Understanding the molecular mechanisms that contribute to dysregulated metabolism in diabetes is essential for developing effective prevention methods and discovering a cure. Over the past 10 years, substantial evidence supports the mitochondrial overload theory of overnutrition-induced metabolic dysregulation, including a specific role for dysregulated branched-chain amino acid (BCAA) metabolism. The goal here is to identify how acyl-CoA species derived from BCAA metabolism induce protein and histone modifications, and to assess how protein hyperacylation affects metabolic regulation in the setting of overnutrition. We recently discovered a class of highly reactive acyl-CoA species derived from leucine oxidation that modify enzymes involved in BCAA catabolism. We also uncovered a novel enzymatic activity of the mitochondrial sirtuin SIRT4 to remove these modifications, thereby regulating leucine catabolic flux. These discoveries define a new paradigm of protein acylation and deacylation, and identify an unexpected level of control over BCAA metabolism and nutrient homeostasis. In this project, we will build upon these findings and focus on the following Specific Aims: 1) To determine how alterations in nutrient flux lead to changes in mitochondrial protein acylation; 2) To determine the consequence of mitochondrial protein hyperacylation on BCAA enzyme function in the setting of over-nutrition; and 3) To determine how metabolites derived from nutrient metabolism are sensed and integrated into the epigenome. Together, these studies combine a comprehensive experimental design and an innovative conceptual framework in order to determine how intermediary metabolites derived from central carbon metabolism drive specific nutrient-sensing responses. Furthermore, this study will build a foundation of knowledge to further how these pathways contribute to the pathophysiology of diabetes. Ultimately, these studies will deepen our understanding of emergent, novel metabolic control mechanisms, and have the potential to inform the development of new therapies and prevention methods.