Leptin signaling as a therapeutic target of obesity.

Review

Leptin signaling as a therapeutic target of obesity Neira Saínz, Carlos J Gonza´lez-Navarro, J Alfredo Martıńez & Maria J Moreno-Aliaga†

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Introduction

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Leptin signaling pathways

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Role of leptin in the regulation

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of body weight and lipid and

Expert Opin. Ther. Targets Downloaded from informahealthcare.com by Queen's University on 03/10/15 For personal use only.

glucose metabolism 4.

Regulation of leptin secretion

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Leptin resistance

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Leptin in human obesity: pathophysiology and therapeutics

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Conclusion

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Expert opinion

University of Navarra, Department of Nutrition, Food Sciences and Physiology, C/Irunlarrea, Pamplona, Spain

Introduction: Leptin is a hormone with a key role in food intake and body weight homeostasis. Congenital leptin deficiency (CLD) is a rare disease that causes hyperphagia and early severe obesity. However, common obesity conditions are associated with hyperleptinemia and leptin resistance. Areas covered: The main signaling pathways activated by leptin as well as the mechanisms underlying the regulatory actions of leptin on food intake and on lipid and glucose metabolism are reviewed. The potential mechanisms involving leptin resistance and the main regulatory hormonal and nutritional factors controlling leptin production/functions are also analyzed. The pathophysiology of leptin in human obesity, and especially the trials analyzing effects of leptin replacement therapy in patients with CLD or in subjects with common obesity and in post-obese weight-reduced subjects are also summarized. Expert opinion: The use of drugs or specific bioactive food components with anti-inflammatory properties to reduce the inflammatory state associated with obesity, especially at the hypothalamus, may help to overcome leptin resistance. Research should also be focused on investigating dietary strategies, food supplements or drugs capable of avoiding or reversing the leptin fall during weight management, in order to promote sustained body weight lowering and weight loss maintenance. Keywords: inflammation, leptin, nutrients, obesity Expert Opin. Ther. Targets [Early Online]

1.

Introduction

Obesity is a major health problem in industrialized and transition societies caused by the dysregulation of the energy balance. The adipocyte-derived hormone, leptin, is essential for the maintenance of body weight by inhibiting food intake and increasing energy expenditure. Indeed, defects in leptin production cause hyperphagia and severe obesity. Circulating concentrations of leptin reflect the size of fat depots, and nutritional status regulates leptin production. Thus, leptin levels are reduced in fasted animals and are increased by re-feeding [1]. The leptin system is especially sensitive to leptin deprivation [2]. Chronic high-fat feeding increases leptin levels, but in this situation leptin does not prevent hyperphagia and obesity. Indeed, obesity is related to hyperleptinemia and leptin resistance development, which impair the physiological functions of leptin [3]. In addition, after weight loss leptin levels are decreased and the anorectic responses to leptin are reduced [4]. Interestingly, leptin production as well as the sensitivity to leptin at central and peripheral tissues is modulated by various hormonal, metabolic factors and nutrients [5,6].

10.1517/14728222.2015.1018824 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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Article highlights. .

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Congenital leptin deficiency (CLD) causes severe obesity, but common obesity is associated with hyperleptinemia and leptin resistance. The drop in leptin that occurs in post-obese subjects could contribute to hunger, lowered metabolic rate and further weight regain. Leptin replacement therapy is effective in leptin deficiency states, including congenital deficiency and lipoatrophy. Obesity-associated inflammation disrupts leptin signaling, favoring leptin resistance. Understanding the factors (nutrients/drugs) that regulate leptin production/functions, especially during weight loss and leptin sensitivity, is the key.

This box summarizes key points contained in the article.

2.

Leptin signaling pathways

Leptin is transported to various regions of the brain across the blood--brain barrier (BBB), and most of the actions of this adipokine on body weight are attributable to effects in the hypothalamus [7]. However, leptin has additional pleiotropic functions in peripheral tissues [8]. Leptin effects are mediated by binding to its receptor (ObR). Leptin receptors are expressed not only throughout the hypothalamus, the cortex and several other brain areas but also in peripheral tissues such as adipose tissue, heart, muscle, lung, intestine, liver and breast [9-11]. Alternative splicing of the ObR mRNA and/or post-translational processing generates at least six isoforms of ObR: four short isoforms (ObRa, ObRc, ObRd and ObRf) with shortened intracellular tails, the secreted isoform (ObRe) that does not carry a transmembrane domain and the long isoform (ObRb). This isoform is considered the main functional receptor of leptin, since it is the isoform with the greater signaling capacity. Leptin binding to ObRb induces a series of intracellular signaling cascades, such as Janus tyrosine kinase family (JAK)/signal transducer and activator of transcription (STAT), Ras/Raf/MAPK, phosphatidylinositol 3-kinase (PI3K)/IRS and 5¢-AMP-activated protein kinase (AMPK)/ acetyl-CoA carboxylase (ACC) (Figure 1). Furthermore, ObRb is abundant in the hypothalamus and is also present at lower levels elsewhere [9,10]. The most known function of leptin, the inhibition of food intake, occurs through the activation of any of these signaling pathways in the hypothalamus [7]. However, leptin is able to signal through other ObR isoforms. In particular, leptin activates the JAK2, IRS1 and extracellular signal-regulated kinase (ERK) through the ObRa isoform, ubiquitously expressed and known as the short isoform before the discovery of the ObRc-f isoforms [9,10]. The JAK/STAT signaling cascade is triggered by the phosphorylation of a JAK2 and subsequent phosphorylation and recruitment of STAT3 binding [12], although STAT1, 2

STAT5 and STAT6 may be activated by leptin as well. STAT3 forms dimers that translocate into the nucleus to induce the expression of genes involved in the regulation of food intake [10]. Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin-induced JAK/STAT pathway that inhibits tyrosine phosphorylation of ObR. Other negative molecules of this cascade have been described, such as protein inhibitor of activated STAT 3, which physically interacts with STAT proteins to block their binding to the response elements in the DNA, and the cytosolic protein tyrosine phosphatase non-receptor type 1 (PTP-1B), which negatively regulates leptin pathway by dephosphorylating JAK2 and STAT3 proteins [10]. Leptin also activates the Ras/Raf/MAPK signaling cascade by ObRb. The binding of leptin to its receptor leads to the phosphorylation of Src homology-2 tyrosine phosphatase (SHP2) that along with the growth factor receptor-bound protein 2 (Grb2) activates ERK. Independently of the phosphorylation of ObRb, JAK2 is also associated with Grb2 and SHP2 and this complex activates further signaling steps [13]. The PI3Ks are heterodimeric complexes composed of regulatory and catalytic subunits. The leptin receptor activation promotes the interaction and formation of the complex SH2B/JAK2/IRS1, causing subsequent activation of downstream targets such as protein kinase B (Akt) [10]. Furthermore, AMPK is also activated by leptin. AMPK is the downstream component of a protein kinase cascade that plays a major role in maintaining energy homeostasis. AMPK is a heterotrimeric enzyme that functions as an energy sensor, which is activated by a rise in the AMP:ATP ratio that occurs following a fall in ATP levels. Activation of AMPK requires phosphorylation of the catalytic subunit by either serine/threonine kinase 1 or calcium/calmodulin-dependent protein kinase kinase b. Then phospho-AMPK inhibits the activity of ACC in lipid utilization [14]. Thus, leptin activates different intracellular signaling pathways through which multiple functions are exerted at central and peripheral levels. Leptin negatively regulates feeding in the hypothalamus and enhances the oxidation of fatty acids in peripheral tissues by signaling through the JAK/STAT, MAPK, PI3K and AMPK pathways. Leptin also stimulates glucose uptake and sympathetic activity through the phosphorylation of the MAPK, PI3K and AMPK proteins. Furthermore, leptin evidences a role in the control of the immune function by activating the JAK/STAT, MAPK and PI3K signaling pathways (Figure 1). Importantly, obesity is a risk factor for different types of cancers, and leptin and ObR are expressed in tumor cells such as breast cancer cells. It has been suggested that leptin induced proliferation of breast cancer cell lines, by activating JAK2--STAT3, PI3K--Akt--glycogen synthase kinase 3, ERK1/2 and AP-1 pathways [15].

Expert Opin. Ther. Targets (2015) 19(7)

Leptin signaling as a therapeutic target of obesity

Leptin


MAPK

PI3K

AMPK

JAK/STAT

Food intake

Food intake

Food intake

Food intake

Fatty acid oxidation




Glucose uptake

Glucose uptake

Glucose uptake

Sympathetic activity



Immune function

Immune function

Immune function

Figure 1. Illustration showing the main signaling pathways activated by leptin.

Role of leptin in the regulation of body weight and lipid and glucose metabolism

3.

Leptin regulates body weight homeostasis by inhibiting food intake and increasing thermogenesis via sympathetic innervation of brown adipose tissue [16]. Thus, genetic deficiency of leptin (ob/ob mouse) or ObRb (db/db mouse) results in obesity due to hyperphagia and reduced energy expenditure [17]. Indeed, leptin replacement increases energy expenditure in ob/ob mice [18]. However, leptin-deficient humans show normal resting energy expenditure but are markedly hyperphagic [19]. Leptin promotes negative energy balance through various signaling pathways in the hypothalamus. The primary site of action of leptin through JAK/STAT3 is the hypothalamus, where it stimulates the transcription of the anorectic neuropeptide proopiomelanocortin (POMC) and suppresses the transcription of the orexigenic neuropeptide Y (NPY) to inhibit appetite. Furthermore, the disruption of the leptin receptor--STAT3 pathway causes hyperphagia in mice and dysregulation of energy expenditure [20], evidencing that STAT3 is important for the control of food intake by leptin. Similarly, leptin activates the MAPK pathway to stimulate the POMC neurons and inhibits the agouti-related protein (AgRP) and NPY-expressing neurons. Through the activation of the PI3K pathway, leptin induces the phosphorylation and inhibition of Forkhead box protein O1 (FOXO1) activity, which is a negative regulator of POMC transcription. Phosphorylation of FOXO1 results in an export from the nucleus

and allows p-STAT3 to bind to neuropeptide promoters, stimulating the expression of POMC and inhibiting AgRP neuropeptides. In addition, the anorectic response to leptin can be reversed by blockade of PI3K [21]. Finally, leptin modulation of AMPK activity in the hypothalamus also plays a role in feeding. An increase in the AMPK activity promotes feeding, whereas a reduction in the activation of AMPK decreases the food intake through the stimulation of ACC activity [2]. Several in vitro and in vivo studies have demonstrated other important physiological functions of the hormone in peripheral tissues, inducing a key role on the regulation of glucose and lipid metabolism in liver, skeletal muscle and adipose tissue independently of its effect on satiety [22]. Previous in vitro studies have shown a dose-dependent lipolytic effect of leptin in white adipocytes of rodents [23,24]. Moreover, leptin inhibits de novo lipogenesis through the hypothalamic PI3K signaling pathway [25] and activates fatty acid oxidation in skeletal muscle of rodents by activating AMPK. Phosphorylation of AMPK inhibits the synthesis of fatty acids, reducing the activity of ACC, and stimulates the fatty acid oxidation de-inhibiting carnitine palmitoyltransferase 1 [26]. Furthermore, leptin administration improves insulin sensitivity and can reverse the obese and diabetic phenotype of rodents [27]. In addition, ex vivo and in vitro studies have shown that leptin increases both basal and insulin-stimulated glucose uptake and oxidation in isolated muscles of rodents [28]. In contrast to the hypothalamus, in peripheral tissues, activation of


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AMPK promotes catabolic processes, and a close relationship between skeletal muscle AMPK activity and sensitivity to leptin has been demonstrated. Leptin-induced AMPK activation regulates the glycolysis and the gluconeogenesis in skeletal muscle, improving insulin sensitivity. Overexpression of leptin increases the phosphorylation of AMPK and ACC, a major component of the insulin-independent AMPK signaling pathway, in soleus muscle, thus improving insulin sensitivity [29]. Leptin also regulates glucose homeostasis and improves insulin sensitivity in peripheral tissues, such as muscle, through the PI3K signaling pathway [30]. Leptin increases protein levels of glucose transporter type (GLUT)4 and reduces the expression and activity of the key negative regulators of GLUT4, TBC1 domain family member 1 (TBC1D1) and member 4 (TBC1D4), in skeletal muscle of leptindeficient mice [31]. Interestingly, the activation of the MAPK signaling pathway by leptin promotes the sympathetic activity in brown adipose tissue of rats [32], which is essential to maintain body weight stability, given that a component of resting energy expenditure is dependent on the sympathetic nervous system. 4.

Regulation of leptin secretion

Regulation of leptin production by other hormones and cytokines

4.1

Several factors modulate the expression and secretion of leptin, including insulin, glucocorticoids, estrogens and proinflammatory cytokines, which promote leptin secretion. In contrast, catecholamines and adrenergic agonists, thyroid hormones, androgens, thiazolidinediones and agonists of PPAR-g inhibit leptin production [5]. In vitro and in vivo studies examining the effects of the proinflammatory cytokine TNF-a on leptin production evidence controversial results. Thus, TNF-a acutely enhances leptin production from 3T3-L1 [33] and primary murine adipocytes [34], whereas long-term exposure to TNF-a inhibits this formation [35]. In addition, insulin and glucocorticoids also stimulate leptin gene expression by adipose tissue [36]. On the contrary, thyroid hormones (T3, T4) decrease leptin levels [37], as well as growth hormone (GH), in rats. Indeed, GH administration to GH-deficient humans (9 months) decreases body weight, fat mass and circulating levels of leptin; however it may reflect the reduction of the adipose fat depot size [38]. Contrarily, in vivo administration of GH induced gene expression of leptin in adipose tissue of male cattle (3 days), whereas no direct effect on leptin production was found in adipose tissue explants incubated with GH (24 h) [36]. These controversial studies of GH on leptin production may be partly explained by an indirect effect of GH on leptin production. Also, the specific differences between human and animal samples, the in vitro and in vivo studies and the duration of the treatment may contribute to understand the different findings observed in these studies. On the other hand, the orexigenic neuropeptide galanin, 4

whose levels are increased by exercise, reduces gene expression and secretion of leptin in rat adipose tissue [39]. Nutritional regulation of leptin The nutritional status of the body is an important stimulus for the production of leptin to regulate body weight homeostasis. Thus, leptin levels decline during fasting and increase on refeeding [40,41]. There are strong evidences that glucose is a key regulator of leptin production by adipocytes. Thus, increases in leptin mRNA after glucose administration in mice are more closely related to plasma glucose concentrations than to plasma insulin concentrations [1]. In this context, it has been demonstrated that insulin-stimulated glucose metabolism rather than insulin per se is a major determinant of leptin production in both rodents and humans [42-44]. Moreover, leptin secretion by cultured adipocytes is more sensitive to the amount of glucose utilized during the culture period than to the extracellular insulin concentration, and in the presence of 2-deoxy-D-glucose, a competitive inhibitor of glucose uptake and phosphorylation, the effects of insulin on leptin are abolished [42,43]. In this context, it has been demonstrated that Sp1-mediated transcription is involved in the induction of leptin by insulin-stimulated glucose metabolism [45]. Furthermore, it is important to mention that glucose not only regulates leptin production but also plays a key role in leptin signaling. Thus, the study of Su et al. [46] suggests that glucose and/or its metabolites play a permissive role in leptin signaling, enhancing leptin sensitivity, in part by attenuating the ability of AMPK to inhibit leptin signaling. In addition, glucosamine also increases leptin gene and protein expression in adipose and muscle cells. So, it has been proposed that hexosamine biosynthetic pathway is a mechanism through which cells ‘sense’ nutrient flux to regulate leptin release [47]. Dietary fat also regulates secretion and action of leptin. In vitro and in vivo studies have shown that triglycerides impair leptin transport through the BBB, inducing leptin resistance development [48], thus suggesting that the reduction of triglycerides intake may improve leptin sensitivity. On the other hand, fatty acids exert differential effects on the ability of adipocytes to produce leptin. Thus, some n-6 polyunsaturated fatty acids (PUFAs), such as arachidonic acid, linoleic acid and conjugated linoleic acid, reduce gene expression and secretion of leptin in primary rat adipocytes [49-51]. The n-3 PUFAs also modulate leptin production both in vitro and in vivo [52,53]. Thus, a trial evidenced an increase in plasma leptin levels after dietary supplementation with n-3 PUFAs (cod oil 7/100 g of diet, 2 months) in sucrose-fed obese and insulin-resistant rats [54]. Similarly, both leptin circulating levels and leptin gene expression in adipose tissue (epididymal and retroperitoneal) were enhanced by supplementation with n-3 PUFAs (fish oil MaxEPA 14/100 g of diet, 3 -- 6 weeks) in sucrose-fed insulin-resistant rats after 3 and 6 weeks of treatment [55]. Some studies have suggested differential effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the regulation of leptin and other 4.2




adipokines such as adiponectin. Thus, a recent study showed that EPA increased leptin release from young adipocytes, whereas DHA did not have any significant effect [56]. In this context, previous studies observed that the n-3 PUFA EPA (100 -- 200 µM) can stimulate leptin production and can increase the oxidative metabolism of glucose in primary cultured adipocytes [52,57]. In addition, in vivo EPA supplementation (1 g/kg, 5 weeks) reduced food intake, fat accumulation and increased serum and adipose leptin levels in overweight rats, whereas it decreased leptin circulating levels in lean rats fed on a standard diet [58]. Other studies have reported no changes or even decreases of n-3 PUFAs on leptin both in vitro and in vivo [52]. Regarding the regulation of leptin in human studies by n-3 PUFAs a recent meta-analysis concluded that n-3 PUFAs supplementation could moderately reduce circulating levels of leptin in lean but not in obese subjects [53]. In fact, omega-3 associated increases in leptin levels have been observed in obese subjects [59]. In this context, a recent study has evidenced that EPA supplementation (1300 mg/day, 10 weeks) prevents the fall of leptin during weight loss in overweight/obese humans, suggesting that EPA could contribute to prevent weight regain in healthy weight-reduced subjects [60]. Taken together, these data highlight the relevance of the dose and duration of the treatment as well as the composition of the dietary fish oil on leptin production. Moreover, the current evidence suggests that n-3 PUFAs action on leptin seems to be dependent on the physiological and metabolical status of the subjects, as well as emphasizes the need to perform longer randomized controlled trials with n-3 PUFAs followed by sustained weight maintenance periods. Moreover, vitamin D has been demonstrated to regulate energy metabolism and leptin production [61,62]. Administration of vitamin D (1.5 µg/kg, 1 week) upregulated circulating levels and gene expression of leptin in adipose tissue of mice [61]. Consistently, addition of vitamin D (0.1 -- 100 nM, 24 h) to explants of murine epididymal fat pad increases leptin production [61], whereas inhibition of leptin secretion has been observed in human omental adipose tissue explants treated with vitamin D (1 -- 100 nM, 48 -- 96 h) [62]. The discrepancy between these studies could be due to differences between species and fat pad depots or to the different experimental conditions, in particular the duration of treatments. Vitamin D is a key regulator of calcium and phosphate metabolism. Interestingly, inorganic phosphate (Pi) is an abundant dietary element that is emerging as an important signaling molecule capable of modulating multiple cellular functions by altering signal transduction pathways [63]. Relevantly, excess of Pi has been recently implicated in the development of cancer, hypertension and also obesity [64]. Indeed, a high intake of Pi has been associated with hyperplasia of adipocytes, enhanced synthesis of triglycerides and leptin levels [64]. Further investigation is required to better establish the potential significance of high phosphate diets on the leptin system and obesity.

Furthermore, a-lipoic acid (LA), a naturally occurring antioxidant, has been shown to decrease leptin production in cultured adipocytes as well as after dietary supplementation both in rodents and humans [60,65,66]. Thus, addition of LA inhibited leptin production in cultured 3T3-L1 (250 µM, 24 h) [65] and human subcutaneous adipocytes (100 -- 250 µM, 24 h) [66]. Consistently, dietary supplementation with LA (0.25/100 g diet, 56 days) to low- and high-fat-fed rats also decreased circulating leptin levels and gene expression of leptin in epididymal fat [65]. A recent study has also revealed a reduction in plasma leptin levels in overweight and obese women following an energy-restricted diet supplemented with LA (300 mg/day, 10 weeks) [60]. Furthermore, chitosan supplementation, a natural polysaccharide comprising copolymers of glucosamine and N-acetylglucosamine, reduced body weight and food intake but increased leptin concentrations in pigs [67]. Thereby, dietary composition or bioactive food compounds might be used as a therapeutic strategy in leptinresistant states associated with obesity to reduce body fat while preserving leptin sensitivity and leptin levels and, consequently, the inhibitory effect of the hormone on appetite. 5.

Leptin resistance

Human obesity due to congenital leptin deficiency (CLD) is rare [68]. Physiological levels of leptin are observed in some cases of obesity in spite of the high fat pad depots. However, in most cases, hyperleptinemia is a characteristic of human obesity, which has suggested the existence of resistance to the hormone at central and peripheral levels [3,4]. Thereby, the high levels of leptin could be a mechanism to compensate for the lack of response to the hormone. The concept of leptin resistance is usually used to define states of hyperleptinemia and lack of response to the hormone. However, this view is used in different frameworks and a more precise definition of leptin resistance is needed [69]. The mechanisms leading to the development of leptin resistance are unclear, but several possibilities have been postulated: i) a failure of the hormone to cross the BBB and to act on its target neurons [70], ii) a reduction in the expression of leptin receptors, and iii) a failure on the leptin signaling pathway at central and peripheral levels (Figure 2) [71]. However, other factors such as inflammatory mediators and eating behavior have been shown to contribute to the development of leptin resistance [6,72]. Obesity is often characterized by low chronic inflammatory state, and several in vitro and in vivo studies have reported that inflammatory factors enhance leptin production contributing to the hyperleptinemia present in obesity and to the development to leptin resistance. Inflammatory mediators such as TNF-a, C reactive protein (CRP) and lipopolysaccharide (LPS) increase circulating leptin concentrations in rodents and humans [33], thus suggesting that these molecules may be involved in the leptin resistance onset. Interestingly, leptin


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Central leptin resistance a)

Central and peripheral leptin resistance

Failure to cross the BBB

BBB b) Impaired expression of leptin receptors 1. Low gene expression


Leptin

2. Gene mutation

Leptin

c) Impaired leptin signaling pathway Leptin

SOCS3

STAT3

PTP-1B NPY

POMC

Figure 2. Illustration showing the potential mechanisms underlying leptin resistance. BBB: Blood--brain barrier; NPY: Neuropeptide Y; PTP-1B: Protein tyrosine phosphatase, non-receptor type 1; POMC: Proopiomelanocortin peptide; SOCS3: Suppressor of cytokine signaling 3; STAT3: signal transducer and activator of transcription 3.

resistance has been partly attributed to an interaction between CRP and leptin that may inhibit the binding of leptin to membrane receptors [73]. On the other hand, SOCS3 appears to be the key in the central leptin resistance development. The anorectic effects of LPS through the activation of STAT3 in the hypothalamus are abolished by leptin resistance in highfat-fed rats through the increased expression of SOCS3 [74]. In addition, high-fat feeding in rodents induces the expression of SOCS3 and resistance to STAT3 activation by leptin in POMC [75] and AgRP-expressing [76] neurons. Interestingly, switching from high- to low-fat diet reduces SOCS3 expression in AgRP neurons, thus suggesting that these neurons may be more responsive than POMC neurons to the circulating levels of leptin [76]. Accordingly to these studies, silencing the hypothalamic SOCS3 protects against the development of diet-induced obesity in rodents [77]. Thereby, these studies support that several mediators of inflammation are related with hyperleptinemia and leptin resistance development in states of diet-induced obesity. The occurrence of hyperleptinemia is highly correlated with dietary obesity [6,78]. High-fat diet consumption triggers central and peripheral leptin resistance as has been extensively demonstrated in rodent models of diet-induced obesity. However, some studies have observed that hyperphagic obese rats develop hyperleptinemia but not apparently central or peripheral leptin resistance [79]. In contrast, leptin-deficient ob/ob mice develop leptin resistance after high-fat diet consumption, independently from leptin levels [80]. So, several 6

controversial outcomes have been reported in this field. Indeed, caloric restriction did not improve the impaired leptin downstream signaling in central and peripheral tissues of hyperphagic rats, suggesting that hyperleptinemia may be an adaptive mechanism to overcome leptin response and diet-induced obesity [79]. Another study revealed that obese animals with constant and low circulating levels of leptin remain highly sensitive to exogenous leptin, even after long-term exposure to a high-fat diet, indicating that dietary fat alone is not capable of blocking the response to leptin [81,82]. Other investigations have reported that the development of leptin resistance may be dependent on the type and the duration of diet. In this sense, dietary fat and sugars have reported different effects on leptin response and a possible beneficial effect of sugars within a fat diet has been suggested. Thus, rats with a short-term high-fructose and low-fat feeding were leptin-resistant, whereas rats fed on a high-fructose and high-fat diet or a high-glucose and high-fat diet were sensitive to intraperitoneal leptin administration (4 weeks) [83]. In contrast, a long-term high-fructose diet (6 months) induced leptin resistance in rats [84] and, similarly, another long-term trial (134 days) has also suggested a key role of fructose in the induction of leptin resistance [85]. On the other hand, dietary habits not only modulate leptin production but, interestingly, also alter the composition of the intestinal microbiota. Relevantly, recent studies have implicated gut microbiome in obesity and leptin resistance




development by decreasing the expression of anti-obesity neuropeptides and increasing SOCS3 in rodents [86,87]. In this regard, diet-induced obese animals present alterations in the intestinal microbiota, similar to those of the leptin-deficient signaling mice (ob/ob and db/db). Interestingly, weight reduction altered gut microbial community of diet-induced obese animals and the differences in relative bacterial abundance of microbiome were related to circulating levels of leptin [86]. Moreover, a recent study has reported that leptin signaling modulates the composition of the intestinal microbiota independently of food intake [88].

Leptin in human obesity: pathophysiology and therapeutics

6.

Trials in congenital leptin-deficient subjects CLD occurs as a consequence of missense mutations in the leptin gene. However, this is a very rare case of severe early onset obesity, since only four homozygous mutations in the leptin gene in a total of 14 individuals have been so far described: i) a frameshift mutation in the Lep gene (c398delG D133G), resulting in a truncated, unsecreted protein [19]; ii) a recessive missense mutation (c313C > T Arg105Trp; R105W) [89]; iii) a missense mutation N103K, resulting from the substitution of asparagines (AAC) by lysine (AAA) at codon 103 [90], and finally; and iv) a homozygous transition (TTA to TCA) in exon 3 of the Lep gene resulting in a L72S replacement in the leptin protein [91]. CLD is characterized by severe early onset obesity due to hyperphagia and impaired satiety and can be accompanied by insulin resistance and type 2 diabetes, hyperlipidemia, immunological differences, hypogonadotropic hypogonadism and abnormal pubertal development and liver steatosis [92]. Administration of recombinant human leptin to congenital leptin-deficient subjects was aimed to restore the physiological levels of the hormone. Table 1 summarizes clinical trials with recombinant leptin in obese subjects with CLD, describing the characteristic of participants and treatments (doses, duration) as well as the main outcomes and benefits obtained [93-106]. All clinical trials in leptin-deficient subjects carrying some of the missense mutations (D133G, R105W or L72S) have demonstrated that recombinant methionyl human leptin (also named metreleptin) effectively decreases body fat mass and body weight. This effect is mainly due to a reduction of food intake, since no significant changes in energy expenditure have been found (Table 1). This outcome contrasts with findings in ob/ob mice, where leptin replacement was able not only to reduce hyperphagia but also to increase energy expenditure [17]. Leptin replacement in leptin-deficient obese subjects improves insulin resistance and decreases type 2 diabetes manifestations as well as hyperlipidemia [94,97,105]. Recombinant human leptin administration was also capable of reversing the reduced numbers of circulating CD4+ T cells and 6.1

impaired T-cell proliferation and cytokine release associated with leptin deficiency [94]. Moreover, the hypothalamic hypothyroidism associated with leptin deficiency is also reversed by leptin replacement [94,95]. In addition, the administration of leptin also normalized the pulsatile secretion of folliclestimulating hormone, luteinizing hormone (LH) and counteracted the hypogonadotropic hypogonadism observed in congenital deficient subjects [94,97,106]. In fact, leptin treatment appropriately facilitated timed pubertal development and the induction of menstrual cycles [94,106]. Leptin replacement also normalized the circulating levels of IGF-1 and the pulsatile nocturnal GH secretion, in connatal leptin-deficient humans [106]. There is strong evidence that in addition to the homeostatic control of food intake, leptin also influences hedonic aspects of feeding in part by regulating the mesolimbic dopaminergic system [22]. Several studies using functional magnetic resonance images to obtain blood oxygen level-dependent signals have demonstrated changes in the activation of different brain areas in states of leptin deficiency and after leptin replacement as well as the pattern of brain activation in response to food and non-food images (Table 1) [96,101,102]. Trials in lean and normal (non-leptin-deficient) obese subjects

6.2

In common obesity, leptin levels are directly proportional to the amount of body fat and therefore obesity is usually associated with hyperleptinemia and leptin resistance. Several trials have addressed the efficacy of leptin administration in lean and obese subjects (alone or as adjuvant of hypocaloric diets). Although the first trial suggested that leptin could promote a moderate weight and fat mass loss at the highest supraphysiological doses (0.3 mg/kg/day) tested [107], in most of the subsequent trials, leptin has shown a very limited efficacy as a therapeutic agent for losing weight in patients with common obesity. Table 2 summarizes the outcomes obtained in clinical trials after recombinant leptin administration in lean and common obese (hyperleptinemic) subjects with or without hypocaloric diet [107-114]. Because, recombinant methionyl human leptin has a half-life of ~ 4 h in humans and requires daily administration to obtain sustained blood level, some trials were performed with long-acting PEGylated recombinant leptin (PEG-OB). However, neither in this form, leptin was able to significantly promote fat mass reduction and to counteract the neuroendocrine adaptations to semi-starvation induced by a very low energy diet [108-112]. Efficacy of leptin replacement in obese subjects after weight loss

6.3

After weight loss, significant changes in the circulating levels of several peripheral hormones involved in the homeostatic regulation of body weight take place, accompanying fat lowering. Indeed, weight-reduced subjects exhibit acute compensatory changes, including profound reductions in energy


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Table 1. Clinical trials with recombinant leptin in obese subjects with congenital leptin deficiency. Study design One child with congenital leptin deficiency (D133G)

One girl homozygous for the D133G mutation

Recombinant leptin (r-metHuLeptin) 0.028 mg/kg/day lean mass s.c. for 12 months r-metHuLeptin (initial dose calculated to achieve 10% predicted serum leptin concentration based on age, gender and percentage of body fat) s.c. for up to 4 years r-metHuLeptin (0.19 -- 0.06) s.c. for up to 4 years

One boy and one girl with congenital leptin deficiency (D133G)

r-metHuLeptin for 7 days Test: fMRI with presentation of food cues

Three morbidly obese leptindeficient adult patients (R105W)

r-metHuLeptin (0.01 -- 0.04 mg/kg/day) s.c. for 18 months

Three morbidly obese leptindeficient adult patients (R105W)

r-metHuLeptin (0.01 -- 0.04 mg/kg/d) s.c. for 15 weeks

Three non-sibling adults homozygous for a Lep mutation (R105W)

Daily s.c. r-metHuLeptin at doses predicted to achieve blood concentrations typical of men with body fat of 20% and women with body fat of 30% for 18 months Test session 1: 57 months after the initial start of leptin replacement; Test session 2: After 33 days of discontinued treatment; Test session 3: 14 days after treatment resume. Test: fMRI with presentation of food cues 5 and 6 years leptin-treated patients. Session 1: each year occurred after 10 months of continuous daily replacement. Session 2: after 33 -- 37 days without leptin. Session 3: 14 -- 23 days after restoration of daily replacement. Test: BOLD-whole brain fMRI r-metHuLeptin (0.02 -- 0.04 mg/kg/day) for 19 weeks for leptin-deficient subjects; controls were under low-calorie diet (890 kcal/day) Metreleptin (0.6 mg/twice a day ~ 0.024 mg/kg lean body mass) for 3 days, 6, 12 and 24 months

Three children with congenital leptin deficiency (D133G)


Leptin administration

Three leptin-deficient obese adults (R105W)

Three leptin-deficient obese adults (R105W)

Three leptin-deficient obese adults and three obese controls (R105W) Leptin-deficient adolescent girl with a novel homozygous mutation in the leptin gene (L72S) accompanied by liver steatosis and hypogonadotropic hypogonadism

Outcomes

Ref.

# Appetite, fat mass and body weight $ Energy expenditure

[93]

# Appetite, fat mass and body weight # Hyperinsulinemia and hyperlipidemia " Thyroid hormone " T-cell responsiveness $ Restore normal pubertal development # fat mass and body weight # Hyperinsulinemia # Triglycerides " HDL-cholesterol Restore abnormal thyroid function # Appetite # Activation in the nucleus accumbenscaudate and putamen--globus pallidus regions induced by food stimuli # Food intake, fat mass and body weight # Hyperinsulinemia and hyperlipidemia " 24 h levels of LH, testosterone and cortisol # Food intake, fat mass and body weight # Appetite and " satiety and modify eating behavior # Fat mass and body weight Normalized fat partitioning in human

[94]

# Brain activation in regions linked to hunger (insula, parietal and temporal cortex) " Activation in regions linked to inhibition and satiety (prefrontal cortex)

[100]

Longer duration of replacement was associated with more activation by food images in a ventral portion of the posterior lobe of the cerebellum. Simultaneous decreases in body mass were associated with decreased activation in a more dorsal portion of the same lobe

[101]

Leptin replacement prevents weight lossinduced decrease in energy expenditure and fat oxidation

[104]

Acute and long-term changes in homeostatic, reward and food-related brain areas # Fat mass and body weight # Liver fat content and transaminases # Cholesterol and insulin resistance " LH and FSH and induce menstrual cycles Normalized GH and IGF-1 levels

[102,103]

[95]

[96]

[97]

[98]

[99]

[105,106]

BOLD: Blood oxygen level-dependent; fMRI: Functional magnetic resonance images; FSH: Follicle-stimulating hormone; GH: Growth hormone; HDL: High-density lipoprotein; LH: Luteinizing hormone; r-metHuLeptin: Recombinant methionyl human leptin (also named metreleptin); s.c.: Subcutaneous.

8



Table 2. Clinical trials with recombinant leptin in lean and common obese (hyperleptinemic) subjects with or without hypocaloric diet. Study design


Randomized, doubled-blind, placebo-controlled multicenter trial in 54 lean and 73 obese subjects

Randomized to a doubleblind treatment in 30 healthy obese men

Randomized to a double-blind treatment in 28 healthy obese (16 women; 12 men) A randomized double-blind placebo-controlled trial in 24 overweight male subjects

Randomized, placebocontrolled trial in obese subjects with newly diagnosed type 2 diabetes Twenty-four overweight and obese adults (16 women and 8 men)


Outcomes

r-metHuLeptin (0, 0.01 -- 0.03, 0.10 or 0.30 mg/kg/day) s.c. for 4 weeks in lean and obese (part A). In part B, obese subjects continued for additional 20 weeks. Lean subjects: eucaloric diet; obese subjects: hypocaloric diet (-500 kcal/day) from amount needed to maintain stable weight Weekly s.c. injections of 20 mg PEG-OB or placebo for 12 weeks, in addition to a hypocaloric diet (deficit, 2 MJ/day)

Weekly s.c. injections of 60 mg PEG-OB or placebo for 8 weeks, in addition to a mildly hypoenergetic diet Weekly s.c. injections of 80 mg PEG-OB or placebo for 46 days, in addition to a very low energy diet (2.1 MJ/day)

Placebo, low-dose (30 mg/day), or high-dose (80 mg/day) r-metHuLeptin for 14 days Metreleptin (10 mg/day self-injected s.c.) or placebo for 6 months + hypocaloric diet (-500 kcal/day)

Ref.

# Fat mass and body weight (dosedependent) in some obese and lean subjects

[107]

$ Weight loss or percentage body fat $ Sleeping metabolic rate $ Respiratory quotient Moderate (n.s.) reduction in triglycerides # Subjective appetite and hunger before breakfast $ Weight loss or percentage body fat $ Metabolic status $ CRP, soluble TNF-a receptors

[108,109]

# Body weight (2.8 kg) Do not reverse the fasting-induced changes in the thyroid, corticotropic, somatotropic axes and sympathetic nervous system activity Attenuated the drop in LH $ Changes in insulin, glucose and adiponectin No changes on insulin-mediated suppression of glucose, glycerol or palmitate rates of appearance into plasma $ Weight loss $ Changes in circulating hormones of the thyroid and IGF axes

[111]

[110]

[112] [113]

[114]

CRP: C reactive protein; LH: Luteinizing hormone; PEG-OB: PEGylated recombinant leptin; r-metHuLeptin: recombinant methionyl human leptin (also named metreleptin); s.c.: Subcutaneous.

expenditure accompanied by decreases in thyroxine and triiodothyronine levels and increases in ghrelin and appetite, all of which promote weight regain [115]. Circulating leptin levels also markedly decrease during weight loss, and it has been suggested that this drop in leptin could contribute to hunger, a lowered metabolic rate, and further weight regain. In fact, many of the metabolic and neurohormonal profiles of weight-reduced individuals are remarkably similar to those of leptin-deficient humans and rodents [116], and the weightreduced state may be regarded as a condition of relative leptin insufficiency [117]. Therefore, leptin replacement therapy could prevent subsequent resting metabolic rate fall after weight loss and the weight regain in weight-reduced subjects. Table 3 summarizes clinical trials with leptin replacement in previously weigh-reduced lean and obese subjects [116-122]. Most of trials support the efficacy of leptin in this circumstances, since the replacement with the hormone

is able to promote further weight and fat mass loss by restoring energy expenditure, skeletal muscle work efficiency, sympathetic nervous system tone and circulating T3 and T4 to pre-weight-loss levels [117,118,123]. Moreover, leptin administration reverses weight loss-induced changes in regional neural activity responses to visual food stimuli [120] and increased satiation [116]. However, a recent study showed that the injection of leptin to women who had an important reduction of weight after gastric bypass did not have any additional effect on body weight, fat mass, resting energy expenditure, thyroid hormones or cortisol levels [122]. 7.

Conclusion

Physiological leptin signaling is essential for the maintenance of body weight and circulating concentrations of leptin are proportional to fat pad depots. However, inflammation and


9

N. Saínz et al.

Table 3. Effects of leptin administration in post-obese weight-reduced subjects.


Study design


Outcomes

Thirty healthy obese subjects (7 males and 23 females)

Mildly energy-restricted diet for 21 days, and then treated with recombinant human leptin (10 mg s.c. once [n = 15] or twice [n = 6] daily) or placebo (n = 9) for 12 weeks.

[119]

One obese and three lean subjects

Experimental phases: i) usual body weight; ii) while stable at 10% reduced body weight; and iii) during a 5-week period at 10% reduced body weight while receiving twice per day leptin injections that restored 08.00 h circulating leptin concentrations to those seen at usual body weight Three experimental conditions: i) maintaining usual weight; ii) maintaining a 10% reduced weight; iii) receiving twice-daily s.c. doses of leptin sufficient to restore 08.00 h circulating leptin concentrations to pre-weight-loss levels and remaining on the same formula diet required to maintain 10% reduced weight Three experimental conditions: i) maintaining usual weight; ii) after a 10% reduced weight; iii) 5 weeks of twice-daily s.c. injections of saline or recombinant methionyl human leptin in doses required to achieve circulating concentrations of leptin at 08.00 h equal to those measured at initial (around 0.08 and 0.14 mg/kg fat mass).

" Weight loss Positive correlation between changes in leptin during the initial 21 days and the loss of body weight following leptin treatment. Inverse association between leptin levels prior to the initiation of rLeptin therapy and the amount of body weight lost in response to intervention Leptin replacement restores the decrease in circulating thyroid hormones T3, T4 and leptin that occurs after weight loss Leptin replacement promotes fat mass loss and increases total energy expenditure Leptin restores energy expenditure, skeletal muscle work efficiency, sympathetic nervous system tone and circulating T3 and T4 to pre-weightloss levels

[117]

Leptin reverses the increases observed after weight loss in neural activity in response to visual food cues in the brainstem, parahippocampal gyrus, culmen, inferior and middle frontal gyrus, middle temporal gyrus and lingual gyrus Leptin reverses the decreases in activity in response to food cues in the hypothalamus, cingulate gyrus and middle frontal gyrus Leptin did not reverse the drop in bone alkaline phosphatase induced by weight loss Leptin increases N-terminal telopeptides No changes by weight loss or leptin on PTH, calcium and 25D Leptin administration to weightreduced subjects increases satiation (post-meal feelings of fullness and the perception of how much food was eaten)

[120]

Leptin administration did not have any additional effect on body weight, fat mass, resting energy expenditure, thyroid hormones or cortisol levels

[122]

Ten inpatient subjects (5 males, 5 females [3 never-obese,7 obese])

Six obese subjects (2 male, 4 female)

Prospective, single-blinded study of 12 subjects (8 women,4 men; 2 non-obese, 10 obese)

i) Maintaining their usual weight and ii) weight loss, and iii) during maintenance of 10% weight loss while receiving twice daily injections of either a placebo or replacement doses of leptin

Ten obese humans (4 men, 6 women). Satiation was studied 3 h after ingestion of 300 kcal of liquid-formula diet

Three time periods: i) maintaining usual weight; ii) after weight reduction and stabilization at 10% below initial weight iii) while they received 5 wk of twice-daily injections of placebo or ‘replacement doses’ of leptin in a single-blind crossover design with a 2-week washout period between treatments Leptin or placebo via s.c. injection twice daily for 16 weeks, then crossed over to receive the alternate treatment for 16 weeks.

Randomized, double-blind, placebo-controlled crossover study of 27 women after at least 18 months post-RYGB and lost on average 30.8% of their pre-surgical body weight

25D: 25-hydroxy vitamin D; PTH: Parathyroid hormone; RYGB: Roux-en-Y gastric bypass.

10


Ref.

[118]

[121]

[116]



hyperleptinemia are common features of diet-induced obesity, which lead to leptin resistance development and to the loss of the anorectic response to leptin. In addition, after weight loss the leptin levels are reduced and the inhibitory effect of leptin on appetite is lost, which may lead to the recovery of body weight. In this sense, eating behavior may modulate leptin sensitivity and expression in peripheral tissues. Thereby, specific nutrients and dietary compound with anti-inflammatory properties may be used as a therapeutic strategy to reduce the inflammatory state associated with obesity and to regulate leptin production and sensitivity after weight loss. It is no doubt that leptin replacement therapy has brought great benefits in CLD patients. In addition, most of these trials suggest that although leptin administration may not promote substantial weight loss in obese leptin-resistant subjects, leptin therapy might be useful in maintaining the weight loss achieved by dieting. 8.

Expert opinion

It has been proposed that leptin deficiency should be considered as a clinical syndrome similar to other hormone deficiency states [2]. As previously reported, treatment with leptin in congenital leptin-deficient obese subjects corrects not only hyperphagia but also all the metabolic, neuroendocrine and immune disorders associated. For these few patients, leptin is available through a compassionate-use program from Amylin Pharmaceuticals. Leptin treatment is also beneficial in other non-obese pathologies that are accompanied by leptin deficiency such as lipoatrophy. In fact, recently the FDA has approved leptin as a therapeutic for the treatment of severe congenital lipodystrophies [124]. Leptin replacement could be also effective in relatively hypoleptinemic obese subjects as a consequence of being heterozygotes for leptin gene mutations [125]. In obese people, the weight-reduced state after a hypocaloric diet could also be considered as a situation of relative leptin deficiency. Although some trials have reported positive effects, the efficacy and safety of leptin administration in this state should be further explored. Research should also be focused to look for dietary strategies, food supplements or drugs capable of avoiding or reversing the fall in leptin during weight loss programs, which would help to promote sustained body weight loss and to better approach weight loss maintenance. Taking into consideration that the vast majority of obese individuals are hyperleptinemic and resistant to leptin, it was reasonable that most of trials found a lack of efficacy of leptin as a monotherapy for common obesity. Therefore, efforts should be taken for overcoming leptin resistance in obesity. In this context, some recent trials have focused in analyzing the efficacy of the coadministration of leptin with leptin sensitizers such as amylin. Amylin is a pancreatic b-cell-derived hormone that mimics leptin effects on the reduction of body weight in diet-induced obese and leptinresistant animals [109]. Also, some clinical trials in obese

subjects showed that the combination of leptin and an amylin analog pramlintide is more effective in promoting weight loss than any of the agents alone [126,127]. However, this study was suspended due to the development of anti-metreleptin antibodies [128]. Some preclinical trials have evaluated the efficacy of other potential leptin sensitizers such as exendin-4 and fibroblast growth factor 21 in obese mice [129]. Future clinical trials in humans are needed in order to test if they can also restore leptin responsiveness in hyperleptinemic obese. There is growing evidence that low-grade chronic inflammatory state associated with obesity could play a key role in disrupting leptin signaling favoring leptin resistance development. In fact, hypothalamic inflammation seems to be an important etiological factor in the pathophysiology of obesity. Therefore, the use of drugs or specific nutrients with antiinflammatory properties to reduce the inflammatory state may help improve leptin sensitivity in the organism. In this context, a recent study has shown that the administration of a plant terpenoid compound, ginsenoside Rb1, with antiinflammatory properties, reduced hypothalamic inflammation and restored the anorexic effect of leptin in high-fat-fed mice as well as leptin pSTAT3 signaling in the hypothalamus [130]. Several studies in rodents and humans have supported the ability of marine n-3 PUFAs to ameliorate chronic inflammation associated with obesity. In fact, n-3 PUFAs alleviate the characteristic inflammation observed in white adipose tissue in obese rodents and humans by reducing the activation of NK-kb and other transcription factors involved in the inflammatory response and by decreasing the recruitment of type 1 macrophages, in part through the stimulation of the G-protein-coupled receptor 120 (GPR120) [131-133]. Interestingly, a recent study has also demonstrated the anti-inflammatory actions of n-3 PUFAs on immortalized hypothalamic neurons through the activation of GPR120 [134]. It is well established that n-3 PUFAs also decrease inflammation by inhibiting the formation of n-6 fatty acidsderived proinflammatory eicosanoids and especially by serving as substrates for the formation of potent specialized pro-resolving lipid mediators (SPM), such as resolvins (Rv), protectins (PD) and maresins [135]. Interestingly, several studies have demonstrated that the administration of some of these SPMs derived from EPA and DHA such as RvE1, RvD1, 17-HDHA and PDX are able to counteract adipose tissue inflammation and several metabolic disorders like insulin-resistance both in leptin-deficient or hyperleptinemic obese rodents [136-140]. Importantly, neuroprotectin D1 is also capable of inducing homeostatic regulation of neuroinflammation and cell survival [141]. Therefore, n-3 PUFA-derived SPM have emerged as a promising novel therapeutic strategy for combating inflammation and metabolic disorders associated with obesity. However, the efficacy of these SPMs to resolve hypothalamus inflammation and resolution of leptin resistance is unclear and deserve future research.


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On the other hand, as it has been stated above, most attempts at using recombinant leptin or leptin analogous have been unsuccessful due, among several reasons, to an impaired transport of leptin across the BBB. In fact, human and rodent studies indicate that the major cause of this resistance arises from an impaired ability of leptin to cross the BBB [142]. Thus, several efforts have been made to improve the poor response to peripherally administered leptin in moderately obese patients. Some of these approaches, summarized by Yi et al. [143], include modifications of leptin (using PEG or transactivating transcriptional activator) and the use of engineered Fc--leptin fusion proteins or leptin peptide mimics carrying a carbohydrate moiety. Unfortunately, these modifications have only been partially successful when evaluated in animal models but have failed in clinical trials. Nevertheless, this latter author has recently reported that the use of leptin--pluronic conjugates improved peripheral bioavailability and increased brain uptake. Remarkably, conjugates containing multiple pluronic chains might cross the BBB Bibliography

independently of the leptin transporter, thus bypassing one of the main reasons for resistance to peripheral leptin and opening a new research pathway [143]. Hence, these new strategies might lead to upcoming leptin-based treatments for obesity but there is still a long way from these promising but preliminary results to their application in clinical practice.

Declaration of interest The authors were supported by the Ministry of Economy and Competitiveness (AGL 2009-10873/ALI and BFU201236089) and CIBERobn of the government of Spain, Navarra government and Linea Especial ‘Nutrition Obesidad y Salud’ University of Navarra. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Papers of special note have been highlighted as either of interest () or of considerable interest () to readers.

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of the blood--brain barrier and its potential for the treatment of obesity.


Affiliation Neira Saínz1, Carlos J Gonza´lez-Navarro1,2, J Alfredo Martıńez1,2,3 & Maria J Moreno-Aliaga†1,2,3 † Author for correspondence 1 University of Navarra, Centre for Nutrition Research, School of Pharmacy, C/Irunlarrea 1, 31008 Pamplona, Spain 2 University of Navarra, Department of Nutrition, Food Sciences and Physiology, C/Irunlarrea 1, 31008 Pamplona, Spain Tel: +34 948 42 5600 Ext. 6558; E-mail: [email protected] 3 Instituto de Salud Carlos III, CIBER Fisiopatologıá de la Obesidad y la Nutricioń (CIBERobn), 28029 Madrid, Spain


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Chronic Oxytocin Administration as a Treatment Against Impaired Leptin Signaling or Leptin Resistance in Obesity.

Angiogenesis as a therapeutic target for obesity and metabolic diseases.

TrkA Signaling as a Therapeutic Target for Pain.

IL-11 signaling as a therapeutic target for cancer.

Macrophage Metabolism As Therapeutic Target for Cancer, Atherosclerosis, and Obesity.

Oncogenic role and therapeutic target of leptin signaling in colorectal cancer.

GABAergic signaling as therapeutic target for autism spectrum disorders.

Prion protein signaling as therapeutic target in human tumors.

Blocking CD40-TRAF6 signaling is a therapeutic target in obesity-associated insulin resistance.

KRAS as a Therapeutic Target.

Exploiting the therapeutic potential of leptin signaling in cachexia.

Leptin signaling molecular actions and drug target in hepatocellular carcinoma.

Leptin-STAT3-G9a Signaling Promotes Obesity-Mediated Breast Cancer Progression.

Serotonin as a New Therapeutic Target for Diabetes Mellitus and Obesity.

Adiponectin, a Therapeutic Target for Obesity, Diabetes, and Endothelial Dysfunction.

Adipose ABCG1: A potential therapeutic target in obesity?

Dynamics of wound healing signaling as a potential therapeutic target for radiation-induced tissue damage.

Evaluation of Cysteinyl Leukotriene Signaling as a Therapeutic Target for Colorectal Cancer.

Evaluating DAPK as a therapeutic target.

Mitochondrial Quality Control as a Therapeutic Target.

Ror2 as a therapeutic target in cancer.

Heme as a Target for Therapeutic Interventions.

Prostanoid receptor EP2 as a therapeutic target.

Memory as a new therapeutic target.