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，瘦素的产生是在翻译水平上调节的，并且其5' UTR内的元件刺激报告基因的翻译，而其3' UTR抑制报告基因的翻译。本申请的主要目标是鉴定响应于营养状态的变化而调节瘦素翻译的顺式元件和反式作用因子。本申请的目的是：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）确定体内饥饿/进食和体外用胰岛素和β-肾上腺素能激动剂急性处理对RBP表达及其与瘦素mRNA相互作用的影响; 3）使用敲低和过表达研究来评估特定RBP在瘦素mRNA翻译中的功能作用;和4）描述导致肥胖症中高基础和胰岛素抵抗瘦素翻译的信号传导机制。瘦素3 'UTR包括富含AU的序列和用于结合已知刺激（HuR）或抑制（TIA-1，TIAR）特异性mRNA翻译的RBP的基序。因此，我们将确定是否瘦素mRNA“在体内”与RBPs“在体内”通过免疫沉淀核糖核蛋白复合物从3 T3-L1脂肪细胞和大鼠脂肪细胞的胞质提取物。使用生物素化的瘦素UTR探针的下拉测定将验证感兴趣的RBP的相互作用并指向所涉及的基序。我们将测试的假设，胰岛素在体外或营养状况在体内（饥饿，喂养）影响特定的RBP瘦素mRNA的结合，并涉及其表达和/或核质穿梭的改变。最后，我们将检验以下假设：与肥胖相关的慢性高胰岛素血症通过激活应激/能量感应途径（例如mTOR、AMPK和MAPK）和/或通过改变特定RBP与瘦素mRNA的表达或结合来在翻译水平上增加瘦素的产生。总的来说，拟议的研究将增强对瘦素生物学的理解，并拓宽脂肪细胞如何作为内分泌细胞发挥作用的知识，该内分泌细胞响应营养提示协调多种激素和细胞因子的产生。

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.