Search

Mice lacking the functional cystinosin gene ( Ctns -/- ), a model of infantile nephropathic cystinosis (INC), exhibit the cachexia phenotype with adipose tissue browning and muscle wasting. Elevated leptin signaling is an important cause of chronic kidney disease-associated cachexia. The pegylated leptin receptor antagonist (PLA) binds to but does not activate the leptin receptor. We tested the efficacy of this PLA in Ctns -/- mice. We treated 12-month-old Ctns -/- mice and control mice with PLA (7 mg/kg/day, IP) or saline as a vehicle for 28 days. PLA normalized food intake and weight gain, increased fat and lean mass, decreased metabolic rate and improved muscle function. It also attenuated perturbations of energy homeostasis in adipose tissue and muscle in Ctns -/- mice. PLA attenuated adipose tissue browning in Ctns -/- mice. PLA increased gastrocnemius weight and fiber size as well as attenuated muscle fat infiltration in Ctns -/- mice. This was accompanied by correcting the increased expression of muscle wasting signaling while promoting the decreased expression of myogenesis in gastrocnemius of Ctns -/- mice. PLA attenuated aberrant expressed muscle genes that have been associated with muscle atrophy, increased energy expenditure and lipolysis in Ctns -/- mice. Leptin antagonism may represent a viable therapeutic strategy for adipose tissue browning and muscle wasting in INC.

Leptin, a multifunctional hormone primarily, but not exclusively, secreted in adipose tissue, is implicated in a wide range of biological functions that control different processes, such as the regulation of body weight and energy expenditure, reproductive function, immune response, and bone metabolism. In addition, leptin can exert angiogenic and mitogenic actions in peripheral organs. Leptin biological activities are greatly related to its interaction with the leptin receptor. Both leptin excess and leptin deficiency, as well as leptin resistance, are correlated with different human pathologies, such as autoimmune diseases and cancers, making leptin and leptin receptor important drug targets. The development of leptin signaling modulators represents a promising strategy for the treatment of cancers and other leptin-related diseases. In the present manuscript, we provide an update review about leptin-activity modulators, comprising leptin mutants, peptide-based leptin modulators, as well as leptin and leptin receptor specific monoclonal antibodies and nanobodies.

Objective: Metabolic syndrome is characterized by abdominal obesity, hypertriglyceridemia and hyperglycemia. Fatty acid binding protein 4 (FABP4), as a member of intracellular lipid chaperones, is not only engaged in lipid transport but involved in inflammation and insulin resistance. The present study was to investigate the effects of BMS309403, a specific FABP4 inhibitor, on metabolic syndrome and its possible molecular mechanisms in islets., Materials and Methods: Leptin receptor knockout (Lepr-/-) rat, a novel and representative animal model of metabolic syndrome, was adopted in this study. Lepr-/- male rats and their wild littermates were grouped and intragastrically administered with BMS309403. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were performed on all rats. Serum insulin was detected by ELISA. The metabolic characters, as well as liver and kidney functions, were evaluated by serum biochemical assay. Immunohistochemistry and Western blot were adopted to detect the expression levels of FABP4, CD68, GRP78, ATF6, p-IRE1a, and Cleaved caspase-3., Results: Lepr-/- rats showed prominent characteristics of metabolic syndrome with increased FABP4, inflammatory infiltration, ER stress and apoptosis in islets. BMS309403 administration attenuated inflammation, ER stress and apoptosis in Lepr-/- rat islets while stimulating insulin secretion as well as improving manifestation of metabolic syndrome without hepatic and renal toxicity., Conclusions: FABP4 increased in Lepr-/- rat islets and might be involved in the regulation of islet inflammation and apoptosis via ER stress. FABP4 inhibitor BMS309403 could ameliorate islet inflammation and apoptosis in metabolic syndrome through suppressing ER stress.

The purpose of this study was to explore the potential contracting effect of leptin on isolated guinea pig tracheal smooth muscle (TSM), the possible mechanism, and the impact of epithelium denudation or allergen sensitization, respectively. An in vitro experiment investigated the effect of leptin at a concentration of 250-1000 nmol/L on isolated guinea pig TSM with an intact or denuded epithelium. Ovalbumin and IgE were used to test the impact of active and passive sensitization. The isolated TSM strips were incubated in Krebs solution and aerated with carbogen (95% O 2 and 5% CO 2 ) via an automated tissue organ bath system ( n = 4 for each group). Isometric contractions were recorded digitally using iox2 data acquisition software. The possible mechanism of leptin-induced TSM contraction was examined by preincubation with leptin receptor (Ob-R) antagonist. Leptin had significant concentration-dependent contraction effects on guinea pig TSM ( p < 0.05). Epithelium denuding and active or passive sensitization significantly increased the potency of the leptin. Preincubation with a leptin receptor (Ob-R) antagonist significantly reduced the contraction effects, suggesting an Ob-R-mediated mechanism. Leptin had a contracting effect on airway smooth muscles potentiated by either epithelium denuding or sensitization, and the Ob-R mechanism was a possible effect mediator.

Leptin links body energy stores to high energy demanding processes like reproduction and immunity. Based on leptin's role in autoimmune diseases and cancer, several leptin and leptin receptor (LR) antagonists have been developed, but these intrinsically lead to unwanted weight gain. Here, we report on the uncoupling of leptin's metabolic and immune functions based on the cross talk with the epidermal growth factor receptor (EGFR). We show that both receptors spontaneously interact and, remarkably, that this complex can partially overrule the lack of LR activation by a leptin antagonistic mutein. Moreover, this leptin mutant induces EGFR phosphorylation comparable to wild-type leptin. Exploiting this non-canonical leptin signalling pathway, we identified a camelid single-domain antibody that selectively inhibits this LR-EGFR cross talk without interfering with homotypic LR signalling. Administration in vivo showed that this single-domain antibody did not interfere with leptin's metabolic functions, but could reverse the leptin-driven protection against starvation-induced thymic and splenic atrophy. These findings offer new opportunities for the design and clinical application of selective leptin and LR antagonists that avoid unwanted metabolic side effects.

Background/aim: A common finding in cancer cells is the overexpression of histone deacetylases (HDACs), leading to altered expression and activity of numerous proteins involved in carcinogenesis. Considering that leptin can modulate the levels of HDACs, we hypothesised that leptin receptor antagonists can alter HDAC expression., Materials and Methods: HDAC expression in cells exposed to leptin and leptin receptor antagonists (SHLA and Lan2) were evaluated in ovarian epithelial (OVCAR-3, CaOV3) and folliculoma (COV434, KGN) cells., Results: Higher HDAC expression was found in epithelial compared to folliculoma cells. Leptin increased class I and II HDACs only in OVCAR-3 cells, and SHLA was more potent then Lan-2. In folliculoma cells, leptin only increased class II HDAC expression, Lan-2 was more potent than SHLA in the COV434 and neither antagonist affected the KGN cells., Conclusion: SHLA and Lan2 eliminate the negative effects of leptin on HDAC expression in a cell-type-dependent manner. This is the first report testing leptin receptor blockers as HDAC inhibitors in ovarian cancer cells., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Widespread expression of leptin and its receptor in developing cartilage and bone suggests that leptin may regulate bone growth and development in the fetus. Using microcomputed tomography, this study investigated the effects of exogenous leptin and leptin receptor antagonism on aspects of bone structure in the sheep fetus during late gestation. From 125 to 130 days of gestation (term ~145 days), chronically catheterized singleton sheep fetuses were infused intravenously for 5 days with either saline (0.9% saline, n = 13), recombinant ovine leptin at two doses (0.6 mg·kg -1 ·day -1 LEP1, n = 10 or 1.4 mg·kg -1 ·day -1 LEP2, n = 7), or recombinant superactive ovine leptin receptor antagonist (4.6 mg·kg -1 ·day -1 SOLA, n = 6). No significant differences in plasma insulin-like growth factor-I, osteocalcin, calcium, inorganic phosphate, or alkaline phosphatase were observed between treatment groups. Total femur midshaft diameter and metatarsal lumen diameter were narrower in male fetuses treated with exogenous leptin. In a fixed length of femur midshaft, total and bone volumes were reduced by the higher dose of leptin; nonbone space volume was lower in both groups of leptin-treated fetuses. Leptin infusion caused increments in femur porosity and connectivity density, and vertebral trabecular thickness. Leptin receptor antagonism decreased trabecular spacing and increased trabecular number, degree of anisotrophy, and connectivity density in the lumbar vertebrae. The increase in vertebral porosity observed following leptin receptor antagonism was greater in the malecompared with female, fetuses. Therefore, leptin may have a role in the growth and development of the fetal skeleton, dependent on the concentration of leptin, sex of the fetus, and bone type examined.

Excess adiposity in obese inhibits negatively impacts immune function and host defence. Obesity is characterized by a state of low-grade, chronic inflammation in addition to disturbed levels of circulating nutrients and metabolic hormones. The impact of metabolic abnormalities on obesity-related co-morbidities has undergone intense scrutiny over the past decades. Thus, treatment of obesity and its associated immune-mediated diseases is challenging due to impaired function of leptin system. These disorders are managed through antibiotics and by cytokines replacement. However, the effectiveness of cytokines coupled to the complexity of the cytokine network leads to severe side-effects, which can still occur after careful preclinical evaluation. In addition, synthetic immunotherapeutics carry a degree of risk, time-consuming and expensive. Hence, the complexity of existing therapy and adverse effects emphasizes the need for an alternative approach for the management of immune dysfunction associated with obesity. Computer-aided small molecule antibody technology has been successful in the design of novel biologicals for the diagnosis of diseases and therapeutic interventions. In this study, the crystal structure of leptin receptor (LEPR) complex with monoclonal antibody (9F8 Fab) was explored to predict Ag-Ab interactions using bioinformatics tools. The LEPR of complementarity-determining region (CDR) loops were mutated with published positive control residues of Ser, Thr, Tyr, Trp, and Phe to design a set of 678 peptides which were evaluated through Ag-peptide docking, binding free-energies, and interaction energies. Thus, hypothesized novel peptides can be explored as clinically applicable antagonists for the treatment of obesity and associated immune-mediated diseases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Leptin antagonists (L39A/D40A/F4lA mutants) of mouse, human, rat and ovine leptins were developed in our laboratory by rational mutagenesis, expressed in Escherichia coli, refolded and purified to homogeneity. Pegylation of these antagonists resulted in long-acting reagents suitable for in-vivo studies. Further selection of high-affinity leptin antagonists was achieved by random mutagenesis of the whole open reading frame followed by yeast- surface display; an additional mutation (D23L) increased their affinity toward leptin receptor 60-fold. This superactive pegylated mouse leptin antagonist (PLA) exhibited a strong orexigenic effect, leading, in 10-14 days, to a 40% increase in body weight resulting mainly from obesity; this was reversed once PLA treatment was ceased. Cachexia is common in patients with Chronic Kidney Disease (CKD). Our studies suggested that leptin mediates cachexia by decreasing food intake while increasing energy consumption in CKD mice. We showed that PLA ameliorates CKD-associated cachexia in mice. Leptin may also contribute to the development of muscle and renal fibrosis in CKD, serious complications associated with increased morbidity and mortality. Transforming growth factor (TGF)-β signaling may be the most potent mediator of fibrogenesis in multiple organs, and leptin is a co-activator of TGF-β. Muscle fibrosis was evident in our CKD mice and PLA treatment significantly reduced the mRNA levels of TGF- β1 and its downstream targets in their muscle and renal tissues. PLA may offer a novel therapeutic strategy for CKD-associated cachexia, muscle and renal fibrosis to improve CKD patients' survival and quality of life., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Objective: We asked if leptin and its cognate receptor were present in normal and diseased parathyroid glands, and if so, whether they had any functional effects on parathyroid hormone (PTH) secretion in parathyroid neoplasms., Background: The parathyroid glands acting through PTH play a critical role in the regulation of serum calcium. Based on leptin's recently discovered role in bone metabolism, we hypothesized these glands were the sites of a functional interaction between these 2 hormones., Methods: From July 2010 to July 2011, 96 patients were enrolled in a prospective study of leptin and hyperparathyroidism, all of whom were enrolled based on their diagnosis of hyperparathyroidism, and their candidacy for surgical intervention provided informed consent. Immediately after parathyroidectomy, 100 to 300 mg of adenomatous or hyperplastic diseased parathyroid tissue was prepared and processed according to requirements of the following: in situ hybridization, immunohistochemistry, immunofluorescence by conventional and spinning disc confocal microscopy, electron microscopy, parathyroid culture, whole organ explant, and animal model assays., Results: Leptin, leptin receptor (long isoform), and PTH mRNA transcripts and protein were detected in an overlapping fashion in parathyroid chief cells in adenoma and hyperplastic glands, and also in normal parathyroid by in situ hybridization, qRT-PCR, and immunohistochemistry. Confocal microscopy confirmed active exogenous leptin uptake in cultured parathyroid cells. PTH secretion in explants increased in response to leptin and decreased with leptin receptor signaling inhibition by AG490, a JAK2/STAT3 inhibitor. Ob/ob mice injected with mouse leptin exhibited increased PTH levels from baseline., Conclusions: Taken together, these data suggest that leptin is a functionally active product of the parathyroid glands and stimulates PTH release.

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Here, we report the role of the adipokine leptin as master regulator of anti-myeloma tumor immunity by modulating the invariant natural killer T (iNKT) cell function. A marked increase in serum leptin levels and leptin receptor (LR) expression on iNKT cells in MM patients and the 5T33 murine MM model was observed. MM cells and leptin synergistically counteracted anti-tumor functionality of both murine and human iNKT cells. In vivo blockade of LR signaling combined with iNKT stimulation resulted in superior anti-tumor protection. This was linked to persistent IFN-γ secretion upon repeated iNKT cell stimulation and a restoration of the dynamic antigen-induced motility arrest as observed by intravital microscopy, thereby showing alleviation of iNKT cell anergy. Overall our data reveal the LR axis as novel therapeutic target for checkpoint inhibition to treat MM.

Purpose: There are no data showing a direct correlation between obesity and increased blood leptin levels with folliculoma. Moreover, folliculoma is not the best studied among other ovarian cancer types. We investigated whether oestradiol can modulate ObR expression in some oestrogen-responsive tissues and that leptin exerts its activity not only via the leptin receptor but also through cross talk with other signalling systems. We hypothesise that blocking ObR expression could be a novel treatment for gonadal ovarian cancer., Methods: We evaluated the effect of SHLA, Lan1 and Lan2 blockers on cell proliferation (BrdU incorporation assay), ObR and ERα/β gene expression (qPCR), oestradiol secretion (ELISA) and cell cycle protein expression (Western blot) in the non-cancerous cell line HGrC1 and two granulosa cancer cell lines: the juvenile form (COV434) and the adult form (KGN)., Results: ObR gene expression in cancer cell lines was 50% higher than in the non-cancer cells. Lan-1 and Lan-2 decreased ObR expression in COV434, while it had no effect in KGN cells. Higher ERβ expression in non-cancer and higher ERα expression in both cancer cell lines was noted. SHLA and Lan-1 changed the ratio towards greater expression of ERβ, characteristic of non-cancer granulosa cells. All ObR antagonists in HCrC1 and KGN but only Lan-2 in COV434 reversed leptin-stimulated proliferation. In both non-cancer and cancer granulosa cells, leptin acts as a cyclinD/cdk4, cyclin A/cdk2 and E2F inhibitor., Conclusion: These results indicate that SHLA and Lan2 are promising leptin receptor inhibitors that can eliminate the negative effects of leptin. These compounds should be considered in further ex vivo studies on the cancer microenvironment.

Introduction: A number of leptin receptor antagonists have been synthesised for therapeutic use, with pre-clinical tests suggesting their future use in anticancer therapy. To our knowledge, there are no data concerning the possible application of leptin receptor blockers in ovarian cancer., Methods: In this study, we evaluated two leptin receptor antagonists: superactive human leptin antagonist (SHLA) and quadruple leptin mutein, Lan-2 (L39A/D40A/F41A/I42A), on cell proliferation (Alamar Blue test, BrdU assay), cell cycle gene (qPCR) and protein expression (Western blot) and cell signalling pathways (Western blot) in three different types of cell lines: OVCAR-3, CaOV-3 and HOSEpiC., Results: Both receptor blockers had no effect on non-cancerous HOSEpiC cell line proliferation; however, both reversed the stimulatory effect of leptin on CaOV-3 cell line proliferation to control levels and to below control levels in OVCAR-3 cells. In metastatic carcinoma CaOV-3, both ObR antagonists had an inhibitory effect on the cdk2/cyclin D1 complex, while in serous carcinoma, OVCAR-3, they only had an effect on cdk2 and cdk4 protein expression. SHLA had an inhibitory effect on all investigated signalling pathways in OVCAR-3, while only on Stat3 in CaOV-3. Lan-2 had an inhibitory effect on Stat3 and ERK1/2 in CaOV-3, while in OVCAR-3 it only affected Akt protein phosphorylation., Conclusion: Based on these results, we conclude that SHLA and Lan-2 are promising leptin receptor inhibitors which could be used to block leptin activity, eliminating its negative effects on activities related to carcinogenesis. However, the selection of a specific antagonist should be related to tumour type., Competing Interests: The authors declare no conflicts of interest.

High fat diet (HFD)-induced hypertension in rabbits is neurogenic and caused by the central action of leptin, which is thought to be dependent on activation of α-melanocortin-stimulating hormone (α-MSH) and neuropeptide Y-positive neurons projecting to the dorsomedial hypothalamus (DMH) and ventromedial hypothalamus (VMH). However, leptin may act directly in these nuclei. Here, we assessed the contribution of leptin, α-MSH, and neuropeptide Y signaling in the DMH and VMH to diet-induced hypertension. Male New Zealand white rabbits were instrumented with a cannula for drug injections into the DMH or VMH and a renal sympathetic nerve activity (RSNA) electrode. After 3 weeks of an HFD (13.3% fat; n=19), rabbits exhibited higher RSNA, mean arterial pressure (MAP), and heart rate compared with control diet-fed animals (4.2% fat; n=15). Intra-VMH injections of a leptin receptor antagonist or SHU9119, a melanocortin 3/4 receptor antagonist, decreased MAP, heart rate, and RSNA compared with vehicle in HFD rabbits (P<0.05) but not in control diet-fed animals. By contrast, α-MSH or neuropeptide Y injected into the VMH had no effect on MAP but produced sympathoexcitation in HFD rabbits (P<0.05) but not in control diet-fed rabbits. The effects of the leptin antagonist, α-MSH, or neuropeptide Y injections into the DMH on MAP or RSNA of HFD rabbits were not different from those after vehicle injection. α-MSH into the DMH of control diet-fed animals did increase MAP, heart rate, and RSNA. We conclude that the VMH is the likely origin of leptin-mediated sympathoexcitation and α-MSH hypersensitivity that contribute to obesity-related hypertension., (© 2016 American Heart Association, Inc.)

After its discovery in 1994, it soon became clear that leptin acts as an adipocyte-derived hormone with a central role in the control of body weight and energy homeostasis. However, a growing body of evidence has revealed that leptin is a pleiotropic cytokine with activities on many peripheral cell types. Inappropriate leptin signaling can promote autoimmunity, certain cardiovascular diseases, elevated blood pressure and cancer, which makes leptin and the leptin receptor interesting targets for antagonism. Profound insights in the leptin receptor (LR) activation mechanisms are a prerequisite for the rational design of these antagonists. In this review, we focus on the molecular mechanisms underlying leptin receptor activation and signaling. We also discuss the current strategies to interfere with leptin signaling and their therapeutic potential., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ruminants remain productive during the energy insufficiency of late pregnancy or early lactation by evoking metabolic adaptations sparing available energy and nutrients (e.g. higher metabolic efficiency and induction of insulin resistance). A deficit in central leptin signaling triggers these adaptations in rodents but whether it does in ruminants remains unclear. To address this issue, five mature ewes were implanted with intracerebroventricular (ICV) cannula in the third ventricle. They were used in two experiments with an ovine leptin antagonist (OLA) when well-conditioned (average body condition score of 3.7 on a 5 point scale). The first experiment tested the ability of OLA to antagonize leptin under in vivo conditions. Ewes received continuous ICV infusion of artificial cerebrospinal fluid (aCSF), ovine leptin (4 µg/h) or the combination of ovine leptin (4 µg/h) and its mutant version OLA (40 µg/h) for 48 h. Dry matter intake (DMI) was measured every day and blood samples were collected on the last day of infusion. ICV infusion of leptin reduced DMI by 24% (P < 0.05), and this effect was completely abolished by OLA co-infusion. A second experiment tested whether a reduction in endogenous leptin signaling in the brain triggers metabolic adaptations. This involved continuous ICV infusions of aCSF or OLA alone (40 µg/h) for 4 consecutive days. The infusion of OLA did not alter voluntary DMI over the treatment period or on any individual day. OLA did not affect plasma variables indicative of insulin action (glucose, non-esterified fatty acids, insulin and the disposition of plasma glucose during an insulin tolerance test) or plasma cortisol, but tended to reduce plasma triiodothyronine and thyroxine (P < 0.07). Overall, these data show that a reduction of central leptin signaling has little impact on insulin action in well-conditioned mature sheep. They also raise the possibility that reduced central leptin signaling plays a role in controlling thyroid hormone production.

There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation., (Copyright © 2015 the American Physiological Society.)

Muscle- and liver-derived IGF-1 play important roles in muscle anabolism throughout growth and aging. Yet, prolonged food restriction is thought to increase longevity in part by lowering levels of IGF-1, which in turn reduces the risk for developing various cancers. The dietary factors that modulate IGF-1 levels are, however, poorly understood. We tested the hypothesis that the adipokine leptin, which is elevated with food intake and suppressed during fasting, is a key mediator of IGF-1 levels with aging and food restriction. First, leptin levels in peripheral tissues were measured in young mice fed ad libitum, aged mice fed ad libitum, and aged calorie-restricted (CR) mice. A group of aged CR mice were also treated with recombinant leptin for 10 days. Later, aged mice fed ad libitum were treated with saline (VEH) or with a novel leptin receptor antagonist peptide (Allo-aca) and tissue-specific levels of IGF-1 were determined. On one hand, recombinant leptin induced a three-fold increase in liver-derived IGF-1 and a two-fold increase in muscle-derived IGF-1 in aged, CR mice. Leptin also significantly increased serum growth hormone levels in the aged, CR mice. On the other, the leptin receptor antagonist Allo-aca did not alter body weight or muscle mass in treated mice compared to VEH mice. Allo-aca did, however, produce a significant (20%) decline in liver-derived IGF-1 as well as an even more pronounced (>50%) decrease in muscle-derived IGF-1 compared to VEH-treated mice. The reduced IGF-1 levels in Allo-aca treated mice were not accompanied by any significant change in growth hormone levels compared to VEH mice. These findings suggest that leptin receptor antagonists may represent novel therapeutic agents for attenuating IGF-1 signaling associated with aging, and could potentially mimic some of the positive effects of calorie restriction on longevity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Despite evidence that leptin may play a role in the pathogenesis of endometriosis, the specific function of leptin in the migration and invasion of endometriotic cells is not well characterized. In this study, we investigated the effect of leptin on the migration, invasion and matrix metalloproteinase (MMP) expression levels of human endometriotic cells. We found that leptin stimulated the migration and invasion of endometriotic cells (11Z, 12Z and 22B) in a dose-dependent manner. Leptin receptor (ObR) siRNA significantly inhibited the migration and invasion induced by leptin in 11Z and 12Z cells. Leptin-induced migration and invasion were significantly attenuated by pretreatment with SB-3CT, a specific gelatinase (MMP-2 and MMP-9) inhibitor. In addition, leptin-induced increases in the mRNA and protein expression and enzyme activity of MMP-2 in 11Z and 12Z cells. Selectively inhibiting MMP-2 using siRNA and an inhibitor (GM6003), impaired the ability of leptin to stimulate the migration and invasion of endometriotic cells, suggesting that MMP-2 plays an essential role in leptin-induced migration and invasion. Janus Kinase 2/Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) inhibitor (AG490) significantly inhibited the migration, invasion and MMP-2 expression induced by leptin in endometriotic cells. Furthermore, the Extracellular signal-Regulated Kinase inhibitor PD98059 neutralized the migration and invasion promoting effects of leptin. Taken together, these results suggest that leptin may contribute to the migration and invasion abilities of endometriotic cells via the up-regulation of MMP-2 through an ObR-dependent JAK2/STAT3 signaling pathway., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Obesity is characterized by hyperleptinemia and decreased response to exogenous leptin. This has been widely attributed to the development of leptin resistance, a state of impaired leptin signaling proposed to contribute to the development and persistence of obesity. To directly determine endogenous leptin activity in obesity, we treated lean and obese mice with a leptin receptor antagonist. The antagonist increased feeding and body weight (BW) in lean mice, but not in obese models of leptin, leptin receptor, or melanocortin-4 receptor deficiency. In contrast, the antagonist increased feeding and BW comparably in lean and diet-induced obese (DIO) mice, an increase associated with decreased hypothalamic expression of Socs3, a primary target of leptin. These findings demonstrate that hyperleptinemic DIO mice retain leptin suppression of feeding comparable to lean mice and counter the view that resistance to endogenous leptin contributes to the persistence of DIO in mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Obesity is a significant risk factor for the development of different cancer types and has been associated with poorer response to oncotherapies and linked to earlier recurrence of the neoplastic disease. While molecular mechanisms of these associations are still under investigation, functional dysregulation of two major fat tissue-derived adipokines, leptin and adiponectin, appears to play an important role. Leptin is known to activate carcinogenic pathways, while adiponectin appears to exert antineoplastic activities and interfere with leptin-induced processes. Because excess body fat is associated with increased leptin expression and adiponectin downregulation, therapeutic rebalancing of these pathways may benefit cancer patients, especially the obese subpopulations. This review focuses on our novel leptin receptor antagonists and adiponectin receptor agonists designed for therapeutic modulation of obesity-associated pathways in cancer.

The role of the obesity cytokine leptin in breast cancer progression has raised interest in interfering with leptin's actions as a valuable therapeutic strategy. Leptin interacts with its receptor through three different binding sites: I-III. Site I is crucial for the formation of an active leptin-leptin receptor complex and in its subsequent activation. Amino acids 39-42 (Leu-Asp-Phe-Ile- LDFI) were shown to contribute to leptin binding site I and their mutations in alanine resulted in muteins acting as typical antagonists. We synthesized a small peptide based on the wild-type sequence of leptin binding site I (LDFI) and evaluated its efficacy in antagonizing leptin actions in breast cancer using in vitro and in vivo experimental models. The peptide LDFI abolished the leptin-induced anchorage-dependent and -independent growth as well as the migration of ERα-positive (MCF-7) and -negative (SKBR3) breast cancer cells. These results were well correlated with a reduction in the phosphorylation levels of leptin downstream effectors, as JAK2/STAT3/AKT/MAPK. Importantly, the peptide LDFI reversed the leptin-mediated up-regulation of its gene expression, as an additional mechanism able to enhance the peptide antagonistic activity. The described effects were specific for leptin signalling, since the developed peptide was not able to antagonize the other growth factors' actions on signalling activation, proliferation and migration. Finally, we showed that the LDFI pegylated peptide markedly reduced breast tumour growth in xenograft models. The unmodified peptide LDFI acting as a full leptin antagonist could become an attractive option for breast cancer treatment, especially in obese women., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Obesity correlates with an increased risk of developing prostate cancer (PCa) and leptin plays an important role in PCa progression. Since leptin is produced by adipocytes, the serum leptin level is higher in obese than in non-obese individuals. However, the effects of leptin remain controversial and unclear. The aim of the present study was to investigate the effect of leptin on PCa cell aggressiveness. Three human PCa cell lines (LNCaP, DU145 and PC-3) were treated with recombinant leptin for 28 days. Cell proliferation, migration, and invasion were estimated using the WST assay, a wound-healing assay, and a BD Matrigel invasion assay, respectively. The mechanism underlying the proliferative effect of leptin was investigated by cell transfections with small interfering RNA (siRNA) against the leptin receptor (ObR) or forkhead box O1 (FOXO1), and by immunocytochemistry. Long-term exposure of PCa cells to leptin enhanced their proliferation, migration and invasion. Leptin increased ObR expression and enhanced Akt phosphorylation constitutively. Leptin also increased the phosphorylation of FOXO1 via PI3K signaling and FOXO1 gene silencing enhanced PCa cell proliferation. Leptin induced the translocation of FOXO1 from the nucleus to the cytoplasm. Furthermore, the PI3K inhibitor, LY294002 suppressed this translocation. These results suggested that leptin regulated the subcellular localization of FOXO1 and induced Akt phosphorylation. Additionally, we revealed that leptin increased the expression of cyclin D1 and decreased the expression of p21 protein. In conclusion, long-term exposure to leptin increased the cell proliferation, migration, and invasion of PCa cells through inactivation of FOXO1. This inactivation resulted from exclusion of FOXO1 from the nucleus and its restriction to the cytoplasm through PI3K/Akt signaling. Our findings contribute to an understanding of the association between obesity and PCa aggressiveness.

Accumulating evidence indicates that leptin plays an important role in controlling reproductive function. At physiological levels, leptin stimulates steroidogenesis and follicle maturation, whereas supraphysiological concentrations of leptin have been suggested to be an independent risk factor for cyst formation. Since the discovery of the link between leptin and obesity, which is frequently associated with polycystic ovarian syndrome (PCOS), a number of leptin mutants exhibiting antagonistic properties have been developed, opening new avenues for leptin-related research. Here, using a superactive human leptin antagonist (SHLA), we sought to determine whether blocking leptin receptors can reverse the actions of leptin in ovarian follicles. Antral porcine ovarian follicles, collected from prepubertal and mature animals, were exposed to 100, 250 and 500 ng/ml SHLA for 24 hours, after which leptin receptor (ObR), leptin, CYP11A1 and 17β-hydroxysteroid dehydrogenase (17β-HSD) levels in follicles were evaluated by Western blotting. Levels of secreted leptin, progesterone (P4), and testosterone (T) in the follicle incubation medium were measured using enzyme-linked immunosorbent assays. The effects of SHLA on leptin-stimulated P4 and T secretion were also tested by exposing follicles to 40 ng/ml leptin in the presence and absence of SHLA. These experiments revealed that SHLA acted through inhibition of ObR expression and leptin expression and secretion decreased P4 and T secretion by ovarian follicles from both prepubertal and mature animals. Our data further suggest that the mechanism underlying this action of SHLA involves inhibition of CYP11A1 and 17β-HSD protein expression. Importantly, SHLA reversed leptin-induced increases in P4 and T secretion. Collectively, these data indicate that, in addition to their potential application in novel therapeutic strategies in oncology, which has received considerable recent research attention, leptin receptors antagonists might also be beneficial in controlling reproduction.

The decline in cardiovagal baroreflex function that occurs with aging is accompanied by an increase in circulating leptin levels. Our previous studies showed that exogenous leptin impairs the baroreflex sensitivity for control of heart rate in younger rats, but the contribution of this hormone to baroreflex dysfunction during aging is unknown. Thus we assessed the effect of bilateral leptin microinjection (500 fmol/60 nl) within the solitary tract nucleus (NTS) on the baroreflex sensitivity in older (66 ± 2 wk of age) urethane/chloralose anesthetized Sprague-Dawley rats with elevated circulating leptin levels. In contrast to the 63% reduction observed in younger rats, leptin did not alter the baroreflex sensitivity for bradycardia evoked by phenylephrine in older rats (0.76 ± 0.19 baseline vs. 0.71 ± 0.15 ms/mmHg after leptin; P = 0.806). We hypothesized that this loss of sensitivity reflected endogenous suppression of the baroreflex by elevated leptin, rather than cardiovascular resistance to the peptide. Indeed, NTS administration of a leptin receptor antagonist (75 pmol/120 nl) improved the baroreflex sensitivity for bradycardia in older rats (0.73 ± 0.13 baseline vs. 1.19 ± 0.26 at 10 min vs. 1.87 ± 0.32 at 60 min vs. 1.22 ± 0.54 ms/mmHg at 120 min; P = 0.002), with no effect in younger rats. There was no effect of the leptin antagonist on the baroreflex sensitivity for tachycardia, responses to cardiac vagal chemosensitive fiber activation, or resting hemodynamics in older rats. These findings suggest that the actions of endogenous leptin within the NTS, either produced locally or derived from the circulation, contribute to baroreflex suppression during aging., (Copyright © 2014 the American Physiological Society.)

Purpose of Review: The anorexia-cachexia syndrome is a complication of many chronic conditions including cancer, chronic obstructive pulmonary disease, congestive heart failure, and chronic kidney disease (CKD). Leptin levels are significantly elevated in CKD patients and are associated with markers of poor nutritional status as well as mortality and morbidity. This review will focus on the mechanism and exploit the therapeutic potential of leptin signaling in CKD-associated cachexia., Recent Findings: Studies in db/db mice show that the lack of leptin receptor is protective against CKD-induced cachexia. Blockade of leptin's downstream mediators, such as melanocortin-4 receptor, attenuated CKD-associated cachexia. Pegylation of leptin antagonists resulted in a potent and effective long-acting reagents suitable for in-vivo studies or therapies. Pegylated leptin antagonist treatment ameliorates CKD-associated cachexia in mice., Summary: Leptin antagonism may represent a viable therapeutic strategy for cachexia in CKD.

Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The present investigation examined whether leptin stimulation of ventral tegmental area (VTA) or nucleus of the solitary tract (NTS) has a role in body weight homeostasis independent of the medial basal hypothalamus (MBH). To this end, recombinant adeno-associated viral techniques were employed to target leptin overexpression or overexpression of a dominant negative leptin mutant (leptin antagonist). Leptin antagonist overexpression in MBH or VTA increased food intake and body weight to similar extents over 14 days in rats. Simultaneous overexpression of leptin in VTA with antagonist in MBH resulted in food intake and body weight gain that were less than with control treatment but greater than with leptin alone in VTA. Notably, leptin overexpression in VTA increased P-STAT3 in MBH along with VTA, and leptin antagonist overexpression in the VTA partially attenuated P-STAT3 levels in MBH. Interestingly, leptin antagonist overexpression elevated body weight gain, but leptin overexpression in the NTS failed to modulate either food intake or body weight despite increased P-STAT3. These data suggest that leptin function in the VTA participates in the chronic regulation of food consumption and body weight in response to stimulation or blockade of VTA leptin receptors. Moreover, one component of VTA-leptin action appears to be independent of the MBH, and another component appears to be related to leptin receptor-mediated P-STAT3 activation in the MBH. Finally, leptin receptors in the NTS are necessary for normal energy homeostasis, but mostly they appear to have a permissive role. Direct leptin activation of NTS slightly increases UCP1 levels, but has little effect on food consumption or body weight., (© 2014 Society for Endocrinology.)

Intrauterine growth restriction and premature delivery decrease circulating levels of the neurotrophic hormone leptin and increase the risk of adult psychiatric disease. In mouse models, neonatal leptin replacement normalizes brain growth and improves the neurodevelopmental outcomes of growth restricted mice, but leptin supplementation of well-grown mice decreases adult locomotor activity. We hypothesized isolated neonatal leptin deficiency is sufficient to reduce adult brain volumes and program behavioral outcomes, including hyperactivity. C57Bl/6 pups were randomized to daily injections of saline or PEG-leptin antagonist (LX, 12.5 mg/kg) from postnatal day 4 to 14. After 4 months, fear conditioning and open field testing were performed followed by carotid radiotelemetry for the measurement of baseline activity and blood pressure. Neonatal LX did not significantly increase cue-based fear or blood pressure, but increased adult locomotor activity during assessment in both the open field (beam breaks: control 930 ± 40, LX 1099 ± 42, P<0.01) and the home cage (radiotelemetry counts: control 4.5 ± 0.3, LX 5.6 ± 0.3, P=0.02). Follow-up MRI revealed significant reductions in adult frontal cortex volumes following neonatal LX administration (control 45. 1 ± 0.4 mm(3), LX 43.8 ± 0.4 mm(3), P=0.04). This was associated with a significant increase in cerebral cortex leptin receptor mRNA expression. In conclusion, isolated neonatal leptin deficiency increases cerebral cortex leptin receptor expression and reduces frontal cortex volumes in association with increased adult locomotor activity. We speculate neonatal leptin deficiency may contribute to the adverse neurodevelopmental outcomes associated with perinatal growth restriction, and postnatal leptin therapy may be protective., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Epidemiological evidence has linked the development and progression of several cancers including melanoma with obesity. However, whether obesity impinges on responses of cancer cells to treatment remains less understood. Here we report that human adipocytes contribute to resistance of melanoma cells to various therapeutic agents. Exposure to media from adipocyte cultures (adipocyte media) increased cell proliferation and reduced sensitivity of melanoma cells to apoptosis induced by diverse chemotherapeutic drugs, including the DNA-damaging drug cisplatin, the microtubuletargeting agent docetaxel, and the histone deacetylase inhibitor SAHA. This was associated with increased activation of PI3K/Akt and MEK/ERK signaling, and was attenuated by a PI3K or MEK inhibitor. The effect of adipocyte media on melanoma cells was, at least in part, due to the interaction between the adipokine leptin and its long form receptor OB-Rb, in that immunodepletion of leptin in adipocyte media or siRNA knockdown of OB-Rb in melanoma cells reversed the increase in Akt and ERK activation, enhancement in cell proliferation, and importantly, protection of melanoma cells against the drugs. In support, recombinant leptin partially recapitulated the effect of adipocyte media on melanoma cells. Of note, OB-Rb was increased on the surface of melanoma cells compared to melanocytes, whereas leptin short form receptors appeared to be suppressed post-transcriptionally, suggesting that OB-Rb was selectively upregulated in melanoma cells. Collectively, these results indicate that adipocytes contribute to the resistance of melanoma cells to chemotherapeutic drugs and agents targeting the PI3K/Akt and MEK/ERK pathways, and suggest that inhibition of the leptin/ OB-Rb system may be useful to improve the efficacy of multiple therapeutic approaches in the treatment of melanoma.

Environmental and genetic activation of a brain-adipocyte axis inhibits cancer progression. Leptin is the primary peripheral mediator of this anticancer effect in a mouse model of melanoma. In this study we assessed the effect of a leptin receptor antagonist on melanoma progression. Local administration of a neutralizing nanobody targeting the leptin receptor at low dose adjacent to tumor decreased tumor mass with no effects on body weight or food intake. In contrast, systemic administration of the nanobody failed to suppress tumor growth. Daily intraperitoneal injection of high-dose nanobody led to weight gain, hyperphagia, increased adiposity, hyperleptinemia, and hyperinsulinemia, and central effects mimicking leptin deficiency. The blockade of central actions of leptin by systemic delivery of nanobody may compromise its anticancer effect, underscoring the need to develop peripherally acting leptin antagonists coupled with efficient cancer-targeting delivery.

Endometriosis pain is a very common and extremely disabling condition whose mechanism is still poorly understood. While increased levels of leptin have been reported in patients with endometriosis, their contribution to endometriosis pain has not been explored. Using a rodent model of endometriosis we provide evidence for an estrogen-dependent contribution of leptin in endometriosis-induced pain. Rats implanted with autologous uterine tissue onto the gastrocnemius muscle developed endometriosis-like lesions and local chronic pain. Compared to eutopic uterine tissue, leptin mRNA and protein were up-regulated in the endometriosis-like lesions. Intramuscular injection of recombinant leptin in naive rats produced dose-dependent local mechanical hyperalgesia and nociceptor sensitization to mechanical stimulation. Ovariectomy attenuated the mechanical hyperalgesia induced by recombinant leptin, in rats treated with vehicle compared to those treated with 17β-estradiol replacement, at 1 and 24 h after leptin injection. Finally, intralesional injections of a pegylated leptin receptor (Ob-R) antagonist or of an inhibitor of Janus kinase2, which transduces the Ob-R signal, markedly attenuated pain in the endometriosis model. Taken together these data support the hypothesis that leptin, generated in ectopic endometrial lesions produces mechanical hyperalgesia by acting on nociceptors innervating the lesion. This sensitivity to leptin is dependent on estrogen levels. Thus, interventions targeting leptin signaling, especially in combination with interventions that lower estrogen levels, might be useful for the treatment of endometriosis pain., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Adipose tissue hormone leptin induces endothelium-dependent vasorelaxation mediated by nitric oxide (NO) and endothelium-derived hyperpolarizing factors (EDHF). Previously it has been demonstrated that in short-term obesity the NO-dependent and the EDHF-dependent components of vascular effect of leptin are impaired and up-regulated, respectively. Herein we examined the mechanism of the EDHF-dependent vasodilatory effect of leptin and tested the hypothesis that alterations of acute vascular effects of leptin in obesity are accounted for by chronic hyperleptinemia. The study was performed in 5 groups of rats: (1) control, (2) treated with exogenous leptin for 1 week to induce hyperleptinemia, (3) obese, fed highly-palatable diet for 4 weeks, (4) obese treated with pegylated superactive rat leptin receptor antagonist (PEG-SRLA) for 1 week, (5) fed standard chow and treated with PEG-SRLA. Acute effect of leptin on isometric tension of mesenteric artery segments was measured ex vivo. Leptin relaxed phenylephrine-preconstricted vascular segments in NO- and EDHF-dependent manner. The NO-dependent component was impaired and the EDHF-dependent component was increased in the leptin-treated and obese groups and in the latter group both these effects were abolished by PEG-SRLA. The EDHF-dependent vasodilatory effect of leptin was blocked by either the inhibitor of cystathionine γ-lyase, propargylglycine, or a hydrogen sulfide (H2S) scavenger, bismuth (III) subsalicylate. The results indicate that NO deficiency is compensated by the up-regulation of EDHF in obese rats and both effects are accounted for by chronic hyperleptinemia. The EDHF-dependent component of leptin-induced vasorelaxation is mediated, at least partially, by H2S.

A chicken gene orthologous to human leptin receptor (LEPR) has been characterized and found to be active in leptin signaling in vitro in response to a variety of recombinant leptins and leptin-containing blood samples. However, the endogenous ligand of chicken LEPR (cLEPR) - the putative chicken leptin - has been reported by us and others to be undetectable at the DNA, mRNA, protein and activity levels. These reports have raised questions as to cLEPR's role. Here we analyzed the effects of a pegylated superactive mouse leptin antagonist (PEG-SMLA) in chicken. We showed that the leptin antagonist efficiently and specifically blocks leptin signaling through the cLEPR in vitro. The effect of the leptin antagonist was then studied in vivo by daily administration of 10 mg kg(-1) for 10 consecutive days to white leghorn female chickens (Gallus gallus) at the age of 2 weeks. Despites the efficient attenuation of the cLEPR in vitro, no effect was observed on body mass, feed intake, feed efficiency or fat accumulation in the treated birds. Because similar treatment in rodents leads to a highly pronounced increase in appetite and body mass that are observed from the first day of treatment, it is concluded that the cLEPR is not implicated in the control of appetite or adipose homeostasis in chickens.

Epidemiological evidence strongly links excess body weight with an increased risk to develop atherothrombotic complications. Obesity is frequently associated with systemic and local inflammation as well as elevated circulating leptin levels, and clinical studies found hyperleptinemia to correlate not only with measures of adiposity, but also with circulating biomarkers of an increased metabolic and cardiovascular risk or surrogate markers of subclinical atherosclerosis. Moreover, experimental studies in mice with systemic disruption of the leptin-leptin receptor system as well as after administration or neutralization of the adipokine demonstrated that leptin promotes both arterial and venous thrombosis. In addition to directly binding to and activating platelets and thus potentiating their aggregation in response to agonist stimulation, leptin enhances the expression of prothrombotic and anti-fibrinolytic proteins in vascular and inflammatory cells. On the other hand, its ability to mobilize and recruit vascular progenitor cells from the bone marrow to sites of vascular injury was found to be impaired in hyperleptinemic, obese humans and rodents. Thus, leptin promotes thrombus formation and resolution by several different mechanisms involving primary hemostasis, the coagulation cascade as well as the integrity of the vessel wall. Dissection of the molecular mechanisms underlying each of its actions may pave the road for novel therapeutic options in targeting the increased risk of thrombosis associated with obesity, keeping in mind unresolved issues of a cell-specific leptin resistance as well as individual differences in the responsiveness to leptin.

Elevated serum leptin levels correlate with inflammation and predict changes in lean body mass in patients with CKD, and activation of the melanocortin system by leptin signaling mediates the pathophysiology of CKD-associated cachexia. We tested whether treatment with a pegylated leptin receptor antagonist (PLA) attenuates cachexia in mice with CKD. CKD and Sham mice received vehicle or PLA (2 or 7 mg/kg per day). At these doses, PLA did not influence serum leptin levels in mice. Treatment with 7 mg/kg per day PLA stimulated appetite and weight gain, improved lean mass and muscle function, reduced energy expenditure, and normalized the levels of hepatic TNF-α and IL-6 mRNA in mice with CKD. Furthermore, treatment with 7 mg/kg per day PLA attenuated the CKD-associated increase in the transcriptional and protein abundance of uncoupling proteins that mediates thermogenesis, and it normalized the molecular signatures of processes associated with muscle wasting in CKD, including proteolysis, myogenesis and muscle regeneration, and expression of proinflammatory muscle cytokines, such as IL-1α, -1β, and -6 and TNF-α. Our results suggest that leptin antagonism may represent a viable therapeutic strategy for cachexia in CKD.

Leptin and its receptor are involved in breast carcinogenesis as mitogenic factors. Therefore, they could be considered as targets for breast cancer therapy. Expression of the leptin receptor gene could be modulated by leptin secretion. Silibinin and curcumin are herbal compounds with anti-cancer activity against breast cancer. The aim of this study was to assess their potential to inhibit of expression of the leptin gene and its receptor and leptin secretion. Cytotoxic effects of the two agents on combination on T47D breast cancer cells was investigated by MTT assay test after 24h treatment. With different concentrations the levels of leptin, leptin receptor genes expression were measured by reverse-transcription real-time PCR. Amount of secreted leptin in the culture medium was determined by ELISA. Data were statistically analyzed by one-way ANOVA test. The silibinin and curcumin combination inhibited growth of T47D cells in a dose dependent manner. There were also significant difference between control and treated cells in leptin expression and the quantity of secreted leptin with a relative decrease in leptin receptor expression. In conclusion, these herbal compounds inhibit the expression and secretion of leptin and it could probably be used as drug candidates for breast cancer therapy through leptin targeting in the future.

Random mutagenesis of mouse leptin antagonist (L39A/D40A/F41) followed by selection of high-affinity mutants by yeastsurface display indicated that replacing residue D23 with a non-negatively charged amino acid (most specifically with Leu) leads to dramatically enhanced affinity of leptin toward LEPR leading to development of superactive mouse, human, ovine and rat leptin antagonists (D23L/L39A/D40A/F41A). Superactive leptin antagonist mutants of mouse, human, rat or ovine leptins were developed in our laboratory, expressed in E. coli, refolded and purified to homogeneity as monomeric proteins. Pegylation of leptin antagonists resulted in potent and effective long-acting reagents suitable for in vivo studies or therapies. In the present review we explain the mechanism of leptin inhibition and summarize the possible use of leptin antagonists as possible leptin blockers in various human pathologies such as antiinflammatory and anti-autoimmune diseases, uremic cachexia, and cancer. We also suggest the use of leptin antagonists as research reagents for creation of a novel, fast and reversible model of T2DM in mice.

Obesity is a world health problem that increases the risk for developing type 2 diabetes, cardiovascular disease, fatty liver, and some types of cancer. In postmenopausal women, it represents an important risk factor for the development of breast cancer (BC). Leptin is an adipokine that is secreted by fatty tissue, and high leptin levels are observed both in mouse models of obesity and in obese subjects. High levels of leptin promote the proliferation and progression of various types of cancer, including BC. This review provides a general overview of the biology of leptin, important laboratory studies, and animal and clinical models that have provided evidence for an active role of leptin in the proliferation, progression, and survival of mammary tumors. Finally, this review addresses the most recent studies on the use of leptin receptor antagonists as a novel therapeutic treatment for BC.

The first adipokine, leptin, discovered almost 20 years ago, is secreted into circulation mainly from adipose tissue and acts both centrally and peripherally. Leptin regulates energy metabolism, reproductive function, bone metabolism, and immune response. However in some physiological or pathological situations such as enhancement of undesired immune responses in autoimmune diseases, tumorigenesis, elevated blood pressure, and certain cardiovascular pathologies, leptin activity may be harmful. In this review we screen different approaches to blocking leptin action, in vitro and in vivo. The recent development of superactive leptin muteins exhibiting antagonistic properties, and other leptin-action-blocking peptides, proteins, monoclonal antibodies, and nanobodies, opens new perspectives for their use in research, and eventually, therapy for cachexia, autoimmune disease, cancer, and other pathologies.

Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.

Background: Emerging evidence suggests that angiogenic and pro-inflammatory cytokine leptin might be implicated in ocular neovascularization. However, the potential of inhibiting leptin function in ophthalmic cells has never been explored. Here we assessed mitogenic, angiogenic, and signaling leptin activities in retinal and corneal endothelial cells and examined the capability of a specific leptin receptor (ObR) antagonist, Allo-aca, to inhibit these functions., Methods and Results: The experiments were carried out in monkey retinal (RF/6A) and bovine corneal (BCE) endothelial cells. Leptin at 50-250 ng/mL stimulated the growth of both cell lines in a dose-dependent manner. The maximal mitogenic response (35±7 and 27±3% in RF6A and BCE cells, respectively) was noted at 24 h of 250 ng/mL leptin treatments. Leptin-dependent proliferation was reduced to base levels with 10 and 100 nM Allo-aca in BCE and RF6A cells, respectively. In both cell lines, leptin promoted angiogenic responses, with the maximal increase in tube formation (163±10 and 133±8% in RF6A and BCE cultures, respectively) observed under a 250 ng/mL leptin treatment for 3 h. Furthermore, in both cell lines 250 ng/mL leptin modulated the activity or expression of several signaling molecules involved in proliferation, inflammatory activity and angiogenesis, such as STAT3, Akt, and ERK1/2, COX2, and NFκB. In both cell lines, leptin-induced angiogenic and signaling responses were significantly inhibited with 100 nM Allo-aca. We also found that leptin increased its own mRNA and protein expression in both cell lines, and this autocrine effect was abolished by 100-250 nM Allo-aca., Conclusions: Our data provide new insights into the role of leptin in ocular endothelial cells and represent the first original report on targeting ObR in ophthalmic cell models.

Increased hepatic vascular resistance mainly due to elevated vascular tone and to fibrosis is the primary factor in the development of portal hypertension in cirrhosis. Leptin, a hormone associated with reduction in nitric oxide bioavailability, vascular dysfunction, and liver fibrosis, is increased in patients with cirrhosis. We aimed at evaluating whether leptin influences the increased hepatic resistance in portal hypertension. CCl4-cirrhotic rats received the leptin receptor-blocker ObR antibody, or its vehicle, every other day for 1 wk. Hepatic and systemic hemodynamics were measured in both groups. Hepatic nitric oxide production and bioavailability, together with oxidative stress, nitrotyrosinated proteins, and liver fibrosis, were evaluated. In cirrhotic rats, leptin-receptor blockade significantly reduced portal pressure without modifying portal blood flow, suggesting a reduction in the intrahepatic resistance. Portal pressure reduction was associated with increased nitric oxide bioavailability and with decreased O2(-) levels and nitrotyrosinated proteins. No changes in systemic hemodynamics and liver fibrosis were observed. In conclusion, the present study shows that blockade of the leptin signaling pathway in cirrhosis significantly reduces portal pressure. This effect is probably due to a nitric oxide-mediated reduction in the hepatic vascular tone.

Perinatal leptin impairment has long-term consequences on energy homeostasis leading to body weight gain. The underlying mechanisms are still not clearly established. We aimed to analyze the long-term effects of early leptin blockade. In this study, newborn rats received daily injection of a pegylated rat leptin antagonist (pRLA) or saline from day 2 (d2) to d13 and then body weight gain, insulin/leptin sensitivity, and expression profile of microRNAs (miRNAs) at the hypothalamic level were determined at d28, d90, or d153 (following 1 month of high-fat diet (HFD) challenge). We show that pRLA treatment predisposes rats to overweight and promotes leptin/insulin resistance in both hypothalamus and liver at adulthood. pRLA treatment also modifies the hypothalamic miRNA expression profile at d28 leading to the upregulation of 34 miRNAs and the downregulation of four miRNAs. For quantitative RT-PCR confirmation, we show the upregulation of rno-miR-10a at d28 and rno-miR-200a, rno-miR-409-5p, and rno-miR-125a-3p following HFD challenge. Finally, pRLA treatment modifies the expression of genes involved in energy homeostasis control such as UCPs and AdipoRs. In pRLA rat muscle, Ucp2/3 and Adipor1/r2 are upregulated at d90. In liver, pRLA treatment upregulates Adipor1/r2 following HFD challenge. These genes are known to be involved in insulin resistance and type 2 diabetes. In conclusion, we demonstrate that the impairment of leptin action in early life promotes insulin/leptin resistance and modifies the hypothalamic miRNA expression pattern in adulthood, and finally, this study highlights the potential link between hypothalamic miRNA expression pattern and insulin/leptin responsiveness.

The obesity hormone leptin has been implicated in the development and progression of different cancer types, and preclinical studies suggest that targeting leptin signaling could be a new therapeutic option for the treatment of cancer, especially in obese patients. To inhibit pro-neoplastic leptin activity, we developed leptin receptor (ObR) peptide antagonists capable of blocking leptin effects in vitro and in vivo. Our lead compound (Allo-aca), however, crosses the blood-brain-barrier (BBB), inducing undesirable orexigenic effects and consequent weight gain. Thus, redesigning Allo-aca to uncouple its central and peripheral activities should produce a superior compound for cancer treatment. The aim of this study was to generate novel Allo-aca analogs and test their biodistribution in vivo and anti-neoplastic activity in vitro in breast and colorectal cancer cells. Examination of several Allo-aca analogs resulted in the identification of the peptidomimetic, d-Ser, that distributed only in the periphery of experimental animals. d-Ser inhibited leptin-dependent-proliferation of ObR-positive breast and colorectal cancer cells in vitro at 1nM concentration without exhibiting any partial agonistic activity. d-Ser efficacy was demonstrated in monolayer and three-dimensional cultures, and its antiproliferative action was associated with the inhibition of several leptin-induced pathways, including JAK/STAT3, MAPK/ERK1/2 and PI3K/AKT, cyclin D1, and E-cadherin. In conclusion, d-Ser is the first leptin-based peptidomimetic featuring peripheral ObR antagonistic activity. The novel peptide may serve as a prototype to develop new therapeutics, particularly for the management of obesity-related cancers., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We previously reported elevated adipose leptin expression, plasma leptin concentrations, and adrenocortical leptin receptor expression in the long-term hypoxic (LTH) ovine fetus. This study addressed whether leptin antagonist (LA) administration to LTH fetal sheep altered expression of key genes governing cortisol synthesis. Ewes were maintained at high altitude (3,820 meters) from 40 to 130 days gestation (dG), returned to Loma Linda University, and implanted with a maternal tracheal catheter. Reduced Po2 was maintained by nitrogen infusion. On 132 dG, LTH (n = 11) and age-matched, normoxic control (n = 11) fetuses underwent vascular catheter implantation. At 138 dG, fetuses were continuously infused with either saline or the LA (1.5 mg·kg(-1)·day(-1)) for 4 days and samples collected for blood gases, ACTH, and cortisol. Fetal adrenal cortex was collected for determination of steriodogenic acute regulatory protein (StAR), ACTH, and leptin receptor, cholesterol side-chain cleavage (CYP11A1), cytochrome P-450 11β-hydroxylase (CYP11B1), 17α-hydroxylase (CYP17), 21-hydroxylase (CYP21), signal transducer and activator of transcription 3 (STAT3), pSTAT3, and 17β-hydroxysteroid dehydrogenase (HSD3B) expression. In the saline-infused LTH fetuses, StAR, ACTH receptor, CYP11A1, and CYP17 expression was significantly lower compared with control (P < 0.05), whereas levels of CYP11B1, CYP21, and HSD3B mRNA were similar between groups. LA infusion restored expression of StAR, pSTAT3, CYP11A1, and CYP17, but not ACTH receptor, to normal ontogenic levels in the LTH group while having no effect on control fetuses. Neither fetal plasma ACTH nor cortisol concentrations were altered by LA infusion. We speculate that while leptin plays a role in governing expression of key enzymes and StAR in response to LTH, other factors play a role in modulating cortisol synthesis in these fetuses.

Leptin regulates energy balance. However, knowledge of the critical intracellular transducers of leptin signaling remains incomplete. We found that Rho-kinase 1 (ROCK1) regulates leptin action on body weight homeostasis by activating JAK2, an initial trigger of leptin receptor signaling. Leptin promoted the physical interaction of JAK2 and ROCK1, thereby increasing phosphorylation of JAK2 and downstream activation of Stat3 and FOXO1. Mice lacking ROCK1 in either pro-opiomelanocortin (POMC) or agouti-related protein neurons, mediators of leptin action, displayed obesity and impaired leptin sensitivity. In addition, deletion of ROCK1 in the arcuate nucleus markedly enhanced food intake, resulting in severe obesity. Notably, ROCK1 was a specific mediator of leptin, but not insulin, regulation of POMC neuronal activity. Our data identify ROCK1 as a key regulator of leptin action on energy homeostasis.

Leptin plays a central role in maintaining energy balance, with multiple other systemic effects. Despite leptin importance in peripheral regulation of mesenchymal stem cells (MSC) differentiation, little is known about its expression mechanism. Leptin is often described as adipokine, while it is expressed by other cell types. We have recently shown an in vitro leptin expression, enhanced by glucocorticoids in synovial fibroblasts (SVF). Here, we investigated leptin expression in MSC from bone marrow (BM-MSC) and umbilical cord matrix (UMSC). Results showed that BM-MSC, but not UMSC, expressed leptin that was strongly enhanced by glucocorticoids. Transforming growth factor β1 (TGF-β1) markedly inhibited the endogenous- and glucocorticoid-induced leptin expression in BM-MSC. Since TGF-β1 was shown to signal via ALK-5-Smad2/3 and/or ALK-1-Smad1/5 pathways, we analyzed the expression of proteins from both pathways. In BM-MSC, TGF-β1 increased phosphorylated Smad2 (p-Smad2) expression, while ALK-5 inhibitor (SB431542) induced leptin expression and significantly restored TGF-β1-induced leptin inhibition. In addition, both prednisolone and SB431542 increased p-Smad1/5 expression. These results suggested the ALK-5-Smad2 pathway as an inhibitor of leptin expression, while ALK-1-Smad1/5 as an activator. Indeed, Smad1 expression silencing induced leptin expression inhibition. Furthermore, prednisolone enhanced the expression of TGF-βRII while decreasing p-Smad2 in BM-MSC and SVF but not in UMSC. In vitro differentiation revealed differential osteogenic potential in SVF, BM-MSC, and UMSC that was correlated to their leptin expression potential. Our results suggest that ALK-1/ALK-5 balance regulates leptin expression in MSC. It also underlines UMSC as leptin nonproducer MSC for cell therapy protocols where leptin expression is not suitable.

The adipocyte-derived hormone leptin plays an important role in regulation of energy homeostasis and the innate immune response against bacterial infections. Leptin's actions are mediated by signaling events initiated by phosphorylation of tyrosine residues on the long form of the leptin receptor. We recently reported that disruption of leptin receptor-mediated STAT3 activation augmented host defense against pneumococcal pneumonia. In this report, we assessed leptin receptor-mediated ERK activation, a pathway that was ablated in the l/l mouse through a mutation of the tyrosine 985 residue in the leptin receptor, to determine its role in host defense against bacterial pneumonia in vivo and in alveolar macrophage (AM) antibacterial functions in vitro. l/l mice exhibited increased mortality and impaired pulmonary bacterial clearance after intratracheal challenge with Klebsiella pneumoniae. The synthesis of cysteinyl-leukotrienes was reduced and that of PGE(2) enhanced in AMs in vitro and the lungs of l/l mice after infection with K. pneumoniae in vivo. We also observed reduced phagocytosis and killing of K. pneumoniae in AMs from l/l mice that was associated with reduced reactive oxygen intermediate production in vitro. cAMP, known to suppress phagocytosis, bactericidal capacity, and reactive oxygen intermediate production, was also increased 2-fold in AMs from l/l mice. Pharmacologic blockade of PGE(2) synthesis reduced cAMP levels and overcame the defective phagocytosis and killing of bacteria in AMs from l/l mice in vitro. These results demonstrate that leptin receptor-mediated ERK activation plays an essential role in host defense against bacterial pneumonia and in leukocyte antibacterial effector functions.