Recombinant Mouse Leptin Research Articles

OR12-06 Hypothalamic Adgrl1 Is A Potential Glucoreceptor Involved In Regulating Energy Balance, Glucose Homeostasis, And Leptin Sensitivity In Mice

Abstract Disclosure: D.J. Samuel: None. T.S. Faniyan: None. M.U. Raza: None. S. Bathina: None. K.H. Chhabra: None. The hypothalamus regulates energy and glucose homeostasis by influencing glucose-stimulated insulin secretion, insulin sensitivity, and activating the hypoglycemia counterregulatory response. Although hypothalamic glucose transporters coupled with glucose metabolism are involved in mediating some of these effects, it is unknown whether a plasma membrane receptor that directly binds to glucose can contribute to hypothalamic glucose signaling. We used affinity chromatography and biotin-labeled glucose to enrich glucose-binding plasma membrane proteins/receptors in the mouse hypothalamus and then performed proteomic analyses. Adhesion G-protein Coupled Receptor L1 (Adgrl1) was a top candidate as a potential glucoreceptor. Therefore, we generated Adgrl1loxp/loxp mice to knockout Adgrl1 in the ventromedial hypothalamus (VMH; a region known for glucose sensing) using the Cre-Lox system to determine the role of Adgrl1VMH in glucose homeostasis, energy balance, and leptin sensitivity. We measured body weight, food intake, 6h fasting blood glucose and plasma insulin levels. We also determined leptin sensitivity in the Adgrl1VMH deficient mice. We observed that 24 weeks old male Adgrl1VMH deficient mice had higher (p<0.05) body weight (49 ±3.6 vs 35 ±1.3 g), blood glucose (210 ±4 vs 180 ±5 mg/dl), plasma insulin levels (2.92 ±0.24 vs 1.63 ±0.09 ng/ml), and leptin levels (21.11 ±1.72 vs 8.38 ±0.80 ng/ml) compared to their control littermates. Hyperinsulinemia and hyperleptinemia persisted in the Adgrl1VMH knockout mice even when they were weight-matched to their littermate controls, indicating the direct role of Adgrl1VMH in regulating plasma insulin and leptin levels. Ovariectomy in female mice was necessary to unmask the effects of Adgrl1VMH deficiency on energy and glucose homeostasis. To assess acute leptin sensitivity, we injected Adgrl1VMH deficient mice and their littermate control mice with recombinant mouse leptin (5 mg/Kg, i.p., twice a day for two days), and monitored their body weight and food intake. Male Adgrl1VMH deficient mice were more susceptible (p<0.05) to leptin mediated decrease in food intake (-1 ±0.1 vs -0.5 ±0.005, g/day; these values are relative to each group’s corresponding baseline food intake) than their littermate controls. Decrease in body weight was similar in both groups with this acute leptin treatment. These findings suggest that Adgrl1VMH is essential in regulating energy and glucose homeostasis in a sex-specific manner. Moreover, Adgrl1VMH contributes to leptin’s acute effects on food intake. Therefore, hypothalamic Adgrl1 could be a target to enhance leptin sensitivity in future therapies for treating type 2 diabetes and obesity. Presentation: Friday, June 16, 2023

A synergy of estradiol with leptin modulates the long non-coding RNA NEAT1/ mmu-miR-204-5p/IGF1 axis in the uterus of high-fat-diet-induced obese ovariectomized mice

Obesity increases the risk of developing cancers for both males and females. This study investigated potential crosstalk between estradiol and leptin signaling pathways within the endometrium of high-fat-diet-induced obese ovariectomized mice to gain insight into possible links between obesity and endometrial cancer. We administered 17-β estradiol (0.2 μg/mouse subcutaneously) and/or recombinant mouse leptin (1 μg/g Bwt intraperitoneally.,) for 20 h to high-fat-diet-induced obese ovariectomized mice. The uterine tissues of experimental animals after treatments were studied by histological, immunohistochemical, quantitative real-time PCR (gene/miRNAs), and methylation-specific PCR analyses. Quantitative real-time PCR analysis revealed significantly increased expression of Cyclin d1, Esr1, Igf1, Igfbp2, Vegf, Oct4, and Pgr after estradiol and leptin co-treatment. Methylation-specific PCR results indicated that the hormonal dependent transcriptional regulation of Vegf, Igf1, and Pgr is independent of promoter methylation. The decreased expression of mmu- miR-204-5p after estradiol and leptin treatments correlated with the increased expression of long non-coding RNA Neat1. Insilico analysis confirmed the interaction of Neat1 and mmu- miR-204-5p and gene targets of mmu-miR-204-5p, including Igf1 were analyzed in this study. Immunohistochemical analyses revealed subcellular localization and increased expression of ESR, VEGF, phospho-Estrogen Receptor-α (pTyr537), and LEPR proteins following estradiol and leptin exposure. Overall, the data from our in vivo studies suggest the regulation of Neat1-mmu-miR-204−5p- Igf1 axis and associated gene expression changes in uterine tissue after estradiol and leptin co-treatment. In humans, long-term exposure to estradiol and leptin can alter endometrial homeostasis through the NEAT1-miR-204−5p-Igf1 axis and favor carcinogenic pathways, which provide mechanistic insight into the obesity-associated endometrial cancer.

Gene expression changes and promoter methylation with the combined effects of estradiol and leptin in uterine tissue of the ovariectomized mice model of menopause.

Substantial epidemiological studies have shown an association of obesity with the common gynecological malignancy, endometrial cancer. The relevant interactions and contribution of estradiol and the adipose cytokine, leptin, in endometrial lesions are not completely understood. Suitable animal models to understand the physiological response of uterine tissue to the combined effects of estradiol-leptin are lacking. To investigate the effect of estradiol-leptin crosstalk on gene expression and associated altered pathways, we established an ovariectomized mouse model, treated with 17-β estradiol (0.1µg/mouse subcutaenously., for every 12h) and/or recombinant mouse leptin (1μg/g Bwt intraperitoneally., for every 12h) for 4h, 20h, and 40h. Gene expressions by semi-quantitative RT-PCR, uterine tissue protein phosphorylation status by western blotting and promoter methylation were analyzed in estradiol, progesterone insufficient animals. Semi-quantitative RT-PCR demonstrated significantly increased expression of Esr, Igf1, Igfbp3, Vegfr1, and Vegf, and significantly decreased expression of Mmp9 after co-treatment with estradiol and leptin, indicating a common transcriptional network regulated by the treatments. Ovariectomy-induced histomorphological changes were only reversed by estradiol. Methylation-specific PCR, analyzing methylation of CpG sites of Vegfa, Pgr, and Igf1, revealed that transcriptional regulation after hormonal treatments is independent of methylation at the examined CpG sites. Western blot confirmed the increased expression of PSTAT-3 (Ser-727) and PERK1/2 proteins after estradiol + leptin treatment, confirming the estradiol + leptin cross-talk hypothesis. In conclusion, our in vivo studies determined specific gene expression and signaling protein changes, and further unraveled the molecular targets of estradiol + leptin that may perturb endometrial homeostasis and lead to endometrial hyperplasia development in the chronic stimulated state.

Abstract 128: Leptin Accelerates Autoimmune Disease Progression and the Development of Hypertension in an Experimental Model of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that predominately affects women and is associated with high rates of hypertension and cardiovascular disease. SLE is characterized by B and T lymphocyte dysfunction and altered cytokine production, including elevated levels of the adipocytokine leptin. Leptin promotes the expansion of proinflammatory T helper (T H ) 1 and T H 17 cells and inhibits the differentiation of regulatory T cells (T REG ). Because T cells are mechanistically linked to the development of hypertension in SLE, we hypothesized that administration of leptin would alter T H subsets and accelerate the development of hypertension in a female mouse model of SLE. To test this hypothesis, 30 week old female SLE (NZBWF1, n=21) and control (NZW, n=25) mice were implanted with minipumps to continuously deliver recombinant mouse leptin (0.5 mg/kg/day) or vehicle for four weeks. At the conclusion of the study, splenic CD4 + T H subsets were analyzed by flow cytometric analyses and no significant alterations in T H 1, T H 2, T H 17, or T REG cells were detected; however, circulating T REG in SLE mice were lower after leptin administration (1.1±0.2% SLE-vehicle vs. 0.52±0.1% SLE-leptin, p<0.05). Circulating IgG isotypes were also analyzed to correlate with T H subset activities and circulating levels of IgG2a were higher in SLE mice administered leptin (0.79±0.2 mg/mL SLE-vehicle vs. 1.2±0.05 mg/mL SLE-leptin, p<0.05), indicating increased T H 1 activity. In further support of inflammatory T cell activity, circulating levels of TNF-α and IL-17 were elevated in SLE mice administered leptin (TNF-α: 18.6±8.4 pg/mL SLE-vehicle vs. 80.5±15.9 pg/mL SLE-leptin, p<0.01) (IL-17: 0.46±0.1 pg/mL SLE-vehicle vs. 1.68±0.5 pg/mL, p<0.01). Mean arterial pressure (MAP; mmHg), measured in conscious mice by carotid catheter, was higher in SLE mice than in control mice (113±3 Control-vehicle vs. 128±3, p=0.06), and leptin further increased blood pressure in SLE mice (SLE-leptin: 134±3; p<0.01 vs. Control-vehicle and Control-leptin). In conclusion, these data suggest that leptin plays a pathogenic role in the development of hypertension in autoimmunity, in part by altering the balance of T H 1, T H 17, and T REG activity.

Systemic leptin administration alters callus VEGF levels and enhances bone fracture healing in wildtype and ob/ob mice

IntroductionLeptinʼs 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. MethodsStandardized 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 findingsThe 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. ConclusionLow-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.

Leptin Accelerates Autoimmune Disease Progression in an Experimental Model of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a prototypic multisystem autoimmune disorder that is characterized by a loss of immunological tolerance and the expansion of autoreactive T and B lymphocytes, leading to the production of autoantibodies. The autoantibody production leads to downstream chronic inflammation resulting in high rates of hypertension, renal injury, and cardiovascular disease in patients with SLE. The immunomodulatory adipocytokine leptin plays a key role in the maintenance and development of inflammation, in part by promoting the expansion of proinflammatory helper T cells and inhibiting the differentiation of regulatory T cells (TREG). Circulating levels of leptin are elevated in patients with SLE, but it is unclear whether leptin plays a direct role in the pathogenesis of SLE. In the present study, we hypothesized that administration of leptin will accelerate the progression of disease in a female mouse model of SLE. To test this hypothesis, 30 week old female SLE (NZBWF1, n=10) and control (NZW, n=14) mice were implanted with microosmotic pumps to continuously deliver recombinant mouse leptin at a rate of 0.5 mg/kg/day or vehicle (0.9% NaCl) for four weeks. Plasma leptin levels, as measured by ELISA, were significantly increased in both control (10.6±1.6 ng/mL Control‐vehicle vs. 29.5±5.4 ng/mL Control‐leptin, p<0.01) and SLE mice who received leptin (21.0±6.4 ng/mL SLE‐vehicle vs. 50.3±17.5 ng/mL SLE‐leptin, p<0.05). Leptin administration significantly decreased food intake in SLE mice (4.0±0.15 g/day SLE‐vehicle vs. 3.2±0.10 g/day SLE‐leptin, p<0.05), but did not alter food intake in control mice (3.7±0.16 g/day Control‐vehicle vs. 3.3±0.25 g/day Control‐leptin, p=0.33). Body composition was assessed using Echo MRI and fat mass, as a percentage of body weight, was not changed in control mice (19.8±1.3% Control‐vehicle vs. 20.8±1.6% Control‐leptin p=0.99), but was significantly lower in SLE‐leptin treated mice (24.7±2.4% SLE‐vehicle vs. 12.2±4.8% SLE‐leptin, p<0.05). Circulating levels of anti‐dsDNA autoantibodies, a marker of SLE disease activity, were higher in SLE mice compared to controls (0.56±0.16 Control‐vehicle vs. 0.98±0.16 SLE‐vehicle OD450, p<0.05), and these levels were further increased in leptin infused SLE mice compared to vehicle‐infused controls (0.98±0.16 SLE‐vehicle vs. 1.3±0.12 SLE‐leptin OD450, p<0.05). Circulating CD4+FoxP3+ TREG, as assessed by flow cytometry, were lower in SLE mice than in control mice (2.88±0.44% Control‐vehicle vs. 1.44±0.16% SLE‐vehicle, p<0.05), as previously reported by our laboratory. Leptin administration decreased the levels of circulating TREG in both control and SLE animals. Leptin infusion also increased the prevalence of mice with albuminuria (60% of SLE mice administered leptin vs. 25% of vehicle‐treated mice), as measured by dipstick assay. Taken together, these data suggest that SLE mice may have enhanced sensitivity to leptin and that increased leptin levels exacerbate disease activity. In addition, therapeutics aimed at modulating leptin activity could have potential benefit for patients with SLE.Support or Funding InformationResearch supported by: VA merit award # I01BX002604‐01A2 to MJR and NHLBI F32HL137393 to EBT.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Photoperiod-Induced Increases in Bone Mineral Apposition Rate in Siberian Hamsters and the Involvement of Seasonal Leptin Changes.

The adipokine leptin regulates energy balance, appetite, and reproductive maturation. Leptin also acts on bone growth and remodeling, but both osteogenic and anti-osteogenic effects have been reported depending on experimental conditions. Siberian hamsters (Phodopus sungorus) have natural variation in circulating leptin concentrations, where serum leptin is significantly decreased during the short day (SD)-induced winter state. In summer long day (LD) photoperiods, appetite and body adiposity increase with associated central leptin insensitivity. This natural change in leptin secretion was exploited to investigate leptin’s effect on bone growth. Hamsters were injected with calcium-chelating fluorescent dyes to measure bone mineral apposition rate (MAR). Measurements were initially obtained from 5-week and 6-month-old animals maintained in low leptin (SD) or high leptin (LD) states. A further study investigated effects of chronic administration of recombinant mouse leptin to hamsters housed in SD and LD conditions; growth plate thickness and bone density were also assessed. As expected, a reduction in body mass was seen in hamsters exposed to SD, confirming the phenotype change in all studies. Serum leptin concentrations were significantly reduced in SD animals in all studies. MAR was reproducibly and significantly increased in the femurs of SD animals in all studies. Vitamin D and growth plate thickness were significantly increased in SD animals at 6 months. No effect on bone density was observed in any study. Taken together these data suggest that bone growth is associated with the low leptin, winter, lean state. In leptin-treated animals, there was a significant interaction effect of leptin and photoperiod. In comparison to their vehicle counterparts, SD animals had decreased and LD animals had increased MAR, which was not apparent prior to leptin administration. In conclusion, increased MAR was associated with low serum leptin levels in early life and sustained over 6 months, implying that leptin has a negative effect on bone growth in this model. The unexpected finding that MAR increased after peripheral leptin administration in LD suggests that leptin exerts different effects on bone growth dependent on initial leptin status. This adds further weight to the hypothesis that leptin-treated LD animals display central leptin resistance.

Determination of the half-life of circulating leptin in the mouse.

BackgroundThe adipokine hormone, leptin, is a major component of body weight homeostasis. Numerous studies have been performed administering recombinant mouse leptin as an experimental reagent; however, the half life of circulating leptin following exogenous administration of recombinant mouse leptin has not been carefully evaluated.MethodsExogenous leptin was administered (3 mg leptin/kg body weight) to ten week old fasted non-obese male mice and plasma was serially collected at seven time points; plasma leptin concentration was measured by ELISA at each time point to estimate the circulating half life of mouse leptin.ResultsUnder the physiological circumstances tested, the half life of mouse leptin was 40.2 (+/− 2.2) minutes. Circulating leptin concentrations up to one hour following exogenous leptin administration were 170-fold higher than endogenous levels at fasting.ConclusionsThe half life of mouse leptin was determined to be 40.2 minutes. These results should be useful in planning and interpreting experiments employing exogenous leptin. The unphysiological elevations in circulating leptin resulting from widely used dosing regimens for exogenous leptin are likely to confound inferences regarding some aspects of the hormone’s clinical biology.

The development of depression-like behavior is consolidated by IL-6-induced activation of locus coeruleus neurons and IL-1β-induced elevated leptin levels in mice

Many studies have supported the cytokine hypothesis as the underlying pathophysiology of depressive disorder. We previously reported that lipopolysaccharide (LPS)-induced depression-like behavior is abrogated by the α1-adrenoceptor antagonist prazosin. Since cytokines are involved in LPS effects on the brain, we investigated the effects of cytokines on noradrenergic neurons in the locus coeruleus (LC) and whether central α1-adrenoceptors can cause the development of depression-like behavior. Adult male CD1 mice were treated with LPS (1mg/kg, i.p.) or saline and sacrificed 2h later for immunofluorescence studies of c-fos and tyrosine hydroxylase (TH) expression in LC neurons. Serum cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Another group of mice were implanted with intracerebroventricular (i.c.v.) cannulae and given artificial cerebrospinal fluid (CSF) (control), interleukin (IL)-1β (0.5μg), IL-6 (1μg), or tumor necrosis factor (TNF)-α (1μg), and sacrificed 2h later for c-fos and TH immunofluorescence analysis. Serum samples were analyzed for leptin levels. In addition, tail suspension test (TST), forced swimming test (FST), and sucrose preference (SP) test were conducted in a separate group of mice treated i.c.v. with cytokines, recombinant mouse leptin (5μg) or phenylephrine (40μg). These effects were countered by i.c.v. administration of prazosin and a leptin antagonist. LPS increased c-fos expression in TH-positive neurons. Central administration of IL-6 and IL-1β increased c-fos immunoreactivity and serum leptin levels. Phenylephrine, an α1-adrenoceptor agonist, given i.c.v., increased the immobility time during FST and decreased SP, but had no effect on TST. Central leptin administration increased immobility time during FST but did not affect TST or SP. The combination of phenylephrine and leptin increased immobility time during FST and TST, and decreased SP. Induction of depression-like behavior by co-administration of IL-1β and IL-6 was prevented by pretreatment with prazosin alone. These results suggest that IL-6-dependent LC neuronal activation induced depression-like behavior and IL-1β-induced increase in leptin levels enhanced α1-adrenoceptor-mediated depression-like behavior.

Leptin increases mitotic index and regeneration ratio in hepatectomized rats

BackgroundThe aim of this study was to evaluate the potential effect of intraperitoneal administration of leptin on the hepatic regeneration and the mitotic index.Material/Methods56 Sprague-Dawley rats were divided into 7 groups each containing 8 rats. Group 1 was evaluated as the sham group and no surgical procedure was performed on animals. The rats in groups 2, 3, and 4 (named C24, C48, C72, respectively) were given intraperitoneal injection of 2 ml/kg normal saline 60 minutes before the surgical procedure consisting of laparotomy and 70% hepatectomy. These groups were used as controls at 24, 48, and 72 hours. The rats in groups 5, 6, and 7 (named L24, L48, and L72, respectively) were given intraperitoneal injection of 20 μg/kg doses of recombinant mouse leptin 60 minutes before the same surgical procedure. These groups were evaluated as the experiment groups at 24, 48, and 72 hours. Blood samples were collected for aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the remaining tissue samples were obtained for liver histopathology, regeneration rate, and mitotic index (MI). The weights of the remaining livers were also noted.ResultsThe values of AST and ALT were higher in the groups that were administered leptin and they had significantly higher mitotic index than the other groups. Leptin also significantly increased the regeneration ratio as compared to the control group. The weights of the remaining livers were also higher in the leptin groups.ConclusionsIntraperitoneal administration of leptin was observed to increase liver regeneration and mitotic rate in 70% hepatectomized rats.

Effect of exogenous leptin on thrombotic and metabolic profiles of FVB/B6 lipodystrophic mice

The adipokine leptin appears to play a pro‐thrombotic role in mice, a function that has not been examined in lipodystrophic animals. This study was designed to examine the thrombotic and metabolic effects of leptin administration in a mouse model of lipodystrophy. Eight‐week‐old, male FVB(AZIP/F1)/B6 mice (Transgenic, Tg and Wild‐type, Wt) received recombinant mouse leptin (Tg‐T, n=4; Wt‐T, n=4) or vehicle (Tg‐C, n=3; Wt‐C, n=5) via osmotic pumps over 12 days. Tg mice had a prolonged time to occlusive thrombosis compared to Wt mice (p<0.05), and leptin therapy had no effect on this trait. At baseline/control, Tg mice were heavier than Wt mice (p<0.05), had larger livers (p<0.0001), higher serum insulin (p<0.01) and plasma and liver triglyceride (p<0.01 and p<0.05, respectively), and undetectable leptin (p<0.0001). Tg‐T mice lost weight (p<0.05) and had smaller livers (p<0.05), higher leptin levels (p=0.01) and reduced insulin levels (p<0.05), compared to Tg‐C mice. Elevated HOMA‐IR scores in Tg‐C mice (p<0.05) verified insulin resistance, ameliorated by leptin therapy. These findings confirm the hepatomegaly, insulin resistance and hyperlipidemia seen in lipodystrophic mice and the effectiveness of leptin therapy while also suggesting that lipodystrophy, at least in the AZIP/F1 mouse, exerts an anti‐thrombotic phenotype independent of the lack of leptin. Funding source: American Diabetes Association.

Leptin Mediates the Pathogenesis of Severe 2009 Pandemic Influenza A(H1N1) Infection Associated With Cytokine Dysregulation in Mice With Diet-Induced Obesity

Obesity is associated with a high circulating leptin level and severe 2009 pandemic influenza A virus subtype H1N1 (A[H1N1]pdm09) infection. The mechanism for severe lung injury in obese patients and the specific treatment strategy remain elusive. We studied the pathogenesis of A(H1N1)pdm09 infection in a mouse model of diet-induced obesity. Obese mice had significantly higher initial pulmonary viral titer and mortality after challenge with A(H1N1)pdm09, compared with age-matched lean mice. Compared with lean mice, obese mice had heightened proinflammatory cytokine and chemokine levels and more severe pulmonary inflammatory damage. Furthermore, obese mice had a higher preexisting serum leptin level but a lower preexisting adiponectin level. Recombinant mouse leptin increased the interleukin 6 (IL-6) messenger RNA expression in mouse single-lung-cell preparations, mouse macrophages, and mouse lung epithelial cell lines infected with A(H1N1)pdm09. Administration of anti-leptin antibody improved the survival of infected obese mice, with associated reductions in pulmonary levels of the proinflammatory cytokines IL-6 and interleukin 1β but not the pulmonary viral titer. Our findings suggest that preexisting high levels of circulating leptin contribute to the development of severe lung injury by A(H1N1)pdm09 in mice with diet-induced obesity. The therapeutic strategy of leptin neutralization for the reduction of proinflammatory responses and pulmonary damage in obese patients warrants further investigations.

Leptin and leucine synergistically regulate protein metabolism in C2C12 myotubes and mouse skeletal muscles

Leucine and leptin play important roles in regulating protein synthesis and degradation in skeletal muscles in vitro and in vivo. However, the objective of the present study was to determine whether leptin and leucine function synergistically in regulating protein metabolism of skeletal muscles. In the in vitro experiment, C2C12 myotubes were cultured for 2 h in the presence of 5 mm-leucine and/or 50 ng/ml of leptin. In the in vivo experiment, C57BL/6 and ob/ob mice were randomly assigned to be fed a non-purified diet supplemented with 3 % L-leucine or 2·04 % L-alanine (isonitrogenous control) for 14 d. Ob/ob mice were injected intraperitoneally with sterile PBS or recombinant mouse leptin (0·1 μg/g body weight) for 14 d. In C57BL/6 mice, dietary leucine supplementation increased (P< 0·05) plasma leptin, leptin receptor expression and protein synthesis in skeletal muscles, but reduced (P< 0·05) plasma urea and protein degradation in skeletal muscles. Dietary leucine supplementation and leptin injection increased the relative weight of the gastrocnemius and soleus muscles in ob/ob mice. Moreover, leucine and leptin treatments stimulated (P< 0·05) protein synthesis and inhibited (P< 0·05) protein degradation in C2C12 myotubes and skeletal muscles of ob/ob mice. There were interactions (P< 0·05) between the leucine and leptin treatments with regard to protein metabolism in C2C12 myotubes and soleus muscles of ob/ob mice but not in the gastrocnemius muscles of ob/ob mice. Collectively, these results suggest that leptin and leucine synergistically regulate protein metabolism in skeletal muscles both in vitro and in vivo.

In vivo effects of leptin on lymphocyte subpopulations in mice

Leptin, a hormone-cytokine mainly produced by the adipose tissue, has pleitropic effects on many biological system including metabolic, endocrine, and immune system. Although it is well known that leptin controls food intake on hypothalamic regions of brain, the role of leptin in hematopoietic and immune processes has been mainly investigated with in vitro and transgenic mouse studies. The aim of this study was to investigate the effects of peripheral leptin on lymphocyte subpopulation. Initially forty male Swiss albino mice were divided into five groups. Mice in group I (Control) were given serum physiologic (SP) and group L100, group L250, group L500, and group L1000 were given 100, 250, 500 and 1000μg/kg/day recombinant mouse leptin, respectively. Leptin or SP was injected subcutaneously for the next 6 days. Daily food/water intake was recorded for each group. At the end of the study, whole blood samples (500μl) were obtained via intracardiac punction in anesthetized mice. Leptin levels and lymphocyte subpopulations in blood samples were analyzed. We show that no in vivo dose-dependent effect of leptin is existed on lymphocyte subpopulations count in mice. Treatment of mice with high-dose leptin led to increase only CD4+ cells (P<0.05). In addition, high-dose leptin slightly increased CD3+ cells but this was not statistically confirmed (P=0.08). Notably, it was found that leptin caused insignificant changes on body weight and food intake in normal body weight mice. The data support that high-dose leptin has proliferative effect on CD4+ cells in vivo. However, more in vivo study needs to be examined to clarify how leptin affect lymphocyte subpopulations.

Leptin Therapy Reverses Hyperglycemia in Mice With Streptozotocin-Induced Diabetes, Independent of Hepatic Leptin Signaling

OBJECTIVELeptin therapy has been found to reverse hyperglycemia and prevent mortality in several rodent models of type 1 diabetes. Yet the mechanism of leptin-mediated reversal of hyperglycemia has not been fully defined. The liver is a key organ regulating glucose metabolism and is also a target of leptin action. Thus we hypothesized that exogenous leptin administered to mice with streptozotocin (STZ)-induced diabetes reverses hyperglycemia through direct action on hepatocytes.RESEARCH DESIGN AND METHODSAfter the induction of diabetes in mice with a high dose of STZ, recombinant mouse leptin was delivered at a supraphysiological dose for 14 days by an osmotic pump implant. We characterized the effect of leptin administration in C57Bl/6J mice with STZ-induced diabetes and then examined whether leptin therapy could reverse STZ-induced hyperglycemia in mice in which hepatic leptin signaling was specifically disrupted.RESULTSHyperleptinemia reversed hyperglycemia and hyperketonemia in diabetic C57Bl/6J mice and dramatically improved glucose tolerance. These effects were associated with reduced plasma glucagon and growth hormone levels and dramatically enhanced insulin sensitivity, without changes in glucose uptake by skeletal muscle. Leptin therapy also ameliorated STZ-induced hyperglycemia and hyperketonemia in mice with disrupted hepatic leptin signaling to a similar extent as observed in wild-type littermates with STZ-induced diabetes.CONCLUSIONSThese observations reveal that hyperleptinemia reverses the symptoms of STZ-induced diabetes in mice and that this action does not require direct leptin signaling in the liver.

Role of leptin in improvement of oocyte quality by regulation of ovarian angiogenesis

Ovarian angiogenesis plays an important role in folliculogenesis. An active blood supply via ovarian angiogenesis seems to be essential for the induction of oocytes with good quality. Leptin is an angiogenic factor which regulates VEGF expression. This study was aimed to investigate whether leptin administration during superovulation influences ovarian response, oocyte quality and VEGF expression in the ovary using different aged mice model. C57BL inbred female mice of two age groups (18–21, and 29–31 weeks) were superovulated by intraperitoneal co-injection with 5 IU of pregnant mare's serum gonadotropin (PMSG) supplemented with recombinant mouse leptin at various doses (0.01, 0.1, 1 μg), followed by injection with 5 IU of human chorionic gonadotropin (hCG) approximately 48 h later. Then, the mice were immediately paired with an individual male. The control group was superovulated with PMSG and hCG without leptin. After 18 h, one-cell embryos were flushed and cultured for 4 days. Proteins were extracted from ovaries removed just after the retrieval of one-cell embryos and VEGF expression was examined by Western blot. Treatment of 0.1 μg and 1 μg leptin significantly increased the number and embryo development rate of one-cell embryos retrieved compared to the control group. This positive effect of leptin was more significant with advancing female age. Ovarian VEGF expression was also significantly increased in 0.1 and 1 μg leptin-treated groups compared to the control group in both age groups ( P < 0.05). Our present study showed that leptin administration with gonadotropins during superovulation in aged mice increased the ovarian response, developmental competence of oocytes and ovarian VEGF expression. This research may have potential clinical implications in the treatment of age-related decline of fertility.

Early Maternal Hyperleptinemia Programs Adipogenic Phenotype in Rats

We have previously reported on the treatment of maternal rats with leptin during the three last days of lactation program for overweight and leptin hypothalamic resistance in the offspring. Here we have investigated whether treatment of maternal rats with leptin in the first ten days of lactation can program metabolic dysfunctions on the adult offspring. Lactating rats were divided into 2 groups: rats (LEP) injected with recombinant mouse leptin (8 microg/100 g/body weight, daily during the first 10 days of lactation) and control group (C) that received the same volume of saline. After weaning, all pups had free access to normal diet, their body weight and food intake were monitored at 4 days interval until 180 days, when they were tested for food intake and response to either leptin (0.5 mg/kg body weight, ip) or saline. The offspring from leptin-treated mothers gained more weight from day 69 onward and had higher food intake from day 145 onward, higher amount of visceral adipose tissue (57%), higher serum glucose (10%), and higher serum leptin (135%) at 180 days compared to control group. The food intake was not reduced as expected after acute injection of leptin in these animals, suggesting resistance to the anorexigenic effect of leptin. We conclude that maternal hyperleptinemia in early lactation programed higher food intake, body weight gain due to higher total and visceral fat mass, and resistance to anorexigenic effect of leptin in the adult offspring even when this hyperleptinemia occurred at the beginning of lactation.

Up-regulation of apolipoprotein E by leptin in the hypothalamus of mice and rats

Apolipoprotein E (apoE) is a satiation factor, playing an important role in the regulation of food intake and body weight. We previously reported that apoE was present in the hypothalamus, but it is unclear which type of the cells in this brain area expressing apoE. In addition, hypothalamic apoE mRNA levels were significantly reduced in both genetically obese ob/ob (leptin deficient) mice and high-fat diet-induced obese (leptin resistant) rats, raising the possibility that deficient leptin signaling might be related to the change in apoE gene expression. In the present studies, using double-staining immunohistochemistry, we demonstrated that apoE is mainly present in astrocytes. To characterize the effect of leptin on apoE gene expression, ob/ ob and db/db mice were treated with recombinant mouse leptin (3 μg/g daily, i.p.) or vehicle for 5 days. We found that the increased hypothalamic apoE mRNA levels occurred only in leptin-treated ob/ob, but not in pair-fed ob/ob, or db/db, mice, indicating that leptin up-regulated hypothalamic apoE gene expression depends upon an intact leptin receptor, and this effect is not related to the changes in food intake and body weight. The reduced apoE gene expression caused by fasting, which also results in relatively lower leptin level, is restored by intracerebroventricular administration of leptin. In addition, leptin was significantly less efficacious in apoE KO mice because these animals consumed more food and lost less weight following leptin treatment, compared with wild-type controls. These observations imply that apoE signaling, at least partially, mediates the inhibitory effects of leptin on feeding.

Beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and a high-fat diet

We have previously demonstrated the therapeutic usefulness of leptin in lipoatrophic diabetes and insulin-deficient diabetes in mouse models and could also demonstrate its dramatic effects on lipoatrophic diabetes in humans. The aim of the present study was to explore the therapeutic usefulness of leptin in a mouse model of type 2 diabetes with increased adiposity. To generate a mouse model mimicking human type 2 diabetes with increased adiposity, we used a combination of low-dose streptozotocin (STZ, 120 microg/g body weight) and high-fat diet (HFD, 45% of energy as fat). Recombinant mouse leptin was infused chronically (20 ng [g body weight](-1) h(-1)) for 14 days using a mini-osmotic pump. The effects of leptin on food intake, body weight, metabolic variables, tissue triacylglycerol content and AMP-activated protein kinase (AMPK) activity were examined. Low-dose STZ injection led to a substantial reduction of plasma insulin levels and hyperglycaemia. Subsequent HFD feeding increased adiposity and induced insulin resistance and further augmentation of hyperglycaemia. In this model mouse mimicking human type 2 diabetes (STZ/HFD), continuous leptin infusion reduced food intake and body weight and improved glucose and lipid metabolism with enhancement of insulin sensitivity. Leptin also decreased liver and skeletal muscle triacylglycerol content accompanied by an increase of alpha2 AMPK activity in skeletal muscle. Pair-feeding experiments demonstrated that leptin improved glucose and lipid metabolism independently of the food intake reduction. This study demonstrates the beneficial effects of leptin on glycaemic and lipid control in a mouse model of type 2 diabetes with increased adiposity, indicating the possible clinical usefulness of leptin as a new glucose-lowering drug in humans.

Elongin B/C Recruitment Regulates Substrate Binding by CIS

SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.