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. 2020 Jul 9;9:e55388. doi: 10.7554/eLife.55388

Figure 5. Resetting of hypothalamic clocks by ADIPOQ.

(A) Representative normalized Bmal1-luc luminescence rhythms of dexamethasone (DEX) synchronized hypothalamus-derived mHypo-N44 (N44) cells. Inset depicts raw luminescence data of the same set. (B) Normalized Bmal1-luc luminescence rhythms of N44 cells after treatment with ADIPOQ (Adn; red and blue) peptide or PBS (black) at the depicted time points after synchronization. Shown are averages ± SEM of 3 traces each. (C) Phase response curve for ADIPOQ-mediated resetting of N44/Bmal1-luc cells (n = 3 per time point; treatment time given in degrees with 90 °=maximal luminescence and 270 °=minimal luminescence – see (A) for reference). (D) Normalized PER2::LUC luminescence rhythms of primary hypothalamic neurons after treatment with ADIPOQ (Adn; red and blue) peptide at the depicted time points after synchronization. Shown are averages ± SEM of 3 traces each. Inset shows quantification of phase shifts (p<0.0001; unpaired Student’s t-test). (E) Normalized PER2::LUC luminescence rhythms of organotypic MBH slices after treatment with ADIPOQ (Adn; red and blue) peptide at the depicted time points. Shown are averages ± SEM of 3 traces each. (F) Phase response curve for ADIPOQ-mediated resetting of PER2::LUC MBH slices (treatment time given in degrees with 90 °=maximal luminescence and 270 °=minimal luminescence – see (E) for reference.

Figure 5—source data 1. Raw data of experiments shown in Figure 5.
elife-55388-fig5-data1.xlsx (113.2KB, xlsx)

Figure 5.

Figure 5—figure supplement 1. ADIPOQ resets hypothalamic clocks in a dose-dependent manner.

Figure 5—figure supplement 1.

(A, B) Sample traces (A) and quantification (B) of resetting of N44/Bmal1-luc cells by increasing doses of ADIPOQ (Adn) peptide added at 23 hr after synchronization. (C, D) Sample traces (C) and quantification (D) of resetting of N44/Bmal1-luc cells by increasing doses of ADIPOQ (Adn) peptide added at 13 hr after synchronization. All data are means ± SEM (n = 3 per condition). Small letters depict significantly different groups (p<0.05; 1-way ANOVA). p-values: (B) 0.0121 (1 µg/ml vs. PBS), 0.0005 (10 µg/ml vs. PBS), ANOVA dF = F(2, 6)=33.44; (D) 0.0095 (10 µg/ml vs. PBS), ANOVA dF = F(2, 6)=11.43.
Figure 5—figure supplement 2. Hormonal resetting of clocks in N44/Bmal1-luc cells.

Figure 5—figure supplement 2.

(A–F) Normalized Bmal1-luc luminescence rhythms of N44 cells after treatment with leptin (Lep; A), visfatin (Vis; B), insulin (Ins; C), ghrelin (Ghr; D), glucagon (Gcg; E), resistin (Res; F) or PBS at the depicted time points after synchronization. Shown are averages ± SEM of 3 traces each.
Figure 5—figure supplement 3. Resetting of clocks in N44/Bmal1-luc cells by dexamethasone and forskolin.

Figure 5—figure supplement 3.

(A, B) Normalized Bmal1-luc luminescence rhythms of N44 cells after treatment with dexamethasone (Dex; A) or forskolin (Fors; B) or PBS at the depicted time points after synchronization. Shown are averages ± SEM of 3 traces each. Bottom panels: phase response curves for dexamethasone (left) and forskolin (right). Shown are averages of three experiments per time point each with sine wave regressions (treatment time given in degrees with 90 °=maximal luminescence and 270 °=minimal luminescence – see (Figure 5A) for reference.