Organelle associations and metabolic rewiring of size-defined hepatic lipid droplets

Abstract Lipid droplet (LD) size is a key determinant of hepatic lipid metabolism, yet the molecular programming routing size-defined subpopulations to distinct fates remains unclear. Here we show that a fractionation workflow resolves hepatic LDs by size and organelle association, defining a high-confidence intrinsic LD proteome. Moreover, comparative proteomics of fasted livers reveals a clear ‘division of labor’: Large LDs (L-LDs) function as specialized mitochondrion-coupled ‘putative fueling hubs’, whereas small LDs (S-LDs) serve as lysosome-coupled ‘putative trafficking and turnover hubs’. This interaction map is highly plastic; metabolic stress (fasting or steatosis) triggers a programmatic remodeling where lysosomal machinery is inducibly recruited to S-LDs. Crucially, in vivo stress recruits mitochondria to both pools, supporting a ‘parallel programs’ model where S-LD-associated mitochondria are hypothesized to power the energy-intensive degradative machinery. These findings establish that metabolic state leverages LD size to direct organelle partnerships, nominating the small-LD lysosomal axis as a critical regulatory node in metabolic-associated fatty liver disease (MAFLD).