Project Summary Obesity is a growing global health burden that strongly associates with several comorbidities including non- alcoholic fatty liver disease, hypertriglyceridemia, insulin resistance, and type 2 diabetes (T2D), reducing life expectancy and quality. Fish oil is an important dietary component that provides essential omega-3 fatty acids (Ω-3 FA) and is effective reducing severe hypertriglyceridemia. To better understand fish oil metabolites and their effects, a metabolomics approach on urine and serum of healthy volunteers taking Lovaza, a clinical grade FDA-approved omega-3 formulation, was performed. The furan CMPF was the major metabolite, followed by several other furans and their glucuronides. To evaluate its effects, mice fed a high-fat diet were administered CMPF to achieve levels equivalent to the ones observed in humans treated with Lovaza. CMPF lowered plasma and liver triglycerides and reduced hepatic steatosis, recapitulating the effects reported both in animals and in humans for Lovaza and fish oil treatments. The presence of furan fatty acids was confirmed in both fish oil and Lovaza. Moreover, we found furan fatty acid to bind and inhibit hepatic acetyl-CoA carboxylase, providing a mechanism of action. Based on these highly provocative preliminary results and current understanding, we hypothesize that fish oil furans are significant bioactive compounds responsible for fish oil-induced changes in hepatic lipid metabolism through acute modulation of metabolic pathways, in particular, inhibition of acetyl-CoA carboxylase. This hypothesis will be tested by pursuing the following Specific Aims Aim 1: Define FuFA levels in Lovaza and FO, dose, exposure and pharmacokinetics. Aim 2: Establish the role of FuFA on Lovaza-dependent TG reduction and NAFLD protection. Aim 3: Define hepatic protein targets, metabolic effects and role of ACC on FuFA protective effects. The development of mass spectrometry-based strategies to identify and characterize furans, application of thermal shift assay to determine binding partners, synthetic strategies, lipidomic studies and metabolomic assessments support a solid approach to definitively identify and characterize fish oil-derived bioactive furans. Synthetic furan fatty acids will be tested in a rat model of diet-induced obesity and evaluated in the context of Ω-3 FA. A successful completion of this proposal will have defined the participation and role of furans in the beneficial effects described for fish oil. These are potent dietary ACC inhibitors present in fish oil at pharmacologically relevant concentrations and are qualified impurities with a safe toxicological profile. This proposal is designated to fill critical gaps in knowledge in Ω-3 FA therapeutics and can potentially re- define current paradigms related to the impact of Ω-3 FA supplementation on lipogenesis, lipolysis, and glycolysis.