Reversal of intramyocellular lipid accumulation by lipophagy and a p62-mediated pathway

Abstract

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).

Therapeutic targeting of PFKFB3 with a novel glycolytic inhibitor PFK158 promotes lipophagy and chemosensitivity in gynecologic cancers

“…The reciprocal relationship between LDs and autophagy was first described by Singh et al . where they showed that the presence of exogenous lipids leads to inhibition of autophagy and accumulation of triglycerides promoting lipotoxic‐mediated cell death . This is in contrast to what we observed in our study.…”

“…Another report highlighted the role of ATGL‐mediated LD catabolism in the liver that involved SIRT1 as the primary mechanism for lipophagy and fatty acid oxidation . Pertinent to our observation, a recent study highlighted the role of p62/SQSTM1 in promoting lipophagy by binding to ADRP on the LD . In our study, we showed that p62/SQSTM1 interacts with cPLA2 and LC3B on LDs.…”

Classical and alternative roles for autophagy in lipid metabolism

“…A separate study reveals rapamycin-driven autophagy promotes localization of p62 on lipid droplets via interaction with perilipin 2 (PLIN2), a lipid droplet membrane protein. The recruitment of p62 to lipid droplets decreases lipid accumulation in myocytes efficiently, suggesting that p62 may direct autophagosomes to the lipid droplets and accelerate lipid turnover by lipophagy [18]. Interestingly, mutations in the protein huntingtin which associates with organelle membranes lead to accumulation of dysfunctional autophagosomes as well as cytoplasmic lipid droplets in multiple cell types [19].…”

Exercise-Induced Autophagy in Fatty Liver Disease

“…As the liver is a primary tissue controlling both gluconeogenesis and ketogenesis and starvation is a powerful autophagy inducer in the liver and muscle as well, it is plausible to speculate that muscle autophagy could directly impact the liver or vice versa . Although defective autophagy in the muscle causes accumulation of intramyocellular triglycerides and enhanced autophagy facilitates removal of lipids from muscle cells (28), Takagi et al recently demonstrated in tissue-specific ATG5 knockout mice that the mice lacking ATG5 in both the liver and muscle indeed exhibited the improvement of metabolic profile, as compared to liver-specific knockout counterparts, suggesting that autophagy in the skeletal muscle may be metabolically distinct from that in the liver (46). In an independent study, skeletal muscle–specific ATG7 knockout mice also showed lower lipid accumulation and higher expression of β-oxidation–related genes, as compared to control mice (26).…”