Brown Adipose Tissue Activation in Humans Increases Plasma Levels of Lipid Mediators.

CONTEXT: Activation of brown adipose tissue (BAT) thermogenesis improves insulin sensitivity and is beneficial in obesity. Emerging evidence indicates that BAT activation increases lipid mediators that play autocrine and endocrine roles to regulate metabolism and inflammation. OBJECTIVE: The goal of the study was to determine the relationship between 2 distinct approaches of BAT activation (cold exposure and mirabegron treatment) with lipid mediators in humans. METHODS: Healthy female subjects (n = 14) were treated with the ß3-adrenergic receptor agonist mirabegron (100 mg) daily for 28 days. A subset of female subjects (n = 8) was additionally exposed to cold temperatures (14-16 °C) for 2 hours using a cooling vest prior to initiating mirabegron treatment. A panel of lipid mediators was assessed in plasma using targeted liquid chromatography-tandem mass spectrometry, and their relationship to anthropometric and metabolic parameters was determined. RESULTS: Activation of BAT with cold exposure acutely increased levels of lipoxygenase and cyclooxygenase products, including 12-hydroxyeicosapentaenoic acid, 12-hydroxyeicosatetraenoic acid (HETE), 5-HETE, 14-hydroxydocosahexaenoic acid (HDHA), an isomer of maresin 2 (MaR2), 17-HDHA, protectin D1 (PD1), and prostaglandin E2. Mirabegron treatment similarly increased these products acutely, although levels of some mediators were blunted after chronic mirabegron treatment. Selected lipid mediators, including an MaR2 isomer, 17-HDHA, 5-HETE, and 15-HETE, positively correlated with nonesterified fatty acids and negatively correlated with the respiratory quotient, while PD1, 15-HETE, and 5-HETE positively correlated with adiponectin. CONCLUSION: These results indicate that selected lipid mediators may serve as biomarkers of BAT activation.

Mapping the transcriptional landscape of human white and brown adipogenesis using single-nuclei RNA-seq.

Adipogenesis is key to maintaining organism-wide energy balance and healthy metabolic phenotype, making it critical to thoroughly comprehend its molecular regulation in humans. By single-nuclei RNA-sequencing (snRNA-seq) of over 20,000 differentiating white and brown preadipocytes, we constructed a high-resolution temporal transcriptional landscape of human white and brown adipogenesis. White and brown preadipocytes were isolated from a single individual's neck region, thereby eliminating inter-subject variability across two distinct lineages. These preadipocytes were also immortalized to allow for controlled, in vitro differentiation, allowing sampling of distinct cellular states across the spectrum of adipogenic progression. Pseudotemporal cellular ordering revealed the dynamics of ECM remodeling during early adipogenesis, and lipogenic/thermogenic response during late white/brown adipogenesis. Comparison with adipogenic regulation in murine models Identified several novel transcription factors as potential targets for adipogenic/thermogenic drivers in humans. Among these novel candidates, we explored the role of TRPS1 in adipocyte differentiation and showed that its knockdown impairs white adipogenesis in vitro. Key adipogenic and lipogenic markers revealed in our analysis were applied to analyze publicly available scRNA-seq datasets; these confirmed unique cell maturation features in recently discovered murine preadipocytes, and revealed inhibition of adipogenic expansion in humans with obesity. Overall, our study presents a comprehensive molecular description of both white and brown adipogenesis in humans and provides an important resource for future studies of adipose tissue development and function in both health and metabolic disease state.

Brown adipose tissue-derived MaR2 contributes to cold-induced resolution of inflammation.

Obesity induces chronic inflammation resulting in insulin resistance and metabolic disorders. Cold exposure can improve insulin sensitivity in humans and rodents, but the mechanisms have not been fully elucidated. Here, we find that cold resolves obesity-induced inflammation and insulin resistance and improves glucose tolerance in diet-induced obese mice. The beneficial effects of cold exposure on improving obesity-induced inflammation and insulin resistance depend on brown adipose tissue (BAT) and liver. Using targeted liquid chromatography with tandem mass spectrometry, we discovered that cold and ß3-adrenergic stimulation promote BAT to produce maresin 2 (MaR2), a member of the specialized pro-resolving mediators of bioactive lipids that play a role in the resolution of inflammation. Notably, MaR2 reduces inflammation in obesity in part by targeting macrophages in the liver. Thus, BAT-derived MaR2 could contribute to the beneficial effects of BAT activation in resolving obesity-induced inflammation and may inform therapeutic approaches to combat obesity and its complications.

Signaling Lipidomic Analysis of Thermogenic Adipocytes.

Brown adipose tissue is a thermogenic organ that possesses anti-diabetic and anti-obesogenic potential. There has recently been growing interest on the secretory role of brown adipose tissue in regulating whole-body metabolism. Several signaling lipids, including 12-HEPE and 12,13-diHOME, have been shown to be secreted by brown adipose tissue and have demonstrated roles in regulating whole-body energy metabolism. Lipidomics platforms that broadly characterize the signaling lipidome can deconvolute the underlying biology of the lipid metabolites having a broad systemic impact on physiology. Herein, we describe how to perform and analyze LC-MS/MS signaling lipidomics on mature brown adipocytes.

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.

Narrating the story ARC of adipose tissue aging.

In this issue of Developmental Cell, Nguyen et al. use scRNA-seq to explore the changing cellular landscape of subcutaneous adipose tissue during aging. In the process, they discover a new population of cells called age-dependent regulatory cells (ARC), which contributes to age-related adipose tissue dysfunction that drives metabolic disease.

Selection of Potent Inhibitors of Soluble Epoxide Hydrolase for Usage in Veterinary Medicine.

The veterinary pharmacopeia available to treat pain and inflammation is limited in number, target of action and efficacy. Inhibitors of soluble epoxide hydrolase (sEH) are a new class of anti-inflammatory, pro-resolving and analgesic drugs being tested in humans that have demonstrated efficacy in laboratory animals. They block the hydrolysis, and thus, increase endogenous concentrations of analgesic and anti-inflammatory signaling molecules called epoxy-fatty acids. Here, we screened a library of 2,300 inhibitors of the sEH human against partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six very potent sEH inhibitors (IC50 < 1 nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in solvents suitable for the formulation of drugs. The trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB, 1,728) appears to be the best compromise between stability and potency across species. Thus, it was selected for further testing in veterinary clinical trials of pain and inflammation in animals.

Cytochrome P450-derived linoleic acid metabolites EpOMEs and DiHOMEs: a review of recent studies.

Linoleic acid (LA) is the most abundant polyunsaturated fatty acid found in the Western diet. Cytochrome P450-derived LA metabolites 9,10-epoxyoctadecenoic acid (9,10-EpOME), 12,13-epoxyoctadecenoic acid (12,13-EpOME), 9,10-dihydroxy-12Z-octadecenoic acid (9,10-DiHOME) and 12,13-dihydroxy-9Z-octadecenoic acid (12,13-DiHOME) have been studied for their association with various disease states and biological functions. Previous studies of the EpOMEs and DiHOMEs have focused on their roles in cytotoxic processes, primarily in the inhibition of the neutrophil respiratory burst. More recent research has suggested the DiHOMEs may be important lipid mediators in pain perception, altered immune response and brown adipose tissue activation by cold and exercise. The purpose of this review is to summarize the current understanding of the physiological and pathophysiological roles and modes of action of the EpOMEs and DiHOMEs in health and disease.

Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2.

Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.

Drug-Target Residence Time Affects in Vivo Target Occupancy through Multiple Pathways.

The drug discovery and development process is greatly hampered by difficulties in translating in vitro potency to in vivo efficacy. Recent studies suggest that the long-neglected drug-target residence time parameter complements classical drug affinity parameters (K I, K d, IC50, or EC50) and is a better predictor of in vivo efficacy. Compounds with a long drug-target residence time are often more efficacious in vivo. The impact, however, of the drug-target residence time on in vivo efficacy remains controversial due to difficulties in experimentally determining the in vivo target occupancy during drug treatment. To tackle this problem, an in vivo displacement assay was developed using soluble epoxide hydrolase as a biological model. In this report, we experimentally demonstrated that drug-target residence time affects the duration of in vivo drug-target binding. In addition, the drug-target residence time plays an important role in modulating the rate of drug metabolism which also affects the efficacy of the drug.

Lipokines and Thermogenesis.

Adaptive thermogenesis is a catabolic process that consumes energy-storing molecules and expends that energy as heat in response to environmental changes. This process occurs primarily in brown and beige adipose tissue. Thermogenesis is regulated by many factors, including lipid derived paracrine and endocrine hormones called lipokines. Recently, technologic advances for identifying new lipid biomarkers of thermogenic activity have shed light on a diverse set of lipokines that act through different pathways to regulate energy expenditure. In this review, we highlight a few examples of lipokines that regulate thermogenesis. The biosynthesis, regulation, and effects of the thermogenic lipokines in several families are reviewed, including oloeylethanolamine, endocannabinoids, prostaglandin E2, and 12,13-diHOME. These thermogenic lipokines present potential therapeutic targets to combat states of excess energy storage, such as obesity and related metabolic disorders.

12-Lipoxygenase Regulates Cold Adaptation and Glucose Metabolism by Producing the Omega-3 Lipid 12-HEPE from Brown Fat.

Distinct oxygenases and their oxylipin products have been shown to participate in thermogenesis by mediating physiological adaptations required to sustain body temperature. Since the role of the lipoxygenase (LOX) family in cold adaptation remains elusive, we aimed to investigate whether, and how, LOX activity is required for cold adaptation and to identify LOX-derived lipid mediators that could serve as putative cold mimetics with therapeutic potential to combat diabetes. By utilizing mass-spectrometry-based lipidomics in mice and humans, we demonstrated that cold and ß3-adrenergic stimulation could promote the biosynthesis and release of 12-LOX metabolites from brown adipose tissue (BAT). Moreover, 12-LOX ablation in mouse brown adipocytes impaired glucose uptake and metabolism, resulting in blunted adaptation to the cold in vivo. The cold-induced 12-LOX product 12-HEPE was found to be a batokine that improves glucose metabolism by promoting glucose uptake into adipocytes and skeletal muscle through activation of an insulin-like intracellular signaling pathway.

Role of epoxy-fatty acids and epoxide hydrolases in the pathology of neuro-inflammation.

Neuroinflammation is a physiologic response aimed at protecting the central nervous system during injury. However, unresolved and chronic neuroinflammation can lead to long term damage and eventually neurologic disease including Parkinson's disease, Alzheimer's disease and dementia. Recently, enhancing the concentration of epoxyeicosatrienoic acids (EETs) through blocking their hydrolytic degradation by soluble epoxide hydrolase (sEH) has been applied towards reducing the long-term damage associated with central neurologic insults. Evidence suggests this protective effect is mediated, at least in part, through polarization of microglia to an anti-inflammatory phenotype that blocks the inflammatory actions of prostaglandins and promotes wound repair. This mini-review overviews the epidemiologic basis for using sEH inhibition towards neuroinflammatory disease and pharmacologic studies testing sEH inhibition in several neurologic diseases. Additionally, the combination of sEH inhibition with other eicosanoid signaling pathways is considered as an enhanced approach for developing potent neuroprotectants.

Design and Potency of Dual Soluble Epoxide Hydrolase/Fatty Acid Amide Hydrolase Inhibitors.

Fatty acid amide hydrolase (FAAH) is responsible for regulating concentrations of the endocannabinoid arachidonoyl ethanolamide. Multiple FAAH inhibitors have been developed for clinical trials and have failed to demonstrate efficacy at treating pain, despite promising preclinical data. One approach toward increasing the efficacy of FAAH inhibitors is to concurrently inhibit other targets responsible for regulating pain. Here, we designed dual inhibitors targeting the enzymes FAAH and soluble epoxide hydrolase (sEH), which are targets previously shown to synergize at reducing inflammatory and neuropathic pain. Exploration of the sEH/FAAH inhibitor structure-activity relationship started with PF-750, a FAAH inhibitor (IC50 = 19 nM) that weakly inhibited sEH (IC50 = 640 nM). Potency was optimized resulting in an inhibitor with improved potency on both targets (11, sEH IC50 = 5 nM, FAAH IC50 = 8 nM). This inhibitor demonstrated good target selectivity, pharmacokinetic properties (AUC = 1200 h nM, t 1/2 = 4.9 h in mice), and in vivo target engagement.

Indomethacin Enhances Brown Fat Activity.

Indomethacin, a nonsteroidal anti-inflammatory drug, has been shown to induce white adipocyte differentiation; however, its roles in brown adipocyte differentiation and activation in brown adipose tissue (BAT) and obesity are unknown. To address this issue, we treated mouse brown preadipocytes with different doses of indomethacin, and delivered indomethacin to interscapular BAT (iBAT) of obese mice using implanted osmotic pumps. Indomethacin dose dependently increased brown preadipocyte differentiation and upregulated both mRNA and protein expression of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor (PPAR) γ coactivator 1-alpha. The mechanistic study showed that indomethacin significantly activated the reporter driven by the PPAR response element, indicating that indomethacin may work as a PPARγ agonist in this cell line. Consistently, indomethacin significantly decreased iBAT mass and fasting blood glucose levels in high-fat diet-induced obesity (DIO) mice. Histologic analysis showed that brown adipocytes of indomethacin-treated mice contained smaller lipid droplets compared with control mice, suggesting that indomethacin alleviated the whitening of BAT induced by the high-fat diet. Moreover, indomethacin significantly increased UCP1 mRNA expression in iBAT. Taken together, this study indicates that indomethacin can promote mouse brown adipocyte differentiation, and might increase brown fat and glucose oxidation capacity in DIO mice.

Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency towards fatty acid amide hydrolase.

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.

Development of amide-based fluorescent probes for selective measurement of carboxylesterase 1 activity in tissue extracts.

Carboxylesterases are well known for their role in the metabolism of xenobiotics. However, recent studies have also implicated carboxylesterases in regulating a number of physiological processes including metabolic homeostasis and macrophage development, underlying the need to quantify them individually. Unfortunately, current methods for selectively measuring the catalytic activity of individual carboxylesterases are not sufficiently sensitive to support many biological studies. In order to develop a more sensitive and selective method to measure the activity of human carboxylesterase 1 (hCE1), we generated and tested novel substrates with a fluorescent aminopyridine leaving group. hCE1 showed at least a 10-fold higher preference for the optimized substrate 4-MOMMP than the 13 other esterases tested. Because of the high stability of 4-MOMMP and its hydrolysis product, this substrate can be used to measure esterase activity over extended incubation periods yielding a low picogram (femtomol) limit of detection. This sensitivity is comparable to current ELISA methods; however, the new assay quantifies only the catalytically active enzyme facilitating direct correlation to biological processes. The method described herein may allow hCE1 activity to be used as a biomarker for predicting drug pharmacokinetics, early detection of hepatocellular carcinoma, and other disease states where the activity of hCE1 is altered.

Reduced carboxylesterase 1 is associated with endothelial injury in methamphetamine-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.

Parabens inhibit fatty acid amide hydrolase: A potential role in paraben-enhanced 3T3-L1 adipocyte differentiation.

Parabens are a class of small molecules that are regularly used as preservatives in a variety of personal care products. Several parabens, including butylparaben and benzylparaben, have been found to interfere with endocrine signaling and to stimulate adipocyte differentiation. We hypothesized these biological effects could be due to interference with the endocannabinoid system and identified fatty acid amide hydrolase (FAAH) as the direct molecular target of parabens. FAAH inhibition by parabens yields mixed-type and time-independent kinetics. Additionally, structure activity relationships indicate FAAH inhibition is selective for the paraben class of compounds and the more hydrophobic parabens have higher potency. Parabens enhanced 3T3-L1 adipocyte differentiation in a dose dependent fashion, different from two other FAAH inhibitors URB597 and PF622. Moreover, parabens, URB597 and PF622 all failed to enhance AEA-induced differentiation. Furthermore, rimonabant, a cannabinoid receptor 1 (CB1)-selective antagonist, did not attenuate paraben-induced adipocyte differentiation. Thus, adipogenesis mediated by parabens likely occurs through modulation of endocannabinoids, but cell differentiation is independent of direct activation of CB1 by endocannabinoids.

Anti-Ulcer Efficacy of Soluble Epoxide Hydrolase Inhibitor TPPU on Diclofenac-Induced Intestinal Ulcers.

Proton pump inhibitors such as omeprazole (OME) reduce the severity of gastrointestinal (GI) ulcers induced by nonsteroidal anti-inflammatory drugs (NSAIDs) but can also increase the chance of dysbiosis. The aim of this study was to test the hypothesis that preventive use of a soluble epoxide hydrolase inhibitor (sEHI) such as TPPU can decrease NSAID-induced ulcers by increasing anti-inflammatory epoxyeicosatrienoic acids (EETs). Dose- [10, 30, and 100 mg/kg, by mouth (PO)] and time-dependent (6 and 18 hours) ulcerative effects of diclofenac sodium (DCF, an NSAID) were studied in the small intestine of Swiss Webster mice. Dose-dependent effects of TPPU (0.001-0.1 mg/kg per day for 7 days, in drinking water) were evaluated in DCF-induced intestinal toxicity and compared with OME (20 mg/kg, PO). In addition, the effect of treatment was studied on levels of Hb in blood, EETs in plasma, inflammatory markers such as myeloperoxidase (MPO) in intestinal tissue homogenates, and tissue necrosis factor-α (TNF-α) in serum. DCF dose dependently induced ulcers that were associated with both a significant (P < 0.05) loss of Hb and an increase in the level of MPO and TNF-α, with severity of ulceration highest at 18 hours. Pretreatment with TPPU dose dependently prevented ulcer formation by DCF, increased the levels of epoxy fatty acids, including EETs, and TPPU's efficacy was comparable to OME. TPPU significantly (P < 0.05) reversed the effect of DCF on the level of Hb, MPO, and TNF-α Thus sEHI might be useful in the management of NSAID-induced ulcers.