Nutritional regulation of leptin production

描述（由申请人提供）：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节，其5' UTR内的元件刺激和3' UTR抑制报告基因的翻译。本应用程序的主要目的是确定顺式元件和反式因子，调节瘦素翻译响应营养状态的变化。本应用程序的目的是：1)鉴定与瘦素5‘和3’ utr相互作用的RNA结合蛋白（RBP），并确定所涉及的顺式元件；2)研究体内饥饿/摄食以及体外胰岛素和β -肾上腺素能激动剂急性治疗对rbp表达的影响及其与瘦素mRNA的相互作用；3)通过敲低和过表达研究评估特异性rbp在瘦素mRNA翻译中的功能作用；4)阐明肥胖中导致高基础和胰岛素抵抗瘦素翻译的信号机制。瘦素3'UTR包含富含au的序列和结合rbp的基序，这些基序已知可刺激（HuR）或抑制（TIA-1， TIAR）特定mrna的翻译。因此，我们将通过免疫沉淀来自3T3-L1脂肪细胞和大鼠脂肪细胞的胞质提取物的核糖核蛋白复合物来确定瘦素mRNA是否与rbp在体内相关。使用生物素化瘦素UTR探针的下拉试验将验证感兴趣的rbp的相互作用，并指出所涉及的基序。我们将验证体外胰岛素或体内营养状况（饥饿、喂养）影响特异性rbp与瘦素mRNA结合的假设，并涉及其表达和/或核细胞质穿梭的改变。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应激/能量感应通路（如mTOR、AMPK和MAPK）和/或通过改变特异性rbp对瘦素mRNA的表达或结合，在翻译水平上增加瘦素产生的假设。总的来说，拟议的研究将加强对瘦素生物学的理解，并拓宽对脂肪细胞作为内分泌细胞如何协调多种激素和细胞因子的产生以响应营养线索的认识。

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.