Weller, Shaun G.

The liver stores substantial numbers of neutral lipid organelles termed lipid droplets (LDs) that accumulate within hepatocytes in response to chronic ethanol (EtOH) consumption leading to hepatic steatosis. Mass spectrometry analysis of LDs isolated from EtOH-damaged rat livers revealed a substantial reduction in the valosin-containing protein ATPase (VCP/p97) that acts to remove targeted proteins from cellular membranes for degradation. Experimental disruption of VCP function resulted in an increase in LD content in hepatocytes and mouse livers along with a marked increase in LD-associated hydroxysteroid dehydrogenase (HSD17β13) known to contribute to hepatic steatosis. Surprisingly, treatment of hepatocytes with the proteasome inhibitor MG132 had no effect on HSD17β13 levels, while a disruption of lysosome function and chaperone-mediated autophagy increased cellular HSD17β13 levels substantially. These findings provide new insights into the cellular mechanisms by which the liver regulates its lipid stores and how this is disrupted by chronic EtOH exposure.

Background:Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5.Methods and Results:Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH.Conclusions:These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a “docking” site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.

Steatotic hepatocytes accumulate profound numbers of large neutral lipid‐storage organelles called lipid droplets (LDs). The process by which the formation or utilization of these organelles are perturbed during NAFLD and NASH is poorly defined. EtOH consumption can also exacerbate these conditions markedly by yet unknown mechanisms. Thus, there is a critical need to understand lipolytic mechanisms for LD catabolism in hepatocytes. In adipocytes, catecholamines induce lipolysis through a β‐adrenergic/cAMP pathway that activates cytosolic lipases ATGL and HSL to catabolize LD triglycerides. Importantly, β‐adrenergic induction of LD catabolism has not been demonstrated in hepatocytes. Thus, the GOAL of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP‐mediated lipolysis in response to β‐adrenergic (β‐AR) stimulation. First, treatment of primary rat hepatocytes or Hep3B human hepatoma cells with the β‐AR agonist isoproterenol caused a 20–40% reduction in total cellular LD content within 24 hours. β‐AR induced LD loss was mimicked by the cAMP agonist forskolin/IBMX and was abrogated by the cAMP‐dependent protein kinase (PKA) inhibitor H89. In correlation, PKA activation following β‐AR stimulation was confirmed by FRET biosensors and biochemical analysis. β‐AR stimulation directly activated the lipolytic pathway, as evidenced by cAMP/PKA‐mediated phosphorylation of hormone sensitive lipase (HSL) and adipose triglyceide lipase (ATGL). In addition, pharmacological inhibitors against ATGL or HSL each blocked β‐AR induced LD loss, suggesting that lipase activation is critical for this process in stimulated cells. To examine whether β‐AR/cAMP stimulation triggered lipase recruitment directly to the LD, live‐cell imaging of Hep3B cells expressing either ATGL‐EGFP or HSL‐sfGFP showed that within 60 min of stimulation, each lipase was significantly displaced from the cytosol and recruited to the LD surface. This recruitment was blocked by the PKA inhibitor H89. Finally, to test if EtOH exposure attenuates the activation of lipolysis in hepatocytes, we isolated primary hepatocytes from rats fed a 6‐week ethanol‐containing Lieber‐DeCarli diet. Compared with controls, ethanol‐exposed hepatocytes showed a drastic inhibition in lipase activation and LD breakdown following β‐AR/cAMP stimulation. Remarkably, in ethanol‐metabolizing VA13 hepatocytes, ethanol insult completely prevented isoproterenol‐induced translocation of ATGL‐EGFP to the LD. In summary , these results show that β‐adrenergic stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic ethanol insult leading to fatty liver. Manipulation of this regulatory cascade could be useful in developing new therapies to attenuate fatty liver disease. Support or Funding Information This study was generously supported by funding from NIAAA 5R01AA020735 (MAM and CAC) and NIDDK 5R37DK044650 (MAM), T32DK007352‐36 (MBS), and P30DK084567 (Mayo Clinic Center for Cell Signaling in Gastroenterology).

ABSTRACT Lipophagy is a central cellular process for providing the cell with a readily utilized, high energy source of neutral lipids. Since its discovery over a decade ago, we are just starting to understand the molecular components that drive lipophagy, how it is activated in response to nutrient availability, and its potential as a therapeutic target in disease. In this Cell Science at a Glance article and the accompanying poster, we first provide a brief overview of the different structural and enzymatic proteins that comprise the lipid droplet (LD) proteome and reside within the limiting phospholipid monolayer of this complex organelle. We then highlight key players in the catabolic breakdown of LDs during the functionally linked lipolysis and lipophagy processes. Finally, we discuss what is currently known about macro- and micro-lipophagy based on findings in yeast, mammalian and other model systems, and how impairment of these important functions can lead to disease states.

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. Methods Using 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. Results Confocal microscopy of endogenous ATGL revealed a preferential association with large LDs with an average size of 4.0 μm 2 , 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 μm 2 ). Consistent with this observation, siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm 2 ), whereas knockdown of LAL left cells filled with numerous small LDs (1.5 μm 2 ). 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. Conclusion Hepatocytes 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 Information Supported 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 .

Fatty liver disease affects 80–100 million Americans and is a precursor to irreversible and sometimes fatal liver damage. The initial stages of fatty liver disease are marked by the hepatocellular accumulation of fat‐storage organelles known as lipid droplets (LDs) that are degraded by two pathways: lipolysis that involves the recruitment of cytosolic lipases (ATGL, HSL) to the LD surface, and lipophagy that relies on autophagic membranes to target and traffic LDs for lysosomal degradation. Importantly, the mechanisms underlying a cooperation between these seemingly distinct pathways are not well understood. Previous studies have shown that the autophagic engulfment of cytosolic cargo is size‐restricted. Because of this, we HYPOTHESIZE that lipolysis and lipophagy occur as sequential processes initiated by cytosolic lipases to reduce LD size and allow for subsequent engulfment by autophagic membranes. Methods In AML12 hepatocytes, immunofluorescence microscopy and biochemical approaches were used to determine the enrichment of lipolysis vs lipophagy machinery on large vs. small LDs. Live‐cell microscopy was used for the first time to monitor LDs transitioning from lipolysis to lipophagic engulfment. Results Confocal microscopy of AML12 cells immunolabeled for adipose triglyceride lipase (ATGL) revealed a preferential association with large LDs with an average area of 4.0 μm 2 , seven‐fold larger than LDs not associated with ATGL. In striking comparison, LDs associated with components of the autophagic machinery (LC3) were two‐fold smaller in area (0.39 μm 2 ). Consistent with this observation was the finding that siRNA knockdown of ATGL resulted in the persistence of large LDs (2.8 μm 2 ), whereas knockdown of lysosomal acid lipase (LAL) left cells filled with many LDs of a smaller size (1.5 μm 2 ). These observations were mimicked by inhibitors of ATGL (atglistatin) and lysosomal acidification (chloroquine). 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 and the autophagosome marker LC3‐II. Most interesting are time lapse movies of live cells showing engulfment of small LDs by a LAMP3‐positive late endosomal/lysosomal compartment, suggesting that these organelles play an important role in the direct targeting and engulfment of LDs for lipophagy. Conclusion Hepatocytes appear to catabolize LDs in a tandem stepwise process that first utilizes cytosolic lipases to substantially reduce LD size, thereby allowing for subsequent engulfment by autophagosomes as well as late endosomes and lysosomes. Support or Funding Information This study was generously supported by funding from NIAAA R01AA020735, NIDDK T32DK007352, and the Mayo Clinic Robert and Arlene Kogod Center on Aging. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Significance Lipid droplets (LDs) are specialized fat-storage organelles that can be used as a fuel source by many types of cells when nutrients are scarce. One mechanism used by hepatocytes (the functional cells of the liver) for the catabolism of these energy reserves is the lysosome-directed process of autophagy. Traditionally, autophagy necessitates the enclosure of cargo within a double-membrane autophagosome before delivery to the lysosome for degradation. Here, we use live-cell and electron microscopy to demonstrate that stable contacts between LDs and lysosomes in hepatocytes can result in the transfer of both proteins and lipids from LDs directly into the lysosome in the absence of an autophagosomal intermediate. These findings reveal a mechanism used for lipid homeostasis in the liver.

Lipid droplets (LDs) are fat‐storage organelles that play critical roles in energy homeostasis, but can excessively accumulate in metabolic diseases such as obesity and fatty liver where hepatocytes become grossly engorged with excess lipid. The catabolism of LDs aids in disease resolution and occurs in part by lipophagy, a form of selective autophagy, but the molecular and vesicle trafficking events that facilitate this process are not fully understood. During lipophagy, autophagosomes are thought to target LDs prior to fusion with acidic lysosomes, where neutral lipids are terminally degraded by lysosomal acid lipase. Ultrastructural viewing of hepatocytes showing LDs contained within electron‐dense acidic vesicles is consistent with this concept. In addition to canonical autophagy, we find that most LDs undergoing degradation are encased within single‐membrane endo‐lysosomes, and few LDs are observed within traditional double‐membrane autophagosomes. Further, studies of LD catabolism in yeast have shown that LDs can be directly internalized by the large acidic vacuole through a process known as microlipophagy. Thus, the GOAL of this study was to test if similar microlipophagy mechanisms occur in mammalian cells such as hepatocytes, and whether this process involves autophagy. METHODS To test this, we used microscopic fatty acid tracing studies and time‐lapse confocal microscopy to determine the role of autophagosomes versus endo‐lysosomes in LD catabolism. To trace the fate of LDs into terminal degradative vesicles, cells were first loaded with the fluorescent lipid marker Bodipy C12, then treated for 24h with LAListat, an inhibitor of lysosomal acid lipase. RESULTS We observed a ~5‐fold higher incidence of LDs within endo‐lysosomes (marked with the tetraspanin CD63), compared with autophagosomes (marked by the autophagy protein LC3). Furthermore, siRNA knockdown of macroautophagy proteins (Atg5, Atg7, and Fip200) did not inhibit LD internalization by endo‐lysosomes, suggesting that autophagy is not a prerequisite for this process. In addition, time‐lapse microscopy revealed a direct internalization of cytosolic LDs by endo‐lysosomes positive for lysotracker or CD63‐mCherry. CONCLUSION These findings suggest that hepatocytes utilize a distinct form of microautophagy that entails the direct envelopment of cytoplasmic LDs by endocytic vesicles such as multivesicular bodies and lysosomes/late endosomes. In addition to lipolysis and canonical macroautophagy, this process plays an important role in the catabolic utilization of LDs by the hepatocyte. Support or Funding Information This study was supported by NIH R01DK044650 (to M.A.M.), RO1AA020735 (to M.A.M.), and the K99AA020735 (to M.B.S.).

Background and Aims Hepatocytes play a central role in storage and utilization of fat by the liver. Selective breakdown of lipid droplets (LDs) by autophagy (also called lipophagy) is a key process utilized to catabolize these lipids as an energy source. How the autophagic machinery is selectively targeted to LDs, where it mediates membrane engulfment and subsequent degradation, is unclear. Recently, we have reported that two distinct GTPases, the mechanoenzyme, dynamin2 (Dyn2), and the small regulatory Rab GTPase, Rab10, work independently at distinct steps of lipophagy in hepatocytes. Approach and Results In an attempt to understand how these proteins are regulated and recruited to autophagic organelles, we performed a nonbiased biochemical screen for Dyn2‐binding partners and found that Dyn2 actually binds Rab10 directly through a defined effector domain of Rab10 and the middle domain of Dyn2. These two GTPases can be observed to interact transiently on membrane tubules in hepatoma cells and along LD‐centric autophagic membranes. Most important, we found that a targeted disruption of this interaction leads to an inability of cells to trim tubulated cytoplasmic membranes, some of which extend from lipophagic organelles, resulting in LD accumulation. Conclusions This study identifies a functional, and direct, interaction between Dyn2 and a regulatory Rab GTPase that may play an important role in hepatocellular metabolism.

Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.