CLOCK 蛋白调节大鼠胰岛β细胞中与 L 型钙通道相关的胰岛素分泌。
The CLOCK protein regulates insulin secretion related with L-type calcium channels in rat pancreatic beta cells.
机构信息
NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300134, China; Tianjin Children's Hospital, Tianjin, 300134, China.
NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, 300134, China.
出版信息
Biochem Biophys Res Commun. 2022 Jan 22;589:116-122. doi: 10.1016/j.bbrc.2021.12.016. Epub 2021 Dec 8.
BACKGROUND
Circadian locomotor output cycles kaput protein (CLOCK) plays a crucial role in glucose homeostasis and controlling insulin secretion. However, the mechanism of the CLOCK regulating rhythmic insulin secretion has not been fully understood.
METHODS
Rhythmic expression of the CLOCK in rat pancreatic beta cell was detected. INS-1 cells were transfected with siRNAs to knockdown the CLOCK before the cells were incubated with different concentrations of glucose. Insulin secretion was analyzed by ELISA method. Expression of the L-type calcium channel protein (Cav1.2, Cacna1c) was determined both in the CLOCK-knockdown cells and the control cells. Calcium influx was probed by fluorescent. Chromatin immunoprecipitation (ChIP) test and dual-luciferase reporter gene experiments were applied to verify the relationship between the CLOCK and Cav1.2.
RESULTS
The CLOCK is abundantly expressed in rat pancreatic beta cells. Transcription level of the CLOCK showed rhythmicity in the beta cells. Compared to the control group, insulin release was significantly impaired with 25 mM glucose incubation in the CLOCK-knockdown group, but not showed with 2.5 mM glucose incubation. The expression of Cav1.2 and the influx of calcium were significantly decreased in the CLOCK-knockdown group with 25 mM glucose incubation. ChIP test indicted that the CLOCK bound to -444∼-454 region of the Cacna1c promoter of the INS-1 cells, but the binding was significantly reduced following the CLOCK-knockdown. Luciferase experiment was in accordance with the finding of ChIP.
CONCLUSIONS
The CLOCK mediating Cav expression may point out a potential pathway of circadian rhythm affecting insulin secretion.
背景
生物钟输出周期 kaput 蛋白（CLOCK）在葡萄糖稳态和控制胰岛素分泌中起着至关重要的作用。然而，CLOCK 调节节律性胰岛素分泌的机制尚未完全阐明。
方法
检测大鼠胰岛β细胞中 CLOCK 的节律性表达。在不同浓度葡萄糖孵育前，用 siRNA 转染 INS-1 细胞以敲低 CLOCK。通过 ELISA 法分析胰岛素分泌。在 CLOCK 敲低细胞和对照细胞中均测定 L 型钙通道蛋白（Cav1.2、Cacna1c）的表达。通过荧光测定钙内流。应用染色质免疫沉淀（ChIP）试验和双荧光素酶报告基因实验验证 CLOCK 与 Cav1.2 之间的关系。
结果
CLOCK 在大鼠胰岛β细胞中大量表达。β 细胞中 CLOCK 的转录水平呈节律性。与对照组相比，CLOCK 敲低组在 25mM 葡萄糖孵育时胰岛素释放明显受损，但在 2.5mM 葡萄糖孵育时则没有受损。在 25mM 葡萄糖孵育时，CLOCK 敲低组 Cav1.2 的表达和钙内流明显降低。ChIP 试验表明，CLOCK 结合于 INS-1 细胞 Cacna1c 启动子的-444∼-454 区域，但在 CLOCK 敲低后结合明显减少。荧光素酶实验与 ChIP 的结果一致。
结论
CLOCK 介导的 Cav 表达可能指出了生物钟节律影响胰岛素分泌的潜在途径。
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