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

Figure 6. Adiponectin signaling modulates hypothalamic appetite regulation in male mice.

(A) Transient upregulation of Bmal1-luc activity in non-synchronized N44 cells after adiponectin (Adn) treatment. Shown are averages ± SEM of 3 traces each. (B–H) Induction of mRNA levels in N44 cells after Adn treatment and in the mediobasal hypothalamus (MBH) after i.v. Adn injection in wild-type mice; (B) Bmal1, (C) Per1, (D) Per2, (E) Per2, (F) Rev-erb alpha, (G) Npy, (H) Agrp (n = 5 per condition). (I–K) Induction of Npy (I), Agrp (J) and Per2 (K) mRNA levels in Bmal1-deficient N44 cells after Adn treatment and in the mediobasal hypothalamus (MBH) after i.v. Adn injection in Bmal1 knockout mice (n = 3 per condition). */**/***: p<0.05/0.01/0.001, unpaired Student’s t-test. (L) Luciferase promotor assay for Npy in HEK293T cells after transient expression of increasing doses of Clock/Bmal1 or Cry1 (n = 6–10). */***: p<0.05/0.001 vs. -/-, ###: p<0.001 vs. 40/-, 1-way ANOVA. (M) Expression of Adipor1/2 mRNA levels after shRNA-mediated knockdown of Adipor1 (blue) or Adipor2 (green) in N44 cells (n = 3 per condition). (N, O) Expression of Bmal1 (N) and Npy (O) mRNA after Adipor1/2 knockdown in N44 cells (n = 3–4 per condition). (P) Adiponectin-induced phase shift of N44/Bmal1-luc rhythms after Adipor1/2 knockdown in N44 cells (n = 4 per condition). */**/***: p<0.05/0.01/0.001 vs. Con, 1-way ANOVA. (Q) Expression of Adipor1 mRNA in the mediobasal hypothalamus (MBH; n = 7–10) and of ADIPOR1 protein in the arcuate nucleus (ARC; inset; n = 6–9) after shRNA-mediated viral knockdown of Adipor1. (R, S) Daily (LD; R) and circadian (DD; S) food intake profiles of mice after shRNA-mediated viral knockdown of Adipor1 in the MBH (KD) and of scramble-AAV treated control animals (Con) (n = 9–13 per group). (T) Relative light phase food intake in LD (left) and rest phase food intake in DD (right) in Adipor1-KD animals (KD) and controls (Con) (n = 9–13 per group). */**/***: p<0.05/0.01/0.001 vs. Con, unpaired Student’s t-test (Q, T) or 2-way ANOVA with Sidak’s multiple comparisons (R, S). All data are averages ± SEM. Box plots show medians, quartiles and min/max. p-values: (B) 0.0084 (N44, +/- Adn), 0.0309 (MBH, +/- Adn); (G) < 0.001 (N44, +/- Adn and MBH, +/- Adn); (H) 0.0002 (N44, +/- Adn), 0.0059 (MBH, +/- Adn); (L) 0.019 (10 /- vs. -/-),<0.001 (20 /- and 40 /- vs. -/-),<0.001 (40/12, 40/25 and 40/50 vs. 40/-), ANOVA dF = F(7, 51)=15.63; (M) < 0.001 (Adipor1 KD, Adipor1 and Adipor2 vs. control and Adipor2 KD, Adipor2), ANOVA dF = F(4, 10)=68.39; (N) 0.002 (Adipor1 KD vs. control),<0.001 (Adipor2 KD and Adipor1/2 KD vs. control), ANOVA dF = F(3, 10)=68.50; (O) 0.010 (Adipor1 KD vs. control), 0.005 (Adipor2 KD vs. control), 0.016 (Adipor1/2 KD vs. control), ANOVA dF = F(3, 8)=31.95; (P) < 0.001 (Adipor1 KD and Adipor1/2 KD vs. Con), ANOVA dF = F(3, 12)=25.72; (Q) 0.006 (mRNA) and <0.001 (protein), unpaired Student’s t-test; (R) 0.037 (KD vs. Con, ZT12-18), ANOVA dF = F(3, 54)=2.359 (interaction)/37.82 (time)/0.56 (knockdown); (S) 0.002 (KD vs. Con, ZT12-18), ANOVA dF = F(3, 66)=5.337 (interaction)/87.32 (time)/1.43 (knockdown); (T) 0.0454 (LD, Con vs. KD) and 0.0199 (DD, Con vs. KD), unpaired Student’s t-test.

Figure 6—source data 1. Raw data of experiments shown in Figure 6.

Figure 6.

Figure 6—figure supplement 1. Adiponectin induces Bmal1 expression in synchronized N44 cells.

Figure 6—figure supplement 1.

(A) Raw luminescence data of N44/Bmal1-luc cells treated with ADIPOQ (Adn) or PBS at hr 63 after synchronization (black arrow). Shown are means ± SEM of 3 traces. (B) Normalized luminescence changes during 24 hr after Adn treatment at hr 63 (n = 3). (C) Bmal1 mRNA (left) and BMAL1 protein expression at 3 hr (mRNA) or 6 hr (protein) after Adn treatment at hr 63 (n = 3 for mRNA and four for protein). Right panel: representative Western blot for BMAL1 and ß-tubulin as loading control. All data are means ± SEM; */**: p<0.05/0.01, unpaired Student’s t-test. p-values: (B) 0.004 (Adn vs. PBS); (C) 0.003 (mRNA, Adn vs. PBS) and 0.014 (protein, Adn vs. PBS).