PROJECT SUMMARY Targeting brown adipose tissue (BAT) function to increase energy expenditure represents an attractive strategy to treat obesity and the associated type 2 diabetes. BAT and the related beige fat express uncoupling protein 1 (UCP1), a mitochondrial membrane protein that uncouples respiration from ATP synthesis and promotes thermogenesis. Paradoxically, UCP1 null mice are only obese when maintained at thermoneutrality, suggesting the existence of alternative mechanisms of thermogenesis. Consistent with this notion, several UCP1- independent ATP-consuming futile cycles have been identified in brown and beige adipocytes. However, the relative contribution of these pathways to the regulation of whole-body energy metabolism remains unclear. Our preliminary studies suggest that peroxisomes, organelles specialized for lipid metabolism, are involved in an alternative mechanism of adipose tissue thermogenesis based on peroxisomal metabolism of branched chain fatty acids (BCFA). Peroxisomes account for up to 20% of total cellular oxygen consumption. Peroxisomal respiration, unlike mitochondrial oxygen consumption, is not linked to ATP synthesis and instead generates heat. Monomethyl (mm) BCFA are synthesized via de novo lipogenesis using a precursor derived from catabolism of branched chain amino acids (BCAA). Our results reveal that cold treatment increases the gene expression of factors involved in mmBCFA synthesis and beta-oxidation in thermogenic fat. A thermogenic stimulus promotes translocation of BCFA synthetic proteins to peroxisomes, the site of BCFA beta-oxidation. Upregulation of BCFA beta oxidation raises the intracellular temperature in brown adipocytes and increases oxygen consumption rate in WT and UCP1 KO brown adipocytes. Together, these results lead us to hypothesize that peroxisomes are involved in a UCP1-independent mechanism of thermogenesis characterized by a futile process of BCFA synthesis and beta-oxidation. Since de novo synthesis of fatty acids is a highly energy-demanding process and peroxisomal beta-oxidation is not linked to ATP production, we further hypothesize that this futile cycle promotes negative energy balance, leading to protection against obesity and insulin resistance. To test this hypothesis, we propose two specific aims. The first aim will use biochemical, cell culture-based, and in vivo approaches to implicate peroxisomes in a futile process of BCFA metabolism. The second aim will use loss-of-function and gain-of-function mouse models of BCFA beta-oxidation to study its role in thermogenesis and whole-body energy metabolism.