CLOCK基因调节大鼠胰岛β细胞胰岛素分泌分子机制的研究

The occurrence of type 2 diabetes (T2D) is closely associated with the circadian rhythm disorders. The various complications of T2D also reflect the obvious time rhythmicity. CLOCK gene is the core of controlling the body’s rhythm steady state. It can regulate the exocytosis of insulin granules in pancreatic beta cells, and maintain the dynamic balance of blood sugar. But the exact molecular mechanism is still unclear. The preliminary research of our project suggested several evidences of our hypothesis. The expression level of CLOCK gene were positively correlated with the levels of several major calcium ion channels (Cav) in human islets. The CLOCK gene expression in the diabetes human islets were lower than the non-diabetes human islets at different time points, and a variety of Cav expression levels were consistent with the CLOCK genes The insulin secretion in SD rat pancreatic cells showed an obvious rhythm, and the rhythm is closely related to the activity of Cav. Therefore we plan to establish a diabetic rats model with circadian rhythm disorders, to extract the islets from specific islet CLOCK gene knock out rats and specific islet CLOCK gene overexpression of rats. So we could identify the target Cav of the CLOCK gene in pancreatic beta cells, verify the relationship between the disorder of the CLOCK gene which is caused by the biological rhythm disorders and the variation of the expression and activity of the Cav. We plan to prove the hypothesis both in vivo and in vitro. A variety of techniques will be used, such as the patch clamp technique and high-definition calcium imaging technology. So we could reveal some new pathogenesis and pathophysiological basis of diabetes.

2型糖尿病的发生与昼夜节律紊乱密切相关，且其多种并发症也体现出明显的时间节律性。CLOCK基因是控制机体节律稳态的核心基因，其可在胰岛β细胞中调控胰岛素颗粒的胞吐作用，维持血糖动态平衡，但其分子机制尚不明确。本课题的前期研究表明，人胰岛中CLOCK基因的表达和多种钙离子通道(Cav)基因呈正相关。糖尿病人的胰岛中，多个时间点CLOCK基因的表达明显下调，且多种Cav的表达水平与CLOCK基因一致。在SD大鼠胰岛中胰岛素分泌作用存在明显的时间节律，且这一节律与Cav的活性密切相关。因此拟建立昼夜节律紊乱的糖尿病大鼠模型，并提取胰岛特异性CLOCK基因敲除及过表达大鼠的胰岛，采用膜片钳技术、高清晰钙成像技术等手段，明确CLOCK基因在胰岛β细胞中所调节的靶向性Cav，验证生物节律紊乱导致的CLOCK基因表达紊乱及其造成的Cav表达、活性的变异与糖尿病发病的联系，揭示糖尿病发病新的病理生理基础。

2型糖尿病的发生与昼夜节律紊乱密切相关，且其多种并发症也体现出明显的时间节律性。CLOCK基因是控制机体节律稳态的核心基因，其可在胰岛β细胞中调控胰岛素颗粒的胞吐作用，维持血糖动态平衡，但其分子机制尚不明确。本课题研究表明，在大鼠中CLOCK基因分布具有组织特异性，在INS-1细胞中有振荡节律性。抑制CLOCK基因内源性表达后，2.8mM葡萄糖孵育下胰岛素分泌无明显变化，在16.7mM高糖刺激下胰岛素释放量及钙离子含量明显减少。钙离子通道Cacna1c mRNA及蛋白表达量明显降低，有统计学意义；Cacna1d mRNA及蛋白表达量降低但无统计学意义。通过生物信息网站预测CLOCK基因作为转录因子在Ca1.2的结合位点并设计特异性引物，利用CHIP技术进行检测，结果表明Clock蛋白可结合于Cav1.2基因启动子区域（-444～-454bp）。本研究还发现抑制INS-1细胞中Clock内源性表达后，在高糖刺激时细胞内cAMP含量和CREB磷酸化水平明显减少，提示其影响胰岛素分泌可能与经典通路cAMP/PKA/CREB有关。动物实验上构建了慢性睡眠剥夺小鼠，结果表明慢性睡眠紊乱可导致小鼠胰岛时钟基因的表达紊乱，可能是引起小鼠糖代谢异常的重要原因之一。本研究揭示了CLOCK基因影响胰岛素分泌分泌的作用机制，为治疗2型糖尿病提供新靶点。