2.

1 Leptin Leptin (from the Greek leptos, meaning thin) is a protein hormone with important effects in regulating body weight, metabolism and reproductive function. The protein is approximately 16 kDa in mass and encoded by the obese (ob) genE.1 Leptin, discovered through positional cloning 15 years ago is an adipocyte-secreted hormone with a key role in energy homeostasis.2 2.1.1 Biosynthesis Leptin is a 167 amino acid protein which belongs to the cytokine family.3 Human leptin gene is located on chromosome 7.4 It is manufactured primarily in the adipocytes of white adipose tissue, and the level of circulating leptin is directly proportional to the total amount of fat in the body. In addition to white adipose tissue, the major source of leptin, it can also be produced by brown adipose tissue, placenta (syncytiotrophoblasts), ovaries, skeletal muscle, stomach (lower part of fundic glands), mammary epithelial cells, bone marrow, pituitary and liver.Leptin play key role in food intake, energy balance, and adiposity as well as in immune and endocrine system. It acts as feedback loop to maintain the constant store of body fat.5 Leptin levels are pulsatile and follow a circadian rhythm, with highest levels between midnight and early morning and lowest levels in the early- to mid- afternoon . Specifically, the concentration of circulating leptin may be up to 75.6% higher during the night as compared to afternoon trough levels. The pulsatile characteristics of leptin secretion are similar in obese and lean individuals, except the obese have higher pulse amplitudes. Leptin concentration reflects the amount of energy stored in body fat. Circulating leptin levels are directly proportional to the amount of body fat and fluctuate with acute changes in caloric intake. This system is especially sensitive to energy deprivation. Women tend to have higher leptin levels than men, although women experience a significant decline in the amount of circulating leptin after menopause. This sexual dimorphism is largely independent of body mass index (BMI), and is due in part to differences in sex hormones, fat mass, and body fat distribution. Women tend to accumulate body fat peripherally whereas men are prone to an abdominal or android distribution of fat. Subcutaneous fat expresses more leptin mRNA than omental fat, and this may partially explain

the higher leptin concentrations in women compared to men. Hormones and cytokines other than sex steroids also affect leptin secretion but to a smaller degree (Table 1).6 Table 1 Factors that regulate circulating leptin levels6 Factors promoting leptin secretion
* *

Excess energy stored as fat (obesity) Overfeeding

Glucose Insulin Glucocorticoids Estrogens Inflammatory cytokines, including Tumor Necrosis Factor- and Interleukin-6 (acute effect) Factors inhibiting leptin secretion
* *

Low energy states with decreased fat stores (leanness)

Fasting

Catecholamines and adrenergic agonists Thyroid hormones Androgens Peroxisome Proliferator-activated Receptor- (PPAR) agonists Inflammatory cytokines, including Tumor Necrosis Factor- (prolonged effect)

2.1.2 Mechanism of action Leptin binds to leptin receptors (ObRs) located throughout the central nervous system and several peripheral tissues. At least six variations or isoforms of the leptin receptor have been identified (ObRa, ObRb, ObRc, ObRd, ObRe, and ObRf). These isoforms have homologous extracellular domains but distinct intracellular domains, which vary by length and sequence due

to alternative mRNA splicing. The short isoforms ObRa and ObRc are thought to play important roles in transporting leptin across the bloodbrain barrier (BBB). The long leptin receptor isoform ObRb is primarily responsible for leptin signaling.5 The long leptin receptor isoform is expressed abundantly in the hypothalamus and activates the Janus kinase signal transducer and activator of trascription (JAK-STAT) system to alter the expression of hypothalamic neuropeptides.This functional leptin receptor ObRb, expressed in several organs, is strongly expressed throughout the central nervous system but particularly in the hypothalamus, where it regulates energy homeostasis and neuroendocrine function described further below. In the db/db mouse model, the ObRb is dysfunctional, resulting in obesity and the metabolic syndrome.6

Figure 1 Leptin's action in the brain during states of energy excess and energy deficiency.6 During states of leptin and energy excess, leptin's access to the hypothalamus and other brain areas is impaired and leptin's action is blunted. In states of leptin and energy deficiency, neuropeptides that are normally inhibited by leptin are elevated (+) and neuropeptides stimulated by leptin are suppressed (-). A change in the concentrations of these neuropeptides leads to

alterations in neuroendocrine function and energy homeostasis. Alterations in leptin levels may also affect the hedonic aspects of feeding behavior. The role of leptin in the brain is discussed in greater detail in the text. Abbreviations: ACTH, adrenocorticotropic hormone; AgRP, agouti-related peptide; ARC, arcuate nucleus; BDNF, brain-derived neurotrophic factor; CART, cocaine- and amphetamineregulated transcript; CCK, cholecystokinin; CRH, corticotropin-releasing hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GLP-1, glucagon-like peptide 1; GnRH, gonadotropin-releasing hormone; IGF-1, insulin-like growth factor 1; LH, luteinizing hormone; LHA, lateral hypothalamic area; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; NST, nucleus of the solitary tract; PO, preoptic area; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SN, substantia nigra; TRH, thyrotropin-releasing hormone; TSH, thyrotropin-stimulating hormone; VMH, ventromedial hypothalamus; VTA, ventral tegmental area.

Activation of ObRb sets off a cascade of several signal transduction pathways (Table 2), of which the best studied pathway is the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. STAT3 has been shown to mediate the transcription of several genes that affect a number of cellular processes. Leptin controls energy homeostasis and body weight primarily by activating ObRb in the hypothalamus. The ObRb activate numerous JAK2/STAT3-dependent and -independent signaling pathways that act in coordination as a network to fully mediate leptin's action. The activation of individual pathways in the leptin signaling network appears to be differentially regulated in discrete subpopulations of ObRbexpressing neurons. These pathways are also likely to be regulated by various other hormonal, neuronal, and metabolic signals that cross-talk with leptin. Hence, it is important to fully determine whether and how positive and negative regulators of ObRb signaling, metabolic state, and/or neuronal activity regulate leptin signaling networks in a cell/tissue type-specific manner and how activation of these signaling pathways mediates leptin's effects in humans.6

Table 2 Leptin Signaling6 Signaling Pathway Primary Site of Action Known Mechanisms of Action Stimulates transcription of POMC and suppresses Regulates appetite and, thus, body weight May also contribute to neuroendocrine function Clinical Results

JAK-STAT3 Hypothalamus

transcription of NPY

as neural-specific STAT3 deletion results in

decreased linear growth and infertility P13K Hypothalamus Stimulates POMC neurons Inhibits inhibitor transcription, FOXO1, of to an POMC increase Regulates appetite and body weight. May contribute to leptin resistance in obesity, given the overlapping pathway with insulin May mediate the stimulation of sympathetic outflow MAPK Hypothalamus, liver, Stimulates POMC neurons pancreas, adipose and inhibits AgRP/NPY Regulates appetite and body weight Increases sympathetic activity to brown adipose tissue Increases fatty acid oxidation in peripheral tissues Promotes cardiomyocyte

POMC expression

tissue, and myocytes

neurons

Signaling Pathway

Primary Site of Action

Known Mechanisms of Action

Clinical Results

hypertrophy AMPK Hypothalamus, muscle Stimulates ACC activity in the hypothalamus to Regulates weight Stimulates fatty-acid oxidation in muscle and may sensitize muscle to insulin Regulates weight appetite and appetite and

regulate food intake and weight Inhibits ACC activity in muscle mTOR Hypothalamus Induces phosphorylation of S6K1 to regulate protein synthesis

Abbreviations: ACC, acetyl coenzyme A carboxylase; AMPK, 5'adenosine monophosphateactivated protein kinase; ATP, adenosine triphosphate; FOXO1, forkhead box O1; JAK-STAT3, janus kinase-signal transducers and activator of transcription 3; K+, potassium; MAPK, mitogenactivated protein kinase; mTOR, mammalian target of rapamycin; NPY, neuropeptide Y; POMC, pro-opiomelanocortin; P13K, phosphatidylinositol 3-kinase; S6K1, S6 Kinase 1.

2.1.3 Effects in organs Leptin acts on receptors in the hypothalamus of the brain where it inhibits appetite by (1) counteracting the effects of neuropeptide Y (a potent feeding stimulant secreted by cells in the gut and in the hypothalamus); (2) counteracting the effects of anandamide (another potent feeding stimulant that binds to the same receptors as THC), and (3) promoting the synthesis of MSH, an appetite suppressant. This inhibition is long-term, in contrast to the rapid inhibition of eating by cholecystokinin (CCK) and the slower suppression of hunger between meals mediated by PYY3-36.7 The absence of leptin (or its receptor) leads to uncontrolled food intake and resulting obesity. Several studies have shown that fasting or following a very-low-calorie diet (VLCD) lowers leptin levels. It might be that, in the short-term, leptin is an indicator of energy

balance. This system is more sensitive to starvation than to overfeeding; leptin levels change more when food intake decreases than when it increases.8 There is some controversy regarding the regulation of leptin by melatonin during the night. One research group suggested that increased levels of melatonin caused a downregulation of leptin.9 However, in 2004, Brazilian researchers found that melatonin increases leptin levels in the presence of insulin, therefore causing a decrease in appetite during sleeping.10 2.1.3.1 Clinical significance Leptin has traditionally been regarded as a link between fat mass, food intake, and energy expenditure. This link originally arose from animal research findings, but its application to describing human systems has since been challenged.11 In humans, there are many instances where leptin dissociates from the strict role of communicating nutritional status between body and brain and no longer correlates with body fat levels:

Leptin levels decrease after short-term fasting (2472 hours), even when changes in fat mass are not observed. 12

In the obese patients with obstructive sleep apnea (OSA), Leptin is increased, but decreases after administration of a CPAP.13 In non-obese individuals, however, restful sleep (i.e., 812 hours of unbroken sleep) can increase leptin within normal ranges.

Serum levels of Leptin are reduced by sleep deprivation.14 Increased by perceived emotional stress.15 Decreased by testosterone and increased by estrogen. 16 Chronically affected by exercise training; it decreases leptin levels.16

2.1.3.2 Adiposity signal To date, only leptin and insulin are known to act as an adiposity signal. In general, Leptin circulates at levels proportional to body fat. It enters the central nervous system (CNS) in proportion to its plasma concentration. Its receptors are found in brain neurons involved in regulating energy intake and expenditure. It controls food intake and energy expenditure by acting on receptors in the mediobasal hypothalamus. 17

2.1.3.3 Interaction with amylin Co-administration of two neurohormones known to have a role in body weight control, amylin (produced by beta cells in the pancreas) and leptin (produced by fat cells), results in sustained, fat-specific weight loss in a leptin-resistant animal model of obesity. 18 2.1.3.4 Satiety Leptin binds to neuropeptide Y (NPY) neurons in the arcuate nucleus, in such a way that decreases the activity of these neurons. Leptin signals to the brain that the body has had enough to eat, producing a feeling of satiety. A very small group of humans possess homozygous mutations for the leptin gene that leads to a constant desire for food, resulting in severe obesity. This condition can be treated somewhat successfully by the administration of recombinant human leptin. However, extensive clinical trials using recombinant human leptin as a therapeutic agent for treating obesity in humans have been inconclusive because only the most obese subjects who were given the highest doses of exogenous leptin produced statistically significant weight loss. It was concluded that large and frequent doses are needed to provide only modest benefit because of leptins low circulating half-life, low potency, and poor solubility. Circulating leptin levels give the brain input regarding energy storage so it can regulate appetite and metabolism. Leptin works by inhibiting the activity of neurons that contain neuropeptide Y (NPY) and agouti-related peptide (AgRP), and by increasing the activity of neurons expressing -melanocyte-stimulating hormone (-MSH). The NPY neurons are a key element in the regulation of appetite; small doses of NPY injected into the brains of experimental animals stimulates feeding, while selective destruction of the NPY neurons in mice causes them to become anorexic. On the converse, -MSH is an important mediator of satiety, and differences in the gene for the receptor at which -MSH acts in the brain are linked to obesity in humans. 19 2.1.3.5 Circulatory system The role of leptin/leptin receptors in modulation of T cell activity in immune system was shown in experimentation with mice. It modulates the immune response to atherosclerosis, which is a predisposing factor in patients with obesity. 20

In some epidemiological studies, hyperleptinemia is considered as a risk factor. However, recently a handful of animal experiments demonstrated that systemic hyperleptinemia produced by infusion or adenoviral gene transfer decreases blood pressure in rats. 21,22 2.1.3.6 Lung surfactant activity In fetal lung, leptin is induced in the alveolar interstitial fibroblasts ("lipofibroblasts") by the action of PTHrP secreted by formative alveolar epithelium (endoderm) under moderate stretch. The leptin from the mesenchyme, in turn, acts back on the epithelium at the leptin receptor carried in the alveolar type II pneumocytes and induces surfactant expression, which is one of the main functions of these type II pneumocytes. 23 2.1.3.7 Reproduction In mice, leptin is also required for male and female fertility. Leptin has a lesser effect in humans. In mammals such as humans, ovulatory cycles in females are linked to energy balance (positive or negative depending on whether a female is losing or gaining weight) and energy flux (how much energy is consumed and expended) much more than energy status (fat levels). When energy balance is highly negative (meaning that a woman is starving) or energy flux is very high (meaning that a woman is exercising at extreme levels, but still consuming enough calories), the ovarian cycle stops and females stop menstruating. Only if a female has an extremely low body fat percentage does energy status affect menstruation. Some studies have indicated that leptin levels outside an ideal range can have a negative effect on egg quality and outcome during IVF.24 The body's fat cells, under normal conditions, are responsible for the constant production and release of leptin. This can also be produced by the placenta.25 Leptin levels rise during pregnancy and fall after parturition (childbirth). Leptin is also expressed in fetal membranes and the uterine tissue. Uterine contractions are inhibited by leptin. 26 2.1.3.8 Effects on bone It is now well established that leptin can affect bone metabolism via direct signalling from the brain and that although leptin acts to reduce cancellous bone, it conversely increases cortical bone. A number of theories suggesting that increased leptin during obesity may represent

a mechanism for enlarging bone size and thus bone resistance to cope with increased body weight.27 Bone metabolism is under direct control of the brain and thus nerve fibres are present in bone tissue. A number of brain signalling molecules (neuropeptides and neurotransmitters) have been found in bone including adrenaline, noradrenaline, serotonin, calcitonin gene-related peptide, vasoactive intestinal peptide and neuropeptide Y. This evidence supports a direct signalling system between the brain and bone with accumulating evidence suggesting that these molecules are directly involved in the regulation of bone metabolism. Leptin, once released from fat tissue, can cross the blood-brain barrier and bind to its receptors in the brain where it acts through the sympathetic nervous system to regulate bone metabolism. It is also possible that, in addition to its effects through the brain, leptin may act directly on cells in the bone to regulate bone metabolism. In reality, leptin probably signals to bone on multiple levels, with local and systemic checks and balances impacting the final outcome. As a result, the clinical utility of leptin for treatment of bone diseases remains open but ongoing research may yet provide much needed therapies for stimulating bone formation. 28 2.1.3.9 Inflammatory marker Factors that acutely affect leptin levels are also factors that influence other markers of inflammation, e.g., testosterone, sleep, emotional stress, caloric restriction, and body fat levels. While it is well-established that leptin is involved in the regulation of the inflammatory response, it has been further theorized that leptin's role as an inflammatory marker is to respond specifically to adipose-derived inflammatory cytokines. 29 In terms of both structure and function, leptin resembles IL-6 and is a member of the cytokine superfamily. Circulating leptin seems to effect the HPA axis, suggesting a role for leptin in stress response. Elevated leptin concentrations are associated with elevated white blood cell counts in both men and women. 30 Similar to what is observed in chronic inflammation, chronically-elevated leptin levels are associated with obesity, overeating, and inflammation-related diseases including hypertension, metabolic syndrome, and cardiovascular disease. However, while leptin is associated with body fat mass, the size of individual fat cells, and the act of overeating, it is

interesting that it is not affected by exercise (for comparison, IL-6 is released in response to muscular contractions). 31 2.1.4.0 Obesity and leptin resistance Although leptin is a circulating signal that reduces appetite, obese individuals generally exhibit an unusually high circulating concentration of leptin.These people are said to be resistant to the effects of leptin, in much the same way that people with type 2 diabetes are resistant to the effects of insulin. The pathway of leptin control in obese people might be flawed at some point so the body does not adequately receive the satiety feeling subsequent to eating. A signal-to-noise ratio theory has been proposed to explain the phenomenon of leptin resistance. In healthy individuals, baseline leptin levels are between 1-5 ng/dl in men and 713 ng/dl in women. A large intake of calories triggers a leptin response that reduces hunger, thereby preventing an overload of the inflammatory response induced by caloric intake. It has been theorized that, in obese individuals, the leptin response to caloric intake is blunted due to chronic, low-grade hyperleptinemia depressing the signal-to-noise ratio such that acute leptin responses have less of a physiological effect on the body. The mere fact that leptin resistance is extremely common in obese individuals suggests that it may simply be an adaptation to excess body weight. It has been suggested that the major physiological role of leptin is not as a satiety signal to prevent obesity in times of energy excess, but as a starvation signal to maintain adequate fat stores for survival during times of energy deficit, and that leptin resistance in overweight individuals is the standard feature of mammalian physiology, which possibly confers a survival advantage. 31

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