Soluble leptin receptor represents the main leptin binding activity in human blood

In human blood leptin circulates both free and bound to high molecular weight proteins. Hypothesising that these proteins may modulate ligand bioavailability and bioactivity of leptin, we investigated their molecular nature. Therefore, leptin binding activity was partially purified from human plasma using a leptin affinity column. Subjecting this preparation to size exclusion chromatography (SEC) we observed a coelution of leptin binding activity with levels of the soluble leptin receptor (sOB-R) determined by a newly developed ligand immunofunctional assay. In Western blot analysis the partially purified leptin binding activity exhibited sOB-R immunoreactivity in two bands of 110 and 140 kD. Following N-deglycosylation these bands were replaced by two bands with the molecular weight of 90 and 60 kD, suggesting two isoforms which are capable of leptin binding, as determined by cross-linking. Furthermore, different ratios of these isoforms were detectable in fractions of the leptin binding activity after separation by SEC. These findings indicate the formation of heterodimers and homodimers complexed with and without leptin. As the two sOB-R bands from Western blot analysis correspond to only two specific bands in cross-linking experiments with 125l-leptin, the role of both isoforms as leptin binding proteins appears to be exclusive. Therefore, our results indicate that sOB-R is the major leptin binding protein in the circulating human blood.     

小鼠胚胎附植前后瘦素长型受体在下丘脑及消化器官的表达

目的对长型瘦素受体在雌性昆明鼠生殖周期中的表达情况进行研究。方法采用蛋白免疫印迹实验方法对下丘脑、胃、十二指肠组织中的瘦素受体进行了分析,并用免疫组织化学染色法对雌性KM鼠生殖周期不同阶段的下丘脑、胃、十二指肠组织中表达的瘦素长型受体进行定位分析。结果与结论瘦素受体六个亚型的分子量大约分别为(120、90、77、66.2、54、47)×103;下丘脑神经元胞质中有棕褐色阳性颗粒,且数目随妊娠日龄增加而增加;胃底腺中下部分胞质和细胞核有棕褐色阳性颗粒,且阳性率随妊娠日龄的增加而增加;十二指肠腺中胞质和细胞核有棕褐色阳性颗粒,且阳性率随妊娠日龄的增加而增加。     

瘦素在哺乳动物体重调节、繁殖和免疫中的作用

瘦素(Leptin) 主要是由白色脂肪细胞分泌的、肥胖基因编码的、分子量为16 KD 的蛋白类激素。其N 端具有信号肽序列, 引导蛋白质进入分泌途径, 信号肽被切除后成为有生物学功能的成熟蛋白质。瘦素在动物的体重调节、发育与生殖、免疫和糖代谢等方面有重要作用。瘦素已经不仅仅是脂肪细胞分泌的蛋白类激素, 而是一个在许多方面发挥作用的神经内分泌调节因子。本文综述了瘦素在哺乳动物体重调节、繁殖和免疫中的作用及其调控机制, 主要包括: 动物血清瘦素浓度的季节性变化; 光周期、温度和食物等环境因子对瘦素浓度的影响; 瘦素与解偶联蛋白(Uncoupling proteins , UCPs) 在能量代谢和产热中的互作; 瘦素与下丘脑神经肽Y (Hypothalamus neuropeptide Y, NPY) 在体重调节和产热作用中的拮抗; 瘦素与甲状腺激素和胰岛素在能量代谢中的互作以及瘦素在生殖和免疫中的作用。     

Overeating of palatable food is associated with blunted leptin and ghrelin responses

Palatable food is rich in fat and/or sucrose. In this study we examined the long-term effects of such diets on food intake, body weight, adiposity and circulating levels of the satiety peptide leptin and the hunger peptide ghrelin. The experiments involved rats and mice and lasted 5 weeks. In rats, we examined the effect of diets rich in fat and/or sucrose and in mice the effect of a high fat diet with or without sucrose in the drinking water. Animals fed with the palatable diets had a larger intake of calories, gained more weight and became more adipose than animals fed standard rat chow. Fasted animals are known to have low serum leptin and high serum ghrelin and to display elevated serum leptin and lowered serum ghrelin postprandially. With time, a sucrose-rich diet was found to raise the fasting level of leptin and to lower the fasting level of ghrelin in rats. A fat-rich diet suppressed serum ghrelin without affecting serum leptin; high sucrose and high fat in combination greatly reduced serum ghrelin and raised serum leptin in the fasted state. The mRNA expression of leptin in the rat stomach was up-regulated by sucrose-rich (but not by fat-rich) diets, whereas the expression of ghrelin seemed not to be affected by the palatable diets. Mice responded to sucrose in the drinking water with elevated serum leptin (fasted state) and to all palatable diets with low serum ghrelin. The expression of both leptin and ghrelin mRNA in the stomach was suppressed in fasted mice that had received a high fat diet for 5 weeks. We conclude that the expression of leptin mRNA in stomach and the concentration of leptin in serum were elevated in response to sucrose-rich rather than fat-rich diets, linking leptin with sucrose metabolism. In contrast, the expression of ghrelin and the serum ghrelin concentration were suppressed by all palatable diets, sucrose and fat alike. In view of the increased body weight and adiposity neither elevated leptin nor suppressed ghrelin were able to control/restrain the overeating that is associated with palatable diets.     

牛血清中27kDa蛋白的免疫学特性研究

为探讨牛血清中存在的HBsAg样蛋白的性质,给HBV的发病机制、治疗、预防等研究提供依据,我们采集93份牛血清,以SDS-PAGE、Westem Blot对血清中HBsAg样蛋白进行研究,发现其分子量约为27kDa：以SDS-PAGE分离牛血清中的HBsAg样蛋白,经皮下多点免疫小鼠,可产生与人HBV编码蛋白HBsAg反应的抗体。表明该27kDa蛋白可能存在与HBsAg相似的免疫学特性。     

Regulation of free and bound leptin and soluble leptin receptors during inflammation in mice

Leptin, an appetite-regulating hormone/cytokine, circulates both free and bound to soluble leptin receptors (s-leptin R). An electrochemiluminescence (ECL) assay for the quantitative measurement of murine s-leptin R was developed. The absence of s-leptin R immunoreactivity in the serum of db(pas)/db(pas)mice demonstrated the specificity of the assay, which detected s-leptin R both in the free form and complexed with leptin. The distribution of free vs bound leptin and the regulation of s-leptin R were evaluated in mice following administration of the pro-inflammatory stimuli endotoxin (100 microg/mouse) and turpentine (100 microl/mouse). Both endotoxin and turpentine significantly increased serum leptin and s-leptin R levels compared to control mice. The distribution of free vs bound leptin was not altered by administration of endotoxin or turpentine. In fact, approximately 50% of total leptin was present in the free form in either control, endotoxin- or turpentine-injected mice. On the contrary, during the hyperleptinemia of pregnancy, only 10% of total leptin was present in the free form. We conclude that inflammation leads to the increase of both bound and free leptin. Therefore, the total amount of bioactive leptin is increased during acute inflammation, suggesting that leptin participates in the host response to inflammation.     

Deletion of the serotonin 2c receptor from transgenic mice overexpressing leptin does not affect their lipodystrophy but exacerbates their diet-induced obesity

The binding of leptin to hypothalamic neurons elicits inhibition of orexigenic NPY/AgRP neurons and stimulation of anorexigenic POMC/CART neurons. Projections of serotonergic neurons onto POMC neurons suggest that leptin and serotonin converge onto POMC neurons to regulate body weight. We probed the interaction of these pathways by generating transgenic mice overexpressing leptin (LepTg) without 5HT2c receptors. On a chow diet, the lean phenotype of LepTg mice was unaffected by the absence of 5HT2c receptors, whereas on a high fat diet, LepTg/5HT2c receptors knockout mice showed an exacerbation of diet-induced obesity. POMC mRNA levels were low in LepTg, 5HT2c receptors knockout and LepTg/5HT2c receptors knockout mice, demonstrating that perturbations of the 5HT2c receptor and leptin pathways, either alone or in combination, negatively impact on POMC expression. Thus, on a chow diet, leptin action is independent of 5HT2c receptors whereas on a high fat diet 5HT2c receptors are required for the attenuation of obesity.     

Reduced Body Weight and Increased Energy Expenditure in Transgenic Mice Over-Expressing Soluble Leptin Receptor

Background

Soluble leptin receptor (OBRe), one of several leptin receptor isoforms, is the only bona fide leptin binding protein in plasma. Our earlier studies demonstrated that OBRe modulates leptin levels in circulation. Both clinical and in vitro studies have shown that OBRe expression is inversely correlated to body weight and leptin levels. However, it is not clear whether OBRe plays an active role, either in collaboration with leptin or independently, in the maintenance of body weight.

Methodology/Principal Findings

To investigate the function of OBRe in the regulation of energy homeostasis, we generated transgenic mice that express OBRe under the control of human serum amyloid P (hSAP) component gene promoter. The transgene led to approximately doubling of OBRe in circulation in the transgenic mice than in wild type control mice. Transgenic mice exhibited lower body weight at 4 weeks of age, and slower rate of weight gain when compared with control mice. Furthermore, transgenic mice had lower body fat content. Indirect calorimetry revealed that transgenic mice had reduced food intake, increased basal metabolic rate, and increased lipid oxidation, which could account for the differences in body weight and body fat content. Transgenic mice also showed higher total circulating leptin, with the majority of it being in the bound form, while the amount of free leptin is comparable between transgenic and control mice.

Conclusions

These results are consistent with the role of OBRe as a leptin binding protein in regulating leptin''s bioavailability and activity.     

Modulation of the Leptin Receptor Mediates Tumor Growth and Migration of Pancreatic Cancer Cells

Obesity has been implicated as a significant risk factor for development of pancreatic cancer. In the setting of obesity, a systemic chronic inflammatory response is characterized by alterations in the production and secretion of a wide variety of growth factors. Leptin is a hormone whose level increases drastically in the serum of obese patients. High fat diet induced obesity in mice leads to an overall increased body weight, pancreatic weight, serum leptin, and pancreatic tissue leptin levels. Here we report the contribution of obesity and leptin to pancreatic cancer growth utilizing an in vivo orthotopic murine pancreatic cancer model, which resulted in increased tumor proliferation with concomitant increased tumor burden in the diet induced obese mice compared to lean mice. Human and murine pancreatic cancer cell lines were found to express the short as well as the long form of the leptin receptor and functionally responded to leptin induced activation through an increased phosphorylation of AKT473. In vitro, leptin stimulation increased cellular migration which was blocked by addition of a PI3K inhibitor. In vivo, depletion of the leptin receptor through shRNA knockdown partially abrogated increased orthotopic tumor growth in obese mice. These findings suggest that leptin contributes to pancreatic tumor growth through activation of the PI3K/AKT pathway, which promotes pancreatic tumor cell migration.     

瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达的影响

目的研究瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体(Ob-R)mRNA表达的影响。方法采用六孔板培养皿培养鼠平滑肌细胞,5个孔作为一个浓度组,通过半定量的RT-PCR方法,分别观察了不同浓度的大鼠瘦素、胰岛素及IL-6对平滑肌细胞瘦素受体mRNA表达水平的影响。结果通过半定量RT-PCR,我们检测了短型瘦素受体(Ob-Ra)和长型瘦素受体(Ob-Rb)的mRNA表达水平,瘦素受体(Ob-R)mRNA表达水平随着瘦素及IL-6浓度增加而上升,随着胰岛素的浓度增加而下降。结论瘦素及IL-6可在体外上调平滑肌细胞瘦素受体的表达,胰岛素可下调平滑肌细胞瘦素受体的表达。这三个因子参与了瘦素受体的表达调控,对我们了解瘦素抵抗的发生机制有重要意义。     

Identification of a murine pregnancy-associated serum protein (alpha 1-PAP) a female-specific acute-phase reactant

A murine pregnancy-associated protein (alpha 1-PAP) with alpha 1-electrophoretic mobility and an estimated molecular weight of 150 000 was present in serum from pregnant C57BL/10 mice but could not be detected in serum of mature non-pregnant females and males. During pregnancy alpha 1-PAP was first detected on Day 7, rose to maximum levels between Days 12 and 14, and thereafter declined during the remainder of pregnancy and was undetectable by Day 8 post partum. The protein was also detected in the serum of females, but not males, subjected to an inflammatory stimulus. Examination of the alpha 1-PAP levels during an acute-phase response in females demonstrated that the protein behaved as a typical classical acute-phase reactant, although the levels were only 10% of those observed during Days 12 to 14 of pregnancy. alpha 1-PAP therefore appears to represent a hitherto undescribed female-specific acute phase reactant in the mouse.     

Lactation-dependent down regulation of leptin production in mouse mammary gland

Lactation-dependent regulation of leptin expression in mouse mammary gland and parametrial adipose tissue was estimated by RT-PCR analysis for virgin, pregnant, lactating and post-lactating mice, and the serum and milk leptin levels of these mice were also determined by ELISA. Leptin gene expression in mammary gland as well as in adipose tissue was obviously detected before pregnancy, markedly decreased to 30-50% after parturition and kept at the low level during lactation period, and restored to the original level after weaning. The leptin concentration of milk collected just before weaning was about two-fold higher than that of the milk collected at mid-lactating stages. The serum leptin levels of the mid- and late-lactating mice were not significantly higher than those of non-pregnant mice. These results suggested that the lactation-induced down regulation of leptin was associated with autocrine/paracrine action of leptin in mammary and adipose tissues, and that the milk leptin, especially at the latter stages of lactation, was not only ascribed to diffusive transport from maternal blood stream, but also regional production and secretion by mammary epithelial cells. This possible production of leptin by mammary epithelial cells was further supported by the fact that leptin was expressed by cultured cells of mammary epithelial cell line, COMMA-1D, in a manner negatively dependent on the lactogenic hormones.     

Depression of Cellular Immunity in Pregnancy due to a Serum Factor

Lymphocytes from pregnant women and non-pregnant individuals were cultured under the stimulus of phytohaemagglutinin in the presence of their own and heterologous (allogeneic) sera. The results indicate that heterologous sera have an inhibitory effect on the lymphocyte transformation rate and suggest that the inhibitory property is more powerful in pregnant and fetal sera. Conversely, the addition of heterologous non-pregnant sera to cultures of pregnant lymphocytes increases their transformation rate. These findings suggest that there is a serum inhibitor in pregnancy and this finding may be relevant to the survival of the fetal allograft.     

血清瘦素和胰岛素与犊牛初生重相关性

本实验旨在研究分娩奶牛(Bos taurus)、犊牛和脐静脉血瘦素、胰岛素之间的相关性及与犊牛初生重的相关性,为探究瘦素、胰岛素对犊牛初生重的影响机理提供理论数据。实验选取规模化养殖场正常分娩奶牛54头,按犊牛初生重划分为A组(≤40 kg)9头、B组(40~45 kg)25头、C组(≥45 kg)20头共3组,分别采集分娩奶牛、犊牛和脐静脉血,ELISA法检测血清瘦素、胰岛素含量。多组间采用单因素方差分析和双变量Pearson分析瘦素和胰岛素在各部位静脉血中表达的相关性及与犊牛初生重的相关性。结果表明:(1)奶牛静脉血瘦素、胰岛素含量极显著高于犊牛静脉血和脐静脉血(P0.01),奶牛静脉血瘦素、胰岛素含量与犊牛静脉血和脐静脉血瘦素、胰岛素相关性均不显著(P0.05);(2)瘦素、胰岛素含量在犊牛静脉血与脐静脉血之间均差异不显著(P0.05),犊牛静脉血瘦素、胰岛素含量与脐静脉血瘦素、胰岛素均分别呈极显著正相关(P0.01);(3)奶牛静脉血和脐静脉血中瘦素与胰岛素含量均呈显著正相关(P0.05),而犊牛静脉血中瘦素与胰岛素相关性不显著(P0.05);(4)犊牛初生重与奶牛静脉血瘦素、胰岛素含量均不相关(P0.05),但与犊牛静脉血和脐静脉血瘦素含量显著正相关(P0.05),与犊牛静脉血和脐静脉血胰岛素含量极显著正相关(P0.01);(5)奶牛静脉血、犊牛静脉血和脐静脉血瘦素和胰岛素含量在公犊牛与母犊牛间均无显著差异(P0.05)。可见,奶牛静脉血、犊牛静脉血和脐静脉血均有瘦素、胰岛素表达,且其含量奶牛静脉血显著高于犊牛静脉血和脐静脉血。奶牛静脉血瘦素、胰岛素含量与犊牛初生重相关性不显著,而犊牛静脉血和脐静脉血瘦素、胰岛素含量与犊牛初生重显著正相关性。     

Obesity-induced upregulation of myocardial endothelin-1 expression is mediated by leptin

Several studies have shown that leptin, the product of the obese gene, may link obesity with cardiovascular diseases, and in particular with cardiac hypertrophy. In vitro studies suggest that the mechanism by which leptin causes cardiac hypertrophy involves the upregulation of endogenous endothelin-1 (ET-1), a potent vasoconstrictor and mitogen. Whether obesity-associated hyperleptinemia causes an increase in myocardial ET-1 expression in vivo remains unclear. To address this issue, we fed mice with a high-fat diet and analyzed serum levels of ET-1 and ET-1 mRNA in the heart. We found that in mice fed a high-fat diet, serum ET-1, myocardial ET-1, leptin and leptin receptor mRNA were all elevated. In contrast, in leptin-deficient obese (ob/ob) mice, both serum and myocardial ET-1 levels were not higher than in wild type mice. These findings suggest that upregulation of myocardial ET-1 by obesity is mediated by leptin.     

Cafeteria diet-induced obesity plus chronic stress alter serum leptin levels

Obesity is a disease that has become a serious public health issue worldwide, and chronic stressors, which are a problem for modern society, cause neuroendocrine changes with alterations in food intake. Obesity and chronic stress are associated with the development of cardiovascular diseases and metabolic disorders. In this study, a rat model was used to evaluate the effects of a hypercaloric diet plus chronic restraint stress on the serum leptin and lipids levels and on the weight of specific adipose tissue (mesenteric, MAT; subcutaneous, SAT and visceral, VAT). Wistar rats were divided into the following 4 groups: standard chow (C), hypercaloric diet (HD), stress plus standard chow (S), and stress plus hypercaloric diet (SHD). The animals in the stress groups were subjected to chronic stress (placed inside a 25 cm × 7 cm plastic tube for 1 h per day, 5 days per week for 6 weeks). The following parameters were evaluated: the weight of the liver, adrenal glands and specific adipose tissue; the delta weight; the Lee index; and the serum levels of leptin, corticosterone, glucose, total cholesterol, and triglycerides. The hypercaloric diet induced obesity in rats, increasing the Lee index, weight, leptin, triglycerides, and cholesterol levels. The stress decreased weight gain even in animals fed a hypercaloric diet but did not prevent a significant increase in the Lee index. However, an interaction between the independent factors (hypercaloric diet and stress) was observed, which is demonstrated by the increased serum leptin levels in the animals exposed to both protocols.