Autophagic Processes and Hepatocellular Steatosis

About This Grant

PROJECT SUMMARY/ABSTRACT Metabolic dysfunction-associated steatotic liver disease (MASLD) affects over 30% of the US population and has been identified as a leading cause of type II diabetes, hepatocellular carcinoma, and is now the most prevalent disease leading to liver transplantation. A defining feature of MASLD 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 MASLD 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 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 found that macropinocytosis (MP; “large cellular drinking”) plays a significant role in regulating hepatocellular lipid stores by forming large macropinosomes from the plasmalemma that traffic into the cell to mediate LD-lysosome fusion. We have demonstrated that important components of these essential cellular processes are large and small GTPases. These include the Ras-like Rab GTPases that control nearly all membrane-trafficking processes in the hepatocyte while the dynamin (Dyn2) family of large GTPase mechanoenzymes mediate membrane scission/fusion throughout the cell. Further, we have compelling evidence that this MiLi process occurs at the ER surface to catabolize nascent LDs as they form. Equally exciting to us is our finding that Mayo patients with MASLD possess mutant variants of these proteins that cause hepatocellular steatosis in culture. 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 Rabs (Rab8a/10) and Dyn2 GTPases, which are altered and disrupted during steatosis. The strategy of this proposal utilizes state of the art hepatocellular imaging approaches, coupled with electron microscopy, molecular methods, and membrane biochemistry. This is correlated with data gleaned from patients, and 4 distinct and novel conditional knock out mouse models we have designed. Aim 1 will define the physiological contributions of MP to hepatocellular lipid stores and steatosis by testing how MP drives MiLi via a novel protein complex of Rab 8a/10, the large GTPase Dyn2, and a new endocytic adapter (SH3D19) we have identified. Aim 2 will define the mechanisms of a novel process we have observed that is focused on lysosomal targeting and catabolism of nascent LDs at the ER as they form that utilizes the actin cytoskeleton and ER-phagy/autophagy receptors. Completion of these studies will provide valuable insights into hepatocellular lipid metabolism, the underlying basis for hepatic steatosis, and potential novel strategies for therapeutic intervention in MASLD.