PROJECT SUMMARY Emerging evidence suggests that brown adipose tissue (BAT) functions as a significant metabolic-sink for glucose and fatty acids, but also branched-chain amino acids (BCAA; valine, leucine, and isoleucine). Our recent study shows that cold-activated BCAA catabolism in the BAT promotes systemic BCAA clearance in mice and humans, and that this metabolic-sink action is tightly coupled with its ability to improve glucose tolerance and insulin sensitivity. The notion of BAT being a metabolic-sink for BCAA provides new insights into the epidemiological observations that increased circulating BCAA levels are associated with insulin resistance and type 2 diabetes, conditions under which BAT mass/activity is reduced. However, the mechanisms remain insufficiently understood because the gatekeeper of mitochondrial BCAA transport, i.e., mitochondrial BCAA transporter that determines BCAA fate in the mitochondria vs. cytosol, was unknown for many years. We recently identified the first mitochondrial BCAA transporter, SLC25A44, in mammals. Our preliminary data suggest that SLC25A44 is required for mitochondria BCAA oxidation, BAT thermogenesis, and systemic glucose homeostasis. Accordingly, this proposal aims to determine the mechanisms by which SLC25A44 loss causes systemic glucose intolerance and insulin resistance. First, we will determine the metabolic organ that is primarily responsible for the diabetic phenotype through characterization of the newly developed BAT-specific SLC25A44 deficient mice. Second, we will employ metabolomics and biochemical approaches to determine the molecular mechanisms by which SLC25A44 loss alters mitochondrial function and intracellular signaling pathways. Lastly, we aim to examine the regulatory mechanisms of SLC25A44 expression and function. The work resulting from this application will establish a conceptual framework to understand the regulation of intracellular BCAA fate, and also provide a new roadmap to reverse disease phenotypes that stem from dysregulation in the BCAA catabolic processes.