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Poster Abstract Presentations
Session Title: Poster Session 3

Abstract 322: Reversal of Intramuscular Lipotoxicity is Mediated by a Novel p62-lipophagy Pathway

Originally publishedhttps://doi.org/10.1161/res.117.suppl_1.322Circulation Research. 2015;117:A322

    Background and Hypothesis: In the mammalian heart, autophagy is considered 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 hypothesize that autophagy is impaired in the muscle in chronic lipid overload. Therefore, we investigated whether activation of autophagy protects myocytes from lipotoxicity.

    Methods: We incubated rat L6 myocytes 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. Following the pharmacologic treatments, glucose uptake was measured using [3H] 2-Deoxy-D-glucose, and insulin sensitivity was assessed. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining and immunofluorescence and was quantified enzymatically. 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. In the same cells, rapamycin treatment increased glucose uptake, in response to insulin (100mM).

    Conclusions: Autophagy promotes the clearance of lipids from myocytes, improves insulin sensitivity, and switches to an alternative p62 mediated pathway of lipophagy in the context of chronic lipid overload. Moreover, lipophagy promotes metabolic adaptation and myocyte survival.

    Footnotes

    Author Disclosures: T.N. Lam: None. R. Harmancey: None. H. Vasquez: None. N. Patel: None. B. Gilbert: None. H. Taegtmeyer: None.

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