ANK1基因变异与2型糖尿病遗传易感的分子机制

Type 2 diabetes (T2D) is a chronic metabolic disease with multifactorial pathogenesis, and genetic contribution to this disease is well recognized. Increasing case-control studies have substantiated the association of some single nucleotide polymorphisms (SNPs) with T2D, and proposed possible underlying physiological basis with more elaborated glucose metabolic measures. .We have screened and validated single nucleotide polymorphisms (SNPs) in risk ANK1 locus among two case-control studies, and revealed a significant association of three SNPs rs508419, rs515071, rs516946 with T2D, of which rs508419 was firstly identified in this study. Fine mapping showed that rs508419 was located at the promoter of muscle-specific small ankyrin 1 isoforms (sAnk1). Measurement of allele-specific mRNA and protein levels confirmed that C allele was associated with an increased expression of sAnk1 in human skeletal muscle. Luciferase assay showed increased transcriptional activity of rs508419-C allele in murine skeletal muscle C2C12 myoblasts. Electrophoretic mobility shift assays demonstrated an altered formation of DNA-protein complexes of rs508419, and TR4 is invoved in this process. The underlying mechanism involves an effect of sAnk1, present in the sarcoplasmic reticulum fraction of skeletal muscle, where it is physically associated with sarco/endoplasmic reticulum calcium ATPase (SERCA) proteins. We further demonstrated the association between sAnk1 and SERCA expression. .We, therefore, hypothesize that increased sAnk1 expression in skeletal muscle, mediated by TR4, might be one molecular genetic contributor to T2D susceptibility at this GWAS locus. A high sAnk1 expression increased SERCA activity, which resulted in decreased cytosolic calcium. Increased uptake of cytosolic calcium led to CaMKKβ-dependent activation of AMPK to impair glucose metabolism. To test this hypothesis, we will study the effect of TR4 on sAnk1 transcription with rs508419-C or –T alleles in vitro, using EMSA, Luciferase assay and real-time PCR. We will also study the effect of sAnk1 on diabetes development, with mice lacking or over-expressing sAnk1 induced by high-fat diet (HFD) feeding. We will further study the effect of sAnk1 on glucose uptake in vivo, using mutant or transfer muscle cells lacking or over-expressing sAnk1 from diabetic or non-diabetic mice. In addition, we will demonstrate the mechanism regarding SERCA activity, Ca2+ balance and AMPK signal pathway. .In a word, this project is planned to demonstrate the molecular mechanism underlying genetic association with diabetes. We design to understand the sAnk1 function form blood suger metabolism. It will give a bridge between gene function and genetics, and provide a new insight into the possible effect besides a new class of muscles.

2型糖尿病（T2D）是遗传和环境因素共同作用的复杂疾病，在全球爆发流行的态势下，寻找易感基因、探讨分子机制有其重要的意义。我们前期在ANK1基因上发现了一个新的易感位点，位于骨骼肌异构体sAnk1的启动子区，影响该启动子与转录因子TR4的结合及其活性，进而影响骨骼肌sAnk1的转录以及骨骼肌的糖代谢。由此我们推测，sAnk1可能直接参与调节骨骼肌的糖代谢过程；而影响TR4转录调控正是此ANK1变异增加T2D易感的分子基础。本研究将利用分子和细胞实验明确TR4对sAnk1的转录调控，探讨ANK1风险变异的作用机制；利用差异表达的动物和细胞模型探讨sAnk1参与骨骼肌糖代谢的作用；并基于前期观察，探讨sAnk1对钙离子ATP酶的作用，研究其调节糖代谢的可能机制。本项目从分子、细胞和整体层面，系统研究ANK1基因骨骼肌异构体sAnk1的转录调控和作用机理，为阐明T2D的遗传易感机制提供理论依据。

2型糖尿病（T2D）是最常见的复杂代谢性疾病，我国糖尿病患病人数居全球首位。β细胞功能缺陷和周边组织对胰岛素利用障碍是T2D主要特征，而全身胰岛素抵抗（IR）是T2D发展的最终途径。骨骼肌是机体消耗利用葡萄糖的重要外周组织和胰岛素主要效应器官，并且骨骼肌胰岛素抵抗先于β细胞功能衰竭前发生。我们前期的工作证实了rs508419位于sAnk1启动子区域，并证实其与骨骼肌sAnk1的高表达相关，进而引起骨骼肌胰岛素抵抗，参与T2D的发病过程。其具体机制并不清楚。既往研究提示，sAnk1主要表达于肌肉等组织细胞内，与骨骼肌结构和功能密切相关。 而GPSM1是先前发现的与肥胖相关的基因。近年来全基因组关联分析（GWAS）研究发现GPSM1与亚洲人群T2D相关。有研究发现GPSM1 rs11787792是骨骼肌胰岛素抵抗可能的遗传调控位点。沿着已有的技术平台，探讨这个新些骨骼肌胰岛素抵抗的调控节点，帮助明晰T2D的发病机制有着重要的理论和实际意义。.本研究第一部分主要探讨风险位点rs508419对sAnk1转录调控的机制，并阐明sAnk1参与T2D的发病机制：1）Nono与rs508419-C结合调控sAnk1转录表达；2）构建骨骼肌高表达sAnk1转基因小鼠，证实TgsAnk1小鼠基础代谢较野生对照略降低；3）高脂饮食下，TgsAnk1小鼠糖脂代谢较野生对照明显降低； 4）进一步高通量测序结合基因富集分析发现高表达sAnk1导致AMPK通路活性抑制，并且骨骼肌SERCA1表达及钙离子水平增加。 第二部分主要利用已发表的全基因组关联数据进行精细定位鉴定新的糖尿病易感位点，并探讨其可能的作用机制：1）rs28539249在亚洲人群中与T2D关联；2）GPSM1表达与糖尿病相关；3）rs28539249-C结合CTCF调控基因转录。.本研究围绕骨骼肌sAnk1参与2型糖尿病的易感性及后GWAS的精细定位分为两个研究部分，研究发现有一定的理论与实现意义。本项目还推进了与意大利实质性的合作与交流，开展双方的互访，共同合作推进研究进展并共享研究成果。

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