PROJECT SUMMARY Metabolic syndrome is a pandemic driven by poor nutrition and sedentary lifestyles that is associated with being overweight or obese. Its pathology is complex, and its comorbidities—including type 2 diabetes, cardiovascular disease, NAFLD, and cancer—are devastating. While better diets and exercise can improve prognosis, this alone typically cannot overcome the synergy of genetics, environment, and food engineering that collectively caused this epidemic. The health care and human costs of this pandemic are astronomical, and thus, innovative clinical strategies are needed. What if we could burn off excess calories when at rest, or in combination with lifestyle changes or other therapeutics? Such energy expenditure is the normal function of brown adipose tissue (BAT). Active BAT can convert large quantities of calories into heat (rather than storing them as fat)—a process called non-shivering thermogenesis. BAT is naturally stimulated by cold exposure, by certain high fat diets, and by beta-adrenergic agonists. The presence of BAT in adult humans also protects against metabolic diseases. For this reason, brown fat is often called healthy fat, and studying its biology and therapeutic strategies to stimulate it are now key focus areas of metabolic disease research. Glucose is a major brown fuel and it has been proposed that BAT could function therapeutically as a “glucose sink.” It is often assumed that BAT completely metabolizes glucose to provide energy for thermogenesis despite historical literature arguing that only a small percentage of the glucose BAT consumes is directly oxidized. This raises a fundamental unanswered question in BAT biology—what else is glucose doing? In fact, very little is known about BAT metabolic fluxes in general due to technical limitations in studying in vivo organ metabolism. Here, we combine state-of-the-art technologies in mass spectrometry (MS) coupled with in vivo stable isotope tracing and genetics to overcome previous barriers to understanding the biochemistry of BAT metabolism. In Aim 1, we take advantage of protocols we developed to quantitatively explore how glucose and other metabolites are used by BAT. We also explore how BAT metabolic “fluxes” are affected by environment, diet, and gender. In Aim 2, we explore a specific auxiliary pathway that we discovered through unbiased metabolomics to be upregulated in active BAT. Quantitatively defining the biochemistry of brown fat metabolism and its interplay with other organs is an essential step towards reaching the ultimate goal of harnessing brown fat’s calorie burning power to reverse obesity trends.