Discovering and dissecting new regulators of insulin production in beta cells

描述(申请人提供)：全球糖尿病患病率史无前例的上升，使我们无法阻止这种潜在疾病的发展变得更加令人担忧。我的职业目标是指导一个专注于开发新的糖尿病疗法的基础研究项目。使用RNA干扰(RNAi)筛选，我已经确定了一种新的G蛋白偶联受体(GPCR)，它对胰岛素的产生和分泌很重要。我的目标如下：目的一、明确这种新型GPCRs作用于胰岛素启动子和胰岛素分泌的分子机制。我将确定G蛋白信号转导途径(S)是这个GPCR对胰岛素启动子和胰岛素分泌的影响所必需的。我还将确定受GPCR调控的胰岛素启动子中的转录因子和调控位点。还将采用一种使用GQ/11耦合工程GPCR的并行方法。目的II.确定该GPCR在体内β细胞发育和胰岛素产生中的作用。我将为这一GPCR研究一只基因敲除小鼠，重点是β细胞发育和成年β细胞功能。目的III.鉴定胰岛素启动子活性所需的新基因。在用于识别AIM I中确定的候选基因的RNAi屏幕上进行扩展，我将使用高通量系统来筛选针对所有已知基因的siRNAs，以找到胰岛素启动子的其他调节因子。这些目标将为我的第一次R01申请提供必要的培训和独立研究方向。具体地说，我将学习使用糖尿病的小鼠模型，以及全基因组RNAi屏幕的设计和性能。为了获得这种经验，我加入了迈克尔·麦克马努斯博士的实验室，他是一名RNA干扰筛查和非编码RNA方面的专家，以及紧邻的迈克尔·德恩博士的实验室，他是一名内科科学家，在体外和体内都研究胰腺β细胞。我还有两位出色的合作者--布鲁斯·康克林博士，GPCR信号专家和米歇尔·阿金博士，加州大学旧金山分校高通量筛查设施的负责人。加州大学旧金山分校和加州大学旧金山分校糖尿病中心是年轻研究人员的极佳培训环境。我将利用他们丰富的资源。为了帮助AIM III，我将报名参加加州大学旧金山分校生物医学信息学项目关于大数据集统计分析的课程。对于职业发展，我还将参加关于负责任地进行研究、撰写补助金和撰写科学文章的课程和研讨会。我将出席几个以糖尿病、RNAi或非编码RNAs为重点的研讨会系列和联合实验室会议，并展示我的研究。重要的是，我在加州大学旧金山分校的职位允许我将100%的精力投入到我的培训中。综上所述，我建议研究一种新的GPCR在胰岛素产生中的机制，并确定其他在β细胞功能中重要的新基因。这笔拨款不仅可以让我完成这些研究，还可以让我过渡到一名独立的糖尿病研究人员。

DESCRIPTION (provided by applicant): The unprecedented rise in worldwide diabetes prevalence is made even more alarming by our inability to halt the progression of this insidious disease. My career goal is to direct a basic research program focused on developing new diabetes therapies. Using an RNA interference (RNAi) screen, I have identified a novel G- protein coupled receptor (GPCR) that is important for insulin production and secretion. My aims are as follows: Aim I. Define the molecular mechanism of this novel GPCR's action on the insulin promoter and insulin secretion. I will identify what G protein signaling pathway(s) are required for this GPCR's effect on the insulin promoter and insulin secretion. I will also identify the transcription factors and the regulatory sites in the insulin promoter that are regulated by this GPCR. A parallel approach using a Gq/11-coupled engineered GPCR will also be undertaken. Aim II. Determine the role of this GPCR in beta cell development and insulin production in vivo. I will study a knockout mouse for this GPCR focusing on beta cell development and adult beta cell function. Aim III. Identify novel genes required for insulin promoter activity. Expanding on the RNAi screen used to identify the candidate identified in Aim I, I will use a high throughput system to screen siRNAs targeting all known genes to find additional regulators of the insulin promoter. These aims will provide the training and independent research direction necessary for my first R01 application. Specifically, I will learn to use mouse models of diabetes and the design and performance of whole genome RNAi screens. To gain this experience, I have joined the laboratory of Dr. Michael McManus, an expert on RNA interference screening and non-coding RNAs, and the immediately adjacent laboratory of Dr. Michael German, a physician scientist who studies the pancreatic beta cell both in vitro and in vivo. I also have two excellent collaborators -- Dr. Bruce Conklin, an expert in GPCR signaling, and Dr. Michelle Arkin, the leader of the UCSF High Throughput Screening facility. UCSF and the UCSF Diabetes Center are excellent training environments for young investigators. I will capitalize on their rich resources. To assist with Aim III, I will enroll in the UCSF Biomedical Informatics Program's class on the statistical analysis of large data sets. For career development, I will also participate in classes and workshops on the responsible conduct of research, grant writing, and scientific writing. I will attend and present my research in several seminar series and joint lab meetings focused on diabetes, RNAi or non- coding RNAs. Importantly, my position at UCSF allows 100% of my effort to be devoted to my training. In summary, I propose to study the mechanism of a novel GPCR in insulin production and to identify other novel genes important in beta cell function. This grant will not only allow the completion of these studies but will also allow me to transition into an independent diabetes investigator.