Schott, Micah B.

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.).

ABSTRACT Epithelial cells, such as liver-resident hepatocytes, rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rab proteins can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports the mechanistic target of rapamycin complex 1 (mTORC1) signaling under basal and amino acid-stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase, and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling. This article has an associated First Person interview with the first author of the paper.

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.

The liver performs numerous vital functions, including the detoxification of blood before access to the brain while simultaneously secreting and internalizing scores of proteins and lipids to maintain appropriate blood chemistry. Furthermore, the liver also synthesizes and secretes bile to enable the digestion of food. These diverse attributes are all performed by hepatocytes, the parenchymal cells of the liver. As predicted, these cells possess a remarkably well-developed and complex membrane trafficking machinery that is dedicated to moving specific cargos to their correct cellular locations. Importantly, while most epithelial cells secrete nascent proteins directionally toward a single lumen, the hepatocyte secretes both proteins and bile concomitantly at its basolateral and apical domains, respectively. In this Beyond the Cell review, we will detail these central features of the hepatocyte and highlight how membrane transport processes play a key role in healthy liver function and how they are affected by disease.

Alcohol consumption is a well‐established risk factor for the onset and progression of fatty liver disease. An estimated 90% of heavy drinkers are thought to develop significant liver steatosis. For these reasons, an increased understanding of the molecular basis for alcohol‐induced hepatic steatosis is important. It has become clear that autophagy, a catabolic process of intracellular degradation and recycling, plays a key role in hepatic lipid metabolism. We have shown that Rab7, a small guanosine triphosphatase known to regulate membrane trafficking, acts as a key orchestrator of hepatocellular lipophagy, a selective form of autophagy in which lipid droplets (LDs) are specifically targeted for turnover by the autophagic machinery. Nutrient starvation results in Rab7 activation on the surface of the LD and lysosomal compartments, resulting in the mobilization of triglycerides stored within the LDs for energy production. Here, we examine whether the steatotic effects of alcohol exposure are a result of perturbations to the Rab7‐mediated lipophagic pathway. Rats chronically fed an ethanol‐containing diet accumulated significantly higher levels of fat in their hepatocytes. Interestingly, hepatocytes isolated from these ethanol‐fed rats contained juxtanuclear lysosomes that exhibited impaired motility. These changes are similar to those we observed in Rab7‐depleted hepatocytes. Consistent with these defects in the lysosomal compartment, we observed a marked 80% reduction in Rab7 activity in cultured hepatocytes as well as a complete block in starvation‐induced Rab7 activation in primary hepatocytes isolated from chronic ethanol‐fed animals.Conclusion: A mechanism is supported whereby ethanol exposure inhibits Rab7 activity, resulting in the impaired transport, targeting, and fusion of the autophagic machinery with LDs, leading to an accumulation of hepatocellular lipids and hepatic steatosis. (Hepatology Communications2017;1:140‐152)

A novel protein complex facilitates lipid droplet autophagy.