Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver

Cody N. Rozeveld, Bridget Mehall, Carol Casey, Mark McNiven, Schott Micah

Research output: Contribution to journalArticlepeer-review

Abstract

INTRODUCTION AND OBJECTIVE: Alcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol-related death is alcohol-associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid-degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD-lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well-known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy. MATERIALS AND METHODS: To this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high-resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD-lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat. RESULTS: First, confocal microscopy revealed that GFP-Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2-fold) and LD number (1.6-fold), and also blocked the catabolism of LDs in lipid-loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH-fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult. SIGNIFICANCE/IMPLICATION: This study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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