Single-Cell Metabolic Imaging Reveals Glycogen Driven-Adaptations in Endothelial Cells

Endothelial dysfunction (ED) is a defining feature of diabetes mellitus (DM) and a key contributor to many metabolic and cardiovascular diseases. Endothelial cells (ECs) are known to be highly glycolytic and primarily rely on glucose to meet their energy demands. However, the role of glycogen metabolism in ECs remains poorly characterized due to a lack of suitable tools. Here, we utilize stimulated Raman scattering (SRS) microscopy to investigate subcellular glycogen metabolism in live ECs under stress conditions associated with highly prevalent diabetes and diabetic complications. We demonstrate that ECs exposed to a diabetes-mimicking milieu- high glucose and tumor necrosis factor (TNF-)- divert excess glucose toward subcellular glycogen storage, and that this storage capacity is significantly enhanced by the inhibition of glycogen synthase kinase 3 (GSK3). Pulse-chase experiments uncover glycogen dynamics and reveal that glycogen is rapidly mobilized under glucose starvation, highlighting its role as an immediate energy reserve in ECs. We further extend the capabilities of SRS metabolic imaging to visualize glutamine and lactate metabolism for the first time, directly showcasing the reliance of ECs on these alternative carbon substrates during glucose deprivation. Our results indicate that ECs containing glycogen exhibit a reduced immediate metabolic demand for these gluconeogenic substrates in the absence of extracellular glucose. These findings suggest that glycogen may play a broader role beyond energy reserves in ECs by modulating stress-responsive metabolic adaptations and may offer potential therapeutic opportunities to address diabetes-induced ED and related cardiometabolic diseases.