LFS-14372; No of Pages 7 Life Sciences xxx (2015) xxx–xxx

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Leptin resistance in obesity: An epigenetic landscape Ana B. Crujeiras a,b,1, Marcos C. Carreira a,b,1, Begoña Cabia a,b, Sara Andrade a,b, Maria Amil a,b, Felipe F. Casanueva a,b,⁎ a Laboratory of Molecular and Cellular Endocrinology, Instituto de Investigación Sanitaria (IDIS), Complejo Hospitalario Universitario de Santiago (CHUS) and Santiago de Compostela University (USC), Santiago de Compostela, Spain b CIBER Fisiopatología de la Obesidad y la Nutrición (CIBERobn), Madrid, Spain

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Article history: Received 30 March 2015 Received in revised form 2 May 2015 Accepted 12 May 2015 Available online xxxx Keywords: Appetite DNA methylation Obesity pharmacotherapy Leptin

a b s t r a c t Leptin is an adipocyte-secreted hormone that inhibits food intake and stimulates energy expenditure through interactions with neuronal pathways in the brain, particularly pathways involving the hypothalamus. Intact functioning of the leptin route is required for body weight and energy homeostasis. Given its function, the discovery of leptin increased expectations for the treatment of obesity. However, most obese individuals and subjects with a predisposition to regain weight after losing it have leptin concentrations than lean individuals, but despite the anorexigenic function of this hormone, appetite is not effectively suppressed in these individuals. This phenomenon has been deemed leptin resistance and could be the result of impairments at a number of levels in the leptin signalling pathway, including reduced access of the hormone to its receptor due to changes in receptor expression or changes in post-receptor signal transduction. Epigenetic regulation of the leptin signalling circuit could be a potential mechanism of leptin function disturbance. This review discusses the molecular mechanisms, particularly the epigenetic regulation mechanisms, involved in leptin resistance associated with obesity and the therapeutic potential of these molecular mechanisms in the battle against the obesity pandemic. © 2015 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Pharmacological perspectives of leptin in obesity treatment. . . . . . . . . . . . 3. Molecular mechanisms involved in leptin resistance . . . . . . . . . . . . . . . 4. Epigenetic regulation of leptin-related signalling pathways: role in leptin resistance. 5. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authors’ contributions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Twenty years ago, the discovery of leptin, the product of the ob gene [1] and the first adipose signalling molecule implicated in the regulation ⁎ Corresponding author at: Molecular and Cellular Endocrinology Area (Lab. 2), Instituto de Investigación Sanitaria (IDIS), Complejo Hospitalario Universitario de Santiago (CHUS), C/ Choupana, s/n. 15706 Santiago de Compostela, Spain. E-mail address: [email protected] (F.F. Casanueva). 1 Both authors equally contributed to this work.

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of energy balance, changed the knowledge of energy homeostasis and changed the view of adipose tissue as an energy depot to a highly active endocrine organ. To date, more than 600 adipokines have been discovered [2]. Leptin is mainly, but not exclusively, produced by adipose tissue, and its circulating levels are correlated with the amount of body fat and reflect energy status. Conversely, emerging research has also suggested that leptin plays an important role in energy-deficient states, such as fasting, diet or exercise-induced amenorrhea and lipoatrophy, which are associated with leptin deficiency and with infertility and

http://dx.doi.org/10.1016/j.lfs.2015.05.003 0024-3205/© 2015 Elsevier Inc. All rights reserved.

Please cite this article as: A.B. Crujeiras, et al., Leptin resistance in obesity: An epigenetic landscape, Life Sci (2015), http://dx.doi.org/10.1016/j. lfs.2015.05.003

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2. Pharmacological perspectives of leptin in obesity treatment

Fig. 1. Schematic representation of leptin-activated intracellular signalling pathway.

neuroendocrine abnormalities, metabolic dysfunction, depressed immune function and bone loss [3]. Many of the actions of leptin are attributable to its effects on the central nervous system (CNS), as it crosses the blood–brain barrier (BBB) through a receptor-mediated endocytosis mechanism [4]. This is a critical step in the actions of leptin and is a possible target for clinical applications. Leptin acts predominantly in hypothalamic regions and the arcuate nucleus (ARC); they express the active leptin receptor (OBR), which regulates the synthesis of different neuropeptides implicated in the control of food intake and energy homeostasis, such as neuropeptide Y (NPY) and proopiomelanocortin (POMC) [4–8] (Fig. 1). Leptin binding to OBR results in the activation of JAK 2, which in turn phosphorylates tyrosine residues in the cytoplasmic domain of the receptor. Phosphorylation and activation of intracellular Tyr1138 result in the recruitment of STAT3 to OBR and its phosphorylation (pSTAT3) and the activation of JAK2 [9,10]. The activation of STAT3 induces its dimerization and translocation into the nucleus where it mediates changes in the expression of several genes, including suppressor of cytokine signalling 3 (SOCS3), an inhibitor of OBR signalling [11], and coordinates the regulation of energy homeostasis and food intake by altering the expression of NPY, AgRP and POMC. In addition, phosphorylation of Tyr985 recruits protein tyrosine phosphatase 2 (SHP2, PTPN1) to OBR, inducing the activation of the ERK signalling pathway and also serving as another binding site for SOCS3. Administration of leptin to obese rodents and humans with congenital leptin deficiency (extremely rare in humans) decreases body weight and food intake [12–14]. Leptin treatment has also been effective in patients with lipodystrophy, characterized by an absence of fat mass and very low secretion of leptin, with excessive food intake which is stored in the liver and muscle as fat, causing high blood lipid levels and type II diabetes. However, in diet-induced obese mice and the most greatly obese humans who are not leptin-deficient, leptin administration is totally inefficient, dispelling the idea of using leptin to treat obesity [15]. Additionally, patients who have a propensity to regain the weight lost after an energy restriction treatment exhibit higher circulating leptin levels than patients who show successful weigh maintenance [16,17]. These observations led to the concept of leptin resistance, which is used to define states of obesity with hyperleptinaemia and/or a decreased response to leptin administration. This represents one of the major challenges in obesity research and in the possibility of using leptin as an antiobesity drug.

Because of its important role in the control of satiety and body weight, the discovery of leptin generated great interest in its potential use to treat obesity. Congenital leptin deficiency due to a homozygous mutation of the leptin gene is extremely rare in humans and is associated with consanguineous effects [18]. This deficiency results in early-onset morbid obesity, mainly due to profound hyperphagia, hyperinsulinaemia and type II diabetes, reproductive dysfunction and advanced bone ageing [19]. In these cases, daily subcutaneous leptin administration decreases appetite, food intake, weight and insulin level to facilitate pubertal maturation [18,20]. However, the hope that leptin can be used as an antiobesity therapy was lost at the end of the 20th century, after leptin failed to induce significant weight loss in overweight and obese persons with leptin resistance [21]. Recently, leptin therapy showed promising results when combined with reduced caloric intake. Although the effects were minimal and it appears that leptin replacement is not sufficient by itself, these results suggest that a multitherapy of leptin in combination with other molecules with known physiological roles in the regulation of body weight may be able to target several mechanisms related to obesity at the same time to reduce compensation to leptin treatment. In this sense, a combination of leptin with leptin-sensitizing molecules is a promising pharmacological approach for weight loss [22]. For example, amylin and leptin, triinfusion of cholecystokinin, leptin and amylin and glucagon-like peptide 1 and leptin therapies result in greater inhibition of food intake and body weight loss than leptin monotherapy [23–27]. Another strategy is the use of leptin-related analogues capable of binding and activating OBR, which avoids some problems associated with natural leptin, such as low stability and short half-life, and the issue of leptin being inactive. Different approaches have produced several generations of conjugated compounds with different linker properties, and different types of leptin sequences or leptin fragments with hydrophilic polymers, such as polyethylene glycol [22]. Given the role of negative regulators of leptin signalling in leptin resistance, such as SOCS3 and the phospho-tyrosine protein phosphatase PTP1B, several authors have also investigated blocking these negative regulators to improve the leptin response in obese persons [22,28,29]. PTP1B inhibitors, which are based on pTyr mimetics, such as thiazolidinedione derivatives, suppress weight gain and improve lipidic parameters in high fat diet mice [30], and the selective allosteric inhibitor of PTP1B, trodusquemine, reduces food intake, fat mass and body weight in diet-induced obese mice [31]. SOCS3 inhibitors do not exist, but blocking the interaction of SOCS3 with OBR increases JAK2 and OBR phosphorylation, increasing the activation of the leptin intracellular signalling pathway. Direct action on OBR could also be used to improve leptin activity. The leptin receptor is mainly found in the cytoplasm rather than at the cell surface. It has high constitutive activity and a low recycling rate [32], resulting in low levels of receptor expression at the cell surface of hypothalamic neurons (5–25%), which is critical for overall leptin binding sensitivity. Several reports suggested that OBR endocytosis and intracellular trafficking could be used to treat obesity without altering or affecting OBR neosynthesis or degradation. The ubiquitin ligase RNF41 controls OBR trafficking, increasing OBR recycling into the plasma membrane [33]. In addition, endospanin 1, a protein encoded by the db gene through an alternative splicing mechanism, interacts directly with OBR to retain a significant level of OBR in intracellular compartments [34]. In vitro and in vivo models of endospanin 1 knock-down, significantly increase leptin-induced STAT3 phosphorylation in the hypothalamus and are efficient in preventing or reversing the development of obesity in DIO mice [34,35]. To reach OBR in the hypothalamus, leptin must cross the BBB through a specific and saturable transporter [36], and in obesity, that transporter is continuously activated and saturated by the high levels of leptin present, leading to leptin resistance. Increased leptin entry

Please cite this article as: A.B. Crujeiras, et al., Leptin resistance in obesity: An epigenetic landscape, Life Sci (2015), http://dx.doi.org/10.1016/j. lfs.2015.05.003

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into the brain constitutes one of the most promising tools in the potential therapeutic use of leptin to treat obesity. However, leptin is a protein and is too large to cross the BBB by transmembrane diffusion in an efficient manner. Therefore, other strategies are necessary, such as improving leptin pharmacokinetics, attaching leptin molecules to another compound with the ability to cross the BBB and modifying leptin to reach the brain by vesicular endocytosis independent of the leptin transporter.

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leptin sensitivity. Low-fructose and high-fat diets induce leptin resistance, but animals fed high-fructose and high-sugar diets are sensitive to exogenous leptin administration, suggesting a beneficial effect of sugars in comparison to fat [55,56]. Conversely, a lack of leucine in the diet improves leptin signalling and the leptin response in animals fed with a standard diet and recovers leptin sensitivity in animals fed a sustained high-fat diet [57]. Other possible explanations for leptin resistance and obesity development may be related to epigenetic mechanisms [58].

3. Molecular mechanisms involved in leptin resistance Leptin resistance is involved in the pathogenesis of diet-induced obesity (DIO) [37], which is the main cause of obesity in humans. High fat diet consumption triggers central and peripheral leptin resistance and, in this sense, hyperleptinaemia due to the expansion of adipose tissue, which is a key player in the development of leptin resistance. The lack of response to leptin affects food intake regulation, nutrient absorption, metabolism and insulin sensitivity, leading to energy balance dysregulation. The molecular mechanisms underlying leptin resistance are not currently clear, but several possibilities have been hypothesized: 1) inability of leptin to cross the BBB, which limits its access to the CNS and prevents it from reaching its neuronal targets [36,38]; and 2) inhibition of the leptin intracellular signalling pathway within specific neurons at the level of desensitization and/or downregulation of the leptin receptor and/or intracellular downstream signalling proteins [39]. Although leptin resistance at both the BBB and specific areas in the brain is very potent in advanced obesity, resistance at the level of the BBB is predominant in the earlier states of obesity [15]. The molecular mechanism underlying leptin resistance at the BBB is due to mediation of the transport of serum leptin from adipose tissue into the brain by a saturable transporter, and as serum leptin levels increase during obesity, the leptin transporter is increasingly saturated [40]. It has been suggested that the leptin transporter is saturated at 50% at a serum leptin level of 10 ng/ml, which is the concentration associated with ideal body weight in rodents [41]. In addition, the transporter is at full capacity at a leptin concentration in the order of 40 ng/ml, blocking the access of leptin into the CNS and resulting in continuous weight gain. In addition, other circulating factors are involved in the regulation of the leptin transporter. Leptin transporter level is increased by epinephrine, glucose, ethanol and insulin and decreased by triglycerides [41–43]. Therefore, resistance at the BBB level is multifactorial, with two of the phenomena that appear during obesity, hypertriglyceridemia and hyperleptinaemia, being important. At the brain level, there is no clear evidence regarding the different areas of the brain and the molecules implicated in the process of leptin resistance. Some studies have shown that the anorectic effects of leptin are not specific to a brain region, suggesting that different regions can work in a coordinated manner and that leptin resistance in one brain region may be compensated by another brain region [44–49]. In this sense, the ARC and ventral segmental area (VTA) appear to be important areas in leptin action. High fat diet activates the suppressor of cytokine signalling 3 (SOCS3) with subsequent inactivation of the leptin-activated STAT 3 intracellular signalling pathway in the ARC, agouti-related protein (AgRP) and pro-opiomelanocortin (POMC) expressing neurons [39,46,50,51]. In agreement with these results, gene silencing of SOCS3 in the hypothalamus protects against obesity in mice fed a high fat diet [52,53], and SOCS3 expression in AgRP neurons is reduced after switching from a high-fat to a low-fat diet [51]. Leptin resistance is a common characteristic of DIO, but obese animals with low plasma leptin levels remain sensitive to exogenous leptin before and after exposure to a high-fat diet, suggesting that fat content is not the only cause of leptin resistance [52]. This effect could be explained by different nutritional aspects, as the type and duration of a diet as well as dietary compounds affect the development of leptin resistance [54]. In this sense, fats and sugars have different effects on

4. Epigenetic regulation of leptin-related signalling pathways: role in leptin resistance Epigenetics is the biological regulatory system through which organisms respond to environmental pressures [59]. Epigenetic modifications refer to mitotically and/or meiotically heritable changes in gene expression that occur without altering DNA sequences [60,61]. Such mechanisms play important roles in many biological processes that occur over a person's lifetime [59]. The epigenetic machinery involves several levels of regulation, including DNA methylation, post-translational histone modifications, nucleosome positioning, and non-coding RNAS [62–64]. Among these mechanisms, DNA methylation occurs in certain areas of the genome with high concentrations of CpG dinucleotides (“CpG islands”). In addition, this epigenetic modification also occurs in areas of low CpG density (b10 CpG/100 bp) named “CpG deserts”. Although the previous dogma is that epigenetic modifications in CpG islands or shores with the highest CpG density are critical, these “CpG deserts” may be especially important for gene regulation, as reflected by the maintenance of small CpG clusters in these deserts, despite the high mutation rates observed in CpG sites [65,66]. As all levels of epigenetic regulation appear to have wide-ranging effects on development and health and are reversible, epigenetic marks might explain the link between lifestyle and risk for disease. They have been proposed to be sensitive biomarkers of disease and potential therapeutic targets for disease management [67]. In this context, recent studies have revealed a key component of the epigenetic network in the control of adipogenesis, food intake and energy homeostasis, indicating that the role of epigenetic modifications in human nutrition and obesity is a relevant area of study to determine molecular mechanisms underlying metabolic disorders [68]. Indeed, since it is known that environmental factors like nutrition accelerate the onset of metabolic disease by altering the epigenome, several studies investigated if lifestyle interventions such as exercise would conversely improve metabolic health by re-tuning the epigenome as described in a recent review [69]. DNA methylation is the most well-known epigenetic modification that regulates gene expression and it causes the silencing of both coding and non-coding genes. The strong evidence that complex diseases, such as metabolic disorders, are under the influence of epigenetic modifications even in early life is opening up exciting new avenues for the identification of DNA methylation biomarkers associated with these disorders and for estimation of future disease risk [70]. Many tissue types are suitable for the identification of DNA methylation biomarkers including cell-based samples such as blood cells and tissue material and cell-free DNA samples such as plasma [70]. As blood is easily accessible and is routinely sampled in clinical and large-scale studies, peripheral blood cells are the most frequently used source of DNA for epigenetic studies. However, epigenetic changes may be more tissue specific and the blood cell methylation profile may not necessarily report the epigenetic state in other tissues [71]. Nowadays a huge effort is being carried out to get a better insight in tissue-specific epigenetic signatures and their role in disease development. Importantly, for diagnostic purposes in a clinical setting, such as young children or to study inaccessible tissues such as brain tissue, epigenetic marks should be detectable in easily accessible samples, such as peripheral blood. Regarding leptin function and biology, it was established that methylation of a proximal region of LEP promoter constitutes a significant

Please cite this article as: A.B. Crujeiras, et al., Leptin resistance in obesity: An epigenetic landscape, Life Sci (2015), http://dx.doi.org/10.1016/j. lfs.2015.05.003

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determinant of leptin expression in human adult tissues since that DNA methylation has a role in long-term gene silencing and that demethylation is able to increase and reactive leptin expression [72]. Moreover, several studies have demonstrated that methylation of a CpG island in the leptin promoter plays an important role in leptin expression during pre-adipocyte differentiation [73,74] and may be involved in obesityrelated leptin upregulation [58,75,76]. A recent study showed that DNA methylation of the leptin promoter varied with obesity. In the adipose tissue of diet induced obese (DIO) mice, the methylated fraction of the leptin promoter was decreased by feeding a high-fat diet at the initial stage; however, it increased after 12 weeks of feeding a high-fat diet compared to control and low-fat diet mice [77]. In peripheral blood samples from human obese adolescents, methylation of the LEP promoter was shown to be negatively associated with BMI [78]. Moreover, obese patients who responded to a low-calorie-diet treatment with a weight loss higher than 5% exhibited lower leptin methylation levels in subcutaneous adipose tissue than those who lost less than 5% of their initial body weight [79]. These data suggest that leptin methylation level could be used as an epigenetic biomarker for the response of obese patients to a low-calorie diet [79]. Additionally, recent studies show that leptin DNA methylation is a plausible factor to be involved in foetal metabolic programming. Maternal glycaemia is part of causal pathways influencing offspring leptin epigenetic regulation [80]. Moreover, it was demonstrated that LEP promoter DNA methylation is influenced by perinatal factors including: maternal obesity, infant growth, genotype and gender in a tissuespecific manner [81]. Thus, leptin epigenetic control may be influenced by perinatal factors. This metabolic foetal programming could contribute to the rising obesity epidemic and may have multigenerational implications. Importantly, regarding leptin regulatory machinery, previous research on adult male rodent hypothalamus has shown that hypermethylation of the promoter region of the pro-opiomelanocortin (POMC) gene interferes with the binding of transcription factors and blocks the effect of high leptin levels found in obesity [82,83]. A similar effect has been demonstrated in nulliparous female rats fed a high fat diet for a long period; despite significantly higher body weight and plasma leptin levels in these rats, they had relatively low POMC mRNA expression, which was associated with hypermethylation of the POMC promoter [84]. POMC neurons in the arcuate nucleus (ARC) regulate energy homeostasis by secreting α-melanocyte-stimulating hormone (α-MSH), which is derived from a POMC precursor, in response to leptin signalling. Therefore, this epigenetic regulation in POMC neurons may be a mechanism of leptin resistance. In fact, mice lacking methyl-CpGbinding protein 2 (MeCP2, a nuclear protein essential for neuronal function) in POMC neurons showed increased POMC promoter DNA methylation, which, in turn, downregulated POMC expression. This regulation of the POMC promoter in POMC neurons led to obesity and an increase in leptin resistance in these mice [85]. In line with these results, neonatal overfeeding, which leads to rapid early weight gain and results in a metabolic syndrome phenotype (i.e., obesity, hyperleptinaemia, hyperglycaemia, hyperinsulinaemia) and an increased insulin/glucose ratio, is accompanied by low methylation levels (i.e., b 5%) in the promoter of the main orexigenic neurohormone, neuropeptide Y (NPY). In contrast, in overfed rats, the hypothalamic gene promoter of the main anorexigenic neurohormone, POMC, showed hypermethylation of CpG dinucleotides within the two Sp1-related binding sequences (Sp1 and NF-kappaB), which are essential for the mediation of leptin and insulin effects on POMC expression. Thus, in overfed rats POMC expression was not upregulated despite hyperleptinaemia and hyperinsulinaemia [86]. Similar results were recently reported in newborn rats reared on a high-carbohydrate (HC) milk formula; they developed hyperinsulinaemia in the post-weaning period and adult-onset obesity compared with mother-fed rats, suggesting that epigenetic modifications contribute to the altered expression of the NPY and POMC genes in the hypothalami of overfed individuals. This could be a

mechanism that leads to hyperphagia and the development of obesity in adulthood [87]. Additionally, the obesogenic phenotype induced in the offspring of dams supplemented with a high multivitamin gestational diet and methyl vitamins was associated with altered hypothalamic gene expression, causing increased food intake concurrent with DNA methylation and leptin and insulin receptor signalling dysfunction [88]. Similarly, it has been demonstrated that obese men who regained the weight they had lost after an energy restriction treatment exhibited higher methylation levels of POMC and lower methylation levels of the NPY promoter region in blood leukocytes before the nutritional intervention than obese men that did not regain the dietary-induced weight loss (Fig. 2) [89]. Therefore, the methylation levels of NPY observed in patients who maintained weight loss could indicate a poorer efficiency in promoting appetite in these patients. This fact, together with the decreased methylation of POMC, suggests that successful weight maintenance after a dietary weight loss programme is mediated by epigenetic regulation of the appetite-regulatory neuropeptides that confers a protective mechanism against weight regain [89]. This epigenetic regulation of the leptin signalling pathways could be modified or reverted by nutrients and food compounds. In fact, numerous studies have demonstrated the effects of alcohol, B vitamins, proteins, micronutrients, functional food components and general nutritional status on DNA methylation, as described in a recent review [90]. For example, high vitamin A in post-weaning diets was reported to reduce post-weaning weight gain and food intake and to modify gene expression pathways related to food intake and reward, and this was associated with high DNA methylation of the POMC gene in the hypothalamus of male rats born to dams fed a high multivitamin diet [91]. Additionally, leptin treatment during the suckling period was shown to promote epigenetic modifications in the POMC promoter, which confers protection from an obesogenic environment in adulthood [92].

5. Conclusion Overall, the current review shows that leptin resistance is a relevant factor in the pathogenesis of obesity and that, among the molecular mechanisms involved, epigenetic modifications could contribute to leptin expression and signalling disturbances in obesity. Considering that epigenetic marks are reversible and modifiable by the environment and could be involved in leptin resistance associated with obesity, the epigenetic marks in the leptin gene and genes in its signalling pathways could constitute a therapeutic target for the design of leptin sensitizers to counteract the leptin resistance commonly observed in obesity. Nutrients and food compounds that have been shown to slightly modify the epigenetic patterns of different cell lines and tissues [93] could help to induce leptin sensitivity and to improve the obesogenic phenotype. Therefore, understanding the influence of the obesity-related microenvironment on the epigenetic regulation of appetite and the energy homeostasis machinery will provide new tools to improve the management and the prevention of metabolic disorders. Further studies are needed to elucidate if blood cell samples are suitable to reflect the epigenetic regulation of leptin and other genes related to leptin regulatory pathways in human studies.

Authors’ contributions A.B.C. and M.C.C. designed the review, interpreted the data, supervised the writing of the manuscript and wrote the final version of the manuscript. B.C.F., S.A. and M.A. performed the literature search, extracted the data and drafted the article. F.F.C. designed the review, interpreted the data and critically reviewed the complete manuscript. All authors provided final approval of the manuscript.

Please cite this article as: A.B. Crujeiras, et al., Leptin resistance in obesity: An epigenetic landscape, Life Sci (2015), http://dx.doi.org/10.1016/j. lfs.2015.05.003

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Fig. 2. Epigenetic regulation of NPY and POMC in weight regain. A) Pre-treatment DNA methylation status (%) of 28 CpG sites of NPY in regainers and non-regainers of diet-induced weight loss. The examined CpG sites of the NPY promoter region are within nucleotides −211 bp to −646 bp in respect to the transcription start site (+1 bp). A circle on a pole indicates that the site was quantified. Broken lines on circles and underscore numbers indicate CpGs whose methylation levels could not be measured independently by EpiTyper. Shaded boxes indicate CpGs whose methylation levels were significantly different between regainers and non-regainers and correlated with other measurements (*p b 0.05; Mann–Whitney U test). B) Associations between pre-treatment DNA methylation levels and diet-induced weight loss, weight regain and the appetite-related hormones ghrelin, leptin and insulin. C) Comparison of the pre-treatment total DNA methylation levels (%) of NPY between the non-regainer and the regainer groups. The methylation levels are presented as the means (SE). D–F) Correlation analyses of pre-treatment NPY total methylation with body weight regain 6 months after finishing the dietary intervention and with circulating ghrelin levels and leptin/ghrelin ratios at the beginning of the restriction diet. G) Comparison of the pre-treatment DNA methylation levels (%) of POMC CpG 10_11 (CpG sites +136 bp and +138 bp) between non-regainers and regainers. The methylation levels are presented as the means (SE). H) Correlation analysis between pre-treatment DNA methylation levels (%) of POMC CpG 10_11 and body weight regain 6 months after finishing the dietary intervention. The POMC promoter region examined, including CpG sites within nucleotides +15 to +274 bp with respect to the transcription start site.

Conflict of interest statement The authors have no potential conflicts of interest in regard to the publication of this manuscript.

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Acknowledgements The author's laboratory work was supported by Instituto de Salud Carlos III (ISCIII), Spain with CIBERobn (CB06/003), the PI10/02464 (INTRASALUD programme) and PI14/01012 research projects, and the Xunta de Galicia, (GRC2014/034) Spain. B. Cabia was funded by a Santiago de Compostela University (USC)-Campus Vida predoctoral contract (ref. 011-020). References [1] Y. Zhang, R. Proenca, M. Maffei, M. Barone, L. Leopold, J.M. Friedman, Positional cloning of the mouse obese gene and its human homologue, Nature 372 (1994) 425–432. [2] S. Lehr, S. Hartwig, H. Sell, Adipokines: a treasure trove for the discovery of biomarkers for metabolic disorders, Proteomics Clin. Appl. 6 (2011) 91–101. [3] M.B. Allison, M.G. Myers Jr., 20 years of leptin: connecting leptin signaling to biological function, J. Endocrinol. 223 (2014) T25–T35. [4] P.L. Golden, T.J. Maccagnan, W.M. Pardridge, Human blood–brain barrier leptin receptor. Binding and endocytosis in isolated human brain microvessels, J. Clin. Invest. 99 (1997) 14–18. [5] J. Korner, S.C. Chua Jr., J.A. Williams, R.L. Leibel, S.L. Wardlaw, Regulation of hypothalamic proopiomelanocortin by leptin in lean and obese rats, Neuroendocrinology 70 (1999) 377–383. [6] S.J. Lee, S. Verma, S.E. Simonds, M.A. Kirigiti, P. Kievit, S.R. Lindsley, A. Loche, M.S. Smith, M.A. Cowley, K.L. Grove, Leptin stimulates neuropeptide Y and cocaine amphetamine-regulated transcript coexpressing neuronal activity in the

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