Assessing the Safety of MA-[D-Leu-4]-OB3, a Synthetic Peptide Leptin Mimetic: Two Pre-Clinical Toxicity Studies in Male and Female C57BL/6 Mice.

Although the regulatory influence of leptin on energy balance, glycemic control, immunity, reproduction, and cognition is well established, its clinical application to common obesity and its co-morbidities has been limited by impaired transport across the blood-brain barrier, and tendencies to induce adverse side effects. To circumvent these drawbacks, MA-[D-Leu-4]-OB3, a leptin-related synthetic peptide that mimics the metabolic and neurotrophic effects of leptin in mouse models of genetic and non-genetic obesity, diabetes, and cognitive dysfunction, has been developed. This report presents the results of our initial efforts to assess the safety of orally delivered MA-[D-Leu-4]-OB3. Two pre-clinical studies were done in male and female C57BL/6 mice: a short-term study with a high dose of MA-[D-Leu-4]-OB3 (50 mg/kg/100 µL/day) and a dose-response study with 3 increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/100 µL/day). Body weight, food and water intake, glucose tolerance, and episodic memory were evaluated. Once-daily cage-side clinical observations were conducted to detect any physical or behavioral indicators of toxicity. Our results indicate that all metabolic and neurologic endpoints tested were either unaffected or improved by MA-[D-Leu-4]-OB3, and no clinical indicators of toxicity were evident. Together with our previously reported efficacy data, these results provide additional evidence supporting further development of this novel synthetic peptide leptin mimetic as a first-in-class peptide drug candidate for the treatment of a number of metabolic and/or cognitive dysfunctions in humans.

Globular adiponectin antagonizes leptin-induced growth of cancer cells by modulating inflammasomes activation: Critical role of HO-1 signaling.

Accumulating evidence suggests that adipokines, a group of hormones secreted from adipose tissue, modulate tumor growth in a complicated manner. Among diverse adipokines, adiponectin exerts potent anti-tumor activities, whereas leptin exhibits pro-tumorigenic properties. Herein, we have examined the opposing effect of adiponectin on leptin-induced growth of cancer cells and investigated the underlying mechanisms, particularly in the context of inflammasomes activation, which plays a role in the growth of cancer cells. Globular adiponectin (gAcrp) significantly suppressed leptin-induced growth of human breast (MCF-7) and hepatic (HepG2) cancer cells by modulating both cell cycle and apoptosis. To elucidate the underlying mechanisms, we examined the modulatory effects of gAcrp and leptin on inflammasomes. Herein, we showed that gAcrp substantially abolished leptin-induced inflammasomes activation, as evidenced by suppression of IL-1ß maturation, caspase-1 activation, and downregulation of inflammasomes components, including NLRP3 and ASC, in both MCF-7 and HepG2 cancer cells. Interestingly, suppression of inflammasomes activation by gAcrp was almost completely restored by blockade of heme oxygenase-1 (HO-1) signaling. In addition, suppressive effects of gAcrp on ROS production and NADPH oxidase activation, both of which critically contribute to leptin-induced inflammasomes activation, disappeared by inhibition of HO-1 signaling. Moreover, gAcrp downregulated estrogen receptor-α (ER-α) expression and blocked leptin-induced ER-α activation, which also plays an important role in inflammasomes activation. Finally, the opposing effects of gAcrp on leptin-induced inflammasomes activation and tumor growth were further confirmed in MCF-7 tumor xenografts. In summary, treatment with gAcrp prevents leptin-induced cancer cell growth by modulating inflammasome activation, which is mediated, at least in part, via HO-1 induction and modulation of ER-α signaling.

Leptin stimulates the release of pro-inflammatory cytokines in hypothalamic astrocyte cultures from adult and aged rats.

Leptin is an adipose tissue-derived hormone that acts on the hypothalamus in order to maintain energy homeostasis. However, leptin can also induce an inflammatory response. Increasing evidence has highlighted a critical role of astrocytes in the effects of leptin on the hypothalamus. In addition, astrocytes participate in neuroinflammation by producing and releasing a wide range of inflammatory mediators. In this study, we aimed to investigate the age-dependent effect of leptin on pro- and anti-inflammatory cytokines released by the hypothalamic astrocyte cultures obtained from newborn, adult, and aged Wistar rats. In hypothalamic astrocytes from newborn rats, leptin did not change the release of pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). On the other contrary, leptin increased the release of both TNF-α and IL-1ß in astrocyte cultures from adult and aged animals. Regarding the anti-inflammatory cytokine interleukin 10 (IL-10), we did not observe any change in response to leptin. In conclusion, our data suggests a pro-inflammatory action of leptin on the hypothalamus during aging. This in turn may be related to the triggering of metabolic disorders, as both of these conditions are associated with neuroinflammation.

Leptin induces SIRT1 expression through activation of NF-E2-related factor 2: Implications for obesity-associated colon carcinogenesis.

Leptin, a representative adipokine secreted from the white adipose tissue, is considered as a potential linker between obesity and cancer. SIRT1 is an NAD+-dependent histone/protein deacetylase speculated to function as an oncogene. In the present study, we found that leptin signaling-defective ob/ob and db/db mice had lower colonic expression of SIRT1 compared with leptin signaling-intact C57BL/6J mice, implying that leptin signaling is crucial for SIRT1 expression in vivo. Moreover, leptin induced up-regulation of SIRT1 in human colon cancer (HCT-116) cells. Leptin stimulated migration and invasion of cultured HCT-116 cells and tumor growth in the xenograft assay, and these effects were abrogated by a SIRT1 inhibitor sirtinol, suggesting that SIRT1 plays a role in leptin-induced colon carcinogenesis. Leptin-induced SIRT1 expression was regulated by the redox-sensitive transcription factor NF-E2-related factor 2 (Nrf2). Leptin stimulated nuclear accumulation of Nrf2 as well as its binding to the antioxidant response elements located in the SIRT1 promoter. Moreover, siRNA knockdown of Nrf2 abrogated the leptin-induced SIRT1 expression. Notably, SIRT1 was significantly reduced in colon tissues of Nrf2-null mice, lending further support to Nrf2-dependent SIRT1 expression. Expression of leptin, Nrf2 and SIRT1 was coordinately increased in human colon tumor tissues. In conclusion, leptin might play a role in colon carcinogenesis by inducing Nrf2-dependent SIRT1 overexpression.

Leptin suppresses microRNA-122 promoter activity by phosphorylation of foxO1 in hepatic stellate cell contributing to leptin promotion of mouse liver fibrosis.

Adipocytokine leptin promotes hepatic stellate cell (HSC) activation (a key step in liver fibrogenesis) and liver fibrosis. microRNA-122 (miR-122) is the most abundant liver-specific miRNA and was demonstrated to inhibit liver fibrosis and reduced HSC proliferation. Our previous study revealed that leptin reduced miR-122 level in HSCs. This study was aimed to investigate whether leptin affected miR-122 promoter and the underlying mechanisms in HSCs. Results showed that leptin inhibited miR-122 promoter activity. Forkhead box protein O1(FoxO1) bound to miR-122 promoter at a site around - 56 and thus promoted miR-122 promoter activity, which could be suppressed by leptin-induced phosphorylation of FoxO1 at serine 256. The PI3K/Akt signaling pathway was involved in leptin-induced phosphorylation of FoxO1 and the effect of leptin on miR-122 expression. Furthermore, FoxO1 increased miR-122 and pri-miR-122 (primary miR-122) levels in HSCs in vivo, and reduced leptin-induced HSC activation and liver fibrosis in ob/ob mouse (leptin deficient) model. In conclusion, leptin suppressed microRNA-122 expression by PI3K/Akt/foxO1 axis in HSCs. These results have potential implications for clarifying the mechanisms for liver fibrogenesis in obese patients with hyperleptinaemia.

Leptin accelerates the pathogenesis of heterotopic ossification in rat tendon tissues via mTORC1 signaling.

Leptin, an adipocyte-derived cytokine associated with bone metabolism, is believed to play a critical role in the pathogenesis of heterotopic ossification (HO). The effect and underlying action mechanism of leptin were investigated on osteogenic differentiation of tendon-derived stem cells (TDSCs) in vitro and the HO formation in rat tendons. Isolated rat TDSCs were treated with various concentrations of leptin in the presence or absence of mTORC1 signaling specific inhibitor rapamycin in vitro. A rat model with Achilles tenotomy was employed to evaluate the effect of leptin on HO formation together with or without rapamycin treatment. In vitro studies with TDSCs showed that leptin increased the expression of osteogenic biomarkers (alkaline phosphatase, runt-related transcription factor 2, osterix, osteocalcin) and enhanced mineralization of TDSCs via activating the mTORC1 signal pathway (as indicated by phosphorylation of p70 ribosomal S6 kinase 1 and p70 ribosomal S6). However, mTORC1 signaling blockade with rapamycin treatment suppressed leptin-induced osteogenic differentiation and mineralization. In vivo studies showed that leptin promoted HO formation in the Achilles tendon after tenotomy, and rapamycin treatment blocked leptin-induced HO formation. In conclusion, leptin can promote TDSC osteogenic differentiation and heterotopic bone formation via mTORC1 signaling in both vitro and vivo model, which provides a new potential therapeutic target for HO prevention.

Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc.

Exposure to excess estradiol or leptin during pregnancy increases mammary cancer risk and prevents parity-induced protective genomic changes in rats.

Using a preclinical model, we investigated whether excess estradiol (E2) or leptin during pregnancy affects maternal mammary tumorigenesis in rats initiated by administering carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) on day 50. Two weeks later, rats were mated, and pregnant dams were treated daily with 10 µg of 17ß-estradiol, 15 µg of leptin, or vehicle from gestation day 8 to 19. Tumor development was assessed separately during weeks 1 to 12 and 13 to 22 after DMBA administration, because pregnancy is known to induce a transient increase in breast cancer risk, followed by a persistent reduction. Parous rats developed less (32%) mammary tumors than nulliparous rats (59%, P < 0.001), and the majority (93%) of tumors in the parous rats appeared before week 13 (vs. 41% in nulliparous rats), indicating that pregnancy induced a transient increase in breast cancer risk. Parous rats exposed to leptin (final tumor incidence 65%) or E2 (45%) during pregnancy developed mammary tumors throughout the tumor-monitoring period, similar to nulliparous control rats, and the incidence was significantly higher in both the leptin- and E2-exposed dams after week 12 than in the vehicle-exposed parous dams (P < 0.001). The mammary glands of the exposed parous rats contained significantly more proliferating cells (P < 0.001). In addition, the E2- or leptin-treated parous rats did not exhibit the protective genomic signature induced by pregnancy and seen in the parous control rats. Specifically, these rats exhibited downregulation of genes involved in differentiation and immune functions and upregulation of genes involved in angiogenesis, growth, and epithelial-to-mesenchymal transition.

Supraphysiological leptin levels shift the profile of steroidogenesis in porcine ovarian follicles toward progesterone and testosterone secretion through increased expressions of CYP11A1 and 17b-HSD: a tissue culture approach.

Evidence from both clinical and animal studies suggests that exposure to excess androgens results in cyst formation. The present in vitro study assessed the effects of supraphysiological concentrations of leptin (20 and 40 ng/ml) on progesterone (P(4)), androstenedione androstendione (A(4)), testosterone and estradiol (E(2)) secretion by ELISA and the expression of CYP11A1, CYP17, 17b-hydroxysteroid dehydrogenase (17b-HSD) and CYP19 by western blot to answer the question of whether leptin could be independent risk factor for cystformation in pigs. Small- and medium-sized ovarian follicles were collected from prepubertal and cycling pigs. Increased P(4) and testosterone secretions were observed in both small- and medium-sized follicles in prepubertal and cycling animals whereas there was no change in E2 secretion. Leptin treatment resulted in an increase in CYP11A1 and 17b-HSD protein expression but had no effect on CYP17 and CYP19 expression in follicles of either size from prepubertal and cycling pigs. Results of presented data suggest that leptin in elevated doses, by stimulatory effect on CYP11A1 and 17b-HSD protein expression resulting in elevated P(4) and testosterone secretions could be an independent risk factor for cyst formation in both prepubertal and cycling pigs.

Targeting the lateral but not the third ventricle induces bone loss in ewe: an experimental approach to generate an improved large animal model of osteoporosis.

BACKGROUND: Osteoporosis is a chronic disease characterized by bone loss and increased skeletal fragility. Large animal models are required for preclinical testing of new therapeutic approaches. We have recently demonstrated that continuous intracerebroventricular (ICV) application of leptin into the lateral ventricle (LV) induces bone loss in ewe. On the basis of these findings, we reasoned that the third ventricle (TV) is an even better target because of its closer location to the hypothalamus that mediates leptin effects on bone. METHODS: Corriedale sheep were randomly mixed to four groups of four ewe each: control entire (control), ovarectomy plus ICV application of cerebrospinal fluid (OVX), OVX plus ICV application of leptin into the LV (leptin-LV); and ICV application of leptin into the TV (leptin-TV). After 3 months, histomorphometric characterization and bone turnover parameters were analyzed. RESULTS: Highly significant loss of trabecular bone was observed only in leptin-LV group. Increased osteoclast indices and urinary cross-lap excretion were observed in OVX and leptin-TV group. In contrast, serum parameters of osteoblast activity were only significantly decreased in leptin-LV group. Autopsy of ewe brain showed fibrosis around the stainless steel cannula in leptin-TV group. CONCLUSIONS: ICV application of leptin into the LV strongly reduces bone formation and leads to a highly significant trabecular bone loss in ewe. In contrast, ICV application of leptin into the TV is technically more demanding and results are unpredictable, because the required use of stainless steel cannula induces peri-implant fibrosis that might prevent leptin to enter the cerebrospinal fluid.

Up-regulation of renal Na+, K+-ATPase: the possible novel mechanism of leptin-induced hypertension.

Hyperleptinemia may be involved in the pathogenesis of obesity-associated hypertension, however, the mechanism of hypertensive effect of leptin has not been elucidated. We investigated the effect of experimental hyperleptinemia on renal function, renal Na(+), K(+)-ATPase and ouabain-sensitive H(+), K(+)-ATPase activities in the rat. Leptin administered for 7 days (0.25 mg/kg twice daily sc) decreased food intake on 6th and 7th day of treatment but had no effect on body weight. Systolic blood pressure was 30.5% higher in leptin-treated animals. Urinary excretion of sodium decreased by 35.0% following leptin treatment. Leptin had no effect on potassium and phosphate excretion as well as on creatinine clearance. The activity of Na(+), K(+)-ATPase in the renal cortex and medulla was higher in leptin-treated rats by 32.4% and 84.2%, respectively. In contrast, leptin had no effect on either cortical or medullary ouabain-sensitive H(+), K(+)-ATPase. In pair-fed group, in which food intake was reduced to the level observed in leptin-treated group, no changes in sodium metabolism and renal Na(+), K(+)-ATPase were observed. Leptin decreased urinary excretion of nitric oxide metabolites by 55.0% and urinary excretion of cGMP by 26.3%. Plasma concentration of atrial natriuretic peptide tended to be higher and urinary excretion of urodilatin was 64.9% higher in leptin-treated animals. These data suggest that hyperleptinemia decreases natriuresis by up-regulating Na(+), K(+)-ATPase and stimulating tubular sodium reabsorption. This effect is mediated, at least in part, by deficiency of nitric oxide (NO). Abnormal renal sodium retention and vasoconstriction associated with NO deficiency may contribute to leptin-induced hypertension and to blood pressure elevation in hypertensive obese individuals.