ABSTRACT This project aims to understand of how mitochondrial carbon trafficking and bioenergetics are regulated by pyruvate dehydrogenase kinase 4 (PDK4), a protein that is highly responsive to nutrient and energetic cues and one that has received much attention as a potential therapeutic target. PDK4 is a member of a family of pyruvate dehydrogenase kinase enzymes (PDK1-4) that phosphorylate and inactive the mitochondrial pyruvate dehydrogenase complex (PDC). By converting pyruvate to acetyl-CoA, the PDC connects glycolysis to the tricarboxylic acid cycle (TCAC), which generates reducing equivalents needed for ATP synthesis. Notably, PDK4 is one of the most robustly induced genes/proteins in response to acute energy stresses–such as fasting, exercise and consumption of a high fat meal. PDK4 is also strongly induced by acute exposure to fatty acids and/or other ligands that activate the PPAR family of transcription factors. This remarkable level of nutrient/energy-induced regulation is unique to PDK4 (as compared with PDKs1-3), raising the possibility that PDK4 has distinct metabolic functions. The major conceptual innovation and central premise of this proposal stems from new and exciting evidence from our laboratory that PDK4 phosphorylates and regulates proteins beyond the PDC. Preliminary studies used mass spectrometry-based proteomics to assess the phospho- proteome of hearts and/or skeletal muscles from mice in which the PDK4 gene was overexpressed or ablated. In aggregate, the findings support a working model wherein PDK4 phosphorylates and regulates multiple mitochondrial enzymes and proteins in response to lipid stress. Accordingly, the project seeks to test hypothesis that PDK4 plays a central role in mediating lipid-induced phosphorylation of mitochondrial proteins beyond the PDC, which in turn modulates carbon trafficking and bioenergetics in manner that confers metabolic resilience. To test this hypothesis, we will combine gain- and loss-of-function mouse models with several state-of-the-art molecular profiling tools (mass spectrometry-based proteomics, phospho-proteomics, metabolomics and stable isotope metabolic flux analysis), a sophisticated mitochondrial diagnostics platform, and comprehensive physiological assessments to delineate the PDK4 interactome and its critical physiological functions.

RELEVANCE STATEMENT Mitochondria are tiny engines that generate the chemical power that cells use to grow and function. This project aims to understand how an enzyme known as PDK4 helps mitochondria to maintain high rates of energy production in during normal stresses, such as food restriction and exercise. These studies will delineate the precise role of PDK4 in regulating fuel selection and use in tissues such as muscle and heart. The results could provide new approaches to treating metabolic disorders such as diabetes and heart disease.