Glycolytic ATP production enables rapid mammalian cell growth

Proliferating cells must produce ATP rapidly enough to meet the energy demands of growth and maintenance. While microbes show a linear coupling between ATP production rate and growth, whether this principle holds in mammalian cells has remained unclear and it has been suggested that most ATP is allocated to cell maintenance, regardless of growth rate. Here, we quantified lactate production, oxygen consumption, and proliferation across twelve mammalian cell lines and found a strong linear relationship between total ATP production and growth with the majority of ATP allocated to macromolecular synthesis. By inhibiting glycolysis, inhibiting respiration, or reducing translation, cells shift along this ATP-growth line in predictable directions, indicating bidirectional coupling between ATP supply and demand. A genetically encoded ATP hydrolysis sink increased ATP turnover yet slowed proliferation, demonstrating that ATP production capacity can limit growth. Together, these results show that respiration alone cannot generate enough ATP to support the growth rates of rapidly dividing cells, whereas glycolysis can. Our results provide a quantitative rationale for the Warburg Effect, where cells rely on glycolysis to achieve doubling times faster than 30 hours. Our results establish ATP production rate as a quantitative constraint on growth across species.