Effects of 17,18-Epoxyeicosatetraenoic Acid and 19,20-Epoxydocosapentaenoic Acid Combined with Soluble Epoxide Hydrolase Inhibitor t-TUCB on Brown Adipogenesis and Mitochondrial Respiration

Abstract

Background/Objectives: 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive metabolites produced from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively, by CYP450s. These metabolites are unstable and quickly metabolized by auto-oxidation, esterification, β-oxidation, or hydrolysis by soluble epoxide hydrolase (sEH). 17,18-EEQ or 19,20-EDP combined with a potent sEH inhibitor t-TUCB differentially activated brown adipose tissue in diet-induced obesity. In the current study, we investigated whether these n-3 epoxy fatty acids with t-TUCB directly promote brown adipocyte differentiation and their thermogenic capacities. Methods: Murine brown preadipocytes were treated with 17,18-EEQ or 19,20-EDP with t-TUCB during and post differentiation. Brown marker protein expression and mitochondrial respiration were measured. In addition, the activation of PPARγ and suppression of NFκB reporter by 17,18-EEQ or 19,20-EDP alone or with t-TUCB were assessed, and the roles of PPARγ were evaluated with PPARγ knockdown and GW9662. Results: 17,18-EEQ or 19,20-EDP with t-TUCB promoted brown adipogenesis and mitochondrial respiration and uncoupling. Moreover, with t-TUCB, both epoxides improved mitochondrial respiration, but only 17,18-EEQ with t-TUCB significantly increased mitochondrial uncoupling (and heat production) in the differentiated adipocytes. PPARγ may be required for the effects of epoxides on differentiation but not on the thermogenic function post differentiation. Conclusions: The results demonstrate that, with t-TUCB, 17,18-EEQ and 19,20-EDP promote brown adipogenesis and mitochondrial respiration and uncoupling. 17,18-EEQ also promotes thermogenesis in differentiated brown adipocytes. Together, the results suggest thermogenic potentials of tested n-3 epoxides, especially 17,18-EEQ with t-TUCB. Translational studies of these n-3 epoxides on human brown adipocyte differentiation and functions are warranted.

Keywords: 17,18-EEQ; 19,20-EDP; brown adipogenesis; n-3 epoxy fatty acid; soluble epoxide hydrolase; soluble epoxide hydrolase inhibitor t-TUCB; thermogenesis.

Figure 1

17,18-EEQ and 19,20-EDP combined with t-TUCB promoted murine brown adipogenesis. Murine brown preadipocytes were differentiated in the presence or absence of DMSO (the vehicle control), t-TUCB alone (T), or with 17,18-EEQ (T+EEQ) or 19,20-EDP (T+EDP) for 6 days. ORO-stained brown adipocyte morphology (A) and ORO absorbance (B) were shown. Protein expression of brown adipocyte marker genes, PGC1α, CD36, and UCP1 and the loading control ERK1/2 is shown in (C). Quantification of each protein expression by densitometry is shown on the right in (C). Data = Mean ± SEM (n = 3). One-way ANOVA was used in (B). a, p < 0.05, aaa, p < 0.001 compared to DMSO; b, p < 0.05, bbb, p < 0.001 compared to T; ccc, p < 0.001 compared to T+EEQ 1; ddd, p < 0.001 compared to T+EDP 1. Scale bar = 100 µM.

Figure 2

When combined with t-TUCB, 17,18-EEQ in a dose-dependent manner increased mitochondrial respiration and proton-leak coupled OCRs in differentiating murine brown adipocytes. Murine brown preadipocytes were differentiated in the presence of DMSO, t-TUCB alone (T), or 17,18-EEQ (T+EEQ) at 1 or 10 µM, as indicated for 4 days. Then, the cells were reseeded onto a 24-well XFe assay plate at 2.0 × 10⁴ cells per well. After 24 h, the cells were subjected to real-time OCR measurements. OCRs during mitochondrial stress tests (A) and bar graphs of basal respiration, maximal respiration, ATP production- and proton leak-coupled OCRs, and coupling efficiency (%) (B) are shown. Data = Mean ± SEM (n = 3–4). One-way ANOVA was used in (B). a, p < 0.05, aa, p < 0.01, aaa, p < 0.001 compared to DMSO; b, p < 0.05, bb, p < 0.01, bbb, p < 0.001 compared to T; c, p < 0.05, ccc, p < 0.001 compared to T+EEQ 1.

Figure 3

When combined with t-TUCB, 19, 20-EDP in a dose-dependent manner increased mitochondrial respiration and proton-leak coupled OCRs in differentiating murine brown adipocytes but is less potent compared to 17,18-EEQ. Murine brown preadipocytes were differentiated in the presence of DMSO, t-TUCB alone (T), or 19,20-EDP at 1 or 10 µM, as indicated for 4 days. Then, the cells were reseeded onto a 24-well XFe assay plate at 2.0 × 10⁴ cells per well. After 24 h, the cells were subjected to real-time OCR measurements. OCRs during mitochondrial stress tests (A) and bar graphs of basal respiration, maximal respiration, ATP production- and proton leak-coupled OCRs, and coupling efficiency (%) (B) are shown. Data = Mean ± SEM (n = 3–4). One-way ANOVA was used to compare among (-), DMSO, T, T+EDP 1, and T+EDP 10 in (B). Unpaired t-tests were used to compare T+EDP 10 and T+EEQ 10 in (B). aa, p < 0.01, aaa, p < 0.001 compared to DMSO; bb, p < 0.01, bbb, p < 0.001 compared to T; d, p < 0.05, dd, p < 0.01, ddd, p < 0.001 compared to T+EDP 1; *, p < 0.05, ***, p < 0.001, compared to T+EDP 10.

Figure 4

Combined with t-TUCB, 17,18-EEQ and 19,20-EDP differentially regulated protein expression and thermogenic function in differentiated brown adipocytes. Murine brown preadipocytes were differentiated for 6 days and then were treated with DMSO, t-TUCB alone (T), or 17,18-EEQ (T+EEQ) or 19,20-EDP (T+EDP) for 3 days. The protein expression of PGC1α, CD36, and UCP1 and the loading control ERK1/2 is shown in (A). Quantification of each protein expression by densitometry is shown on the right in (A). OCRs during mitochondrial stress tests were shown in (B), and bar graphs of basal respiration, maximal respiration, ATP production- and proton leak-coupled OCRs, and coupling efficiency (%) are shown in (C). Data = Mean ± SEM (n = 3–4). Unpaired t-tests were used in (A). One-way ANOVA was used in (C). a, p < 0.05, aaa, p < 0.001 compared to DMSO; b, p < 0.05, bb, p < 0.01, bbb, p < 0.001 compared to T; *, p < 0.05, ***, p < 0.001 compared to T+EDP 10.

Figure 5

17,18-EEQ and 19,20-EDP activated PPARγ and inhibited LPS-induced NFκB activation in murine brown preadipocytes. Murine brown preadipocytes were seeded onto 24-well plates at 5.0 × 10⁴ cells per well. On the next day, cells were transiently transfected with murine PPARγ (A) or NFκB (B) transactivation reporters and β-gal plasmid for 24 h. The cells were treated with the DMSO, 17,18-EEQ (1 or 10 μM), or 19,20-EDP (1 or 10 μM) in the presence or absence of t-TUCB (1 μM) for 24 h in (A) or were pre-treated with DMSO. 17,18-EEQ (10 µM) or 19,20-EDP (10 µM) in the presence or absence of t-TUCB (1 μM) for 1 h, then co-treated with LPS for 18 h in (B). Reporter gene assays were performed and normalized to β-gal activities. Relative luciferase activities were expressed as folds of the (-) (set as 1). Data = Mean ± SEM (n = 3). Two-way ANOVA was used in (A,B). a, p < 0.05, aa, p < 0.01, aaa, p < 0.001 compared to DMSO, ccc, p < 0.001, compared to EEQ 1; ddd, p < 0.001 compared to EDP 1; **, p < 0.01 compared to (-). ns, not significantly different.

Figure 6

The effects of PPARγ knockdown on the brown adipocyte differentiation treated by t-TUCB alone or combined with 17,18-EEQ and 19,20-EDP. PPARγ-KD and SCR cells were differentiated in the presence or absence of DMSO, t-TUCB alone (T), and with 17,18-EEQ (T+EEQ) or 19,20-EDP (T+EDP). ORO-stained brown adipocyte morphology is shown in (A). Protein expression of brown adipocyte markers PPARγ, CD36, and UCP1 and the loading control ERK1/2 are shown in (B). Quantification of each protein expression by densitometry is shown below. Data = Mean ± SEM (n = 3 of technical replicates). One-way ANOVA was used to analyze the effects of T alone or combined with epoxides within each cell type. Two-way ANOVA was used to analyze the knockdown effects. aa, p < 0.01, aaa, p < 0.001 compared to DMSO; b, p < 0.05, bbb, p < 0.001 compared to T; *, ***, p < 0.05 and p < 0.001 compared to T+EDP 10; ###, significant differences between the SCR and PPARγ-KD cells. Scale bar = 100 µM.

Figure 7

The effects of PPARγ antagonist GW9662 on protein expression and thermogenic function induced by t-TUCB alone or combined with epoxides in differentiated brown adipocytes. Murine brown preadipocytes were differentiated, pre-treated with GW9662 or DMSO, and then co-treated with DMSO, t-TUCB alone (T), or with 17,18-EEQ (T+EEQ) or 19,20-EDP (T+EDP) for 3 days. Protein expression of PGC1α, CD36, and UCP1 and the loading control ERK1/2 are shown in (A). Quantification of each protein expression by densitometry is shown below, and Data = Mean ± SEM (n = 3 of technical replicates) (A). Bar graphs of basal respiration, maximal respiration, ATP production- and proton leak-coupled OCRs, and coupling efficiency (%) of the DMSO- or GW9662-treated cells are shown, and Data = Mean ± SEM (n = 3–6) in (B,C), respectively. One-way and two-way ANOVA were used in (A) and one-way ANOVA was used in (B,C). a, p < 0.05, aa, p < 0.01, aaa, p < 0.001 compared to DMSO; b, p < 0.05, bb, p < 0.01, bbb, p < 0.001 compared to T; **, p < 0.01, ***, p < 0.001 compared to T+EDP 10. ###, significant differences between the DMSO- and GW9662-treated cells.