Intracellular metabolic gradients dictate dependence on exogenous pyruvate

During developmental transitions, cells frequently remodel metabolic networks, including changing reliance on metabolites such as glucose and glutamine to fuel intracellular metabolic pathways. Here we used embryonic stem (ES) cells as a model system to understand how changes in intracellular metabolic networks that characterize cell state transitions affect reliance on exogenous nutrients. We find that ES cells in the naive ground state of pluripotency increase uptake and reliance on exogenous pyruvate through the monocarboxylate transporter MCT1. Naive ES cells, but not their more committed counterparts, rely on exogenous pyruvate even when other sources of pyruvate (glucose, lactate) are abundant. Pyruvate dependence in naive ES cells is a consequence of their elevated mitochondrial pyruvate consumption at the expense of cytosolic NAD⁺ regeneration. Indeed, across a range of cell types, increased mitochondrial pyruvate consumption is sufficient to drive demand for extracellular pyruvate. Accordingly, restoring cytosolic NAD⁺ regeneration allows naive ES cells to tolerate pyruvate depletion in diverse nutrient microenvironments. Together, these data demonstrate that intracellular metabolic gradients dictate uptake and reliance on exogenous pyruvate and highlight mitochondrial pyruvate metabolism as a metabolic vulnerability of naive ES cells.