Lepr Expression Research Articles

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

Abstract SY28-04: Obesity-associated leptin signaling promotes chemotherapy resistance in triple-negative breast cancer: The role of tumor-initiating cell enrichment

1870-P: Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression

Glucagon is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that GCGR signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in DIO mice; however, the mechanisms underlying this aspect of glucagon biology remains unclear. Investigation of downstream GCGR targets elucidated potent and previously unknown regulation of liver leptin receptor (Lepr) expression. Intriguingly, we found GCGR agonism induces hepatic Lepr expression in vivo and in vitro. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Administration of the selective GCGR agonist IUB288 in both lean and diet-induced obese (DIO) mice increases both total hepatic Lepr (p<0.001) expression and the signaling isoform Lepr-b (p<0.05) in acute and chronic settings. In addition, Lepr-b increased in IUB288 treated primary hepatocytes (p<0.05), suggesting cell-autonomous regulation. Further, GCGR activation potentiates leptin-stimulated phosphorylation of AMPK (Thr172) in a dose-dependent manner, suggesting an increase in hepatic leptin sensitivity. Mice deficient for the hepatic leptin receptor (LeprΔliver) displayed increased hepatic triglycerides on a chow diet alone (p<0.001), which persisted in a DIO state, with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO WT and LeprΔliver mice resulted in similar reductions of liver triglycerides. Glucagon also reduces plasma lipids during oral lipid challenge. Similar to liver triglycerides, there were no differences in response to IUB288 between WT and LeprΔliver mice. Together these data suggest that although hepatic glucagon and leptin signaling have similar lipid targets, these appear to be two distinct pathways. Disclosure S. Nason: None. T. Kim: None. J.P. Antipenko: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. K.M. Habegger: Consultant; Self; Glyscend, Inc., Novo Nordisk Inc. Research Support; Self; Glyscend, Inc. Stock/Shareholder; Self; Glyscend, Inc. Funding National Institutes of Health (T32HL1053491, R011DK112934-01)

491-P: Obesity-Induced Kidney Damage Is Attenuated by Genetic Deletion of miR-379 in Mice

Obesity is significantly associated with diabetes and related complications like kidney disease. Evidence shows dysregulation of certain non-coding RNAs including microRNAs can accelerate diabetic kidney disease (DKD). Recently, we found that miR-379 and its host transcript long non-coding RNA, lncMGC, were regulated by endoplasmic reticulum (ER) stress in glomerular cells, and lncMGC could promote features of DKD in mice. In this study, we examined the in vivo role of miR-379 in obesity-induced kidney disease by testing miR-379-knockout (KO) mice generated by CRISPR-Cas9 editing. Male (M) and female (F) mice were fed with control diet, or high fat diet (HFD) for 24 weeks to induce obesity in wild type (WT) and miR-379KO mice. Kidney function was examined by measuring urinary albumin and creatinine. Expression of miR-379 target genes (Edem3, Fis1), Leptin receptor (LepR) and profibrotic genes (Tgf-β1, Ctgf, Col4a1, Col1a2) was measured in isolated glomerular cells. Cortical sections were stained for histopathology. Blood glucose levels were similar in miR379KO-HFD and WT-HFD. miR379KO-HFD-F mice exhibited significantly lower body weight and body fat composition versus WT-HFD-F. Hyperinsulinemia observed in WT-HFD-F mice was attenuated in miR379KO-HFD-F. LepR expression was significantly decreased in WT-HFD-F but not miR379KO-HFD-F mice relative to controls. Increased expression of profibrotic genes, glomerular hypertrophy and interstitial fibrosis observed in WT-HFD-F were significantly attenuated in miR379KO-HFD-F mice. Similar to females, male miR-379KO-HFD-M mice showed significant reductions in glomerular hypertrophy, fibrosis and ER stress markers vs. WT-HFD-M. However, unlike females, miR379KO-HFD-M mice did not display reduced body weight compared to WT-HFD-M. These results suggest miR-379 deficiency protects mice from HFD-induced obesity and kidney injury, with some gender differences. miR-379 inhibition may be a novel therapy for HFD-induced metabolic disorders. Disclosure M. Abdollahi: None. M. Kato: None. R. Tunduguru: None. L.L. Lanting: None. R. Natarajan: None. Funding National Institutes of Health

Maternal high-fat diet results in cognitive impairment and hippocampal gene expression changes in rat offspring

Consumption of a high-fat diet has long been known to increase risk for obesity, diabetes, and the metabolic syndrome. Further evidence strongly suggests that these same metabolic disorders are associated with an increased risk of cognitive impairment later in life. Now faced with an expanding global burden of obesity and increasing prevalence of dementia due to an aging population, understanding the effects of high-fat diet consumption on cognition is of increasingly critical importance. Further, the developmental origins of many adult onset neuropsychiatric disorders have become increasingly clear, indicating a need to investigate effects of various risk factors, including diet, across the lifespan. Here, we use a rat model to assess the effects of maternal diet during pregnancy and lactation on cognition and hippocampal gene expression of offspring. Behaviorally, adult male offspring of high-fat fed dams had impaired object recognition memory and impaired spatial memory compared to offspring of chow-fed dams. In hippocampus, we found decreased expression of Insr, Lepr, and Slc2a1 (GLUT1) among offspring of high-fat fed dams at postnatal day 21. The decreased expression of Insr and Lepr persisted at postnatal day 150. Together, these data provide additional evidence to suggest that maternal exposure to high-fat diet during pregnancy and lactation can have lasting effects on the brain, behavior, and cognition on adult offspring.

Tanycyte-Independent Control of Hypothalamic Leptin Signaling.

Leptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.

Cynometra cauliflora Linn. Attenuates metabolic abnormalities in high-fat diet-induced obese mice

Ethnopharmacological relevanceCynometra cauliflora Linn. belongs to the Fabaceae family and is known locally in Malaysia as nam-nam. Traditionally, a decoction of the C. cauliflora leaves is used for treating hyperlipidemia and diabetes. Aim of the studyThis study aims to investigate the anti-obesity and lipid lowering effects of ethanolic extract of C. cauliflora leaves and its major compound (vitexin) in C57BL/6 obese mice induced by high-fat diet (HFD), as well as to further identify the molecular mechanism underlying this action. Methods and materialMale C57BL/6 mice were fed with HFD (60% fat) for 16 weeks to become obese. The treatment started during the last 8 weeks of HFD feeding and the obese mice were treated with C. cauliflora leaf extract at 200 and 400 mg/kg/day, orlistat (10 mg/kg) and vitexin (10 mg/kg). ResultsThe oral administration of C. cauliflora (400 and 200 mg/kg) and vitexin significantly reduced body weight, adipose tissue and liver weight and lipid accumulation in the liver compared to control HFD group. Both doses of C. cauliflora also significantly (P ≤ 0.05) decreased serum triglyceride, LDL, lipase, IL-6, peptide YY, resistin levels, hyperglycemia, hyperinsulinemia, and hyperleptinemia compared to the control HFD group. Moreover, C. cauliflora significantly up-regulated the expression of adiponectin, Glut4, Mtor, IRS-1 and InsR genes, and significantly decreased the expression of Lepr in white adipose tissue. Furthermore, C. cauliflora significantly up-regulated the expression of hypothalamus Glut4, Mtor and NF-kB genes. GC-MS analysis of C. cauliflora leaves detected the presence of phytol, vitamin E and β-sitosterol. Besides, the phytochemical evaluation of C. cauliflora leaves showed the presence of flavonoid, saponin and phenolic compounds. ConclusionThis study shows interesting outcomes of C. cauliflora against HFD-induced obesity and associated metabolic abnormalities. Therefore, the C. cauliflora extract could be a potentially effective agent for obesity management and its related metabolic disorders such as insulin resistance and hyperlipidemia.

The Expression of Leptin and Its Receptor During Tumorigenesis of Diffuse Gliomas such as Astrocytoma and Oligodendroglioma- Grade II, III and IV (NOS)

Background:Leptin, an adipocytokine functions via the leptin receptor, OB-Rb that contains an intact intracellular domain and activates the JAK/STAT signalling cascade. It stimulates growth, migration and invasion of cancer cells in vitro potentiating angiogenesis. Recently, the involvement of leptin in tumor progression is being explored. Gliomas exhibit poor prognosis, low survival rates demanding for novel therapeutic regimens resulting in discovery of many potential biomarkers and pharmaceutical targets. We analysed the potential role of leptin and OB-Rb in carcinogenesis of malignant gliomas. Methods:Sixty fresh tissue samples of diffuse gliomas were collected after tumor excision. Real time PCR, immunohistochemical (IHC) analysis and western blot analysis were carried out to assess the expression of leptin and its receptor. Results:The present study demonstrates the expression of leptin and LepR and their involvement in tumor progression. Of the 60 cases, 57 cases (95%) and 53 cases (88.3%) showed amplification for leptin and OB-Rb respectively. The expression of these proteins were measured semi-quantitatively and correlated with degree of malignancy (p<0.05). The bands were visualised on western blot. Conclusion:Leptin may be valued as a pharmaceutical target and anti-leptin compounds could be developed as drugs in mono- or combined therapies for these tumors.

PO-026 Effects of Different Exercise on the Expression of White Fat Browning Proteins in Growing Rats

Objective The prevention of obesity in children and adolescents can reduce the risk of obesity and various chronic diseases in adulthood. In recent years, it has been found that inducing browning of white fat can regulate energy balance and inhibit fat accumulation .Exercise as a healthy intervention can regulate energy balance by affecting the browning of white fat. However, the effect of exercise on the browning of white fat during the critical period of growth and development is not yet known, and the effect of different exercise methods on the browning of different location of white fat is inconsistent. Therefore, this study intends to explore the effects of different exercise methods on the expression of browning-related proteins of peripheral white adipose tissue and browning-related genes of hypothalamus in growing rats through animal experiments, and to explore the relationship between them.&#x0D; Methods 30 Male, 3-week-old SD rats were randomly divided into three groups, C group: Control group (sedentary, n=10);MD group: moderate-intensity training group (MD group, n=10), and high-intensity intermittent training group (HIIT group, n=10). MD group exercise program: 60% VO2max exercise for 60 minutes, 5 times a week for a total of 4 weeks. HIIT group exercise program: 85% ~ 90% VO2max exercise 6min, 50% VO2max exercise interval 4min, repeated 6 times. 5 times a week for a total of 4 weeks. The exercise intensity was adjusted based on the maximal oxygen uptake measurement every 2 weeks. Three groups of rats were weighed once a week. After the experiment, anesthesia was performed with 10% chloral hydrate 50 mg/Kg (body weight) in the abdominal cavity to rapidly separate the hypothalamus、Perirenal and epididymal fat and put into liquid nitrogen and transfer these to -80°C refrigerator for testing. The expression of POMC, NPY, LEPR and GRP78 mRNA in hypothalamus was determined by RT-PCR. The expression of PRDM16 and UCP1 protein in perirenal white fat was measured by Western Blot.&#x0D; Results 1. The weight of the three groups of rats increased continuously within 4 weeks of exercise intervention. Compared with group C,the weight of group E and group H increased slowly from the second week (p&lt;.05). 2. The increase in absolute weight and relative weight of visceral fat in MD group and HIIT group was significantly lower than that in C group (p&lt;.01); the increase in absolute weight and relative weight of visceral fat in MD group was significantly lower than HIIT group(p&lt;.05).3. Compared with group C, the expression of PRDM16 protein in WAT of the kidneys in group MD was significantly increased (p&lt;.01), and the UCP1 protein expression was significantly increased (p&lt;.01). Compared with group C, there was no significant difference in PRDM16 and UCP1 protein expression in WAT of HIIT group (p&gt;.05). 4. Compared with group C, the expression of NPY mRNA in hypothalamus in group MD was significantly decreased,(p&lt;.01), POMC, LEPR, GRP78 mRNA expression had no significant difference (p&gt;.05); Compared with group C, there was no significant difference in the expression of hypothalamus POMC, NPY，LEPR and GRP78 mRNA in HIIT group (p&gt;.05). 5. There was a negative correlation between the expression of NPYmRNA in hypothalamus and the expression of PRDM16 protein in white fat(p&lt;.01).&#x0D; Conclusions Moderate-intensity exercise for 4 weeks in the growth and development period of rats can decrease NPY expression in hypothalamus of rats and induce white-to-brown phenomena in perirenal adipose tissue ,but not in HIIT.

Hepatic leptin receptor expression can partially compensate for IL-6Rα deficiency in DEN-induced hepatocellular carcinoma

ObjectiveThe current obesity pandemic represents a major health burden, given that it predisposes to the development of numerous obesity-associated disorders. The obesity-derived adipokines not only impair systemic insulin action but also increase the incidence of hepatocellular carcinoma (HCC), a highly prevalent cancer with poor prognosis. Thus, worldwide incidences of HCC are expected to further increase, and defining the molecular as well as cellular mechanisms will allow for establishing new potential treatment options. The adipose tissue of obese individuals increases circulating leptin and interleukin-6 (IL-6) levels, which both share similar signaling capacities such as Signal Transducer and Activator of Transcription 3 (STAT3) and Phosphoinositide 3-kinase (PI3K)/Akt activation. While mouse models with deficient IL-6 signaling show an ameliorated but not absent Diethylnitrosamine (DEN)-induced HCC development, the morbid obesity in mice with mutant leptin signaling complicates the dissection of hepatic leptin receptor (LEPR) and IL-6 signaling in HCC development. Here we have investigated the function of compensating hepatic LEPR expression in HCC development of IL-6Rα-deficient mice. MethodsWe generated and characterized a mouse model of hepatic LEPR deficiency that was intercrossed with IL-6Rα-deficient mice. Cohorts of single and double knockout mice were subjected to the DEN-HCC model to ascertain liver cancer development and characterize metabolic alterations. ResultsWe demonstrate that both high-fat diet (HFD)-induced obesity and IL-6Rα deficiency induce hepatic Lepr expression. Consistently, double knockout mice show a further reduction in tumor burden in DEN-induced HCC when compared to control and single LepRL−KO/IL-6Rα knock out mice, whereas metabolism remained largely unaltered between the genotypes. ConclusionsOur findings reveal a compensatory role for hepatic LEPR in HCC development of IL-6Rα-deficient mice and suggest hepatocyte-specific leptin signaling as promoter of HCC under obese conditions.

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels.

Leptin is critical for energy balance, glucose homeostasis, and for metabolic and neuroendocrine adaptations to starvation. A prevalent model predicts that leptin's actions are mediated through pro-opiomelanocortin (POMC) neurons that express leptin receptors (LEPRs). However, previous studies have used prenatal genetic manipulations, which may be subject to developmental compensation. Here, we tested the direct contribution of POMC neurons expressing LEPRs in regulating energy balance, glucose homeostasis and leptin secretion during fasting using a spatiotemporally controlled Lepr expression mouse model. We report a dissociation between leptin's effects on glucose homeostasis versus energy balance in POMC neurons. We show that these neurons are dispensable for regulating food intake, but are required for coordinating hepatic glucose production and for the fasting-induced fall in leptin levels, independent of changes in fat mass. We also identify a role for sympathetic nervous system regulation of the inhibitory adrenergic receptor (ADRA2A) in regulating leptin production. Collectively, our findings highlight a previously unrecognized role of POMC neurons in regulating leptin levels.

Leptin Stimulates Prolactin mRNA Expression in the Goldfish Pituitary through a Combination of the PI3K/Akt/mTOR, MKK3/6/p38MAPK and MEK1/2/ERK1/2 Signalling Pathways.

Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of lepR and major pituitary hormones in different pituitary regions. The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2—but not JAK2/STAT 1, 3 and 5 cascades—were involved in leptin-induced PRL mRNA expression in the goldfish pituitary.

The LepR-mediated leptin transport across brain barriers controls food reward

ObjectiveLeptin is a key hormone in the control of appetite and body weight. Predominantly produced by white adipose tissue, it acts on the brain to inhibit homeostatic feeding and food reward. Leptin has free access to circumventricular organs, such as the median eminence, but entry into other brain centers is restricted by the blood–brain and blood–CSF barriers. So far, it is unknown for which of its central effects leptin has to penetrate brain barriers. In addition, the mechanisms mediating the transport across barriers are unclear although high expression in brain barriers suggests an important role of the leptin receptor (LepR). MethodsWe selectively deleted LepR in brain endothelial and epithelial cells of mice (LepRbeKO). The expression of LepR in fenestrated vessels of the periphery and the median eminence as well as in tanycytes was not affected. ResultsPerfusion studies showed that leptin uptake by the brain depended on LepR in brain barriers. When being fed with a rewarding high-fat diet LepRbeKO mice gained more body weight than controls. The aggravated obesity of LepRbeKO mice was due to hyperphagia and a higher sensitivity to food reward. ConclusionsThe LepR-mediated transport of leptin across brain barriers in endothelial cells lining microvessels and in epithelial cells of the choroid plexus controls food reward but is apparently not involved in homeostatic control of feeding.

Potential Contributions of miR-200a/-200b and Their Target Gene-Leptin to the Sexual Size Dimorphism in Yellow Catfish.

Sexual size dimorphism is the consequence of differential expression of sex-biased genes related to feeding and growth. Leptin is known to regulate energy balance by regulating food intake. In order to investigate the molecular mechanism of sexual size dimorphism in yellow catfish (Pelteobagrus fulvidraco), the expression of leptin (lep) and its functional receptor (lepr) were detected during larval development. Both lep and lepr have lower expression in males than in females during 1–4 weeks post hatching. 17a-Methyltestosterone (MT) treatment resulted in decreased expression of lep and lepr in both male and female larval fish. Interestingly, the mRNA levels of lep and lepr in juvenile male were significantly decreased compared with juvenile female during short-term fasting periods. Lep was predicted to be a potential target of miR-200a and miR-200b that had an opposite expression pattern to lep in male and female larvas. The results of luciferase reporter assay suggested that lep is a target of miR-200a/-200b. Subsequently, male hormone and fasting treatment have opposite effects on the expression of miR-200a/-200b and lep between males and females. In summary, our results suggest that sexual size dimorphism in fish species is probably caused by the sexually dimorphic expression of leptin, which could be negatively regulated by miR-200a/-200b.

OP.8B.05] TRANSGENERATIONAL INHERITANCE OF OBESITY AND HYPERTENSION

Objective: Maternal obesity increases the risk of developing hypertension in the offspring. Circulating leptin is closely linked to the control of sympathetic nerve activity (SNA) and blood pressure (BP) in the central nerve system. The ventromedial hypothalamus (VMH) is a key center of energy homeostasis, hemodynamic and sympathetic tone to renal vasculature. Exposure to overnutrition during early development changes the activity of the neurons and the receptors in the CNS, amplifying sympathetic output which leads to hypertension. We assessed the effects of maternal high fat diet (HFD) feeding during pregnancy on cardiovascular variables and sympathetic nerve activity (SNA) with changes in leptin and insulin signaling pathways in adulthood. Design and method: Breeder rabbits were fed a HFD (13%; HFD) or a control diet (4%; CD) during pregnancy and lactation. Offspring received CD after weaning. All rabbits had a VMH cannula and a renal nerve recording electrode. Mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) were measured at baseline and after receiving increasing doses of αMSH (α-Melanocortin stimulating hormone, 0.3, 1nmol), SHU9119 (melanocortin receptor antagonist, 0.02, 0.04nmol), leptin receptor antagonist (5, 10 μg) or insulin receptor antagonist (0.01, 0.05U). Hypothalamic expression of leptin receptor (LepR) and Phosphoinositide 3-kinase (PI3K) was examined using real-time PCR. Results: Offspring from HFD fed rabbits (mHFD) showed higher MAP (+11%) and RSNA (+42%) than controls in adulthood (P < 0.05). αMSH into the VMH increased MAP, HR and RSNA (+6.6 ± 1mmHg, +23.4 ± 3.45 b/min and +3.66 ± 0.7nu) and SHU9119 reduced MAP and RSNA (−5.6 ± 0.8mmHg, −1.3 ± 0.13 nu) in mHFD rabbits. Leptin receptor antagonist normalized hypertension (−5.8 ± 0.8mmHg) in mHFD rabbits (P < 0.05). Insulin receptor antagonist administration into the VMH showed no changes in MAP, HR and RSNA in both mHFD and mCD rabbits. Offspring from maternal HFD rabbits exhibited an increase in LepR and PI3K expression in the hypothalamus compared to the control group (+47% and +74%). Conclusions: Exposure to over-nutrition during early development leads to permanently altered leptin and MC signaling pathways, due to programming of the key receptors and/or by neuronal plasticity, leading to sympathoexcitation and hypertension in adulthood.

Specific Features of the Hypothalamic Leptin Signaling Response to Cold Exposure Are Reflected in Peripheral Blood Mononuclear Cells in Rats and Ferrets.

Objectives: Cold exposure induces hyperphagia to counteract fat loss related to lipid mobilization and thermogenic activation. The aim of this study was investigate on the molecular mechanisms involved in cold-induced compensatory hyperphagia.Methods: We analyzed the effect of cold exposure on gene expression of orexigenic and anorexigenic peptides, and of leptin signaling-related genes in the hypothalamus of rats at different ages (1, 2, 4, and 6 months), as well as in ferrets. We also evaluated the potential of peripheral blood mononuclear cells to reflect hypothalamic molecular responses.Results: As expected, cold exposure induced hypoleptinemia in rats, which could be responsible for the increased ratio of orexigenic/anorexigenic peptides gene expression in the hypothalamus, mainly due to decreased anorexigenic gene expression, especially in young animals. In ferrets, which resemble humans more closely, cold exposure induced greater changes in hypothalamic mRNA levels of orexigenic genes. Despite the key role of leptin in food intake control, the effect of cold exposure on the expression of key hypothalamic leptin signaling cascade genes is not clear. In our study, cold exposure seemed to affect leptin signaling in 4-month-old rats (increased Socs3 and Lepr expression), likely associated with the smaller-increase in food intake and decreased body weight observed at this particular age. Similarly, cold exposed ferrets showed greater hypothalamic Socs3 and Stat3 gene expression. Interestingly, peripheral blood mononuclear cells (PBMC) mimicked the hypothalamic increase in Lepr and Socs3 observed in 4-month-old rats, and the increased Socs3 mRNA expression observed in ferrets in response to cold exposure.Conclusions: The most outstanding result of our study is that PBMC reflected the specific modulation of leptin signaling observed in both animal models, rats and ferrets, which points forwards PBMC as easily obtainable biological material to be considered as a potential surrogate tissue to perform further studies on the regulation of hypothalamic leptin signaling in response to cold exposure.

Effects of mild calorie restriction on lipid metabolism and inflammation in liver and adipose tissue

Calorie restriction (CR) has been reported to improve lipid metabolism and to decrease inflammatory diseases. However, most existing CR models use 30–50% calorie reduction, which is hard to achieve in humans. We investigated the effects of mild CR on lipid metabolism and inflammatory responses. Male C57BL/6 mice were fed control diet (10% kcal fat, Control) or high fat diet (60% kcal fat, HFD) ad libitum or reduced amount of control diet to achieve 15% CR for 16 wks. Body weights, white adipose tissue weights, liver triacylglycerol levels, and serum fetuin-A levels were lower in CR than in the Control. Serum adiponectin levels were higher in CR and lower in HFD compared with the Control. Liver and adipose tissue Mcp-1 mRNA levels were significantly lower in CR compared with the Control. Adipose tissue mRNA levels of Mcp-1, Il-6, Tnf-α and Socs3 were significantly higher in HFD than in the Control and CR, and levels of these negatively correlated with serum adiponectin levels. CR group had the lowest leptin levels and the highest liver Lepr expression, and Lepr mRNA levels positively correlated with liver Socs3 mRNA levels. Our findings showed that mild CR lowered adiposity which resulted in higher adiponectin and lower fetuin-A levels, and might have contributed to alleviation of inflammatory status in the liver and adipose tissue. Furthermore, mild CR might have affected leptin sensitivity by up-regulating Lepr expression.

Leptin supplementation in vitro improved developmental competence of buffalo oocytes and embryos

The aim of the present study was to determine potential role of leptin on in vitro developmental competence of buffalo oocytes and embryos. Slaughterhouse derived culture grade buffalo cumulus oocyte complexes (COCs) were matured in vitro (IVM) with leptin (10 ng/ml) or without leptin (control). In each experiment, a pool of matured COCs was used for further in vitro embryo production and another pool of COCs was used for cumulus cells and mature oocytes isolation to study the relative mRNA expression of developmentally important genes. Presumptive zygotes were cultured in embryo culture (IVC) media supplemented with leptin (10 ng/ml) or without leptin (control). Cleavage rate was higher (p < 0.05) when leptin was supplemented during IVM + IVC, both, as compared to other groups. Higher cleavage rate was observed in leptin-treated groups, though it was non-significant. Blastocyst rate was higher (p < 0.05) in all the leptin treated groups. The relative mRNA expression of LEPR (Ob-Rb), HAS2 and EGFR was significantly (p < 0.05) up-regulated and the expression of CASPASE3 was down-regulated in cumulus cells of leptin-treated groups. The expression of GDF9, BMP15, GLUT1, LEPR and CASPASE3 transcripts in leptin and non-treated oocytes did not differ. The relative mRNA expression of POU5F1and LEPR transcripts in blastocysts was higher (p < 0.05) in leptin-treated groups; the change in expression of GLUT1, INF-τ and CASPASE3 transcripts was not significant (p > 0.05). Thus, it is concluded that leptin promotes developmental competence of bubaline oocytes by modulating cumulus enabling factors and genes regulating pluripotentcy in the blastocysts.

Restoration of Lepr in β cells of Lepr null mice does not prevent hyperinsulinemia and hyperglycemia

ObjectiveThe adipose-derived hormone leptin plays an important role in regulating body weight and glucose homeostasis. Leptin receptors are expressed in the central nervous system as well as peripheral tissues involved in regulating glucose homeostasis, including insulin-producing β cells of the pancreas. Previous studies assessing the role of leptin receptors in β cells used Cre-loxP to disrupt the leptin receptor gene (Lepr) in β cells, but variable results were obtained. Furthermore, recombination of Lepr was observed in the hypothalamus or exocrine pancreas, in addition to the β cells, and Lepr in non-β cells may have compensated for the loss of Lepr in β cells, thus making it difficult to assess the direct effects of Lepr in β cells. To determine the significance of Lepr exclusively in β cells, we chose to selectively restore Lepr in β cells of Lepr null mice (LeprloxTB/loxTB). Materials and methodsWe used a mouse model in which endogenous expression of Lepr was disrupted by a loxP-flanked transcription blocker (LeprloxTB/loxTB), but was restored by Cre recombinase knocked into the Ins1 gene, which is specifically expressed in β cells (Ins1Cre). We bred LeprloxTB/loxTB and Ins1Cre mice to generate LeprloxTB/loxTB and LeprloxTB/loxTBIns1Cre mice, as well as Leprwt/wt and Leprwt/wtIns1Cre littermate mice. Male and female mice were weighed weekly between 6 and 11 weeks of age and fasting blood glucose was measured during this time. Oral glucose was administered to mice aged 7–12 weeks to assess glucose tolerance and insulin secretion. Relative β and α cell area and islet size were also assessed by immunostaining and analysis of pancreas sections of 12–14 week old mice. ResultsMale and female LeprloxTB/loxTB mice, lacking whole-body expression of Lepr, had a phenotype similar to db/db mice characterized by obesity, hyperinsulinemia, glucose intolerance, and impaired glucose stimulated insulin secretion. Despite restoring Lepr in β cells of LeprloxTB/loxTB mice, fasting insulin levels, blood glucose levels and body weight were comparable between LeprloxTB/loxTBIns1Cre mice and LeprloxTB/loxTB littermates. Furthermore, glucose tolerance and insulin secretion in male and female LeprloxTB/loxTBIns1Cre mice were similar to that observed in LeprloxTB/loxTB mice. Analysis of pancreatic insulin positive area revealed that restoration of Lepr in β cells of LeprloxTB/loxTB mice did not prevent hyperplasia of insulin positive cells nor did it rescue Glut-2 expression. ConclusionCollectively, these data suggest that direct action of leptin on β cells is insufficient to restore normal insulin secretion and glucose tolerance in mice without leptin receptor signaling elsewhere.

Adiponectin Receptors Are Less Sensitive to Stress in a Transgenic Mouse Model of Alzheimer's Disease.

Background: Adiponectin and leptin are implicated in the initiation and pathomechanism of Alzheimer's disease (AD). The serum concentrations of these adipokines has been extensively studied in AD, however little is known about their receptors in this disease.Objective: We developed a novel approach to examine whether the receptors of adiponectin (AdipoR1 and -R2) and/or leptin (LepR) can contribute to AD pathomechanism. To achieve this, we investigated the effect of both genetic and environmental factors associated with AD on the expression of these receptors.Method: We used C57BL/6J (WT) and APP(swe)/Presen(e9d)1 (AD) mice. Both strains were exposed to restraint stress (RS) daily for 6h over different time periods. Then, we measured the mRNA expression of AdipoR1, AdipoR2 and LepR and the level of AdipoR1 and AdipoR2 proteins in the hippocampal and prefrontal cortical areas of each mouse.Results: We detected brain region specific transcriptomic changes of adiponectin receptors induced by APP and PS1 transgenes. Both acute and chronic RS caused significant elevations in AdipoR1 mRNA expression in the hippocampus of WT mice. In the prefrontal cortex, the mRNA expression of AdipoR1 followed a biphasic course. In AD mice, RS did not promote any changes in the expression of AdipoR1 mRNA and AdipoR1 protein levels. AdipoR2 mRNA in AD animals, however, showed a significant increase in the prefrontal cortex during RS. Regarding AdipoR1 and AdipoR2 mRNA and protein expression, relevant changes could be measured during stress exposure in both brain areas. Furthermore, stress exposed groups exhibited little change in LepR mRNA expression.Conclusion: Our findings indicate that carrying the transgenes associated with AD induces modification in the expression of both adiponectin receptors. In the case of a normal genetic background, these receptors also appear to be sensitive to environmental factors, while in a genetically determined AD model less response to stress stimuli could be observed. The results suggest that modification of adipokine receptors could also be considered in the therapeutic approach to AD.