Soluble Epoxide Hydrolase 2 Expression Is Elevated in Obese Humans and Decreased by Physical Activity

Abstract

Epoxide hydrolase 2 (EPHX2) is an emerging therapeutic target in several immunometabolic disorders. EPHX2 metabolizes anti-inflammatory epoxyeicosatrienoic acids into pro-inflammatory diols. The contribution of EPHX2 activity to human obesity remains unexplored. We compared the expression of EPHX2 between lean and obese humans (n = 20 each) in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) using RT-PCR, Western Blot analysis, immunohistochemistry, and confocal microscopy before and after a 3-month physical activity regimen. We also assessed EPHX2 levels during preadipocyte differentiation in humans and mice. EPHX2 mRNA and protein expression were significantly elevated in obese subjects, with concomitant elevated endoplasmic reticulum (ER) stress components (the 78-kDa glucose-regulated protein; GRP78, and the Activating transcription factor 6; ATF6) and inflammatory markers (Tumor necrosis factor-α; TNFα, and Interleukin 6; IL6) as compared to controls (p < 0.05). EPHX2 mRNA levels strongly correlated with adiposity markers. In obese individuals, physical activity attenuated EPHX2 expression levels in both the SAT and PBMCs, with a parallel decrease in ER stress and inflammation markers. EPHX2 expression was also elevated during differentiation of both human primary and 3T3-L1 mouse preadipocytes. Mediators of cellular stress (palmitate, homocysteine, and macrophage culture medium) also increased EPHX2 expression in 3T3-L1 preadipocytes. Our findings suggest that EPHX2 upregulation is linked to ER stress in adiposity and that physical activity may attenuate metabolic stress by reducing EPHX2 expression.

Keywords: EPHX2; diabetes; exercise; obesity; sEH.

Figure 1

Epoxide hydrolase 2 (EPHX2) mRNA expression in subcutaneous adipose tissue (SAT) and peripheral blood mononuclear cells (PBMCs) from human subjects. mRNA expression of EPHX2 (A), the 78-kDa glucose-regulated protein (GRP78) (B), and the Activating transcription factor 6(ATF6) (C) in PBMCs and SAT of participant groups (lean, obese before exercise, and obese after exercise, n = 10 each). mRNA levels were measured by quantitative real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control for normalization. Data are presented as fold changes compared with normal-weight participants. * p < 0.05; ** p < 0.01.

Figure 2

EPHX2 protein levels in SAT and PBMCs from human subjects. Representative immunohistochemical (A) and confocal immunofluorescence (B) images illustrating EPHX2, GRP78, and ATF6 expression and localization in SAT from lean, obese before exercise, and obese after exercise group (n = 5 each). Staining quantification of SAT was performed as detailed in Materials and Methods; data are presented as fold changes compared to the normal-weight group. (C) Protein expression of EPHX2, GRP78, and ATF6 in PBMCs according to groups (at least n = 9 each). Protein levels were measured by Western Blotting. GAPDH was used as an internal control for normalization. Data are presented as fold changes compared to the lean participants. * p < 0.05; ** p < 0.01.

Figure 3

Expression levels of EPHX2 during differentiation of preadipocytes. Protein (A) and mRNA (B) levels were measured by Western Blot and quantitative real-time PCR, respectively, in 3T3-L1 preadipocytes (D0) and adipocytes differentiated for 14 days (D2–D14) from three independent experiments. (C) mRNA levels were measured by quantitative real-time PCR in human visceral (hVAT), subcutaneous (hSAT) preadipocytes (D0), and adipocytes differentiated for 10 days (D4–D10) from two independent experiments. Data are presented as the fold change in differentiated adipocytes compared with preadipocytes (D0). * p < 0.05; ** p < 0.01.

Figure 4

Expression levels of EPHX2 in 3T3-L1 preadipocytes under cellular stressor treatment. The protein levels were measured by Western Blot using 3T3-L1 preadipocytes treated overnight with various concentrations of homocysteine or palmitate or 1 µM Tunicamycin (at least three independent experiments). GRP78 was used as a control for treatment efficacy. * p < 0.05; ** p < 0.01.

Figure 5

Expression levels of EPHX2 during the differentiation of 3T3-L1 preadipocytes and their treatment with a macrophage culture medium. mRNA expression levels of EPHX2 (A), GRP78 (B), and ATF6 (C) were measured by quantitative real-time PCR in 3T3-L1 preadipocytes (D0) and adipocytes differentiated for 8 days (D8) with and without macrophage-conditioned medium (MaCM). Data are presented as fold changes in differentiated adipocytes compared with preadipocytes from three independent experiments. * p < 0.05; ** p < 0.01.