Dissecting the molecular mechanism of metformin action

描述(申请人提供)：2型糖尿病影响着近2500万美国人，是导致死亡和残疾的主要原因。二甲双胍是双胍类药物中的一员，是用于治疗2型糖尿病的一线药物，因其高效、安全，并可降低糖尿病并发症如肾脏疾病、失明和心脏病发作的风险。二甲双胍主要通过降低肝脏葡萄糖输出量来降低糖尿病患者的血糖，也可能增加肌肉中的胰岛素敏感性。尽管它被广泛使用了50多年，但它的完整作用机制尚不清楚，它的直接靶点也是未知的。更好地了解二甲双胍的作用机制可能会导致对2型糖尿病的更智能的治疗。为了确定二甲双胍的分子靶点，我们建议在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线虫的生长和代谢，并且像在哺乳动物中一样，在线虫中的作用部分依赖于AMP激活的蛋白激酶(AMPK)信号。这表明线虫是研究二甲双胍作用的一种简便且遗传上易驯化的模式生物。在线虫中，正向遗传筛选能够识别在任何给定的生物反应中最重要的基因。为了筛选目的，我们使用一个合适的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个独立的抗二甲双胍作用的突变体。同时，我们进行了反向遗传RNAi筛选，鉴定了16个基因，当被敲除时，这些基因会导致对二甲双胍的效果产生抵抗。这16个中有14个具有人类直系同源基因，位于与GWAS中人类糖尿病、肥胖症或心脏代谢性疾病相关的基因座上，这是该数据集与人类疾病相关性的一个令人信服的论点。在目标1中，我们将通过研究二甲双胍处理的线虫的生理学，来确定二甲双胍在线虫体内产生的代谢状态。在目标2中，我们将从基因上确定二甲双胍产生这些代谢影响的主要途径。将通过下一代全基因组测序来鉴定大多数二甲双胍耐药基因突变的原因突变。利用线虫转基因技术、组织特异性RNAi和详细的生理特征，将建立已识别的二甲双胍反应基因的作用机制。最重要的途径将通过分析每条途径对二甲双胍的反应中的额外成员来确定。最后，我们将把每一种二甲双胍反应途径与新出现的人类糖尿病、肥胖症和在GWAS中确定的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，我们希望既能阐明糖尿病发生的可能机制，又能为糖尿病的治疗找到新的靶点。

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.