Metabolic dysfunction-associated fatty liver disease and chronic kidney disease, affecting tens of millions of people in the US alone, exhibit pathological dysregulation of lipid metabolism, which is heavily dependent on the pivotal cofactor CoA. Yet, we do not fully understand how cells and tissues regulate and coordinate the biosynthesis of CoA with the demands of CoA-dependent metabolism, biosynthesis, and homeostasis (e.g. of lipids); nor how CoA synthesis dysregulation and disease progression are related in the kidney and liver. As the major carrier of activated acyl groups, CoA is required for the TCA cycle, lipid synthesis, and fatty acid oxidation among other core metabolic processes. It is synthesized from the essential nutrient vitamin B5 (pantothenate), and a family of pantothenate kinases (PANK1-3 in vertebrates) are rate-determining for the pathway. We have discovered that the highly-conserved PANK4, which has metabolite phosphatase activity, is a rate-limiting suppressor of CoA synthesis, and that PI3K-AKT signaling stimulates CoA synthesis (partially by inhibiting PANK4), thus linking insulin signaling to control of CoA supplies. Our preliminary studies demonstrate that additional regulatory mechanisms for CoA synthesis remain to be characterized. The goal of this proposal is to identify new regulatory inputs that control CoA synthesis in response to the metabolic state of cells and tissues, especially the kidney and liver which play central metabolic roles in humans. To meet this goal, we will leverage the unique and combined advantages of fly and mouse models, mass spectrometry-based metabolomics and lipidomics, metabolic flux analyses through isotope tracing, and protein biochemistry. Completion of our aims will reveal new regulatory inputs for CoA synthesis, advance our mechanistic understanding of the in vivo roles of CoA synthesis enzymes, establish novel animal models, and develop new tools in the CoA metabolism field. Ultimately, our results will provide foundational knowledge for understanding kidney and liver physiology and pathophysiology, and for potentially manipulating CoA synthesis as a new therapeutic strategy.