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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

Published version
Peer-reviewed

Type

Article

Change log

Authors

Biancolin, Andrew D 
Martchenko, Alexandre 
Mitova, Emilia 
Gurges, Patrick 

Abstract

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 regulatory SNARE exocytotic protein that demonstrates circadian expression and is essential for insulin secretion from ß-cell. The objective of the current study was to establish the necessity of the core clock gene Bmal1 and of the SNARE protein, SCGN, as essential regulators of circadian GLP-1 secretion. Methods: Oral glucose tolerance tests were performed at different times of day on 4 hour fasted C57BL/6J, Bmal1 wild-type and Bmal1 knockout mice. Mass spectrometry, RNA-seq, qRT-PCR and/or microarray analyses, as well as immunostaining were conducted on murine and human primary L-cells, and mGLUTag and human (h) NCI-H716 L-cell lines. At peak and trough GLP-1 secretory time points, 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 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. mGLUTag and hNCI-H716 L-cells exhibited circadian rhythms in Scgn expression (p < 0.001). ChIP analysis demonstrated increased binding of BMAL1, only at the peak of Scgn expression (p < 0.01). Immunocytochemistry showed 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 that 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.

Description

Keywords

Bmal1, Circadian, GLP-1, L-cell, Secretagogin, Secretion, ARNTL Transcription Factors, Animals, Circadian Clocks, Female, Glucagon-Like Peptide 1, Glucose Tolerance Test, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Secretagogins

Journal Title

Molecular Metabolism

Conference Name

Journal ISSN

2212-8778
2212-8778

Volume Title

31

Publisher

Elsevier
Sponsorship
Medical Research Council (MC_UU_12012/3)
Medical Research Council (MC_UU_12012/5)
Wellcome Trust (106262/Z/14/Z)
MRC (MC_UU_00014/3)
MRC (MC_UU_00014/5)
Medical Research Council (MC_PC_12012)
Banting Research Foundation Canadian Institutes of Health Research Wellcome Trust (106262/Z/14/Z, 106263/Z/14/Z) UK Medical Research Council (MRC_MC_UU_12012/3)