Roles of the type 2 diabetes (T2D)-associated gene C2cd4a in regulating glucose homeostasis in the mouse

2 型糖尿病 (T2D) 相关基因 C2cd4a 在调节小鼠葡萄糖稳态中的作用

• 批准号: MR/R014329/1
• 负责人: Guy Rutter
• 金额: $ 3万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FR014329%2F1
• 关键词: Roles; type; diabetes; T2D; associated

Type 2 diabetes (T2D) is a strongly hereditary disease and studies over the past decade have highlighted a large number of regions in the human genome which affect disease risk. Identifying the causal gene(s) in each region is now an important goal and may provide targets for new therapies that treat the underlying disease aetiology (i.e. causes and progress), rather than the symptoms alone. "Genome-wide association studies", carried out since 2007 and involving comparisons of the genetic variation in thousands of non-diabetic versus T2D patients, have identified almost 500 coding genes in >100 genomic loci as contributors to type 2 diabetes heredity. Studies in man have also indicated that the vast majority of these variants impact the secretion of insulin from the pancreas rather than the action of the hormone on target tissues (liver, muscle, etc). Our own recent studies, involving post mortem pancreatic islet material from patients, suggest that changes in the expression of genes at a locus on chromosome 15, namely VPS13C, C2CD4A and C2CD4B, may contribute to altered disease risk. Mice deleted selectively in the pancreatic beta-cell (the sole source of circulating insulin in mice and humans) for the homologous Vps13c gene have near-normal blood sugar levels (i.e. they are "normoglycemic") and display no abnormalities in insulin secretion, making this a less likely candidate of the three to confer disease risk. By contrast, silencing of C2cd4a in pancreatic MIN6 beta-cells (which display many lf the properties of primary beta-cells) inhibits glucose-stimulated insulin secretion. Furthermore, C2CD4A expression is strongly induced by high glucose in human islets: in contrast, VPS13C mRNA levels are unaffected. At present, mouse models in which C2cd4a or C2cd4b been deleted have not been studied using the same, detailed, approaches as those deployed for Vps13c null mice. We have therefore recently begun work to examine C2cd4b null mice provided through the IMPC. The present proposal seeks to complete the examination of this locus by using mouse genetics and cellular studies to explore the roles of C2cd4a in controlling insulin secretion and glucose homeostasis, and thus to explore the possibility that it plays a role as causal gene(s) at this genomic locus. Specifically, we will study mice rendered null globally for C2cd4a using CRISPR/Cas9-medited deletion. Firstly, we will explore the ability of the mutant mice to handle a glucose load provided either by injection into the peritoneal cavity or orally (i.e. glucose tolerance tests). Studies will be performed on mice fed (1) a regular chow direct (2) high fat, or (3) high fat/high sucrose diets. The latter resemble western diets in the human population, and lead to obesity and defective insulin secretion in man. In parallel studies we will measure beta cell mass to see whether deletion of either gene impacts the proliferation or survival of these cells. We will also measure insulin secretion into the bloodstream and the processing of the prohormone (proinsulin) to mature insulin - a process which becomes defective in human T2D. Finally, we will perform detailed studies using isolated islets from wild-type and mutant mice of "glucose sensing" by the pancreatic beta-cells. These investigations will involve advanced microscopy approaches and will determine the action of the sugar to stimulate energy metabolism, Ca2+ influx, beta cell-beta cell coordination and the release of stored insulin through regulated exocytosis. These studies will thus provide a deep glycemic phenotyping which goes considerably beyond that undertake through the IMPC pipeline. The majority of the work proposed will be performed by a PhD student, Ms Neda Mousavy, supported by a Diabetes UK Studentship to work on this locus. Her existing funding does not, however, cover the costs of obtaining, maintaining or characterising C2Cd4a mice.

2型糖尿病(T2D)是一种遗传性很强的疾病，过去十年的研究强调了人类基因组中影响疾病风险的大量区域。确定每个区域的致病基因(S)现在是一个重要的目标，并可能为治疗潜在疾病病因学(即病因和进展)的新疗法提供靶点，而不仅仅是症状。自2007年以来开展的“全基因组关联研究”，对数千名非糖尿病患者和T2D患者的基因变异进行了比较，发现&gt；100个基因组基因座上的近500个编码基因是2型糖尿病遗传的贡献者。对人类的研究也表明，这些变异中的绝大多数影响胰腺胰岛素的分泌，而不是激素对靶组织(肝脏、肌肉等)的作用。我们自己最近的研究，涉及患者的死后胰岛材料，表明15号染色体上一个基因的表达变化，即VPS13C，C2CD4A和C2CD4B，可能有助于疾病风险的改变。在胰岛β细胞(小鼠和人类循环胰岛素的唯一来源)中选择性缺失同源Vps13c基因的小鼠的血糖水平接近正常(即它们是“正常血糖”)，并且没有表现出胰岛素分泌的异常，这使得这三个候选小鼠不太可能增加疾病风险。相比之下，胰腺MIN6β细胞中C2cd4a的沉默(表现出许多初级β细胞的特性)抑制了葡萄糖刺激的胰岛素分泌。此外，高糖强烈诱导人胰岛C2CD4A的表达：相比之下，VPS13C的mRNA水平不受影响。目前，C2cd4a或C2cd4b缺失的小鼠模型还没有采用与Vps13c缺失小鼠相同的、详细的方法进行研究。因此，我们最近开始研究通过IMPC提供的C2cd4b缺失小鼠。本研究试图通过小鼠遗传学和细胞学研究来完成对该基因座的检测，以探索C2cd4a在控制胰岛素分泌和血糖稳态中的作用，从而探索其作为因果基因(S)在该基因组座上发挥作用的可能性。具体地说，我们将使用CRISPR/Cas9-Meded缺失来研究C2cd4a全局为空的小鼠。首先，我们将探索突变小鼠处理通过腹腔注射或口服提供的葡萄糖负荷的能力(即葡萄糖耐量测试)。研究将在(1)直接喂食普通食物(2)高脂肪，或(3)高脂肪/高蔗糖饮食的小鼠身上进行。后者类似于人类的西方饮食，会导致人类肥胖和胰岛素分泌缺陷。在平行研究中，我们将测量β细胞质量，看看这两个基因的缺失是否会影响这些细胞的增殖或存活。我们还将测量进入血流的胰岛素分泌和将前激素(胰岛素原)加工成成熟胰岛素的过程--这是一个在人类T2D中变得有缺陷的过程。最后，我们将使用分离自野生型和突变小鼠的胰岛进行详细的研究，这些小鼠通过胰岛β细胞进行“血糖感应”。这些研究将涉及先进的显微镜方法，并将确定糖刺激能量代谢、钙离子内流、β细胞-β细胞协调以及通过调节胞吐释放储存的胰岛素的作用。因此，这些研究将提供一个深入的血糖表型，远远超出通过IMPC管道进行的。建议的大部分工作将由博士生内达·穆萨维女士完成，并得到英国糖尿病学生基金的支持，以研究这一基因座。然而，她现有的资金不包括获得、维护或鉴定C2CD4a小鼠的成本。

项目成果

Sexually dimorphic roles for the type 2 diabetes-associated C2cd4b gene in murine glucose homeostasis.

• DOI: 10.1007/s00125-020-05350-x
• 发表时间: 2021-04
• 期刊: Diabetologia
• 影响因子: 8.2
• 作者: Mousavy Gharavy SN;Owen BM;Millership SJ;Chabosseau P;Pizza G;Martinez-Sanchez A;Tasoez E;Georgiadou E;Hu M;Fine NHF;Jacobson DA;Dickerson MT;Idevall-Hagren O;Montoya A;Kramer H;Mehta Z;Withers DJ;Ninov N;Gadue PJ;Cardenas-Diaz FL;Cruciani-Guglielmacci C;Magnan C;Ibberson M;Leclerc I;Voz M;Rutter GA
• 通讯作者: Rutter GA