Obesity, diabetes, and fatty liver diseases are at endemic proportions. Toxicological data indicates that the development of obesity is caused by more than just overeating, but also chemical exposure. Further, high-fat diet (HFD)-fed Cyp2b-null mice are obese compared to HFD-fed wildtype mice, primarily due to an increase in white adipose tissue (WAT) mass. Additionally, preliminary data demonstrates that the n-3 fatty acid, -linolenic acid (ALA) is a specific substrate for CYP2B6; producing 9-HOTre, an ALA-oxylipin at concentrations >20X more than other PUFA metabolites. ALA reduces triglyceride accumulation in HepG2 cells; however, it increases triglyceride accumulation in CYP2B6-HepG2 cells. This suggests that ALA is being metabolized to a product, probably 9-HOTre, that is signaling for increased fatty acid uptake. In addition, 13- HOTre, a secondary ALA-oxylipin, inhibits PPAR activity in white adipose tissue. We hypothesize that 9- HOTre and 13-HOTre inhibit fatty acid uptake into white adipose tissue, but is not available to do so in Cyp2b- null mice, leading to greater WAT mass and obesity. We will also test whether 9-HOTre increases fatty acid absorption and oxidation into skeletal muscle. Thus, disruption of CYP2B6 by xenobiotics could significantly alter lipid distribution and use by inhibiting the formation of 9-HOTre. In turn we have used ALA-treated HepG2 and CYP2B6-HepG2 cells to screen for chemicals that perturb CYP2B6-mediated triglyceride accumulation. While we have examined obesity in Cyp2b-null mice, we have not investigated fatty liver disease, especially NASH, a pertinent metabolic disease. Therefore, the purpose of this grant is to: (Aim 1) Test whether murine Cyp2b-members play a role in the development of NAFLD and NASH. We will use a methionine-choline deficient diet to determine if Cyp2b-null mice are more susceptible to NASH. RNAseq will be used to determine the mechanism by which Cyp2b provides protection. (Aim 2) Test whether disruption of CYP2B6-mediated metabolism of -linolenic acid (ALA) to 9-HOTre increases hepatic, white adipose tissue (WAT) and skeletal muscle fatty acid uptake or oxidation. We hypothesize that 9-HOTre increases uptake into skeletal muscle and liver and decreases uptake in WAT. In addition, we will test whether CYP2B6 inhibitors and environmental pollutants reverse the effects of ALA by inhibiting the production of 9-HOTre. (Aim 3) We humanized our Cyp2b-null mouse model human CYP2B6. In this aim, we will test whether CYP2B6 protects from toxicant- induced (PFOS-induced) NAFLD in comparison to Cyp2b-null mice. PFOS is a potent inducer of Cyp2b and NAFLD. We hypothesize the induction is protective and therefore CYP2B6 is protective. However, there is conflicting evidence surrounding Cyp2b induction and oxylipin associations with NAFLD, and it is possible that CYP2B6 increases fatty liver disease while providing protection from obesity. This will be ferreted out during the course of these studies. In summary, we propose that inhibition and repression of CYP2B6 plays a role in the NAFLD/obesity epidemic through reduced production of the newly discovered signaling molecule 9-HOTre. Obesity is at endemic proportions in the United States and worldwide, and it increases the risk of other diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), type II diabetes, hypertension, and depression. Recent data indicates that the development of NAFLD and obesity is about more than just overeating, but also chemical exposure and in turn disruption of nutrient signaling. We hypothesize that xenobiotic inhibition of CYP2B6 reduces α-linolenic acid oxylipin signaling and in turn increases NAFLD and obesity.