This proposal represents a bridge funding request (R56) to facilitate our collection of preliminary data that will strengthen a future application of an R01 submission to NIDDK. Its long-term objective has been centered on identifying key cytoskeletal components that orchestrate membrane and receptor trafficking pathways critical to liver physiology such as lipid storage and utilization. Non-alcoholic fatty liver disease (NAFLD), with underlying hepatic steatosis and inflammation, has become a prominent health issue. A defining feature of these diseases is the accumulation of unique triglyceride-rich organelles called lipid droplets (LDs). Understanding the fundamental mechanisms that regulate the hepatocellular storage, breakdown, and catabolism of LDs is essential to effectively prevent, reduce, and treat NAFLD and is the focus of this proposal. Significant evidence, based on our work and others, implicates the selective targeting and breakdown of hepatic LDs by the autophagic machinery during a process called lipophagy. We have recently identified a novel autophagic process termed “microlipophagy” (MiLi), by which lysosomes fuse, engulf, and degrade LDs directly. Our evidence indicates that MiLi is the predominant mechanism by which the hepatocyte catabolizes LDs. We also have found that macropinocytosis (MP; “large cellular drinking”) contributes significantly to hepatocellular lipid stores by a direct internalization of fatty acids. Further, MP appears to play a role in the MiLi process by forming large macropinosomes from the plasmalemma that traffic into the cell to fuse with LDs and the lysosome. We, and others, have demonstrated that important components of these essential cellular process are large (Dynamin) and small guanosine triphosphatases (Rab GTPases). From these observations, the central hypothesis of this proposal predicts that together the MP and MiLi processes play a central role in hepatocellular lipid catabolism and are both supported and regulated by the synergistic actions of specific Rab and Dynamin GTPases, which are altered and disrupted by excess lipids and the steototic condition. The strategy utilizes state of the art hepatocellular imaging approaches, coupled with electron microscopy, and membrane biochemistry, correlated with data gleaned from patients, and novel knock out mouse models. Aim 1 will define the physiological contributions of MP to hepatocellular lipid stores and steatosis by testing the hypothesis that MP drives the uptake of fatty acids, leading to de novo LD biogenesis and MiLi via Rab5, Rab10, the large GTPase Dyn2, and a new endocytic adapter (SH3D19). Aim 2 will define the lysosomal targeting and catabolism of nascent LDs by testing the hypothesis that lysosomes engulf and catabolize nascent LDs forming at the ER by novel mechanisms that utilize the actin cytoskeleton, autophagy receptors, and Rab10. Completion of these studies will provide valuable insights into hepatocellular lipid metabolism, ...