Aerobic glycolysis drives differentiation of unilocular adipocytes.

The hallmark of white adipocytes is a single, large lipid droplet, yet this unilocular morphology has remained challenging to reproduce in vitro due to limited understanding of its molecular drivers. Here, we identify metabolic reprogramming as the key determinant of adipocyte unilocularity in human 3D cultures. Transcriptomic and metabolomic profiling revealed unilocularity to be primarily characterized by enhanced aerobic glycolysis and reduced mitochondrial content. Importantly, this mirrored the metabolic pattern of freshly isolated human white adipocytes but contrasted with that of preadipocytes and 2D cultures. We demonstrate that aerobic glycolysis in adipocytes activates AMP-activated protein kinase (AMPK), which then enhances CD36-mediated fatty acid uptake. Pharmacological inhibition of aerobic glycolysis reduced fatty acid uptake and led to a more multilocular phenotype, which could be rescued by reactivating AMPK. Importantly, limiting mitochondrial activity or activating AMPK in multilocular 3D cultures was enough to promote their unilocularity. These findings establish aerobic glycolysis as a key driver of white adipocyte lipid droplet morphology and size. They also underscore the influence of cellular microenvironment on shaping adipocyte metabolism and function. Taken together, the study provides important insights of adipocyte lipid droplet biology that can be used to improve human adipocyte functionality and prevent disease.