Abstract 19740: Intramuscular Lipid Accumulation is Reversed by a p62-lysosomal-independent Lipophagy Pathway
Background and Hypothesis: In the mammalian heart, autophagy is an adaptive mechanism promoting the removal of protein aggregates or damaged cell organelles. A common feature of non-ischemic heart failure is lipid accumulation. We examined whether autophagy is impaired in the muscle in chronic lipid overload and investigated whether activation of autophagy protects myocytes from lipotoxicity.
Methods: Rat L6 myocytes were incubated over a period of 6 days with or without long chain fatty acids (equimolar mixture of oleate and palmitate, 1.0mM). At day 6, an autophagic inhibitor (bafilomycin A1, 200 nM), or an autophagic activator (rapamycin, 1 μM), or both, were added for 48 hours. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining and immunofluorescence and was quantified colorimetrically. Protein markers of autophagic flux (LC3 and p62) and cell death (Caspase 3 cleavage) were measured by immunoblotting.
Results: Inhibition of autophagy using bafilomycin increased TG accumulation and also increased fatty acid-mediated cell death. Conversely, activation of autophagy using rapamycin reduced both intracellular lipid accumulation and cell death. Unexpectedly, treatment with both rapamycin and bafilomycin resulted in a decrease in lipid accumulation. Immunoblotting indicated p62 degradation (autophagic flux), while immunofluorescence revealed the colocalization of p62 with lipid droplets in these cells. These findings indicate the potential association of p62 with lipid droplet turnover, which is a novel pathway for the breakdown of lipid droplets in muscle cells. TG in the extracellular medium was also assessed. The same cells treated with rapamycin and bafilomycin had higher levels of extracellular TG compared to extracellular TG of cells with other treatments. The results suggest that the p62-mediated pathway for lipid degradation ultimately exports TG’s from the cell by exocytotic mechanisms.
Conclusions: Autophagy promotes the clearance of lipids from myocytes and switches to an alternative, p62-mediated pathway of lipophagy and exocytosis in the context of chronic lipid overload. Moreover, lipophagy promotes metabolic adaptation and myocyte survival.
Author Disclosures: T.N. Lam: None. R. Harmancey: None. H. Vasquez: None. B. Gilbert: None. N. Patel: None.
- © 2015 by American Heart Association, Inc.