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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 increases intracellular synthesis of H2O2 to induce autophagy.A: HUVEC were transfected with AMPK-targeted siRNA for 24 hrs. Then the cells were incubated in EBM media without phenol red and treated with 5 mM of 2-DG for 10 min. CM-H2DCFDA (10 µM; Invitrogen) was added for 30 min before quantification of fluorescence (excitation at 485 nm and emission at 545 nm). (n = 3; two-way ANOVA,* p<0.05, 2-DG vs. vehicle). B: BAEC were pre-treated with 3-amino-1,2,3-triazine (ATZ, 100 µM) and 1-chloro-2,4-dinitrobenzene (DNCB, 100 µM) for 30 min and then treated with 2-DG for 5 or 10 min. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; two-way ANOVA,* p<0.05, ATZ + DNCB vs. vehicle, p<0.05, 2-DG + ATZ + DNCB vs. ATZ + DNCB). C: BAEC were treated with 2-DG for 24 hrs. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; t-test,* p<0.05, 2-DG vs. vehicle). D,E: HUVEC were transfected with Atg4-targeted siRNA or control siRNA for 24 hrs and then treated with 5 mM 2-DG for 24 hrs. Cell lysates were analyzed by Western blot using antibody against LC3 (n = 3; two-way ANOVA, * p<0.05, vehicle vs. 2-DG, p<0.05, Control siRNA vs. Atg4 siRNA).
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pone-0017234-g005: 2-DG increases intracellular synthesis of H2O2 to induce autophagy.A: HUVEC were transfected with AMPK-targeted siRNA for 24 hrs. Then the cells were incubated in EBM media without phenol red and treated with 5 mM of 2-DG for 10 min. CM-H2DCFDA (10 µM; Invitrogen) was added for 30 min before quantification of fluorescence (excitation at 485 nm and emission at 545 nm). (n = 3; two-way ANOVA,* p<0.05, 2-DG vs. vehicle). B: BAEC were pre-treated with 3-amino-1,2,3-triazine (ATZ, 100 µM) and 1-chloro-2,4-dinitrobenzene (DNCB, 100 µM) for 30 min and then treated with 2-DG for 5 or 10 min. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; two-way ANOVA,* p<0.05, ATZ + DNCB vs. vehicle, p<0.05, 2-DG + ATZ + DNCB vs. ATZ + DNCB). C: BAEC were treated with 2-DG for 24 hrs. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; t-test,* p<0.05, 2-DG vs. vehicle). D,E: HUVEC were transfected with Atg4-targeted siRNA or control siRNA for 24 hrs and then treated with 5 mM 2-DG for 24 hrs. Cell lysates were analyzed by Western blot using antibody against LC3 (n = 3; two-way ANOVA, * p<0.05, vehicle vs. 2-DG, p<0.05, Control siRNA vs. Atg4 siRNA).

Mentions: We next determined the effect of 2-DG on ROS production in endothelial cells using CM-H2DCFDA as an intracellular H2O2 probe. It is known that AMPK activation by AICAR reduces the generation of ROS [20]. To circumvent the confounding potential of ROS attenuation by AMPK activation, we detected intracellular H2O2 in 2-DG-stimulated HUVEC transfected with AMPK-targeted siRNA. As expected, the intracellular H2O2 levels in cells in which AMPKα1/2 was knocked down were significantly higher than in cells transfected with the non-targeted control siRNA following a 10–min, 5-mM 2-DG treatment (Fig. 5A). Pre-treatment with a catalase inhibitor 3-amino-1,2,4-triazine (ATZ) and thioredoxin reductase inhibitor 1-chlo-2,4-dinitrobenzene (DNCB) led to a significantly increased intracellular H2O2 level in 2-DG-treated BAEC (Fig. 5B and C).


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 increases intracellular synthesis of H2O2 to induce autophagy.A: HUVEC were transfected with AMPK-targeted siRNA for 24 hrs. Then the cells were incubated in EBM media without phenol red and treated with 5 mM of 2-DG for 10 min. CM-H2DCFDA (10 µM; Invitrogen) was added for 30 min before quantification of fluorescence (excitation at 485 nm and emission at 545 nm). (n = 3; two-way ANOVA,* p<0.05, 2-DG vs. vehicle). B: BAEC were pre-treated with 3-amino-1,2,3-triazine (ATZ, 100 µM) and 1-chloro-2,4-dinitrobenzene (DNCB, 100 µM) for 30 min and then treated with 2-DG for 5 or 10 min. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; two-way ANOVA,* p<0.05, ATZ + DNCB vs. vehicle, p<0.05, 2-DG + ATZ + DNCB vs. ATZ + DNCB). C: BAEC were treated with 2-DG for 24 hrs. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; t-test,* p<0.05, 2-DG vs. vehicle). D,E: HUVEC were transfected with Atg4-targeted siRNA or control siRNA for 24 hrs and then treated with 5 mM 2-DG for 24 hrs. Cell lysates were analyzed by Western blot using antibody against LC3 (n = 3; two-way ANOVA, * p<0.05, vehicle vs. 2-DG, p<0.05, Control siRNA vs. Atg4 siRNA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046135&req=5

pone-0017234-g005: 2-DG increases intracellular synthesis of H2O2 to induce autophagy.A: HUVEC were transfected with AMPK-targeted siRNA for 24 hrs. Then the cells were incubated in EBM media without phenol red and treated with 5 mM of 2-DG for 10 min. CM-H2DCFDA (10 µM; Invitrogen) was added for 30 min before quantification of fluorescence (excitation at 485 nm and emission at 545 nm). (n = 3; two-way ANOVA,* p<0.05, 2-DG vs. vehicle). B: BAEC were pre-treated with 3-amino-1,2,3-triazine (ATZ, 100 µM) and 1-chloro-2,4-dinitrobenzene (DNCB, 100 µM) for 30 min and then treated with 2-DG for 5 or 10 min. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; two-way ANOVA,* p<0.05, ATZ + DNCB vs. vehicle, p<0.05, 2-DG + ATZ + DNCB vs. ATZ + DNCB). C: BAEC were treated with 2-DG for 24 hrs. Intracellular H2O2 levels were detected by fluorescence using CM-H2DCFDA. (n = 3; t-test,* p<0.05, 2-DG vs. vehicle). D,E: HUVEC were transfected with Atg4-targeted siRNA or control siRNA for 24 hrs and then treated with 5 mM 2-DG for 24 hrs. Cell lysates were analyzed by Western blot using antibody against LC3 (n = 3; two-way ANOVA, * p<0.05, vehicle vs. 2-DG, p<0.05, Control siRNA vs. Atg4 siRNA).
Mentions: We next determined the effect of 2-DG on ROS production in endothelial cells using CM-H2DCFDA as an intracellular H2O2 probe. It is known that AMPK activation by AICAR reduces the generation of ROS [20]. To circumvent the confounding potential of ROS attenuation by AMPK activation, we detected intracellular H2O2 in 2-DG-stimulated HUVEC transfected with AMPK-targeted siRNA. As expected, the intracellular H2O2 levels in cells in which AMPKα1/2 was knocked down were significantly higher than in cells transfected with the non-targeted control siRNA following a 10–min, 5-mM 2-DG treatment (Fig. 5A). Pre-treatment with a catalase inhibitor 3-amino-1,2,4-triazine (ATZ) and thioredoxin reductase inhibitor 1-chlo-2,4-dinitrobenzene (DNCB) led to a significantly increased intracellular H2O2 level in 2-DG-treated BAEC (Fig. 5B and C).

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