Abstract 1477 Adaptations in mitochondrial metabolism render lipoate dispensable for human cell proliferation in hypoxia

Lipoate is a conserved mitochondrial cofactor essential for the activity of several central metabolic enzymes including pyruvate dehydrogenase, ɑ-ketoglutarate dehydrogenase, the glycine cleavage system, and multiple amino acid dehydrogenase complexes. Mutations in the lipoate producing enzyme lipoic acid synthetase (LIAS) have been linked clinically to a pediatric form of mitochondria dysfunction. Despite the fundamental role this cofactor plays in mediating nutrient consumption and energy production in mitochondria, little is known about whether and how human cells adapt to the depletion of lipoate in different environmental conditions. Here we describe the unexpected finding that lipoate is completely dispensable for cell proliferation and fitness under low oxygen tensions. We model lipoate depletion by knocking out LIAS in human HepG2 cells. Using a combination of cell culture and quantitative proteomics, we demonstrate that loss of LIAS adversely affects cell proliferation, activates the integrated stress response, and profoundly alters the cellular proteome in standard culture conditions (21% O2). Surprisingly, all these phenotypes are reversible simply by culturing LIAS knockout (KO) cells in hypoxia (1% O2). We further explored the mechanistic basis of this hypoxic rescue using a combination of immunoblotting, 13C-glucose and glutamine tracing, oxygen consumption rate analysis, and glucose uptake and lactate release measurements. Together, these techniques reveal that hypoxia rewires cellular metabolism to allow LIAS KO cells to proliferate and thrive without restoring lipoate levels. This discovery enhances our understanding of mitochondrial metabolic adaptations to altered environments with direct therapeutic implications for patients suffering from lipoate deficiency-based diseases. This work has been supported by the Friedriech's Ataxia Research Alliance (FARA) and the National Science Foundation Graduate Research Fellowship Program. Vamsi K. Mootha is an investigator of the Howard Hughes Medical Institute.