Akt regulation of glycolysis mediates bioenergetic stability in epithelial cells

Cells use multiple feedback controls to regulate metabolism in response to nutrient and signaling inputs. However, feedback creates the potential for unstable network responses. We examined how concentrations of key metabolites and signaling pathways interact to maintain homeostasis in proliferating human cells, using fluorescent reporters for AMPK activity, Akt activity, and cytosolic NADH/NAD+ redox. Across various conditions including metabolic (glycolytic or mitochondrial) inhibition or cell proliferation, we observed distinct patterns of AMPK activity, including stable adaptation and highly dynamic behaviors such as periodic oscillations and irregular fluctuations, indicating a failure to reach a steady state. Fluctuations in AMPK activity, Akt activity, and cytosolic NADH/NAD+redox state were temporally linked in individual cells adapting to metabolic perturbations. By monitoring single-cell dynamics in each of these contexts, we identified PI3K/Akt regulation of glycolysis as a multifaceted modulator of single-cell metabolic dynamics that is required to maintain metabolic stability in proliferating cells.