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iNOS deficiency in ob/ob mice improved liver inflammation and ECM remodeling-related genes, decreasing fibrosis, and metabolic dysfunction. The activation of iNOS by leptin is necessary for the synthesis and secretion of TNC in hepatocytes, suggesting an important role of this alarmin in the development of NAFLD., Competing Interests: The authors declare that they have no conflict of interest.

Leptin and adiponectin, adipokines present in breast milk, have shown immunomodulatory properties. The current study aimed to ascertain whether a nutritional supplementation with leptin or adiponectin in neonatal rats was able to influence the maturation of the systemic immune response in early life. To achieve this, suckling Wistar rats were supplemented with either leptin (0.7 μg/kg/day) or adiponectin (35 μg/kg/day) during the whole suckling period. Plasmatic immunoglobulins were quantified, and spleen lymphocyte composition and their ability to proliferate and release cytokines were evaluated during (day 14) and at the end (day 21) of the suckling period. Rats fed with either adipokine showed higher plasma IgM and IgG1 concentrations and adiponectin supplementation also increased IgG2a at both studied days (P < 0.05). With regard to the lymphocyte composition, both adipokine supplementations increased T cell proportion and both CD4 + and CD8 + T cell subsets after two weeks of supplementation (P < 0.05). Moreover, only leptin administration increased NK and NKT cell proportions at the end of the suckling period. Finally, both adipokines influenced the cytokine secretion pattern by splenocytes. In conclusion, these results suggest that leptin and adiponectin play a role in the maturation of the systemic immune response during the suckling period.

The obesity is a result of energy imbalance and the increase in thermogenesis seems an interesting alternative for the treatment of this disease. The mechanism of energy expenditure through thermogenesis is tightly articulated in the hypothalamus by leptin. The hypothalamic extracellular signal-regulated kinase-1/2 (ERK1/2) is a key mediator of the thermoregulatory effect of leptin and mediates the sympathetic signal to the brown adipose tissue (BAT). In this context, physical exercise is one of the main interventions for the treatment of obesity. Thus, this study aimed to verify the effects of acute physical exercise on leptin-induced hypothalamic ERK1/2 phosphorylation and thermogenesis in obese mice. Here we showed that acute physical exercise reduced the fasting glucose of obese mice and increased leptin-induced hypothalamic p-ERK1/2 and uncoupling protein 1 (UCP1) content in BAT ( P < 0.05). These molecular changes are accompanied by an increased oxygen uptake (VO 2 ) and heat production in obese exercised mice ( P < 0.05). The increased energy expenditure in the obese exercised animals occurred independently of changes in spontaneous activity. Thus, this is the first study demonstrating that acute physical exercise can increase leptin-induced hypothalamic ERK1/2 phosphorylation and energy expenditure of obese mice., (© 2018 Wiley Periodicals, Inc.)

Aim: Metreleptin treatment in lipodystrophy patients improves eating behavior with increased satiety and reduced hunger. However, no data are available whether effects are maintained beyond 52 weeks of treatment., Methods: A prospective study with measurements at baseline and at >150 weeks of metreleptin treatment was performed. Five female lipodystrophy patients with indication for metreleptin were included. Behavioral aspects of hunger- and satiety regulation were assessed by validated eating behavior questionnaires and visual analog scales assessing hunger and satiety feelings before and after a standardized meal., Results: Hunger rated on visual analog scales at 120 min after the meal significantly decreased from 46 ± 10 mm at baseline to 17 ± 6 mm at long-term assessment. Furthermore, satiety at 5 and 120 min after the meal significantly increased from baseline to long-term assessment (5 min: 70 ± 7 mm to 87 ± 3 mm; 120 min: 43 ± 10 mm to 79 ± 8 mm). On the Three Factor Eating Questionnaire, the mean value of factor 3 (hunger) significantly decreased from 9.2 ± 0.2 at baseline to 2.6 ± 1.5 at long-term assessment. In the Inventory of Eating Behavior and Weight Problems Questionnaire, mean values for scale 2 (strength and triggering of desire to eat) and scale 7 (cognitive restraint of eating) significantly decreased from baseline (31.6 ± 4.8 and 11.4 ± 2.2, respectively) to long-term assessment (14.0 ± 2.1 and 10.0 ± 1.9)., Conclusion: First evidence is presented that long-term metreleptin treatment of >150 weeks has sustained effects on eating behavior with increased satiety, as well as reduced hunger and hunger-related measures., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aim: Lipodystrophy syndromes are rare heterogeneous disorders characterized by deficiency of adipose tissue, usually a decrease in leptin levels and, frequently, severe metabolic abnormalities including diabetes mellitus and dyslipidemia., Purpose: To describe the clinical presentation of known types of lipodystrophy, and suggest specific steps to recognize, diagnose and treat lipodystrophy in the clinical setting., Methods: Based on literature and in our own experience, we propose a stepwise approach for diagnosis of the different subtypes of rare lipodystrophy syndromes, describing its more frequent co-morbidities and establishing the therapeutical approach., Results: Lipodystrophy is classified as genetic or acquired and by the distribution of fat loss, which can be generalized or partial. Genes associated with many congenital forms of lipodystrophy have been identified that may assist in diagnosis. Because of its rarity and heterogeneity, lipodystrophy may frequently be unrecognized or misdiagnosed, which is concerning because it is progressive and its complications are potentially life threatening. A basic diagnostic algorithm is proposed. Effective management of lipodystrophy includes lifestyle changes and aggressive, evidence-based treatment of comorbidities. Leptin replacement therapy (metreleptin) has been found to improve metabolic parameters in many patients with lipodystrophy. Metreleptin is approved in the United States as replacement therapy to treat the complications of leptin deficiency in patients with congenital or acquired generalized lipodystrophy and has been submitted for approval in Europe., Conclusions: Here, we describe the clinical presentation of known types of lipodystrophy, present an algorithm for differential diagnosis of lipodystrophy, and suggest specific steps to recognize and diagnose lipodystrophy in the clinical setting.

Background: Obesity is associated with oxidative stress, a major factor in carcinogenesis, and with high leptin concentration. The aim of this study was to determine the effects of leptin on the antioxidant response in three human mammary epithelial cells each presenting a different neoplastic status: healthy human mammary epithelial cells (HMEC), oestrogen-receptor positive MCF-7 cells and triple-negative MDA-MB-231 cells., Methods: This in vitro kinetic study characterized the cell antioxidant response after 1, 6 and 24 h in the presence of leptin (10 or 100 ng/ml).The antioxidant response was defined in terms of cell glutathione content, gene expression and catalytic activity of antioxidant enzymes (i.e. glutathione peroxidase 1 (Gpx1), glutathione reductase (GR), glutathione S transferase (GST), heme-oxygenase 1 (HO-1) and cyclooxygenase-2 (COX-2)). Oxidative stress occurrence was assessed by lipid hydro peroxide (HPLIP) and isoprostane concentrations in culture media at 24 h., Results: At both concentrations used, leptin induced ROS production in all cell models, contributing to various antioxidant responses linked to neoplastic cell status. HMEC developed a highly inducible antioxidant response based on antioxidant enzyme activation and an increase in cell GSH content at 10 ng/ml of leptin. However, at 100 ng/ml of leptin, activation of antioxidant response was lower. Conversely, in tumour cells, MCF-7 and MDA-MB-231, leptin did not induce an efficient antioxidant response, at either concentration, resulting in an increase of lipid peroxidation products., Conclusions: Leptin can modulate the oxidative status of mammary epithelial cells differently according to their neoplastic state. These novel results shed light on oxidative status changes in mammary cells in the presence of leptin.

Objective: To study the effect of the satiety hormone, leptin, in saliva proteome and salivary gland histology and ultrastructure., Design: Increases in blood leptin levels were induced through mini-pump infusion in male Wistar rats, during a period of 7 days. Saliva was collected before and at the end of the experimental period, for proteomic analysis, and major salivary glands were collected, at the end, for biochemical, histological and ultrastructural analysis., Results: Immunohistochemistry revealed the presence of leptin receptors in major salivary glands. Salivary amylase levels and enzymatic activity were decreased in saliva, whereas the enzymatic activity of this protein was increased in the cytosol of parotid gland cells. Transmission electron microscopy allowed the observation of high number of electron-dense granules in cytosol of parotid acinar cells, from leptin treated animals., Conclusions: Increased levels of plasmatic leptin result in changes in saliva composition and salivary glands function. To our knowledge, this is the first study providing evidences for a potential role of leptin in salivary gland secretion and saliva composition. An understanding of how appetite/satiety factors influence saliva composition and how this composition influences food processing in mouth may be relevant in understanding ingestivebehaviour., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca 2+ , and this Ca 2+ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca 2+ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca 2+ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We recently showed that male rats exhibit lower hypophagia and body weight loss compared to female rats following central leptin delivery, suggesting a role for oestradiol in leptin responsiveness. Accordingly, we delivered Ob (leptin) or GFP (control) gene into the brain of male rats that were simultaneously treated with oestradiol or vehicle. In a reciprocal approach, we compared oestradiol-deficient (OVX) with intact females (sham) that received leptin or control vector. Changes in food intake), body weight and body composition were examined. In males, oestradiol and leptin resulted in lower cumulative food intake (15%) and endpoint body weight (5%), although rats receiving dual treatment (oestradiol-leptin) ate 28% less and weighed 22% less than vehicle-control. Changes in food intake were unique to each treatment, with a rapid decrease in vehicle-leptin followed by gradual renormalisation. By contrast, hypophagia in oestradiol-control was of lower amplitude and sporadic. Leptin selectively targeted fat mass and endpoint abdominal fat mass was 65%-80% lower compared to their respective control groups. In females, both leptin groups had lower body weight (endpoint values 20% lower than control groups) with the highest extent in sham animals (endpoint value was 28% less in sham-leptin than in sham-control). OVX rats rapidly started regaining their lost body weight reminiscent of the pattern in males. Leptin rapidly and robustly reduced fat mass with endpoint values 30%-35% less than control treated animals. It appears that leptin and oestradiol decreased food intake and body weight via different mechanisms, with the pattern of oestradiol-leptin being reminiscent of that observed in females and the pattern of OVX-leptin reminiscent of that observed in males. Oestrogen status did not influence initial fat mass loss by leptin. It can be concluded that oestradiol modulates the long-term response to central leptin overexpression, although its actions on energy homeostasis are additive and independent of those of leptin., (© 2018 British Society for Neuroendocrinology.)

Objective: Increasing evidence suggests that leptin is upregulated during allergic reactions in the airway and related to the severity of disease in allergic rhinitis (AR). In this study, we aimed to investigate the expression of leptin during sublingual immunotherapy (SLIT) in AR patients., Methods: Forty AR patients without obesity were recruited in this study. Twenty patients received house dust mite (HDM) allergen extract for SLIT and twenty patients received placebo randomly. Protein expression of leptin in serum and nasal lavage was tested by enzyme-linked immuno sorbent assay (ELISA) 1 and 2 years after SLIT treatment, respectively. Peripheral blood mononuclear cells (PBMCs) and human nasal epithelial cell were prepared and stimulated by recombinant leptin after 24 months' SLIT treatment and the induction of Th2 cytokines (IL-4/IL-5/IL-13) were detected by ELISA., Results: SLIT treatment decreased the expression of leptin protein in serum and nasal lavage significantly compared with placebo group 1 and 2 years after SLIT treatment. Nasal leptin level was correlated to decreased Th2 response (IL-4/IL-5/IL-13) and enhanced Treg (IL-10/TGF-beat) response after 2 years' SLIT. We also found that SLIT decreased the ability of leptin in promoting Th2 cytokines expression by PBMCs and human nasal epithelial cell after 2 years' SLIT treatment., Conclusion: Changes of leptin expression in serum and nasal lavage may be correlated with Th2/Treg regulation during SLIT. Our results suggested that leptin served as an important biomarker during SLIT.

Leptin-mediated DNA-binding activity of STAT3 in hypothalamus plays crucial roles in the maintenance of energy homeostasis in lean mice; however its effects still remains unclear in case of leptin resistance in mice with diet induced obesity (DIO). In this study significant elevation of both basal and exogenously leptin-treated DNA-binding activity of STAT3 was detected using EMSA in the hypothalamus of male C57BL/6J mice fed high-fat diet for 10 wks, in concomitant with hyperleptinemia, high body weight, high fat mass, and hyperphagia as well as decreased POMC expression. The studies in vitro showed that both DNA binding activity and the proximal SBE of POMC promoter was essential to leptin-mediated POMC expression. However, the diminution of STAT3 phosphorylation, achieved by S3I-201 or a FoxO1 mutant, facilitated leptin-mediated POMC expression. The findings here demonstrated excess STAT3 activity negatively regulated POMC expression in hypothalamus of DIO mice, and suggested the limitation of STAT3 activity may promote leptin signaling., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Leptin is an adipocytokine predominantly secreted by adipose tissue that participates in immune modulation. Mast cells are important immune cells that are related to altered sympathetic activity. Previous study has shown that leptin promotes activation of the left stellate ganglion (LSG) directly via the leptin receptor. This study aims to investigate whether mast cells play a key role in indirect activation., Methods: Twenty-eight canines were randomly divided into 3 groups: the control group (saline, n = 8), leptin group (leptin, n = 9), and DSCG group (disodium cromoglycate plus leptin, n = 11). Drugs were locally microinjected into the LSG. The function and neural activity of the LSG were evaluated to investigate LSG activation. Tryptase was adopted to identify activated mast cells in the LSG., Results: Compared with the control group, leptin injection (18 μg) markedly increased the function and neural activity of the LSG. Leptin also upregulated c-fos, nerve growth factor (NGF), and tryptase expression in the LSG. However, these effects of leptin were attenuated by pre-injection of DSCG (25 mg). Additionally, the immunofluorescence analysis revealed that many mast cells were present in the LSG and that those cells were located close to sympathetic neurons. The presence of leptin receptors on the mast cells was verified., Conclusions: Immune mast cells play an important supplementary role in the pathogenesis of leptin-induced LSG activation., (Copyright © 2018. Published by Elsevier B.V.)

Objectives: Female reproductive dysfunction occurs in patients with pathological loss of adipose tissue, i.e. lipodystrophy (LD). However, mechanisms remain largely unclear and treatment effects of adipocyte-derived leptin have not been assessed in LD animals., Methods: In the current study, C57Bl/6 LD mice on a low-density lipoprotein receptor knockout background were treated with leptin or saline for 8 weeks and compared to non-LD controls., Results: The number of pups born was 37% lower in breeding pairs consisting of LD female mice x non-LD male mice (n = 3.3) compared to LD male mice x non-LD female mice (n = 5.2) (p < 0.05). Mean uterus weight was significantly lower in the saline-treated LD group (18.8 mg) compared to non-LD controls (52.9 mg; p < 0.0001) and increased significantly upon leptin treatment (46.5 mg; p < 0.001). The mean number of corpora lutea per ovary was significantly lower in saline-treated LD animals compared to non-LD controls (p < 0.01) and was restored to non-LD control levels by leptin (p < 0.05). Mechanistically, mRNA expression of ovarian follicle-stimulating hormone receptor (p < 0.01) and estrogen receptor β (p < 0.05), as well as of pituitary luteinizing hormone β subunit (p < 0.001) and follicle-stimulating hormone β subunit (p < 0.05), was significantly upregulated in LD mice compared to non-LD controls. In addition, mean time to vaginal opening as a marker of puberty onset was delayed by 12.5 days in LD mice (50.9 days) compared to non-LD controls (38.4 days; p < 0.001)., Conclusions: Female LD animals show impaired fertility which is restored by leptin. Future studies should assess leptin as a subfertility treatment in human leptin-deficiency disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Leptin's role in bone formation has been reported, however, its mechanism of affecting bone metabolism is remaining unclear. In this study, we aimed to test whether leptin has a positive effect on fracture healing through the possible mechanism of increasing vascular endothelial growth factor (VEGF) expression in callus tissue., Methods: Standardized femur fractures were created in leptin-deficient ob/ob and wildtype C57BL/6J mice, and recombinant mouse leptin or its vehicle (physiological saline) was administered intraperitoneally during the study. Body weight, radiological, histologic and immunoblotting analyses were performed at different stages of fracture healing., Key Findings: The results showed that leptin treatment led to lower rate of body weight change in both mice genotypes. Radiological and histological analyses showed that the experimental groups had better fracture healing at 14, 21 and 28 days compared to the control groups. Leptin-treated groups had significantly higher VEGF expression in callus compared with the control groups at 2 and 3 weeks post-fracture except normal mice at 2 weeks, and leptin-deficient mice had higher VEGF levels in calluses than normal mice at the same timepoint., Conclusion: Low-dose systemically-administered leptin has a positive effect on promoting fracture healing during the latter stages in a clinically-relevant mouse bone fracture model, and increase callus VEGF levels., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Vascular disease can manifest as stenotic plaques or ectatic aneurysms. Human abdominal aortic aneurysms (AAA) comprise an inflammatory disease characterized by the predominance of T helper type 2 (Th2) cytokine expression. Leptin has been clearly demonstrated to play an important role in regulating Th0 cell to Th1. So, we hypothesize that leptin has a protective effect on aneurysm formation. In this study, we demonstrated that intraperitoneal injection of leptin attenuated Ang II-induced AAA formation in ApoE-/- mice with no effect on serum lipids and systolic blood pressure. To investigate the mechanisms involved, we found that leptin pretreatment exhibited decreased protein expression of matrix metalloproteinase 2 (MMP-2) and MMP-9 and increased transforming growth factor-β1 (TGF-β1). We also examined potential mechanism of leptin as a modulator of the immune response. Our results proved that pretreatment with leptin downregulated protein expression of Th2 cytokine IL-4 and mRNA levels of GATA-3, the key transcription factor for Th2 polarization, and upregulated Th1 cytokine INF-γ and T-bet, the key transcription factor for Th1 polarization. Taken together, leptin, with the effect of regulation of Th1/Th2 cytokines, may have therapeutic potential for the treatment of AAA. Leptin may constitute a novel therapeutic strategy to prevent AAA formation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Recombinant leptin (metreleptin) ameliorates hyperphagia and metabolic abnormalities in leptin-deficient humans with lipodystrophy. We aimed to determine whether metreleptin improves glucose and lipid metabolism in humans when food intake is held constant., Methods: Patients with lipodystrophy were hospitalized for 19 days, with food intake held constant by a controlled diet in an inpatient metabolic ward. In a nonrandomized, crossover design, patients previously treated with metreleptin (n = 8) were continued on metreleptin for 5 days and then taken off metreleptin for the next 14 days (withdrawal cohort). This order was reversed in metreleptin-naive patients (n = 14), who were reevaluated after 6 months of metreleptin treatment on an ad libitum diet (initiation cohort). Outcome measurements included insulin sensitivity by hyperinsulinemic-euglycemic clamp, fasting glucose and triglyceride levels, lipolysis measured using isotopic tracers, and liver fat by magnetic resonance spectroscopy., Results: With food intake constant, peripheral insulin sensitivity decreased by 41% after stopping metreleptin for 14 days (withdrawal cohort) and increased by 32% after treatment with metreleptin for 14 days (initiation cohort). In the initiation cohort only, metreleptin decreased fasting glucose by 11% and triglycerides by 41% and increased hepatic insulin sensitivity. Liver fat decreased from 21.8% to 18.7%. In the initiation cohort, changes in lipolysis were not independent of food intake, but after 6 months of metreleptin treatment on an ad libitum diet, lipolysis decreased by 30% (palmitate turnover) to 35% (glycerol turnover)., Conclusion: Using lipodystrophy as a human model of leptin deficiency and replacement, we show that metreleptin improves insulin sensitivity and decreases hepatic and circulating triglycerides and that these improvements are independent of its effects on food intake., Trial Registration: ClinicalTrials.gov NCT01778556FUNDING. This research was supported by the intramural research program of the NIDDK.

Insulin potentiates leptin effects on muscle accrual and glucose homeostasis. However, the relationship between leptin's central effects on peripheral insulin sensitivity and the associated structural changes remain unclear. We hypothesized that central leptin infusion modifies muscle size through activation of insulin signaling. Muscle insulin signaling, enzymes of fatty acid metabolism, mitochondrial respiratory chain complexes, proliferating cell nuclear antigen (PCNA) and fiber area were analyzed in the gastrocnemius of chronic central infused (L), pair-fed (PF) and control rats. PCNA-positive nuclei, fiber area, GLUT4 and glycogen levels and activation of Akt and mechanistic target of rapamycin were increased in L, with no changes in PF. Acetyl-CoA carboxylase-β mRNA levels and non-esterified fatty acid and triglyceride content were reduced and carnitine palmitoyltransferase-1b expression and mitochondrial complexes augmented in L. These results suggest that leptin promotes an increase in muscle size associated with improved insulin signaling favored by lipid profile., (Copyright © 2017 Elsevier B.V. All rights reserved.)

In birds little is known about the hormonal signals that communicate nutritional state to the brain and regulate appetitive behaviours. In mammals, the peptide hormones ghrelin and leptin elevate and inhibit consumption and food hoarding, respectively. But in birds, administration of both ghrelin and leptin inhibit food consumption. The role of these hormones in the regulation of food hoarding in avian species has not been examined. To investigate this, we injected wild caught coal tits ( Periparus ater ) with leptin, high-dose ghrelin, low-dose ghrelin and a saline control in the laboratory. We then measured food hoarding and mass gain, as a proxy of food consumption, every 20 min for 2 h post-injection. Both high-dose ghrelin and leptin injections significantly reduced hoarding and mass gain compared with controls. Our results provide the first evidence that hoarding behaviour can be reduced by both leptin and ghrelin in a wild bird. These findings add to evidence that the hormonal control of food consumption and hoarding in avian species differs from that in mammals. Food hoarding and consumptive behaviours consistently show the same response to peripheral signals of nutritional state, suggesting that the hormonal regulation of food hoarding has evolved from the consumption regulatory system., (© 2018 The Author(s).)

Leptin, produced primarily by the adipose tissue, acts as a pro-inflammatory modulator, thereby contributing to the development of obesity-related disease. Although high levels of leptin in the obese are closely related to gastroesophageal reflux disease, the mechanism by which leptin influences esophageal inflammation remains unknown. Macrophage migration inhibitory factor (MIF) is produced by immune cells, such as T lymphocytes and macrophages, and MIF is known to induce the production of tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6). We therefore investigated the mechanism whereby leptin aggravates reflux esophagitis, by focusing on esophageal tissue levels of MIF and CD3+ T lymphocytes, both of which are crucial for the reflux-induced epithelial damage. Esophageal inflammation was surgically induced in male Wistar rats by ligating the forestomach and narrowing the duodenum to facilitate gastroesophageal reflux, followed by administration of leptin or vehicle with an osmotic pump system for 1 week. We demonstrated that the administration of leptin exacerbated the reflux esophagitis with the apparent infiltration of CD3+ T lymphocytes and caused the significant increase in the esophageal tissue levels of MIF. Moreover, the leptin caused increases in the esophageal tissue levels of TNF-α, IL-1β and IL-6, downstream targets of MIF. Importantly, the increases in these pro-inflammatory cytokines were accompanied by increased protein levels of phospho-STAT3 and phospho-AKT, pivotal molecules of leptin signaling pathways. In conclusion, through enhancing the MIF-induced inflammatory signaling, leptin could contribute to the development of gastroesophageal reflux disease.

Background: Leptin is a peptide hormone produced by adipose tissue whose basic function is regulating energy balance and sympathetic outflow. Previous studies have shown that increased nerve activity of the left stellate ganglion (LSG) promotes ventricular arrhythmia (VA)., Objective: The purpose of this study was to investigate whether leptin could facilitate VA through activation of the LSG., Methods: Sixteen pentobarbital-anesthetized dogs were divided into a control group (saline; n = 8) and a leptin group (leptin; n = 8). Microinjections of either 0.1 mL saline or leptin (18 μg) were injected into the LSG. Action potential duration (APD) of the myocyte and the function and neural activity of the LSG were measured at different time points. VA induced by occlusion of the left anterior descending branch was continuously measured for 1 hour. At the end of the experiment, the LSG tissues were collected for molecular detections., Results: Compared with the control group, leptin microinjection resulted in (1) significant enhancement in the incidence of VA; (2) significant decrease in APD and increase in APD dispersion; and (3) significant increase in the function and neural activity of the LSG. Mechanistically, the leptin receptor was found in the LSG, and its signaling was significantly activated in the leptin-injected group. Additionally, leptin microinjection markedly increased the expression of proinflammatory cytokines., Conclusion: LSG activation induced by leptin microinjection promotes ischemia-induced VAs. Activated leptin receptor signaling and up-regulation of proinflammatory cytokines in the LSG may be responsible for these effects., (Copyright © 2017 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

We evaluated the contribution of brown adipose tissue (BAT) sympathetic innervation on central leptin-mediated weight loss. In a short- and long-term study, F344BN rats were submitted to either a denervation of interscapular BAT (Denervated) or a sham operation (Sham). Animals from each group received the Ob (Leptin) or green fluorescent protein (GFP; Control) gene through a single injection of recombinant adeno-associated virus delivered centrally. Changes in body weight were recorded for 14 or 35 days, after which adipose tissues and skeletal muscles were weighed. In both studies, hypothalamic phosphorylated STAT3 (P-STAT3) was significantly higher in Sham-Leptin and Denervated-Leptin groups compared with their respective Control groups ( P < 0.01), indicating that leptin signaling was enhanced at the end point. We measured uncoupling protein 1 (UCP1), a marker of BAT thermogenic activity, and found a significant induction in Leptin in Sham animals ( P < 0.001) but not in Denervated animals, demonstrating that BAT UCP1 protein was only induced in Sham rats. Both Sham-Leptin and Denervated-Leptin rats lost ~15% of their initial body weight ( P < 0.001) by day 14 and reached a maximum of 18% body weight loss that stabilized over week 3 of treatment, indicating that sympathetic outflow to BAT is not required for leptin-mediated weight loss. In summary, interscapular BAT (iBAT) denervation did not prevent body weight loss following central leptin gene delivery. The present data show that sympathetic innervation of iBAT is not essential for leptin-induced body weight loss.

Celia's encephalopathy (progressive encephalopathy with/without lipodystrophy, PELD) is a recessive neurodegenerative disease that is fatal in childhood. It is caused by a c.985C>T variant in the BSCL2/seipin gene that results in an aberrant seipin protein. We evaluated neurological development before and during treatment with human recombinant leptin (metreleptin) plus a dietary intervention rich in polyunsaturated fatty acids (PUFA) in the only living patient. A 7 years and 10 months old girl affected by PELD was treated at age 3 years with metreleptin, adding at age 6 omega-3 fatty acid supplementation. Her mental age was evaluated using the Battelle Developmental Inventory Screening Test (BDI), and brain PET/MRI was performed before treatment and at age 5, 6.5, and 7.5 years. At age 7.5 years, the girl remains alive and leads a normal life for her mental age of 30 months, which increased by 4 months over the last 18 months according to BDI. PET images showed improved glucose uptake in the thalami, cerebellum, and brainstem. This patient showed a clear slowdown in neurological regression during leptin replacement plus a high PUFA diet. The aberrant BSCL2 transcript was overexpressed in SH-SY5Y cells and was treated with docosahexaenoic acid (200 µM) plus leptin (0.001 mg/ml) for 24 h. The relative expression of aberrant BSCL2 transcript was measured by qPCR. In vitro studies showed significant reduction (32%) in aberrant transcript expression. This therapeutic approach should be further studied in this devastating disease.

Subjects spending much time sitting have increased risk of obesity but the mechanism for the antiobesity effect of standing is unknown. We hypothesized that there is a homeostatic regulation of body weight. We demonstrate that increased loading of rodents, achieved using capsules with different weights implanted in the abdomen or s.c. on the back, reversibly decreases the biological body weight via reduced food intake. Importantly, loading relieves diet-induced obesity and improves glucose tolerance. The identified homeostat for body weight regulates body fat mass independently of fat-derived leptin, revealing two independent negative feedback systems for fat mass regulation. It is known that osteocytes can sense changes in bone strain. In this study, the body weight-reducing effect of increased loading was lost in mice depleted of osteocytes. We propose that increased body weight activates a sensor dependent on osteocytes of the weight-bearing bones. This induces an afferent signal, which reduces body weight. These findings demonstrate a leptin-independent body weight homeostat ("gravitostat") that regulates fat mass., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

The role of central leptin in regulating the heart from lipid accumulation in lean leptin-sensitive animals has not been fully elucidated. Herein, we investigated the effects of central leptin infusion on the expression of genes involved in cardiac metabolism and its role in the control of myocardial triacylglyceride (TAG) accumulation in adult Wistar rats. Intracerebroventricular (icv) leptin infusion (0.2 µg/day) for 7 days markedly decreased TAG levels in cardiac tissue. Remarkably, the cardiac anti-steatotic effects of central leptin were associated with the selective upregulation of gene and protein expression of peroxisome proliferator-activated receptor β/δ (PPARβ/δ, encoded by Pparb/d ) and their target genes, adipose triglyceride lipase (encoded by Pnpla2 , herefater referred to as Atgl ), hormone sensitive lipase (encoded by Lipe , herefater referred to as Hsl ), pyruvate dehydrogenase kinase 4 ( Pdk4 ) and acyl CoA oxidase 1 ( Acox1 ), involved in myocardial intracellular lipolysis and mitochondrial/peroxisomal fatty acid utilization. Besides, central leptin decreased the expression of stearoyl-CoA deaturase 1 ( Scd1 ) and diacylglycerol acyltransferase 1 ( Dgat1 ) involved in TAG synthesis and increased the CPT-1 independent palmitate oxidation, as an index of peroxisomal β-oxidation. Finally, the pharmacological inhibition of PPARβ/δ decreased the effects on gene expression and cardiac TAG content induced by leptin. These results indicate that leptin, acting at central level, regulates selectively the cardiac expression of PPARβ/δ, contributing in this way to regulate the cardiac TAG accumulation in rats, independently of its effects on body weight., (© 2018 Society for Endocrinology.)

Synaptic transmission controls brain activity and behaviors, including food intake. Leptin, an adipocyte-derived hormone, acts on neurons located in the lateral hypothalamic area (LHA) to maintain energy homeostasis and regulate food intake behavior. The specific synaptic mechanisms, cell types, and neural projections mediating this effect remain unclear. In male mice, using pathway-specific retrograde tracing, whole-cell patch-clamp recordings and post hoc cell type identification, we found that leptin reduces excitatory synaptic strength onto both melanin-concentrating hormone- and orexin-expressing neurons projecting from the LHA to the ventral tegmental area (VTA), which may affect dopamine signaling and motivation for feeding. A presynaptic mechanism mediated by distinct intracellular signaling mechanisms may account for this regulation by leptin. The regulatory effects of leptin depend on intact leptin receptor signaling. Interestingly, the synaptic regulatory function of leptin in the LHA-to-VTA neuronal pathway is highly sensitive to energy states: both energy deficiency (acute fasting) and excessive energy storage (high-fat diet-induced obesity) blunt the effect of leptin. These data revealed that leptin may regulate synaptic transmission in the LHA-to-VTA neurocircuitry in an inverted "U-shape" fashion dependent on plasma glucose levels and related to metabolic states. SIGNIFICANCE STATEMENT The lateral hypothalamic area (LHA) to ventral tegmental area (VTA) projection is an important neural pathway involved in balancing whole-body energy states and reward. We found that the excitatory synaptic inputs to both orexin- and melanin-concentrating hormone expressing LHA neurons projecting to the VTA were suppressed by leptin, a peptide hormone derived from adipocytes that signals peripheral energy status to the brain. Interestingly, energy states seem to affect how leptin regulates synaptic transmission since both the depletion of energy induced by acute food deprivation and excessive storage of energy by high-fat diet feeding dampen the suppressive effect of leptin on synaptic transmission. Together, these data show that leptin regulates synaptic transmission and might be important for maintaining energy homeostasis., (Copyright © 2017 the authors 0270-6474/17/3711854-13$15.00/0.)

Interleukin (IL)-6 - / - mice develop mature onset obesity, whereas i.c.v. injection of IL-6 decreases obesity in rodents. Moreover, levels of IL-6 in cerebrospinal fluid (CSF) were reported to be inversely correlated with obesity in humans. Tanycytes lining the base of the third ventricle (3V) in the hypothalamus have recently been reported to be of importance for metabolism. In the present study, we investigated whether tanycytes could respond to IL-6 in the CSF. With immunohistochemistry using a well characterised antibody directed against the ligand binding receptor for IL-6, IL-6 receptor α (IL-6Rα), it was found that tanycytes, identified by the two markers, vimentin and dopamine- and cAMP-regulated phosphoprotein of 32 kDa, contained IL-6Rα. There were fewer IL-6Rα on another type of ventricle-lining cells, ependymal cells, as identified by the marker glucose transporter-1. To demonstrate that the immunoreactive IL-6Rα were responsive to IL-6, we injected IL-6 i.c.v. This treatment increased immunoreactive phosphorylated signal transducer and activator of transcription-3 (pSTAT3) in tanycytes after 5 minutes and in cells in the medial part of the arcuate nucleus after 5 and 15 minutes. Intracerebroventricular injection of leptin exerted similar effects. As expected, i.p. injection of leptin also induced pSTAT3 staining in the hypothalamus, whereas i.p. IL-6 injection had little effect on this parameter. Intracerebroventricular or i.p. injection of vehicle only had no effect on pSTAT3-immunoreactivity. In summary, there are functional IL-6Rα on tanycytes at the bottom of the 3V, in agreement with the possibility that ventricular administration of IL-6 decreases obesity in mice via an effect on this cell type., (© 2017 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd. on behalf of British Society for Neuroendocrinology.)

The hypothalamic-pituitary-adrenal (HPA) axis has been postulated to play a major role in mediating the antidiabetic effects of leptin. We tested if the pituitary is essential for the chronic central nervous system mediated actions of leptin on metabolic and cardiovascular function in insulin-dependent diabetic and non-diabetic rats. Male 12-week-old hypophysectomized Sprague-Dawley rats (Hypo, n = 5) were instrumented with telemetry probes for determination of mean arterial pressure (MAP) and heart rate (HR) 24-hrs/day and an intracerebroventricular (ICV) cannula was placed into the brain lateral ventricle for continuous leptin infusion. In additional groups of Hypo and control rats (n = 5/group), diabetes was induced by single injection of streptozotocin (50 mg/kg, IP). Hypo rats were lighter, had lower MAP and HR (83±4 and 317±2 vs 105±4 mmHg and 339±4 bpm), with similar caloric intake per kilogram of body weight and fasting plasma glucose levels (84±4 vs 80±4 mg/dl) compared to controls. Chronic ICV leptin infusion (7 days, 0.62 μg/hr) in non-diabetic rats reduced caloric intake and body weight (-10%) in Hypo and control rats and markedly increased HR in control rats (~25 bpm) while causing only modest HR increases in Hypo rats (8 bpm). In diabetic Hypo and control rats, leptin infusion reduced caloric intake, body weight and glucose levels (323±74 to 99±20 and 374±27 to 108±10 mg/dl), respectively; however, the effects of leptin on HR were abolished in Hypo rats. These results indicate that hypophysectomy attenuates leptin's effect on HR regulation without altering leptin's ability to suppress appetite or normalize glucose levels in diabetes.

Aim: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action., Methods: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured., Results: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia., Conclusions: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.

Leptin is released in response to increased triglyceride storage in adipocytes and impacts body weight, but has drawbacks such as poor therapeutic effect and side effects when delivered systemically. Leptin also modifies adipocyte sensitivity to insulin to inhibit lipid accumulation. Here, light-triggered degradation of hydrogels was used to improve accuracy and effectiveness for sustained and controllable release. In our approach, leptin was entrapped within methylcellulose (MC)-based hydrogels, with incorporation of gold nanoparticles (NP). The incorporation of gold NP into MC hydrogels led to a tunable light irradiation response that dictated the hydrogel release rate of leptin. This manuscript demonstrates feasibility in designing tunable thermosensitive hydrogels for loading multimodality therapeutic agents to enhance the bioactivity of leptin for obesity therapy., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Obesity, diabetes and fatty liver disease are extremely common in leptin-resistant patients. Dysfunction of leptin or its receptor is associated with obesity. The present study aimed to assess the effects of intramuscular injection of exogenous leptin or its receptor on fat deposition and leptin-insulin feedback regulation. Forty-five 40-day old female Sprague Dawley (SD) rats were injected thrice with leptin or its receptor intramuscularly. Adiposity and fat deposition were assessed by assessing the Lee's index, body weight, food intake, and total cholesterol, high density lipoprotein, low density lipoprotein, and triglyceride levels, as well as histological properties (liver and adipose tissue). Serum glucose, leptin, and insulin amounts were evaluated, and glucose tolerance assessed to monitor glucose metabolism in SD rats; pancreas specimens were analyzed immunohistochemically. Hypothalamic phosphorylated Janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), and phosphatidylinositol-3-kinase (PI3K) signaling, and hepatic sterol regulatory element binding protein-1 (SREBP-1) were qualified by Western blotting. Leptin receptor immunogen reduced fat deposition, increased appetite, and lowered serum leptin levels, enhancing STAT3 signaling in hypothalamus and down-regulating hepatic SREBP-1. In contrast, SD rats administered leptin immunogen displayed significantly increased body weight and fat deposition, with up-regulated SREBP-1, indicating adiposity occurrence. SD rats administered leptin immunogen also showed glucose intolerance, β- cell reduction in the pancreas, and deregulation of JAK2-STAT3/PI3K signaling, indicating that Lep rats were at risk of diabetes. In conclusion, intramuscular injection of exogenous leptin or its receptor, a novel rat model approach, can be used in obesity pathogenesis and therapeutic studies., (Copyright © 2017. Published by Elsevier Inc.)

In common forms of obesity, leptin fails to convey its regulatory effect. This so called "leptin resistance" is not well understood, and solving this puzzle is a key to understanding how obesity develops. In the present study, we investigated the temporal and regional onset of leptin resistance in response to a diet enriched with long-chain saturated fatty acids (high-fat diet; HFD) in mice. Mice were exposed to either a low-fat diet (LFD) or a HFD for 4 hours, 24 hours, 10 days and 28 days. Mice in each group received an i.p. injection of either phosphate-buffered saline or leptin and the number of phosphorylated signal transducer and activator of transcription-3 (pSTAT3) immunoreactive (-IR) cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and dorsomedial nucleus of the hypothalamus (DMH) was analysed 30 or 120 minutes after treatment. In the ARC, as soon as 24 hours of HFD, the molecular leptin response was reduced by 40% (P≤.01). Compared to at 24 hours, after 10 days, the number of leptin-induced pSTAT3-IR cells was elevated after 120 minutes, suggesting a sustained response and a partial return of leptin sensitivity. After 28 days, leptin failed to induce the number of pSTAT3-IR over control levels, suggesting a markedly reduced sensitivity to leptin. In the VMH after 24 hours, we observed a 50% reduction in leptin-induced pSTAT-3-IR cells, followed by a further decline after 10 days. However, after 28 days, there was a significant increase in pSTAT-3-IR cells (P≤.05), indicating partial recovery of leptin sensitivity. By contrast to these two regions, in the DMH, no loss of leptin sensitivity was observed at any time-point. These findings demonstrate that a loss of sensitivity to leptin occurs rapidly after exposure to HFD in the ARC and VMH but not the DMH. However, there appears to be a biphasic pattern of leptin responsiveness, with a partial return of leptin sensitivity occurring after 10 days in the arcuate nucleus, and after 28 days in the VMH. By 28 days, the response to leptin in the arcuate nucleus was completely lost. These findings suggest that the molecular responses to leptin are altered after high-fat feeding in a time- and region-specific manner., (© 2017 British Society for Neuroendocrinology.)

Diet-induced obesity is associated with hypothalamic inflammation and this phenomenon has been proposed to explain leptin resistance. In the present study, we used a short-term high-fat diet (HFD) paradigm for 10 days and analysed the cellular and physiological responses to leptin administration in C57BL6 mice. In parallel, we performed glial fibrillary acidic protein immunostaining to measure the presence of astrocytes in the arcuate nucleus of the hypothalamus (ARH) after 10 days and 20 weeks of HFD. Interestingly, the results obtained demonstrate that the presence of star-like astrocytes is significantly increased after 10 days of HFD, although this is not associated with the absence of cellular and physiological response to leptin administration in mice. Taken together, the results of the present study suggest that star-like astrocytes rapidly increase in numbers in the ARH in response to HFD, although this phenomenon cannot explain the development of leptin resistance by itself., (© 2017 British Society for Neuroendocrinology.)

T-2 toxin and its metabolite HT-2 toxin are one of the most toxic mycotoxins of type A-trichothecenes, which are produced mainly by Fusarium species. Therefore, study of Fusarium toxins T-2 toxin and HT-2 toxin is an essential issue because they could also play role in failures of reproductive functions as well as endocrine system of domestic animals. Assessment of the effect of A-trichothecene mycotoxin HT-2 toxin alone or combined with insulin-like growth factor (IGF-I), leptin and ghrelin on estradiol secretion by rabbit ovarian fragments in vitro was done. Rabbit ovarian fragments were incubated without (control group) or with HT-2 toxin, or its combinations with IGF-I, leptin and ghrelin at various concentrations for 24 h. Secretion of 17beta-estradiol was determined by ELISA. Firstly, HT-2 toxin at the doses 10 and 100 ng.ml(-1), but not at 1 ng.ml(-1) decreased 17beta-estradiol secretion by ovarian fragments. Secondly, 17beta-estradiol secretion was not affected by HT-2 toxin exposure combined with growth factor IGF-I, metabolic hormones leptin and ghrelin. In conclusion, HT-2 toxin has potent direct dose-dependent effects on ovarian steroidogenesis in rabbits. These direct effects of HT-2 mycotoxin on ovarian steroidogenesis could impact negatively on the reproductive performance of rabbits.

We previously reported that low-dose leptin infusions into the third or fourth ventricle that do not affect energy balance when given independently cause rapid weight loss when given simultaneously. Therefore, we tested whether hindbrain leptin enhances the response to forebrain leptin or whether forebrain leptin enhances the response to hindbrain leptin. Rats received fourth-ventricle infusions of saline or 0.01, 0.1, 0.3, or 0.6 μg leptin/day for 13 days. On days 9 and 13 , 0.1 μg leptin was injected into the third ventricle. The injection inhibited food intake for 36 h in saline-infused rats but for 60 h in those infused with 0.6 μg leptin/day. Leptin injection increased intrascapular brown fat temperature in leptin-infused, but not saline-infused, rats. In a separate experiment, rats received third-ventricle infusions of saline or 0.005, 0.01, 0.05, or 0.1 μg leptin/day and fourth-ventricle injections of 1.0 μg leptin on days 9 and 13 Leptin injection inhibited food intake, respiratory exchange ratio, and 14-h food intake in rats infused with saline or the two lowest doses of leptin. There was no effect with higher-dose leptin infusions because food intake, body fat, and lean mass were already inhibited. These data suggest that activation of leptin receptors in the hindbrain enhances the response to third-ventricle leptin, whereas activation of forebrain leptin receptors does not enhance the response to fourth-ventricle leptin, consistent with our previous finding that weight loss in rats treated with fourth-ventricle leptin is associated with indirect activation of hypothalamic STAT3., (Copyright © 2017 the American Physiological Society.)

The objective of this study was to determine the effect of leptin administration on the development, vascularization and function of Corpus luteum (CL) in alpacas submitted to pre-ovulatory fasting. Fourteen alpacas were kept in fasting conditions for 72h and received five doses of o-leptin (2μg/kg e.v.; Leptin group) or saline (Control group) every 12h. Ovulation was induced with a GnRH dose (Day 0). The ovaries were examined every other day by trans-rectal ultrasonography (7.5MHz; mode B and power Doppler) from Day 0 to 13 to determine the pre-ovulatory follicle diameter and ovulation, and then to monitor CL diameter and vascularization until the regression phase. Serial blood samples were taken after GnRH treatment to determine plasma LH concentration; and every other day from Days 1 to 13 to determine plasma progesterone and leptin concentrations. The pre-ovulatory follicle and CL diameter, LH, progesterone and leptin plasma concentrations were not affected by treatment (P>0.05). The vascularization area of the CL was, nevertheless, affected by the treatment (P<0.01) with significant differences between groups at Days 3, 7 and 9 (P<0.05). The Leptin group had a larger maximum vascularization area (0.67±0.1 compared with 0.35±0.1cm 2 ; P<0.05). In addition, there was a positive correlation between CL vascularization, CL diameter and plasma progesterone. The exogenous administration of leptin during pre-ovulatory fasting increased the vascularization of the CL in alpacas in vivo., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Growth factor signaling via insulin receptor (IR) and IGF-1 receptor (IGF1R) plays several important roles in the pathogenesis of metabolic syndrome and diabetes. OSI-906 (linsitinib), an anti-tumor drug, is an orally bioavailable dual inhibitor of IR and IGF1R. To investigate the recovery from metabolic changes induced by the acute inhibition of IR and IGF1R in adult mice, mice were treated with OSI-906 or a vehicle for 7 days and the results were analyzed on the last day of injection (Day 7) or after 7 or 21 days of withdrawal (Day 14 or Day 28). On day 7, the visceral white fat mass was significantly reduced in mice treated with OSI-906 accompanied by a reduced expression of leptin and an increased expression of the lipolysis-related genes Lpl and Atgl. Interestingly, the lipoatrophy and the observed changes in gene expression were completely reversed on day 14. Similarly, liver steatosis and β cell proliferation were transiently observed on day 7 but had disappeared by day 14. Taken together, these results suggest that this model for the acute inhibition of systemic IR/IGF1R signaling may be useful for investigating the recovery from metabolic disorders induced by impaired growth factor signaling.

The circulating concentrations of adiponectin, an antidiabetic adipokine, have been shown to be reduced in obesity, in relation to an increase in inflammation. The aim of the present work was to assess the effect of leptin replacement on adiponectin levels and expression as well as on markers of oxidative stress and inflammation in leptin-deficient ob/ob mice. Twelve-week-old male mice (n = 7-10 per group) were treated with either saline (wild type and ob/ob mice) or leptin (ob/ob mice) for 18 days. A third group of ob/ob mice was treated with saline and pair-fed to the amount of food consumed by the leptin-treated group. Leptin replacement restored values of adiponectin (P < 0.001), reduced circulating 8-isoprostane and serum amyloid A (SAA) levels (P < 0.05 for both), and significantly downregulated the increased gene expression of osteopontin (Spp1, P < 0.05), Saa3 (P < 0.05), Cd68 (P < 0.01), Il6 (P < 0.01) and NADPH oxidase (Nox1 and Nox2, P < 0.01) in the perirenal WAT and Spp1 (P < 0.05) in the liver of ob/ob mice. In cultured adipocytes from ob/ob mice, leptin increased (P < 0.05) the mRNA expression and secretion of adiponectin. We concluded that circulating concentrations of adiponectin are positively regulated by leptin and ameliorate obesity-associated oxidative stress and inflammation in mice.

This study describes the localization of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3, synthetic peptide leptin mimetics, in the hypothalamus of Swiss Webster and C57BL/6J wild-type mice, leptin-deficient ob/ob mice, and leptin-resistant diet-induced obese (DIO) mice. The mice were given [D-Leu-4]-OB3 or MA-[D-Leu-4]-OB3 in 0.3% dodecyl maltoside by oral gavage. Once peak serum concentrations were reached, the mice received a lethal dose of pentobarbital and were subjected to intracardiac perfusion fixation. The brains were excised, post-fixed in paraformaldehyde, and cryo-protected in sucrose. Free-floating frozen coronal sections were cut at 25-µm and processed for imaging by immunofluorescence microscopy. In all four strains of mice, dense staining was concentrated in the area of the median eminence, at the base and/or along the inner wall of the third ventricle, and in the brain parenchyma at the level of the arcuate nucleus. These results indicate that [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 cross the blood-brain barrier and concentrate in an area of the hypothalamus known to regulate energy balance and glucose homeostasis. Most noteworthy is the localization of [D-Leu-4]-OB3 immunoreactivity within the hypothalamus of DIO mice via a conduit that is closed to leptin in this rodent model, and in most cases of human obesity. Together with our previous studies describing the effects of [D-Leu-4]-OB3 and MA-[D-Leu-4]-OB3 on energy balance, glucose regulation, and signal transduction pathway activation, these findings are consistent with a central mechanism of action for these synthetic peptide leptin mimetics, and suggest their potential usefulness in the management of leptin-resistant obesity and type 2 diabetes in humans., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Interactions of hypothalamic signaling pathways that control body temperature (BT), blood pressure (BP), and energy balance are poorly understood. We investigated whether the chronic BP and metabolic actions of leptin are differentially modulated by changes in ambient temperature ( T A ). Mean arterial pressure (MAP), heart rate (HR), BT, motor activity (MA), and oxygen consumption ( V o 2 ) were measured 24 h/d at normal laboratory T A (23°C), at thermoneutral zone (TNZ, 30°C) for mice or during cold exposure (15°C) in male wild-type mice. After control measurements, leptin (4 μg/kg/min) or saline vehicle was infused for 7 d. At TNZ, leptin reduced food intake (-11.0 ± 0.5 g cumulative deficit) and body weight by 6% but caused no changes in MAP or HR. At 15°C, leptin infusion did not alter food intake but increased MAP and HR (8 ± 1 mmHg and 33 ± 7 bpm), while V o 2 increased by ∼10%. Leptin reduced plasma glucose and insulin levels at 15°C but not at 30°C. These results demonstrate that the chronic anorexic effects of leptin are enhanced at TNZ, while its effects on insulin and glucose levels are attenuated and its effects on BP and HR are abolished. Conversely, cold T A caused resistance to leptin's anorexic effects but amplified its effects to raise BP and reduce insulin and glucose levels. Thus, the brain circuits by which leptin regulates food intake and cardiovascular function are differentially influenced by changes in T A -Do Carmo, J. M., da Silva, A. A., Romero, D. G., Hall, J. E. Changes in ambient temperature elicit divergent control of metabolic and cardiovascular actions by leptin., (© FASEB.)

Background: In the present work, we studied the modulatory effect of Leptin (Lep) against pro-inflammatory cytokines, tumour necrosis factor-alpha (TNFα), interleukin 1-beta (IL1β) and interferon-gamma (IFNγ), in primary glial cell cultures., Methods: Glial cultures were treated with pro-inflammatory cytokines (TNFα, 20ng/ml; IL1β, 20ng/ml; IFNγ 20ng/ml). Cells were pre-treated with Lep 500nM, 1h prior to cytokine treatment. NO released from glial cells was determined using the Griess reaction. Cell viability was determined by the MTT method. Protein expression was determined by western blot., Results: Pre-treatment with 500nM Lep produced an inhibitory effect on inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production after glial cells exposure to pro-inflammatory cytokines. Anti-inflammatory effect can be related to a decrease in P38 MAP Kinase (MAPK) pathway activity. Treatment of glial cell cultures with Lep also reduced the intrinsic apoptotic pathway (cytochrome c release and caspase-3 activation)., Conclusions: We suggest that Lep would act as an anti-inflammatory factor in glial cells exposed to pro-inflammatory cytokines, exerting its function on p38 MAPK pathway and reducing NO production., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Leptin receptors (LepRs) expressed in the midbrain contribute to the action of leptin on feeding regulation. The midbrain neurons release a variety of neurotransmitters including dopamine (DA), glutamate and GABA. However, which neurotransmitter mediates midbrain leptin action on feeding remains unclear. Here, we showed that midbrain LepR neurons overlap with a subset of dopaminergic, GABAergic and glutamatergic neurons. Specific removal of vesicular monoamine transporter 2 (VMAT2) in midbrain LepR neurons (KO mice) disrupted DA accumulation in vesicles, but failed to cause a significant change in the evoked release of either glutamate or GABA to downstream neurons. While KO mice showed no differences on chow, they presented a reduced high-fat diet (HFD) intake and resisted to HFD-induced obesity. Specific activation of midbrain LepR neurons promoted VMAT2-dependent feeding on chow and HFD. When tested with an intermittent access to HFD where first 2.5-h HFD eating (binge-like) and 24-h HFD feeding were measured, KO mice exhibited more binge-like, but less 24-h HFD feeding. Interestingly, leptin inhibited 24-h HFD feeding in controls but not in KO mice. Thus, VMAT2-mediated neurotransmission from midbrain LepR neurons contributes to both binge-like eating and HFD feeding regulation.

Leptin, one of the main hormones controlling energy homeostasis, has been associated with different cancer types. In oral cancer, its effect is not well understood. We investigated, through in vitro and in vivo assays, whether leptin can affect the neoplastic behavior of oral squamous cell carcinoma. Expression of genes possibly linked to the leptin pathway was assessed in leptin-treated oral squamous cell carcinoma cells and also in tissue samples of oral squamous cell carcinoma and oral mucosa, including leptin, leptin receptor, hypoxia-inducible factor 1-alpha, E-cadherin, matrix metalloproteinase-2, matrix metalloproteinase-9, Col1A1, Ki67, and mir-210. Leptin treatment favored higher rates of cell proliferation and migration, and reduced apoptosis. Accordingly, leptin-treated oral squamous cell carcinoma cells show decreased messenger RNA caspase-3 expression, and increased levels of E-cadherin, Col1A1, matrix metalloproteinase-2, matrix metalloproteinase-9, and mir-210. In tissue samples, hypoxia-inducible factor 1-alpha messenger RNA and protein expression of leptin and leptin receptor were high in oral squamous cell carcinoma cases. Serum leptin levels were increased in first clinical stages of the disease. In animal model, oral squamous cell carcinoma-induced mice show higher leptin receptor expression, and serum leptin level was increased in dysplasia group. Our findings suggest that leptin seems to exert an effect on oral squamous cell carcinoma cells behavior and also on molecular markers related to cell proliferation, migration, and tumor angiogenesis.

This study tested whether ganglionic blockade or hepatic vagotomy attenuates the chronic central nervous system (CNS)-mediated antidiabetic and cardiovascular effects of leptin. Male Sprague-Dawley rats were instrumented with telemetry probes and arterial and venous catheters for determination of blood pressure (BP), heart rate (HR), blood sampling, and intravenous (iv) infusions. An intracerebroventricular (ICV) cannula was placed into the brain lateral ventricle for infusion of leptin or vehicle. After control measurements, streptozotocin (STZ) was injected iv (50 mg/kg) to induce diabetes, and 5 days later leptin ( n = 6) or saline vehicle ( n = 5) was infused ICV for 12 days via osmotic pumps. Beginning on day 6 of leptin treatment, the ganglionic blocker hexamethonium (15 mg·kg -1 ·day -1 iv) was infused, while leptin infusion was continued, to assess the role of the autonomic nervous system. Induction of diabetes was associated with increases in blood glucose (98 ± 7 to 350 ± 19 mg/dl), food intake (23 ± 3 to 43 ± 3 g/day), decreases in HR (-70 ± 11 beats/min), polyuria, and increased water consumption, which were all completely normalized by ICV leptin infusion. Although hexamethonium attenuated leptin's effect on HR, it failed to impair leptin's ability to restore euglycemia or to prevent the polyuria or increased water intake in STZ-diabetic rats. We also found that after pretreatment with hexamethonium ( n = 8), ICV leptin infusion, during continued ganglionic blockade, completely normalized blood glucose in diabetic rats. In addition, selective hepatic vagotomy did not attenuate leptin's ability to restore euglycemia in diabetic rats. These results suggest that leptin's powerful chronic CNS antidiabetic actions are mediated primarily via nonautonomic mechanisms., (Copyright © 2017 the American Physiological Society.)

It is well established that the maintenance of energy expenditure is linked to active hypothalamic neural mechanisms controlling adaptive stimuli such as food intake. Variations of glucose levels and hormonal (leptin plus orexin-A) parameters, which are involved with energy homeostasis during different behavioral states, have not yet been fully defined. In this first study, behavioral analyses of an unpredictable stress model dealing with the actions of a sub-chronic administration of orexin-A (ORX-A) and the anti-hunger neuropeptide, i.e., leptin (LEP) within the hypothalamic suprachiasmatic (SCH) nucleus, were conducted on the valuable hibernating rodent (hamster; Mesocricetus auratus) model noted for its distinct depression and anxiety states. Treatment with LEP accounted for a notable reduction (p < 0.01) of body weight in stressed hamsters that not only executed very evident (p < 0.001) movements to and from elevated plus maze (EPM) but also spent less time in the dark area of the light-dark box test (LDT). Conversely, ORX-A predominantly evoked anxiogenic effects that were inverted by LEP. Interestingly, the anti-hunger neuropeptide accounted for both down-regulated NPY1 transcripts in mostly lateral-posterior hypothalamic areas while up-regulated levels were detected in the parietal cerebral cortex, hippocampus, and amygdala, which largely behaved in an opposite manner to ORX-A-dependent effects. Overall, the present findings corroborate a predominating LEPergic effect of the SCH toward the reduction of hamster anxiety-like behaviors with respect to that of ORX-A signaling, which may constitute useful therapeutic targets for stress-related obesity states.

Migraine is a disabling brain disorder involving abnormal trigeminovascular activation and sensitization. Fasting or skipping meals is considered a migraine trigger and altered fasting glucose and insulin levels have been observed in migraineurs. Therefore peptides involved in appetite and glucose regulation including insulin, glucagon and leptin could potentially influence migraine neurobiology. We aimed to determine the effect of insulin (10U·kg -1 ), glucagon (100μg·200μl -1 ) and leptin (0.3, 1 and 3mg·kg -1 ) signaling on trigeminovascular nociceptive processing at the level of the trigeminocervical-complex and hypothalamus. Male rats were anesthetized and prepared for craniovascular stimulation. In vivo electrophysiology was used to determine changes in trigeminocervical neuronal responses to dural electrical stimulation, and phosphorylated extracellular signal-regulated kinases 1 and 2 (pERK1/2) immunohistochemistry to determine trigeminocervical and hypothalamic neural activity; both in response to intravenous administration of insulin, glucagon, leptin or vehicle control in combination with blood glucose analysis. Blood glucose levels were significantly decreased by insulin (p<0.001) and leptin (p<0.01) whereas glucagon had the opposite effect (p<0.001). Dural-evoked neuronal firing in the trigeminocervical-complex was significantly inhibited by insulin (p<0.001), glucagon (p<0.05) and leptin (p<0.01). Trigeminocervical-complex pERK1/2 cell expression was significantly decreased by insulin and leptin (both p<0.001), and increased by glucagon (p<0.001), when compared to vehicle control. However, only leptin affected pERK1/2 expression in the hypothalamus, significantly decreasing pERK1/2 immunoreactive cell expression in the arcuate nucleus (p<0.05). These findings demonstrate that insulin, glucagon and leptin can alter the transmission of trigeminal nociceptive inputs. A potential neurobiological link between migraine and impaired metabolic homeostasis may occur through disturbed glucose regulation and a transient hypothalamic dysfunction., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Leptin modulates reproductive activity but its potential influence on LH secretion from anterior pituitary (AP) cells during implantation period in pigs (days 14-16 of pregnancy) remained unexplored. This study focused on determination whether leptin affects basal and GnRH-induced LH secretion and intracellular accumulation and whether leptin receptor (OB-Rb) mRNA is expressed in the AP gland during implantation in pigs. Four individual AP glands were developed into separate primary cultures. 2×105 cells/ml were preincubated (72 h) and next, for 3.5 h, experimentally treated with GnRH (100 ng/ml), leptin (10-11, 10-9, 10-7, 10-6 M) alone, or given in respective combinations with GnRH. In the AP gland, OB-Rb mRNA expression was determined by real-time PCR method. Leptin activated LH secretion and its concentration-dependent effect was observed as stimulation shown in a full range tested (culture 1) and exhibited only at 10-6 M (culture 2). A pooled data analysis revealed that basal LH secretion increased at 10-9, 10-7 and 10-6 M, but GnRH-induced LH release decreased at 10-6 M. Leptin down-regulated GnRH-induced LH secretion in all cultures, but only culture 3 exhibited sensitivity for all concentrations tested. Basal LH accumulation was activated in culture 1 (at 10-11 M) and inhibited in culture 4 (at 10-9 M). In the presence of GnRH leptin up-regulated LH accumulation with individual culture leptin-sensitivity (culture 1-3), while down-regulated LH accumulation in culture 4. Obtained data indicate that OB-Rb mRNA is expressed in the AP gland and leptin alone and in combination with GnRH specifically modulates LH activity during early pregnancy in pigs.

Introduction: The immune system acts on different metabolic tissues that are implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Leptin and linoleic acid have the ability to potentially affect immune cells, whereas curcumin is a known natural polyphenol with antioxidant and anti-inflammatory properties., Aims: This study was designed to evaluate the pro-inflammatory and pro-oxidant effects of leptin and linoleic acid on immune cells from patients with NAFLD and to corroborate the modulatory effects of curcumin and its preventive properties against the progression of NAFLD using a high-fat diet (HFD)-induced NAFLD/nonalcoholic steatohepatitis mouse model., Results: The ex vivo experiments showed that linoleic acid increased the production of reactive oxygen species in monocytes and liver macrophages, whereas leptin enhanced tumor necrosis factor-α (TNF-α) production in monocytes and interferon-γ production in circulating CD4+ cells. Conversely, oral administration of curcumin prevented HFD-induced liver injury, metabolic alterations, intrahepatic CD4+ cell accumulation and the linoleic acid- and leptin- induced pro-inflammatory and pro-oxidant effects on mouse liver macrophages., Conclusion: Our findings provide new evidence for the therapeutic potential of curcumin to treat human NAFLD. However, the development of a preventive treatment targeting human circulating monocytes and liver macrophages as well as peripheral and hepatic CD4+ cells requires additional research.

Objectives: Calorie-restriction during gestation in rats has been seen to produce lasting detrimental effects in the offspring, affecting energy balance control and other related metabolic functions. Our aim was to assess whether leptin supplementation throughout lactation may prevent the dysmetabolic phenotype in adulthood associated with gestational calorie restriction., Methods: Three groups of male Wistar rats were followed: the offspring of ad libitum fed dams (controls); the offspring of 20% calorie-restricted dams during gestation (CR); and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Pups were weaned with a standard diet (SD) until 4 months of age, and then half of the animals of each group were moved to a Western diet (WD) until 6 months of age. Body weight and food intake were recorded. Energy expenditure, locomotive activity, blood parameters, liver triglycerides (TG), and gene expression and specific proteins in liver and white adipose tissue (WAT) were measured in adulthood., Results: Adult CR rats, but not CR-Leptin rats, displayed greater adiposity index and feed efficiency (both under SD) than controls, along with lower locomotive activity and energy expenditure, higher HOMA-IR index and greater circulating TG and leptin levels. CR animals also exhibited increased values of the respiratory exchange ratio and more severe signs of hepatic steatosis under WD than CR-Leptin animals. Gene expression analysis revealed that CR, but not CR-Leptin, animals displayed indicators of lower capacity for WAT expansion, along with decreased lipogenesis and lipolytic capacity under SD, and impaired lipogenic response of the liver to WD feeding, in accordance with diminished insulin sensitivity and WAT leptin signaling., Conclusions: Oral leptin supplementation in physiological doses throughout lactation in rats prevents most of the detrimental effects on energy homeostasis and metabolic alterations in adulthood caused by inadequate fetal nutrition.