Leptin

Malaka B. Jackson and Rexford S. Ahima

Abstract

Recent evidence has shown that adipose tissue is an active participant in maintaining energy and glucose homeostasis and plays crucial roles in controlling neuroendocrine, autonomic, and immune functions. Leptin is a hormone secreted by adipose tissue. Deficiency of leptin or its receptor results in hyperphagia, morbid obesity, insulin resistance, hyperlipidemia, hypothalamic hypogonadism, and immunosuppression. These abnormalities are reversed by leptin treatment in patients with congenital leptin deficiency or lipodystrophy. In contrast, diet-induced obesity is associated with elevated leptin levels and blunted response to leptin. Leptin resistance has been ascribed to the reduced entry of leptin into the brain or impairment of leptin signal transduction. This chapter focuses on the current understanding of leptin's actions, with particular emphasis on transport across the blood-brain barrier, signaling via JAK-STAT and PI3-kinase, neuropeptide targets, and electrophysiological effects.

Key Words: Adipocyte; adipokine; leptin; hypothalamus; neuropeptide; obesity.

1. adipocyte biology and leptin

Adipose tissue is a type of loose connective tissue made up of an extensive network of blood vessels, collagen fibers, fibroblasts, and immune cells that surround lipid-laden cells, known as adipocytes. In humans, the principal form of adipose tissue is white adipose tissue, whose adipocytes have an eccentric nucleus and a single lipid droplet (1). Until recently, adipose tissue was viewed primarily as a specialized tissue for storing energy mainly in the form of triglycerides. However, it is clear that adipose tissue plays an active role in energy homeostasis as well as the control of neuroendocrine, auto-nomic, and immune functions (1). Adipose tissue synthesizes and secretes "adipokines" such as leptin, adiponectin, and resistin. Additionally, adipocytes secrete proinflamma-tory cytokines, such as tumor necrosis factor (TNF)-a and interleukins, and proteins involved in coagulation and vascular function (1). Furthermore, adipose stromal cells mediate the metabolism of glucocorticoids and sex steroids, which exert profound local and systemic effects on adipogenesis, glucose and lipid metabolism, and cardiovascular function (1,2). This chapter will focus on the biology of leptin—in particular, its role in metabolism and control of neuroendocrine function.

As early as the 1950s, it was proposed that that a factor existed whose circulating levels increased with energy stores and signaled the brain to inhibit feeding and decrease body weight and fat (3). The discovery of recessive mutations of obese (ob) and

From: Nutrition and Health: Adipose Tissue and Adipokines in Health and Disease Edited by: G. Fantuzzi and T. Mazzone © Humana Press Inc., Totowa, NJ

diabetes (db) loci in mice, coupled with hypothalamic lesion and parabiosis studies, led to the suggestion that the ob locus encoded a circulating "satiety factor," whereas the db locus was required for the response to this factor (4,5). These predictions were confirmed more than four decades later by the discovery of the leptin (lep) and leptin receptor (lepr) genes (6-9). Leptin is expressed mainly in adipose tissue, although low levels have been found in the placenta, skeletal muscle, gastric fundic mucosa, and mammary epithelium (10). Leptin has a relative mass of approx 16 kDa and circulates as both a bound and a free hormone, the latter likely representing the bioavailable hormone. The primary and complex structures of leptin are highly conserved in mammals (6,10). Leptin concentration is dependent on the quantity of stored energy in fat, as well as the status of energy balance (10). As such, plasma leptin is higher in obese than in lean individuals, falls rapidly during fasting, and increases after feeding. This nutritional regulation of leptin is controlled at least partly by insulin (10).

The concept that leptin acts as an "antiobesity hormone" was based on two key observations: first, rodents and humans deficient in either leptin or its receptors developed insatiable appetite and morbid obesity, associated with insulin resistance, diabetes, and hyperlipidemia (6,11). Second, leptin administered peripherally and more potently via intracerebroventricular (icv) injection in rodents, decreased food intake, body weight and fat content, consistent with the idea of negative feedback action in the brain (12-14). The latter was supported by the localization of leptin receptors in the hypothalamus and various CNS nuclei associated with feeding and energy balance (15). However, it was soon obvious that leptin expression in adipose tissue and plasma leptin levels increased in obesity, and the rise in endogenous leptin levels did not prevent obesity (16). In the absence of obvious leptin receptor abnormalities, leptin treatment in humans and rodents with "common" (diet-induced) obesity has little effect to suppress feeding or reduce weight (16). This blunted response to leptin, termed "leptin resistance," may underlie the progression of obesity, impaired insulin action, diabetes, and elevated lipid levels, characteristic of "metabolic syndrome" (16). Leptin resistance may result from diminution of leptin transport across the blood-brain barrier (BBB) or impairment of leptin signal transduction in the brain (16).

There is strong evidence showing that leptin acts mainly as a "starvation hormone" (17). Leptin falls rapidly during fasting or chronic food deprivation, leading to suppression of thermogenesis and of thyroid, sex, and growth hormones (10,17). Moreover, low leptin levels during fasting mediates immunosuppression, torpor, and activation of the hypothalamic-pituitary-adrenal axis (HPA) in rodents (18,19). These fasting-induced changes mediated by low leptin resemble the metabolic phenotype of congenital leptin deficiency in

Lepob/ob and or lack of leptin response in Lep/^b/db mice, suggesting that leptin deficiency is perceived as a state of unmitigated starvation, leading to adaptations, such as hyperphagia, decreased metabolic rate, and changes in hormones, intended to restore energy balance (16). As predicted, leptin treatment prevents the suppression of reproductive axis, thyroid and growth hormones and energy expenditure, and hyper-phagia typical of food deprivation or sustained weight reduction (17-22).

2. leptin's effects on the neuroendocrine axis

Congenital leptin deficiency in rodents and humans is associated with hypothalamic hypogonadism and lack of pubertal development (10,23,24). Leptin treatment restores gonadotropin and sex steroid levels and puberty, confirming a primary role in reproduction (10,23,24). However, studies in LepPb/ob mice indicate that leptin is not needed for gestation, parturition, or lactation (25). Leptin exerts a permissive action to restore normal hypothalamic-pituitary-gonadal axis function during caloric deprivation (26-29). In rodents and nonhuman primates, these actions involve stimulation of gonadotropins as well as interaction with other metabolic signals (28,29). Women with hypothalamic amenorrhea have low leptin levels associated with blunted luteinizing hormone (LH) pulses (20,30). Leptin administration in these patients restores LH pulses and menstrual cycles, confirming that leptin signals the status of energy stores to the neuroendocrine axis (20).

Lipodystrophy is characterized by generalized or partial loss of adipose tissue, excess lipid accumulation in nonadipose tissues (steatosis), hypoleptinemia, insulin resistance, diabetes, dyslipidemia, hyperandrogenism, and amenorrhea, the last being of a central origin (31). In affected females, treatment with recombinant methionyl human leptin in physiological doses reduced steatosis, insulin resistance, glucose and plasma lipids, and free testosterone, increased sex hormone binding globulin, and produced robust LH pulses and integrated levels (32,33). Importantly, most patients who had amenorrhea prior to leptin therapy developed normal menses after treatment (33). Nonetheless, polycystical ovaries associated with lipodystrophy were not reversed by leptin (33). Serum testosterone tended to increase and sex hormone binding globulin increased following leptin treatment therapy in male lipodystrophic patients (33). In contrast to congenital leptin deficiency, hypoleptinemia did not inhibit pubertal development in lipodystrophy, pointing to a crucial role of other metabolic factors (23,24,33).

The control of thyroid hormone is coupled to energy balance (34). Under normal fed conditions, a fall in thyroid hormone stimulates the synthesis and secretion of thyrotropin-releasing hormone (TRH) and thyroid-stimulating hormone (TSH). Conversely, a rise in thyroid hormone suppresses TRH and TSH. This feedback response is disrupted during fasting and illness, culminating in low thyroxine (T4) and triiodotyro-nine (T3) levels, low or normal TSH, and suppression of TRH. The dampening of the hypothalamic-pituitary-thyroid axis response during caloric deprivation has been termed "euthyroid sick syndrome." Leptin deficiency reduces the response of pituitary thyrotropes to TRH stimulation, whereas leptin treatment reverses the suppression of T3, TSH, and TRH levels in congenital leptin deficiency and fasting (34). Studies indicate that low leptin levels during fasting control the thyroid axis directly through hypophysiotrophic TRH neurons in the paraventricular nucleus (PVN) or indirectly through neurons expressing melanocortin 4 receptors (MC4R) (35-37).

Leptin and growth hormone receptors belong to a family of cytokine receptors coupled to the JAK-STAT pathway (38). In rodents, growth hormone is decreased in states of leptin deficiency (10). Growth hormone pulses are diminished during fasting, and restored by leptin replacement (39,40). In contrast, immunoneutralization of leptin decreased growth hormone secretion in fed rats (40). Leptin receptors and STAT3 have been colocalized with growth hormone-releasing hormone (GHRH) and somatostatin, suggesting a direct interaction (40). Leptin infused into the hypothalamus stimulated growth hormone release more robustly in fasted than fed animals; this was associated with an increase in GHRH and a decrease in somatostatin (41).

Koutkia et al. (41) investigated the regulation of leptin and growth hormone secretion and pulsatility by sampling plasma overnight every 20 min. There was synchronicity between growth hormone and leptin with a lag time of 39 min (41). Ghrelin is a gastric peptide that stimulates appetite, glucose oxidation, and lipogenesis, in contrast with growth hormone, which promotes lipolysis, glucose production, and insulin secretion (42). Plasma ghrelin levels increase at night in lean individuals and in synchrony with leptin (42). This nocturnal rise of ghrelin is blunted in obesity (42). The HPA is activated in leptin-deficient rodents (10,11). Moreover, elevated leptin levels during fasting or immobilization stress lead to corticotropin-releasing hormone (CRH), adrenocorti-cotropin (ACTH), and glucocorticoid inhibition (17,43). Diurnal and pulsatile leptin secretions are inversely related to ACTH and cortisol in humans (44), yet the HPA axis is not significantly altered in congenital leptin deficiency, lipodystrophy, or fasting (21,24).

Although prolactin and leptin are both influenced by body fat content, the link between these hormones in humans is unclear (45). In contrast, a constant infusion of leptin increases prolactin in fed rats, and in particular during fasting (46). Because the leptin receptor is very scant in lactotropes and direct leptin infusion into the arcuate nucleus and median eminence stimulates prolactin secretion, it has been proposed that leptin controls prolactin release through a hypothalamic target (47).

3. leptin receptors and signal transduction in the brain

Soon after it was shown that low doses of leptin, when administered directly into the brain, decreased food intake and body weight (14), leptin receptors were discovered through expression cloning in mouse brain and by positional cloning of the db locus (7,8). The leptin receptor (LEPR) belongs to the cytokine receptor class I superfamily (38). Five alternatively spliced isoforms, a, b, c, d, e, differing in the lengths of their carboxy termini, have been identified (refs. 10,38; Fig. 1). The short leptin receptor isoform, LEPRa, is expressed in several peripheral tissues, choroid plexus, and brain microvessels, and thought to be involved in the transport of leptin across the BBB or efflux from the brain (48-50). The long leptin receptor (LEPRb) that has intracellular domains necessary for signaling via the JAK-STAT pathway is highly expressed in the hypothalamus—e.g., arcuate (Arc), dorsomedial (DMN), ventromedial (VMN), and ventral premamillary nuclei (PMN). Moderate LEPRb expression is found in the periventricular and lateral hypothalamic areas, and nucleus solitarius and various brainstem nuclei (51). The PVN, which integrates energy balance, neuroendocrine function, and glucose homeostasis, has a very low level of LEPRb (51). LEPRb has been colocalized with neuropeptides involved in energy homeostasis (52). Neuropeptide Y (NPY) and agouti-related protein (AGRP), which stimulate feeding, are present in the same neurons in the medial Arc. An increase in leptin directly suppresses NPY and AGRP. Leptin increases the levels of anorectic pep-tides, a-melanin-stimulating hormone (a-MSH) derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART), in the lateral Arc (53). Second-order neurons that synthesize CRH, TRH, and oxytocin in the PVN are controlled indirectly by leptin targets in the Arc, and mediate the inhibitory effects of leptin on food intake, stimulation of thermogenesis, and neuroendocrine secretion (52). Other orexigenic peptides, such as melanin-concentrating hormone (MCH) and orexins, expressed in the lateral hypothalamus, are inhibited indirectly by leptin (51). Outside the hypothalamus, LEPRb mRNA has also been found in the thalamus and cerebellum (52).

  1. 1. Leptin receptors. Five leptin receptor isoforms result from alternate splicing of lepr mRNA transcript. Receptors LEPRa-LEPRd contain identical extracellular ligand binding and cytoplasmic signaling domains. Despite having the same extracellular N-terminal, each isoform has a different C-terminal. However, unlike the other leptin isoforms, LEPRe has neither a transmembrane nor intracellular domains. LEPRa is the principal "short leptin receptor" and lacks the cytoplasmic domain necessary for signaling through the JAK-STAT pathway. LEPRb, the "long leptin receptor," is primarily responsible for the leptin-mediated effects on energy homeostasis and endocrine function through activation of the JAK-STAT pathway. Terminal amino acid residues for each isoform are represented by the alphabetic code.
  2. 1. Leptin receptors. Five leptin receptor isoforms result from alternate splicing of lepr mRNA transcript. Receptors LEPRa-LEPRd contain identical extracellular ligand binding and cytoplasmic signaling domains. Despite having the same extracellular N-terminal, each isoform has a different C-terminal. However, unlike the other leptin isoforms, LEPRe has neither a transmembrane nor intracellular domains. LEPRa is the principal "short leptin receptor" and lacks the cytoplasmic domain necessary for signaling through the JAK-STAT pathway. LEPRb, the "long leptin receptor," is primarily responsible for the leptin-mediated effects on energy homeostasis and endocrine function through activation of the JAK-STAT pathway. Terminal amino acid residues for each isoform are represented by the alphabetic code.

Binding of leptin to LEPRb in hypothalamic and brainstem neurons results in rapid activation of intracellular JAK-2, leading to tyrosine phosphorylation of LEPRb on amino acid residues 985 and 1138, which provide binding motifs for src homology 2 (SH2)-domain containing proteins—i.e., STAT-3 and SH-2-domain-phosphotyrosine phosphatase (SHP-2) (ref. 54; Fig. 2). STAT-3 binds to Y1138, becomes tyrosine-phos-phorylated by JAK-2, then dissociates and forms dimers in the cytoplasm, which are translocated to the nucleus to regulate gene transcription (Fig. 2). The importance of Y1138 has been demonstrated in mice by replacing this amino acid with serine (55). The Y1138S (LeprS1138) mutation disrupted STAT-3 activation, resulting in hyperpha-gia, impairment of thermoregulation, and obesity (55). However, in contrast to Leprdh/db mice, Y1138S mutation did not affect sexual maturation and growth, and glucose levels were lower, pointing to a specific physiological role of this domain (55).

Leptin regulates insulin receptor substrate 1 (IRS-1) and IRS-2, mitogen-activated protein kinase, extracellular-regulated kinase, Akt, and phosphatidylinositol-3 (PI3)-kinase

Fig. 2. Leptin signal transduction. Leptin binding to the hypothalamic LEPRb results in activation and autophosphorylation of JAK-2. Subsequently STAT-3 is phosphorylated then translocated to the nucleus, where it acts together with a variety of transcription factors to regulate the expression of neuropeptides and other genes. In congenital or acquired leptin deficiency or leptin receptor defect, failure to activate LEPRb results in increased orexigenic neuropeptide expression (NPY and AGRP), and decreased anorexigenic neuropeptide expression (POMC), and uncoupling protein (UCP)-1 expression in brown adipose tissue (BAT). The net effect is hyperphagia and a decrease in metabolic rate, resulting in severe obesity, markedly elevated insulin levels and steatosis. Another manifestation of leptin deficiency is hypothalamic hypogonadism. The fall in leptin during fasting triggers hyperphagia, metabolic and neuroendocrine responses similar to congenital leptin deficiency. An increase in lep-tin within the physiological range inhibits feeding and increases the metabolic rate, resulting in weight loss. In contrast, polygenic (diet-induced) obesity results in elevated leptin levels, which promote phos-phorylation of Tyr1138 on the intracellular domain of LEPRb, resulting in inhibition of leptin signaling by induction of SOCS-3 and inhibition of JAK-STAT and SHP-2 pathways. These mechanisms result in "leptin resistance" that blunts the ability of leptin to suppress feeding and increase metabolic rate, resulting in obesity. The degree of obesity, steatosis, insulin resistance, and hyperlipidemia in diet-induced obesity is less severe than that seen in congenital leptin deficiency or lipodystrophy.

through LEPRb, raising the possibility of crosstalk between leptin and insulin (56). Leptin enhances IRS-2-mediated activation of PI3-kinase in the hypothalamus, concomitant with its ability to inhibit food intake (56,57). In contrast, blockade of PI3-kinase activity prevents the anorectic action of leptin (56).

Bj0rbaek et al. (58,59) first proposed a role of SOCS-3 in leptin signaling, based on the observation that leptin rapidly increased the levels of this cytokine mediator in hypo-thalami of Lepob/ob but not Lepfdb/db mice. In situ hybridization for SOCS-3 mRNA in the rat and mouse brains showed that leptin-induced SOCS-3 expression colocalized with LEPRb and neuropeptides in the hypothalamus (58). Furthermore, expression of SOCS-3 prevented the tyrosine phosphorylation of LEPRb and downstream signaling (59). STAT-3 DNA binding elements are present in the socs-3 promoter, and because the lack of the STAT-3 binding site on LEPRb prevents induction of SOCS-3 mRNA by leptin, this indicates that leptin stimulates socs-3 transcription via the STAT-3 pathway (59). The crucial role of SOCS-3 as a negative regulator of leptin signaling was demonstrated in two studies (60,61). SOCS-3 haploinsufficiency increased leptin sensitivity and prevented diet-induced obesity in mice (60). More specifically, neuron-specific ablation of SOCS-3 enhanced leptin sensitivity, resulting in activation of STAT3, increase in hypothalamic POMC expression, reduction in food intake, and resistance to obesity, hyperlipidemia, and diabetes (61).

Protein tyrosine phosphatase 1B (PTP1B), an insulin receptor phosphatase that inhibits insulin signaling, was implicated in leptin action, based on the finding that mice lacking PTP1B were less hyperphagic and resistant to obesity despite having low serum leptin levels (62-64). In vitro studies revealed that PTP1B directly inhibited JAK2 kinase, and leptin-induced tyrosine phosphorylation of JAK-2 and STAT-3 was attenuated in cells overexpressing PTP1B (63). PTP1B mRNA is colocalized with STAT-3 and neuropeptides in the Arc and various hypothalamic nuclei (64). Importantly, STAT-3 phosphorylation in the hypothalamus is enhanced following leptin treatment in mice lacking PTP1B (PTP1B-/-), suggesting that PTP1B inhibits the signal transduction of leptin in the brain (63,64). This idea was tested by comparing the effects of leptin treatment on energy balance in wild-type (WT) andPTP1B-/-mice.Aspredicted,PTP1B -/and heterozygotes PTP1B+/- mice exhibited greater leptin sensitivity than WT (63).

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