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. 2013 Aug 2;288(31):22693-705.
doi: 10.1074/jbc.M113.477505. Epub 2013 Jun 10.

Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells: A Potential Role for Reducing Lipid Accumulation

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Free PMC article

Epigallocatechin Gallate (EGCG) Stimulates Autophagy in Vascular Endothelial Cells: A Potential Role for Reducing Lipid Accumulation

Hae-Suk Kim et al. J Biol Chem. .
Free PMC article

Abstract

Epigallocatechin gallate (EGCG) is a major polyphenol in green tea that has beneficial effects in the prevention of cardiovascular disease. Autophagy is a cellular process that protects cells from stressful conditions. To determine whether the beneficial effect of EGCG is mediated by a mechanism involving autophagy, the roles of the EGCG-stimulated autophagy in the context of ectopic lipid accumulation were investigated. Treatment with EGCG increased formation of LC3-II and autophagosomes in primary bovine aortic endothelial cells (BAEC). Activation of calmodulin-dependent protein kinase kinase β was required for EGCG-induced LC3-II formation, as evidenced by the fact that EGCG-induced LC3-II formation was significantly impaired by knockdown of calmodulin-dependent protein kinase kinase β. This effect is most likely due to cytosolic Ca(2+) load. To determine whether EGCG affects palmitate-induced lipid accumulation, the effects of EGCG on autophagic flux and co-localization of lipid droplets and autophagolysosomes were examined. EGCG normalized the palmitate-induced impairment of autophagic flux. Accumulation of lipid droplets by palmitate was markedly reduced by EGCG. Blocking autophagosomal degradation opposed the effect of EGCG in ectopic lipid accumulation, suggesting the action of EGCG is through autophagosomal degradation. The mechanism for this could be due to the increased co-localization of lipid droplets and autophagolysosomes. Co-localization of lipid droplets with LC3 and lysosome was dramatically increased when the cells were treated with EGCG and palmitate compared with the cells treated with palmitate alone. Collectively, these findings suggest that EGCG regulates ectopic lipid accumulation through a facilitated autophagic flux and further imply that EGCG may be a potential therapeutic reagent to prevent cardiovascular complications.

Keywords: AMP-activated Kinase (AMPK); Autophagy; CaMKKβ; Calcium; EGCG; Lipid Droplet; Lipophagy; Polyphenols.

Figures

FIGURE 1.
FIGURE 1.
EGCG stimulates autophagy. BAEC were treated with EGCG for the indicated times. Cell lysates were analyzed by Western blot using antibodies against the indicated proteins. A and B, time course of EGCG (10 μm) effect on LC3-II formation. Data are mean ± S.E. *, p < 0.05; **, p < 0.01 versus control (0 time point). C and D, dose-dependent response to EGCG on LC-3-II formation. Data are mean ± S.E. **, p < 0.01; ***, p < 0.001 versus control (no treatment). E and F, BAEC were transfected with control scrambled or ATG5 siRNA and incubated for 48 h. The cells were serum-starved for 2 h and then treated without or with EGCG (10 μm) for 4 h. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Data are mean ± S.E. (**, p < 0.05, versus scrambled siRNA treated).
FIGURE 2.
FIGURE 2.
Electron microscopic images of BAEC treated without or with EGCG. A and B, BAEC were treated without (upper panels) or with (lower panels) 10 μm EGCG for 2 h. Autophagosomes (thin solid arrows), autophagolysosomes (white triangle), and phagophore (black arrow) are indicated. C and D, areas of autophagosome and autophagolysosome were calculated as described under “Experimental Procedures.” Data are mean ± S.E. (***, p < 0.001, versus nontreated (NT) cells).
FIGURE 3.
FIGURE 3.
EGCG enhances autophagic flux. A and B, BAEC were treated without or with EGCG (10 μm, 4 h). The lysosomal inhibitor (NH4Cl (20 mm), Leu (200 μm)) was treated 1 h prior to cell harvest, and cell lysate was collected and analyzed by immunoblotting for LC3-II formation. Three independent experiments were performed, and the density of LC3-II/LC3-I was quantified. Data are mean ± S.E. (***, p < 0.001). C, differences between the absence and presence of NH4Cl/Leu were calculated for the indication of autophagic flux. Three independent experiments were quantified and calculated. Data are mean ± S.E. (*, p < 0.05). D and E, BAEC were treated without or with EGCG (10 μm) for the indicated time points. Cell lysate was harvested and analyzed by immunoblot with anti-SQSTM1(p62) antibody. SQSTM1 was significantly degraded by treatment with EGCG, which indicates autophagic degradation was enhanced. Three independent experiments were performed, and data are mean ± S.E. (***, p < 0.001). NT, not treated.
FIGURE 4.
FIGURE 4.
EGCG-stimulated autophagy is through AMPK and CaMKKβ but not through reactive oxygen species production. A, BAEC were treated with EGCG (10 μm) for the indicated times, and then cell extracts were analyzed by immunoblotting with the indicated antibodies. Three independent experiments were performed and quantified. B and C, BAEC were pretreated with inhibitors for 30 min; 3-methyladenine (3-MA, 5 mm), PD98059 (PD, 25 μm), SB203508 (SB, 10 μm), compound C (CC, 10 μm), or STO-609 (STO, 10 μm), and then treated with EGCG (10 μm, 4 h). Cell extracts were analyzed by Western blot by using the indicated antibodies. Data are mean ± S.E. (*, p < 0.05; **, p < 0.01 versus EGCG alone). D–G, BAEC were pretreated with the indicated dose of N-acetylcysteine and then treated with EGCG (10 μm, 4 h, D and E, or 3 h, F and G). Cell extracts were analyzed by Western blot using the indicated antibodies. Experiments were repeated three times and quantified by densitometry. Data are mean ± S.E., ***, p < 0.001.
FIGURE 5.
FIGURE 5.
CaMKKβ is involved in EGCG-stimulated AMPK, ULK1, and autophagy but not mTOR. A–C, BAEC were pretreated with STO-609 (10 μm) for 30 min and then treated with EGCG (10 μm) for 2 min. Cell lysates were analyzed by immunoblotting with the indicated antibody. The increased fold was calculated by LC3-II/β-actin. Experiments were repeated three times and quantified by densitometry. Data are mean ± S.E.; ns > 0.05, **, p < 0.01, and ***, p < 0.001. D, BAEC were transfected with control scrambled or CaMKKβ siRNA and incubated for 48 h. The cells were serum-starved for 2 h and then treated without or with EGCG (10 μm) for 4 h. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Reduction of CaMKKβ was examined by RT-PCR. Data are mean ± S.E. (**, p < 0.01, and ***, p < 0.001).
FIGURE 6.
FIGURE 6.
Elevation of intracellular calcium is required for EGCG-stimulated autophagy. A, serum-starved BAEC were pre-loaded with fluo-3 (10 μg/ml) and then stimulated with EGCG (10 μm). Cytosolic fluorescence was observed and quantified. The pseudocolor scale is a linear representation of the fluorescence intensity ranging from 200 to 1200 intensity units. Oscillation of calcium signaling was observed in EGCG-treated cells. B, BAEC were pretreated with CPA (20 μm) 30 min prior to EGCG treatment. C–E, pretreatment of BAEC with CPA reduced calcium signaling. C, number of events; D, peak of signaling; and E, cumulative fluorescence were quantified as described under “Experimental Procedures”; F, bovine aortic endothelial cells were pretreated with Ca2+ chelators for 30 min, EGTA (1 mm), or BAPTA-AM (10 μm), and then treated with EGCG (10 μm, 4 h). **, p < 0.01. Cell lysates were harvested and analyzed by immunoblotting. LC3-II bands from three independent experiments were quantified and normalized for β-actin. Data are mean ± S.E. (*, p < 0.05).
FIGURE 7.
FIGURE 7.
Palmitate-induced inhibition of autophagic flux, which was opposed by co-treatment with EGCG. BAEC were treated with BSA (0.1%) or palmitate (200 μm)/BSA conjugate in the presence or absence of EGCG (10 μm) for 4 h. Lysosomal inhibitor (20 mm NH4Cl, 200 μm leupeptin) was treated 1 h prior to cell harvest. Cell lysate was analyzed by immunoblotting for LC3-II formation. Three independent experiments were performed, and density of LC3-II/LC3-I was quantified. Data are mean ± S.E. (*, p < 0.05; ***, p < 0.001). ns, not significant.
FIGURE 8.
FIGURE 8.
EGCG enhances lipophagy. BAEC were seeded on glass coverslips. Cells were treated with BSA (0.1%) (the top two rows in A–D) or palmitate (200 μm)/BSA (0.1%)) conjugate (the bottom two rows in A–D) in the absence or presence of EGCG (10 μm) for 4 h without (A and C) or with (B and D) lysosomal inhibitors (20 mm NH4Cl, 200 μm leupeptin). The accumulation of lipid droplets was stained with BODIPY 493/503 (green). The co-localization of lipid droplets with LC3 (A and B, red) or LAMP-1 (C and D, red) was by immunocytochemistry using anti-LC3 and anti-LAMP-1 antibodies as described under “Experimental Procedures.” Insets show the higher magnification of the areas in the squares. Arrows indicate co-localization events. Nucleus was stained with Hoechst 33342 (blue). E, number of lipid droplets (LDs) was counted, and the average number of lipid droplets per cell was calculated. Data are mean ± S.E. (*, p < 0.05; **, p < 0.01, and ***, p < 0.001, n = 12). The percentage of lipid droplet co-localization with LC3 (F) or LAMP-1 (G) in cells treated with palmitate in the absence or presence of EGCG without or with NH4Cl/leupeptin was quantified. Data are mean ± S.E. (*, p < 0.05, n = 6). H and I, BAEC were incubated with palmitate (200 μm)/BSA (0.1%) conjugate without or with EGCG (10 μm) for 4 h (left column) or preincubated with palmitate for 4 h and then treated without or with EGCG (10 μm) for another 4 h (right column). Cells were fixed with paraformaldehyde and then stained with BODIPY 493/503 (green). Nuclei were stained with Hoechst 33342 (blue). The number of lipid droplets (LDs) was counted, and the average number of lipid droplets per cell was calculated. Data are mean ± S.E. (***, p < 0.001, n = 10).
FIGURE 9.
FIGURE 9.
Schematic diagram of proposed EGCG-stimulated signaling pathway to activate lipophagy. EGCG-induced oscillation of cytosolic Ca2+ levels by stimulating Ca2+ store in ER that activates CaMKKβ/AMPK pathways and facilitation of autophagic flux. Saturated fatty acid (palmitate) causes impairment of autophagic flux, which leads to accumulation of ectopic lipid accumulation. The facilitated autophagic flux by EGCG affects degradation of accumulated lipid droplets in vascular endothelial cells, which may contribute to improvement of cardiovascular function and prevention of cardiovascular disease. This EGCG-stimulated lipophagy may explain the beneficial health effect of green tea consumption.

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