Leptin-Activity Modulators and Their Potential Pharmaceutical Applications.

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.

A Leptin Receptor Antagonist Attenuates Adipose Tissue Browning and Muscle Wasting in Infantile Nephropathic Cystinosis-Associated Cachexia.

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.

FABP4 inhibitor attenuates inflammation and endoplasmic reticulum stress of islet in leptin receptor knockout rats.

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.

Leptin induces a contracting effect on guinea pig tracheal smooth muscle via the Ob-R receptor mechanism: novel evidence.

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% O2 and 5% CO2) 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.

A novel leptin receptor antagonist uncouples leptin's metabolic and immune functions.

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.

Leptin Receptor Antagonists' Action on HDAC Expression Eliminating the Negative Effects of Leptin in Ovarian Cancer.

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.

In silico design of small peptides antagonist against leptin receptor for the treatment of obesity and its associated immune-mediated diseases.

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.

Sex- and bone-specific responses in bone structure to exogenous leptin and leptin receptor antagonism in the ovine fetus.

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.

Preparation of Potent Leptin Receptor Antagonists and Their Therapeutic Use in Mouse Models of Uremic Cachexia and Kidney Fibrosis.

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)-ß signaling may be the most potent mediator of fibrogenesis in multiple organs, and leptin is a co-activator of TGF-ß. Muscle fibrosis was evident in our CKD mice and PLA treatment significantly reduced the mRNA levels of TGF- ß1 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.

Superactive human leptin antagonist (SHLA), triple Lan1 and quadruple Lan2 leptin mutein as a promising treatment for human folliculoma.

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/ß 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 ERß 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 ERß, 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.

Leptin receptor antagonism of iNKT cell function: a novel strategy to combat multiple myeloma.

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.

Leptin Is Produced by Parathyroid Glands and Stimulates Parathyroid Hormone Secretion.

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.

The molecular mechanism of action of superactive human leptin antagonist (SHLA) and quadruple leptin mutein Lan-2 on human ovarian epithelial cell lines.

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.

Origin of Aberrant Blood Pressure and Sympathetic Regulation in Diet-Induced Obesity.

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.

Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

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.

Insensitivity of well-conditioned mature sheep to central administration of a leptin receptor antagonist.

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.

The adipokine leptin mediates muscle- and liver-derived IGF-1 in aged mice.

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.

Leptin promotes human endometriotic cell migration and invasion by up-regulating MMP-2 through the JAK2/STAT3 signaling pathway.

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.

Diet-induced obese mice retain endogenous leptin action.

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.