Nutritional regulation of leptin production

描述(申请人提供)：脂肪细胞激素瘦素调节能量平衡，底物代谢，免疫，骨形成和生殖。我们最近的工作表明，瘦素的产生在翻译水平上受到调控，其5‘UTR区内的元件刺激报告基因的翻译，而其3’UTR区内的元件抑制报告基因的翻译。这项应用的主要目标是确定调节瘦素翻译的顺式元件和反式作用因子，以响应营养状态的变化。本应用的目的是：1)确定与瘦素5‘和3’UTRs相互作用的RNA结合蛋白(RBP)，并确定涉及的顺式元件；2)确定体内饥饿/喂养以及体外胰岛素和β-肾上腺素能激动剂急性治疗对RBP表达的影响及其与Leptin mRNA的相互作用；3)通过基因敲除和过表达研究评估特定RBP在Leptin mRNA翻译中的功能作用；以及4)描述导致肥胖患者高基础和胰岛素抵抗Leptin翻译的信号机制。瘦素3‘非编码区包括富含AU的序列和与限制性商业惯例结合的基序，已知能刺激(Hur)或抑制(TIA-1，TIAR)特定mRNAs的翻译。因此，我们将通过免疫沉淀法从3T3-L1脂肪细胞和大鼠脂肪细胞的胞浆提取液中提取核糖核蛋白复合体，来确定瘦素mRNAs‘体内’是否与RBPs‘体内’相关。使用生物素化的瘦素UTR探针的下拉分析将验证感兴趣的限制性商业惯例的相互作用，并指向所涉及的基序。我们将检验这样一种假设，即体外的胰岛素或体内的营养状态(饥饿、喂养)会影响特定的RBPs与Leptin mRNA的结合，并涉及它们的表达和/或核质穿梭的变化。最后，我们将验证与肥胖相关的慢性高胰岛素血症通过激活压力/能量感知通路(例如mTOR、AMPK和MAPK)和/或通过改变特定限制性商业惯例与瘦素mRNA的表达或结合而在翻译水平上增加瘦素产生的假设。总体而言，拟议的研究将加强对瘦素生物学的理解，并拓宽关于脂肪细胞如何作为内分泌细胞发挥功能的知识，内分泌细胞协调多种激素和细胞因子的产生，以响应营养提示。

DESCRIPTION (provided by applicant): The adipocyte hormone leptin regulates energy balance, substrate metabolism, immunity, bone formation and reproduction. Our recent work demonstrates that leptin production is regulated at the translational level, and that elements within its 5' UTR stimulate and its 3' UTRs inhibit translation of a reporter gene. The major goal of this application is to identify the cis elements and trans acting factors that regulate leptin translation in response to variations in nutritional state. The objectives of this application are: 1) To identify RNA binding proteins (RBP) that interact with the leptin 5' and 3'UTRs, and define the cis elements involved; 2) To determine the effect of starvation/feeding in vivo and acute treatment with insulin and beta-adrenergic agonists in vitro on expression of RBPs and their interaction with leptin mRNA; 3) To assess the functional roles of specific RBPs in leptin mRNA translation using knockdown and overexpression studies; and 4) To delineate the signaling mechanisms that cause high basal and insulin-resistant leptin translation in obesity. The leptin 3'UTR includes AU-rich sequences and motifs for binding of RBPs that are known to stimulate (HuR) or inhibit (TIA-1, TIAR) the translation of specific mRNAs. We will therefore determine if leptin mRNA 'in vivo' associates with RBPs 'in vivo' by immunoprecipitating ribonucleoprotein complexes from cytosolic extracts of 3T3-L1 adipocytes and rat adipocytes. Pull-down assays using biotinylated leptin UTR probes will verify the interactions of RBPs of interest and point to the motifs involved. We will test the hypothesis that insulin in vitro or nutritional status in vivo (starvation, feeding) affects the binding of specific RBPs to leptin mRNA, and involves an alteration in their expression and/or nucleocytoplasmic shuttling. Finally, we will test the hypothesis that the chronic hyperinsulinemia associated with obesity increases leptin production at the translational level through the activation of stress / energy sensing pathways (e.g. mTOR, AMPK, and MAPK) and/or by altering the expression or binding of specific RBPs to leptin mRNA. Overall, the proposed studies will enhance understanding of leptin biology and broaden knowledge of how the adipocyte functions as an endocrine cell that orchestrates the production of numerous hormones and cytokines in response to nutritional cues.