Inhibition of soluble epoxide hydrolase modulates inflammation and autophagy in obese adipose tissue and liver: Role for omega-3 epoxides Cristina López-Vicarioa, José Alcaraz-Quilesa, Verónica García-Alonsoa, Bibiana Riusa, Sung H. Hwangb, Esther Titosa,c, Aritz Lopategia, Bruce D. Hammockb,1, Vicente Arroyoc,d, and Joan Clàriaa,c,e,1 aDepartment of Biochemistry and Molecular Genetics, dLiver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), cCentro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas, and eDepartment of Physiological Sciences I, University of Barcelona, Barcelona 08036, Spain; and bDepartment of Entomology and Comprehensive Cancer Center, University of California, Davis, CA 95616 Contributed by Bruce D. Hammock, December 4, 2014 (sent for review July 25, 2014; reviewed by Karsten Gronert and Steve Watkins) Soluble epoxide hydrolase (sEH) is an emerging therapeutic target levels by targeting the enzyme soluble epoxide hydrolase (sEH) in a number of diseases that have inflammation as a common are currently under investigation. sEH is a cytosolic enzyme with underlying cause. sEH limits tissue levels of cytochrome P450 (CYP) epoxide hydrolase and lipid phosphatase activities that catalyzes epoxides derived from omega-6 and omega-3 polyunsaturated the rapid hydrolysis of EETs, EEQs and EDPs by adding water fatty acids (PUFA) by converting these antiinflammatory mediators to these EpFA and converting them into inactive or less active into their less active diols. Here, we explored the metabolic effects 1,2-diols (10). Accordingly, inhibition of sEH exerts beneficial of a sEH inhibitor (t-TUCB) in fat-1 mice with transgenic expression actions in controlling vascular tone, inflammation, and pain, and of an omega-3 desaturase capable of enriching tissues with endog- this strategy has shown its therapeutic potential for long-term use in hypertension, diabetes, renal disease, organ damage, and enous omega-3 PUFA. These mice exhibited increased CYP1A1, – CYP2E1, and CYP2U1 expression and abundant levels of the vascular remodeling (6, 9 12). The aim of the present study was to investigate the potential omega-3–derived epoxides 17,18-epoxyeicosatetraenoic acid metabolic benefits of sEH inhibition in obesity. Specifically, this (17,18-EEQ) and 19,20-epoxydocosapentaenoic (19,20-EDP) in insulin- study addresses the question as to whether sEH inhibition sensitive tissues, especially liver, as determined by LC-ESI-MS/MS. increases the effectiveness of omega-3–derived epoxides in obese In obese fat-1 mice, t-TUCB raised hepatic 17,18-EEQ and 19,20-EDP – adipose tissue and liver in the context of enriched omega-3 tissue levels and reinforced the omega-3 dependent reduction observed content. fat-1 mice with transgenic expression of the Caeno- t in tissue inflammation and lipid peroxidation. -TUCB also pro- rhabditis elegans omega-3 fatty acid desaturase gene represent fat-1 duced a more intense antisteatotic action in obese mice, as a useful model to address this question because these mice have revealed by magnetic resonance spectroscopy. Notably, t-TUCB abundant tissue omega-3 distribution from their embryonic stage skewed macrophage polarization toward an antiinflammatory M2 phenotype and expanded the interscapular brown adipose Significance tissue volume. Moreover, t-TUCBrestoredhepatic levels of Atg12- Atg5 and LC3-II conjugates and reduced p62 expression, indicating up-regulation of hepatic autophagy. t-TUCB consistently reduced Our study demonstrates that stabilization of cytochrome P-450 endoplasmic reticulum stress demonstrated by the attenuation of epoxides derived from omega-3 polyunsaturated fatty acids IRE-1α and eIF2α phosphorylation. These actions were recapitu- through inhibition of the inactivating enzyme soluble epoxide lated in vitro in palmitate-primed hepatocytes and adipocytes in- hydrolase (sEH) exerts beneficial actions in counteracting cubated with 19,20-EDP or 17,18-EEQ. Relatively similar but less metabolic disorders associated with obesity. In addition, our pronounced actions were observed with the omega-6 epoxide, study sheds more light on the role of sEH in cellular homeo- 14,15-EET, and nonoxidized DHA. Together, these findings identify stasis by providing evidence that omega-3 epoxides and sEH omega-3 epoxides as important regulators of inflammation and inhibition regulate autophagy and endoplasmic reticulum autophagy in insulin-sensitive tissues and postulate sEH as a drug- stress in insulin-sensitive tissues, especially the liver. Therefore, gable target in metabolic diseases. administration of a sEH inhibitor is a promising strategy to prevent obesity-related comorbidities. obesity | inflammation | autophagy | omega-3–derived epoxides | Author contributions: C.L.-V., V.A., and J.C. designed research; C.L.-V., J.A.-Q., V.G.-A., soluble epoxide hydrolase B.R., E.T., A.L., and J.C. performed research; C.L.-V., S.H.H., and B.D.H. contributed new reagents/analytic tools; C.L.-V. and J.C. analyzed data; and C.L.-V., B.D.H., and J.C. wrote ytochrome P450 (CYP) epoxygenases represent the third the paper. Cbranch of polyunsaturated fatty acid (PUFA) metabolism (1). Reviewers: K.G., University of California, Berkeley; and S.W., Lipomics Technologies Inc. CYP epoxygenases add oxygen across one of the four double bonds Conflict of interest statement: B.D.H. and S.H.H. are authors on a patent held by the University of California on the synthesis of soluble epoxide hydrolase (sEH) inhibitors. of PUFA to generate three-membered ethers known as epoxides B.D.H. founded a company, EicOsis, to move these inhibitors to the clinic to treat neuro- (1). In the case of arachidonic acid, CYP epoxygenases convert this pathic and inflammatory pain. The published and freely available sEH inhibitor was pro- omega-6 PUFA into epoxyeicosatrienoic acids (EETs), which act as vided by University of California, Davis to the Claria group in Spain along with additional autocrine or paracrine factors in the regulation of vascular tone, reagents and data. The inhibitor was a key tool to test the hypothesis that the omega-6 and 3 fatty acid epoxides were responsible for biological effects. It is conceivable that use inflammation, hyperalgesia, and organ and tissue regeneration of an sEH inhibitor could be of some benefit to EicOsis or GSK, both of which are working (2, 3). In addition to omega-6s, CYP epoxygenases also convert the to develop these materials clinically. However, numerous papers have been published omega-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic already implicating these inhibitors in diabetes treatment, and other inhibitors of similar acid (DHA) into novel epoxyeicosatetraenoic (EEQs) and epoxy- structure and potency are commercially available from Cayman Chemical and CalBio- chem. B.D.H. is an author on University of California patents in the area, has stock in docosapentaenoic (EDPs) acids, respectively (4, 5). These omega- EicOsis, which has licensed these patents, but has no salary from EicOsis. – 3 derived epoxides also exert salutary actions and are even more 1To whom correspondence may be addressed. Email: [email protected] or bdhammock@ effective and potent than omega-6–derived EETs (4–8). ucdavis.edu. Because the predicted in vivo half-lives of fatty acid epoxides This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (EpFA) are in the order of seconds (9), drugs that stabilize their 1073/pnas.1422590112/-/DCSupplemental. 536–541 | PNAS | January 13, 2015 | vol. 112 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1422590112 Downloaded by guest on September 25, 2021 17,18-EEQ 19,20-EDP WT Fat-1 1.5 A B ** A Liver eWAT Liver eWAT ** Chow 600 Chow HFD Chow HFD Chow HFD Chow HFD HFD EPA DHA *** 1.0 eWAT sEH 400 * CYPs * 0.5 ** β-actin 200 Fold Change vs. Chow 0.0 17,18-EEQ 19,20-EDP Liver eWAT Liver eWAT ng/g of tissue WT Fat-1 COOH COOH 0 WT Fat-1 WT Fat-1 Chow WT Fat-1 B 2500 2200 O O 17,18-EEQ 19,20-EDP HFD * 2000 2000 2000 *** 1500 sEH ** 1800 1000 ** 1000 1000 ** * ng/g tissue 200 500 ** ** ng/g tissue 500 * Liver ** ** * * 0 0 Inactive metabolites 160 t-TUCB - + - + - + - + - + - + t-TUCB - + - + - + - + - + - + * 17,18- 19,20- 5,6- 8,9- 11,12- 14,15- 17,18- 19,20- 5,6- 8,9- 11,12- 14,15- EEQ EDP EET EET EET EET EEQ EDP EET EET EET EET ng/g of tissue 100 * 0 WT Fat-1 WT Fat-1 WT Fat-1 350 800 C 300 250 600 C D 200 400 400 5,6-EET 8,9-EET 11,12-EET 14,15-EET 150 * 100 * 300 ng/g tissue 200 AA * ng/g tissue 50 *** ** 0 0 200 eWAT t-TUCB - + - + - + - + - + - + t-TUCB - + - + - + - + - + - + CYPs 17,18- 19,20- 5,6- 8,9- 11,12- 14,15- 17,18- 19,20- 5,6- 8,9- 11,12- 14,15- DiHETE DiHDPA DHET DHET DHET DHET DiHETE DiHDPA DHET DHET DHET DHET ng/g of tissue 100 ** * Liver eWAT 0 * ** ** ** * 5,6-EET 8,9-EET 11,12-EET 14,15-EET WT Fat-1 WT Fat-1 WT Fat-1 WT Fat-1 Chow D WT Fat-1 E WT Fat-1 WT Fat-1 O O t COOH -TUCB - + - + -+ -+ COOH COOH COOH HFD 17,18-EEQ 19,20-EDP 17,18-EEQ 19,20-EDP O DiHETE DiHDPA DiHETE DiHDPA sEH O 1500 5,6-EET 8,9-EET 11,12-EET 14,15-EET *** *** 14 β-actin *** * sEH 1000 * 12 5 * *** *** *** Liver *** * * 10 4 500 *** *** 8 6 -actin 3 ** Ratio ng/g of tissue * 4 β Inactive metabolites 0.9 ** 2 0 0.6 sEH/ WT Fat-1 WT Fat-1 WT Fat-1 WT Fat-1 *** 1 active metabolites/diols 0.3 0 0.0 WT Fat-1 WT Fat-1 t Fig.