Sequential lipolysis and lipophagy pathways orchestrate lipid droplet breakdown in hepatocytes
Authors: Schott Micah B. , Weller Shaun G. , Schulze Ryan J. , McNiven Mark A. 
Published: June 2021

Neutral lipids are a critical source for cellular energy and are stored within specialized organelles known as lipid droplets (LD). Degradation of LDs for energy utilization is accomplished by two principal pathways: lipolysis that is activated by cAMP to recruit adipose triglyceride lipase (ATGL) to the LD surface, and lipophagy that utilizes the autophagic machinery to target and traffic LDs for degradation by lysosomal acid lipase (LAL). However, the mechanisms underlying synergy between these seemingly distinct pathways are not well understood. Previous studies show that autpohagic membrane cannot effectively engulf large organelles, but is size‐restricted to small cargo. Based on this, we HYPOTHESIZE that lipolysis and lipophagy are sequential pathways whereby lipolysis first reduces the size of large LDs to allow for subsequent autophagic engulfment of small LDs for lipophagy.MethodsUsing light and electron microscopy combined with biochemical approaches, we explored LD size and the sequential nature of lipolysis and lipophagy in primary rat and AML12 hepatocytes. Live‐cell microscopy was used for the first time to monitor LDs transitioning from lipolysis in the cytoplasm to lipophagy within endolysosomal vesicles.ResultsConfocal microscopy of endogenous ATGL revealed a preferential association with large LDs with an average size of 4.0 μm2, seven‐fold larger than LDs not associated with ATGL. In striking comparison, LDs associated with components of the autophagic machinery (LC3, Lamp3) were 50% smaller in size (0.39 μm2). Consistent with this observation, siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm2), whereas knockdown of LAL left cells filled with numerous small LDs (1.5 μm2). These observations were mimicked by inhibitors of ATGL (atglistatin) and LAL (lalistat). In support of these findings, differential centrifugation of AML12 homogenates allowed for separation of two distinct populations of LDs: a buoyant fraction of large LDs enriched in the cytosolic lipase ATGL, and a second collection of smaller, less buoyant LDs enriched in lysosomal enzymes such as Lamp2A, the autophagosome marker LC3‐II, and the endosomal marker Tsg101. Most interesting are time lapse movies of live cells showing engulfment of small LDs by a Lamp3‐positive late endosomal compartment, suggesting that these organelles play an important role in the direct targeting and engulfment of LDs for lipophagy.ConclusionHepatocytes appear to catabolize LDs in a tandem stepwise process that first utilizes cytosolic lipases to substantially reduce LD size, thereby allowing for subsequent engulfment of small LDs by autophagosomes as well as late endosomes and lysosomes.Support or Funding InformationSupported by NIAAA RO1AA020735 to MAM; NIDDK award T32DK007352 to MBS and RJS, and the Mayo Clinic Kogod Aging Fellowship to MBS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.