a. Immunoblots of eWAT from WT mice 12 h after CL-316,243 injection (0.5 mg kg−1) with vehicle or VX765 (50 mg kg−1, twice per day) or IL-1β levels in eWAT from WT mice 3 h after the second CL injection (0.5 mg kg−1 day−1) with vehicle or VX765. (n = 4 per group). P values vehicle vs VX765. b. Chemokines and LTB4 levels from WT PPDIVs pretreated 30 min with VX765 (40 µM), followed by CL-316,243 (1 µM, 3 h). (veh: n = 3, VX765: n = 2, CL: n = 4; VX + CL: n = 3)). P values vehicle vs VX765. c-e. (c) Serum NEFA and glycerol, (d) IL-6 levels and (e) body weight, iWAT tissue weight after CL injection with vehicle or VX765 for the indicated times (CLx2-4: 2-4 days) (NEFA, 0 h/6 h/12 h: n = 4; 24 h: n = 8,7. Glycerol, 0 h: n = 3; 6 h: n = 7). P values vehicle vs VX765. f. Paired serum glycerol levels from the same mice at 30 min and 1 h after the second CL injection with vehicle or VX765 for the indicated times (n = 8 per group). P values 30 min vs 1 h. g. Analysis of DT-treated mice after the second CL injection. Serum glycerol and immunoblots of eWAT from 15 min after second CL injection. (0 min: n = 5,4; 15 min: n = 6,5; 30 min: n = 7,5; 60 min: n = 6,5). P values Neu-WT vs Neu-DTR. h. Glycerol release from ex vivo cultured eWAT of WT mice. eWAT was collected 24 h after CL injection with vehicle or VX765. Then treated in culture with vehicle or CL-316,243 (1 µM, 30 min). (0 h: n = 6,5; 0.5 h: n = 6,5). P values vehicle vs VX765. i. Fatty acid oxidation genes in both eWAT and iWAT 3 h after the second CL-injection with vehicle or VX765. (veh: n = 7,8; VX: n = 7,8). P values vehicle vs VX765. j-k. WT PPDIVs were pretreated 18 h with IL-1β (5 ng ml−1) or vehicle, followed by CL-316,243 (1 µM, 0.5 h). (j) Immunoblots and (k) glycerol release and fold change compared to vehicle or IL-1β. (n = 4 per group). P values vs groups. l-p. Genes expression in (l) 3T3-L1 adipocytes or (m, p) WT PPDIVs treated with IL-1β (5 ng ml−1, 3 h) or CL-316,243 (1 µM, 3 h) or TNFa (5 ng ml−1, 3 h). (veh: n = 2, IL-1β: n = 3, TNFa: n = 2; CL: n = 3). P values vs vehicle. n. Beta 3 adrenergic receptor gene in eWAT from WT mice 3 h after the second CL injection with vehicle or VX765. (n = 4 per group). P values vehicle vs VX765. o. Beta 3 adrenergic receptor gene expression in eWAT from DT-treated Neu-WT and Neu-DTR mice 6 h after the second CL injection. (n = 3,4 per group). P values Neu-WT vs Neu-DTR. Data are mean ± s.e.m. Each dot (n) represents one biological replicate. Data are merged from three (c-d), two (f-i) or representative of two (a, b, g (blot), j-p) independent experiments. Statistics: two-way ANOVA with Sidak’s post hoc test (c, d, g, h); two-way ANOVA with Fisher’s (e); one-way ANOVA with Tukey’s (b, n, o); Welch’s one-way ANOVA with Dunnett’s T3 (b (cxcl1), k); unpaired two-sided Student’s t-test (f); unpaired two-sided Welch’s t-test (a); two-sided Mann Whitney U test (i).
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