Dissecting the molecular mechanism of metformin action

描述（由申请人提供）：2型糖尿病影响近2500万美国人，是死亡和残疾的主要原因。二甲双胍是双胍类药物的一员，是用于治疗2型糖尿病的一线药物，因为它非常有效，安全，并降低糖尿病并发症的风险，如肾脏疾病，失明和心脏病发作。二甲双胍主要通过降低肝葡萄糖输出来降低糖尿病患者的血糖，并且还可以增加肌肉中的胰岛素敏感性。尽管二甲双胍已被广泛使用超过50年，但其完整的作用机制尚不清楚，二甲双胍的直接靶点也未知。更好地了解二甲双胍的作用机制可能会导致2型糖尿病更智能的治疗。为了确定二甲双胍的分子靶点，我们建议使用经典遗传学和基因组学在C。优雅我们已经发现二甲双胍影响C. elegans的作用呈剂量依赖性，二甲双胍对C.与哺乳动物一样，秀丽隐杆线虫在遗传上部分依赖于AMP活化蛋白激酶（AMPK）信号传导。这表明C.线虫是研究二甲双胍作用的一种简单易行且遗传学上易于处理的模式生物。In C.正向遗传筛选能够识别任何给定生物反应中最重要的基因。使用最佳剂量来减缓野生型蠕虫的生长以用于筛选目的，我们进行了大规模的300，000个单倍体基因组正向遗传筛选，产生了30个对二甲双胍作用具有抗性的独立突变体。与此同时，我们进行了反向遗传RNAi筛选，确定了16个基因，当敲除这些基因时，它们会导致对二甲双胍作用的耐药性。这16个中的14个具有位于GWAS中与人类糖尿病、肥胖症或心脏代谢疾病相关的基因座中的人类直系同源物，这是该数据集与人类疾病相关性的令人信服的论据。在目标1中，我们将定义二甲双胍在C中产生的代谢状态。通过研究二甲双胍处理的C.优雅在目标2中，我们将从遗传学上确定二甲双胍产生这些代谢效应的主要途径。大多数二甲双胍耐药基因突变体中的因果突变将通过下一代全基因组测序进行鉴定。将使用C.线虫转基因，组织特异性RNAi，并通过详细的生理特性。通过分析二甲双胍应答中各途径的其他成员，确定最重要的途径。最后，我们将把每个二甲双胍反应途径与GWAS中鉴定的新出现的人类糖尿病、肥胖和二甲双胍反应基因相关联。通过对二甲双胍作用的基础研究，我们希望阐明糖尿病发展的可能机制，并确定糖尿病治疗的新靶点。

DESCRIPTION (provided by applicant): Type 2 diabetes mellitus affects almost 25 million Americans, and is a leading cause of death and disability. Metformin, a member of the biguanide class of drugs, is a first-line medication used to treat type 2 diabetes mellitus, as it s highly effective, safe, and reduces the risk of diabetic complications such as kidney disease, blindness, and heart attack. Metformin lowers blood sugar in diabetes predominantly by lowering hepatic glucose output, and may also increase insulin sensitivity in muscle. Despite its widespread use for more than 50 years, the full mechanism of action of metformin is not understood, and the direct target of metformin is unknown. Better understanding of the mechanism of metformin action may lead to more intelligent therapies for type 2 diabetes mellitus. To determine the molecular targets of metformin, we propose the use of classical genetics and genomics in C. elegans. We have found that metformin affects both growth and metabolism of C. elegans in a dose dependent manner, and metformin's action in C. elegans, as it is in mammals, is partially genetically dependent upon AMP-activated protein kinase (AMPK) signaling. This indicates that C. elegans is a facile and genetically tractable model organism to study metformin's action. In C. elegans, forward genetic screening enables identification of the most important genes in any given biological response. Using an optimal dose to slow the growth of wild-type worms for screening purposes, we conducted a large-scale, 300,000 haploid-genome forward genetic screen, yielding 30 independent mutants resistant to the effects of metformin. In parallel, we conducted reverse genetic RNAi screening, identifying 16 genes, which when knocked down, lead to resistance to metformin's effects. Fourteen of these 16 have human orthologs which lie in loci associated with human diabetes, obesity or cardiometabolic disease in GWAS, a compelling argument for the relevance of this data set to human disease. In Aim 1 we will define the metabolic state produced by metformin in C. elegans by studying the physiology of metformin-treated C. elegans. In Aim 2, we will genetically identify major pathways through which metformin exacts these metabolic effects. Causal mutations in the most metformin resistant genetic mutants will be identified by next-generation whole genome sequencing. The mechanism of action of identified metformin response genes will be established using C. elegans transgenics, tissue specific RNAi, and through detailed physiologic characterization. The most important pathways will be identified by analysis of additional members of each pathway in the response to metformin. Finally, we will correlate each metformin response pathway with emerging human diabetes, obesity and metformin response genes identified in GWAS. Through fundamental study of metformin's action, we hope both to illuminate possible mechanisms of the development of diabetes and to identify new targets for diabetes treatment.