomeal timing in man [288]; and the evidence that circu-lating leptin levels increase within 4 h of natural feedingin rat [359] suggest that leptin may be involved in dailyfood intake and short-term regulation of body weight.Paradoxically, in the majority of cases, human obesitycannot be attributed to defects in leptin or its receptor[58,65,66,115,209,220], and serum leptin levels are sig-nificantly higher in obese humans relative to non-obesehumans [43,64,65,129,193,202,292], and leptin adminis-tration shows very limited effects in obese people [134],suggesting a state of leptin-resistant in obese individuals.

In addition to its role in normal regulation of foodintake and body weight, leptin treatment correctsobesity related disorders, including hyperglycemia,hyperinsulinemia, and sterility in ob/ob mice[14,42,49,124,243], and blunts the starvation-inducedabnormalities in the gonadal, adrenal, and thyroid axesin lean mice [1]. Furthermore, leptin!s role in repro-duction is becoming increasingly apparent. For exam-ples, leptin has been shown to accelerate puberty in mice[2,50]. Also transgenic mice over expressing leptin dis-play accelerated puberty [364]. Leptin reverses the sup-pression of sexual maturation induced by fasting inrodents [54], and the effects of fasting on pulsatile se-cretion of luteinizing hormone [226]. Leptin also stim-ulates gonadotropin-releasing hormone secretion in vivo[346]. However, leptin!s role in primate puberty appearsto be permissive, because there is no evidence of in-creased circulating leptin before the onset of puberty[247–250,274]. In a recent study, we have shown thatcontinuous peripheral infusion of leptin failed to inducegonadotropin-releasing hormone secretion in pre-pubertal monkeys [15]. Other central action of leptinincludes regulation of bone formation [83,157] and an-giogenesis [36,309].

In most part, leptin!s role in various physiologicalfunctions, including food intake and body weight regu-lation, reproduction, bone formation, and angiogenesisappears to be mediated through the hypothalamus. Inthis review, I will however focus on the mechanisms ofleptin action in the hypothalamus with regard to foodintake and body weight regulation, obesity, and leptinresistance.

2. Hypothalamus as the major site of leptin action

From the lesion studies by Hetherington and Ranson[133], and by Anand and Brobeck [7], it has been es-tablished that lesion in the ventromedial hypothalamuscauses hyperphagia and obesity, and lesion in the lateralhypothalamus causes aphagia and even death by star-vation. These studies clearly suggested the hypothala-mus as the primary center for regulation of food intakeand body weight with the ventromedial nucleus as the‘‘satiety center’’ and the lateral hypothalamus as the‘‘feeding center.’’ Since then a large body of evidencesuggest that neural circuitry comprising of orexigenicand anorectic signals reside in the hypothalamus, andintricate regulation of this circuitry is critical for normalfood intake and body weight in the individual. It ap-pears that this circuitry senses the status of body energystores from peripheral signals, such as leptin and insulin,and modifies its activity accordingly.

Along this line, accumulated evidence clearly indi-cates that hypothalamus is one of the major sites ofleptin action. First, central injection of leptin is more

226 A. Sahu / Frontiers in Neuroendocrinology 24 (2004) 225–253

potent than peripheral administration in reducing foodintake and body weight [42]. Within the hypothalamus,leptin is most effective in the arcuate nucleus (ARC) andventromedial nucleus (VMN) areas in reducing foodintake and body weight [146,279]. Second, leptin re-ceptors are present in the hypothalamus and choroidplexus [199,203,291,327]. In this regard, the long-formof the Ob receptor (Ob-Rb) that is thought to be crucialfor intracellular leptin signal transduction [51,182,328]has been localized in various hypothalamic sites, in-cluding ARC, VMN, dorsomedial nucleus (DMN), lat-eral hypothalamus (LH) and paraventricular nucleus(PVN), which are known to regulate food intake andenergy homeostasis [51,214]. Systemic or central injec-tions of leptin also activate neurons in these hypotha-lamic sites [91,92,122,214,291]. Third, inhibition ofleptin signaling in the hypothalamus due to mutation inleptin receptors is the cause of obesity in db/db mice[56,328] and Zucker fa/fa rats [56,246]. Fourth, leptincan cross the blood–brain barrier [12,292]. Fifth, lesionsin the hypothalamus make the animal become obese andunresponsive to exogenous leptin [80,168,280]. Sixth,neuronal specific knockout of Ob-Rb results in obesity[59]. Finally, leptin receptor mutations (although rare)in humans lead to morbid obesity [58]. Thus, the leptinsignal to the hypothalamus is obligatory for normalfood intake and body weight regulation; and any alter-ation in leptin action in the hypothalamus due either todefect in leptin transport and or leptin resistance inleptin target neurons would lead to dysregulation ofbody weight seen in obesity.

3. Neuronal targets of leptin action

The hypothalamus produces an array of orexigenicand anorectic peptides that constitute a major part ofthe neural circuitry regulating ingestive behavior andbody weight [153,272,294,357]. Evidence accumulatedduring the last several years suggest that leptin!s effectsare mediated through the activity of several neuropept-idergic neurons of both orexigenic and anorectic in na-ture in specific site of the hypothalamus. Leptin sensitiveneurons include those that produce neuropeptide Y(NPY), agouti-related protein (AgRP), melanin con-centrating hormone (MCH), galanin, orexin, a-mela-nocyte stimulating hormone (a-MSH), neurotensin(NT), corticotropin-releasing hormone (CRH), and co-caine- and amphetamine-regulated transcript (CART),etc.

3.1. Orexigenic peptide-producing neurons as leptintargets

Among the orexigenic neuronal systems, NPY hasbecome a prime candidate implicated in mediating leptin

action in the hypothalamus, because: (a) NPY is themost potent endogenous orexigenic signal in variousmammals, including rat [152,153,263,272,357]; (b) NPYneuronal activity is enhanced in hyperphagia observedin experimental diabetes [264,266,348,349], as well as inseveral experimental and genetic models of obesity[153,278,294,357], and (c) continuous or repeated cen-tral administration of NPY produces hyperphagia, bodyweight gain and ultimately, obesity [47,315,366]. Ac-cordingly, leptin decreases hypothalamic NPY gene ex-pression [267,291,317] and NPY release from thehypothalamic explants [317], leptin opposes the actionof NPY on feeding [268,310], and NPY may act an-tagonistically against anorectic effect of leptin [16,96].Furthermore, NPY neurons express Ob-Rb [215] andsignal transducer and activator of transcription 3(STAT3) [121], suggesting a direct action of leptin onthese neurons. Finally, genetic knockout of NPY re-duces hyperphagia and obesity in ob/ob mice indicatingthat full response to leptin deficiency requires NPYsignaling [97]. Interestingly, mice lacking NPY show noabnormality in food intake and body weight regulation,and in fact these mice are more sensitive to leptin [96],suggesting further the antagonism of leptin action byNPY. This also indicates redundant signaling mecha-nisms in the hypothalamus, such that, in the absence ofNPY, leptin acts through other pathways to maintainnormal food intake and body weight.

NPY neurons also co-express AgRP [39,120]; and likeNPY, AgRP over expression results in obesity [116] andAgRP is up-regulated in obese and diabetic mutant mice[308]. Importantly, AgRP is an endogenous melano-cortin antagonist [239], and leptin decreases AgRPmRNA levels in the hypothalamus [84,167,218,355].These findings suggest that AgRP inhibition may be animportant mechanism by which leptin can enhance itsanorectic effect in the hypothalamus.

Recently, MCH (melanin-concentrating hormone)neurons have received significant attention with regardto feeding and body weight regulation, and therefore fora target of leptin signaling. MCH is primarily expressedin the lateral hypothalamus (LH). Central MCH ad-ministration stimulates feeding [258], MCH synthesis inthe hypothalamus is elevated by both energy restrictionand leptin deficiency [252], MCH-knockout mice arehypophagic and have increased metabolic rate, despitelow leptin levels, and are excessively lean [304]. MCHoverexpression in the hypothalamus causes obesity[197], and MCH knockout in ob/ob mice results in de-creased body weight mainly due to increased energyexpenditure without any change in food intake [298].Along this line, our study shows that leptin not onlydecreases MCH gene expression but also reduces foodintake induced by MCH in rat [267,268]. MCH is also afunctional melanocortin antagonist in the hypothalamus[196]. Functional interactions between MCH, NPY, and

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anorectic peptides occurs in the hypothalamus [332].Thus, MCH neurons appear to function as feedingstimulant downstream of leptin signaling.

Among other orexigenic neurons, galanin, and orexinproducing neurons have been shown to play importantrole in food intake and body weight regulation[183,276,319]. Galanin and orexin can both elicitstrong feeding behavior when injected centrally[81,175,176,268,276,287,320] and are expressed in hy-pothalamic areas associated with feeding and metabolicregulations [48,78,13,183,245,276,301]. However, no ormodest effect of orexin on feeding has also been reported[85,130]. While galanin-positive neurons are present inthe PVN, PFH, LH, and ARC [48,301], orexin-positivecells are localized in the LH and zona incerta[78,138,245]. Similar to NPY neurons, orexin and gala-nin producing neurons exhibit reciprocal changes intheir activities in response to fasting and refeeding[183,219,276]. Furthermore, galanin and orexin neuronsexpress leptin receptors [122,123,138] and STAT3 [123];and leptin administration decreases galanin and orexinmRNA in the hypothalamus [194,267]. Our study alsoshows that leptin not only decreases galanin gene ex-pression [267], it also reduces food intake induced bygalanin [268]. Other evidences, such as (a) orexin neu-rons establish synaptic contacts with NPY and POMCproducing neurons in the ARC [38,88,118,138] and theseARC neurons establish reciprocal contact with the or-exin cells [38,88]; (b) orexin and NPY interacts with eachother in stimulating feeding [82,145,147,271,361], (c)orexin and MCH neurons make synaptic contacts in theLH [21]; and d) galanin stimulates NPY secretion fromhypothalamic neurons [27], suggest that interactionsbetween the neurons of the ARC, LHA, and PVN play acritical role in energy homeostasis, and consequentlyleptin signaling in the hypothalamus.

There are some evidences that suggest a potential roleof recently identified galanin-like peptide (GALP) inmediating leptin action in the hypothalamus. GALP, a60 amino acid peptide, was isolated from porcine hy-pothalamus by Ohtaki et al. [237] on the basis of itsability to bind and activate galanin receptors in vitro.Amino acids 9–21 of GALP are 100% homologous tothe biologically active N-terminal (1–13) portion ofgalanin [237]. The role of GALP in energy homeostasishas been recently revealed. GALP is highly expressed inthe ARC [149,150,161,179,323]. Although central ad-ministration of GALP has been shown to increase foodintake [180,208], and PVN GALP stimulates feeding[301] and increases NPY release [302], GALP is alsoreported to decrease food intake [170]. Nevertheless,GALP neurons express leptin receptor [73,323], leptinincreases GALP-expressing cells in the ob/ob mice [150],fasting decreases the number of GALP-expressing neu-rons and leptin administration restores GALP express-ing cells in fasted rats [149]. In addition, leptin induces

GALP mRNA levels in the hypothalamus [174], andGALP mRNA levels are decreased in Zucker rats and indb/db and ob/ob mice [174]. These findings clearly sug-gest that leptin modify GALP neuronal activity in thehypothalamus. Recent reports also suggest that GALPco-expresses with a-MSH [324] and orexin receptor-1[325] in some of the ARC neurons. Thus, GALP mayplay an important role in regulation of feeding behavior,and therefore, regulation of GALP by leptin may bepotentially important in energy homeostasis.

Recent demonstrations that ghrelin, a 28-amino acidpeptide produced predominantly in the stomach [229], isalso produced in a group of neurons adjacent to the thirdventricle between the dorsal, ventral, paraventricular,and ARC, and that these neurons send efferents ontoNPY, AgRP, POMC, and CRH neurons suggest thatcentral ghrelin may play an important role in the neuralcircuitry controlling energy homeostasis [71]. In addi-tion, peripheral or central injection of ghrelin inducesfood intake [181,229,333,358], and ghrelin receptors arelocalized in the hypothalamus, particularly in the NPYneurons [353]. Ghrelin induces food intake by engagingNPY/AgRP neurons in that antibodies or antagonists ofNPY or AgRP reverses the effect of ghrelin on feeding[155,229,306], and ghrelin induces NPY/AgRP gene ex-pression [9,154,155,300] and c-Fos expression in theNPY neurons [345]. Recent evidence also suggest thatorexin pathway may mediate ghrelin!s action in the hy-pothalamus [331]. Importantly, ghrelin blocks the effectsof leptin on feeding, and prior leptin administration at-tenuates the effect of ghrelin on feeding [306], suggestinga functional interaction between leptin and ghrelin.Furthermore, Kohno et al. [164] have demonstratedthat ghrelin directly interacts with NPY neurons in theARC to induce Ca2þ signaling, and leptin attenuatesghrelin-induced Ca2þ increase. Thus, it appears that theregulation of ghrelin!s effect on hypothalamic neurons,particularly NPY/AgRP neurons, may be one of theimportant mechanisms by which leptin controls foodintake and body weight. It is however unknown whetherleptin modify ghrelin gene expression in the hypothala-mus—another possible mechanism by which leptin maytransduce its anorectic action in the hypothalamus.

3.2. Anorectic peptide-producing neurons as leptin targets

One of the major neural systems involved in leptinsignaling in the hypothalamus has been the melanocortinsystem. Because the CNS melanocortin system exertseffects of opposite to NPY, this system has been studiedextensively with regard to food intake and body weightregulation and therefore to the leptin signaling [35,61,62,69,70,98,153,217,293,329]. The endogenous melanocor-tin implicated most strongly in the control of food intakeand body weight is a-MSH, a product of POMC neurons[61,294], which binds with high affinity to melanocortin


receptor-3 (MC3) and MC4 [61,62]. Furthermore, MC3and MC4 receptors are highly expressed in the hypo-thalamus [223], mice lacking MC4 receptor become ob-ese [142], MC4 receptor mutation causes obesity[142,223,336,337,362], and MC4 antagonist reverses theeffect of leptin on feeding [297]. The regulation of POMCneurons by leptin has been evident in rats and mice[293,294,329]. Accordingly, fasting that decreases leptininduces POMC gene expression [293,329], POMCmRNA levels are reduced in ob/ob mice and leptin ad-ministration to these animals reverses this effect [217].However, a small decrease [267] or no change [339] inhypothalamic POMC mRNA levels following centraladministration of leptin has been reported in ad lib fedrats, although leptin increases POMC mRNA levels inFD rats [293]. Also, chronic sc leptin infusion had noeffect on POMC mRNA levels in ad lib fed rats [3]. Be-cause POMC gene produces both a-MSH and b-endor-phin, which have opposite effects on feeding, one beinganorectic and the other orexigenic, the effect of leptin onPOMC mRNA could vary depending on the experi-mental situation. Nevertheless, POMC producing neu-rons express leptin receptor [53] and STAT3 [121], andleptin induces suppressor of cytokine signaling-3(SOCS3) and c-Fos in these neurons [90], suggesting di-rect action of leptin on POMC neurons. One of themechanisms by which leptin acts on the POMC neuronsis by reducing inhibitory c-aminobutyric acid (GABA)release from the NPY neurons [70]. In addition, NPY/GABA cells innervate POMC neurons [137], suggestinginteraction between NPY/GABA and POMC neurons.As mentioned previously, since NPY neurons co-expressAgRP [39,120], an endogenous melanocortin antagonist[239], it appears that stimulation of POMC neurons byleptin is a result of direct action as well as by inhibition ofNPY/AgRP neurons. Because orexin also excites GAB-Aergic neurons in the ARC [40], it is possible that leptin!seffect on POMC could also be mediated indirectly bydecreasing orexin neuronal activity. Recent studies fur-ther show that POMC neurons are glucose responsiveand express K-ATP channels [144]; and leptin activatesK-ATP channel in the POMC neurons [70]. These find-ings along with the demonstration that mutation inPOMC gene results in obesity [173], provide furtherevidence in support of a significant role of POMC inmediating leptin action and energy homeostasis.

The importance of leptin regulation of POMC neu-rons in the hypothalamus is further demonstrated by thefact that POMC neurons express CART, a potent in-hibitor of food intake [89,172,178,341]. CART mRNAin the hypothalamus is reduced in the leptin-deficient ob/ob mice [172] and fasted rats [172]; and leptin normalizesCART mRNA in these animals [172]. Leptin inducesSOCS3 mRNA in CART neurons. CART neurons ex-press leptin receptors [89]. Leptin induces Fos expres-sion in the hypothalamic CART neurons [89]. Thus,

these findings altogether suggest a major role of POMC/CART neurons in mediating leptin!s action in the hy-pothalamus. In a recent study, Xu et al. [360] havedemonstrated that brain-derived neurotrophic factor(BDNF) regulates energy balance downstream of MC4receptor. Because central BDNF administration de-creases food intake [228], and because MC4 agonist,MT11, increases BDNF mRNA in the VMH of fooddeprived mice [360], it is likely that MC4 mediated leptinaction may involve BDNF.

Because NT is an important centrally acting anorecticsignal [24,45,162,186,198,260,314,352,354] and it is lo-calized in those areas of the hypothalamus that are im-plicated in food intake and body weight regulation[94,143,151], we investigated the role of NT neurons inmediating leptin action in the hypothalamus. We dem-onstrated that daily icv injection of leptin significantlyincreased NT gene expression in the hypothalamus[267]. We also observed that administration of NT-an-tibody or specific NT receptor antagonist, SR48692,reversed the suppressive effects of leptin on food intakein rats [269]. In a recent study, leptin has been shown toinduce NT gene expression in a hypothalamic cell line[72]. These findings plus the observations of synergisticaction between leptin and NT in feeding [25] stronglysuggest that this anorectic peptide has a significant rolein mediating leptin action on feeding in the hypothala-mus. In addition, NT also stimulates activity of theneurons producing corticotrophin-releasing hormone(CRH) [231,261], a potent anorectic signal [222].

The role of CRH in mediating leptin action has beeninvestigated [291,334]. CRH is localized in the PVN [63].Central administration of CRH inhibits food intake[8,169]. The findings that leptin increases CRH mRNAexpression [140] and CRH release in the PVN [140]; andleptin!s satiety action is attenuated by pre-treatment witha-helical CRH (a-CRH), a specific CRH antagonist, orwith anti-CRH antibody [105,238,334]; and the recentdemonstrations that treatment with a-CRH markedlyattenuated leptin-induced c-fos expression in the PVNand the VMH [206], and attenuated leptin-induceduncoupling protein-1 (UCP1) expression in the brownadipose tissue [206] clearly suggest that CRH is an im-portant mediator of leptin signaling in the hypothalamusin regulation of food intake and energy expenditure.Although it remains to be established whether NT an-tagonist reverses the effects of leptin on CRH or CRHantagonist reverses the effects of leptin on NT neuronalactivity, the reports that NT increases CRH release [261]and NT antagonist, SR48692, reduces CRH mRNAlevels in the PVN [231], suggest that leptin!s action onCRH may be a direct and/or indirectly through theactivation of NT neurons in the hypothalamus.

In total, accumulated evidence including those citedabove strongly suggest that leptin!s action is mediatedthrough a large number of orexigenic and anorectic


neurons that are localized in the arcuate, LH/PFH- andPVN areas of the hypothalamus. Furthermore, it ap-pears that leptin not only modifies the synthesis and re-lease of the peptides, it also modifies (antagonistic orsynergistic effect) the action of the peptides after beingsecreted (Fig. 1). Because of the established morpho-logical and functional communications among theorexigenic and anorectic signal-producing neurons in theARC–PVN–LH/PFH axis, compromise in interactionsbetween orexigenic peptides or in their effects on ano-rectic peptides could be one of the major mechanisms ofleptin action in the hypothalamus. It is also important toconsider the possibility of unidentified orexigenic andanorectic peptide-producing neurons that could beinvolved in mediating leptin action in the hypothalamusin regulation of food intake and body weight.

4. Leptin signal transduction pathways in the hypothala-mus

4.1. Leptin receptor

Leptin receptor is a member of the class I cytokinereceptor family [327,328]. Of the several alternativespliced isoforms (a–f, as well as others) of the leptinreceptor (Ob-R), the Ob-Rb, which has the longest cy-toplasmic domain (302 amino acids), is expressed in high

levels in the hypothalamus [51,182,214,328], and hasbeen clearly demonstrated to be capable of initiatingsignal transduction [51,102,107,182,213,335]. Otherforms of the Ob-R appear to have no (Ob-Re) or shortcytoplasmic domains, but share the common extracel-lular domain [51,102,328], and their role in leptin sig-naling is not clear. The hypothalamus has the highestratio of Ob-Rb to Ob-Ra [107], consistent with its role inmediating the effects of leptin on feeding and energybalance. Ob-Ra and Ob-Rc are highly expressed inchoroid plexus and microvessels [135], suggesting theirrole in blood–brain barrier transport.

Hypothalamic leptin binding [17] and leptin receptorgene expression [16,190,273] are up regulated followingfasting, suggesting that reduction in circulating leptinseen during fasting may be involved in modifying leptinreceptor expression. However, although leptin decreasesOb-R mRNA expression in the ob/ob mice [16], icv ad-ministration of leptin [242] or hyperleptinemia producedby adenovirus [273] does not alter Ob-R mRNA ex-pression in rats. Thus, besides leptin, there may be othersignals that modulate leptin receptor expression. Avail-able evidence suggests that insulin may be involved inleptin receptor regulation [46,75].

4.2. JAK2–STAT3 pathway

Early recognition of leptin receptor as a member ofthe class 1 cytokine receptor super-family [328] resultedin prompt identification of the JAK–STAT pathway asthe major pathway of leptin signaling [20,28,107,108,257,335,350, see Fig. 6]. In leptin-signaling cascade, thebinding of leptin to the receptor results in phosphory-lation and activation of JAK2. Activated JAK2, in turn,mediates phosphorylation at the specific receptor tyro-sine residue, which then serves as a docking site forSTAT3. STAT3 becomes phosphorylated, and phos-phorylated STAT3 becomes dimerized and translocatedto the nucleus where they bind and regulate expressionfrom target promoter [76]. Although leptin inducesJAK2 phosphorylation in BaF3 cells transfected withOb-Rb [108], McCowen et al. [210] reported that a singleiv injection of leptin, which induced STAT3 activation,failed to induce JAK2 phosphorylation in the rat hy-pothalamus. However, we observed an increased JAK2phosphorylation in the hypothalamus following 2–4days of continuous central leptin infusion [240].Amongst several STAT proteins, leptin only activatesSTAT3 in the hypothalamus [210,335] including in theARC, LH, VMN, and DMN areas [141]; indicatingSTAT3 as one of the major intracellular mediators ofleptin signaling in the hypothalamus. Several leptintarget neurons including NPY, POMC, galanin, andorexin neurons have been shown to express STAT3[121,123], and it is likely that other leptin sensitiveneurons that express leptin receptor do also express

Fig. 1. Schematic presentation of leptin action on hypothalamic pep-tides governing feeding. In this model, decrease in circulating leptinlevels during fasting or deficiency in leptin action due to absence ofleptin, leptin receptor mutation or leptin resistance would increasegene expression, peptide release, and action of orexigenic neuropep-tides, such as NPY, MCH, GAL, and orexin; and decrease synthesis,release of anorectic peptides, such as a-MSH, NT, CRH, etc. resultingin increased food intake. Similarly, increased circulating leptin levelswould inhibit not only the synthesis and release of the orexigenicpeptides, but it would modify the action of these peptides after beingreleased, and enhance activity of anorectic peptides including synthe-sis, release, and postsynaptic action, resulting in decreased food intake.We hypothesize that acute inhibition of food intake that occurs withinan hour of leptin injection may be due to modification of postsynapticaction of orexigenic and anorectic neuropeptides.


STAT3, unless leptin signaling is mediated by a mech-anism that does not involve STAT3 activation in theseneurons. Although Munzberg et al. [224] demonstratedleptin induction of STAT3 phosphorylation in hypo-thalamic POMC neurons, leptin-induced activation ofSTAT3 in other neurons including those producingNPY, MCH, orexin, and galanin is yet to be docu-mented. It is also known that Tyr 1138 of Ob-Rb me-diates activation of STAT3 during leptin action[11,20,313,350]. Recently, Bates et al. [19] have shownthat Ob-Rb-STAT3 signaling is required for leptin reg-ulation of energy balance but not reproduction. Specif-ically, these authors showed that in transgenic mice inwhich Tyr 1138 of Ob-Rb was replaced with a serineresidue, STAT3 activation by leptin was impaired andcaused obesity without affecting reproduction. Further-more, disruption of Ob-Rb-STAT3 signaling resulted indysregulation of leptin action on POMC neurons with-out compromising the effects of leptin on NPY neurons,suggesting the inhibition of NPY by leptin may be in-dependent of STAT3 signaling [19]. Because leptin ac-tivates SOCS3 in the NPY neurons [90] and becauseinduction of SOCS3 is dependent on STAT3 activation[11] and NPY neurons express STAT3 [121], it is criticalto demonstrate whether leptin induces STAT3 in NPYneurons. Nevertheless, other pathways of leptin signal-ing (see below) including regulation of cAMP may playcrucial role in leptin!s action on NPY and possibly inmany other neurons.

Cumulative evidence suggests that JAK–STATpathway of cytokine signaling is under the negativefeedback control of a family of SOCS proteins[95,136,227,316]. The member of SOCS family of pro-teins contains an Src-homology (SH2) domain and a C-terminal SOCS box [136]. SOCS proteins are induced bya variety of cytokines and act as a negative regulator ofcytokine signaling. Among eight SOCS proteins[171,230], leptin specifically induces SOCS3 mRNAlevels in the hypothalamus [18,29,31,90] and activatesSOCS3 expression in NPY and POMC neurons [18,90].In mammalian cell lines, over expression of SOCS3blocked leptin-induced signal transduction by inhibitingleptin-induced JAK2 phosphorylation [31,32]. Further-more, SOCS3 also inhibits leptin signaling by binding tophosphorylated Tyr-985 [11]. SH2-containing phospha-tase-2 (SHP-2), another mediator of leptin signaling [32],also competes with SOCS3 for p-Tyr-985 of Ob-Rb [31].Thus, an alteration in any of these mechanisms couldcompromise inhibitory feedback action of SOCS3during leptin signaling.

4.3. PI3K–PDE3B–cAMP pathway, an alternativepathway of leptin signaling

Recent studies in peripheral tissues (pancreatic b-cells, hepatocytes and adipocytes) have demonstrated

that leptin induces an insulin-like signaling pathwayinvolving PI3K-dependent activation of PDE3B andeventual reduction in cAMP levels [369,370]. Intracel-lular cAMP levels are regulated by adenylyl cyclase andcAMP phosphodiesterase (PDEs) [74,119]. Cyclic nu-cleotide PDEs are a large super family of enzymesconsisting currently of 20 different genes sub-groupedinto 11 different PDE families [23,67,79,204,311].PDE3B, one of the two members of type 3 PDE familyof genes [23], exhibits high affinities for both cAMP andcGMP, but prefer cAMP as the substrate [23,67,79,204].In addition to its localization in several peripheral tis-sues such as adipose tissue, liver, pancreatic b-cells,kidney and testes, PDE3B is also localized in the CNS[211,216,225,255,322] including the hypothalamus.PDE3B plays a major role in regulation of intracellularcAMP levels by several hormones. For example, PDE3Bis activated by insulin in adipocytes [368] and hepato-cytes [79], resulting in decreased cAMP levels. Fur-thermore, the inhibition of glucagon-like peptide1-stimulated insulin secretion by leptin in pancreaticb-cells, and the anti-glycogenolytic function of leptin inthe rat primary hepatocytes are mediated through aP13K-dependent activation of PDE3B and subsequentreduction of cAMP [369,370].

The idea that regulation of hypothalamic cAMPlevels could play a critical role in feeding and bodyweight regulation, and therefore in leptin signaling in thehypothalamus, is supported by the following evidence.First, central injection of either a cAMP analog, or theagents that increase endogenous cAMP, stimulatesfeeding in satiated rats [109–111]. Second, intracere-broventricular dibutyryl cAMP administration induceshypothalamic levels of NPY [5]. Finally, leptin alsomodifies cAMP response element (CRE)-mediated geneexpression including that of NPY neurons in thehypothalamus [305].

Thus, we assessed whether PDE3B-activation de-pendent reduction in cAMP levels is involved in ano-rectic and body weight reducing effects of leptin [371].First, we observed that while cilostamide, a specificPDE3 inhibitor [22], reversed the anorectic effect ofcentral administration of leptin on food intake, R0-20-1724, a specific PDE4 inhibitor [22], failed to reverse thiseffect of leptin (Fig. 2). In addition, body weight re-ducing effect of daily leptin injection for three consecu-tive days was completely reversed by the PDE3antagonist. Second, we demonstrated that within 45minof leptin injection to the overnight fasted rats, PBE3Bactivity was significantly increased in association with areduction in cAMP levels in the hypothalamus (Fig. 3).These findings suggested that activation of the PDE3B–cAMP pathway is an important mechanism of leptinsignaling in the hypothalamus. If PDE3B–cAMP path-way plays a critical role in leptin signaling, then it wasnecessary to demonstrate whether this pathway interacts


with the JAK2–STAT3 pathway of leptin signaling inthe hypothalamus. To demonstrate this we examined theeffects of PDE3B inhibition by cilostamide on STAT3activation in the hypothalamus. We reasoned that ifthese two pathways crosstalk then cilostamide shouldreverse the effects of leptin on STAT3 activation. Wefound indeed that this was the case, because cilostamidereversed the effects of leptin on p-STAT3 levels andDNA-binding activity of STAT3 in the hypothalamus(Fig. 4). These findings together with our unpublishedobservation that PDE3B is localized in the hypothala-mus, particularly in the ARC, VMN, DMN, PVN, LH,and PFH areas (Fig. 5), which are implicated in foodintake and body weight regulation, strongly suggest thatthe PDE3B–cAMP pathway plays a very important rolein transducing leptin action in the hypothalamus. Invarious non-neuronal tissues, PI3K, in association withthe insulin receptor substrate IRS1/2, is an upstreamregulator of PDE3B [4,253,256,370]. We also observedan activation of IRS1-associated PI3K activity in thehypothalamus within 45min of leptin injection [371, seeFig. 3]; an observation that was also supported byNiswender et al. [232]. These authors further showedthat PI3K inhibitor reversed the effect of leptin on foodintake [232]. While in non-neuronal tissues, PKB hasbeen demonstrated to be an upstream regulator ofPDE3B [79,370], it is yet to be examined whether this istrue for the hypothalamus.

Overall, these findings all together indicate that aPI3K–PDE3B–cAMP pathway interacting with theJAK2–STAT3 pathway constitutes a critical component

of leptin signaling in the hypothalamus (Fig. 6). Wehypothesize that PI3K–PDE3B–cAMP signaling path-way may mediate leptin!s action in the hypothalamusin general. A further understanding of this signaltransduction pathway would therefore be critical tounraveling the molecular mechanisms of hypothalamicaction of leptin in normal states and during thedevelopment of leptin resistance seen in obesity andrelated disorders.

Fig. 2. Cilostamide, a PDE3 inhibitor, reverses the satiety action ofleptin in male rats that are fed ad libitum, but RO-20-1724 (RO-20), aPDE4 inhibitor, does not. Rats were first injected ICV into the thirdventricle with artificial cerebrospinal fluid (aCSF) or 4 lg of leptin anddimethyl sulphoxide (DMSO) or one of the PDE inhibitors; then wereinjected 1 h later with DMSO or the inhibitors. Data are means#SEMfor the number (n) of animals in parentheses. $p < 0:05, relative toothers except the groups with * or **. (Adapted from [371].)

Fig. 3. Leptin activates PI3K (A), and PDE3B (B) and reduces cAMP(C) levels in rat hypothalamus. For PI3K activity, rats fasted for 24 hwere injected ICV with 4lg of leptin or vehicle (aCSF) and 45minlater, the medial basal hypothalamus (MBH) were processed for theassay of PI3-kinase activity. An aliquot of the hypothalamic homog-enate (%1mg) was pre-cleared with 40 ll of 50% protein-G–agaroseconjugate for 30min before incubated under shaking with an antibodyagainst IRS-1 overnight at 4 "C. The assay of PI3-kinase activity as-sociated with the IRS-1 immunoprecipitate and subsequent thin-layerchromatography were carried out according to a previously describedprotocol (Avanti Polar Lipids, AL) with phosphatidylinositol as asubstrate in the presence of [c-32P]ATP. The phosphatidylinositol 3-phosphate (PI–P3) and PI-P2 products were quantitated using densi-tometer and NIH image 1.6 software. PDE3B activity and cAMPlevels were examined in the similar experiments. PDE3B activity wasmeasured by previously described method [369,370], using 1lM cAMPas a substrate; and the activity was adjusted to the quantity of im-munoprecipitated PDE3B as shown on western blot, and expressed aspmol hydrolyzed cAMP/min/density unit of PDE3B. cAMP levels weremeasured by RIA kit (NEN) and expressed as relative (%) to vehiclegroup. Data are means# SEM for the number (n) of animals in pa-rentheses. $p < 0:05 versus vehicle treated group. (Adapted from[371].)


Because of the recent reports of stimulation of PI3Kby insulin in the hypothalamus [235], reversal of insulin!sanorectic action by PI3K inhibitor [235] and localizationof PI3K immunoreactive neurons in the hypothalamus[233], it appears that stimulation of PI3K may be acommon pathway for both leptin and insulin signalingin the hypothalamus [234]. In this regard, as seen inperipheral tissues [127,321], activation of PI3K has beenproposed to mediate acute membrane effect of leptin andinsulin [234] including the activation of ATP-sensitivepotassium channel in the hypothalamus [128,253,312].Since insulin stimulates STAT3 in the hypothalamus[46], it remains to be determined whether, like leptinsignaling [371], insulin signaling through STAT3 path-way also requires PDE3B activation dependent reduc-

tion in cAMP levels. As mentioned above, since cAMPstimulates food intake [109–111] and cAMP analog in-duces NPY gene expression in the hypothalamus [5], wehypothesize that PDE3B activation-dependent reduc-tion in cAMP levels by leptin may be responsible formodifying NPY gene expression [267,317] and NPY!saction on feeding [268]. Similarly, insulin!s inhibitoryaction on NPY neuronal activity [265,289,290,342] mayinvolve the activation of PI3K–PDE3B–cAMP pathwayof intracellular signal transduction.

4.4. Other potential pathways

Among other potential pathways, an SHP2–GRB2(growth receptor bound 2)-Ras–Raf–MAPK/ERK (mi-togen-activated protein kinase/extracellular signal regu-lated kinase) pathway has been proposed in leptinreceptor signaling [11,32]. In support of this pathway isthe demonstration that leptin induces MAPK/ERK ac-tivity and egr-1 mRNA levels in the hypothalamus [32].In addition, the findings that: (i) ERK activation is re-quired for Ob-Rb mediated c-fos gene expression in cellline (11), (ii) central or peripheral administration ofleptin induces c-fos in the hypothalamus [93,338,356,363], and (iii) leptin induces c-fos in POMC neurons [89],suggest a potential role of this pathway in leptin signal-ing. Furthermore, an ERK dependent STAT3 Ser727phosphorylation and DNA binding activity by leptin hasbeen recently documented in macrophages [236]. It ishowever interesting to note that disruption of Ob-Rb-STAT3 signaling does not compromise ERK activationin the hypothalamus, suggesting that ERK activationmay be independent of STAT3 signaling [19]. Never-theless, the physiological role of ERK-egr-1 activation inmediating leptin action in the hypothalamus is yet to beestablished. One other important issue is whether ERKactivation in the hypothalamus is mediated by SHP2. Ingrowth factor receptor signaling, such as platelet-derivedgrowth factor receptor, tyrosine phosphorylated SHP2acts as an adaptor molecule that recruits Grb2 and Sos,members of the Ras/MAPK/ERK signaling pathway.Using dominant negative SHP2 construct, Bjorbaek et al.[32] reported that SHP2 is essential for leptin-inducedMAPK/ERK phosphorylation by Ob-Rb. However,Carpenter et al. [44] reported that activation of leptinreceptor induces tyrosine phosphorylation of SHP2, butSHP2 appears to act as negative regulator of STAT3-mediated transcription. Similarly, Li and Friedman [189]observed that activation of SHP2 by the leptin receptorresulted in decrease phosphorylation of JAK2, andthereby, SHP2 acts as negative regulator of leptinsignaling. All these studies have been conducted in celllines, therefore, although SHP2 has been found in thebovine and mouse hypothalamus [189], precise role ofSHP2 in leptin signaling in the hypothalamus is yet to beresolved.

Fig. 4. Cilostamide reverses the effect of leptin on STAT3 activation inthe hypothalamus. Fasted (24 h) rats were injected ICV with DMSO orcilostamide (10lg) followed 30min later by leptin (4lg) or aCSF. (A)Top, western blot of STAT3 and p-STAT3 in the mediobasal hypo-thalamic (MBH) extracts. Bottom, densitometric analysis of the im-munoreactive bands for p-STAT3 and expressed as relative (%) tovehicle group (DMSO+aCSF). (B) Top, DNA binding activity ofSTAT3 in the MBH as determined by an electrophoretic mobility shiftassay using a 32P-labeled M67-SIE oligonucleotide probe. Top right,DNA binding activity is specific to p-STAT3 because a "supershift! didnot occur in the presence of anti-SOCS3 antibody. Bottom, resultsobtained by phosphor imaging and expressed as relative (%) to vehicle.Data are means#SEM for the number (n) of animals in parentheses.$p < 0:05 as compared to all other groups. The reversal of STAT3activation by PDE3B inhibition implies a crosstalk between the JAK2-STAT3 and PDE3B–cAMP pathways in transducing leptin action inthe hypothalamus. (Adapted from [371].)


Protein tyrosine phosphatase 1B (PTP1B) has recentlybeen shown to regulate leptin signal transduction in vivo[52,365] and in vitro [158,365]. It is suggested that PTP1Bacts as a negative inhibitor of leptin receptor signalingprimarily via dephosphorylation of JAK2 [52,365]. Be-cause PTP1B is localized in the brain including the hy-pothalamic areas where Ob-Rb is localized [365], andbecause PTP1B knockout mice are resistant to diet-in-duce obesity [87,163] and become more sensitive to leptin[52], PTP1B appears to play a significant role in leptinsignaling in the hypothalamus. It is interesting to notethat SOCS3 is also a negative regulator of cytokine sig-naling, and as described above, SOCS3 inhibits leptinsignaling by binding to JAK2 and leptin receptor[11,31,32] and SOCS3 also competes with SHP2 for itsbinding to Ob-Rb [31]. Thus, while SHP2, SOCS3, andPTP1B seem to play important role in regulation ofleptin signaling, it is critical to understand the interac-tions among these and other unidentified negative regu-lator(s) of leptin signaling in the hypothalamus to furtherour understanding on leptin signaling in normal and

during the development of leptin resistance. It is un-doubtedly one of the most interesting areas of futureinvestigation in leptin signal transduction mechanism.Thus, intracellular leptin signal transduction mechanismis much more complicated than it was originally thought.

5. Mechanisms underlying the central Leptin resistance

Because human obesity, in the majority of cases,cannot be attributed to defects in leptin or its receptor[54,64,65,115,209], and because obese humans are hy-perleptinimic [43,57,64,129,193,202,292], it is suggested

Fig. 5. Bright-field photograph showing phosphodiesterase 3B(PDE3B) immunoreactive cells in rat hypothalamus. Adult male ratanesthetized with pentobarbital was perfused intracardially with 0.9%saline kept at room temperature, followed by ice-cold 4% parafor-maldehyde in 0.1M phosphate buffer, The brain was post-fixed in thesame fixative for 4–5 h, and then kept in 25% sucrose solution at 4 "Cuntil it sank. Thereafter, brain was frozen on dry ice and coronal 25 lmfree-floating sections were cut through the hypothalamus on a freezingmicrotome, and stored in cryoprotectant at )20 "C until use. PDE3B-ircells in the sections were detected by immunocytochemical (ICC)method, using tyramide amplification kit (NEN), primary PDE3Bantibody (1:1000 dilution, SC-11838, Santa Cruz, CA), biotinylatedanti-goat secondary antibody (1:200, BA-500, Vector Laboratories Inc,Burlingame, CA) and the avidin–biotin–horseradish peroxidase com-plex (Vector elite kit, Vector Laboratories), and were visualized with5min incubation with diaminobenzidine hydrochloride (Sigma) in thepresence of hydrogen peroxide. (A) PBE3B-ir positive cells in the pa-raventricular nucleus (PVN), lateral hypothalamus (LH) and supra-optic nucleus (SON). (B) Immunocytochemical reaction withoutprimary antibody in a section through the median eminence (ME)-arcuate (ARC) area; (C) PDE3B-ir positive cells in the ARC, ventro-medial nucleus (VMN), dorsomedial nucleus (DMN), and parafornicalhypothalamic area (PFH). PDE3B-ir was completely blocked in thepresence of PDE3B blocking peptide (SC-11838P, Santa Cruz) (datanot shown). Localization of PDE3B in the ARC–VMN–DMN–PVN–LH axis along with activation of PDE3B in the hypothalamus (see Fig.3) strongly suggests a physiological role of PDE3B in leptin signalingin the hypothalamus.

Fig. 6. Schematic of leptin intracellular signal transduction in the hy-pothalamus. Leptin binding to it!s receptor (Ob-Rb) induces activationof Janus kinase (JAK), receptor dimerization, and JAK-mediatedphosphorylation of the intracellular part of the receptor, followed byphosphorylation and activation of signal transducer and activators oftranscription-3 (STAT3). Activated STAT3 dimerizes, translocates tothe nucleus and tans-activates target genes, including suppressor ofcytokine signaling-3 (SOCS3), neuropeptide Y (NPY) and proopio-melanocortin (POMC). Our evidence suggests that leptin also activatesphosphatidylinositol 3-kinase (PI3K), and phosphodiesterase 3B(PDE3B) and reduces cAMP levels in the hypothalamus, and that thePI3K–PDE3B–cAMP pathway interacting with the JAK2–STAT3pathway constitutes a critical component of leptin signaling in thehypothalamus. We hypothesize that defects in either one or both of thesignaling pathways may be responsible for the development of leptinresistance seen in obesity. Other potential signaling pathways includingthe involvement of SHP2–GRB2–Ras–Raf–MAPK/ERK pathwayand PTP1B in regulating leptin action in the hypothalamus are left outof this scheme to avoid complication in the figure. Furthermore, therole of SHP2–GRB2–Ras–Raf–MAPK/ERK pathway in leptin sig-naling in the hypothalamus is not clearly understood. Also the roleof cofactors and co-activators, such as p300/CBP and NcoA/SRC1a,in STAT3 transcriptional activity is yet to be established in thehypothalamus.


that obese individuals are, in general, leptin-resistant[57,202]. Obese humans, and mice made obese by dietarymanipulation, have elevated levels of circulating leptinbut maintain a normal food intake [57,125,202]. Thus, itis likely that an extended period of exposure of thebrain, especially the hypothalamus, to a high level ofleptin may result in the development of central leptinresistance.

5.1. Defective leptin transport through blood–brainbarrier (BBB) in obesity

A defective leptin transport to the hypothalamus isthought to be one of the many defects in obesity. Theevidence in support of this notion has come from thefindings that: (i) the cerebrospinal fluid: plasma leptinratio is lower in obese individuals compared to leancontrols [43,292]; (ii) peripheral leptin administration tohyperleptinimic DIO mice does not have any effect onfood intake or body weight, while central injection ofleptin is effective in decreasing food intake and bodyweight in these mice [340], (iii) although peripheral ad-ministration of leptin to DIO mice does not activateSTAT3 in the hypothalamus, icv administration doesinduce STAT3 activation, albeit 75% reduction thancontrol [86], and (iv) leptin transport through BBB isreduced in several models of obesity including the DIO[13,41]. The molecular mechanism behind apparent de-fect in leptin access or transport is not clearly under-stood. While short isoforms of the leptin receptor havebeen implicated in leptin transport through BBB[30,114], and both Ob-Ra and Ob-Rc are highly ex-pressed in cerebral microvessels, no change in mRNAlevels in either of these isoforms has been reported inDIO mice [86,135]. However, in DIO rats, there is anincreased expression of Ob-Ra mRNA levels in cerebralmicrovessels [33], Nevertheless, it is necessary to deter-mine whether a decrease in brain uptake of leptin is thecause or consequence of obesity. Thus, a time coursestudy in DIO animals before and during the develop-ment of obesity is of significant importance.

5.2. Central leptin resistance in DIO animals

Using male Wistar rats, Widdowson et al. [351]demonstrated that while leptin injection into the lateralcerebroventricle resulted in a dose dependent decrease infood intake in the animals that were in normal labora-tory diet, the anorectic effect of leptin was attenuated inthe diet-induced obese rats. This is probably the firstdemonstration of the occurrence of central leptin in-sensitivity in DIO animals. Recently, reduced sensitivityto central leptin in decreasing food intake has also beenreported in DIO prone Sprague–Dawley rats [185].Halaas et al. [125] demonstrated that AKR/J mice, thatare lean on a chow diet but have a heritable disposition

toward developing obesity, when fed high-fat (HF) diet,were sensitive to peripheral leptin after 14 weeks on HFdiet. These authors also demonstrated that AY micedevelop central leptin resistance. In other studies, a re-duced sensitivity to central leptin administration hasbeen reported in mice that were on HF-diet for 19weeks, but not during one or 8 weeks of HF dieting[192]. In a recent study, Bowen et al. [37] have demon-strated that 6- to 7-weeks-old mice that have beenweaned onto a HF diet became obese and showed at-tenuated response to central effects of leptin on foodintake and body weight without a compromise in pe-ripheral effect of leptin. Furthermore, obesity due to HFdiet decreases lumber sympathetic nerve activity andcardiovascular responses to intracerebroventricularleptin in female rats [195]. These findings strongly sug-gest that central leptin resistance also contributes to thedevelopment of diet-induced obesity and related disor-ders, and therefore understanding the mechanisms ofcentral leptin resistance has been the subject of intensiveresearch.

One of the obvious assumptions is that leptin resis-tance is due to a defective leptin receptor signaling in thehypothalamus. As described above, Ob-Rb, the longsignaling from of the leptin receptor, is highly expressedin the hypothalamus and modulated by nutritional sta-tus in that fasting increases Ob-Rb gene expression[16,190,273] and leptin binding in the hypothalamus[17]. Also hypothalamic leptin receptor mRNA levelsare increased in ob/ob [16,139] and db/db [16] mice,models of obesity that are characterized by a lack ofleptin and functional leptin receptors, respectively.Fasting effect on hypothalamic leptin receptor mRNAlevels seen in lean mice is absent in the ob/ob mice[139,190], and leptin decreases hypothalamic leptin re-ceptor mRNA expression in ob/ob mice [16]. Thus al-tered leptin levels and or defective leptin action areassociated with changes in leptin receptor gene expres-sion in the hypothalamus. However, the reports onleptin receptor gene expression in the hypothalamusduring diet-induced obesity are variables. For example,El-Hasami et al. [86] reported that there was no changein leptin receptor gene expression in the mice feeding HFdiet for 18 weeks. By contrast, Lin et al. [192] reportedthat the mice on HF diets for 19 weeks had less OB-RbmRNA levels but those on HF diets for only 8 weekshad increased Ob-Rb mRNA levels in the ARC. Wehave also shown that leptin receptor gene expressiondoes not change in rats on HF diets for 9 weeks [273].However, despite unaltered Ob-R gene expression in thehypothalamus, there have been reports of decreasedreceptor protein levels in DIO rats [201] and reducedleptin signaling in DIO mice [86]. The discrepancies seenin data for leptin receptor gene expression in DIO ani-mals in different studies could be due to the techniquesused for mRNA measurement (in situ hybridization,


RT-PCR, ribonuclease protection assay), strain differ-ence, period of treatment, or due to the data collectedfor whole hypothalamus versus specific hypothalamicnuclei.

Because these studies examined changes in Ob-R geneexpression in DIO animals in the fed state [86,192,201]and because leptin receptor gene expression is modifiedby nutritional status [16,190,273], we examined whetherthere is any defect in nutritional regulation of leptinreceptor in DIO. We observed that rats fed with HFdiets for 9 weeks had similar levels of hypothalamic Ob-Rb mRNA and Ob-Rtot (all receptor isoforms) mRNAlevels in the fed state, as reported by others [86,201,242].However, in the fasted (18-h) state, Ob-Rb mRNAlevels were significantly increased in standard chow fedrats as compared to DIO rats (Fig. 7). This differencewas due to a failure of Ob-RB mRNA to increase inresponse to an overnight fast in DIO [273]. Thus thefinding that fasting was unable to induce leptin receptorgene expression in the hypothalamus of DIO animalssuggests a defective leptin signaling in the hypothalamusof these animals. This notion is supported by the studyof El-Hasami et al. [86] demonstrating approximately75% reduction in hypothalamic STAT3 DNA bindingactivity in response to icv administration of leptin inovernight fasted DIO mice. While it is not clear whether

increased hypothalamic leptin binding seen in normalanimals after fasting [17] is also altered in DIO animals,our study clearly suggests that leptin resistance in theDIO animals may be due to insensitivity of leptin re-ceptor gene expression to fasting. Although there is nodefinitive study addressing whether nutritional regula-tion of leptin receptor gene expression plays any physi-ological role in mediating leptin action on food intakeand body weight, in ad lib fed rats leptin receptormRNA levels in the hypothalamus increase at the timeof lights off (onset of ingestive behavior) and decreasedgradually thereafter [359]. These results suggest thatincreased leptin receptor gene expression before theonset of feeding in normal ad lib fed animals may playan important role in mediating action of postprandialincreased leptin [359]. Thus, impaired nutritional regu-lation of leptin receptor gene expression could alter foodintake and body weight in DIO.

Since STAT3 activation is one of the importantmechanisms of leptin signaling, the demonstration of%75% reduction in leptin-induced STAT3 activation inDIO mice reported by El-Hasami et al. [86] suggests thata defective STAT3 signaling in the hypothalamus maybe responsible for central leptin resistance in DIO. Al-though a defective nutritional regulation of leptin re-ceptor gene expression [273] may be one of the reasonsof reduced STAT3 signaling [86], it is not clear whetherthere is a defect in leptin receptor phosphorylation andor JAK2 activity. While overexpression of SOCS3 inmammalian cells antagonizes proximal leptin signaling[31], and protein inhibitor of activated STAT (PIAS3)antagonizes STAT3 DNA binding activity [57], El-Hasami et al. [86] reported no increase in either SOCS3or PIAS3 mRNA levels in the hypothalamus following18 weeks of HF dieting. Similarly, Peiser et al. [242]reported no change in either SOCS3 or PIAS3 geneexpression in rats on HF diet for 15 weeks. However, itremains to be determined whether there is any change inprotein levels of SOCS3 or PIAS3 in the hypothalamusof DIO animals. It is quite possible that interactionsamong several negative regulators of STAT3 signaling,such as SOCS3, PIAS3, PTP1B, and SHP2, could bealtered and thus may produce reduced leptin response inthe hypothalamus without noticeable change in theirprotein levels.

Because leptin resistance may be due to a defect inleptin signaling in leptin target neurons, many studieshave examined changes, if any, in various leptin targetneurons in DIO animals. Levin and Dunn-Meynell[184,185] reported that DIO-prone SD rats maintainedon high-energy (HE, 31% fat) diet had significantly in-creased levels of ARC NPY mRNA within 2 weeksdespite hyperleptinemia. However, after 12 weeks onHE diet, when the rats were more obese, ARC NPYmRNA levels were decreased. The authors suggestedthat the elevated ARC NPY expression in DIO-prone

Fig. 7. Diet-induced obesity (DIO) is associated with a defective nu-tritional regulation of leptin receptor gene expression. Male Wistar ratswere fed a standard chow diet (SC) or were fed a high fat diet for 9weeks to induce DIO. At the end of this period, animals underwent an18-h overnight fast (SC-FAST) and DIO-FAST) or continued feedingad libitum (SC-FED and DIO-FED). Subsequently, all animals werekilled and the hypothalami were processed for ribonuclease protectionassay. Representative phosphor images showing the level of Ob-RbmRNA, Ob-Rtot mRNA (all isoforms of the leptin receptor) and b-actin mRNA in the hypothalamus of SC-FED and SC-FAST (A) andDIO-FED and DIO-FAST (C). Graphical representation of the totaldataset showing the fold difference of Ob-Rtot mRNA and Ob-RbmRNA between SC-FAST and SC-FED (B) and between DIO-FASTand DIO-FED (D). Data are means# SEM for the number of animalsshown in parentheses. $p < 0:05 between the SC-FAST and SC-FED.Note that while fasting induced Ob-Rb gene expression in SC rats, itfailed to induce Ob-Rb gene expression in DIO rats, suggesting anutritional defect in leptin receptor gene expression in DIO, and thismay contribute to central leptin resistance. (Adapted from [273].)


rats predisposes them to become obese in the presence ofHE diet, but decrease ARC NPY expression may play arole in defending the body weight once they becomeobese. Ziotopoulou et al. [372] demonstrated that duringthe first 2 days of HF (42% fat) feeding, C57BL/6J miceshowed hyperphagia and hyperleptinimia in associationwith a decrease in hypothalamic NPY and AgRPmRNA levels. However, after 1 week, both NPY andAgRP mRNA levels were comparable to control mice.Furthermore, while there was no change in POMCmRNA levels during the first week of HF feeding; after2 weeks, POMC mRNA levels were increased in asso-ciation with an increased calorie intake [372]. Againthese authors also concluded that increase in POMCmRNA levels might be a second defense against obesity.Lin et al. [192] reported that at 8 weeks of HF (59% fat)feeding, ARC NPY mRNA levels were significantlydecreased, without any change in POMC mRNA levels;however at 19 weeks, both NPY and POMC mRNAlevels were significantly decreased. In another study,Wang et al. [343] showed that SD rats fed 60% fat dietfor 8 weeks had significantly increased CART mRNAlevels in the hypothalamus. Furthermore, increase cir-culating leptin levels with adenovirus-leptin treatmentdid not alter CART mRNA levels or food intake inthese HF rats.

Since CART is an anorectic signal [172,178], andCART is known to be up-regulated by leptin [89,172]and is also co-localized with POMC in the ARC[89,341], increased CART mRNA levels in DIO ratscould also be involved in second defense against obesity.It is to be noted that the diet used in these studies hadvariable amount of fat (31–60%), which may modifygene expression of NPY/AGRP or POMC/CART in-dependent of circulating leptin levels. Increase in NPYand AgRP mRNA levels, but not in MCH mRNA, inassociation with hyperleptinemia has also been reportedin DIO rats on HF diet for 6 months [103]. In anotherstudy, rats on HF diets for 2–8 weeks had increasedAgRP levels in the hypothalamus without any change ina-MSH or POMC levels [126]. Torri et al. [330] reportedthat DIO as a result of feeding cafeteria diet was asso-ciated with increased hypothalamic POMC mRNA inrats. Furthermore, Bergen et al. [26] showed that themice (A/J mice) that are resistance to DIO, when sub-jected to 14 weeks of HF feeding, had increased POMCmRNA and decreased NPY mRNA levels; however, HFfeeding for 14 weeks had no effect on either NPY orPOMC gene expression in C57BL/6J mice. Overall, itappears that changes in NPY/AGRP and POMC neu-rons are dependent on species, strain and duration ofdieting. Furthermore, the changes seen following HFdiet are most likely due to several factors, includingcirculating levels of leptin, insulin, ghrelin and otherfactors and fat content in the food and duration of ex-posure to HF. While all these studies have examined the

effects of DIO on some of the orexigenic and anorecticneuropeptides, and there may be changes in other neu-ropeptidergic neurons; it is important to demonstratewhether leptin sensitivity to these neurons are altered,and how and when does it occur during the developmentof DIO.

5.3. Leptin resistance following chronic central leptininfusion

The increase in leptin levels as early as day-1 of highfat feeding [372], suggests that hypothalamus is sub-jected to gradual increase in circulating leptin levelsduring the development of DIO. Thus, central leptinresistance seen in various studies [37,125,185,191,195,351] could be due to a consequence of chronic ex-posure of high levels of leptin to the hypothalamus, inaddition to a defect in leptin transport. To address thisissue, we developed a rat model of chronic central leptininfusion [270]. This was based on a mouse model de-veloped by Friedman!s group at the Rockefeller Uni-versity [125]. As shown in mice [125], we observed thatin rats chronic central leptin infusion (160 ng/h) via Al-zet pump resulted in an initial marked decrease in foodintake followed by a recovery to the normal levels bytwo weeks of infusion, and food intake remained nor-malized throughout the rest of the 4 weeks of leptininfusion. These results suggest that rats develop resis-tance to the satiety action of leptin. In contrast, bodyweight was gradually decreased to reach a nadir by 10–12 days of infusion and thereafter, it remained stabilizedat reduced level despite the normalization in food in-take. However, withdrawal of chronic leptin infusionresulted in hyperphagia, and body weight was mostlynormalized by 22 days post leptin (Fig. 8). Although, thestabilization of body weight at a reduced level in theface of normal food intake is of considerable interest,the underlying mechanism behind this remains to bedetermined.

Because chronic leptin infusion resulted in the de-velopment of resistance to the satiety action of thispeptide, we reasoned that this rat model could be usedto decipher some of the underlying mechanisms ofcentral leptin resistance. Because NPY plays an impor-tant role in food intake and body weight regulation, andbecause NPY is one of the primary targets of leptinaction in the hypothalamus, we tested the hypothesisthat resistance to the satiety action of leptin seen duringchronic central leptin infusion was due to the develop-ment of leptin resistance in the NPY neurons. This hy-pothesis predicted that NPY gene expression should bedecreased during early period of leptin infusion whenfood intake remained decreased, and normalization offood intake on days 14–16 of infusion should be ac-companied by unaltered NPY gene expression. Indeed,we observed that NPY gene expression was decreased


on days 3–4 of leptin infusion, and this decrease wasprimarily in the rostral and middle part of the arcuatenucleus. On the other hand, on days 13–16 of leptininfusion, NPY gene expression in the hypothalamus wasnot significantly different from that observed in thecontrol rats (Fig. 9). This effect of leptin on NPY geneexpression was also not due to a reduced food intake,because the rats that were paired-fed to those of theleptin group exhibited increased NPY gene expression.Furthermore, our preliminary study [241] shows thathypothalamic POMC and NT gene expression is in-creased on day 4 of leptin infusion, but remains unal-tered as compared to those of control on day 16 of leptininfusion. Thus, normalization of food intake followingchronic leptin infusion may be due to relative increase inNPY gene expression and or a relative decrease inPOMC and NT gene expression. These results clearlysuggest the development of leptin resistance in the hy-pothalamus, particularly in the NPY, POMC, and NTneurons following chronic central leptin infusion andsupport that this rat model may be used to further ourunderstanding on central leptin resistance.

To understand the mechanisms of leptin resistance inthe hypothalamus, we next tested whether a defect inhypothalamic leptin receptor activity and associatedsignal transduction through JAK2–STAT3 pathwayunderlies the development of leptin resistance in NPY,POMC NT and other leptin sensitive neurons followingchronic central leptin infusion, that could contribute tonormalization in food intake after an initial decrease.However, we found that JAK2–STAT3 pathway ofleptin signaling was operative normally during chronicleptin infusion [240] in that phosphorylated leptin re-ceptor and phosphorylated STAT3 remained elevated inassociation with a sustained elevation in DNA-bindingactivity of STAT3 in the hypothalamus throughout 16-day period of leptin infusion (Fig. 10). In addition,phosphorylated JAK2 levels were increased during ini-tial period but not day 16 of leptin infusion. AlthoughSOCS3 has been thought to be involved in leptin resis-tance [29,31], we observed that while hypothalamicSOCS3 mRNA levels were increased throughout leptininfusion (Fig. 11), SOCS3 leptin levels were increased onday 16 of leptin infusion. These findings clearly suggest a

Fig. 8. Effect of chronic central leptin infusion followed by leptin withdrawal on body weight (upper panel) and food intake (lower panel) in rats. Ratswere infused with artificial cerebrospinal fluid (aCSF) via Alzet osmotic minipump (1 ll/h) for 7 days before infusion with either recombinant mouseleptin in phosphate-buffered saline (PBS) at a dose of 160 ng/0.5ll or PBS alone for 28 days (experimental period). Thereafter, leptin was withdrawnand aCSF was infused for approximately 3 weeks. Note that after the initial decrease in food intake, rats developed resistance to the satiety action ofleptin, and withdrawal of the chronic leptin infusion resulted in hyperphagia. Using this rat model, we have demonstrated that NPY neurons developleptin resistance (see Fig. 9) despite a sustained elevation of the JAK–STAT pathway of leptin signaling throughout 16 days of chronic leptin infusion(see Fig. 10). Thus, this rat model can be used to decipher some of the underlying mechanisms of central leptin resistance. (Adapted from [273].)


sustained elevation in hypothalamic leptin receptor sig-naling through JAK–STAT pathway despite an in-creased expression of SOCS3 during chronic centralleptin infusion [240].

Because increased SOCS3 was unable to inhibitJAK2–STAT3 pathway, it appears that continuous ex-posure of leptin may modify the mechanism, such asSOCS3 binding to Ob-Rb [11] and JAK2 [31,32], by

which SOCS3 regulates JAK2–STAT3 pathway of lep-tin signaling in the hypothalamus. Furthermore, leptinresistance in the NPY, POMC, and NT neurons in the

Fig. 9. Representative dark-field photographs (left panel) of coronal sections through rostral part of the hypothalamic arcuate nucleus of rats showchanges in NPY mRNA expression (determined by in situ hybridization and denoted by silver grains) following continuous central leptin infusion.Note that while neuropeptide Y mRNA expression was decreased on day 3 of leptin infusion, mRNA expression remained unchanged on day 13 ofleptin infusion compared to that of control. Relative optical density of NPY mRNA (as determined by in situ hybridization) in rostral, middle andcaudal division of the arcuate nucleus after 3 or 13 days of leptin infusion (right panel). Data are means# SEM of five rats in each group and arerepresented relative to aCSF controls. $p < 0:05 versus aCSF control group. (Adapted from [270].)

Fig. 10. DNA-binding activity of STAT3 in the hypothalamus is in-creased after 2, 4, or 16 days of central leptin infusion in rat. (A) DNA-binding activity of STAT3 in the MBH extracts as determined by anEMSA using a 32P-labeled M67-SIE oligonucleotide probe. (B) resultsobtained by phosphorimaging and expressed as relative to aCSFgroup. Values represent the mean#SEM for the number of animalsindicated in parentheses. $p < 0:01 vs. all other groups; a, p < 0:01 vs.d 2 and d 4 leptin groups. PF, pair fed. (Adapted from [240].)

Fig. 11. SOCS3 gene expression (as determined by ribonuclease pro-tection assay) in the hypothalamus is increased after 2, 4, or 16 days ofcentral leptin infusion in rat. (A) representative phosphor imagesshowing the level of SOCS3 mRNA and b-actin mRNA in the MBH.(B) results obtained by phosphorimaging showing the changes inSOCS3 mRNA levels. The values are first normalized to b-actinmRNA and then expressed as relative to aCSF group. Values representthe mean#SEM for the number of animals indicated in the paren-theses. $p < 0:01 vs. corresponding aCSF groups. C, aCSF control,PF, pair fed; L, leptin. (Adapted from [240].)


presence of elevated JAK2-STAT3 signaling duringchronic leptin infusion provides evidence in support of acritical role of other pathways including the PI3K–PDE3B–cAMP pathway [371] in transducing leptin ac-tion in these neurons. In this regard, our preliminarystudy [275] shows that continuous central leptin infusionresulted in decreased hypothalamic cAMP levels in as-sociation with an increased PDE3B activity on day 2 ofinfusion, when NPY, NT or POMC neurons were re-sponsive to central leptin infusion; but this effect wasabolished on day 16 of infusion in association with thedevelopment of resistance in these neurons. This finding,although preliminary, suggests a defective regulation ofPDE3B–cAMP pathway of leptin signaling followingcontinuous exposure of the hypothalamus to leptin, andthis may be responsible for the development of resis-tance in NPY, POMC or NT neurons. However, it isalso critical to identify if STAT3 transcriptional activityis altered after chronic leptin infusion and may be in-volved in leptin resistance. In this regard, recent evi-dence suggests that upon reaching the nucleustranscriptional activity of STAT proteins may be de-pendent on its interaction with other DNA-bindingprotein or co-activators [187]. Furthermore, STAT3transcriptional activity can be regulated by other factors(co-activator), such as p300/CBP (cAMP response ele-ment binding protein-binding protein) and steroid re-ceptor co-activators 1 (NcoA/SRC1a) [77,112,113,254].Because both CBP and SRC1 are localized in the hy-pothalamus [212,318], any alteration in these coactiva-tors could compromise STAT3 mediated leptin actiondespite an activation of STAT3. This is an interestingpossibility and requires future investigation. Althoughthis rat model of chronic leptin infusion may not nec-essarily be comparable to that of DIO, our evidence ofthe development of leptin resistance in the NPY, POMCand NT neurons following only 2 weeks of leptin infu-sion strongly suggests that this rat model provides aninteresting opportunity to decipher the mechanisms ofleptin resistance in the hypothalamus. In addition, sinceleptin withdrawal resulted in hyperphagia, this ratmodel can be used to examine the hypothalamicmechanism behind this phenomenon.

5.4. Central leptin resistance in ageing associated obesity

Late-onset obesity is characterized by a steady in-crease in body weight and adiposity as adults age untilearly senescence, after which body weight declines [295].This aging-associated obesity is different from DIO inthat the later occurs rapidly with HF diet, while theformer occurs slowly but steadily over a long period.Using F-344&BN rats, Scarpace and colleagues haveprovided very strong evidence in support of the devel-opment of both central and peripheral leptin resistanceduring age-related obesity [282]. Adiposity and serum

leptin levels are increased with age; and like late-onsetobesity in humans, F-344&BN rats exhibit a steady in-crease in body fat into early senescence, followed bydecline [188,281,303]. Although subcutaneous leptin in-fusion for 7 days decreased food consumption by 50% inyoung rats as compared to the saline control, but it onlydecreased 20% of food intake in aged rats; oxygenconsumption was increased by leptin in young but not inold rats; and hypothalamic NPY mRNA levels weredecreased by leptin in young but not in old rats [281].These results clearly suggest the development of leptinresistance in aged-rats. The effects of central leptin in-jection on food intake, UCP1 gene expression in theBAT and hypothalamic NPY mRNA levels were sig-nificantly reduced in aged rats as compared to that inyoung rats, demonstrating resistance to the anorexic andthermogenic effects of centrally administered leptin[303].

In addition, STAT3-DNA binding activity in re-sponse to central leptin was greatly reduced in aged ratsas compared to young rats; and old rats had reducedprotein levels of the long-form of the leptin receptor,suggesting that diminished leptin receptor protein maybe involved in reduced STAT3 activation in the hypo-thalamus of aged rats [283]. In a recent study, Scarpaceet al. [284] reported that both the anorexic and ther-mogenic responses to rAAV mediated central leptingene delivery were completely attenuated in aged-obeserats sometime after day 9 of 46-day period of the ex-periment without compromising leptin signal transduc-tion through STAT3 activation. In addition, even onday 9 when food intake was reduced, NPY and POMCneurons did not respond to central leptin gene therapyin the aged obese rats [284]. Furthermore, despite a re-sistance to anorexic action of leptin, body weight in theobese rats remained lowered after %20 days of centralgene delivery. This is somewhat similar to our finding ofthe maintenance of reduced body weight despite nor-malization in food intake in a rat model of chroniccentral leptin infusion [270]. However, in young rats,leptin signaling through STAT3 activation remainedelevated, albeit at reduced level, at both day 9 and day46 of leptin gene delivery; and leptin action on NPY andPOMC neurons, and anorexic (although reduced by50%) and thermogenic responses to leptin were not de-sensitized with prolonged elevated central leptin [284].In another study, this group showed that in 18-month-old mildly obese rats, the anorexic and energy-expen-diture responses were attenuated at 25 and 83 days,respectively, and STAT3 remained elevated following138 days of central leptin gene therapy [285]. Thus, thesestudies altogether suggest that in rats the developmentof leptin-induced leptin resistance is accelerated by theextent of obesity [285].

Central leptin gene therapy for 138 days has been re-ported to increase hypothalamic SOCS3 gene expression


in 18-month-old mildly obese rats, although STAT3activation was normal in these rats [285]. We also ob-served an increase in both STAT3 activation and SOCS3gene expression following chronic central leptin infusionin rat [240]. Thus, the role of SOCS3 in the developmentof leptin resistance in the hypothalamus is yet to bedemonstrated.

Similar to F344&BN rats, in old Wistar rats, ano-rectic and body weight-reducing responses to centralleptin injections are attenuated [101], suggesting thedevelopment of central leptin resistance. Also leptinuptake in the hypothalamus is reduced in associationwith decreased hypothalamic Ob-Rb mRNA and pro-tein levels in 24-month-old Wistar rats [100]. Foodrestriction decreases adiposity and recovers leptin re-sponsiveness in these aged rats. Aging also increasesSOCS3 expression in the hypothalamus, and food re-striction partially reverts the increases in SOCS3 mRNAlevels associated with aging [244]. Increased SOCS3 ex-pression has also been reported in 18-month-old leanwild type (+/+) Zucker diabetic fatty rats [344]. Becausein these studies STAT3 activation was not examined, itis not clear whether increased SOCS3 is involved incentral leptin resistance in these aged rats.

Overall, age-associated obesity appears to be, at leastpartly, due to the development of central leptin resis-tance. This is most likely due to decreased leptin uptake,and down regulation of leptin receptor signalingthrough the JAK–STAT pathway in the hypothalamus.Although SOCS3 is increased in aged rats, the role ofSOCS3 in hypothalamic leptin resistance during aging isnot clear and should be explored more mechanistically.

5.5. Leptin resistance during pregnancy

During pregnancy, leptin levels are elevated in serumduring human and rodent gestation [55,106,159,177,207]. The source of increased circulating leptinduring pregnancy may be an increased leptin productionin adipose tissue and/or placenta, or increased level ofleptin-binding protein [6,106,159, 207,299]. However,food intake either increases [259] or remains unchanged[307] during pregnancy despite hyperleptinimia, sug-gesting that the pregnancy induces a state of leptin re-sistance most likely in the hypothalamus. Leptinsignaling in the hypothalamus during pregnancy is in-completely understood. Garcia et al. [104] reported adecrease in Ob-Rb mRNA levels in the hypothalamus ofpregnant rats on day 18 of gestation as compared tonon-pregnant animals. Seeber et al. [296] reported thathypothalamic Ob-Rb mRNA expression was elevatedon day 7 of pregnancy but returned to pre-pregnancylevels by mid gestation and remained stable thereafter.Because the status of leptin receptor phosphorylation orSTAT3 activation was not examined in either of thesestudies, the interpretation of small changes in OB-Rb

levels becomes difficult. Nevertheless, better under-standing of the mechanism of leptin signaling duringhyperleptinimia seen in pregnancy may shed some lighton the mechanism of central leptin resistance.

5.6. Selective leptin resistance

Recently, Mark et al. [205] have proposed an inter-esting concept of ‘‘selective leptin resistance.’’ This hascome from their findings that while agouti yellow obese(Ay) mice develop resistance to the satiety and weight-reducing effects of central and peripheral treatment ofleptin, the sympathoexcitatory action of leptin is pre-served in these animals [68]. Similar preservation ofsympathetic activation despite the resistance to meta-bolic action of leptin has also been reported in DIO miceby these authors [205]. Since intracerebroventricularadministration of leptin, like peripheral administration,produces regional sympathetic stimulation, and lesionsin the arcuate nucleus prevent the sympathetic responsesto peripheral leptin administration, it is suggested thatthe sympathoexcitatory action of leptin originates in thehypothalamus [131]. Although human obesity is asso-ciated with hyperleptinimia, suggesting leptin resistance,but it is often associated with hypertension [156] andincreased sympathetic activity [117,262,286]. It will beinteresting whether the concept of ‘‘selective leptin re-sistance’’ is applicable to human obesity. In this regard,it is to be noted that in Wistar rat obesity due to HF dietdecreases the sympathetic nervous and cardiovascularresponses to icv leptin [195], suggesting resistance also atthe level of sympathetic activity. Nevertheless, the con-cept of selective leptin resistance is very interesting andshould be carefully interpreted and requires furtherinvestigation in different models of obesity.

6. Summary and conclusion

In this review, available data on the mechanisms ofleptin signaling in the hypothalamus in regulation offood intake and body weight in normal condition andthat during the development of leptin resistance in DIO,ageing-associated obesity and following continuouscentral leptin infusion have been summarized. It appearsthat in addition to the conventional JAK2–STAT3pathway, an alternative insulin-like signaling pathway,involving activation of PI3K and PDE3B and reductionin cAMP levels, mediates leptin intracellular signaltransduction in the hypothalamus. A crosstalk betweenthe JAK2–STAT3 and PI3K–PDE3B–cAMP pathwaysmay be critical for normal leptin signaling in the hypo-thalamus. At the neuronal level, leptin action is medi-ated by orexigenic and anorectic signal producingneurons in the ARC–PVN–LH/PFH axis. Because ofmorphological connections between the neurons in this


axis, and the evidence of functional interactions amongthese neurons, it is no wonder that pharmacologicalintervention of activity of any one of them, in mostcases, reverses the anorectic effect of leptin on food in-take. While it is clear that NPY/AgRP and POMC/CART neurons may represent the major neuronal sys-tems for mediating leptin action on food intake andbody weight, it is important to consider the contributionof other members of the neural circuitry that regulatesenergy homeostasis. Furthermore, one of the importantmechanisms by which leptin acts in the hypothalamusmay be by modifying the action of the neural signal afterit is being produced, and therefore action at the post-synaptic level could still occur without having any effecton gene expression. This mechanism is most likely in-volved in mediating effect of leptin on food intake withinhour of central injection, because leptin!s effect on geneexpression of target neurons cannot be achieved withinthis short time period. One of the other mechanisms bywhich leptin might be acting is by derailing the inter-actions among orexigenic signals and or enhancing in-teractions among anorectic signals in the hypothalamus.

It has been a matter of debate whether obesity isassociated with central leptin resistance. The evidencepresented in this review clearly suggests that centralleptin resistance contributes to the development of DIOand aging-associated obesity. Among the possiblemechanisms, a defective nutritional regulation of leptinreceptor, defective STAT3 signaling and or a defect atdown stream of leptin receptor signaling in specificneurons such as NPY, POMC or NT have been impli-cated for central leptin resistance. Interestingly, leptinresponsive neurons (e.g., NPY and POMC) developleptin resistance in response to central leptin infusiondespite sustained activation of the JAK2–STAT3 path-way. Thus, we hypothesize that besides the JAK2–STAT3 pathway, defect in other pathways of leptinsignaling, such as PI3K–PDE3B–cAMP pathway, couldplay very important role in the development of leptinresistance in the hypothalamus. Furthermore, sustainedelevation of JAK2-STAT3 signaling in the presence ofincreased SOCS3 expression and the development ofleptin resistance in leptin-sensitive neurons duringchronic central leptin infusion challenge the role ofSOCS3 in central leptin resistance. However, if SOCS3is involved in leptin resistance, it can be predicted thatablation of SOCS3 in the hypothalamus will result indecreased body weight, and the animals will be hyper-sensitive to leptin and would become resistant to DIO asseen in PTP1B knockout mice. It is also likely that co-operation between different negative regulators of cy-tokine signaling such as SHP2, PTP1B, and SOCS3 iscritical in proper leptin signaling in the hypothalamus.An important caution about the concept of leptin re-sistance in DIO, whether it is central or peripheral, thatthese animals are fertile, and sympathoexcitatory effect

of leptin is preserved. Therefore, some action of leptin isstill intact in the hypothalamus, and it is possible thatpartial leptin signaling seen in these animals could beresponsible for transducing these effects.

In conclusion, PI3K–PDE3B–cAMP pathway inter-acting with the JAK2–STAT3 pathway of signal trans-duction constitutes a critical component of leptinsignaling in the hypothalamus, engaging various orexi-genic and anorectic signal-producing neurons in theARC–PVN–LH/PFH axis. Resistance or attenuatedresponse to specific action of leptin, particularly foodintake and body weight regulation, may occur due todefect in any of the steps in the signal transductionmechanism and or a defect at downstream of signaling,such as other co-factors/co-activators, resulting in thedevelopment of leptin resistance in target neurons. Un-derstanding the key signaling mechanisms that are beingaltered during the development of leptin resistance iscritical for the drug discovery to treat and or preventobesity and related disorders.

Acknowledgments

The author is indebted to Drs. Allan Zhao andRobert O!Doherty for their collaboration in somestudies. This work was supported by US Public HealthService Grants DK 54484 and DK 61499.

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