sig

a*,

s & M

arch

stand

r, stu

topoi

the

other hormonal abnormalities [65]. This so-called adiposus This model was supported by studies in rodents, in which

forced overfeeding resulted in inhibition of voluntary feed-

[107] provided further insights into the link between adipose

Physiology & Behavior 81 (2dual center model of feeding regulation. It was postu-

lated that a satiety center was present in the ventrome-

dial hypothalamus while a feeding center was present

in the LHA [6,108]. However, the idea of discrete brain

centers for regulation of body weight was controversial, as

precise lesions of hypothalamic nuclei did not reproduce

the above phenotypes [65]. Nonetheless, these classic

tissue and the brain. He showed that cross-circulation

(parabiosis) between obese VMH-lesioned and normal (non-

lesioned) rats resulted in suppression of feeding and weight

loss in the normal rat, while the VMH-lesioned partner

gained weight. In contrast, parabiosis of a pair of obese

VMH-lesioned rats did not prevent hyperphagia or weight

gain in either rat. These findings suggested that a circulatingobservations provided an anatomic framework for theinsufficiency; however, later studies pointed to disruption

of hypothalamic pathways [6,65,108]. Lesions of the

ventromedial hypothalamic (VMH) region resulted in

hyperphagia and morbid obesity, while lesions of the

lateral hypothalamic area (LHA) prevented spontaneous

feeding, leading to death from starvation [6,108]. These

ing, whereas food deprivation or surgical removal of adi-

pose tissue stimulated food intake until body weight was

restored [74,101103]. Although it was proposed that a

factor emanating from adipose tissue signaled the brain to

regulate body weight and fat content, the chemical nature of

this substance remained elusive. Experiments by Herveygenitalis syndrome, was initially attributed to pituitaryA connection between the brain and regulation of body

weight was first postulated, based on the observation that

tumors encroaching on the base of the brain caused

voracious appetite, morbid obesity, hypogonadism and

expenditure. Based on this observation, Kennedy [121]

proposed the existence of a physiologic system designed

to match energy intake to expenditure, with the goal of

keeping body weight, specifically fat, at a constant level.Leptin

Rexford S. Ahim

Department of Medicine, Division of Endocrinology, Diabete

415 Curie Boulevard, 764 Clinical Rese

Abstract

The discovery of leptin was a major breakthrough in our under

Leptin was initially thought to act mainly to prevent obesity; howeve

fasting, regulation of neuroendocrine and immune systems, hema

signaling pathways which mediate these diverse effects of leptin in

D 2004 Elsevier Inc. All rights reserved.

Keywords: Leptin; Obesity; Feeding; Hypothalamus; Neuropeptide

1. Early ideas on energy homeostasisexperiments demonstrated a significant role of the brain

in energy homeostasis.

0031-9384/$ see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.physbeh.2004.02.014

* Corresponding author. Tel.: +1-215-573-1872; fax: +1-215-573-

5809.

E-mail address: ahima@mail.med.upenn.edu (R.S. Ahima).naling

Suzette Y. Osei

etabolism, University of Pennsylvania School of Medicine,

Building, Philadelphia, PA 19104, USA

ing of the role of adipose tissue as a storage and secretory organ.

dies have demonstrated profound effects of leptin in the response to

esis, bone and brain development. This review will focus on the

brain and other physiologic systems.

Humans and most mammals maintain a constant body

weight despite short-term fluctuations in feeding and energy

004) 223241satiety factor related to adipose tissue acted at the VMH to

suppress feeding and prevent obesity [107].

The notion that adipose tissue played an active role in

energy homeostasis gained further credence as a result of the

discovery of spontaneous mutations, ob (obese) and db

(diabetes), which caused hyperphagia and morbid obesity

in mice. In his classic experiments, Coleman [44,45] ob-

served that parabiosis of ob/ob and lean (wild-type) mice

among adipose tissue, the nervous system and peripheral

organs.

Chronic glucocorticoid exposure increases leptin synthesis

and release from cultured adipocytes and in vivo

[53,62,126,132,142,149]. A sexual dimorphism of leptin

has been demonstrated in several species [177,190]. In

humans, leptin is higher in females than males matched for

age, and the gender difference has been attributed to higher

leptin production in subcutaneous adipose tissue, stimulation

of leptin by estrogen in females and suppression of leptin by

testosterone in males [32,33,61,120,173,175,177]. Unlike

humans, leptin is higher in male rodents compared with

females [160]. The reasons for these species differences in

leptin are unclear.

Leptin is elevated during acute infection, and in response

to endotoxin and proinflammatory cytokines (Table 1)

[22,78,119,180]. In contrast, cold exposure, catecholamines

and melatonin decrease leptin [57,132,169,189,200,215].

There have been conflicting reports regarding the effects

ogy &2. Control of leptin production

The murine Lepob gene was discovered through posi-

tional cloning [5,220]. In mice, the leptin gene encodes a

4.5 kilobase mRNA transcript with a highly conserved 167-

amino acid open reading frame [220]. Leptin is remarkably

similar across species [93,105,117,118,220]. It is synthe-

sized mainly by adipose tissue and is released into the

blood [220]. Various regulatory elements have been iden-

tified within the leptin promoter, e.g., cAMP and glucocor-

ticoid response elements, and CCATT/enhancer and SP-1

binding sites, suggesting a direct regulation of leptin

expression through membrane and transcriptional pathways

[93,102,105,118,220]. Leptin is produced, albeit at lower

levels in other tissues, such as gastric epithelium, skeletal

muscle and placenta [7,143,144,206]. Studies have sug-

gested physiologic roles of leptin in these tissues. For

example, leptin mRNA and protein levels are increased in

skeletal muscle following glucosamine treatment, consistent

with involvement in energy metabolism [206] (Table 1).

Leptin expression in the stomach is stimulated by feeding,

cholecystokinin and gastrin, suggesting a role in regulation

of energy balance [7] (Table 1). Placenta leptin is stimu-

lated by hypoxia, elevated in eclampsia and may influence

fetal outcome [143,144] (Table 1). Furthermore, de novoresulted in suppression of feeding and weight loss in the ob/

ob mice. In contrast, body weight was drastically reduced in

wild-type or ob/ob mice when parabiosed with db/db mice,

whereas the latter continued to gain weight [44,45]. These

seminal findings suggested that the ob locus encoded a

circulating satiety factor, while the db locus mediated the

tissue response [44,45]. More than four decades later, the ob

(Lep) gene was discovered, and its product missing in ob/ob

mice was named leptin (from the Greek root leptos

meaning thin), because it suppressed feeding and decreased

body weight when administered in mice [25,99,161,220].

On the other hand, obesity in db/db mice was linked to a

defect of the leptin receptor (LEPR) [38,129,199]. The

discovery of leptin has shed light on the complex biology

of adipose tissue [84]. Contrary to the prevailing view of

adipose tissue as merely a storage depot for triglyceride, we

now know that adipose tissue is composed of specialized

fat-storing cells (adipocytes) as well as vascular and im-

mune cells which mediate various physiologic processes

[84,85]. Adipose tissue secretes leptin, adiponectin, resistin,

proinflammatory cytokines, complement factors, steroid

hormones and other molecules which actively regulate

energy balance, endocrine, immune and cardiovascular

systems [84,85]. An understanding of the biology of leptin

offers significant insights into the complex interrelationships

R.S. Ahima, S.Y. Osei / Physiol224leptin synthesis has been demonstrated in the brain, sug-

gesting a paracrine or autocrine action; however, thephysiologic relevance of brain-derived leptin remains to

be ascertained [147,212].

In ad libitum fed animals, the levels of leptin mRNA and

protein in adipose tissue and plasma are positively correlat-

ed to body fat and adipocyte size [47,83,138]. Thus, obese

persons have higher leptin mRNA and protein levels than

lean individuals. Leptin secretion appears to occur mainly

via a constitutive mechanism, although the levels can be

regulated by various physiologic states. For example, leptin

falls during fasting, out of proportion to the decrease in

body fat [4,21,179]. Conversely, leptin mRNA and protein

are increased several hours after eating [122,179]. The

effects of nutrition are mediated, at least in part by insulin,

as shown by a direct stimulation of leptin synthesis and

release when adipocytes are cultured in the presence of

insulin [13,19,137,172]. In both humans and rodents, the

postprandial rise in leptin follows the peak insulin secretion

[79,182,188]. In contrast, insulin deficiency results in rapid

reduction of leptin mRNA and protein levels [122,179].

Leptin is regulated by steroid hormones (Table 1).

Table 1

Factors implicated in leptin regulation

Increase leptin Decrease leptin

Adipose tissue Adipose tissue

Overfeeding Fasting

Obesity (except ob/ob mutation) Cold exposure

Insulin h-adrenergic agonistGlucocorticoids Testosterone

Acute infection

Proinflammatory cytokines (TNF-a, IL-1)Placenta Stomach

Insulin Feeding

Glucocorticoids Cholecystokinin

Hypoxia/eclampsia

Skeletal muscle

Glucose

Glucosamine

Lipids

Behavior 81 (2004) 223241of thyroid and growth hormone on leptin. While some

studies have reported a rise in leptin in thyroid deficiency,

have not observed a significant change in postnatal leptin

levels, or an association between leptin and reproductive

ogy &others have demonstrated an increase in leptin in response to

hyperthyroidism or no significant effect of thyroid hormone

on leptin [69,80,140,159,163]. Similarly, the link between

growth hormone and leptin remains controversial [80,90].

Leptin level is increased in growth hormone deficiency

(GHD), presumably as a result of increased body fat [80].

However, this association has not been consistent with other

studies. For example, growth hormone treatment has been

reported to stimulate leptin, or have no significant effect on

leptin [80,90].

A nocturnal rise in leptin occurs under ad libitum fed

conditions [3,179,182]. In rodents, the increase in leptin

mRNA level and plasma leptin is prevented by fasting

[179]. Moreover, restriction of feeding to the light cycle

shifts the peak plasma leptin level from nocturnal to

diurnal [3,179]. The shift in leptin is accompanied by a

parallel shift in insulin and corticosterone; however, it is

doubtful that the latter is mediated by leptin, because a

diurnal rhythm of corticosterone occurs in ob/ob mice

despite a total absence of leptin [3,179]. As in rodents,

leptin peaks at night and declines during the day in

humans [133,134,182,188]. This pattern is thought to be

regulated mainly by insulin [182,188]. Interestingly, the

diurnal leptin rhythm appears to be blunted with aging,

and has been associated with an increase in visceral

adiposity and insulin resistance [135].

An ultradian leptin rhythm has been demonstrated fol-

lowing frequent blood sampling in humans [133,134].

Leptin is secreted in pulses that are inversely associated

with ACTH and cortisol, and positively correlated to gona-

dotropins, estradiol and thyrotropin [133,134]. Obesity is

associated not only with higher basal leptin level, but also a

blunted diurnal rhythm and dampened pulsatility [135,187].

Healthy men and women have similar leptin pulse frequen-

cy; however, leptin pulse amplitude is more than twice as

high in women [135]. The gender difference appears to be

influenced mainly by the mass or amount of leptin released

or removed per unit time, suggesting that women may be

more resistant to leptin feedback than men [135]. Potential-

ly, this may underlie the greater susceptibility to disorders of

feeding and body weight regulation in females.

To test the hypothesis that changes in plasma leptin were

related to the levels of luteinizing hormone (LH) and

estradiol, Licinio et al. [134] sampled plasma from six

healthy women every 7 min for 24 h. Cross-correlation

analysis revealed a strong association between leptin and

LH release, with a lag time of 4284 min. The ultradian

pattern of leptin was synchronous with LH and estradiol.

Moreover, the nocturnal leptin peak was positively corre-

lated to LH pulses of longer duration, higher amplitude and

larger area. The nocturnal synchronicity of LH and leptin

was associated with significant coupling with estradiol,

suggesting a functional link between leptin and the hypo-

thalamicpituitarygonadal axis [134]. The latter is con-

R.S. Ahima, S.Y. Osei / Physiolsistent with the diminution of leptin amplitude and

frequency in patients with hypothalamic amenorrhea [128].development [139]. So far, it is not known whether the

changes in circulating leptin with age are determined by

leptin synthesis or clearance.

Leptin gene mutations are rare. In C57Bl/6J mice, a

frameshift mutation (C-to-T) results in a stop codon at

position 105 instead of arginine, leading to production of

a truncated protein that cannot be secreted [220]. Leptin

mRNA is increased in ob/ob mice, suggesting a short

negative feedback regulation of leptin synthesis [220].

Leptin gene mutations have been identified in highly

consanguineous human families [146,158,192]. Affected

members of a Pakistani family have a deletion of guanine

in codon 133, resulting in synthesis of a truncated protein

which is degraded [146,170]. A missense leptin gene

mutation (C-to-T in codon 105) in a Turkish family results

in production of a mutant protein which cannot be secreted

[158,192]. In these cases, a lack of bioactive leptin culmi-

nates in hyperphagia, morbid obesity, hypothalamic hypo-

gonadism and immune suppression, similar to ob/ob mice.

Moreover, heterozygousity of the leptin gene has been

associated with increased body fat in both rodents and

humans, indicating a dose effect of leptin on body fat

[41,72,104]. Nonetheless, there are significant differences

between leptin-deficient humans and rodents, as some

characteristics of leptin deficiency in C57Bl/6J mice, such

as impaired thermoregulation, elevated glucocorticoids, in-

sulin resistance and diabetes, have not been observed in

leptin-deficient humans [70,146,158,168]. It is possible that

these disparate responses to leptin deficiency are due to

species differences in energy substrate fluxes, as well as

brown adipose tissue metabolism which is prominent in

rodents [112].

3. Leptin receptors

The first LEPR was isolated from mouse choroid plexus

by expression cloning [199]. However, because this receptor

was present in db/db mice, it was apparent that other LEPRs

had to exist [199]. To date, six splice variants of the LEPR,

a to f, have been identified [5,198] (Fig. 1). LEPR

belongs to a family of class I cytokine receptors, which

typically contains a cytokine receptor homologous domainThe timing of leptin production varies according to age.

In rodents, leptin is expressed widely during the prenatal

period. Some studies have indicated that leptin mRNA and

protein levels decrease rapidly after birth, followed by a

transient increase in the neonatal period and a steady

increase in adults [3,55,145,190]. Similar changes in plasma

leptin have been observed in longitudinal studies in prepu-

bertal boys, in whom leptin is thought to exert a permissive

effect on sexual maturation [86,139]. However, other studies

Behavior 81 (2004) 223241 225in the extracellular region. Two conserved disulfide links are

present in the N-terminus, and a WSXWS motif is present in

ino a

iffer a

smem

ogy &the C-terminus. LEPR shares highest sequence similarity

with receptors for interleukin-6 (IL-6), leukemia inhibitory

Fig. 1. Domain structure of alternatively sliced LEPR isoforms. Terminal am

Leptin receptors share a common extracellular leptin-binding domain, but d

intracellular motifs necessary for JAK-STAT signaling. LEPRe lacks a tran

receptor.

R.S. Ahima, S.Y. Osei / Physiol226factor (LIF), granulocyte-colony stimulating factor (GCSF)

and oncostatin [198]. LEPR isoforms have a similar extra-

cellular ligand-binding domain at the amino terminus, but

differ at the intracellular carboxy-terminal domain. LEPRa,

LEPRb, LEPRc, LEPRd and LEPRf have transmembrane

domains; however, only the long receptor, LEPRb, has

intracellular motifs necessary for activation of the JAK-

STAT signal transduction pathway. LEPRe lacks both trans-

membrane and intracellular domains and circulates as a

soluble receptor [198].

The db/db mutation is caused by insertion of a premature

stop codon in the 3V-end of LEPRb mRNA transcript,resulting in synthesis of LEPRa [38,129,198]. As expected,

db/db mice are hyperphagic, morbidly obese, sexually

immature, exhibit cold intolerance and elevated glucocorti-

coids, and do not respond to leptin treatment [38,129].

However, the phenotype of db/db mice is influenced by

genetic background. For example, breeding on C57BlKS/J

background results in early-onset severe diabetes, due to

apoptosis of pancreatic h cells, and a shorter life span. Incontrast, the C57Bl/6J background protects against diabetes

and promotes longevity in db/db mice. Mice homozygous

for Leprdb3J mutation fail to express all membrane LEPRs

[124]. This mutant is hyperphagic, cold intolerant, obese,

insulin resistant and infertile. Expression of a neuron-spe-

cific enolase (NSE)-LEPRb transgene restored the ability to

activate the JAK-STAT pathway in both db3J/db3J and db/

db mice, partially reversed hyperphagia, obesity, glucoseintolerance and infertility in males, and rescued the cold

intolerance in both sexes [124]. Importantly, NSE-LEPRb

cid residues for various LEPR isoforms are denoted by the alphabet code.

t the carboxy-terminus intracellular domain. The long isoform, LEPRb, has

brane domain (TM) and intracellular domains and circulates as a soluble

Behavior 81 (2004) 223241was expressed mainly in the brain, confirming the impor-

tance of this organ as a target for leptin [124]. Analysis of

gene expression revealed that NSE-LEPRb restored the

ability to regulate proopiomelanocortin (POMC), agouti

gene-related protein (AGRP) and neuropeptide Y (NPY),

consistent with a significant role of these neuropeptides as

mediators of leptin action [124].

LEPR mutations have been discovered in rats [40,51,

195,212,215]. Substitution of Gln for Pro at amino acid

position 269 in the extracellular domain results in drastic

reduction of cell surface expression of LEPR and reduced

binding to leptin in Zucker fatty (fa/fa) rats [40,51,211].

These mutant rats are hyperphagic, obese and hyperlipi-

demic, and have increased glucocorticoids and hyperglyce-

mia [40,51]. When expressed in Chinese hamster ovary

(CHO) cells, the fa/fa receptor not only exhibited a reduction

in leptin-binding affinity, but also performed reduced signal

transduction, as evidenced by induction of the immediate

early genes, c-fos, c-jun, and jun-B in CHO cells

[40,52,211]. Moreover, fa/fa rats are capable of responding

to high doses of leptin administered by intracerebroventric-

ular injection, consistent with a partial function of the

receptor [51]. The obese Koletsky rat (SHROB, fak) has a

point mutation of LEPR at amino acid 763, resulting in a

premature stop codon in the extracellular domain and ab-

sence of all cell surface LEPRs [195,215]. Plasma leptin

concentration is greater than lean spontaneous hypertensive

(SHR) littermates, suggesting severe leptin resistance. Kolet-

conserved box 1 and 2 motifs in the intracellular domain of

LEPRb (Fig. 1). In mice, the box 1 motif (amino acids 6

ogy &17) is critical for JAK2 activation, and box 2 motif (amino

acids 4960) is required for maximal activation of LEPRb.

Binding of leptin to LEPRb results in autophosphorylation

of JAK1 and JAK2, and tyrosine phosphorylation of the

cytoplasmic domain of LEPRb and downstream transcrip-

tion factors, named STATs. These signaling molecules are

highly expressed in hypothalamic, brainstem and other

brain regions which control food intake, autonomic and

neuroendocrine function [98].

LEPRb has three conserved tyrosine residues in the

intracellular domain, corresponding to Y985, Y1077 and

Y1138 in mice. Leptin treatment results in phosphorylation

of the latter site, and recruitment of STAT3 via its SH2

domain. Tyrosyl-phosphorylated STAT3 undergoes homo-

dimerization and nuclear translocation, and transactivates

target genes by binding to specific promoter elements [150].

The essential role of Y1138 was demonstrated in mice bysky rats are hyperphagic, morbidly obese and have various

hormonal abnormalities [195,215]. However, in contrast to

fa/fa rats, obese Koletsky rats do not respond to leptin

treatment [215].

Leptin receptor mutations are rare in humans. Affected

members of a French family have a single nucleotide

substitution (G-to-A) in the splice donor site of exon 16,

resulting in encoding of a LEPR lacking both transmem-

brane and intracellular domains [42]. The mutant receptor

circulates at high concentrations bound to leptin [42]. As is

the case in rodents, LEPR null humans are hyperphagic,

morbidly obese and fail to undergo normal sexual matura-

tion [42]. Furthermore, these patients failed to respond

normally to thyrotropin-releasing hormone (TRH) and

growth-hormone-releasing hormone (GHRH) testing, sug-

gesting a critical role of leptin in neuroendocrine regulation

[42].

4. Intracellular signal transduction of leptin

Leptin circulates as a 16-kD protein partially bound to

plasma proteins [113,187]. Most likely, protein-bound lep-

tin exists in equilibrium with free leptin, and the latter

represent the bioactive hormone. Studies have shown that

the ratio of bound-to-free leptin is increased in obesity,

pregnancy and LEPR mutation [42,113,187]. The rise in

serum leptin in pregnancy and LEPR null humans is due to

binding to LEPRe [42,187]. An additional pool of leptin

may exist in various tissues, and contribute to the mainte-

nance of plasma leptin [111]. As with other class I cytokine

receptors, e.g., IL-6, LIF, oncostatin M, ciliary neurotrophic

factor, growth hormone and prolactin, the leptin signal is

thought to be transmitted mainly by the JAK-STAT path-

way [8,14,88,150,203]. JAKs associate constitutively with

R.S. Ahima, S.Y. Osei / Physiolreplacing this residue with serine [14]. Y1138S knock-in

mice (LeprS1138) were unable to activate STAT3 [14]. Likedb/db mice [89], LeprS1138 homozygous mice became hy-

perphagic and obese. However, in contrast to db/db mice,

LeprS1138 homozygotes attained normal sexual maturation,

fertility and body length [14]. Moreover, LeprS1138 homo-

zygotes were less hyperglycemic [14]. Expression of NPY

in hypothalamus was elevated in db/db but not LeprS1138

homozygotes, whereas melanocortin expression was sup-

pressed in both mutants [14]. These findings suggest that the

LEPRb-STAT3 signaling is required for energy balance and

regulation of melanocortins; however, a separate LEPRb

pathway, possibly involving other STATs, is likely to control

reproduction, linear growth, glucose and hypothalamic NPY

mRNA level [14].

Leptin-activated LEPRb regulates well-known insulin

targets, such as IRS-1, MAP kinase, ERK, Akt, AMP kinase

and PI3-kinase, raising the possibility that leptin pathways

act in concert with insulin to control energy metabolism and

other cellular processes [154,165]. This idea is supported by

the coexistence of LEPR, JAKs, STATs, insulin receptor and

its substrates in a variety of tissues, e.g., neurons, adipo-

cytes, pancreatic islets, immune cells and adrenal cortex.

Leptin is able to induce the tyrosine phosphorylation of the

SH2-containing protein SHC, which associates with the

adaptor protein, Grb2. The formation of this complex may

directly link tyrosine phosphorylation events to Ras activa-

tion, and serve as a critical step in mediating the effects of

leptin and insulin on cell proliferation and differentiation

[8,20,28,150,154]. Studies have also shown that leptin and

insulin responses in the brain can both be disrupted by

inhibition of PI3 kinase, providing further proof for an

overlapping signaling pathway [154].

Although leptin enters the brain via a saturable process,

the exact structures responsible for leptin transport are

unknown [9,10]. Based on experience with other polypep-

tide hormones, it had been suggested that leptin was trans-

ported by receptor-mediated transcytosis across the blood

brain barrier [160]. Because short LEPRs are widely present

in brain microvessels, kidney, liver, lung and gonads, and

capable of binding, internalizing and translocating leptin, it

was suggested that these receptors mediate leptin transport

[19,20,92,202]. Cerebrospinal fluid (CSF) leptin is present

but markedly reduced in obese Koletsky rats which totally

lack membrane LEPRs, indicating that other factors besides

LEPRs are involved in brain leptin transport [195,217].

Furthermore, it is doubtful that CSF is a significant source

of leptin for neurons, because leptin concentration in CSF is

lower than plasma leptin and below the dissociation con-

stant of the LEPR [20,27,81,92,183].

Despite the widespread distribution of LEPRs in the

brain and peripheral organs, there is little evidence in

support of an involvement of these receptors in energy

homeostasis or neuroendocrine control. Leprdb homozygous

mice lacking LEPRb but possessing a full complement of

short LEPR isoforms, develop hyperphagia, cold intoler-

Behavior 81 (2004) 223241 227ance, obesity, insulin resistance and infertility, as is the case

with Leprdb3J homozygotes that are null for all isoforms of

prone to DIO [194]. In contrast, POMC, the precursor of

the anorexigenic neuropeptide a-MSH, is elevated in

ogy &LEPR [124]. In contrast, transgenic expression of NSE-

LEPRb capable of activating JAK-STAT, partially reversed

obesity, hyperphagia, glucose and cold intolerance in male

and female db3J/db3J mice, and restored fertility in male

db3J/db3J mice, confirming the importance of LEPRb

[124].

Leptin binds to LEPRs in kidney epithelium, and the

complex is internalized and degraded [202]. A functional

role of LEPRs in leptin clearance is suggested by the

elevation of plasma leptin in patients with renal impair-

ment [185]. Long and short LEPRs are coexpressed in

some tissues, raising the possibility that heterodimers of

these receptors may signal leptin response through the

JAK-STAT pathway. However, chimeric receptor hetero-

dimers of LEPRa and LEPRb failed to activate JAK-

STAT, whereas receptor dimers of LEPRb gave rise to

the expected ligand-dependent activation of JAK2, phos-

phorylation of STAT3, and increased STAT3-dependent

promoter activity [8,150]. Furthermore, site-directed mu-

tagenesis has revealed that two hydrophobic residues

(Leu896 and Phe897) not present in LEPRa were essential

for leptin signal transduction [8].

The leptin signal is terminated by induction of SOCS-3, a

member of a family of proteins which inhibits the JAK-STAT

signaling cascade [17,66]. SOCS proteins have a variable N-

terminal domain, a central SH2 domain and a C-terminal

domain, termed SOCS-box motif. They are induced by

cytokines and act in a negative feedback loop to inhibit the

receptor. Overexpression of SOCS-3 inhibits leptin-mediated

tyrosine phosphorylation of JAK-2 [17,18,66]. Protein

tyrosine phosphastase (PTP)-1B is a critical downstream

regulator of leptin signal transduction [218]. PTP-1B recog-

nizes a specific substrate motif within JAK2. Overexpression

of PTP-1B decreased phosphorylation of JAK2 and blocked

leptin-induced transcription of SOCS-3 and c-fos. In con-

trast, deletion of the PTP-1B gene enhanced leptin sensitivity

in mice, thereby preventing obesity [218]. Hypothalamic

STAT-3 phosphorylation was also enhanced in PTP-1B-null

mice in response to leptin treatment, confirming the impor-

tance of PTP-1B as a mediator of in vivo leptin signaling

[183].

While these findings suggest an important role of the

JAK-STAT cascade in leptin signaling, there have been

reports of rapid effects of leptin that cannot be explained

by gene expression [49,91,114,191]. For example, leptin

inhibits NPY secretion from hypothalamic explants [91].

Application of leptin to hypothalamic slices hyperpolarizes

arcuate hypothalamic NPY neurons and depolarizes POMC

neurons [49]. In the latter case, POMC neurons are activated

in part through disinhibition by leptin-responsive NPY

neurons in the same nucleus [49]. Electrophysiologic studies

have also revealed an inhibitory response to leptin in the

supraoptic nucleus and modulation of vagal afferents in the

gut [114]. Furthermore, leptin is able to rapidly regulate

R.S. Ahima, S.Y. Osei / Physiol228glucose-sensitive neurons in the brain and insulin secretion

from pancreatic islets [191]. These effects appear to involveobesity-resistant A/J and SWR/J mice [15]. Expression of

genes which mediate adaptive thermogenesis, e.g., UCP-1,activation of ATP-sensitive potassium channels or other

membrane receptors.

5. Role of leptin in energy homeostasis

5.1. Leptin as an antiobesity hormone

At the time of its discovery, it was thought that leptin

acted as an afferent signal in the brain to suppress feeding

and increase energy expenditure [5,220]. This view was

largely based on the observation that obese (leptin-deficient)

rodents developed hyperphagia and morbid obesity, which

were reversed by leptin treatment, consistent with a feed-

back loop from adipose tissue to the brain [25,99,161].

However, the initial studies clearly demonstrated that leptin

replete wild-type mice were less sensitive to exogenous

leptin [25,99,161]. Subsequently, leptin mRNA and protein

levels were noted to be markedly elevated in obese rodents

(apart from ob/ob mice), and yet, the rise in leptin was

unable to suppress feeding or weight gain [33,83,138,205].

Likewise, diet-induced obesity (DIO) in humans is associ-

ated with increased leptin level and reduced sensitivity to

leptin treatment [47,109,138]. Akin to hyperinsulinemia and

insulin resistance, it has been postulated that the hyper-

leptinemia is indicative of leptin resistance [81].

DIO may arise from defective brain leptin transport, as

evidenced by reduced plasma-to-brain leptin transport in

obese rodents [9]. The CSF: plasma leptin ratio is reduced in

obesity compared with anorexia nervosa, and may underlie

the false perception of satiety in the latter [27,183]. Leptin

response is decreased in aged rodents, suggesting that leptin

resistance may be acquired [9]. Although no apparent

defects of LEPRb has been demonstrated in the vast

majority of obese animals, abnormalities of distal leptin

signaling molecules have been reported [17,18,28,63,66].

For example, DIO mice are unable to activate STAT-3 in the

hypothalamus following peripheral leptin injection, whereas

the response to intracerebroventricular leptin treatment is

preserved [63]. Leptin resistance may result from induction

of SOCS-3, and/or activation of SHP-2 and PTP-1B

[17,18,28,63,66,218]. SOCS-3 mRNA expression is higher

in the hypothalamus of obese agouti (Ay/a) mice and

thought to mediate leptin resistance [18]. However,

SOCS-3 is not consistently elevated in DIO, and its signif-

icance in the latter remains uncertain [63].

Susceptibility to DIO may be determined by differences

in the levels of hypothalamic neuropeptide targets of leptin

[15,167,194]. For example, the orexigenic hypothalamic

neuropeptide, NPY, is increased in C57Bl/6J mice, a strain

Behavior 81 (2004) 223241UCP-3 and PGC-1, is increased in A/J and SWR/J mice,

and may prevent obesity in these strains [167,194]. Obe-

ogy &sity-resistant SWR/J mice are more sensitive to leptin,

compared with obesity-prone C57Bl/6J mice [194]. More-

over, susceptibility to obesity in C57Bl/6J mice is posi-

tively correlated with failure to suppress hypothalamic

NPY mRNA and blunting of brown adipose tissue UCP-

1 expression [167,194]. Whether these factors are involved

in the pathogenesis of DIO in humans and other primates

remains to be determined.

Reduced leptin sensitivity in DIO and aged animals

predisposes to lipid accumulation in nonadipose tissues

[201]. This condition, known as steatosis, is characterized

by excessive triglyceride accumulation in liver, pancreatic

h-cells, myocardium and skeletal muscle, resulting in lip-otoxic insulin deficiency, diabetes, and impairment of

myocardium and other organs, characteristic of aging and

obesity [201]. The increase in extraadipose tissue lipid is

primarily the result of enhanced lipogenesis, although a

decrease in fatty acid oxidation also contributes (reviewed in

Ref. [201]). Consistent with this idea, pancreatic islets and

liver express high levels of lipogenic transcription factors,

e.g., SREBP-1c and PPARg, and their target genes, e.g.,acetyl coA carboxylase (ACC), fatty acid synthase (FAS)

and glycerol phosphate acyl transferase (GPAT), as a result

of impaired leptin signaling [130,201]. Leptin slows the

progression of steatosis and its sequelae, by stimulating lipid

oxidation and preventing toxic metabolites, such as ceram-

ide, from accumulating [130,201].

5.2. Leptin as a starvation signal

There is strong evidence showing that the dominant

action of leptin is to act as a starvation signal. Leptin

declines rapidly during fasting, and triggers a rise in

glucocorticoids, and reduction in thyroxine (T4), sex and

growth hormones [2,4]. Moreover, the characteristic de-

crease in thermogenesis during fasting and postfast hyper-

phagia is mediated, at least in part, through a decline in

leptin level [2,5]. The reduction in leptin during fasting

stimulates expression of NPY and AGRP, and suppresses

CART and POMC [2]. These fasting-induced responses

resemble the phenotypes of ob/ob and db/db mice [65].

Therefore, we reasoned that leptin deficiency was perceived

as a state of unmitigated starvation, leading to compensatory

responses, such as hyperphagia, decreased metabolic rate

and changes in hormone levels, designed to restore energy

balance [2,4,81]. In contrast to the low insulin levels

characteristic of fasting, ob/ob and db/db mice have ex-

tremely high insulin levels. Perhaps, the elevation in insulin

in these mice is commensurate with high energy efficiency,

and may contribute to excessive fat storage [81].

Chan et al. [34] have examined the role of leptin in

regulating neuroendocrine and metabolic function in fasted

humans. Placebo, low-dose recombinant-methionyl human

leptin (r-metHuLeptin) or replacement-dose r-metHuLeptin

R.S. Ahima, S.Y. Osei / Physiolwas administered during 72-h fasting. Replacement-dose

leptin prevented the starvation-induced changes in sexhormones and partially prevented the suppression of hypo-

thalamicpituitarythyroid axis and IGF-1 binding capac-

ity. However, unlike rodents, leptin replacement during

acute fasting did not affect fuel utilization, glucocorticoids

or growth hormone levels in humans [34]. An earlier study

by Rosenbaum et al. [176] demonstrated that chronic leptin

treatment fully prevented the reduction in energy expendi-

ture and thyroid hormone during sustained weight reduction

in humans [150]. Taken together, these data support the idea

that leptin plays an important role in controlling the neuro-

endocrine and metabolic response to caloric depletion.

Studies have suggested that low leptin may predispose to

obesity in apparently healthy populations [72,84]. For

example, family members heterozygous for a leptin gene

mutation have partial leptin deficiency and excess body fat

compared with wild-type patients [72]. Similarly, mice with

heterozygous mutations of the leptin gene have increased

body fat compared with wild-type littermates [41,104].

Presumably, the reduction in leptin level signals the brain

and other targets to enhance energy storage. It has been

reported that leptin is decreased in obesity-prone Pima

Indians [171]. Moreover, cross-sectional studies have sug-

gested that leptin is inappropriately low in 1020% of obese

individuals, suggesting that partial leptin deficiency may

promote obesity by stimulating appetite, decreasing energy

expenditure and creating the hormone mellieu necessary for

obesity [84]. More importantly, it is possible that these

obese patients with low leptin could benefit from leptin

supplementation [84].

NPY is increased in the hypothalamus in response to

leptin deficiency, and postulated to stimulate feeding and

weight gain [5]. Although the original report discounted a

role for NPY in the leptin-mediated response to fasting, later

studies have revealed a blunted postfast hyperphagia and

weight gain in NPY-deficient mice [11,184]. Moreover,

deletion of the NPY gene partly attenuated hyperphagia,

cold intolerance, obesity and infertility in leptin-deficient

ob/ob mice, confirming the importance of NPY as a sensor

of low leptin [68]. NPY acts via a variety of receptors in the

brain and peripheral tissues. Crossing the Y2 receptor

knockout mouse onto ob/ob background attenuated obesity,

hyperglycemia and high glucocorticoids, but did not alter

hyperphagia or hypogonadism in ob/ob mice [152,153]. In

contrast, deletion of Y4 receptor did not prevent obesity,

diabetes or excess glucocorticoids, but restored sexual

maturation and fertility in ob/ob mice [152].

The fall in leptin triggers a suppression of the immune

system during starvation [136]. Conversely, leptin treatment

stimulates the immune response, e.g., reversal of splenic and

thymic atrophy, delayed hypersensitivity and lipopolysac-

charide-mediated cytokine production and mortality [136].

The machinery for leptin signal transduction, i.e., LEPRb,

JAK and STAT, is present in immune cells, and leptin is

capable of directly regulating lymphocyte proliferation and

Behavior 81 (2004) 223241 229differentiation. Based on the robust responses to leptin

deficiency, it has been suggested that leptin may have

preceding the rise in testosterone [139]. A transient increase

in leptin has also been noted in boys aged 510 years [86].

ogy &evolved as a critical signal linking adipose energy stores and

the brain and peripheral targets, as a safeguard against the

threat of starvation [81]. Reduced leptin levels promote

energy intake and limit the high energy cost of reproduction,

thyroid thermogenesis and immune response [81]. While the

leptin-mediated adaptation to energy deficiency is likely to

have been beneficial in times of food shortage, this tendency

towards efficient energy metabolism may have contributed

to the current epidemic of obesity in an environment where

food is abundant [81].

5.2.1. Lipodystrophy

Lipodystrophic syndromes comprise of a heterogeneous

group of disorders characterized by partial or generalized

loss of adipose tissue depots, and commonly associated with

severe insulin resistance, diabetes, dyslipidemia and steato-

sis [87]. Adipocyte-secreted proteins, e.g., leptin and adi-

ponectin, are decreased in lipodystrophy [87]. By far the

commonest cause of acquired lipodystrophy is highly active

antiretroviral therapy (HAART)-induced lipodystrophy in

HIV patients [37]. HIV lipodystrophy results in loss of

facial and peripheral fat, preservation of visceral fat, insulin

resistance and lipid abnormalities [37]. Given the well-

known association between these metabolic alterations and

atherosclerosis, there is concern that the beneficial effect of

antiretroviral treatment would be offset by premature coro-

nary artery disease [37]. The striking similarities between

the metabolic syndrome of obesity and lipodystrophy

have stimulated a search for common underlying mecha-

nisms. Earlier studies attributed the metabolic changes in

lipodystrophy to the absence of adequate adipocyte storage

capacity in lipodystrophy, resulting in triglyceride accumu-

lation in liver, skeletal and cardiac muscle, and in the

pancreatic h-cell, and culminating in impaired insulin ac-tion, diabetes and lipid abnormalities [87]. This idea was

supported by studies showing that insulin sensitivity im-

proved following fat transplantation in mice with general-

ized lipodystrophy [88]. However, fat transplantation from

leptin-deficient ob/ob mice failed to reverse the metabolic

disturbance [46]. Rather, infusion or transgenic delivery of

leptin alone or in combination with adiponectin, improved

insulin resistance, glucose and lipids in lipodystrophic mice

[59,186,216]. These findings suggested that a deficiency in

adipose secreted factors, rather than decreased adipose mass

per se, contributed to the metabolic abnormalities in lip-

odystrophy [186,216].

Further support for a role of leptin in carbohydrate and

lipid metabolism came from experiments showing that

leptin replacement partially reversed insulin resistance,

steatosis and lipid abnormalities in lipodystrophic patients

[155,156,162]. Importantly, leptin replacement was more

effective than the standard-of-care plasmapheresis, in reduc-

ing hepatic steatosis and intramyocellular triglycerides, and

improving insulin sensitivity [155,156,162]. Interestingly,

R.S. Ahima, S.Y. Osei / Physiol230leptin replacement restored the pituitarygonadal axis in

lipodystrophic patients, confirming the importance of leptinIn the same study, plasma leptin was higher in girls;

however, there was no prepubertal increase [86]. Interest-

ingly, a nocturnal rise in leptin precedes the prepubertal

increase in pulsatile LH release in monkeys [193]. This

observation is contrary to an earlier report in which there

was no change in peripubertal leptin levels in relation to the

rise in LH, FSH and testosterone [164]. Possible reasons for

these disparate results include the timing of sample collec-

tion (i.e., daytime vs. nighttime), variability of LH release

and whether intact or castrated animals were studied [110].

Leptin stimulates the synthesis and release of LH andas a modulator of reproduction [155]. Molecular targets for

leptin include a reduction in fatty acyl-CoA, and induction

of hepatic and muscle lipid oxidation via activation of

AMP-activated protein kinase activation [216]. In rodents,

these effects of leptin are mediated centrally through the

sympathetic nervous system and peripherally through

LEPRb [216]. The beneficial effects of leptin on glucose

and lipids occur independently of regulation of food intake

and metabolic rate per se, and have given impetus for

consideration of leptin treatment in lipodystrophy as well

as obese patients with relatively low leptin levels.

6. Leptins effects on classical hormones

6.1. Reproduction

As discussed earlier, total leptin deficiency or insensitiv-

ity is associated with hypothalamic hypogonadism in

humans and rodents. In mice, the effect of leptin deficiency

on sexual maturation is modified by genetic background, as

evidenced by spontaneous pubertal development in ob/ob

mice bred onto Balb/c background [35,70]. Similarly, men-

strual cycles occurred spontaneously in a patient with leptin

gene mutation, while family members bearing the same

leptin gene mutation failed to undergo normal pubertal

development [158]. Leptin treatment restored LH secretion

and pubertal development in leptin-deficient patients, con-

firming its critical role in reproduction [73]. However, while

leptin is essential to puberty and reproductive cycles, studies

in ob/ob mice have indicated that it is not required for

gestation, paturition or lactation [148]. Based on studies in

rodents and nonhuman primates, leptin appears to exert a

permissive action to restore normal hypothalamicpitui-

tarygonadal axis function during starvation [12,39,96].

These actions are likely to be mediated through stimulation

of gonadotropins, in concert with other metabolic signals

[207,214].

The link between leptin and puberty in normal animals

remains controversial [1,36,110,164]. A longitudinal study

in boys revealed elevation of prepubertal leptin levels,

Behavior 81 (2004) 223241FSH [29,79,151,210,217]. Moreover, leptin stimulates

GnRH synthesis and potentiates the effect of insulin on

ogy &GnRH release [207]. Ovarian follicular cells are regulated

directly by leptin [219], indicating that leptin is able to

control the hypothalamicpituitarygonadal axis at multi-

ple levels. Although leptin restores reproductive function in

food-deprived rodents and humans, and accelerates the

onset of sexual maturation (vaginal opening) in ad libitum

fed postnatal mice [1,36,39,96], there are no published

studies showing direct effects of leptin reproductive func-

tion in healthy humans. Current knowledge is based pri-

marily on associations between leptin and reproductive

hormones. For example, frequent blood sampling has

revealed a positive and strong correlation between leptin

pulsatility and LH and estradiol levels in normally cycling

women [134]. In contrast, mean leptin level and diurnal

leptin rhythm are impaired in hypothalamic amenorrhea

[128]. Although leptin is elevated in association with

obesity in patients with polycystic ovarian syndrome, it

does not appear to account for menstrual abnormalities in

this population [127].

6.2. Hypothalamicpituitaryadrenal axis

Leptin deficiency or insensitivity in rodents is character-

ized by elevated glucocorticoid levels [3]. Leptin injection

decreases corticosterone levels in ob/ob mice before signif-

icant weight loss occurs [3], indicating that leptin is able to

control the hypothalamicpituitaryadrenal (HPA) axis

independently of its role in energy balance. However, unlike

ob/ob and db/db mice, humans null for leptin or LEPR

genes have normal levels of cortisol and do not exhibit

abnormalities in basal or corticotropin-releasing hormone

(CRH)-stimulated response [42]. In rats, leptin blunts the

rise in ACTH and corticosterone during restraint stress and

inhibits glucocorticoid synthesis and secretion in the adrenal

cortex [106]. Moreover, leptin prevents ACTH-stimulated

glucocorticoid secretion in adrenal cortex [22,23]. Paradox-

ically, intracerebroventricular leptin injection increases noc-

turnal glucocorticoid levels [166,204].

An interaction between leptin and the HPA axis is further

evident in the temporal relationship between plasma leptin

and glucocorticoids. Cortisol in humans and corticosterone

in rodents peak at night, coincident with the leptin nadir and

vice versa [3,4,132]. This reciprocal relationship between

leptin and the HPA axis is dependent on the feeding cycle.

Hence, a change in the timing of feeding results in a parallel

shift in glucocorticoids [3,182]. However, leptin is not

essential for establishment of the diurnal glucocorticoid

rhythm, because ob/ob mice maintain a normal rhythm,

albeit with higher basal corticosterone levels [3].

There have been conflicting reports regarding the inter-

action between leptin and CRH. Leptin stimulated basal

CRH secretion from hypothalamic fragments [48]; however,

another study demonstrated an inhibition of hypoglycemia-

induced CRH secretion from hypothalamic explants [106].

R.S. Ahima, S.Y. Osei / PhysiolMoreover, it has been reported that leptin increased CRH

mRNA expression in the paraventricular hypothalamic nu-cleus (PVN) in fasted rats, but did not alter CRH levels in

ob/ob mice [116]. These discrepancies may be explained by

differential effects of leptin on subsets of CRH neurons in

the PVN [5,65].

6.3. Thyroid hormone

T4 and triidotyronine (T3) are both subject to negative

feedback regulation. A fall in thyroid hormone stimulates

the synthesis and secretion of TRH and TSH. Conversely, a

rise in thyroid hormone suppresses TRH and TSH. This

feedback response is disrupted during fasting and illness,

culminating in low T4 and T3 levels, low or normal TSH

and suppression of TRH. The blunting of the hypothalam-

icpituitarythyroid axis response during caloric depriva-

tion or illness has been termed euthyroid sick syndrome. It

has been suggested that the dampening of hypophysiotropic

TRH neuron attenuation of the rise in TSH and T3 may have

evolved to limit energy expenditure and prevent protein

catabolism during starvation [81]. Leptin deficiency has

been associated with impairment of thyrotrope response to

TRH stimulation, while leptin replacement in leptin null

humans and during food restriction reverses the suppression

of T3, TSH and TRH mRNA levels in PVN [2,37,73,131].

Because ablation of the arcuate nucleus abolished the effect

of low leptin on PVN TRH mRNA expression, we surmised

that leptin acted indirectly via NPY, AGRP and POMC

neurons in the arcuate nucleus [131]. The latter neurons act

through melanocortin receptors (MCRs) in PVN and other

areas of the hypothalamus [75,76]. However, subsequent

studies revealed a colocalization of TRH and LEPR in PVN,

as well as direct regulation of TRH promoter activity by

leptin [100], indicating that leptin regulates thyroid function

via multiple hypothalamic circuits.

6.4. Growth hormone

Leptin and growth hormone act through a family of

cytokine receptors coupled to the JAK-STAT pathway

[198]. In rodents, growth hormone synthesis/secretion is

impaired in states of leptin deficiency or leptin insensitivity

[5,42]. Pulsatile growth hormone secretion is markedly

blunted during fasting, and restored by leptin replacement

[197], while immunoneutralization of leptin decreased

growth hormone secretion in fed rats [30,31,71,197]. To

analyze the in vivo effects of leptin on growth hormone

release, Watanobe and Habu [208] infused leptin into the

hypothalamus. Leptin was more potent in stimulating

growth hormone release in fasted than fed animals, as

manifested by increased pulse amplitudes without signifi-

cant changes in the pulse frequency. Leptin increased

GHRH in fed animals, while decreasing somatostatin level

[208]. Leptin receptors and STAT3 have been colocalized

with GHRH and somatostatin, providing strong anatomical

Behavior 81 (2004) 223241 231evidence for interaction between leptin and the somatotropic

axis [97,98]. Moreover, LEPRb is expressed in somato-

ogy &trophs and stimulates growth hormone release from isolated

pituitary gland [217]. In contrast, ovine leptin acts directly

on primary cultured somatotropes, by reducing the mRNA

levels encoding growth hormone and GHRH receptor [174].

In contrast to rodents, growth secretion in humans is

enhanced by fasting and impaired in obesity and aging.

Because obesity is associated with high plasma levels of

leptin, it has been postulated that the inhibitory action of

obesity on growth hormone may be mediated by leptin

[157]. Ozata et al. [157] compared patients with missense

mutation of the leptin gene with obese and nonobese

controls. The secretion of growth hormone in response to

GHRH and GHRP-6 was negatively affected by adiposity,

but not influenced by leptin levels. Growth hormone peaks

were negatively correlated with body mass index in control

(wild-type) patients as well as leptin-deficient patients,

indicating that other adiposity factors besides leptin con-

trolled growth hormone. Leptin is increased in GHD and

decreased in response to growth hormone treatment [5,71].

This inverse relationship is maintained in short prepubertal

children treated with growth hormone [125]. Serum leptin

concentrations were significantly reduced after 1, 3 and 12

months of growth hormone treatment. Importantly, the

growth response correlated negatively with the change in

serum leptin concentration, suggesting that short-term

changes in leptin levels in response to growth hormone

could be useful markers of growth response [125].

The effect of growth hormone on leptin levels has been

compared between patients with growth hormone insensi-

tivity (GHI) as a result of E180 splice mutation, and

idiopathic GHD [141]. Insulin-like growth factor I (IGF-I)

and IGFBP-3 levels were lower in homozygous GHI and

GHD patients compared with either normal controls or GHI

heterozygotes. Leptin was significantly higher in homozy-

gous GHI patients than normal controls and heterozygous

GHI and GHD patients. Leptin levels were best predicted by

gender (higher in females) and body mass index in both

homozygous GHI and normal patients [141].

6.5. Ghrelin

Ghrelin, a 28-amino acid octanoylated peptide, was

identified in the rat stomach as an endogenous ligand for

the growth hormone secretagogue receptor. Plasma ghrelin

is reduced in obesity and elevated in anorexia nervosa and

thin patients (reviewed in Ref. [115]). In contrast, leptin is

decreased in anorexia nervosa and thin patients. Both

plasma ghrelin and leptin levels return to control values in

anorexia patients after renutrition. Thus, the inverse rela-

tionship between plasma leptin and ghrelin is dependent on

body fat mass as well as nutritional status. In addition to

growth hormone-releasing properties in rodents, ghrelin

stimulates feeding following systemic or intracerebroven-

tricular administration. Systemic ghrelin administration in-

R.S. Ahima, S.Y. Osei / Physiol232creased Fos expression in leptin-sensitive neurons in the

arcuate nucleus, suggesting an interaction between theseligands [50,115]. Subsequent electrophysiologic analysis

revealed that ghrelin increased the electrical activity of the

majority of hypothalamic cells that were inhibited by leptin

[50]. Thus, the opposite effects of leptin and ghrelin on

feeding may be mediated through similar neuronal targets in

the arcuate nucleus.

There has been compelling evidence in support of

endogenous ghrelin production in the hypothalamus

[50,144]. Ghrelin-positive cells lie adjacent to the third

ventricle between the dorsal, ventral, paraventricular and

arcuate hypothalamic nuclei. These neurons send efferent

projections to NPY, AGRP, POMC and CRH neurons.

Ghrelin is bound mostly on presynaptic terminals of NPY

neurons, and stimulates the activity of arcuate NPY projec-

tions to the paraventricular nucleus [50,144]. Hence, ghrelin

produced in the hypothalamus may modulate energy bal-

ance by interacting with well-known leptin target neurons.

6.6. Prolactin

Prolactin has a major role in influencing the deposition

and mobilization of fat. The prolactin receptor belongs to

the same family as LEPR [198]. In humans, obesity dimin-

ishes the prolactin response to insulin-hypoglycemia and

thyrotrophin-releasing hormone stimulation [123]. More-

over, the spontaneous 24-h release of prolactin is dampened

in obesity [123]. Weight reduction, with accompanying

decrease in plasma insulin, improves prolactin responses

in some but not all cases [123]; hence, the molecular link

between prolactin and increased adiposity remains elusive.

Acute leptin treatment did not affect prolactin levels in fed

or fasted rats [209]. In contrast, a constant infusion of leptin in

fed rats prevented the fall in prolactin [209]. Moreover,

higher doses of leptin led to further increases in prolactin in

fasted animals. Thus, as with other pituitary hormones,

prolactin is more responsive to leptin deficiency during

fasting [2,151,197]. LEPR is very scant in lactotropes,

arguing against a significant direct effect of leptin. Moreover,

because leptin infusion into the arcuate nucleus and median

eminence complex stimulates prolactin secretion, it is likely

that leptin controls prolactin release via a hypothalamic target

[208]. Conversely, prolactin has been shown to stimulate

leptin secretion from rat adipose tissue [94].

6.7. Melatonin

Melatonin declines with aging in humans and rat, while

visceral fat, insulin and leptin levels increase [169]. In

contrast, melatonin treatment reversed the aging-associated

increase in retroperitoneal and epididymal fat, plasma insu-

lin and leptin levels to youthful levels [169]. In the same

study, corticosterone and T4 were not significantly altered

by aging or melatonin treatment. Moreover, while plasma

testosterone, IGF-I and T3 declined by middle age, these

Behavior 81 (2004) 223241changes were not affected by melatonin treatment. Interest-

ingly, melatonin decreased visceral adiposity, leptin and

remains to be determined.

implications for osteoporosis and other bone diseases.

the caudal regions of the nucleus ventral to the pars

compacta. LEPRb mRNA is localized mainly to the dorso-

medial division of the ventromedial nucleus (VMN) with

much less hybridization in the ventrolateral VMN [64]. In

contrast, LEPRb is prominent throughout the arcuate nucle-

us, extending from the retrochiasmatic region to the poste-

rior periventricular region. Moderate expression of LEPRb

is also detectable in the periventricular hypothalamic nucle-

us, medial mammillary nucleus and posterior hypothalamic

nucleus. A low level of LEPRb mRNA is detectable within

the parvicellular division of the PVN and LHA [64].

Unlike LEPRb, short LEPR isoforms are distributed

widely in the choroid plexus, meninges and surrounding

blood vessels in the brain parenchyma [19,64]. The presence

of LEPR mRNA in the meninges and microvessels raises

the possibility that LEPRs are responsible for transporting

ogy & Behavior 81 (2004) 223241 2338. Central neuronal circuitry for leptin

The findings discussed above indicate that leptin has

profound effects on energy homeostasis and neuroendocrine

systems. Leptin regulates specific neuronal groups within

the hypothalamus, brainstem and other regions of the brain

[5,65,95]. Here, we will focus mainly on leptin targets in the

hypothalamus. The long LEPR and LEPRb is enriched in

the hypothalamus, especially in ventrobasal hypothalamic

nuclei implicated in feeding behavior, thermogenesis and

hormone regulation [64,98]. For example, LEPRb mRNA is

present in the arcuate, dorsomedial, ventromedial and ven-7. Other actions of leptin

Leptin exerts acute and long-term systemic effects,

independent of its role in body weight regulation (reviewed

in Ref. [5]). For example, peripheral or intracerebroventric-

ular leptin administration rapidly decreases glucose and

insulin in ob/ob mice before weight loss. Leptin also

regulates glucose and lipids in wild-type rodents in part

through stimulation of gluconeogenesis and increased lipol-

ysis. Expression of leptin in the stomach is believed to act

locally to influence satiety, through regulation of cholecys-

tokinin and gastrin. Placental leptin increases in response to

hypoxia, and is strongly correlated with low birthweight.

Leptin regulates skeletal muscle metabolism, hematopoiesis,

immune function, angiogenesis, wound healing and brain

development. Many of these tissues express LEPRb and

downstream leptin gene targets, suggesting a direct effect of

leptin. Surprisingly, leptin deficiency is associated with

increased bone mass in rodents, despite hypogonadism

and high glucocorticoids which are well known to decrease

bone mass [58,196]. Studies have suggested that the effect

of leptin on bone in rodents is mediated through central

sympathetic neuronal pathways [196]. This finding, if

confirmed in humans, would have enormous therapeuticinsulin without altering food intake [213]. Taken together

with the ability of pinealectomy to increase leptin, these

findings suggest that melatonin exerts an inhibitory effect on

leptin release [26].

A rare condition known as the night-eating syndrome

(NES) may provide a link between body fat, leptin and

melatonin. NES patients are typically obese, and have

morning anorexia, evening hyperphagia and insomnia

[16]. Analysis of their neuroendocrine profile has revealed

higher cortisol level, as well as attenuation of the nocturnal

increase in plasma melatonin and leptin levels [16]. The

molecular basis of these behavioral and hormonal alterations

R.S. Ahima, S.Y. Osei / Physioltral premamillary hypothalamic nuclei. Within the dorso-

medial nucleus (DMN), intense hybridization is present inleptin in or out of the brain. Leptin may enter the brain

through circumventricular organs, i.e., regions lacking a

bloodbrain barrier, including the median eminence, sub-

fornical organ, organum vasculosum of the lamina termi-

nalis, median eminence and area postrema [19]. Because the

arcuate nucleus lies adjacent to the median eminence, it is

possible that leptin diffuses to neurons in this region through

the median eminence. However, transport via the circum-

ventricular organs cannot explain how leptin reaches deeper

structures, such as the cerebellum and thalamus, where

LEPRs have been localized [19,64]. Rather, it has been

suggested that LEPRs located in the brain microvasculature

and choroid plexus mediate leptin transport [19,64].

Hypothalamic neuropeptides involved in leptin action

have been classified into two major groups (Table 2).

Orexigenic peptides stimulate appetite, and are inhibited

by leptin and increase in response to leptin deficiency.

Anorexigenic peptides, which inhibit feeding, are stimu-

lated by leptin and decrease in response to leptin deficien-

cy. Orexigenic peptides include NPY, AGRP, melanin-

concentrating hormone (MCH) and orexins (ORX), while

a-MSH (derived from POMC), CART and CRH are major

Table 2

Neurotransmitters and peptide targets of leptin

Stimulate feeding Inhibit feeding

Neuropeptide Y (NPY) Alpha-melanocyte

stimulating hormone (a-MSH)Agouti-related

peptide (AGRP)

Cocaine and

amphetamine-regulated

transcript (CART)

Melanin-concentrating

hormone (MCH)

Corticotropin-releasing

hormone (CRH)

Orexins Neurotensin

Ghrelin Urocortin

Galanin Serotonin

Growth

hormone-releasing

hormone (GHRH)

Cholecystokinin (CCK)

Opioid peptides Glucagon-like peptide-1 (GLP-1)g-Aminobutyric acid(GABA)

Bombesin

t neur

arcuat

tem nu

utflow

R.S. Ahima, S.Y. Osei / Physiology & Behavior 81 (2004) 223241234anorexigenic neuropeptides (Table 2). NPY, AGRP and

LEPRb mRNAs are coexpressed in the arcuate nucleus

(Figs. 2 and 3). Ablation of the arcuate nucleus disrupts

leptin response [54]. Importantly, targeted ablation of

neuronal LEPRb produced a phenotype similar to db/db

Fig. 2. A schematic drawing showing the connections between leptin targe

inhibits NPY/AGRP neurons and stimulates a-MSH/CART neurons in theLHA. The PVN receives input from the gastrointestinal tract via the brains

(LPB), and regulates feeding, hormone synthesis/secretion and autonomic omice, suggesting that this LEPR mediates most of the

metabolic and hormonal actions of leptin in the brain [43].

Although NPY is a major leptin target, deletion of the

NPY or its receptors had little effect or did not complete-

Fig. 3. Leptin, ghrelin, NPY and melanocortin target neurons in the hypothalamu

arcuate nucleus. NPY stimulates feeding via Y1 and Y5 receptors. The Y2 receptor

The effect of NPY is modulated by ghrelin derived from the circulation or produce

receptors, resulting in appetite stimulation, reduced energy expenditure and weigly reverse the obese phenotype in ob/ob mice, indicating

that other neuropeptides and neurotransmitters play sig-

nificant roles in the transmission of the leptin signal

[67,68,152,153,178].

POMC neurons in the arcuate nucleus coexpress LEPRb

ons in the hypothalamus, brainstem and peripheral targets. Leptin directly

e nucleus. These neurons project to second order neurons in the PVN and

clei, e.g., nucleus tractus solitarius (NTS) and lateral parabrachial nucleus

.[5,19] (Fig. 3). The POMC gene product, a-MSH, is apotent anorectic peptide, which acts as an agonist of

MCRs in the PVN and other regions of the hypothalamus.

AGRP (colocalized with NPY) is distributed to similar

s. Leptin directly regulates NPY/AGRP and POMC/CART neurons in the

acts presynaptically to regulate NPY release at the POMC (a-MSH) neuron.d locally in the hypothalamus. AGRP antagonizes a-MSH action at MC4/3ht gain. GHS-R: growth hormone secretagogue receptor.

ogy &hypothalamic regions, such as PVN, perifornical and LHA,

and acts as an antagonist of a-MSH. Neurons containingMC4Rs localize to the PVN (Fig. 3), DMN and LHA [65]

(Fig. 3). MC4R is thought to mediate appetite suppression,

whereas MC3R decreases body weight through stimulation

of thermogenesis. Additional molecules that contribute to

the regulation of feeding include CART, galanin, MCH

and ORX, ghrelin, GLP-1, CCK and monoamines [65]

(Table 2).

We have addressed the question of whether different

populations of hypothalamic neurons respond differently to

changes in plasma leptin concentration [2]. Leptin was

infused by constant subcutaneous infusion in ad libitum

fed rodents to mimic the rise in plasma leptin as would

occur during overfeeding and obesity [2]. Conversely, we

administered leptin by constant subcutaneous infusion to

prevent the characteristic fall in plasma leptin with fasting

[2]. Chronic leptin elevation to the mildly obese range

elicited a transient suppression of feeding and sustained

reduction in body weight. NPY mRNA expression in the

arcuate hypothalamic nucleus decreased in a dose-related

manner. Insulin, T4 and testosterone were not affected.

Moreover, major anorexigenic peptides, e.g., CRH, POMC

and CART mRNA levels, were not affected by a rise in

leptin from fed to obese levels [2]. In contrast, leptin

replacement during fasting markedly blunted the suppres-

sion of T4 and testosterone, as well as the rise in

glucocorticoids and changes in hypothalamic NPY, POMC

and CART mRNA levels [2]. Postfast hyperphagia and

weight gain were also potently attenuated by leptin re-

placement. Taken together, these results suggest that the

sensing of the leptin by hypothalamic neurons is skewed

towards detection of low levels during starvation [2,81].

The rise in orexigenic peptides in conjunction with re-

duced expression of anorexigenic peptides is likely aimed

at optimizing food intake during starvation. Leptin-sensi-

tive hypothalamic peptides are also likely to couple go-

nadal, adrenal and thyroid function with alterations in

energy stores [2,81].

The PVN is uniquely positioned to transduce the leptin

signal during periods of changing energy availability, as it

possesses chemically specific projections to autonomic and

endocrine control sites involved in maintenance of homeo-

stasis (reviewed in Refs. [5,65]; Figs. 2 and 3). For example,

the parvicellular neurons in the medial PVN control secre-

tion of hormones, including TSH, growth hormone and

ACTH. The PVN has also been implicated in control of

feeding behavior, as lesions of the PVN induce hyperphagia

and obesity. The PVN expresses low levels of LEPR, but is

richly innervated by leptin-sensitive neurons in the arcuate

nucleus, DMN and brainstem [5,65]. Neurons in the dorsal,

ventral and lateral PVN provide autonomic preganglionic

neurons projection to the medulla and spinal cord, to control

the gastrointestinal system and brown adipose tissue [5,65].

R.S. Ahima, S.Y. Osei / PhysiolThe largest number of leptin-activated neurons that

project to the PVN is located in the DMN [5,64,65]. Thisnucleus lies caudal to the PVN and dorsal to the VMN, and

has been implicated in regulation of ingestive behavior,

insulin secretion and cardiovascular and neuroendocrine

systems. A major target of DMN efferents is the PVN,

specifically the dorsal, ventral and lateral parvicellular

subdivisions that directly innervate parasympathetic and

sympathetic preganglionic in the medulla and spinal cord.

Lesions of the DMN alter pancreatic neural activity, while

stimulation of the DMN increases glucose, presumably

through interactions with the parasympathetic (dorsal motor

nucleus of the vagus) and sympathetic (intermediolateral

cell column of the spinal cord) preganglionic neurons.

Because the DMN contains LEPRs, expresses SOCS-3

mRNA and Fos-immunoreactive cells following leptin ad-

ministration, and heavily innervates the PVN, it is plausible

that this nuclear group contributes significantly to leptins

effects on body weight, and control of the neuroendocrine

axis, insulin and glucose levels, blood pressure and body

temperature [5,65].

Ablation of VMN abolishes leptin response [181]. How-

ever, because relatively few cells in this region express

LEPR, it is likely that leptin engages the VMN via an

indirect pathway [64]. Fos immunoreactivity, a marker of

neuronal activation, is induced in the dorsomedial VMN in

response to leptin injection [64]. The dorsomedial VMN

projects to the subparaventricular zone (SPVZ) that receives

a dense innervation from the suprachiasmatic nucleus, the

circadian pacemaker of the mammalian brain [5]. The SPVZ

also interacts with PVN. Thus, input from the VMN to

SPVZ may couple leptin-mediated regulation of feeding to

sleepwake cycles to hormone rhythms, as manifested by

the link between nutrition and circadian glucocorticoid

rhythm [3,179,182]. VMN neurons also respond to glucose,

and could provide an interphase between long-term regula-

tion of body weight by leptin and short-term effects of

nutrients [65].

The LHA is well known to regulate feeding; however,

there are very few, if any, LEPR positive cells in this

region [64]. Detailed anatomic studies have revealed that

arcuate hypothalamic NPY/AGRP and POMC/CART neu-

rons, which respond directly to leptin, innervate the LHA,

adjacent perifornical area and zona incerta [56,64] (Figs.

2 and 3). The LHA contains two major neuropeptides,

MCH and the ORX (also called hypocretins), expressed

in separate neuronal populations [24]. Both cell groups

contribute to the lateral hypothalamic neuronal projections

from the cerebral cortex to the spinal cord to regulate

complex physiologic functions. The levels of MCH and

ORX are increased by leptin deficiency and decreased in

response to leptin treatment [65]. Apart from regulating

feeding and body weight, both MCH and ORX also

influence sleepwake cycles, and are likely to integrate

the latter with energy balance [5,65]. Ultimately, these

diverse mechanisms need to be connected to neural

Behavior 81 (2004) 223241 235networks producing specific behavioral effects of leptin,

e.g., reduction in meal size [60,82], regulation of brain

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9. Conclusion

Advances in molecular biology and genetics have ex-

tended our understanding of mechanisms underlying feed-

ing behavior, energy homeostasis, neuroendocrine

regulation and other complex physiologic systems. Here,

we have discussed the studies leading to the discovery of

leptin and its receptors, control of leptin production and

transport, cellular signaling and neuronal pathways for

leptin action in the brain. We have discussed how leptin

might improve glucose and lipids, aside from regulating

food intake and metabolic rate. The diverse mechanisms

linking leptin to the brain and peripheral tissues will clarify

the pathogenesis of obesity and associated diseases, and

facilitate the development of rationale therapeutic strategies.

Acknowledgements

This work was supported by grant P30DK19525 from

the National Institutes of Health.

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