Soluble Epoxide Hydrolase Deletion Limits High-Fat Diet-Induced Inflammation

The soluble epoxide hydrolase (sEH) enzyme is a major regulator of bioactive lipids. The enzyme is highly expressed in liver and kidney and modulates levels of endogenous epoxy-fatty acids, which have pleiotropic biological effects including limiting inflammation, neuroinflammation, and hypertension. It has been hypothesized that inhibiting sEH has beneficial effects on limiting obesity and metabolic disease as well. There is a body of literature published on these effects, but typically only male subjects have been included. Here, we investigate the role of sEH in both male and female mice and use a global sEH knockout mouse model to compare the effects of diet and diet-induced obesity. The results demonstrate that sEH activity in the liver is modulated by high-fat diets more in male than in female mice. In addition, we characterized the sEH activity in high fat content tissues and demonstrated the influence of diet on levels of bioactive epoxy-fatty acids. The sEH KO animals had generally increased epoxy-fatty acids compared to wild-type mice but gained less body weight on higher-fat diets. Generally, proinflammatory prostaglandins and triglycerides were also lower in livers of sEH KO mice fed HFD. Thus, sEH activity, prostaglandins, and triglycerides increase in male mice on high-fat diet but are all limited by sEH ablation. Additionally, these changes also occur in female mice though at a different magnitude and are also improved by knockout of the sEH enzyme.

Changes of Morphology, Chemical Compositions, and the Biosynthesis Regulations of Cuticle in Response to Chilling Injury of Banana Fruit During Storage

The plant cuticle covers almost all the outermost surface of aerial plant organs, which play a primary function in limiting water loss and responding to the environmental interactions. Banana fruit is susceptible to thermal changes with chilling injury below 13°C and green ripening over 25°C. Herein, the changes of surface morphology, chemical compositions of cuticle, and the relative expression of cuticle biosynthesis genes in banana fruit under low-temperature storage were investigated. Banana fruit exhibited chilling injury rapidly with browned peel appearance stored at 4°C for 6 days. The surface altered apparently from the clear plateau with micro-crystals to smooth appearance. As compared to normal ones, the overall coverage of the main cuticle pattern of waxes and cutin monomers increased about 22% and 35%, respectively, in browned banana stored under low temperature at 6 days. Fatty acids (C16–C18) and ω-OH, mid-chain-epoxy fatty acids (C18) dominated cutin monomers. The monomers of fatty acids, the low abundant ω, mid-chain-diOH fatty acids, and 2-hydroxy fatty acids increased remarkably under low temperature. The cuticular waxes were dominated by fatty acids (> C19), n-alkanes, and triterpenoids; and the fatty acids and aldehydes were shifted to increase accompanied by the chilling injury. Furthermore, RNA-seq highlighted 111 cuticle-related genes involved in fatty acid elongation, biosynthesis of very-long-chain (VLC) aliphatics, triterpenoids, and cutin monomers, and lipid-transfer proteins were significantly differentially regulated by low temperature in banana. Results obtained indicate that the cuticle covering on the fruit surface was also involved to respond to the chilling injury of banana fruit after harvest. These findings provide useful insights to link the cuticle on the basis of morphology, chemical composition changes, and their biosynthesis regulations in response to the thermal stress of fruit during storage.

Relative Importance of Soluble and Microsomal Epoxide Hydrolases for the Hydrolysis of Epoxy-Fatty Acids in Human Tissues

Epoxy-fatty acids (EpFAs) are endogenous lipid mediators that have a large breadth of biological activities, including the regulation of blood pressure, inflammation, angiogenesis, and pain perception. For the past 20 years, soluble epoxide hydrolase (sEH) has been recognized as the primary enzyme for degrading EpFAs in vivo. The sEH converts EpFAs to the generally less biologically active 1,2-diols, which are quickly eliminated from the body. Thus, inhibitors of sEH are being developed as potential drug therapeutics for various diseases including neuropathic pain. Recent findings suggest that other epoxide hydrolases (EHs) such as microsomal epoxide hydrolase (mEH) and epoxide hydrolase-3 (EH3) can contribute significantly to the in vivo metabolism of EpFAs. In this study, we used two complementary approaches to probe the relative importance of sEH, mEH, and EH3 in 15 human tissue extracts: hydrolysis of 14,15-EET and 13,14-EDP using selective inhibitors and protein quantification. The sEH hydrolyzed the majority of EpFAs in all of the tissues investigated, mEH hydrolyzed a significant portion of EpFAs in several tissues, whereas no significant role in EpFAs metabolism was observed for EH3. Our findings indicate that residual mEH activity could limit the therapeutic efficacy of sEH inhibition in certain organs.

Biosynthesis of New Epoxy Fatty Acids from C18 Polyunsaturated Fatty Acids by Linoleate 9-Lipoxygenase from Sphingopyxis macrogoltabida

Epoxy fatty acids (EFAs), which exist in the human body, are signaling molecules that maintain homeostasis. They are involved in anti-inflammation and are precursors of dihydroxy fatty acids. EFAs derived from the C20 polyunsaturated fatty acid (PUFA) arachidonic acid by lipoxygenases, such as 5,6-epoxy-7E,9E,11Z,14Z-eicosatetraenoic acid and 8,9-epoxy-5Z,10E,12E,14Z-eicosatetraenoic acid, are found in humans and mice, respectively. However, EFAs derived from C18 PUFAs by lipoxygenases have not been identified to date. In this study, the putative lipoxygenase gene of Spingopyxis macrogoltabida was cloned and expressed in Escherichia coli. The activity and catalytic efficiency (k/K) of the recombinant enzyme were the highest for linoleic acid among the C18 PUFAs, including also α-linolenic acid and γ-linolenic acid. The product obtained from the conversion of linoleic acid by the putative lipoxygenase was identified as 9-hydroxy-10E,12Z-octadecadienoic acid (9-HODE) by high-performance liquid chromatography using 9-HODE and 13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE) standards. These results indicate that the enzyme is a linoleate 9-lipoxygenase. The enzyme converted linoleic acid, α-linolenic acid, and γ-linolenic acid into 9-HODE, 13-hydroxy-9Z,11E,15Z-octadecatrienoic acid, and 9-hydroxy-6Z,10E,12Z-octadecatrienoic acid, respectively. Moreover, the enzyme also converted the three C18 PUFAs into 9,10-epoxy-11E,13E-octadecadienoic acid, 12,13-epoxy-8E,10E,15Z-octadecatrienoic acid, and 9,10-epoxy-6Z,11E,13E-octadecatrienoic acid, respectively, which were identified as new EFAs by liquid chromatography-mass spectrometry/mass spectrometry. To our knowledge, this is the first report on the biosynthesis of EFAs from C18 PUFAs via a lipoxygenase.

Optimization of Partial Epoxidation on Jatropha curcas Oil Based Methyl Linoleate Using Urea-hydrogen Peroxide and Methyltrioxorhenium Catalyst

Natural epoxy fatty acids such as Coronaric acid (9,10-epoxy-12Z-octadecenoic acid) and vernolic acid (12,13-epoxy-9Z-octadecenoic acid) are rich in of Vernolia galamensis, Vernolia anthelmintica and Chrysanthemums coronanium. The two fatty acids each contains an oxirana ring and a double bond C = C. The oil or its derivatives are suitable for industrial usage as reactive diffluent of alkyd resins, plasticizers and stabilizers, surface coatings, surfactants and lubricants, as intermediates in chemical reactions for making linear epoxides of composite materials and polymers. However, the use of such oils on an industrial scale is impossible due to limited resources. Therefore, epoxidation reactions need to be carried out to overcome the demand for partial epoxide fatty acids. Partially epoxidation of methyl linoleate at room temperature (30°C) in the presence of pyridine, methyltrioxorhenium (MTO) as catalyst and urea-hydrogen peroxide (UHP) as oxidant was studied by using response surface methodology (RSM). A five-level-four-factors central composite rotatable design (CCRD) was used to optimize the partially epoxidation conditions and study the effect of MTO, UHP, pyridine and reaction time on relative conversion to oxirane (RCO). Quadratic polynomial model was employed to generate response surface plots for RCO. At optimal condition, 79.05% monoepoxide was formed at the RCO of 58.15% under condition of 0.75 mol% mole ratio of MTO, 300 mol% mole ratio of UHP and 9 mol% of pyridine at 120 min reaction time. It can be concluded that the effect of UHP mole ratios was the dominant factor to control the degree of partial epoxidation of methyl linoleate followed by mole ratio of MTO, reaction time and mole ratio of pyridine to formed methyl 12,13-epoxy-9Z-octadecenoate or/and methyl 9,10-epoxy-12Z-octadecenoate.

Harmonized procedures lead to comparable quantification of total oxylipins across laboratories

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Epoxy fatty acid dysregulation and neuroinflammation in Alzheimer’s disease is resolved by a soluble epoxide hydrolase inhibitor

Neuroinflammation has been increasingly recognized to play critical roles in Alzheimer’s disease (AD). The epoxy fatty acids (EpFAs) are derivatives of the arachidonic acid metabolism with anti-inflammatory activities. However, their efficacy is limited due to the rapid hydrolysis by the soluble epoxide hydrolase (sEH). We found that sEH is predominantly expressed in astrocytes where its levels are significantly elevated in postmortem human AD brains and in β-amyloid mouse models, and the latter is correlated with drastic reductions of brain EpFA levels. Using a highly potent and specific small molecule sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), we report here that TPPU treatment potently protected against LPS-induced inflammation in vitro and in vivo. Long-term administration of TPPU to the 5xFAD mouse model via drinking water reversed microglia and astrocyte reactivity and immune pathway dysregulation, and this is associated with reduced β–amyloid pathology and improved synaptic integrity and cognitive function. Importantly, TPPU treatment reinstated and positively correlated EpFA levels in the 5xFAD mouse brain, demonstrating its brain penetration and target engagement. These findings support TPPU as a novel therapeutic target for the treatment of AD and related disorders.One Sentence SummaryWe show that soluble epoxide hydrolase is upregulated in AD patients and mouse models, and that inhibition of this lipid metabolic pathway using an orally bioavailable small molecule inhibitor is effective in restoring brain epoxy fatty acids, ameliorating AD neuropathology and improving synaptic and cognitive function.