Lipid droplet membrane proteome remodeling parallels ethanol-induced hepatic steatosis and its resolution.

Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol (EtOH) consumption, subsequent abstinence from EtOH, or fasting differentially affect LD membrane proteome content, and that these changes influence how LDs interacts with other intracellular organelles. Here, male Wistar rats were pair-fed liquid control or EtOH diets for 6 weeks, then randomly-chosen animals from both groups were refed either a control diet for 7 days or were fasted for 48 hr before sacrifice. From all groups, LD membrane proteins from purified liver LDs were analyzed immunochemically and by mass spectrometry-proteomics. Liver LD numbers and sizes were greater in EtOH-fed rats than in pair-fed control, 7 day refed or fasted rats. Compared with control rats, EtOH feeding markedly altered the LD membrane proteome, enriching LD structural perilipins and proteins involved in lipid biosynthesis, while lowering LD lipase levels. EtOH feeding also lowered LD-associated mitochondrial and lysosomal proteins. In seven day refed (i.e. EtOH-abstained) or fasted-EtOH-fed rats we detected distinct remodeling of the LD proteome, as judged by lower levels of lipid biosynthetic proteins, and enhanced LD interaction with mitochondria and lysosomes. Our study reveals evidence of significant remodeling of the LD membrane proteome that regulates EtOH-induced steatosis, its resolution after withdrawal and abstinence, and changes in LD interactions with other intracellular organelles.