IPC knockout, 14 day old female flies, 48h yeast feeding

Background Insulin signaling pathway is conserved from worms to humans. In Drosophila, three insulin-like peptides (dilps) are expressed in the insulin producing cells (IPCs) in the brain. In order to identify target genes of insulin signaling, the IPCs of female flies were destroyed during development by expressing apoptosis inducing factors reaper and head involution defective under control of the promoter region of dilp3. These insulin-deficient flies were analysed using high density microarrays. Results From the genes that appeared to be regulated in the absence of IPCs, the strongest difference was found for a gene involved in carbohydrate metabolism. Sequence analysis revealed that it is an alpha-glucosidase, which is able to break down disaccharides and/or glycogen. Conclusions We could identify an alpha-glucosidase, involved in carbohydrate metabolism, to be strongly affected in the context of decreased insulin signaling. The striking feature of this regulation was not only that it was the highest regulated, but it was the only gene regulated to this degree. Nutrient Conditions and Fly handling Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. Flies were shock frozen in liquid nitrogen and stored at - 80°C for further handling. Keywords: IPC knockout, 14 day old female flies, 48h yeast feeding Insulin producing cell deficient flies were compared to control flies. Two independent biological repeats were performed. From the two repeats, 5 chips were hybridised, including dye swap Experimental and control female flies were collected after hatching, then kept on fly maintenance food and separated from males after 11 days. After one day of recovery from carbon dioxide treatment, females were put in cages with PBS agar plates and supplied with yeast paste as only food source for 48 hours. The microarray was scanned 3 times: low scan (suffix _L in data table) with a low amplification / intensity to avoid saturated spots for proper data analysis, high scan (suffix _H in data table) with a high amplification / intensity to detect also weak fluorescent spots which are missed in the low scan and medium scan which lies between high and low intensity and gives additional data points for subsequent calculations. Both copies and all hybridisation repeats were used for normalisation, VALUE therefore has the same value for these copies in all relevant hybridisations. For additional information see publication and web link. Keywords = insulin, aging, longevity, nutrient dependance Lot batch = FP7

背景 胰岛素信号通路（insulin signaling pathway）从线虫到人类均高度保守。在果蝇（Drosophila）中，脑内的胰岛素分泌细胞（insulin producing cells, IPCs）可表达三种胰岛素样肽（insulin-like peptides, dilps）。为鉴定胰岛素信号通路的靶基因，研究人员通过在dilp3启动子区域调控下表达凋亡诱导因子reaper与head involution defective，在发育阶段破坏了雌性果蝇的IPCs。随后利用高密度微阵列（high density microarrays）对这些胰岛素缺陷型果蝇进行分析。
结果 在IPCs缺失状态下呈现表达调控变化的基因中，参与碳水化合物代谢（carbohydrate metabolism）的某一基因表现出最显著的表达差异。序列分析表明，该基因为α-葡萄糖苷酶（alpha-glucosidase），可降解二糖与/或糖原。
结论 本研究鉴定出一种参与碳水化合物代谢的α-葡萄糖苷酶，其表达在胰岛素信号通路减弱的背景下受到显著调控。该调控的显著特征不仅在于其为表达变化幅度最大的基因，更是唯一达到该调控程度的基因。
营养条件与果蝇饲养 实验与对照雌性果蝇在羽化后收集，置于果蝇维持培养基中饲养，并在11天后与雄性果蝇分离。经二氧化碳处理后恢复1天，将雌性果蝇转移至装有PBS琼脂平板的笼养装置中，仅以酵母膏作为唯一食物来源饲养48小时。随后将果蝇在液氮（liquid nitrogen）中速冻，并保存于-80℃以备后续实验操作。
关键词：IPC敲除（IPCs knockout）、14日龄雌性果蝇、48小时酵母饲喂 胰岛素分泌细胞缺陷型果蝇与对照果蝇进行对比。本研究完成了两次独立的生物学重复（biological repeats）。两次重复中共完成5张微阵列芯片的杂交，包括染料互换（dye swap）实验。
实验与对照雌性果蝇在羽化后收集，置于果蝇维持培养基中饲养，并在11天后与雄性果蝇分离。经二氧化碳处理后恢复1天，将雌性果蝇转移至装有PBS琼脂平板的笼养装置中，仅以酵母膏作为唯一食物来源饲养48小时。
微阵列扫描共进行3次：低强度扫描（数据表中后缀为_L）采用低放大/强度参数，以避免信号点饱和，保障数据分析的准确性；高强度扫描（后缀为_H）采用高放大/强度参数，以检测低强度扫描中遗漏的弱荧光信号点；中等强度扫描介于高低强度之间，可为后续计算提供额外的数据点。所有重复样本与所有杂交重复均用于标准化（normalisation）分析，因此在所有相关杂交实验中，这些样本的VALUE值均保持一致。更多详细信息可参见发表论文与网络链接。
关键词=胰岛素、衰老、寿命、营养依赖性
批次编号=FP7