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).

Figure 8

Modulation of autophagy and experimental design. (a) Autophagy is triggered by various stresses, including, but not limited to, nutrient deprivation, oxidative stress, and metabolic stress. To assess autophagic flux, we measured levels of two proteins: LC3, microtubule-associated protein light chain 3, and p62/SQSTM1 (Sequestosome1 complex), an adapter protein which acts as a substrate for the autophagy pathway. Two drugs, rapamycin and bafilomycin A1, were utilized to activate and inhibit the autophagy pathway, respectively. The autophagy pathway proceeds through six major steps: (1) Initiation, (2) Induction, (3) Elongation, (4) Autophagosome formation, (5) Autolysosome formation, and (6) Vesicle breakdown and degradation. (b) Summary of experimental design. Cells were treated with various fatty acid concentrations (0.0 mM oleate/palmitate=Control; 0.5 mM oleate/palmitate=Physiologic; 1.0 mM oleate/palmitate=Diabetic) and either rapamycin (1 uM), bafilomycinA1 (200 nM) drugs, or a combination of both.

Figure 1

Activation of autophagy in L6 myocytes as measured by LC3-II and p62 protein levels; autophagic flux is higher in 48 h treated versus 24 h treated cells. (a–c) Immunofluorescence of cells treated with 1.0 mM oleate/palmitate fatty acids and without drugs (Ctrl) (a), with rapamycin (b), or with bafilomycin (c). All immunofluorescence images were taken at ×20 magnification. (d) LC3-II protein levels for these treatments. (e) p62 protein levels for these treatments. (f and g) LC3-II:p62 ratios of protein levels at 24 h (f) and 48 h (g). Ratios for the bafilomycin treatment in each graph represent inhibition of autophagy while ratios for control represent basal autophagy. Values in between are representative of autophagic flux. Data are expressed in fold change of protein levels versus BSA control at 0.0 mM oleate/palmitate (NS=not significant, *P<0.05, **P<0.01, ***P<0.001).

Figure 2

TG levels increase with bafilomycin and decrease with rapamycin treatment; combination treatment decreases TG levels as well. (a–g) Measurement of intracellular TG for cells treated with drugs for 24 h and (h–l) for 48 h. L6 myocytes were treated with changing levels of fat and measured by colorimetric assay (a and h) and assessed by Oil Red O staining (b–g) and (i–l). (b–d) All cells treated with BSA (0.0 mM) with no drugs (b), with rapamycin (c), or with bafilomycin (d). (e–g) and (i–l) All cells treated with 1.0 mM oleate/palmitate fatty acids with no drugs (e and i), with rapamycin (f and j), with bafilomycin (g and k), or with both rapamycin and bafilomycin (l). All images were taken at ×20 magnification (*P<0.05, **P<0.01, ****P<0.0001).

Figure 3

Rapamycin decreases whereas bafilomycin promotes cell death in the presence of fat. (a–c) Immunofluorescence of same cells treated with (a) no drugs, (b) rapamycin, and (c) bafilomycin. (d) Cleaved caspase-3 protein levels for cells treated with (1.0 mM) or without (0.0 mM) oleate/palmitate fatty acids with no drug, rapamycin, or bafilomycin. DAPI stains for nuclei while FITC stains for cleaved caspase-3 proteins. All immunofluorescence images were taken at ×20 magnification. Immunoblotting data are expressed in fold change of protein levels versus BSA control at 0.0 mM oleate/palmitate (*P<0.05).

Figure 4

Combination of rapamycin and bafilomycin treatment suggest a new mechanism for clearing intramyocellular lipids. L6 myocytes were treated with rapamycin, bafilomycin, and a combination of both rapamycin and bafilomycin. (a) LC3-II, (b) p62, and (c) cleaved caspase-3 protein levels. Data are expressed in fold change of protein levels versus BSA control at 0.0 mM oleate/palmitate (*P<0.05; **P<0.01; ***P<0.001; NS, not significant).

Figure 5

p62 associates with lipid droplets with the combination treatment of both rapamycin and bafilomycin. Immunofluorescence of cells stained with (b) BODIPY 493/503 (neutral lipid stain) and (c) TRITC (p62). (a) DAPI was used for nuclear staining. Cells were treated with both rapamycin and bafilomycin. White arrowheads in panel (d) indicate areas of colocalization between p62 and lipid droplets. All images were taken at ×40 magnification. (e) Immunoblotting for co-immunoprecipitation between p62 and ADRP in the presence of rapamycin, bafilomycin, or both. IgG groups represent negative controls; immunoblotting for Arf-1, known co-immunoprecipitate of ADRP, represent the positive controls. All cells were treated with 1.0 mM oleate/palmitate (**P<0.01).

Figure 7

A proposed pathway for lipid removal from the myocyte. Muscle cells have been shown to breakdown lipid droplets through the classical pathway of lipolysis (A) or more recently by lipophagy (B). When cells are treated with high amounts of fat for a long period of time or lipophagy is impaired by a lysosomal defect, a new lysosomal-independent pathway for lipid removal is activated. P62 binds to lipid droplets (C1) and eventually leads to the export of TGs into the extracellular space (C3). The mechanism by which the TGs are being transported outside the cell is unknown and is currently under investigation (C2).

Figure 6

Extracellular TG reveals potential mechanism for export of TG; ATGListatin does not alter effects of autophagy drugs. Amount of TGs (a and b) in the cells and (c and d) in the extracellular space after treatment with the various drugs. Intracellular and extracellular TG were measured from cells treated with ATGListatin in addition to autophagy drugs (b and d). Data are expressed in fold change of protein levels versus corresponding 1.0 mM oleate/palmitate Ctrl value intracellular (a) or extracellular (c) (*P<0.05, **P<0.01).