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The core clock gene, Bmal1, and its downstream target, the SNARE regulatory protein secretagogin, are necessary for circadian secretion of glucagon-like peptide-1.
Molecular Metabolism ( IF 8.1 ) Pub Date : 2019-11-21 , DOI: 10.1016/j.molmet.2019.11.004
Andrew D Biancolin 1 , Alexandre Martchenko 1 , Emilia Mitova 1 , Patrick Gurges 1 , Everan Michalchyshyn 1 , Jennifer A Chalmers 1 , Alessandro Doria 2 , Josyf C Mychaleckyj 3 , Alice E Adriaenssens 4 , Frank Reimann 4 , Fiona M Gribble 4 , Manuel Gil-Lozano 1 , Brian J Cox 5 , Patricia L Brubaker 6
Affiliation  

Objectives

The incretin hormone glucagon-like peptide-1 (GLP-1) is secreted from intestinal L-cells upon nutrient intake. While recent evidence has shown that GLP-1 is released in a circadian manner in rats, whether this occurs in mice and if this pattern is regulated by the circadian clock remain to be elucidated. Furthermore, although circadian GLP-1 secretion parallels expression of the core clock gene Bmal1, the link between the two remains largely unknown. Secretagogin (Scgn) is an exocytotic SNARE regulatory protein that demonstrates circadian expression and is essential for insulin secretion from β-cells. The objective of the current study was to establish the necessity of the core clock gene Bmal1 and the SNARE protein SCGN as essential regulators of circadian GLP-1 secretion.

Methods

Oral glucose tolerance tests were conducted at different times of the day on 4-hour fasted C57BL/6J, Bmal1 wild-type, and Bmal1 knockout mice. Mass spectrometry, RNA-seq, qRT-PCR and/or microarray analyses, and immunostaining were conducted on murine (m) and human (h) primary L-cells and mGLUTag and hNCI-H716 L-cell lines. At peak and trough GLP-1 secretory time points, the mGLUTag cells were co-stained for SCGN and a membrane-marker, ChIP was used to analyze BMAL1 binding sites in the Scgn promoter, protein interaction with SCGN was tested by co-immunoprecipitation, and siRNA was used to knockdown Scgn for GLP-1 secretion assay.

Results

C57BL/6J mice displayed a circadian rhythm in GLP-1 secretion that peaked at the onset of their feeding period. Rhythmic GLP-1 release was impaired in Bmal1 knockout (KO) mice as compared to wild-type controls at the peak (p < 0.05) but not at the trough secretory time point. Microarray identified SNARE and transport vesicle pathways as highly upregulated in mGLUTag L-cells at the peak time point of GLP-1 secretion (p < 0.001). Mass spectrometry revealed that SCGN was also increased at this time (p < 0.001), while RNA-seq, qRT-PCR, and immunostaining demonstrated Scgn expression in all human and murine primary L-cells and cell lines. The mGLUTag and hNCI-H716 L-cells exhibited circadian rhythms in Scgn expression (p < 0.001). The ChIP analysis demonstrated increased binding of BMAL1 only at the peak of Scgn expression (p < 0.01). Immunocytochemistry showed the translocation of SCGN to the cell membrane after stimulation at the peak time point only (p < 0.05), while CoIP showed that SCGN was pulled down with SNAP25 and β-actin, but only the latter interaction was time-dependent (p < 0.05). Finally, Scgn siRNA-treated cells demonstrated significantly blunted GLP-1 secretion (p < 0.01) in response to stimulation at the peak time point only.

Conclusions

These data demonstrate, for the first time, that mice display a circadian pattern in GLP-1 secretion, which is impaired in Bmal1 knockout mice, and that Bmal1 regulation of Scgn expression plays an essential role in the circadian release of the incretin hormone GLP-1.



中文翻译:

核心时钟基因Bmal1及其下游靶标SNARE调节蛋白secretagogin是昼夜分泌胰高血糖素样肽1所必需的。

目标

摄入营养素后,肠L细胞分泌肠降血糖素激素胰高血糖素样肽-1(GLP-1)。尽管最近的证据表明GLP-1在大鼠中以昼夜节律的方式释放,但这种现象是否在小鼠中发生以及这种模式是否由昼夜节律的时钟调节仍有待阐明。此外,尽管昼夜节律的GLP-1分泌与核心时钟基因Bmal1的表达平行,但两者之间的联系仍然未知。Secretagogin(Scgn)是一种胞外SNARE调节蛋白,具有昼夜节律性表达,是β细胞分泌胰岛素所必需的。本研究的目的是确定核心时钟基因Bmal1和SNARE蛋白SCGN作为昼夜节律性GLP-1分泌的必要调节剂的必要性。

方法

在一天中的不同时间对禁食4小时的C57BL / 6J,Bmal1野生型和Bmal1基因敲除小鼠进行口服葡萄糖耐量测试。在鼠(m)和人(h)原代L细胞以及mGLUTag和hNCI-H716 L细胞系上进行了质谱分析,RNA-seq,qRT-PCR和/或微阵列分析以及免疫染色。在高峰和谷底GLP-1分泌时间点,将mGLUTag细胞共染色用于SCGN和膜标记,使用ChIP分析Scgn启动子中的BMAL1结合位点,通过共免疫沉淀法测试蛋白质与SCGN的相互作用, siRNA被用于敲除Scgn用于GLP-1分泌测定。

结果

C57BL / 6J小鼠的GLP-1分泌昼夜节律在进食期开始时达到峰值。与野生型对照相比,Bmal1基因敲除(KO)小鼠的节律性GLP-1释放在峰值处(p <0.05)受到了抑制,但在谷底分泌时间点没有受到破坏。在GLP-1分泌的高峰时间点,微阵列将SNARE和转运囊泡途径在mGLUTag L细胞中高度上调(p <0.001)。质谱分析表明,此时的SCGN也有所增加(p <0.001),而RNA-seq,qRT-PCR和免疫染色证明了Scgn在所有人类和鼠类原代L细胞和细胞系中的表达。mGLUTag和hNCI-H716 L细胞在Scgn表达中表现出昼夜节律(p <0.001)。ChIP分析表明,仅在Scgn表达的峰值处,BMAL1的结合增加(p <0.01)。免疫细胞化学显示刺激后仅在高峰时间点SCGN易位至细胞膜(p <0.05),而CoIP显示SNAP25和β-肌动蛋白将SCGN下拉,但仅后者是时间依赖性的(p <0.05)。最后,Scgn siRNA处理的细胞仅在峰值时间点响应刺激,显示GLP-1分泌明显减弱(p <0.01)。

结论

这些数据首次证明,小鼠在GLP-1分泌中显示出昼夜节律模式,这在Bmal1基因敲除小鼠中受损,并且Bmal1对Scgn表达的调节在肠降血糖素激素GLP-的昼夜节律释放中起着至关重要的作用。 1。

更新日期:2019-11-21
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