Obesity, a major risk factor for multiple diseases including diabetes and cardiovascular disease, has reached pandemic level and is still increasing alarmingly in the US and worldwide. Despite the recent breakthrough with GLP-1 based anti-obesity drugs, the causes of obesity remain unknown. To develop effective preventive interventions, the causal factors and physiological mechanisms of obesity need to be better understood. One known determinant is diet composition, but how it determines body weight remains unclear. Our rationale is that to ultimately understand the problem we first need to better understand how diet composition affects metabolic processes in energy metabolism, including those in energy expenditure and energy excretion where our preliminary data revealed major knowledge gaps. Determining how metabolic processes function requires quantifying metabolic fluxes, the rate at which nutrients and metabolites are produced or consumed. This in turn requires a complex interdisciplinary approach and, as a result, has only been done for limited number of cases. The long-term goal of this research team is to determine the physiological mechanisms of body-weight dependence on diet composition. Toward this goal, this proposal will leverage their fluxomics expertise to determine the effects of dietary fat and carbohydrate content on fundamental processes of energy metabolism. Their in vivo flux quantification approach integrates isotope tracer infusion into animals, mass spectrometry and stable isotope gas measurements, and mathematical modeling. They will quantify the fluxes of metabolic processes that lie within two main areas of energy metabolism that have been shown to be dependent on dietary fat and carbohydrate composition: energy expenditure and energy excretion. Addressing these areas, the specific aims of the project are 1) to determine the dietary dependence of tissue fuel selection, 2) to determine the dietary dependence of systemic metabolic cycling, and 3) to determine the dietary dependence of fecal nutrient content. The proposed study is innovative because it will generate previously unavailable metabolic flux information on the effects of dietary fat and carbohydrate composition on important energy metabolic processes. The proposed research is significant because it will reveal the specific metabolic processes that underly the changes in energy metabolism on different diets. The results will move us forward to understanding the physiological mechanisms of how diet composition affects body weight, and ultimately contribute to the development of effective and sustained therapeutical and preventive strategies for obesity.