Leptin signaling

Physiology & Behavior 81 (2004) 223 – 241

Leptin signaling Rexford S. Ahima*, Suzette Y. Osei Department of Medicine, Division of Endocrinology, Diabetes & Metabolism, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 764 Clinical Research Building, Philadelphia, PA 19104, USA

Abstract The discovery of leptin was a major breakthrough in our understanding of the role of adipose tissue as a storage and secretory organ. Leptin was initially thought to act mainly to prevent obesity; however, studies have demonstrated profound effects of leptin in the response to fasting, regulation of neuroendocrine and immune systems, hematopoiesis, bone and brain development. This review will focus on the signaling pathways which mediate these diverse effects of leptin in the brain and other physiologic systems. D 2004 Elsevier Inc. All rights reserved. Keywords: Leptin; Obesity; Feeding; Hypothalamus; Neuropeptide

1. Early ideas on energy homeostasis A 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 other hormonal abnormalities [65]. This so-called adiposus genitalis syndrome, was initially attributed to pituitary insufficiency; 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 observations provided an anatomic framework for the ‘‘dual center’’ model of feeding regulation. It was postulated that a ‘‘satiety center’’ was present in the ventromedial 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 experiments demonstrated a significant role of the brain in energy homeostasis.

* Corresponding author. Tel.: +1-215-573-1872; fax: +1-215-5735809. E-mail address: [email protected] (R.S. Ahima). 0031-9384/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.physbeh.2004.02.014

Humans and most mammals maintain a constant body weight despite short-term fluctuations in feeding and energy 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. This model was supported by studies in rodents, in which forced overfeeding resulted in inhibition of voluntary feeding, whereas food deprivation or surgical removal of adipose tissue stimulated food intake until body weight was restored [74,101 – 103]. 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 Hervey [107] provided further insights into the link between adipose tissue and the brain. He showed that cross-circulation (parabiosis) between obese VMH-lesioned and normal (nonlesioned) 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 circulating satiety 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] observed that parabiosis of ob/ob and lean (wild-type) mice

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resulted 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 immune 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 among adipose tissue, the nervous system and peripheral organs.

2. Control of leptin production The murine Lepob gene was discovered through positional cloning [5,220]. In mice, the leptin gene encodes a 4.5 kilobase mRNA transcript with a highly conserved 167amino acid open reading frame [220]. Leptin is remarkably similar across species [93,105,117,118,220]. It is synthesized mainly by adipose tissue and is released into the blood [220]. Various regulatory elements have been identified within the leptin promoter, e.g., cAMP and glucocorticoid 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 suggested 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 stimulated by hypoxia, elevated in eclampsia and may influence fetal outcome [143,144] (Table 1). Furthermore, de novo leptin synthesis has been demonstrated in the brain, suggesting a paracrine or autocrine action; however, the

Table 1 Factors implicated in leptin regulation Increase leptin

Decrease leptin

Adipose tissue Overfeeding Obesity (except ob/ob mutation) Insulin Glucocorticoids Acute infection Proinflammatory cytokines (TNF-a, IL-1) Placenta Insulin Glucocorticoids Hypoxia/eclampsia Skeletal muscle Glucose Glucosamine Lipids

Adipose tissue Fasting Cold exposure h-adrenergic agonist Testosterone

Stomach Feeding Cholecystokinin

physiologic 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 correlated 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). 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 of thyroid and growth hormone on leptin. While some studies have reported a rise in leptin in thyroid deficiency,

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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 following frequent blood sampling in humans [133,134]. Leptin is secreted in pulses that are inversely associated with ACTH and cortisol, and positively correlated to gonadotropins, 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 frequency; 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]. Potentially, 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 42 –84 min. The ultradian pattern of leptin was synchronous with LH and estradiol. Moreover, the nocturnal leptin peak was positively correlated 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 hypothalamic– pituitary – gonadal axis [134]. The latter is consistent with the diminution of leptin amplitude and frequency in patients with hypothalamic amenorrhea [128].

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The 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 prepubertal boys, in whom leptin is thought to exert a permissive effect on sexual maturation [86,139]. However, other studies have not observed a significant change in postnatal leptin levels, or an association between leptin and reproductive 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 culminates in hyperphagia, morbid obesity, hypothalamic hypogonadism 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, insulin 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 domain in the extracellular region. Two conserved disulfide links are present in the N-terminus, and a WSXWS motif is present in

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Fig. 1. Domain structure of alternatively sliced LEPR isoforms. Terminal amino acid residues for various LEPR isoforms are denoted by the alphabet code. Leptin receptors share a common extracellular leptin-binding domain, but differ at the carboxy-terminus intracellular domain. The long isoform, LEPRb, has intracellular motifs necessary for JAK-STAT signaling. LEPRe lacks a transmembrane domain (TM) and intracellular domains and circulates as a soluble receptor.

the C-terminus. LEPR shares highest sequence similarity with receptors for interleukin-6 (IL-6), leukemia inhibitory factor (LIF), granulocyte-colony stimulating factor (GCSF) and oncostatin [198]. LEPR isoforms have a similar extracellular 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 JAKSTAT signal transduction pathway. LEPRe lacks both transmembrane 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 glucocorticoids, 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. In contrast, 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-specific 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, glucose

intolerance and infertility in males, and rescued the cold intolerance in both sexes [124]. Importantly, NSE-LEPRb was expressed mainly in the brain, confirming the importance 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 hyperlipidemic, and have increased glucocorticoids and hyperglycemia [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 intracerebroventricular 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 absence of all cell surface LEPRs [195,215]. Plasma leptin concentration is greater than lean spontaneous hypertensive (SHR) littermates, suggesting severe leptin resistance. Kolet-

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sky 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 transmembrane 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 maturation [42]. Furthermore, these patients failed to respond normally to thyrotropin-releasing hormone (TRH) and growth-hormone-releasing hormone (GHRH) testing, suggesting 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 leptin 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 maintenance 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 pathway [8,14,88,150,203]. JAKs associate constitutively with conserved box 1 and 2 motifs in the intracellular domain of LEPRb (Fig. 1). In mice, the box 1 motif (amino acids 6 – 17) is critical for JAK2 activation, and box 2 motif (amino acids 49– 60) 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 transcription 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 homodimerization and nuclear translocation, and transactivates target genes by binding to specific promoter elements [150]. The essential role of Y1138 was demonstrated in mice by replacing this residue with serine [14]. Y1138S knock-in mice (LeprS1138) were unable to activate STAT3 [14]. Like

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db/db mice [89], LeprS1138 homozygous mice became hyperphagic and obese. However, in contrast to db/db mice, LeprS1138 homozygotes attained normal sexual maturation, fertility and body length [14]. Moreover, LeprS1138 homozygotes were less hyperglycemic [14]. Expression of NPY in hypothalamus was elevated in db/db but not LeprS1138 homozygotes, whereas melanocortin expression was suppressed 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, adipocytes, 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 activation, 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 polypeptide hormones, it had been suggested that leptin was transported 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 constant 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 intolerance, obesity, insulin resistance and infertility, as is the case with Leprdb3J homozygotes that are null for all isoforms of

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LEPR [124]. In contrast, transgenic expression of NSELEPRb 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 impairment [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 heterodimers of LEPRa and LEPRb failed to activate JAKSTAT, whereas receptor dimers of LEPRb gave rise to the expected ligand-dependent activation of JAK2, phosphorylation of STAT3, and increased STAT3-dependent promoter activity [8,150]. Furthermore, site-directed mutagenesis 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 Nterminal 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 recognizes 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 contrast, 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 importance 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 glucose-sensitive neurons in the brain and insulin secretion from pancreatic islets [191]. These effects appear to involve

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 feedback 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 associated 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 hyperleptinemia 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 significance 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 prone to DIO [194]. In contrast, POMC, the precursor of the anorexigenic neuropeptide a-MSH, is elevated in obesity-resistant A/J and SWR/J mice [15]. Expression of genes which mediate adaptive thermogenesis, e.g., UCP-1, UCP-3 and PGC-1, is increased in A/J and SWR/J mice, and may prevent obesity in these strains [167,194]. Obe-

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sity-resistant SWR/J mice are more sensitive to leptin, compared with obesity-prone C57Bl/6J mice [194]. Moreover, susceptibility to obesity in C57Bl/6J mice is positively correlated with failure to suppress hypothalamic NPY mRNA and blunting of brown adipose tissue UCP1 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 ‘lipotoxic’ 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 ceramide, 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 decrease in thermogenesis during fasting and postfast hyperphagia 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 extremely 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 was administered during 72-h fasting. Replacement-dose leptin prevented the starvation-induced changes in sex

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hormones and partially prevented the suppression of hypothalamic –pituitary –thyroid axis and IGF-1 binding capacity. 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 expenditure 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 neuroendocrine 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 suggested that leptin is inappropriately low in 10 – 20% 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 lipopolysaccharide-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 differentiation. Based on the robust responses to leptin deficiency, it has been suggested that leptin may have

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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 steatosis [87]. Adipocyte-secreted proteins, e.g., leptin and adiponectin, 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 wellknown association between these metabolic alterations and atherosclerosis, there is concern that the beneficial effect of antiretroviral treatment would be offset by premature coronary artery disease [37]. The striking similarities between the ‘‘metabolic syndrome’’ of obesity and lipodystrophy have stimulated a search for common underlying mechanisms. Earlier studies attributed the metabolic changes in lipodystrophy to the absence of adequate adipocyte storage capacity in lipodystrophy, resulting in triglyceride accumulation in liver, skeletal and cardiac muscle, and in the pancreatic h-cell, and culminating in impaired insulin action, diabetes and lipid abnormalities [87]. This idea was supported by studies showing that insulin sensitivity improved following fat transplantation in mice with generalized 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 lipodystrophy [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 reducing hepatic steatosis and intramyocellular triglycerides, and improving insulin sensitivity [155,156,162]. Interestingly, leptin replacement restored the pituitary– gonadal axis in lipodystrophic patients, confirming the importance of leptin

as 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. Leptin’s effects on classical hormones 6.1. Reproduction As discussed earlier, total leptin deficiency or insensitivity 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, menstrual 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, confirming 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 hypothalamic – pituitary – gonadal 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, preceding the rise in testosterone [139]. A transient increase in leptin has also been noted in boys aged 5– 10 years [86]. In the same study, plasma leptin was higher in girls; however, there was no prepubertal increase [86]. Interestingly, 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 collection (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 and FSH [29,79,151,210,217]. Moreover, leptin stimulates GnRH synthesis and potentiates the effect of insulin on

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GnRH release [207]. Ovarian follicular cells are regulated directly by leptin [219], indicating that leptin is able to control the hypothalamic – pituitary– gonadal axis at multiple 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 function in healthy humans. Current knowledge is based primarily 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. Hypothalamic –pituitary –adrenal axis Leptin deficiency or insensitivity in rodents is characterized by elevated glucocorticoid levels [3]. Leptin injection decreases corticosterone levels in ob/ob mice before significant weight loss occurs [3], indicating that leptin is able to control the hypothalamic – pituitary – adrenal (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]. Paradoxically, intracerebroventricular leptin injection increases nocturnal 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 interaction between leptin and CRH. Leptin stimulated basal CRH secretion from hypothalamic fragments [48]; however, another study demonstrated an inhibition of hypoglycemiainduced CRH secretion from hypothalamic explants [106]. Moreover, it has been reported that leptin increased CRH mRNA expression in the paraventricular hypothalamic nu-

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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 hypothalamic –pituitary – thyroid axis response during caloric deprivation 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 significant 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 evidence for interaction between leptin and the somatotropic axis [97,98]. Moreover, LEPRb is expressed in somato-

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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 controlled 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 insensitivity (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 homozygous 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 relationship 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 intracerebroventricular administration. Systemic ghrelin administration increased Fos expression in leptin-sensitive neurons in the arcuate nucleus, suggesting an interaction between these

ligands [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 projections to the paraventricular nucleus [50,144]. Hence, ghrelin produced in the hypothalamus may modulate energy balance 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 diminishes the prolactin response to insulin-hypoglycemia and thyrotrophin-releasing hormone stimulation [123]. Moreover, 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 insulin 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 changes were not affected by melatonin treatment. Interestingly, melatonin decreased visceral adiposity, leptin and

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insulin 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 remains to be determined.

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 intracerebroventricular 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 lipolysis. Expression of leptin in the stomach is believed to act locally to influence satiety, through regulation of cholecystokinin 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 therapeutic implications for osteoporosis and other bone diseases.

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the caudal regions of the nucleus ventral to the pars compacta. LEPRb mRNA is localized mainly to the dorsomedial division of the ventromedial nucleus (VMN) with much less hybridization in the ventrolateral VMN [64]. In contrast, LEPRb is prominent throughout the arcuate nucleus, extending from the retrochiasmatic region to the posterior periventricular region. Moderate expression of LEPRb is also detectable in the periventricular hypothalamic nucleus, 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 leptin in or out of the brain. Leptin may enter the brain through circumventricular organs, i.e., regions lacking a blood –brain barrier, including the median eminence, subfornical organ, organum vasculosum of the lamina terminalis, 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 circumventricular 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 stimulated by leptin and decrease in response to leptin deficiency. Orexigenic peptides include NPY, AGRP, melaninconcentrating 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

8. Central neuronal circuitry for leptin

Neuropeptide Y (NPY)

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 ventral premamillary hypothalamic nuclei. Within the dorsomedial nucleus (DMN), intense hybridization is present in

Agouti-related peptide (AGRP)

Alpha-melanocyte stimulating hormone (a-MSH) Cocaine and amphetamine-regulated transcript (CART) Corticotropin-releasing hormone (CRH) Neurotensin Urocortin Serotonin Cholecystokinin (CCK)

Melanin-concentrating hormone (MCH) Orexins Ghrelin Galanin Growth hormone-releasing hormone (GHRH) Opioid peptides g-Aminobutyric acid (GABA)

Glucagon-like peptide-1 (GLP-1) Bombesin

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Fig. 2. A schematic drawing showing the connections between leptin target neurons in the hypothalamus, brainstem and peripheral targets. Leptin directly inhibits NPY/AGRP neurons and stimulates a-MSH/CART neurons in the arcuate nucleus. These neurons project to second order neurons in the PVN and LHA. The PVN receives input from the gastrointestinal tract via the brainstem nuclei, e.g., nucleus tractus solitarius (NTS) and lateral parabrachial nucleus (LPB), and regulates feeding, hormone synthesis/secretion and autonomic outflow.

anorexigenic 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 mice, 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-

ly reverse the obese phenotype in ob/ob mice, indicating that other neuropeptides and neurotransmitters play significant roles in the transmission of the leptin signal [67,68,152,153,178]. POMC neurons in the arcuate nucleus coexpress LEPRb [5,19] (Fig. 3). The POMC gene product, a-MSH, is a potent 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

Fig. 3. Leptin, ghrelin, NPY and melanocortin target neurons in the hypothalamus. Leptin directly regulates NPY/AGRP and POMC/CART neurons in the arcuate nucleus. NPY stimulates feeding via Y1 and Y5 receptors. The Y2 receptor acts presynaptically to regulate NPY release at the POMC (a-MSH) neuron. The effect of NPY is modulated by ghrelin derived from the circulation or produced locally in the hypothalamus. AGRP antagonizes a-MSH action at MC4/3 receptors, resulting in appetite stimulation, reduced energy expenditure and weight gain. GHS-R: growth hormone secretagogue receptor.

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hypothalamic regions, such as PVN, perifornical and LHA, and acts as an antagonist of a-MSH. Neurons containing MC4Rs 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 suppression 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 replacement. 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 reduced expression of anorexigenic peptides is likely aimed at optimizing food intake during starvation. Leptin-sensitive hypothalamic peptides are also likely to couple gonadal, 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 homeostasis (reviewed in Refs. [5,65]; Figs. 2 and 3). For example, the parvicellular neurons in the medial PVN control secretion 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]. The largest number of leptin-activated neurons that project to the PVN is located in the DMN [5,64,65]. This

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nucleus 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 administration, and heavily innervates the PVN, it is plausible that this nuclear group contributes significantly to leptin’s 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]. However, 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 sleep – wake 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 regulation 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 neurons, 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 sleep – wake cycles, and are likely to integrate the latter with energy balance [5,65]. Ultimately, these diverse mechanisms need to be connected to neural networks producing specific behavioral effects of leptin, e.g., reduction in meal size [60,82], regulation of brain

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reward responses [77] and coordination of neuroendocrine responses [5].

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