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

Figure 7. PGC1a-mediated induction of Bmal1 by ADIPOQ.

(A) Circadian profile of Pgc1a mRNA levels in the MBH of WT (black) and Adipoq deficient (MT) mice (red; n = 3 per time point). (B) Induction of Pgc1a mRNA levels in N44 cells after Adn treatment and in the mediobasal hypothalamus (MBH) after i.v. Adn injection in wild-type mice (n = 5). (C) Expression of Pgc1a in N44 cells and in the MBH after shRNA-mediated knockdown of Adipor1 or Adipor2 (n = 3 for N44 cells and 7 (Con)/10 (KD) in MBH). (D) mRNA levels of Pgc1a (left) and Bmal1 (right) in non-synchronized N44 cells after shRNA-mediated knockdown of Pgc1a (n = 4). (E) Bmal1 mRNA levels in unsynchronized N44 cells in response to Adn with (right) or without (left) prior knockdown of Pgc1a (n = 4). (F) Bmal1 mRNA levels in unsynchronized N44 cells in response to Adn with (right) or without (left) prior treatment with 5 µM VPR66 (n = 4). (G) ChIP-qPCR for PGC1a binding to Bmal1 RORE (and Bmal1 3’-UTR as negative control) in unsynchronized N44 cells in response to Adn treatment (n = 3). (H, I) Response of Bmal1-luc luminescence rhythms in synchronized N44 cells to Adn treatment at 23 hr after synchronization (arrow) with or without prior knockdown of Pgc1a. (H) Normalized luminescence data (n = 3 per condition). (I) Phasing of the first and second peak after Adn treatment relative to solvent (n = 8). All data are means ± SEM. Box plots show medians, quartiles and min/max. p-values: (B) 0.0116 (N44, +Adn vs. -Adn), ANOVA dF = F(1, 16)=0.299 (interaction)/0.294 (tissue)/15.60 (treatment); (C) 0.0001 (N44, Adipor1 KD vs. Con), 0.0020 (MBH, Adipor1 KD vs. Con), ANOVA dF = F(1, 19)=0.028 (interaction)/0.044 (tissue)/40.90 (treatment); (D) < 0.0001 (Pgc1a, Pgc1a KD vs. Con),<0.0001 (Bmal1, Pgc1a KD vs. Con), ANOVA dF = F(1, 12)=9.693 (interaction)/8.566 (gene)/151.5 (treatment). (E) 0.0019 (Con, +Adn vs. -Adn), ANOVA dF = F(1, 12)=4.894 (interaction)/37.05 (genotype)/15.39 (treatment); (F) 0.0087 (PBS, +Adn vs. -Adn), ANOVA dF = F(1, 12)=2.044 (interaction)/9.281 (inhibition)/12.45 (treatment); (G) 0.0013 (RORE, +Adn vs. -Adn), ANOVA dF = F(1, 8)=13.17 (interaction)/87.25 (site)/16.20 (treatment); (I) < 0.0001 (peak 1, Con vs. KD), 0.0258 (peak 2, Con vs. KD), ANOVA dF = F(1, 24)=2.809 (interaction)/3.192 (peak)/29.93 (genotype).

Figure 7—source data 1. Raw data of experiments shown in Figure 7.

Figure 7.

Figure 7—figure supplement 1. Knockdown of Pgc1a affects circadian rhythms in N44 cells.

Figure 7—figure supplement 1.

(A) Representative Bmal1-luc luminescence traces of N44 cells after transfection with Pgc1a shRNA (purple) or scramble controls (black; n = 3). (B) Timing of the first peak (n = 18), (C) dampening rates (n = 18) and (D) period length (n = 18). All data are means ± SEM; **/***: p<0.01/0.001, unpaired Student’s t-test. p-values: (B) 0.0002; (C) 0.0011; (D) < 0.0001.