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. 2020 Jan:31:124-137.
doi: 10.1016/j.molmet.2019.11.004. Epub 2019 Nov 21.

The core clock gene, Bmal1, and its downstream target, the SNARE regulatory protein secretagogin, are necessary for circadian secretion of glucagon-like peptide-1

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
Affiliations

The core clock gene, Bmal1, and its downstream target, the SNARE regulatory protein secretagogin, are necessary for circadian secretion of glucagon-like peptide-1

Andrew D Biancolin et al. Mol Metab. 2020 Jan.

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 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.

Keywords: Bmal1; Circadian; GLP-1; L-cell; Secretagogin; Secretion.

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Figures

Figure 1
Figure 1
Circadian GLP-1 secretion is dependent on the core clock gene Bmal1. (A-B) OGTTs were conducted on 4-hour fasted C57Bl/6J mice at six time points throughout a 24-hour light (open bars)-dark (closed bars) cycle (ZT0 = 06:00), with individual plots for each time point represented in (A) and the 24-hour ΔAUC profile represented in (B). (C-D) OGTTs were performed on 4-hour fasted Bmal1 WT and KO mice at ZT2 and ZT14 with individual plots for each time point shown in (C) and the ΔAUC of each timepoint represented in (D). (A and C) t0 indicates the absolute fasting values for GLP-1 in pg/ml. n = 4–8 mice for all time points in all of the experiments. *p < 0.05.
Figure 2
Figure 2
Identification of potential targets regulating circadian GLP-1 secretion. (A) Heat maps of GO transport vesicle and (B) selected L-cell secretory genes identified by microarray in synchronized the mGLUTag L-cells showing log2 fold-change between the two time points, 4 and 16 h (n = 3 for each time point). (C) Volcano plot of the mass spectrometry results of the synchronized mGLUTag L-cells at peak (8 h) and trough (20 h) of GLP-1 secretion (n = 3 for each time point). SCGN is indicated by the open circle. (D) Pathway enrichment analysis of protein clusters that were up regulated at each time point, with SCGN identified under the secretory granules cluster.
Figure 3
Figure 3
Scgn is expressed in primary and immortalized murine and human L-cells. (A) RNA-seq analysis of Scgn in murine Gcg-Venus L-cells and Venus-negative cells from duodenal, ileal, and colonic sections (n = 2–3 for each cell population). (B) RT-qPCR for Scgn in murine colonic Gcg-Venus L-cells, Venus-negative cells, and mGLUTag L-cells (n = 3 for each cell population). (C) RNA-seq analysis for SCGN in human jejunal L-cells, enteroendocrine cells, and non-endocrine intestinal epithelial cells (n = 11 for each cell population). (D-G) Immunostaining of murine (D) and human (E) ileal sections for SCGN and GLP-1 (representative images of SCGN+:GLP-1+ (top) and SCGN+:GLP-1- (bottom) cells are shown). Cell count histogram for murine (F) and human (G) ileal SCGN and/or GLP-1 stained cells (n = 5 sections for each species; a total of ∼100 cells were counted per section). (H-I) Microarray analysis of mGLUTag cells (n = 6) (H) and RNA-seq analysis of hNCI-H716 cells (n = 2) (I) for proglucagon, clock, and SNARE protein transcripts. (J-K) Immunostaining of mGLUTag (J) and hNCI-H716 (K) L-cells for SCGN; DAPI shows the nuclear stain (representative images of n = 4 are shown). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Scgn expression is circadian in mGLUTag and hNCI-H716 L-cells. (A-C) mRNA and (D-E) protein expression over 48 h in synchronized mGLUTag L-cells for Bmal1 (A), Per2 (B), Scgn (C), BMAL1 (D), and SCGN (E; n = 8, conducted as 4 replicates from each of 2 independent splits; representative blots are shown in (D-E)). (F-G) mRNA expression over 36 h in synchronized hNCI-H716 cells for BMAL1 (F), PER2 (G), and SCGN (H; n = 8, conducted as 4 replicates from each of 2 independent splits).
Figure 5
Figure 5
Time-dependent binding of BMAL1 to the Scgn promoter in mGLUTag L-cells. (A) Three noncanonical BMAL1 E-boxes were identified at 1252, 1176, and 672 base pairs upstream of the transcription start site. (B) ChIP analysis for the BMAL1 binding sites in the Scgn promoter at 4, 8, 16, and 20 h in synchronized mGLUTag L-cells (n = 6, conducted as 3 replicates from each of 2 independent splits). *p < 0.05; ###p < 0.001 vs negative control (-IgG).
Figure 6
Figure 6
Time-dependent recruitment of SCGN to the cell membrane in mGLUTag L-cells. (A-B) 8 h and (C-D) 20 h after synchronization, mGLUTag L-cells were treated with 10−7 M GIP or vehicle for 0, 10, and 60 min, followed by staining for SCGN and a membrane marker (with wheat germ agglutinin). Co-localization of SCGN with the cell membrane was determined by Pearson's correlation coefficient. (n = 4, conducted as 2 replicates from each of 2 independent splits; representative images are shown). *p < 0.05.
Figure 7
Figure 7
Time-dependent interactions of SCGN with β-actin but not SNAP25 in mGLUTag L-cells. mGLUTag L-cells were synchronized and then 8 or 20 h later treated with 10−7 M GIP or vehicle for 2 h. (A) Immunoprecipitation of SNAP25 and immunoblotting for SCGN. (B) Immunoprecipitation of SCGN and immunoblotting for β-actin. (n = 4, conducted as 2 replicates from each of 2 independent splits; representative blots are shown). *p < 0.05.
Figure 8
Figure 8
Scgn is essential for peak GLP-1 secretion in mGLUTag L -cells. mGLUTag L-cells were treated with scRNA or Scgn siRNA, synchronized and then 8 or 20 h later treated with 10−7 M GIP or vehicle for 2 h. The cells were then analyzed for Bmal1 (A), Per2 (B), Scgn (C), SCGN (a representative blot is shown) (D), and GLP-1 secretion (E) (n = 8, conducted as 4 replicates from each of 2 independent splits). *p < 0.05, **p < 0.01, ***p < 0.001.
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