Reversal of intramyocellular lipid accumulation by lipophagy and a p62-mediated pathway.
We have previously observed the reversal of lipid droplet deposition in skeletal muscle of morbidly obese patients following bariatric surgery. We now investigated whether activation of autophagy is the mechanism underlying this observation. For this purpose, we incubated rat L6 myocytes over a period of 6 days with long-chain fatty acids (an equimolar, 1.0 mM, mixture of oleate and palmitate in the incubation medium). At day 6, the autophagic inhibitor (bafilomycin A1, 200 nM) and the autophagic activator (rapamycin, 1 μM) were added separately or in combination for 48 h. Intracellular triglyceride (TG) accumulation was visualized and quantified colorimetrically. Protein markers of autophagic flux (LC3 and p62) and cell death (caspase-3 cleavage) were measured by immunoblotting. Inhibition of autophagy by bafilomycin increased TG accumulation and also increased lipid-mediated cell death. Conversely, activation of autophagy by rapamycin reduced both intracellular lipid accumulation and cell death. Unexpectedly, treatment with both drugs added simultaneously resulted in decreased lipid accumulation. In this treatment group, immunoblotting revealed p62 degradation (autophagic flux), immunofluorescence revealed the colocalization of p62 with lipid droplets, and co-immunoprecipitation confirmed the interaction of p62 with ADRP (adipose differentiation-related protein), a lipid droplet membrane protein. Thus the association of p62 with lipid droplet turnover suggests a novel pathway for the breakdown of lipid droplets in muscle cells. In addition, treatment with rapamycin and bafilomycin together also suggested the export of TG into the extracellular space. We conclude that lipophagy promotes the clearance of lipids from myocytes and switches to an alternative, p62-mediated, lysosomal-independent pathway in the context of chronic lipid overload (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
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T
Lam
University of Mississippi School of Medicine , Jackson, MS, USA.
R
R
Harmancey
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
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H
Vasquez
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
B
B
Gilbert
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
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N
Patel
Davidson College , Davidson, NC, USA.
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V
Hariharan
Keck School of Medicine of USC , Los Angeles, CA, USA.
A
A
Lee
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
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M
Covey
Internal Medicine/Cardiology, McGovern Medical School at The University of Texas Health Science Center at Houston, TX, USA.
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Taegtmeyer