Mechanisms of Leptin Signaling in the Hypothalamus

描述(由申请者提供)：这个项目的长期目标是了解负责食物摄入量和体重调节的神经生物学机制。瘦素是肥胖基因的产物，是最重要的外周饱足因子之一，主要作用于大脑的下丘脑区域，从而抑制食物摄取和体重。然而，这种激素在下丘脑中作用的分子机制还没有很好地确定。在这一点上，由于瘦素受体是细胞因子受体家族的成员，瘦素激活了下丘脑中的JAK-STAT信号通路。我们最近的研究表明，瘦素可能通过另一种信号途径发挥作用，包括通过激活磷酸二酯酶PDE3B来降低cAMP水平。具体地说，瘦素诱导PDE3B活性并降低下丘脑中cAMP水平，PDE3抑制剂西洛斯塔胺逆转瘦素对大鼠摄食量和体重的影响。虽然下丘脑中的几个瘦素敏感的食欲神经元和厌食神经元组成了控制食物摄入量和体重调节的神经回路，但这些神经元中PDE3B的激活在瘦素信号中的变化程度尚不清楚。目前尚不清楚哪些细胞对西洛胺有特别的反应，或者哪些细胞对瘦素的反应改变了cAMP水平。因此，为了进一步证明PDE3B-cAMP通路是下丘脑中瘦素信号的一个组成部分，将针对以下特定目标进行研究：目的1：确定依赖PDE3B的瘦素信号的初级下丘脑部位：检测在特定下丘脑部位抑制PDE3对瘦素作用的影响。目的2：确定介导PDE3B激活依赖的瘦素信号的神经元网络。目的3：确定导致瘦素诱导PDE3B活化和cAMP减少的上游信号成分。PDE3B和PI3K的作用将被脑室内给予特定的抑制剂所阻断。用酶法检测PDE3B、PDE3A、PDE4、PKB、PI3K的活性。环磷酸腺苷水平将用放射免疫法测定。采用核糖核酸酶保护试验和原位杂交技术，检测PDE3B、NPY、MCH、GAL、增食欲素、POMC和NT基因表达的变化。蛋白印迹法检测JAK2、STAT3和PKB蛋白水平。用凝胶迁移率改变分析法检测STAT3的DNA结合活性。这些研究将进一步加深我们对下丘脑中瘦素信号与进食的关系的理解，因此将与饮食障碍治疗方法的发展相关。

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.