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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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Time-course and dose-response for 2-DG-induced AMPK activation.A: Confluent BAEC monolayers were treated with 2-DG (5 mM) for the indicated times. Cell lysates were analyzed by Western blot using antibody against p-AMPK and AMPKα1/2 (n = 3; one-way ANOVA: *p<0.05 vs. control). B: Confluent BAEC monolayers were treated with the indicated concentration of 2-DG for 10 min (n = 3; one-way ANOVA: *p<0.05 vs. control). C: BAEC were incubated with 5 mM of 2-DG for the indicated times. AMPK was immunoprecipitated from cell lysis with an antibody against AMPKα bound to Protein A/G agarose overnight at 4°C. AMPK activity was determined by SAMS phosphorylation using a [32P]ATP assay (n = 3; one-way ANOVA, *p<0.05 vs. control).
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pone-0017234-g002: Time-course and dose-response for 2-DG-induced AMPK activation.A: Confluent BAEC monolayers were treated with 2-DG (5 mM) for the indicated times. Cell lysates were analyzed by Western blot using antibody against p-AMPK and AMPKα1/2 (n = 3; one-way ANOVA: *p<0.05 vs. control). B: Confluent BAEC monolayers were treated with the indicated concentration of 2-DG for 10 min (n = 3; one-way ANOVA: *p<0.05 vs. control). C: BAEC were incubated with 5 mM of 2-DG for the indicated times. AMPK was immunoprecipitated from cell lysis with an antibody against AMPKα bound to Protein A/G agarose overnight at 4°C. AMPK activity was determined by SAMS phosphorylation using a [32P]ATP assay (n = 3; one-way ANOVA, *p<0.05 vs. control).

Mentions: To further establish if ROS is responsible for 2-DG-induced AMPK activation, we performed a time-course study to determine if ROS production proceeds AMPK activation. Confluent BAEC were stimulated with 5 mM 2-DG for 5 to 120 min, and AMPK activation was measured by monitoring phosphorylation of AMPKα-Thr172. As depicted in Fig. 2A, 2-DG treatment increased the phosphorylation of AMPKα-Thr172 within 5 min, and this reached a maximum of 2.8-fold greater than that in 2-DG untreated cells by 10 min after treatment without affecting the total AMPK levels. After 10 min, phosphorylated AMPKα-Thr172 levels decreased and returned to basal levels by 60 min after treatment. ACC is a well-established downstream target of AMPK and is activated by phosphorylation of Ser79. We measured ACC-Ser79 phosphorylation and observed a similar time course for ACC activation as that for AMPK (Figure S1 in Text S1). Further, 2-DG increased the phosphorylation of AMPK-Thr172 (Fig. 2B) and ACC-Ser 79 (Figure S1 in Text S1) in a dose-dependent manner. 2-DG also increased AMPK activity to 4-fold after 5 min compared to basal activity and reached a peak 5.5-fold higher than basal activity after 10 min (Fig. 2C). These data establish that 2-DG induces AMPK activation in both a time- and dose-dependent manner.


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)

Time-course and dose-response for 2-DG-induced AMPK activation.A: Confluent BAEC monolayers were treated with 2-DG (5 mM) for the indicated times. Cell lysates were analyzed by Western blot using antibody against p-AMPK and AMPKα1/2 (n = 3; one-way ANOVA: *p<0.05 vs. control). B: Confluent BAEC monolayers were treated with the indicated concentration of 2-DG for 10 min (n = 3; one-way ANOVA: *p<0.05 vs. control). C: BAEC were incubated with 5 mM of 2-DG for the indicated times. AMPK was immunoprecipitated from cell lysis with an antibody against AMPKα bound to Protein A/G agarose overnight at 4°C. AMPK activity was determined by SAMS phosphorylation using a [32P]ATP assay (n = 3; one-way ANOVA, *p<0.05 vs. control).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046135&req=5

pone-0017234-g002: Time-course and dose-response for 2-DG-induced AMPK activation.A: Confluent BAEC monolayers were treated with 2-DG (5 mM) for the indicated times. Cell lysates were analyzed by Western blot using antibody against p-AMPK and AMPKα1/2 (n = 3; one-way ANOVA: *p<0.05 vs. control). B: Confluent BAEC monolayers were treated with the indicated concentration of 2-DG for 10 min (n = 3; one-way ANOVA: *p<0.05 vs. control). C: BAEC were incubated with 5 mM of 2-DG for the indicated times. AMPK was immunoprecipitated from cell lysis with an antibody against AMPKα bound to Protein A/G agarose overnight at 4°C. AMPK activity was determined by SAMS phosphorylation using a [32P]ATP assay (n = 3; one-way ANOVA, *p<0.05 vs. control).
Mentions: To further establish if ROS is responsible for 2-DG-induced AMPK activation, we performed a time-course study to determine if ROS production proceeds AMPK activation. Confluent BAEC were stimulated with 5 mM 2-DG for 5 to 120 min, and AMPK activation was measured by monitoring phosphorylation of AMPKα-Thr172. As depicted in Fig. 2A, 2-DG treatment increased the phosphorylation of AMPKα-Thr172 within 5 min, and this reached a maximum of 2.8-fold greater than that in 2-DG untreated cells by 10 min after treatment without affecting the total AMPK levels. After 10 min, phosphorylated AMPKα-Thr172 levels decreased and returned to basal levels by 60 min after treatment. ACC is a well-established downstream target of AMPK and is activated by phosphorylation of Ser79. We measured ACC-Ser79 phosphorylation and observed a similar time course for ACC activation as that for AMPK (Figure S1 in Text S1). Further, 2-DG increased the phosphorylation of AMPK-Thr172 (Fig. 2B) and ACC-Ser 79 (Figure S1 in Text S1) in a dose-dependent manner. 2-DG also increased AMPK activity to 4-fold after 5 min compared to basal activity and reached a peak 5.5-fold higher than basal activity after 10 min (Fig. 2C). These data establish that 2-DG induces AMPK activation in both a time- and dose-dependent manner.

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