Mechanisms of Leptin Signaling in the Hypothalamus

下丘脑瘦素信号传导机制

• 批准号: 6612446
• 负责人: ABHIRAM SAHU
• 金额: $ 27.86万
• 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/6612446
• 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）：该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，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起作用的分子机制尚不清楚。在这方面，由于瘦素受体是细胞因子受体家族的成员，瘦素激活下丘脑的JAK -STAT信号通路。我们最近的研究表明，瘦素可能通过一种替代信号通路起作用，包括通过激活磷酸二酯酶PDE3B来降低cAMP水平。具体来说，瘦素可诱导PDE3B活性并降低下丘脑的cAMP水平，而PDE3抑制剂西洛胺可逆转瘦素对大鼠食物摄入和体重的影响。虽然下丘脑中几个对瘦素敏感的厌氧和厌食神经元组成了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PDE3B激活的改变程度尚不清楚。目前还不清楚哪些细胞对西洛胺有特别的反应，哪些细胞对瘦素有改变的cAMP水平。因此，为了进一步证明PDE3B- camp途径是下丘脑瘦素信号传导的一个组成部分，以下具体目标将被解决：目的1：确定PDE3B依赖性瘦素信号传导的主要下丘脑位点：检查PDE3抑制在特定下丘脑位点对瘦素作用的影响。目的2：确定介导PDE3B激活依赖性瘦素信号的神经网络。目的3：确定导致瘦素诱导的PDE3B激活和cAMP降低的上游信号成分。PDE3B和PI3K的作用会被脑室内特定抑制剂阻断。PDE3B, PDE3A, PDE4， PKB， PI3K的活性将通过酶法测定。循环AMP水平将通过RIA测量。RNAse保护实验和原位杂交将用于检测PDE3B、NPY、MCH、GAL、Orexin、POMC和NT基因表达的变化。Western blotting检测JAK2、STAT3和PKB蛋白水平。STAT3的dna结合活性将通过电泳迁移转移试验进行检测。这些研究将进一步加深我们对下丘脑中与进食有关的瘦素信号的理解，因此将与饮食失调治疗方法的发展有关。