Sugar signaling networks detected by high content fluxomics

描述(由申请人提供)：严格控制血糖水平对人类健康至关重要。细胞不断监测胞外和胞内的糖水平。通过改变葡萄糖转运蛋白和代谢酶的动力学性质，可以使肝脏和外周器官的细胞摄取和释放适应。我们的目标是确定所有参与调节葡萄糖运输、代谢和隔室的基因，并通过通量组学解开通量控制网络。对这些网络的了解为糖尿病、肥胖症或癌症等与健康相关的疾病的干预提供了高级基础。葡萄糖转运蛋白、己糖激酶酶以及与葡萄糖和糖原代谢相关的酶是由基因家族编码的。这些成员的动力学性质不同。我们了解很大一部分间接葡萄糖对胰腺胰岛素释放的影响，以及胰岛素作为器官间调节手段对葡萄糖转运活动的影响；对细胞基础上的糖信号知之甚少。在酵母中，多个平行的糖信号级联控制新陈代谢，它们之间的相互作用还不完全清楚。本项目旨在系统地利用通量组学在酵母和人类中鉴定信号网络。弗罗默实验室开发了基因编码的FRET传感器，用于监测胞浆葡萄糖水平的变化。纳米传感器可用于高通量筛选，以确定遗传变异中的通量，即敲除和siRNA收集。开发了用于静态和动态筛分的微流控和微板平台。该项目将在两个模型中系统地识别影响通量成分的基因：单细胞酵母酵母，以及作为肝癌和癌症模型的人类HepG2细胞。屏幕上识别的命中将使用翻转传感器来表征其他糖、磷酸盐以及运输和代谢物分析。新发现的基因将在包括原代肝细胞在内的不同人类细胞系中进行比较。过表达和双突变/双siRNA将使糖信号网络的比较成为可能。该项目与糖尿病和肥胖症(NIDDK)等代谢性疾病相关，并由于糖对癌症(NCI)细胞增殖的重要性，以及其他因血糖水平变化而引起的疾病。该项目开发的网络和数据库有望为新药提供新的筛选方法，并帮助确定新的药物靶点。

DESCRIPTION (provided by applicant): Tight control over glucose levels is crucial for human health. Cells constantly monitor extracellular and cytosolic sugar levels. Acclimation of cellular uptake and release in liver as well as peripheral organs is achieved by changing the kinetic properties of glucose transporters and metabolic enzymes. Our goal is to identify all genes involved in regulating glucose transport, metabolism and compartmentation, and to unravel the flux control networks by fluxomics. The understanding of the networks provides an advanced base for intervention in health-related conditions such as Diabetes, obesity or cancer. Glucose transporters, hexokinases, and enzymes related to glucose and glycogen metabolism are encoded by gene families. The members differ in kinetic properties. We understand large parts of the indirect glucose effect on pancreatic insulin release and the effect of insulin in glucose transporrt activity as a means of inter-organ regulation; less is known about sugar signaling at the cellular basis. Multiparallel sugar signaling cascades control metabolism in yeast;, their interplay is not understood fully yet. This project intends to systematically advance the identification of the signaling networks using fluxomics in yeast and human. The Frommer lab developed genetically encoded FRET sensors for monitoring changes of cytosolic glucose levels. The nanosensors can be used in high-throughput screens to determine flux in genetic variants, i.e. knock-out and siRNA collections. Microfluidic and microplate platforms are developed for static and kinetic screens. The project will systematically identify genes affecting flux components in two models: the unicellular yeast Saccharomyces, and in human HepG2 cells as a model for liver and cancer. Hits identified in the screen will be characterized using FLIP sensors for other sugars, phosphate and by transport and metabolite analyses. The role of the newly identified genes will be compared in different human cell lines including primary hepatocytes. Overexpression and double mutant/double siRNA will enable comparison of sugar signaling networks. This project is relevant for metabolic diseases such as Diabetes and obesity (NIDDK), and due to the importance of sugars for cell proliferation for cancer (NCI), as well as other diseases that arise as a consequence of altered glucose levels. The networks and databases developed in this project promise to provide novel screening methods for novel drugs as well as help identifying new drug targets.