Increased demand for NAD⁺ relative to ATP drives aerobic glycolysis

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is associated with proliferation across many organisms and conditions. To better understand that association, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the NAD⁺/NADH ratio. This change in NAD⁺/NADH is caused by increased mitochondrial membrane potential that impairs mitochondrial electron transport and NAD⁺ regeneration. Uncoupling respiration from ATP synthesis or increasing ATP hydrolysis restores NAD⁺/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when demand for NAD⁺ to support oxidation reactions exceeds the rate of ATP turnover in cells, NAD⁺ regeneration by mitochondrial respiration becomes constrained, promoting fermentation, despite available oxygen. This argues that cells engage in aerobic glycolysis when the demand for NAD⁺ is in excess of the demand for ATP.