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2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase.

Wang Q, Liang B, Shirwany NA, Zou MH - PLoS ONE (2011)

Bottom Line: AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG).Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress.Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT
Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H(2)O(2) (100 µM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

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2-DG-induced autophagy is ROS-dependent.A: Confluent monolayers of BAEC were treated with 5 mM 2-DG for the indicated times. Cell lysates were analyzed by Western blot using antibody against LC3B. (n = 3; one-way ANOVA: *p<0.05 vs control). B: Confluent monolayers of BAEC were treated with 5 mM 2-DG for 24 hrs in the presence or absence of chloroquine (3 µM) or bafilomycin A (10 nM). Cell lysates were analyzed by Western blot for detection of LC3. (n = 3; two-way ANOVA: *p<0.05 2-DG vs vehicle, 2-DG + chloroquine vs. chloroquine, 2-DG + bafilomycin A vs. bafilomycin A. p<0.05, 2-DG vs. 2-DG + bafilomycin A or 2-DG + chloroquine). C: BAEC expressing GFP-LC3 were treated with chloroquine or 2-DG, and the accumulation of LC3 II (green), localization of LC3 II with lysosomes (red), and DAPI (blue) staining of nuclei in response to treatment were analyzed by fluorescence microscopy. Scale bars, 5 µm. D: Confluent monolayers of BAEC were treated with 100 µM H2O2 for the indicated times. Cell lysates analyzed by Western blot using antibody against LC3 (n = 3; one-way ANOVA: *p<0.05 vs control). E: BAEC were transduced adenovirus vectors encoding SOD1 or catalase for 48 hrs and then exposed to 5 mM 2-DG for 24 hrs (n = 3; two-way ANOVA, * p<0.05, GFP vs. 2-DG, SOD1 vs 2-DG + SOD1, catalase vs 2-DG + catalase, p<0.05, 2-DG vs. 2-DG + catalase overexpression). F: The accumulation of LC3 II (green), localization of LC3 II lysosomes (red), and DAPI (blue) staining of nuclei in response to SOD1 or catalase overexpression and 2-DG treatment were analyzed by fluorescence microscopy.
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pone-0017234-g001: 2-DG-induced autophagy is ROS-dependent.A: Confluent monolayers of BAEC were treated with 5 mM 2-DG for the indicated times. Cell lysates were analyzed by Western blot using antibody against LC3B. (n = 3; one-way ANOVA: *p<0.05 vs control). B: Confluent monolayers of BAEC were treated with 5 mM 2-DG for 24 hrs in the presence or absence of chloroquine (3 µM) or bafilomycin A (10 nM). Cell lysates were analyzed by Western blot for detection of LC3. (n = 3; two-way ANOVA: *p<0.05 2-DG vs vehicle, 2-DG + chloroquine vs. chloroquine, 2-DG + bafilomycin A vs. bafilomycin A. p<0.05, 2-DG vs. 2-DG + bafilomycin A or 2-DG + chloroquine). C: BAEC expressing GFP-LC3 were treated with chloroquine or 2-DG, and the accumulation of LC3 II (green), localization of LC3 II with lysosomes (red), and DAPI (blue) staining of nuclei in response to treatment were analyzed by fluorescence microscopy. Scale bars, 5 µm. D: Confluent monolayers of BAEC were treated with 100 µM H2O2 for the indicated times. Cell lysates analyzed by Western blot using antibody against LC3 (n = 3; one-way ANOVA: *p<0.05 vs control). E: BAEC were transduced adenovirus vectors encoding SOD1 or catalase for 48 hrs and then exposed to 5 mM 2-DG for 24 hrs (n = 3; two-way ANOVA, * p<0.05, GFP vs. 2-DG, SOD1 vs 2-DG + SOD1, catalase vs 2-DG + catalase, p<0.05, 2-DG vs. 2-DG + catalase overexpression). F: The accumulation of LC3 II (green), localization of LC3 II lysosomes (red), and DAPI (blue) staining of nuclei in response to SOD1 or catalase overexpression and 2-DG treatment were analyzed by fluorescence microscopy.

Mentions: To establish whether 2-DG exposure triggered autophagy in endothelial cells, BAEC were exposed for 2 to 24 hrs to 5 mM 2-DG, a dose comparable to the physiologic concentration of D-Glucose, and the conversion of LC3-I to LC3-II was then determined. Exposure of BAEC to 2-DG for 24 hrs markedly increased the conversion of the cytoplasmic LC3-I to the autophagosomal membrane-bound LC3-II, indicating 2-DG might induce autophagy (Fig. 1A). However, rather than inducing autophagy, an increase in LC3-II could instead be due to 2-DG repressing autophagosome fusion with lysosomes and degradation of LC3-II. To determine the mechanism of 2-DG action, we examined the effect of disrupting lysosomal function using chloroquine and bafilomycin A. These compounds increase lysosomal pH and interfere with the function of lysosomal enzymes [7], thereby increasing autophagosome accumulation in the cell. BAECs treated with 2-DG and chloroquine or bafilomycin A had higher levels of LC3-II than cells treated with 2-DG alone (Fig. 1B), which indicates 2-DG acts by inducing autophagosome formation and does not disrupt their downstream maturation into autophagolysosomes.


2-Deoxy-D-glucose treatment of endothelial cells induces autophagy by reactive oxygen species-mediated activation of the AMP-activated protein kinase.

Wang Q, Liang B, Shirwany NA, Zou MH - PLoS ONE (2011)

2-DG-induced autophagy is ROS-dependent.A: Confluent monolayers of BAEC were treated with 5 mM 2-DG for the indicated times. Cell lysates were analyzed by Western blot using antibody against LC3B. (n = 3; one-way ANOVA: *p<0.05 vs control). B: Confluent monolayers of BAEC were treated with 5 mM 2-DG for 24 hrs in the presence or absence of chloroquine (3 µM) or bafilomycin A (10 nM). Cell lysates were analyzed by Western blot for detection of LC3. (n = 3; two-way ANOVA: *p<0.05 2-DG vs vehicle, 2-DG + chloroquine vs. chloroquine, 2-DG + bafilomycin A vs. bafilomycin A. p<0.05, 2-DG vs. 2-DG + bafilomycin A or 2-DG + chloroquine). C: BAEC expressing GFP-LC3 were treated with chloroquine or 2-DG, and the accumulation of LC3 II (green), localization of LC3 II with lysosomes (red), and DAPI (blue) staining of nuclei in response to treatment were analyzed by fluorescence microscopy. Scale bars, 5 µm. D: Confluent monolayers of BAEC were treated with 100 µM H2O2 for the indicated times. Cell lysates analyzed by Western blot using antibody against LC3 (n = 3; one-way ANOVA: *p<0.05 vs control). E: BAEC were transduced adenovirus vectors encoding SOD1 or catalase for 48 hrs and then exposed to 5 mM 2-DG for 24 hrs (n = 3; two-way ANOVA, * p<0.05, GFP vs. 2-DG, SOD1 vs 2-DG + SOD1, catalase vs 2-DG + catalase, p<0.05, 2-DG vs. 2-DG + catalase overexpression). F: The accumulation of LC3 II (green), localization of LC3 II lysosomes (red), and DAPI (blue) staining of nuclei in response to SOD1 or catalase overexpression and 2-DG treatment were analyzed by fluorescence microscopy.
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Related In: Results  -  Collection

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pone-0017234-g001: 2-DG-induced autophagy is ROS-dependent.A: Confluent monolayers of BAEC were treated with 5 mM 2-DG for the indicated times. Cell lysates were analyzed by Western blot using antibody against LC3B. (n = 3; one-way ANOVA: *p<0.05 vs control). B: Confluent monolayers of BAEC were treated with 5 mM 2-DG for 24 hrs in the presence or absence of chloroquine (3 µM) or bafilomycin A (10 nM). Cell lysates were analyzed by Western blot for detection of LC3. (n = 3; two-way ANOVA: *p<0.05 2-DG vs vehicle, 2-DG + chloroquine vs. chloroquine, 2-DG + bafilomycin A vs. bafilomycin A. p<0.05, 2-DG vs. 2-DG + bafilomycin A or 2-DG + chloroquine). C: BAEC expressing GFP-LC3 were treated with chloroquine or 2-DG, and the accumulation of LC3 II (green), localization of LC3 II with lysosomes (red), and DAPI (blue) staining of nuclei in response to treatment were analyzed by fluorescence microscopy. Scale bars, 5 µm. D: Confluent monolayers of BAEC were treated with 100 µM H2O2 for the indicated times. Cell lysates analyzed by Western blot using antibody against LC3 (n = 3; one-way ANOVA: *p<0.05 vs control). E: BAEC were transduced adenovirus vectors encoding SOD1 or catalase for 48 hrs and then exposed to 5 mM 2-DG for 24 hrs (n = 3; two-way ANOVA, * p<0.05, GFP vs. 2-DG, SOD1 vs 2-DG + SOD1, catalase vs 2-DG + catalase, p<0.05, 2-DG vs. 2-DG + catalase overexpression). F: The accumulation of LC3 II (green), localization of LC3 II lysosomes (red), and DAPI (blue) staining of nuclei in response to SOD1 or catalase overexpression and 2-DG treatment were analyzed by fluorescence microscopy.
Mentions: To establish whether 2-DG exposure triggered autophagy in endothelial cells, BAEC were exposed for 2 to 24 hrs to 5 mM 2-DG, a dose comparable to the physiologic concentration of D-Glucose, and the conversion of LC3-I to LC3-II was then determined. Exposure of BAEC to 2-DG for 24 hrs markedly increased the conversion of the cytoplasmic LC3-I to the autophagosomal membrane-bound LC3-II, indicating 2-DG might induce autophagy (Fig. 1A). However, rather than inducing autophagy, an increase in LC3-II could instead be due to 2-DG repressing autophagosome fusion with lysosomes and degradation of LC3-II. To determine the mechanism of 2-DG action, we examined the effect of disrupting lysosomal function using chloroquine and bafilomycin A. These compounds increase lysosomal pH and interfere with the function of lysosomal enzymes [7], thereby increasing autophagosome accumulation in the cell. BAECs treated with 2-DG and chloroquine or bafilomycin A had higher levels of LC3-II than cells treated with 2-DG alone (Fig. 1B), which indicates 2-DG acts by inducing autophagosome formation and does not disrupt their downstream maturation into autophagolysosomes.

Bottom Line: AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG).Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress.Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Medicine, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.

ABSTRACT
Autophagy is a cellular self-digestion process activated in response to stresses such as energy deprivation and oxidative stress. However, the mechanisms by which energy deprivation and oxidative stress trigger autophagy remain undefined. Here, we report that activation of AMP-activated protein kinase (AMPK) by mitochondria-derived reactive oxygen species (ROS) is required for autophagy in cultured endothelial cells. AMPK activity, ROS levels, and the markers of autophagy were monitored in confluent bovine aortic endothelial cells (BAEC) treated with the glycolysis blocker 2-deoxy-D-glucose (2-DG). Treatment of BAEC with 2-DG (5 mM) for 24 hours or with low concentrations of H(2)O(2) (100 µM) induced autophagy, including increased conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II, accumulation of GFP-tagged LC3 positive intracellular vacuoles, and increased fusion of autophagosomes with lysosomes. 2-DG-treatment also induced AMPK phosphorylation, which was blocked by either co-administration of two potent anti-oxidants (Tempol and N-Acetyl-L-cysteine) or overexpression of superoxide dismutase 1 or catalase in BAEC. Further, 2-DG-induced autophagy in BAEC was blocked by overexpressing catalase or siRNA-mediated knockdown of AMPK. Finally, pretreatment of BAEC with 2-DG increased endothelial cell viability after exposure to hypoxic stress. Thus, AMPK is required for ROS-triggered autophagy in endothelial cells, which increases endothelial cell survival in response to cell stress.

Show MeSH
Related in: MedlinePlus