Mechanisms of Leptin Signaling in the Hypothalamus

下丘脑瘦素信号传导机制

• 批准号: 7014495
• 负责人: ABHIRAM SAHU
• 金额: $ 27.06万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7014495
• 关键词: RNase protection assay; appetite regulatory center; biological signal transduction; confocal scanning microscopy; cyclic AMP; cytokine receptors; enzyme induction /repression; enzyme inhibitors; gel mobility shift assay; hypothalamus; immunofluorescence technique; immunoprecipitation; in situ hybridization; laboratory rat; leptin; nutrient intake activity; nutrition related tag; phosphatidylinositol 3 kinase; phosphodiesterases; radioimmunoassay; ultracentrifugation; weight control; western blottings

DESCRIPTION (provided by applicant): The long-term goal of this project is to understand the neurobiological mechanisms that are responsible for food intake and body weight regulation. Leptin, a product of the obese gene, is one of the most important peripheral satiety factors that inhibits food intake and body weight by acting primarily in the hypothalamic region of the brain. However, the molecular mechanisms by which this hormone acts in the hypothalamus are not well defined. In this regard, since the leptin receptor is a member of the family of cytokine receptors, leptin activates the JAK -STAT signaling pathway in the hypothalamus. Our recent study demonstrates that leptin may act via an alternative-signaling pathway, involving reduction of cAMP levels through activation of a phosphodiesterase, PDE3B. Specifically, leptin induces PDE3B activity and reduces cAMP levels in the hypothalamus, and the PDE3 inhibitor, cilostamide, reverses the effect of leptin on food intake and body weight in rats. While several leptin-sensitive orexigenic and anorectic neurons in the hypothalamus comprise the neural circuitry that governs food intake and body weight regulation, but the extent to which PDE3B activation is altered in these neurons in leptin signaling is unclear. It is still unknown which cells in particular are cilostamide responsive or which cells show altered cAMP levels in response to leptin. Thus, to further demonstrate that PDE3B-cAMP pathway is an integral part of leptin signaling in the hypothalamus, following Specific Aims will be addressed: Aim 1: To identify primary hypothalamic sites of PDE3B dependent leptin signaling: examine the effects of PDE3 inhibition in specific hypothalamic sites on leptin action. Aim 2: To identify the neuronal network that mediates PDE3B activation-dependent leptin signaling. Aim 3: To identify upstream signaling components that leads to leptin-induced activation of PDE3B and reduction of cAMP. The action of PDE3B and PI3K will be blocked by intracerebroventricular administration of specific inhibitors. PDE3B, PDE3A, PDE4, PKB, PI3K activity will be measured by enzyme assays. Cyclic AMP levels will be measured by RIA. RNAse protection assays and in situ hybridization will be used to examine changes in PDE3B, NPY, MCH, GAL, Orexin, POMC and NT gene expression. JAK2, STAT3 and PKB protein levels will be measured by Western blotting. DNA-binding activity of STAT3 will be examined by electrophoretic mobility shift assay. These studies will further our understanding of leptin signaling in the hypothalamus in relation to feeding, and therefore will be relevant to the development of therapeutic approaches to eating disorders.

描述（由申请人提供）：本项目的长期目标是了解负责食物摄入和体重调节的神经生物学机制。瘦素是肥胖基因的产物，是最重要的外周饱腹感因子之一，其主要通过作用于大脑的下丘脑区域来抑制食物摄入和体重。然而，这种激素在下丘脑中作用的分子机制还没有很好的定义。在这方面，由于瘦素受体是细胞因子受体家族的成员，瘦素激活下丘脑中的JAK-STAT信号传导途径。我们最近的研究表明，瘦素可能通过一个替代信号通路，包括通过磷酸二酯酶，PDE 3B的激活降低cAMP水平。具体而言，瘦素诱导PDE 3B活性并降低下丘脑中的cAMP水平，并且PDE 3抑制剂西洛酰胺逆转瘦素对大鼠食物摄入和体重的影响。虽然下丘脑中的几个瘦素敏感性食欲和厌食神经元包括控制食物摄入和体重调节的神经回路，但在瘦素信号传导中这些神经元中PDE 3B活化改变的程度尚不清楚。目前还不清楚哪些细胞特别是西洛酰胺响应或哪些细胞显示出响应瘦素的cAMP水平改变。因此，为了进一步证明PDE 3B-cAMP途径是下丘脑中瘦素信号传导的组成部分，将提出以下具体目的：目的1：鉴定PDE 3B依赖性瘦素信号传导的主要下丘脑位点：检查特定下丘脑位点中PDE 3抑制对瘦素作用的影响。目的2：鉴定介导PDE 3B激活依赖性瘦素信号传导的神经元网络。目的3：鉴定导致瘦素诱导的PDE 3B活化和cAMP减少的上游信号传导组分。脑室内给予特异性抑制剂可阻断PDE 3B和PI 3 K的作用。将通过酶试验测量PDE 3B、PDE 3A、PDE 4、PKB、PI 3 K活性。将通过RIA测量环AMP水平。RNA酶保护试验和原位杂交将用于检查PDE 3B、NPY、MCH、GAL、食欲素、POMC和NT基因表达的变化。JAK 2、STAT 3和PKB蛋白水平将通过蛋白质印迹法测量。将通过电泳迁移率变动分析来检查STAT 3的DNA结合活性。这些研究将进一步加深我们对下丘脑中与进食相关的瘦素信号传导的理解，因此将与饮食失调治疗方法的发展相关。