Dissecting the molecular mechanism of metformin action

描述（由申请人提供）：2型糖尿病影响了近2500万美国人，是导致死亡和残疾的主要原因。作为双胍类药物的一员，二甲双胍是用于治疗2型糖尿病的一线药物，因为它非常有效、安全，并且可以降低肾脏疾病、失明、心脏病等糖尿病并发症的风险。二甲双胍主要通过降低肝脏葡萄糖输出来降低糖尿病患者的血糖，也可能增加肌肉的胰岛素敏感性。尽管二甲双胍的广泛应用已超过50年，但其完整的作用机制尚不清楚，二甲双胍的直接作用靶点尚不清楚。更好地了解二甲双胍的作用机制可能会导致更明智的治疗2型糖尿病。为了确定二甲双胍的分子靶点，我们建议在秀丽隐杆线虫中使用经典遗传学和基因组学。我们发现二甲双胍以剂量依赖的方式影响秀丽隐杆线虫的生长和代谢，二甲双胍在秀丽隐杆线虫中的作用与在哺乳动物中的作用一样，部分依赖于amp激活的蛋白激酶（AMPK）信号。这表明秀丽隐杆线虫是一种易于研究二甲双胍作用的模式生物。在秀丽隐杆线虫中，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。为了筛选目的，我们使用最佳剂量来减缓野生型蠕虫的生长，进行了大规模的30万个单倍体基因组正向遗传筛选，产生了30个对二甲双胍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，鉴定了16个基因，当这些基因被敲除时，会导致对二甲双胍的耐药性。这16个基因中有14个具有人类同源基因，它们位于GWAS中与人类糖尿病、肥胖或心脏代谢疾病相关的位点，这是该数据集与人类疾病相关的一个令人信服的论据。在Aim 1中，我们将通过研究二甲双胍处理的秀丽隐杆线虫的生理学来定义二甲双胍在秀丽隐杆线虫中产生的代谢状态。在目标2中，我们将从遗传学角度确定二甲双胍发挥这些代谢作用的主要途径。大多数二甲双胍耐药基因突变的因果突变将通过下一代全基因组测序来确定。已鉴定的二甲双胍应答基因的作用机制将通过秀丽隐杆线虫转基因、组织特异性RNAi和详细的生理表征来确定。最重要的途径将通过分析对二甲双胍反应的每个途径的附加成员来确定。最后，我们将把每个二甲双胍反应途径与新出现的人类糖尿病、肥胖和在GWAS中鉴定的二甲双胍反应基因联系起来。通过对二甲双胍作用的基础研究，我们希望既能阐明糖尿病发展的可能机制，又能找到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.