An Autophagic Flux Probe that Releases an Internal Control.

Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3ΔG, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is degraded by autophagy, while RFP-LC3ΔG remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3ΔG probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.

Genetic deletion of Cyp4f18 disrupts the omega-3 epoxidation pathway and results in psoriasis-like dermatitis.

Cyp4f18 catalyzes the conversion of n-3 polyunsaturated fatty acids (PUFAs) into omega-3 epoxides, such as 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) and 19,20-epoxydocosapentaenoic acid (19,20-EpDPE) from eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), respectively. Cyp4f18-deficient mice spontaneously develop psoriasis-like dermatitis. A significant increase in the number of IL-17A-positive gamma delta (γδ) T cells in the skin and enlargement of draining lymph nodes was observed. These symptoms were drastically suppressed by antibiotic treatment. Cyp4f18 is highly expressed in dendritic cells (DCs), and Cyp4f18-deficient bone marrow-derived dendritic cells (BMDCs) show markedly increased expression levels of cytokines such as IL-23 and IL-1ß in response to lipopolysaccharide (LPS) stimulation. Lipidomic analysis of lymph nodes and BMDCs revealed a significant decrease in a series of omega-3 epoxidized metabolites. Among them, 17,18-dihydroxyeicosatetraenoic acid (17,18-diHETE), a vicinal diol derived from EPA omega-3 epoxidation suppressed IL-23 production in LPS-stimulated BMDCs in Cyp4f18-deficient mice. These results demonstrate that Cyp4f18 endogenously produces omega-3-epoxidized metabolites in the draining lymph nodes, and these metabolites contribute to skin homeostasis by suppressing the excessive activation of the IL-23/IL-17 axis initiated by DCs.

Eosinophils promote corneal wound healing via the 12/15-lipoxygenase pathway.

Lipid mediators play important roles in regulating inflammatory responses and tissue homeostasis. Since 12/15-lipoxygenase (12/15-LOX)-derived lipid mediators such as lipoxin A4 (LXA4 ) and protectin D1 (PD1) protect against corneal epithelial cell damage, the major cell types that express 12/15-LOX and contribute to the corneal wound healing process are of particular interest. Here, we found that eosinophils were the major cell type expressing 12/15-LOX during the corneal wound healing process. Eosinophils were recruited into the conjunctiva after corneal epithelium wounding, and eosinophil-deficient and/or eosinophil-specific 12/15-LOX knockout mice showed delayed corneal wound healing compared with wild-type mice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based mediator lipidomics revealed that a series of 12/15-LOX-derived mediators were significantly decreased in eosinophil-deficient mice and topical application of 17-hydroxydocosahexaenoic acid (17-HDoHE), a major 12/15-LOX-derived product, restored the phenotype. These results indicate that 12/15-LOX-expressing eosinophils, by locally producing pro-resolving mediators, significantly contribute to the corneal wound healing process in the eye.

Omega-3 fatty acid-derived mediators that control inflammation and tissue homeostasis.

Omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, display a wide range of beneficial effects in humans and animals. Many of the biological functions of PUFAs are mediated via bioactive metabolites produced by fatty acid oxygenases such as cyclooxygenases, lipoxygenases and cytochrome P450 monooxygenases. Liquid chromatography-tandem mass spectrometry-based mediator lipidomics revealed a series of novel bioactive lipid mediators derived from omega-3 PUFAs. Here, we describe recent advances on omega-3 PUFA-derived mediators, mainly focusing on their enzymatic oxygenation pathway, and their biological functions in controlling inflammation and tissue homeostasis.

Discovery of Novel SPAK Inhibitors That Block WNK Kinase Signaling to Cation Chloride Transporters.

Upon activation by with-no-lysine kinases, STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) phosphorylates and activates SLC12A transporters such as the Na(+)-Cl(-) cotransporter (NCC) and Na(+)-K(+)-2Cl(-) cotransporter type 1 (NKCC1) and type 2 (NKCC2); these transporters have important roles in regulating BP through NaCl reabsorption and vasoconstriction. SPAK knockout mice are viable and display hypotension with decreased activity (phosphorylation) of NCC and NKCC1 in the kidneys and aorta, respectively. Therefore, agents that inhibit SPAK activity could be a new class of antihypertensive drugs with dual actions (i.e., NaCl diuresis and vasodilation). In this study, we developed a new ELISA-based screening system to find novel SPAK inhibitors and screened >20,000 small-molecule compounds. Furthermore, we used a drug repositioning strategy to identify existing drugs that inhibit SPAK activity. As a result, we discovered one small-molecule compound (Stock 1S-14279) and an antiparasitic agent (Closantel) that inhibited SPAK-regulated phosphorylation and activation of NCC and NKCC1 in vitro and in mice. Notably, these compounds had structural similarity and inhibited SPAK in an ATP-insensitive manner. We propose that the two compounds found in this study may have great potential as novel antihypertensive drugs.

Expression of the tumor suppressive miRNA-23b/27b cluster is a good prognostic marker in clear cell renal cell carcinoma.

PURPOSE: We observed abnormal expression of the microRNA-23b/27b (miR-23b/27b) cluster in our previous study of miRNA expression signatures. However, the relationship between aberrant miRNA expression and clear cell renal cell carcinoma is not well established. We investigated the functional significance of the miR-23b/27b cluster in clear cell renal cell carcinoma cells and evaluated these miRNAs as biomarkers to predict the risk of clear cell renal cell carcinoma. MATERIALS AND METHODS: Expression levels of miR-23b and miR-27b were determined by quantitative real-time reverse transcriptase-polymerase chain reaction. The association between miRNA expression and overall survival was estimated by the Kaplan-Meier method. Gain of function assays were performed using mature miR-23b and miR-27b in the 786-O and A498 renal cell carcinoma cell lines. Targets regulated by these miRNAs were predicted by in silico analysis. RESULTS: Expression of the miR-23b/27b cluster was significantly decreased in clear cell renal cell carcinoma tissue specimens and associated with pathological grade and stage. Significantly shorter overall survival was observed in patients with lower expression of the miR-23b/27b cluster. Restoration of miR-23b and miR-27b significantly inhibited cancer cell proliferation, migration and invasion. CONCLUSIONS: Expression of the miR-23b/27b cluster was frequently decreased in clear cell renal cell carcinoma tissue. Reduced expression of these miRNAs increased the risk of disease progression and predicted poor survival. Thus, miR-23b and miR-27b function as tumor suppressors, targeting several oncogenic genes in clear cell renal cell carcinoma cells.

Synthesis and biological comparison of enantiomers of mepenzolate bromide, a muscarinic receptor antagonist with bronchodilatory and anti-inflammatory activities.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal inflammatory responses and airflow limitations. We recently proposed that the muscarinic antagonist mepenzolate bromide (mepenzolate) would be therapeutically effective against COPD due to its muscarinic receptor-dependent bronchodilatory activity as well as anti-inflammatory properties. Mepenzolate has an asymmetric carbon atom, thus providing us with the opportunity to synthesize both of its enantiomers ((R)- and (S)-mepenzolate) and to examine their biochemical and pharmacological activities. (R)- or (S)-mepenzolate was synthesized by condensation of benzilic acid with (R)- or (S)-alcohol, respectively, followed by quaternization of the tertiary amine. As predicted by computational simulation, a filter-binding assay in vitro revealed that (R)-mepenzolate showed a higher affinity for the muscarinic M3 receptor than (S)-mepenzolate. In vivo, the bronchodilatory activity of (R)-mepenzolate was superior to that of (S)-mepenzolate, whereas anti-inflammatory activity was indistinguishable between the two enantiomers. We confirmed that each mepenzolate maintained its original stereochemistry in the lung when administered intratracheally. These results suggest that (R)-mepenzolate may have superior properties to (S)-mepenzolate as a drug to treat COPD patients given that the former has more potent bronchodilatory activity than the latter.

A lipidome landscape of aging in mice.

Understanding the molecular mechanisms of aging is crucial for enhancing healthy longevity. We conducted untargeted lipidomics across 13 biological samples from mice at various life stages (2, 12, 19 and 24 months) to explore the potential link between aging and lipid metabolism, considering sex (male or female) and microbiome (specific pathogen-free or germ-free) dependencies. By analyzing 2,704 molecules from 109 lipid subclasses, we characterized common and tissue-specific lipidome alterations associated with aging. For example, the levels of bis(monoacylglycero)phosphate containing polyunsaturated fatty acids increased in various organs during aging, whereas the levels of other phospholipids containing saturated and monounsaturated fatty acids decreased. In addition, we discovered age-dependent sulfonolipid accumulation, absent in germ-free mice, correlating with Alistipes abundance determined by 16S ribosomal RNA gene amplicon sequencing. In the male kidney, glycolipids such as galactosylceramides, galabiosylceramides (Gal2Cer), trihexosylceramides (Hex3Cer), and mono- and digalactosyldiacylglycerols were detected, with two lipid classes-Gal2Cer and Hex3Cer-being significantly enriched in aged mice. Integrated analysis of the kidney transcriptome revealed uridine diphosphate galactosyltransferase 8A (UGT8a), alkylglycerone phosphate synthase and fatty acyl-coenzyme A reductase 1 as potential enzymes responsible for the male-specific glycolipid biosynthesis in vivo, which would be relevant to sex dependency in kidney diseases. Inhibiting UGT8 reduced the levels of these glycolipids and the expression of inflammatory cytokines in the kidney. Our study provides a valuable resource for clarifying potential links between lipid metabolism and aging.

Iron overload and glucose abnormalities in chronic hepatitis C virus infection: phlebotomy lowers risk of new-onset diabetes.

BACKGROUND/AIMS: Iron overload and hyperglycemia are common findings in patients with chronic hepatitis C (CHC). The aim of this study was to determine the relation of serum ferritin and hepatic hepcidin expression with glucose metabolic parameters and to evaluate whether long term phlebotomy lowers the risk of new-onset diabetes in CHC patients. METHODOLOGY: Hepatic hepcidin mRNA expression was measured in 28 CHC patients and their relation with clinical parameters and histological findings was evaluated. Ninety-two patients without type 2 diabetes were divided into two groups: a phlebotomy group underwent an initial period of phlebotomy and maintenance phlebotomy was performed; data obtained in CHC patients that declined to receive phlebotomy were used as control. RESULTS: Hepatic hepcidin expression was positively correlated with body mass index and glucose metabolic parameters. A total number of five patients had onset of type 2 diabetes over 38.9 months of follow-up. Long-term phlebotomy tended to lower the risk of new-onset diabetes compared with control CHC patients. In addition, high ferritin levels predicted further episodes of diabetes in control patients. CONCLUSIONS: Iron overload is a risk for the development of diabetes in patients with CHC and that reduction of body iron stores lowers this risk.

Effects of ß-(1,3-1,6)-D-glucan on irritable bowel syndrome-related colonic hypersensitivity.

Irritable bowel syndrome (IBS) is a gastrointestinal disorder characterized by chronic abdominal pain associated with altered bowel habits. Since the prevalence of IBS is very high and thus, involves elevated health-care costs, treatment of this condition by methods other than prescribed medicines could be beneficial. ß-(1,3)-D-glucan with ß-(1,6) branches (ß-glucan) has been used as a nutritional supplement for many years. In this study, we examined the effect of ß-glucan on fecal pellet output and visceral pain response in animal models of IBS. Oral administration of ß-glucan suppressed the restraint stress- or drug-induced fecal pellet output. ß-Glucan also suppressed the visceral pain response to colorectal distension. These results suggest that ß-glucan could be beneficial for the treatment and prevention of IBS.

Therapeutic effect of lecithinized superoxide dismutase on bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is thought to involve inflammatory infiltration of leukocytes, lung injury induced by reactive oxygen species (ROS), in particular superoxide anion, and fibrosis (collagen deposition). No treatment has been shown to improve definitively the prognosis for IPF patients. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) has overcome clinical limitations of SOD, including low tissue affinity and low stability in plasma. In this study, we examined the effect of PC-SOD on bleomycin-induced pulmonary fibrosis. Severity of the bleomycin-induced fibrosis in mice was assessed by various methods, including determination of hydroxyproline levels in lung tissue. Intravenous administration of PC-SOD suppressed the bleomycin-induced increase in the number of leukocytes in bronchoalveolar lavage fluid. Bleomycin-induced collagen deposition and increased hydroxyproline levels in the lung were also suppressed in animals treated with PC-SOD, suggesting that PC-SOD suppresses bleomycin-induced pulmonary fibrosis. The dose-response profile of PC-SOD was bell-shaped, but concurrent administration of catalase restored the ameliorative effect at high doses of PC-SOD. Intratracheal administration or inhalation of PC-SOD also attenuated the bleomycin-induced inflammatory response and fibrosis. The bell-shaped dose-response profile of PC-SOD was not observed for these routes of administration. We consider that, compared with intravenous administration, inhalation of PC-SOD may be a more therapeutically beneficial route of administration due to the higher safety and quality of life of the patient treated with this drug.

Positive role of CCAAT/enhancer-binding protein homologous protein, a transcription factor involved in the endoplasmic reticulum stress response in the development of colitis.

Although recent reports suggest that the endoplasmic reticulum (ER) stress response is induced in association with the development of inflammatory bowel disease, its role in the pathogenesis of inflammatory bowel disease remains unclear. The CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a transcription factor that is involved in the ER stress response, especially ER stress-induced apoptosis. In this study, we found that experimental colitis was ameliorated in CHOP-null mice, suggesting that CHOP exacerbates the development of colitis. The mRNA expression of Mac-1 (CD11b, a positive regulator of macrophage infiltration), Ero-1alpha, and Caspase-11 (a positive regulator of interleukin-1beta production) in the intestine was induced with the development of colitis, and this induction was suppressed in CHOP-null mice. ERO-1alpha is involved in the production of reactive oxygen species (ROS); an increase in ROS production, which is associated with the development of colitis in the intestine, was suppressed in CHOP-null mice. A greater number of apoptotic cells in the intestinal mucosa of wild-type mice were observed to accompany the development of colitis compared with CHOP-null mice, suggesting that up-regulation of CHOP expression exacerbates the development of colitis. Furthermore, this CHOP activity appears to involve various stimulatory mechanisms, such as macrophage infiltration via the induction of Mac-1, ROS production via the induction of ERO-1alpha, interleukin-1beta production via the induction of Caspase-11, and intestinal mucosal cell apoptosis.

Low direct cytotoxicity of loxoprofen on gastric mucosal cells.

Pro-drugs of non-steroidal anti-inflammatory drugs (NSAIDs), such as loxoprofen are widely used for clinical purposes because they are not so harmful to the gastrointestinal mucosa. We recently showed that NSAIDs such as indomethacin and celecoxib have direct cytotoxicity (ability to induce necrosis and apoptosis in gastric mucosal cells) due to their membrane permeabilizing activities, which is involved in NSAID-induced gastric lesions. We show here that under conditions where indomethacin and celecoxib clearly induce necrosis and apoptosis, loxoprofen and its active metabolite loxoprofen-OH, do not have such effects in primary culture of guinea pig gastric mucosal cells. Loxoprofen and loxoprofen-OH induced apoptosis more effectively in cultured human gastric cancer cells than in the primary culture. Loxoprofen and loxoprofen-OH exhibited much lower membrane permeabilizing activities than did indomethacin and celecoxib. We thus consider that the low direct cytotoxicity of loxoprofen observed in vitro is involved in its relative safety on production of gastric lesions in clinical situation.

Successful Kidney Transplantation Alone With Severe Left Ventricular Systolic Dysfunction of Ejection Fraction 14%: A Case Report.

It is well known that correction of uremia by kidney transplantation alone (KTA) improves left ventricular systolic dysfunction (LVSD). However, for kidney transplant candidates with extremely severe LVSD, KTA is considered to be contraindicated because of the high risk of peri-operative management. We report a case of successful kidney transplantation with severe LVSD with an ejection fraction (EF) of 14% and low systolic blood pressure (SBP) of approximately 65 to 80 mm Hg. In this case, in spite of an extremely low EF and SBP, functional capacity was assessed using metabolic equivalents (METs) and showed a level of almost 4. The operation was performed carefully, considering the cardiac, operative, and anesthetic risks. No surgical complications occurred, and the patient received intensive care during the peri-operative period. His postoperative course was almost favorable, and he was discharged on postoperative day 29. The present report concludes that evaluation of METs may expand the indication for KTA in patients with extremely severe LVSD.

Therapeutic effect of lecithinized superoxide dismutase against colitis.

Ulcerative colitis (UC) involves intestinal mucosal damage induced by reactive oxygen species (ROS), in particular, superoxide anion. Superoxide dismutase (SOD) catalyzes dismutation of superoxide anion to hydrogen peroxide, which is subsequently detoxified by catalase. Lecithinized SOD (PC-SOD) is a new modified form of SOD that has overcome previous clinical limitations of SOD. In this study, we examined the action of PC-SOD using an animal model of UC, dextran sulfate sodium (DSS)-induced colitis. DSS-induced colitis was ameliorated by daily intravenous administration of PC-SOD. Unmodified SOD produced a similar effect but only at more than 30 times the concentration of PC-SOD. In vivo electron spin resonance analysis confirmed that the increase in the colonic level of ROS associated with development of colitis was suppressed by PC-SOD administration. The dose-response profile of PC-SOD was bell-shaped, but simultaneous administration of catalase restored the ameliorative effect at high doses of PC-SOD. Accumulation of hydrogen peroxide was observed with the administration of high doses of PC-SOD, an effect that was suppressed by the simultaneous administration of catalase. We also found that either a weekly intravenous administration or daily oral administration of PC-SOD conferred protection. These results suggest that PC-SOD achieves its ameliorative effect against colitis through decreasing the colonic level of ROS and that its ineffectiveness at higher doses is because of the accumulation of hydrogen peroxide. Furthermore, we consider that intermittent or oral administration of PC-SOD can be applied clinically to improve the quality of life of UC patients.

Cell-free and Concentrated Ascites Reinfusion Therapy for Refractory Ascites in Cirrhosis in Post-marketing Surveillance and the Role of Tolvaptan.

Objective Ascites becomes refractory to diuretics in cirrhotic patients, who then require repeated large-volume paracentesis or cell-free and concentrated ascites reinfusion therapy (CART). The objective of this study was to confirm the safety and efficacy of CART, evaluate the actual situations with respect to the prescription of diuretics and determine the role of diuretics after the introduction of CART. Patients and Methods We recruited 34 cirrhotic patients who received CART with concomitant diuretics using furosemide (76.2%), spironolactone (48.5%), thiazide (4.0%) and tolvaptan (53.5%) from a post-marketing surveillance of CART. Results CART improved the tested clinical indices, i.e., body weight, abdominal circumference, performance status, dietary intake, total protein and albumin. The intervals of CART sessions were significantly prolonged in patients who received tolvaptan (mean, 22.5 days) compared to those not receiving tolvaptan (mean, 10.8 days) (p<0.001). The drop-out rate was significantly decreased in patients receiving tolvaptan compared to those not receiving tolvaptan when drop-out was defined as paracentesis (p<0.05). Conclusion We confirmed that CART is an effective treatment for refractory ascites occurring in cirrhotic patients. The administration of tolvaptan in combination with CART leads to a significantly reduced rate of ascites accumulation.

Anatomical Variations of the Left Renal Vein During Laparoscopic Donor Nephrectomy.

OBJECTIVES: The aim of this study is to investigate the outcome of laparoscopic donor nephrectomy with vascular anomalies of the left renal vein. PATIENTS AND METHODS: Between August 2010 and September 2018, a total of 120 laparoscopic donor nephrectomies were performed at Kagoshima University. Among them, we experienced 7 cases of donors with anomalous left renal vein (circumaortic left renal vein n = 5, retroaortic left renal vein n = 1, left renal vein drainage into hemiazygos vein n = 1). We analyzed the following clinical outcomes: pneumoperitoneum time, estimated blood loss, warm ischemia time, length of hospital stay, and serum creatinine level of 1 month after surgery for evaluating the safety of laparoscopic donor nephrectomy. RESULTS: Among the 7 cases, 3 cases underwent transperitoneal approach, and 4 cases underwent retroperitoneal approach. The median pneumoperitoneum time was 168 (108-191) minutes. The median estimated blood loss was 90 (23-170) mL, and no donor required a blood transfusion. Median warm ischemia time was 4 (3-7) minutes. Length of hospital stay was 7 (6-9) days, and no readmission occurred. Median serum creatinine level of 1 month after surgery was 1.19 (0.84-1.74) mg/dL. Kidneys were transplanted successfully, and none of recipients required dialysis. CONCLUSIONS: Laparoscopic donor nephrectomy was safe for donors with an anomalous left renal vein. Preoperative recognition of anomalous left renal vein in computed tomography is important for avoiding hemorrhagic complication.

Efficacy of canagliflozin against nonalcoholic fatty liver disease: a prospective cohort study.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease worldwide and is characterized by insulin resistance, hepatic steatosis and often prediabetes or diabetes. Canagliflozin, a selective sodium glucose cotransporter 2 inhibitor, is a new oral anti-diabetic drug that reduces hyperglycaemia by promoting urinary glucose excretion. Glycosuria produced by canagliflozin is associated with weight loss, mainly due to reduced fat volume and improve insulin resistance. Reduced body weight and improvement of insulin resistance by canagliflozin may be an effective treatment for NAFLD. METHODS: Thirty-five patients with NAFLD (17 men and 18 women) were enrolled and administered canagliflozin (100 mg). Body weight and serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (γ-GTP), low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides (TG), blood sugar (BS), glycated haemoglobin (HbA1C), uric acid (UA) and ferritin, and fibrosis-4 (FIB-4) index values were measured at baseline and at 3-month and 6-month follow-up visits. RESULTS: Body weight and serum levels of AST, ALT, γ-GTP, TG, UA, HbA1C, BS and ferritin decreased significantly after 3 and 6 months of canagliflozin treatment. Serum BS levels and FIB-4 index values decreased slightly following 3 months of treatment; these results reached significance after 6 months. Reduced serum ALT levels at 6 months were significantly correlated with baseline HbA1C and ferritin levels. Moreover, a significant correlation between reduced body weight and serum ALT levels was observed at 6 months. Decreased serum ALT levels were significantly correlated with decreased serum ferritin at 6 months. CONCLUSIONS: Canagliflozin significantly reduced the serum levels of BS, HbA1C, TG, UA and ferritin, as well as FIB-4 index values and body weight, with improved liver function. Sodium glucose cotransporter 2 inhibitors may be an important therapeutic modality for improving liver injury in NAFLD patients.

Identification of Protein Targets of 12/15-Lipoxygenase-Derived Lipid Electrophiles in Mouse Peritoneal Macrophages Using Omega-Alkynyl Fatty Acid.

The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of highest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.