Obesity leads to elevated plasma free fatty acid (FFA) concentration and excessive supply of FFA to the liver and skeletal muscle. The resulting tissue lipid accumulation increases risk for insulin resistance, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes. By directly addressing the problem of excessive lipid uptake in the liver and muscle, interventions that reduce FFA supply could potentially treat these comorbidities of obesity. Albumin is the primary protein in circulation that facilitates the transport of FFA from adipose to other tissues. In evaluating potential targets to prevent insulin resistance and lipid accumulation in metabolic tissues, preliminary experiments demonstrated that albumin knockout mice exhibit reduced plasma FFA, reduced hepatic lipids, and improved insulin sensitivity. While targeting lipolysis to reduce plasma FFA has encountered significant pitfalls in pre-clinical and clinical studies, albumin is a promising therapeutic target. While albumin is a common protein in the body, partial inhibition of FFA-binding to albumin is still expected to be well-tolerated, as even the complete absence of albumin in adults with congenital analbuminemia is typically associated with only mild or complete absence of symptoms. Furthermore, initial studies indicate that the FFA-albumin interaction can be modulated by endogenous metabolites, supporting the notion that FFA- albumin binding can be targeted with small molecule inhibitors. However, critical knowledge gaps remain that must be addressed to develop albumin as a therapeutic target for metabolic disease. It is not yet established if reducing plasma FFA by reducing its binding to albumin can prevent or treat the syndrome of metabolic dysregulation in obesity. It is hypothesized that reducing the abundance of albumin-associated FFA in plasma will decrease the degree of NAFLD pathology, intramuscular lipid, and insulin resistance in obese mice. If the approach is effective, specific inhibitors of FFA-albumin interaction may be beneficial in the future. In Aim-1, the effects of reduced plasma FFA will be studied in obese mouse models through assessment of insulin sensitivity, as well as molecular and biochemical responses in adipose tissue, liver, and muscle. Reduced plasma FFA will be achieved through albumin gene knockout in mice. To provide context for the novel findings, results will be compared to a mouse model that exhibits reduced plasma FFA resulting from blunted lipolysis (adipose tissue-specific knockout of adipose triglyceride lipase). In Aim-2 the same mouse lines will be studied to assess glycemic regulation during fasting and exercise, to assess tolerance to metabolic stressors. In Aim- 3, endogenous small molecules that are potential inhibitors of FFA-albumin binding will be studied using in vitro biochemical assays and molecular docking assessment. This elucidation of the impact of small molecules upon FFA-albumin binding will pave the way for future t...