Fig 2: sEH inhibitor t-TUCB upregulates mitochondrial respiratory chain and lipid metabolic genes in murine brown adipocytes. Murine brown preadipocytes were differentiated in the presence of t-TUCB, as indicated for 6 days. (A) Relative mRNA levels of mitochondrial respiratory chain components (Cox4a and Uqcrh), fatty acid uptake genes (Lpl and Cd36), and lipolytic genes (Plin1, Atgl, and Hsl). mRNA expression was quantified relatively to the loading control 36b4. (B) Protein expression of LPL, CD36, PLIN, and the loading control ERK1/2. Data = Mean+SEM (n = 3). *, **, ***, p < 0.05, p < 0.01, and p < 0.001 compared to t-TUCB 0 samples, respectively.

Fig 3: (+)-1a and (−)-1b regulated the expression of adipogenic and lipogenic proteins in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were incubated with a control vehicle or (+)-1a and (−)-1b (12.5, 25, and 50 μmol/L) for 6 days. (A) Expression of proteins involved in adipogenic differentiation and lipid accumulation (PPARγ and C/EBPα) was determined by Western blot, vinculin was used as a loading control. (B) Expression of proteins involved in adipocyte lipolysis (p-PKA, HSL, p-HSL563, p-HSL660, and Perilipin) was detected by Western blot, vinculin was used as a loading control. The data are expressed as mean ± SD (n = 3). ###P < 0.001 vs vehicle, nsP > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs IDIR.

Fig 4: Agrp neurons promote lipogenesis. a Control (black) and AgrpTrpv1 (blue) mice were injected with capsaicin. b NEFA levels (interaction: F2, 54 = 3.91, P = 0.02; time: F2, 54 = 9.64, P = 0.0003; genotype: F1, 27 = 1.74, P = 0.19; MCT: P80 min = 0.03). c Blood glucose levels (interaction: F2, 54 = 1.47, P = 0.23; time: F2, 54 = 14.6, P < 0.0001; genotype: F1, 27 = 0.67, P = 0.41; MCT: ns). In b and c: control (n = 15) and AgrpTrpv1 mice (n = 14). Statistics: two-way analysis of variance (ANOVA) with time as a repeated measure followed by Holm–Sidak’s multiple comparisons test (MCT). d Gene expression in the WAT. Data are normalized to control levels (dashed line). Statistics: Student’s t-test. *P < 0.05. e Western blotting analysis of phosphorylated HSL in inguinal WAT and retroperitoneal WAT (n = 4 mice per group). Blots were cropped for clarity. Statistics: Student’s t-test. *P < 0.05. f Control and AgrpTrpv1 mice randomly received vehicle or the FAS inhibitor (C75, 10 mg kg−1, intraperitoneal (i.p.)) immediately before capsaicin injection. g–i Respiratory exchange ratio (RER) (interaction: F1, 26 = 4.36, P = 0.04; drug: F1, 26 = 22.28, P < 0.0001; genotype: F1, 26 = 11.73, P = 0.002). j–l Fat utilization (interaction: F1, 26 = 9.97, P = 0.04; drug: F1, 26 = 6.00, P = 0.02; genotype: F1, 26 = 6.56, P = 0.01). m–o Carbohydrate utilization: (interaction: F1, 26 = 12.18, P = 0.001; drug: F1, 26 = 0.76, P = 0.38; genotype: F1, 26 = 4.90, P = 0.03). In g–o, statistics: two-way ANOVA on the mean response after drug and capsaicin injection; genotype and drug as factors for the ANOVA. Holm–Sidak’s multiple comparisons test (MCT) was used and indicated as *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001. Control mice + vehicle (n = 8); control mice + C75 (n = 8); AgrpTrpv1 mice + vehicle (n = 7); AgrpTrpv1 mice + C75 (n = 7). Dashed line indicates time of injections. Colored symbols indicate mean ± SEM. Gray symbols indicate individual values

Fig 5: Transplantation of AUCB-HuBr regulated protein expression involved in lipolysis in the iBAT of the nude mice fed a high-fat diet. Male nude mice were transplanted with HuBr cells differentiated with 5 µM t-AUCB (AUCB 5) or the vehicle control (DMSO) or transplanted with matrix gel only (-). Protein expression of ATGL, HSL, PLIN, phosphorylated HSL, phosphorylated PLIN, and the loading control ERK1/2 in the iBAT of mice in the (-), DMSO, and AUCB 5 groups (A) and their densitometry (B–G) are shown. Bar graphs show the normalized densitometry for ATGL/ERK, HSL/ERK, p-HSL(S563)/ERK, p-HSL(S660)/ERK, PLIN/ERK, and p-PLIN(S517)/ERK. Data = mean ± SEM (N = 6). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. a, p < 0.05 compared to the (-) group; b, p < 0.05 compared to the DMSO group.