Regulation of energy balance in Drosophila

描述（由申请人提供）：禁食触发行为、身体活动和新陈代谢的协调变化，这些变化在进化过程中得到了很好的保护。在哺乳动物中，这样的反应通常是由转录辅激活因子协调的，转录辅激活因子是环境线索调节的靶点。CREB转录因子家族参与多种细胞功能，包括能量稳态。最近，一个新的家族的关键CREB辅激活剂，称为TORC，已被确定。在激活后，TORC蛋白易位到细胞核，在那里它们通过与CREB的直接相互作用来加强基因激活。果蝇有一个单一的TORC家族成员，dTORC，这是诱导禁食，磷酸化和降解后，以胰岛素依赖性的方式喂养。dTORC突变果蝇对饥饿敏感，并且与野生型相比具有显著更低的糖原和脂质储存。dTORC在大脑中发挥作用，在神经内分泌细胞的一个子集中具有活性，以调节未识别的神经元信号的释放，该信号指示脂肪体（能量储存器官）储存糖原和脂质。该提案旨在利用果蝇了解TORC蛋白如何调节能量平衡。使用dTORC突变体表型的拯救测定法，在细胞培养测定法中激活、核转位和结合CREB所需的dTORC的特异性结构域将被突变并在体内测试功能。dTORC在神经内分泌细胞中起作用以产生到脂肪体的信号的假设将通过在这些细胞中特异性地提供dTORC并测定突变表型的拯救来测试。将检查dTORC在神经内分泌细胞内响应于进食和禁食的体内核穿梭。从鉴定dTORC途径的新组分的遗传筛选中，已经发现4个离散的基因组区域增强或抑制dTORC功能，并且因此是包含编码dTORC途径的新组分的基因的极好候选者。这些区域内的基因将使用单基因突变和RNA干扰进行鉴定。将进行影响胰岛素依赖性dTORC磷酸化和降解的基因的筛选，以鉴定dTORC调节剂，从而了解胰岛素信号传导如何调节dTORC活性。考虑到果蝇和哺乳动物之间TORC蛋白结构和功能的保守性，我们希望我们的研究结果能够为哺乳动物TORC蛋白调节能量平衡提供关键见解。公共卫生相关性：肥胖是国家面临的主要健康问题。最近发现TORC蛋白以胰岛素依赖性方式调节葡萄糖和脂质代谢。这些研究将为TORC蛋白如何调节能量平衡提供关键见解。

DESCRIPTION (provided by applicant): Fasting triggers concerted changes in behavior, physical activity and metabolism that are remarkably well conserved through evolution. In mammals, such responses are often coordinated by transcriptional coactivators that are targets for regulation by environmental cues. The CREB family of transcription factors participates in a variety of cellular functions including energy homeostasis. Recently, a new family of key CREB coactivators, called TORCs, has been identified. Upon activation, TORC proteins translocate to the nucleus where they potentiate gene activation via a direct interaction with CREB. Drosophila has a single TORC family member, dTORC, which is induced upon fasting, and phosphorylated and degraded upon feeding in an insulin-dependent manner. dTORC mutant flies are sensitive to starvation and have significantly lower glycogen and lipid stores compared to wild type. dTORC functions in the brain, where it is active in a subset of neuroendocrine cells, to regulate the release of an unidentified neuronal signal that instructs the fat body, the energy storage organ, to store glycogen and lipids. This proposal aims to use Drosophila to understand how TORC proteins function to regulate energy balance. Using a rescuing assay of the dTORC mutant phenotype, the specific domains of dTORC required for activation, nuclear translocation and binding to CREB in cell culture assays will be mutated and tested for function in vivo. The hypothesis that dTORC functions in the neuroendocrine cells to produce the signal to the fat body will be tested by supplying dTORC specifically in these cells and assaying for rescue of the mutant phenotype. In vivo nuclear shuttling of dTORC within the neuroendocrine cells in response to feeding and fasting will be examined. From a genetic screen to identify new components of the dTORC pathway, 4 discrete genomic regions have been found to enhance or suppress dTORC function, and thus are excellent candidates for containing genes that encode novel components of the dTORC pathway. The genes within these regions will be identified using single-gene mutations and RNA interference. A screen for genes that affect insulin-dependent dTORC phosphorylation and degradation will be carried out to identify dTORC regulators, leading to an understanding of how insulin signaling regulates dTORC activity. Given the conservation of TORC protein structure and function between Drosophila and mammals, we expect our results to provide key insights into the regulation of energy balance by mammalian TORC proteins. PUBLIC HEALTH RELEVANCE: Obesity represents a major health problem facing the nation. TORC proteins have recently been found to regulate glucose and lipid metabolism in an insulin-dependent manner. These studies will provide key insights into how TORC proteins regulate energy balance.