大熊猫胃肠道神经肽Y和长型瘦素受体的表达

为了观察神经肽Y(NPY)和瘦素长型受体(OB-Rb)在大熊猫胃肠道的表达分布,并探讨其功能。本实验选用中国保护大熊猫研究中心濒危动物繁殖与保护遗传四川省重点实验室提供的3例大熊猫胃肠组织样品,采用HE和免疫组化SABC法进行组织学、NPY及OB-Rb蛋白的表达研究。HE染色结果显示3例大熊猫胃肠道组织学结构完整,主要表现在单细胞和多细胞黏液腺丰富、小肠绒毛较长,肠道黏膜肌层与肌肉层较厚等。免疫组化方法观察到NPY和OB-Rb阳性产物广泛分布于大熊猫的胃肠道中。NPY阳性神经纤维呈串珠状或点状排列,主要位于黏膜下神经丛和肌间神经丛内,前者阳性神经纤维数量较多。NPY阳性细胞主要分布于黏膜层和肠腺,多呈椭圆形、多边形等。OB-Rb阳性产物主要分布在黏膜层,且在固有层内有大量阳性细胞分布。表明NPY和OB-Rb在大熊猫肠道中的广泛表达,为研究NPY和OB-Rb影响肠道的生长发育、消化吸收、免疫等多种功能奠定了基础。     

Regulation of Jak kinases by intracellular leptin receptor sequences

Leptin signals the status of body energy stores via the leptin receptor (LR), a member of the Type I cytokine receptor family. Type I cytokine receptors mediate intracellular signaling via the activation of associated Jak family tyrosine kinases. Although their COOH-terminal sequences vary, alternatively spliced LR isoforms (LRa-LRd) share common NH(2)-terminal sequences, including the first 29 intracellular amino acids. The so-called long form LR (LRb) activates Jak-dependent signaling and is required for the physiologic actions of leptin. In this study, we have analyzed Jak activation by intracellular LR sequences under the control of the extracellular erythropoeitin (Epo) (Epo receptor/LRb chimeras). We show that Jak2 is the requisite Jak kinase for signaling by the LRb intracellular domain and confirm the requirement for the Box 1 motif for Jak2 activation. A minimal LRb intracellular domain for Jak2 activation includes intracellular amino acids 31-48. Although the sequence requirements for intracellular amino acids 37-48 are flexible, intracellular amino acids 31-36 of LRb play a critical role in Jak2 activation and contain a loose homology motif found in other Jak2-activating cytokine receptors. The failure of short form sequences to function in Jak2 activation reflects the absence of this motif.     

The intracellular domain of the leptin receptor prevents mitochondrial depolarization and mitophagy

Hypothalamic leptin receptor (LR) signaling regulates body weight by balancing food intake and energy expenditure. It is well established that the human LR undergoes ectodomain shedding, but little is known about the fate of the remaining cytosolic domain. This study demonstrates that regulated intramembrane proteolysis (RIP) releases the LR intracellular domain (LR ICD), which translocates to the mitochondria where it binds to SOCS6. This LR ICD-SOCS6 interaction stabilizes both proteins on the mitochondrial outer membrane and requires a functional BC box in SOCS6 for mitochondrial association and a central motif in the LR ICD for SOCS6 binding. The LR ICD prevents CCCP-induced mitochondrial depolarization and mitophagy as shown by lowered Parkin translocation and p62 accumulation. Strict regulation of mitochondrial dynamics in the hypothalamus is known to be essential for body weight homeostasis. This is the first study showing that the LR can directly modulate mitochondrial biology.     

Subcellular localization and internalization of the four human leptin receptor isoforms.

There are four known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains (designated by the number of unique C-terminal amino acids). In cells expressing HLR-5, -15, or -274, 15-25% of the leptin binding sites were located at the plasma membrane. In contrast, in cells expressing HLR-67, only 5% of the total binding sites were at the plasma membrane. Immunofluorescent microscopy showed that all four isoforms partially co-localized with calnexin and beta-COP, markers of the endoplasmic reticulum and the Golgi, respectively. All isoforms were also detected in an unidentified punctate compartment. All isoforms were internalized via clathrin-mediated endocytosis, but at different rates. After 20 min at 37 degrees C, 45% of a bound cohort of labeled ligand had been internalized by HLR-15, 30% by HLR-67, 25% by HLR-274, and 15% by HLR-5. Degradation of internalized leptin occurred in lysosomes. Overnight exposure to leptin down-regulated all isoforms, but to a variable extent. HLR-274 displayed the greatest down-regulation and also appeared to reach lysosomes more quickly than the other isoforms. The faster degradation of HLR-274 may help to terminate leptin signaling.     

Ultrastructural localization of the receptor for leptin in the rat hypothalamus

Ultrastructural localization of the leptin receptor in the rat hypothalamus was studied by immunocytochemistry. The antiserum against the leptin receptor which was used specifically recognized the carboxy terminal of the cytoplasmic domain. Intense leptin receptor immunoreactivity was detected in the arcuate, paraventricular, and ventromedial nuclei of the hypothalamus and in the lateral hypothalamic area. At the ultrastructural level, leptin receptor-like immunoreactivity appeared to be concentrated predominantly in perikarya and dendrites of these areas and strong immunolabeling for the leptin receptor was detected in the plasma membrane, rough endoplasmic reticulum, Golgi apparatus, and cytoplasmic matrix. This study provides the first detailed fine structure of leptin receptor-immunoreactive neurons in the rat hypothalamus. It may help to provide better understanding of the functions of leptin in the rat hypothalamus.     

Effect of metformin on testicular expression and localization of leptin receptor and levels of leptin in the diabetic mice

Diabetes mellitus impairs testicular activity and leads to infertility. Leptin is one of the endogenous regulators of the male reproductive functions, but the role of leptin and its receptor (LEPR/Ob‐R) in the control of testosterone production and testicular proliferation has not been investigated so far, especially in the Type 1 diabetes mellitus (DM1). Metformin is an anti‐hyperglycemic drug which is beneficial for treating the both DM2 and DM1. The aim of this work was to study the possible role of leptin and Ob‐R in the regulation of steroidogenesis and proliferation in the testes of mice with streptozotocin‐induced DM1 (75 mg/kg/day, 4 days) and to estimate the restoring effect of metformin treatment (500 mg/kg, 2 weeks) on the diabetic testes. In the diabetic testes, the plasma and intratesticular leptin levels and plasma testosterone levels were reduced and completely restored by metformin treatment. Metformin also restored the expression of the steroidogenic transport protein steroidogenic acute regulatory protein reduced in DM1. In the diabetic testes, the expression of Ob‐R was downregulated and the immunolocalization of Ob‐R showed weak staining in the Leydig cells, the primary spermatocytes and the round spermatids. The germ cell proliferation was also reduced in DM1, as noticed with proliferating cell nuclear antigen (PCNA) expression. Metformin increased the Ob‐R expression and immunostaining in the different cell types and improved the PCNA expression. Thus, DM1 impairs the testicular steroidogenesis and proliferation by inhibiting the leptin signaling, causing a decrease in leptin levels and Ob‐R expression in the testes of diabetic mice, while metformin improves the leptin signaling and restores testosterone production and testicular proliferation.     

Activation of downstream signals by the long form of the leptin receptor

The adipocyte-derived hormone leptin signals the status of body energy stores by activating the long form of the leptin receptor (LRb). Activation of LRb results in the activation of the associated Jak2 tyrosine kinase and the transmission of downstream phosphotyrosine-dependent signals. We have investigated the signaling function of mutant LRb intracellular domains under the control of the extracellular erythropoietin (Epo) receptor. By using this system, we confirm that two tyrosine residues in the intracellular domain of murine LRb become phosphorylated to mediate LRb signaling; Tyr(985) controls the tyrosine phosphorylation of SHP-2, and Tyr(1138) controls STAT3 activation. We furthermore investigated the mechanisms by which LRb controls downstream ERK activation and c-fos and SOCS3 message accumulation. Tyr(985)-mediated recruitment of SHP-2 does not alter tyrosine phosphorylation of Jak2 or STAT3 but results in GRB-2 binding to tyrosine-phosphorylated SHP-2 and is required for the majority of ERK activation during LRb signaling. Tyr(985) and ERK activation similarly mediate c-fos mRNA accumulation. In contrast, SOCS3 mRNA accumulation requires Tyr(1138)-mediated STAT3 activation. Thus, the two LRb tyrosine residues that are phosphorylated during receptor activation mediate distinct signaling pathways as follows: SHP-2 binding to Tyr(985) positively regulates the ERK --> c-fos pathway, and STAT3 binding to Tyr(1138) mediates the inhibitory SOCS3 pathway.     

Characterization of leptin intracellular trafficking

Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR) is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBR) with an intracellular domain of 303 amino acids and a shorter form (OBR) with an intracellular domain of 34 amino acids. Since OBR, is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3-transferrin containing compartments at 37 degrees C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF) and the lysosomal marker protein lamp-1. The transport of leptin was also shown to engage a monensin and bafilomycin sensitive degradation process in lysosomes. Together, our results provide novel data concerning the uptake, intracellular localization and transport of leptin.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lacta-tion-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Expression and function of leptin and its receptor in mouse mammary gland

Leptin is an autocrine and paracrine factor which affects the development of duct, formation of gland alveolus, expression of milk protein gene and onset involution of mammary gland. In order to know the function and mechanism of leptin in mammary gland, the protein expression and localization of leptin and its long form receptor (OB-Rb) were detected by a confocal laser scanning microscope. To study the impacts of leptin on mammary gland and leptin signal transduction pathway in pregnancy-, lactation-and involution-stage mammary gland, explants were cultured and Western blotting was used. The results showed that in the whole development cycle of mammary gland, the expression of leptin and OB-Rb was in positive correlation. In virgin the leptin expression was the highest and then decreased in pregnancy. In lactation the expression of leptin was low and upgraded in involution, and recovered to the original level about virgin on involution 13 d. The localization of leptin and OB-Rb revealed that leptin induced the expression of OB-Rb specifically and controlled the development and physiological function of the mammary gland by binding to OB-Rb. In pregnancy stage, leptin stimulated proliferation and differentiation of ductal epithelial cells by JAK-MAPK signal pathway. In lactation, leptin induced gene expression of β-casein by JAK-STAT5 signal pathway, and in involution leptin induced mammary epithelial cell apoptosis and mammary gland restitution by JAK-STAT3 signal pathway.     

Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.     

Expression of leptin ligand and receptor and effect of exogenous leptin supplement on in vitro development of porcine embryos

The present study investigated the expression of ligand and receptor for leptin, and the effect of leptin supplementation on preimplantation development of porcine in vitro fertilized (IVF) and somatic cell nuclear transfer (SCNT) embryos. The IVF embryos were produced using frozen boar semen and SCNT embryos were obtained by nuclear transfer of fetal fibroblasts into enucleated oocytes. The protein expression of leptin ligand and receptor was investigated in in vitro matured oocytes, 2-, 4- and 8-cell embryos, morulae and blastocysts derived from IVF and SCNT using immunofluorescence. Both the ligand and receptor were detected in in vitro matured oocytes and all stage of IVF and SCNT embryos. The IVF and SCNT embryos were cultured in modified North Carolina State University (mNCSU)-23 medium supplemented with various concentrations (0, 1, 10, 100 or 1000 ng/mL) of leptin. The rates of cleavage at day 2 and blastocyst formation at day 7, and cell number of blastocysts were monitored as experimental parameters. In SCNT embryos, supplementing with 1000 ng/mL leptin significantly (P<0.05) increased the rate of blastocysts formation (20.2% versus 12.9%) and total cell number (54.6+/-17.4 versus 45.1+/-15.2) compared to the control group. In IVF embryos, leptin supplementation did not affect preimplantation embryo development and cell number in blastocysts. In conclusion, the present study demonstrated the expression of leptin ligand and receptor and the embryotropic effect of leptin in SCNT embryos.     

Expression of the leptin receptor in different types of vascular lesions

Clinical and experimental evidence suggests that the adipokine leptin may be important for the development of cardiovascular complications associated with obesity, possibly through interaction with its receptor on vascular cells. In the present study, we systematically analysed expression of the leptin receptor in normal and diseased vascular specimens using immunohistochemistry, immunofluorescence and quantitative real time-PCR. In particular, human atherosclerotic plaques as well as experimental vascular lesions induced in hypercholesterolemic mice and minipigs, respectively, were examined. Our results demonstrate the presence of the leptin receptor in normal vessel wall segments as well as neointimal or atherosclerotic lesions. In the latter, ObR expressing cells were predominantly localised on the luminal border and within the subintima, and coexpression of von Willebrand factor, VEGF receptor-2 or VE cadherin identified them as endothelial cells. Moreover, CD14-positive monocytes/macrophages were strongly positive for the leptin receptor. In contrast, only few ObR-expressing smooth muscle cells could be detected in human atherosclerotic plaques. The findings of the present study thus support a possible action of leptin on the cardiovascular system by demonstrating expression of the leptin receptor in different types of vascular lesions.     

Expression of leptin receptor isoforms and effects of leptin on the proliferation and hormonal secretion in human pituitary adenomas

OBJECTIVE: To pursue whether leptin regulates anterior pituitary cells, we studied the ex vivo expression of several isoforms of the leptin receptor (OB-R) as well as the in vitro effects of leptin administration in human pituitary adenomas. METHODS: OB-R mRNA expression and in vitro response to leptin were studied in 39 pituitary macroadenomas. RESULTS: All 4 OB-R subtypes were expressed in most adenomas. The expression was significantly more pronounced in GH-secreting adenomas as compared to non-functioning tumor cells (p < 0.05). Leptin administration in vitro did not significantly influence cell proliferation or the secretion of GH, FSH, LH or alpha-subunit. CONCLUSIONS: (1) Several isoforms of the OB-R, including the signal transducing full-length receptor, are expressed in most human pituitary adenomas. (2) This expression ex vivo is not associated with significant effects of leptin in vitro.