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. 2011 Nov;339(2):487-98.
doi: 10.1124/jpet.111.184341. Epub 2011 Aug 19.

Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes

Shuang Mei  1 , Hong-Min Ni, Sharon Manley, Abigail Bockus, Karen M Kassel, James P Luyendyk, Bryan L Copple, Wen-Xing Ding
Affiliations

Differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes

Shuang Mei et al. J Pharmacol Exp Ther. 2011 Nov.

Abstract

Fatty acid-induced lipotoxicity plays a critical role in the pathogenesis of nonalcoholic liver disease. Saturated fatty acids and unsaturated fatty acids have differential effects on cell death and steatosis, but the mechanisms responsible for these differences are not known. Using cultured HepG2 cells and primary mouse hepatocytes, we found that unsaturated and saturated fatty acids differentially regulate autophagy and apoptosis. The unsaturated fatty acid, oleic acid, promoted the formation of triglyceride-enriched lipid droplets and induced autophagy but had a minimal effect on apoptosis. In contrast, the saturated fatty acid, palmitic acid, was poorly converted into triglyceride-enriched lipid droplets, suppressed autophagy, and significantly induced apoptosis. Subsequent studies revealed that palmitic acid-induced apoptosis suppressed autophagy by inducing caspase-dependent Beclin 1 cleavage, indicating cross-talk between apoptosis and autophagy. Moreover, our data suggest that the formation of triglyceride-enriched lipid droplets and induction of autophagy are protective mechanisms against fatty acid-induced lipotoxicity. In line with our in vitro findings, we found that high-fat diet-induced hepatic steatosis was associated with autophagy in the mouse liver. Potential modulation of autophagy may be a novel approach that has therapeutic benefits for obesity-induced steatosis and liver injury.

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Figures

Fig. 1.
Fig. 1.
OA induces autophagy in HepG2 cells. HepG2 cells were first infected with Ad-GFP-LC3 (100 viral particles/cell) overnight and then treated with vehicle control (5% BSA), OA (500 μM), OA plus CQ (20 μM) or CQ (20 μM) alone, or various concentrations of OA (0, 125, 250, and 500 μM) for 6 h followed by fluorescence microscopy. A, representative GFP-LC3 images. B and C, number of GFP-LC3 dots per cell. Data are presented as the mean ± S.E. from three independent experiments by counting more than 20 cells in each individual experiment. *, p < 0.05; #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with vehicle control (5% BSA), OA (500 μM), OA plus CQ (20 μM), CQ (20 μM) alone, OA plus Baf (50 nM), or Baf (50 nM) alone for 6 h. Total cell lysates were subjected to immunoblot analysis with anti-LC3 and anti-β-actin antibodies. Densitometry analysis for the expression level of LC3-II was performed using ImageJ software, which was further normalized with its loading control (β-actin). Digital data are presented as the ratio of LC3-II of the vehicle control (mean ± S.E.) from at least three independent experiments.
Fig. 2.
Fig. 2.
PA fails to induce autophagy in HepG2 cells. HepG2 cells were first infected with Ad-GFP-LC3 overnight and then treated with vehicle control (5% BSA), PA (500 μM), PA plus CQ (20 μM), CQ alone, or various concentrations of PA (0, 125, 250, and 500 μM) for 6 h followed by fluorescence microscopy. A, representative GFP-LC3 images. B and C, number of GFP-LC3 dots per cell. Data are presented as the mean ± S.E. from three independent experiments by counting more than 20 cells in each individual experiment. *, p < 0.05; #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with vehicle control (5% BSA), PA (500 μM), PA plus CQ (20 μM), CQ (20 μM) alone, PA plus Baf (50 nM), or Baf (50 nM) alone for 6 h. Total cell lysates were subjected to immunoblot analysis with anti-LC3 and anti-β-actin antibodies. Densitometry analysis for the expression level of LC3-II was performed using ImageJ software, which was further normalized with its loading control (β-actin). Digital data are presented as the ratio of LC3-II of the vehicle control (mean ± S.E.) from at least three independent experiments.
Fig. 3.
Fig. 3.
OA but not PA increases the number of autophagosomes and lipid droplets in HepG2 cells. A, HepG2 cells were treated with BSA vehicle control (a), PA (500 μM, b), or OA (500 μM, c–f) for 6 h, and the cells were further processed for EM. Arrows denote autophagosomes. N, nuclei; LD, lipid droplet; m, mitochondria. The number of autophagosomes (B) and lipid droplets (C) per cell section was determined (mean ± S.D.) from more than 30 different cells. #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with BSA vehicle control (a), OA (500 μM, b), or PA (500 μM, c) for 6 h and fixed with 4% paraformaldehyde. The cells were further stained with Bodipy 493/503 (0.1 μM) for lipid droplets and Hoechst 33342 (0.5 μg/ml) for the nuclei followed by fluorescence microscopy. E, the number of lipid droplets per cell was quantified, and data are presented as the mean ± S.E. from at least three independent experiments. #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test).
Fig. 4.
Fig. 4.
NAC and 3-MA suppress OA-induced autophagy in HepG2 cells. A, HepG2 cells were first infected with Ad-GFP-LC3 (100 viral particles/cell) overnight and then treated with vehicle control (5% BSA), OA (500 μM), PA (500 μM), OA plus NAC (5 mM), PA plus NAC (5 mM), NAC (5 mM), OA plus 3-MA (10 mM), PA plus 3-MA (10 mM), or 3-MA (10 mM) alone for 6 h followed by fluorescence microscopy. B and C, the number of GFP-LC3 dots per cell (mean ± S.E.) was quantified from three independent experiments and more than 20 cells were counted in each individual experiment. #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with OA or PA (500 μM) for 6, 12, and 24 h, and the expression levels of phosphorylated (p) 4EBP1/total 4EBP1 and p-p70S6k/total p70S6k were determined by immunoblot analysis from at least three independent experiments. E, HepG2 cells were treated with vehicle control (5% BSA) or various concentrations (125, 250, and 500 μM) of OA and PA for 6 h. The expression levels of p-4EBP1/total 4EBP1 and p-p70S6k/Total p70S6k were determined by immunoblot analysis from at least three independent experiments. Densitometry analysis for the expression levels of p-4EBP1 and p-p70S6K was performed using ImageJ software, which was further normalized with its loading control (β-actin).
Fig. 5.
Fig. 5.
Differential effects of OA and PA on apoptosis in HepG2 cells. A, HepG2 cells were treated with vehicle control (5% BSA), OA (500 μM), PA (500 μM), or TNF-α (10 ng/ml) plus ActD (0.2 μg/ml) for 24 h. Apoptotic nuclei were analyzed by nuclear staining with Hoechst 33342 (1 μg/ml) for fragmented or condensed nuclei (arrows). B, HepG2 cells were treated with OA or PA (500 μM) for 6, 12, or 24 h or various concentrations (125, 250, and 500 μM) of OA or PA (C), and the number of apoptotic nuclei was quantified (mean ± S.E., n = 3). #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with vehicle control (5% BSA), OA (500 μM), PA (500 μM), or TNF-α (10 ng/ml) plus ActD (0.2 μg/ml) for 6 h. The cells were loaded with TMRM (50 nM) followed by fluorescence microscopy (arrowheads, cells with partially lost mitochondrial membrane potential; arrows, cells with completely lost mitochondrial membrane potential). E, the number of cells with loss (both partial and complete) of TMRM staining was quantified (mean ± S.E.M.) from at least three independent experiments. #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test).
Fig. 6.
Fig. 6.
PA but not OA induces caspase-mediated Beclin 1 cleavage. A, HepG2 cells were treated with OA or PA (500 μM) for 6, 12, or 24 h. Total cell lysates were subjected to immunoblot analysis using an anti-Beclin 1 antibody. B, HepG2 cells were treated with vehicle control (5% BSA), OA (500 μM), PA (500 μM), or TNF-α (10 ng/ml) plus ActD (0.2 μg/ml) for 24 h. Total cell lysates were subjected to immunoblot analysis for Beclin 1 and caspase 3. C, Total cell lysates (30 μg) were used for caspase-3 activity analysis (mean ± S.E., n = 3). #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). D, HepG2 cells were treated with vehicle control (5% BSA), PA (500 μM), PA (500 μM) plus Z-VAD-fmk (50 μM), TNF-α (10 ng/ml) plus ActD (0.2 μg/ml), or TNF-α (10 ng/ml) plus ActD (0.2 μg/ml) with Z-VAD-fmk (50 μM) for 24 h. Total cell lysates were subjected to immunoblot analysis for Beclin 1 and (E) apoptotic cell death was analyzed by nuclear staining with Hoechst 33342 (mean ± S.E., n = 3). #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test).
Fig. 7.
Fig. 7.
Suppression of autophagy enhances fatty acid-induced cell death and lipid accumulation. A, HepG2 cells were treated with vehicle control (5% BSA), OA (500 μM), OA plus CQ (20 μM), CQ alone, OA plus 3-MA (10 mM), or 3-MA alone for 24 h, and apoptotic cell death was analyzed by nuclear staining with Hoechst 33342 (mean ± S.E., n = 3). #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test). B and C, HepG2 cells were treated with vehicle control (5% BSA) or various concentrations (125, 250, or 500) of OA or PA for 6 h (B) or with OA (500 μM) or PA (500 μM) in the presence or absence of CQ (20 μM) for 6 h (C). Total cell lysates were subjected to immunoblot analysis for perilipin A. Densitometry analysis for the expression level of perilipin was performed using ImageJ software, which was further normalized with its loading control (β-actin). D, HepG2 cells were treated as in C, and cellular TG levels (mean ± S.E., n = 3) were quantified as described under Materials and Methods. #, p < 0.01 (one-way ANOVA with the Scheffé post hoc test).
Fig. 8.
Fig. 8.
A high-fat diet induces steatosis and autophagy in mouse liver. Male C57BL/6J mice were fed either a control diet or a Western diet for 3 months. All the mice were starved for 16 h before they were sacrificed. A, representative photomicrograph of hematoxylin and eosin-stained liver section from a mouse fed a control diet (a) and from a mouse fed the high-fat diet (b). c, enlarged photomicrograph from b showing typical macrovesicular hepatic steatosis (arrows). B, liver samples were processed for EM. a, control diet. b and c, high-fat diet. d and e, enlarged photomicrographs from the boxed areas in c. Arrows, double membrane autophagosomes; N, nuclei, LD, lipid droplets; M, mitochondria. C, total liver lysates were subjected to Western blot analysis using an anti-LC3 antibody. The same membrane was blotted for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the loading control. D, densitometry analysis for the expression level of LC3-II was performed using ImageJ software, which was further normalized with its loading control (GAPDH). Data are presented as the fold of the control diet mouse livers (mean ± S.E., n = 6). #, p < 0.01, Student's t test.
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