Cancer cells depend on environmental lipids for proliferation when electron acceptors are limited.

Production of oxidized biomass, which requires regeneration of the cofactor NAD, can be a proliferation bottleneck that is influenced by environmental conditions. However, a comprehensive quantitative understanding of metabolic processes that may be affected by NAD deficiency is currently missing. Here, we show that de novo lipid biosynthesis can impose a substantial NAD consumption cost in proliferating cancer cells. When electron acceptors are limited, environmental lipids become crucial for proliferation because NAD is required to generate precursors for fatty acid biosynthesis. We find that both oxidative and even net reductive pathways for lipogenic citrate synthesis are gated by reactions that depend on NAD availability. We also show that access to acetate can relieve lipid auxotrophy by bypassing the NAD consuming reactions. Gene expression analysis demonstrates that lipid biosynthesis strongly anti-correlates with expression of hypoxia markers across tumor types. Overall, our results define a requirement for oxidative metabolism to support biosynthetic reactions and provide a mechanistic explanation for cancer cell dependence on lipid uptake in electron acceptor-limited conditions, such as hypoxia.