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dc.contributor.authorBiancolin, Andrew Den
dc.contributor.authorMartchenko, Alexandreen
dc.contributor.authorMitova, Emiliaen
dc.contributor.authorGurges, Patricken
dc.contributor.authorMichalchyshyn, Everanen
dc.contributor.authorChalmers, Jennifer Aen
dc.contributor.authorDoria, Alessandroen
dc.contributor.authorMychaleckyj, Josyf Cen
dc.contributor.authorAdriaenssens, Alice Een
dc.contributor.authorReimann, Franken
dc.contributor.authorGribble, Fionaen
dc.contributor.authorGil-Lozano, Manuelen
dc.contributor.authorCox, Brian Jen
dc.contributor.authorBrubaker, Patricia Len
dc.date.accessioned2019-11-12T01:59:52Z
dc.date.available2019-11-12T01:59:52Z
dc.date.issued2020-01en
dc.identifier.issn2212-8778
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/298800
dc.description.abstractObjectives: 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.
dc.description.sponsorshipBanting 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)
dc.format.mediumPrint-Electronicen
dc.languageengen
dc.publisherElsevier
dc.rightsAll rights reserved
dc.rights.uri
dc.subjectAnimalsen
dc.subjectMice, Inbred C57BLen
dc.subjectMice, Knockouten
dc.subjectMiceen
dc.subjectGlucose Tolerance Testen
dc.subjectFemaleen
dc.subjectMaleen
dc.subjectGlucagon-Like Peptide 1en
dc.subjectARNTL Transcription Factorsen
dc.subjectCircadian Clocksen
dc.subjectSecretagoginsen
dc.titleThe core clock gene, Bmal1, and its downstream target, the SNARE regulatory protein secretagogin, are necessary for circadian secretion of glucagon-like peptide-1.en
dc.typeArticle
prism.endingPage137
prism.publicationDate2020en
prism.publicationNameMolecular metabolismen
prism.startingPage124
prism.volume31en
dc.identifier.doi10.17863/CAM.45855
dcterms.dateAccepted2019-11-01en
rioxxterms.versionofrecord10.1016/j.molmet.2019.11.004en
rioxxterms.versionAM
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2020-01en
dc.contributor.orcidReimann, Frank [0000-0001-9399-6377]
dc.contributor.orcidGribble, Fiona [0000-0002-4232-2898]
dc.identifier.eissn2212-8778
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idMRC (MC_UU_12012/3)
pubs.funder-project-idMRC (MC_UU_12012/5)
pubs.funder-project-idWELLCOME TRUST (106262/Z/14/Z & 106263/Z/14/Z)
rioxxterms.freetoread.startdate2022-11-11


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