Mouse Leptin Research Articles

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

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

Leptin regulates Granzyme-A, PD-1 and CTLA-4 expression in T cell to control visceral leishmaniasis in BALB/c Mice

Visceral leishmaniasis (VL) is responsible for several deaths in malnourished children accompanied by diminished circulating leptin and impaired cell-mediated immunity. Typically, leptin deficiency is associated with the Th2 polarization that markedly coincides with the pathogenesis of VL. The aim of the present study was to unravel the prophylactic role of leptin in malnutrition-coupled VL mice. Interestingly, we observed that L. donovani infection itself reduces the serum leptin levels in malnutrition. Exogenous leptin restored severe body weight loss and parasite load in the spleen and liver of malnourished infected mice compared to controls. Leptin increases functional CD8+ T-cell population, Granzyme-A expression down-regulates anergic T-cell markers such as PD-1 and CTLA-4. It was also noticed that, leptin suppresses GM-CSF mRNA expression in parasite favored monocytes and reduced arginase activity in bone marrow derived macrophage indicate macrophages dependent T-cell activation and proliferation. Leptin-induced IFN-γ, IL-2, and TNF-α cytokines in the culture supernatant of splenocytes upon soluble leishmanial antigen (SLA) stimulation and significantly up-regulates serum IgG2a titers, which help to generate Th1 immune response in VL. Furthermore, leptin induced a granulomatous response and restored L. donovani induced tissue degeneration in the liver. Altogether, our findings suggest the exogenous leptin can restore T cell mediated immunity in malnourished VL mice.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the cardiovascular risk factor obesity may have relevant therapeutic implications.

Abstract 2694: Target obesity-associated inflammation to decrease murine basal-like mammary tumor burden

Abstract Background: Adipose tissue dysregulation, a hallmark of obesity, contributes to a chronic state of low-grade inflammation that promotes cancer growth through multiple signaling pathways. We previously showed that inflammation and basal-like breast cancer (BLBC) growth are increased in chronically obese mice and persist following weight normalization. Purpose: We tested the hypothesis that targeting inflammation in obese mice by treating them with the nonsteroidal anti-inflammatory drug (NSAID) Sulindac would offset the procancer effects of obesity in a mouse model of BLBC. Methods: Mice were administered a control diet (10 kcal % fat; n=34) or diet-induced obesity regimen (DIO, 60 kcal % fat; n=34). After 15 weeks on control or DIO diets, mice were randomized to either receive Sulindac supplementation at 160 ppm in the diet (n=17/diet) or no supplementation (n=17/diet). Twelve weeks later, all mice were orthopically injected with E0771 cells, a model of basal-like breast cancer. Five mice/group were killed at a 4-week interim time-point after injection, and their tissue collected and stored. The remaining 12 mice/group continued in a survival study; these mice were killed when tumor size reached 1.2 cm in diameter in any direction. Results: Sulindac supplementation in DIO mice significantly reduced serum insulin and leptin to levels statistically equivalent to control mice, but had no effect on body weight, body fat percentage, or ex vivo visceral white adipose weight. Sulindac supplementation in DIO mice (but not control) significantly reduced mean tumor volume in the interim tumor study and significantly increased tumor latency in the survival study. Analysis of H&E stained tumor demonstrated that DIO mice had significantly increased adipocytes infiltrating into the tumor (relative to control), but Sulindac supplementation in DIO mice decreased adipocyte infiltration to levels observed in control. Conclusions: Sulindac supplementation significantly reduced insulin and leptin in DIO mice, and increased tumor latency in DIO mice, but had no effects on body weight or fat depots, suggesting that Sulindac offsets some of the pro-tumorigeneic effects of obesity rather than targeting obesity directly. Preliminary analyses of inflammatory surrogates, including circulating cytokines and prostaglandins, mammary gland crown-like structures and cyclooxygenase-2 levels, suggests Sulindac’s effects in obese mice are mediated through its eicosanoid-depressing effects. Citation Format: Emily L. Rossi, Subreen A. Khatib, Laura W. Bowers, Steven S. Doerstling, Andrew J. Dannenberg, Stephen D. Hursting. Target obesity-associated inflammation to decrease murine basal-like mammary tumor burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2694. doi:10.1158/1538-7445.AM2017-2694

Neural Programmatic Role of Leptin, TNFα, Melanocortin, and Glutamate in Blood Pressure Regulation vs Obesity-Related Hypertension in Male C57BL/6 Mice.

Continuous nutritional surplus sets the stage for hypertension development. Whereas moderate dietary obesity in mice is normotensive, the homeostatic balance is disrupted concurrent with an increased risk of hypertension. However, it remains unclear how the obesity-associated prehypertensive state is converted into overt hypertension. Here, using mice with high-fat-diet (HFD)-induced moderate obesity vs control diet (CD)-fed lean mice, we comparatively studied the effects of central leptin and tumor necrosis factor-α (TNFα) as well as the involvement of the neuropeptide melanocortin pathway vs the neurotransmitter glutamate pathway. Compared with CD-fed lean mice, the pressor effect of central excess leptin and TNFα, but not melanocortin, was sensitized in HFD-fed mice. The pressor effect of central leptin in HFD-fed mice was strongly suppressed by glutamatergic inhibition but not by melanocortinergic inhibition. The pressor effect of central TNFα was substantially reversed by melanocortinergic inhibition in HFD-fed mice but barely in CD-fed mice. Regardless of diet, the hypertensive effects of central TNFα and melanocortin were both partially reversed by glutamatergic suppression. Hence, neural control of blood pressure is mediated by a signaling network between leptin, TNFα, melanocortin, and glutamate and changes in dynamics due to central excess leptin and TNFα mediate the switch from normal physiology to obesity-related hypertension.

Effects of voluntary running with defined distances on body adiposity and its associated inflammation in mice fed a high‐fat diet

Sedentary lifestyle contributes to obesity. This study examined the effect of quantitative voluntary running on body adiposity and its associated inflammation in mice fed a high‐fat diet. Male C57BL/6 mice were assigned into six groups and fed the AIN93G (sedentary) or a high‐fat diet (sedentary, unrestricted running, or with 25%, 50%, or 75% restriction in running activity) for 12 weeks. The average daily running distance was 8.3, 6.3, 4.2, and 2.1 km for the unrestricted, 25%, 50%, and 75% restricted runners, respectively. The high‐fat diet increased body fat mass by 46% compared to the AIN93G diet in sedentary mice. Running reduced the fat mass in a dose dependent manner; there was no difference between sedentariness and running at 2.1 km/d. The high‐fat diet significantly increased blood glucose, plasma insulin, HOMA‐IR, and plasma and adipose concentrations of leptin and monocyte chemotactic protein‐1 (MCP‐1) in sedentary mice. Running, despite continued consumption of the high‐diet, reduced these variables in dose‐dependent manners. Running at 8.3 (unrestricted) and 6.3 km/d (25% restricted) had the greatest but similar reductions in, whereas running at 2.1 km/d did not affect (except MCP‐1), these measurements. Running, regardless of daily distance, significantly reduced MCP‐1 in both plasma and adipose tissues. In conclusion, voluntary running at 6.3 km/d is optimal to reduce body adiposity and its associated inflammation and metabolic disturbance in C57BL/6 mice. Results that running at 2.1 km/d reduced MCP‐1 without affecting body fat mass show the benefits of moderate amount of exercise in improving low‐grade inflammation. It suggests that different mechanisms, rather than reducing body adiposity, are responsible for this improvement.Support or Funding InformationThe U.S. Department of Agriculture, ARS, research project 5450‐51000‐045‐00D.

Ghrelin did not change coronary angiogenesis in diet-induced obese mice.

Ghrelin is a 28 amino acids peptide that initially was recognized as an endogenous ligand for growth hormone secretagogue receptor (GHSR). Recently, a number of studies demonstrated that ghrelin is a cardiovascular hormone with a series cardiovascular effect. The main objective of this study was to investigate the effect of systemic ghrelin administration on angiogenesis in the heart and its correlation with serum leptin levels in normal and diet-induced obese mice. 24 male C57BL/6 mice were randomly divided into four groups: normal diet (ND) or control, ND+ghrelin, high-fat-diet (HFD) or obese and HFD+ghrelin (n=6/group). Obese and control groups received HFD or ND, respectively, for 14 weeks. Then, the ghrelin was injected subcutaneously 100µg/kg twice daily. After 10 days, the animals were sacrificed, blood samples were taken and the hearts were removed. The angiogenic response in the heart was assessed by immunohisochemical staining. HFD significantly increased angiogenesis in the heart expressed as the number of CD31 positive cells than standard diet. Ghrelin did not alter angiogenesis in the heart in both obese and control groups, however, it reduced serum nitric oxide (NO) and leptin levels in obese mice. There was a strong positive correlation between the number of CD31 positive cells and serum leptin concentration (r=0.74). Leptin as an angiogenic factor has a positive correlation with angiogenesis in the heart. Although systemic administration of ghrelin reduced serum leptin and NO levels in obese mice, however, it could not alter coronary angiogenesis.

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.

고지방식이 병행섭취 시 연잎, 연 줄기, 연자방 분말가루가 흰쥐의 혈중 생화학적 인자에 미치는 영향

In this study, we analyzed the biochemical factors in lotus (Nelumbo nucifera) leaf, stem, and yeonjabang and their effects on serum factor levels in mice fed a high-fat (HF) diet. The loutus leaf showed <TEX>$9.47{\pm}0.30%$</TEX> moisture content, <TEX>$8.25{\pm}0.39%$</TEX> ash, <TEX>$21.45{\pm}1.25%$</TEX> crude protein, and <TEX>$2.21{\pm}0.13%$</TEX> crude fat content; the lotus stem showed <TEX>$11.84{\pm}0.43%$</TEX> moisture, <TEX>$10.21{\pm}0.64%$</TEX> ash, <TEX>$17.55{\pm}0.92%$</TEX> crude protein, and <TEX>$4.16{\pm}0.23%$</TEX> crude fat content; and the lotus yeonjabang showed <TEX>$11.86{\pm}0.50%$</TEX> moisture, <TEX>$6.81{\pm}0.51%$</TEX> ash, <TEX>$18.71{\pm}1.02%$</TEX> crude protein, and <TEX>$3.95{\pm}0.15%$</TEX> crude fat. Blood triglyceride levels were higher in the HF group (<TEX>$146.43{\pm}38.81mg/dL$</TEX>), and lower in the HF+yeonjabang groups (<TEX>$98.00{\pm}17.18mg/dL$</TEX>). In particular, blood triglyceride levels were significantly lower in the groups that had 10% dry yeonjabang powder added to the high-fat diet. The inclusion of excessive high-fat diet increased concentrations of serum insulin and leptin. Serum leptin concentrations were highest in the HF group mice (<TEX>$3.00{\pm}1.35ng/dL$</TEX>), whereas they were significantly lower in the HF+yeonjabang groups by <TEX>$1.34{\pm}0.52ng/dL$</TEX> (p<0.05). Thus, addition of dry yeonjabang powder to the high-fat diet was more effective in regulating the levels of serum triglycerides and leptin in mice. Additional studies would help in the development of yeonjabang as a functional food.

Abstract 041: mTORC1 is Required for Leptin-induced Sympathetic Activation to the Kidney but Not Brown Adipose Tissue

The adipocyte-derived hormone leptin plays a critical role in the regulation of energy homeostasis through its action in the brain to decrease food intake and promote energy expenditure by increasing sympathetic nerve activity (SNA) to the thermogenic brown adipose tissue (BAT). Leptin also increases SNA to cardiovascular organs including the kidney and raises arterial pressure. However, it is unclear whether leptin controls regional SNA via conserved or distinct molecular mechanisms. Multiple intracellular pathways have been associated with leptin signaling including the mechanistic target of rapamycin complex 1 (mTORC1), which has been proposed as a critical determinant of leptin action. Here, we assessed the contribution of mTORC1 signaling to leptin-evoked regional sympathetic activation. Simultaneous multifiber recording of renal and BAT SNA in anesthetized C57BL/6J mice showed that intracerebroventricular (ICV) administration of leptin (2μg, n=5) increased both renal (170±34%) and BAT (208±37%) SNA. Interestingly, ICV pre-treatment with the mTORC1 inhibitor (rapamycin, 5ng, n=6) abolished the leptin-induced increase in renal (10±6%, P&lt;0.05 vs controls) but not BAT (226±31%) SNA. Next, we used conditional knockout mice that lack the critical mTORC1 subunit, Raptor, specifically in leptin receptor (LRb)-expressing cells (LRb Cre /Raptor fl/fl ) to determine the long-term effects of disrupting mTORC1 signaling on leptin-evoked increase in regional SNA. We confirmed the inability of leptin to activate mTORC1 signaling in LRb-expressing cells of LRb Cre /Raptor fl/fl mice relative to controls using immunohistochemical staining of phosphorylated ribosomal S6, a downstream target of mTORC1. We observed a significant increase in renal SNA in response to ICV leptin in control mice (127±16%, n=9), but not in LRb Cre /Raptor fl/fl mice (-4±15%, n=9, P&lt;0.05 vs controls). Conversely, ICV leptin-induced increase in BAT SNA was not different in LRb Cre /Raptor fl/fl mice (109±27%, n=5) vs. littermate controls (173±52%, n=4). Our data suggest a critical role for mTORC1 signaling in selectively mediating the cardiovascular sympathetic but not the thermogenic actions of leptin, with important implications for obesity-associated hypertension.

Separation of pegylated recombinant proteins and isoforms on CIM ion exchangers.

Protein pegylation is a process of covalent attachment of a polyethylene glycol (PEG) group to the protein tertiary structure that can "mask" the agent from the immune system and also increases the hydrodynamic size of the agent. Usually the pegylation prolongs the protein stability in the organism due to reduced renal clearance and provides superior water solubility to hydrophobic molecules. The mono-pegylated form of protein is usually prefered for medical applications. Different conditions with different PEG reagents have to be tested to find optimal pegylation procedure with specific protein. The goal of this study was to prepare screening method for separation of random mono-pegylated protein. Cytochrome C and beta lactoglobulin were pegylated with four reagents and a complete screening of several chromatographic monoliths in ion exchange mode with different buffers was performed to optimaly separate each mono-pegylated protein. The screening method was developed that produces optimal separation of target pegylated protein on CIM monoliths. Because of short chromatographic run time, CIM monoliths are perfect candidates to test alot of parameters. The results obtained show that each protein has its own unique separation parameters (pH, ionexchange ligand, buffer type). Two biopharmaceuticals were isolated using protocol: super human leptin antagonist (SHLA) was purified from inclusion bodies and mono-pegylated super mouse leptin antagonist (SMLA) from pegylated mixture. During study it was observed that the convective interaction media (CIM) monoliths additionally discriminate between protein isoforms pegylated on different sites in 3D structure of the protein.

Abstract 5245: Sulindac decreases basal-like mammary tumor burden and proinflammatory mediators in obese mice

Abstract Background: A hallmark of the metabolic dysregulation associated with obesity is a pro-inflammatory environment perpetuated by pro-inflammatory mediators including adipokines and growth factor signaling. We previously showed that inflammation and basal-like breast cancer (BLBC) growth are increased in chronically obese mice and persist following weight normalization. Purpose: We tested the hypothesis that Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), can reduce chronic obesity-related inflammation and subsequent BLBC growth. Methods: Mice were administered a control diet (10 kcal% fat) or diet-induced obesity regimen (DIO, 60 kcal% fat). After 15 weeks on diet, DIO mice either continued on DIO diet or were switched to the low fat control diet to induce gradual weight loss, resulting in Formerly Obese (FOb) mice. Half of the mice in all three groups (Control, FOb, DIO, n = 17/group) were randomized to receive Sulindac supplementation at 160 ppm in the diet, which remained constant across diets. Twelve weeks after initiating Sulindac supplementation and weight loss in the FOb groups, all mice were orthopically injected with E0771 cells, a model of basal-like breast cancer. Five mice/group were killed, and their tissue collected and stored at a 4-week intermediate time-point after injection, while 12 mice/group continued in a survival study; these mice were killed when tumor size reached 1.2 cm in diameter in any direction. Results: Sulindac supplementation in DIO mice significantly reduced serum insulin and leptin to levels statistically equivalent to both control and FOb mice. Interestingly, body weight, body fat percentage, and ex vivo visceral white adipose weight were unchanged between all supplemented and non-supplemented counterparts (i.e. DIO vs. DIO+Sulindac). At the 4-week intermediate time point, Sulindac supplementation in DIO mice (but not in control or FOb mice) significantly reduced mean tumor weight relative to their non-supplemented counterparts. In the survival arm of the study, Sulindac significantly increased tumor latency in DIO and FOb groups (but not controls) in comparison to their non-supplemented counterparts. Conclusions: Sulindac supplementation significantly reduced pro-inflammatory mediators insulin and leptin in DIO mice, and increased tumor latency in DIO and FOb mice. Furthermore, Sulindac supplementation did not modulate body weight or fat depots, suggesting that Sulindac indeed offsets some of the pro-tumorigeneic effects of obesity rather than targeting obesity directly. Ongoing analyses of inflammatory surrogates, including circulating cytokines and prostaglandins, mammary gland crown-like structures and cyclooxygenase-2 levels, will help to determine if Sulindac's effects are mediated through its anti-inflammatory activity. Citation Format: Subreen A. Khatib, Emily L. Rossi, Laura W. Bowers, Andrew J. Dannenberg, Stephen D. Hursting. Sulindac decreases basal-like mammary tumor burden and proinflammatory mediators in obese mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5245.

Adult exposure to tributyltin affects hypothalamic neuropeptide Y, Y1 receptor distribution, and circulating leptin in mice.

Tributyltin (TBT), a pesticide used in antifouling paints, is toxic for aquatic invertebrates. In vertebrates, TBT may act in obesogen- inducing adipogenetic gene transcription for adipocyte differentiation. In a previous study, we demonstrated that acute administration of TBT induces c-fos expression in the arcuate nucleus. Therefore, in this study, we tested the hypothesis that adult exposure to TBT may alter a part of the nervous pathways controlling animal food intake. In particular, we investigated the expression of neuropeptide Y (NPY) immunoreactivity. This neuropeptide forms neural circuits dedicated to food assumption and its action is mediated by Y1 receptors that are widely expressed in the hypothalamic nuclei responsible for the regulation of food intake and energy homeostasis. To this purpose, TBT was orally administered at a dose of 0.025mg/kg/day/body weight to adult animals [male and female C57BL/6 (Y1-LacZ transgenic mice] for 4weeks. No differences were found in body weight and fat deposition, but we observed a significant increase in feed efficiency in TBT-treated male mice and a significant decrease in circulating leptin in both sexes. Computerized quantitative analysis of NPY immunoreactivity and Y1-related β-galactosidase activity demonstrated a statistically significant reduction in NPY and Y1 transgene expression in the hypothalamic circuit controlling food intake of treated male mice in comparison with controls. In conclusion, the present results indicate that adult exposure to TBT is profoundly interfering with the nervous circuits involved in the stimulation of food intake.

Human C-reactive protein impedes entry of leptin into the CNS and attenuates its physiological actions in the CNS.

Defective central leptin signalling and impaired leptin entry into the CNS (central nervous system) represent two important aspects of leptin resistance in obesity. In the present study, we tested whether circulating human CRP (C-reactive protein) not only diminishes signalling of leptin within the CNS, but also impedes this adipokine's access to the CNS. Peripheral infusion of human CRP together with co-infused human leptin was associated with significantly decreased leptin content in the CSF of ob/ob mice. Furthermore, following peripheral infusion of human leptin, the CSF (cerebrospinal fluid) concentration of leptin in transgenic mice overexpressing human CRP was sharply lower than that achieved in similarly infused wild-type mice. Administration of LPS (lipopolysaccharide) to human CRP-transgenic mice dramatically elevated the concentrations of human CRP in the CSF. The i.c.v. (intracerebroventricular) delivery of human CRP into the lateral ventricles of ob/ob mice blocked the satiety and weight-reducing actions of human leptin, but not those of mouse leptin. I.c.v. injection of human CRP abolished hypothalamic signalling by human leptin, and ameliorated the effects of leptin on the expression of NPY (neuropeptide Y), AgRP (Agouti-related protein), POMC (pro-opiomelanocortin) and SOCS-3 (suppressor of cytokine signalling 3). Human CRP can impede the access of leptin to the CNS, and elevation of human CRP within the CNS can have a negative impact on the physiological actions of leptin.

Pharmacokinetics of Leptin in Female Mice

Pharmacokinetics of leptin in mammals has received limited attention and only one study has examined more than two time points and this was in ob/ob mice. This study is the first to observe the distribution of leptin over a time course in female mice. A physiologic dose (12 ng) of radiolabelled leptin was injected in adult female mice via the lateral tail vein and tissues were dissected out and measured for radioactivity over a time course up to two hours. Major targets for administered leptin included the liver, kidneys, gastrointestinal tract and the skin while the lungs had high concentrations of administered leptin per gram of tissue. Leptin was also found to enter the lumen of the digestive tract intact from the plasma. Very little of the dose (<1 %) was recovered from the brain at any time. Consequently we confirm that the brain is not a major target for leptin from the periphery, although it may be very sensitive to leptin that does get to the hypothalamus. Several of the major targets (GI tract, skin and lungs) for leptin form the interface for the body with the environment, and given the ability of leptin to modulate immune function, this may represent a priming effect for tissues to respond to damage and infection.

Rapid and efficient identification of the mouse leptin receptor mutation (C57BL/KsJ-db/db) by tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) analysis.

The C57BLKS/J-Leprdb mouse has a point mutation in the leptin receptor gene and is one of the most useful animal model for non-insulin dependent diabetes mellitus in human. Since the homozygote of C57BLKS/J-Leprdb mouse is infertile, detection of point mutation in the leptin receptor gene is important for efficient maintaining strains as well as mass production of homozygotes. To develop a rapid and efficient genotyping method for C57BLKS/J-Leprdb mouse, the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) was used. The 407 and 199 bp PCR products were amplified from normal (+/+) mice; while the 407 and 268 bp PCR products were amplified from homozygotes (db/db) mice; and the 407, 268, and 199 bp PCR products were amplified from heterozygotes (db/+) mice. This result showed that the tetra-primer ARMS-PCR analysis by us is suitable to detect point mutation of the leptin receptor gene. Taken together, our method will dramatically reduce animal use for maintenance of strains as well as production of homozygote in the C57BLKS/J-Leprdb strains.

Dexamethasone treatment alters insulin, leptin, and adiponectin levels in male mice as observed in DIO but does not lead to alterations of metabolic phenotypes in the offspring

Epigenetic inheritance (EI) of metabolic phenotypes via the paternal lineage has been shown in rodent models of diet-induced obesity (DIO). However, the factors involved in soma-to-germline information transfer remain elusive. Here, we address the role of alterations in insulin, leptin, and adiponectin levels for EI of metabolic phenotypes by treating C57BL/6NTac male mice (F0) with the synthetic glucocorticoid dexamethasone and generating offspring (F1) either by in vitro fertilization or by natural fecundation. Dexamethasone treatment slightly alters F0 body composition by increasing fat mass and decreasing lean mass, and significantly improves glucose tolerance. Moreover, it increases insulin and leptin levels and reduces adiponectin levels in F0 fathers as observed in mouse models of DIO. However, these paternal changes of metabolic hormones do not alter metabolic parameters, such as body weight, body composition and glucose homeostasis in male and female F1 mice even when these are challenged with a high-fat diet. Accordingly, sperm transcriptomes are not altered by dexamethasone treatment. Our results suggest that neither increased glucocorticoid, insulin, and leptin levels, nor decreased adiponectin levels in fathers are sufficient to confer soma-to-germline information transfer in EI of obesity via the paternal lineage.Electronic supplementary materialThe online version of this article (doi:10.1007/s00335-015-9616-5) contains supplementary material, which is available to authorized users.

The nutritional composition and anti-obesity effects of an herbal mixed extract containing Allium fistulosum and Viola mandshurica in high-fat-diet-induced obese mice

Obesity is a serious public health issue and plays a critical role in the pathogenesis of hypertension, dyslipidemia, and diabetes [1]. This study investigated the nutritional composition and anti-obesity effects of an herbal extract containing Allium fistulosum and Viola mandshurica (AFE+VME) in high-fat-diet (HFD)-induced obese mice. In traditional oriental medicine, A. fistulosum and V. mandshurica are considered to be effective in promoting blood circulation to remove blood stasis [2]. The nutritional analysis revealed that this mixed extract is high in carbohydrate (72.2 g/100 g) and protein (11.5 g/100 g); low in fat (1.7 g/100 g); rich in vitamins E (4.8 mg/100 g), B1 (14.8 mg/100 g), B2 (1.0 mg/100 g), niacin (7.9 mg/100 g), and folic acid (1.57 mg/100 g); and rich in minerals such as calcium (600 mg/100 g), iron (106.1 mg/100 g), and zinc (5.8 mg/100 g). The oral administration of AFE+VME in mice reduced body weight, tissue weight, adipocyte size, and lipid accumulation in the liver compared with HFD control mice. AFE+VME also decreased serum triglyceride, total cholesterol, and leptin concentrations. Furthermore, AFE+VME markedly increased the mRNA expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), uncoupling protein-2 (UCP-2), and adiponectin and decreased leptin expression in the epididymal adipose tissue. Our results suggest that the extract containing A. fistulosum and V. mandshurica improved lipid metabolism via the up-regulation of PPAR-γ, UCP-2, and adiponectin expression and the down-regulation of leptin in HFD-induced obese mice. Therefore, the extract containing A. fistulosum and V. mandshurica may be a potentially effective therapy for obesity and its related metabolic disorders.

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.

Abstract 085: Role of Suppressor of Cytokines Signaling 3 (Socs3) in Modulating Chronic Metabolic and Cardiovascular Effects of Leptin

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of leptin signaling. Hypothalamic SOCS3 is upregulated in obese animals fed a high-fat diet and has been suggested to contribute to development of resistance to leptin’s anorexic effects. In this study we determined whether deletion of SOCS3 in the entire central nervous system (CNS) amplifies the chronic anorexic and blood pressure (BP) effects of physiological increases in plasma leptin in mice fed a normal diet. SOCS3 flox/flox -Nestin-cre mice were generated by breeding SOCS3 flox/flox with Nestin-cre mice. BP and heart rate (HR) were recorded by telemetry, and oxygen consumption (VO 2 ) was monitored by indirect calorimetry in 22-week-old SOCS3 flox/flox -Nestin-cre (n=4) and control mice (SOCS3 flox/flox , n=4). Compared to controls SOCS3 flox/flox -Nestin-cre mice were lighter (30±1 vs 33±1 g) and normoglycemic (124±7 vs 146±10 mg/dl), consumed less food (3.0±0.4 vs 3.6±0.2 g/day) and had similar VO 2 (77±6 vs 73±3 ml/kg/min). SOCS3 flox/flox -Nestin-cre mice had similar MAP (103±3 vs 107±3 mmHg) but higher HR (666±15 vs 602±17 bpm) compared to control mice. Chronic leptin infusion greatly reduced food in SOCS3 flox/flox -Nestin-cre (46±3 vs 35±4%) and increased MAP (15±3 vs 7±2 mmHg) and VO2 (18±3 vs 14±2%) compared to control mice. No significant changes were observed in HR in either group. Leptin infusion significantly reduced blood glucose levels in both groups (124±7 to 97±7 vs 146±10 to 105±7 mg/dl). These results indicate that SOCS3 deletion in the entire CNS reduces body weight and food intake, and amplifies leptin’s effect on appetite and blood pressure and also suggest the SOCS3 signaling attenuates the chronic actions of leptin on blood pressure as well as appetite regulation even in non-obese mice fed a normal diet. (NHLBI-PO1HL51971, NIGMS P20GM104357 and AHA SDG5680016)