PROJECT SUMMARY Beyond ATP, mitochondria supply a variety of metabolites to the cytosolic compartment. Given the impermeable nature of the mitochondrial inner-membrane, mitochondrial carrier proteins play a central role in metabolite transport between the matrix and the cytosolic compartment. However, a large number of mitochondrial metabolite carriers remain uncharacterized, particularly in the context of metabolic diseases. Recently, our lab identified a liver-specific mitochondrial metabolite carrier, SLC25A47, that is required for energy homeostasis. This is an exceptionally unique carrier among the SLC25A mitochondrial carrier family (53 members known) because it is expressed selectively in hepatocytes. Our recent study found that blockade of SLC25A47 blunted pyruvate-derived hepatic gluconeogenesis under a fasted state, whereas the inhibition led to elevated whole-body energy expenditure even at thermoneutrality. This suggests an exciting opportunity to limit hepatic gluconeogenesis and elevate energy expenditure by targeting this liver-specific mitochondrial carrier. Accordingly, this application aims to determine the following emerging questions: 1) What are the underlying mechanisms of SLC25A47 action? 2) Does inducible SLC25A47 inhibition have a therapeutic effect on reversing hyperglycemia and obesity? 3) How is SLC25A47 expression regulated? Together, the successful completion of this work will offer a liver-specific target through which we can restrict hepatic gluconeogenesis, which is often in excess under hyperglycemic and diabetic conditions, while elevating energy expenditure. Broadly speaking, this work will establish a framework to understand the principle of tissue-specific mitochondrial metabolite transport in physiology and disease.