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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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Activation of AMPK by 2-DG is mediated by mitochondrial ROS independent of NAD(P)H oxidase and xanthine oxidase.A: Confluent BAEC monolayers were pre-treated with 10 µM mito-Tempol for 30 min and then treated with 5 mM of 2-DG for 10 min. Cell lysates were analyzed by Western blot using antibody against p-AMPK (n = 3; two-way ANOVA, *p<0.05, vehicle vs 2-DG, p<0.05, 2-DG vs. 2-DG + mito-Tem). B, C, E: Confluent BAEC monolayers were transduced with adenovirus vectors encoding MnSOD (B), UCP2 (C), or p47phox and p67phox dominant negative mutants (E) for 48 hrs and then treated with 5 mM 2-DG for 10 min. (n = 3; two-way ANOVA, * p<0.05, GFP vs 2-DG + GFP, MnSOD vs 2-DG + Mn SOD, UCP2 vs 2-DG + UCP2, p<0.05 GFP vs. MnSOD or UCP2). D: HUVEC were transfected with control siRNA or UCP-2-targeted siRNA for 24 hrs. Then the cells were treated with 5 mM of 2-DG for 10 min. (n = 3; two-way ANOVA, *p<0.05, 2-DG vs vehicle, p<0.05 control siRNA vs. UCP2 siRNA). F: Confluent BAEC monolayers were pre-treated with allopurinol (100 µM) or oxypurinol (30 µM) for 30 min and then treated with 5 mM of 2-DG for 10 min.
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pone-0017234-g007: Activation of AMPK by 2-DG is mediated by mitochondrial ROS independent of NAD(P)H oxidase and xanthine oxidase.A: Confluent BAEC monolayers were pre-treated with 10 µM mito-Tempol for 30 min and then treated with 5 mM of 2-DG for 10 min. Cell lysates were analyzed by Western blot using antibody against p-AMPK (n = 3; two-way ANOVA, *p<0.05, vehicle vs 2-DG, p<0.05, 2-DG vs. 2-DG + mito-Tem). B, C, E: Confluent BAEC monolayers were transduced with adenovirus vectors encoding MnSOD (B), UCP2 (C), or p47phox and p67phox dominant negative mutants (E) for 48 hrs and then treated with 5 mM 2-DG for 10 min. (n = 3; two-way ANOVA, * p<0.05, GFP vs 2-DG + GFP, MnSOD vs 2-DG + Mn SOD, UCP2 vs 2-DG + UCP2, p<0.05 GFP vs. MnSOD or UCP2). D: HUVEC were transfected with control siRNA or UCP-2-targeted siRNA for 24 hrs. Then the cells were treated with 5 mM of 2-DG for 10 min. (n = 3; two-way ANOVA, *p<0.05, 2-DG vs vehicle, p<0.05 control siRNA vs. UCP2 siRNA). F: Confluent BAEC monolayers were pre-treated with allopurinol (100 µM) or oxypurinol (30 µM) for 30 min and then treated with 5 mM of 2-DG for 10 min.

Mentions: Mitochondria, NAD(P)H oxidase, and xanthine oxidase [37] can generate ROS in both physiological and pathological conditions. We sought to identify the major sources of ROS synthesis in 2-DG-treated endothelial cells. Mito-Tempol is a synthetic Tempol derivate that preferentially scavenges O2− from mitochondria. Pre-treatment of cells with 10 µM mito-Tempol for 30 min dramatically decreased 2-DG-induced phosphorylation of AMPK (Fig. 7A). Further, overexpression of MnSOD, a SOD isoform located in the mitochondrial matrix (Figure S3 in Text S1), attenuated the 2-DG-induced phosphorylation of AMPK and ACC (Fig. 7B).


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)

Activation of AMPK by 2-DG is mediated by mitochondrial ROS independent of NAD(P)H oxidase and xanthine oxidase.A: Confluent BAEC monolayers were pre-treated with 10 µM mito-Tempol for 30 min and then treated with 5 mM of 2-DG for 10 min. Cell lysates were analyzed by Western blot using antibody against p-AMPK (n = 3; two-way ANOVA, *p<0.05, vehicle vs 2-DG, p<0.05, 2-DG vs. 2-DG + mito-Tem). B, C, E: Confluent BAEC monolayers were transduced with adenovirus vectors encoding MnSOD (B), UCP2 (C), or p47phox and p67phox dominant negative mutants (E) for 48 hrs and then treated with 5 mM 2-DG for 10 min. (n = 3; two-way ANOVA, * p<0.05, GFP vs 2-DG + GFP, MnSOD vs 2-DG + Mn SOD, UCP2 vs 2-DG + UCP2, p<0.05 GFP vs. MnSOD or UCP2). D: HUVEC were transfected with control siRNA or UCP-2-targeted siRNA for 24 hrs. Then the cells were treated with 5 mM of 2-DG for 10 min. (n = 3; two-way ANOVA, *p<0.05, 2-DG vs vehicle, p<0.05 control siRNA vs. UCP2 siRNA). F: Confluent BAEC monolayers were pre-treated with allopurinol (100 µM) or oxypurinol (30 µM) for 30 min and then treated with 5 mM of 2-DG for 10 min.
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pone-0017234-g007: Activation of AMPK by 2-DG is mediated by mitochondrial ROS independent of NAD(P)H oxidase and xanthine oxidase.A: Confluent BAEC monolayers were pre-treated with 10 µM mito-Tempol for 30 min and then treated with 5 mM of 2-DG for 10 min. Cell lysates were analyzed by Western blot using antibody against p-AMPK (n = 3; two-way ANOVA, *p<0.05, vehicle vs 2-DG, p<0.05, 2-DG vs. 2-DG + mito-Tem). B, C, E: Confluent BAEC monolayers were transduced with adenovirus vectors encoding MnSOD (B), UCP2 (C), or p47phox and p67phox dominant negative mutants (E) for 48 hrs and then treated with 5 mM 2-DG for 10 min. (n = 3; two-way ANOVA, * p<0.05, GFP vs 2-DG + GFP, MnSOD vs 2-DG + Mn SOD, UCP2 vs 2-DG + UCP2, p<0.05 GFP vs. MnSOD or UCP2). D: HUVEC were transfected with control siRNA or UCP-2-targeted siRNA for 24 hrs. Then the cells were treated with 5 mM of 2-DG for 10 min. (n = 3; two-way ANOVA, *p<0.05, 2-DG vs vehicle, p<0.05 control siRNA vs. UCP2 siRNA). F: Confluent BAEC monolayers were pre-treated with allopurinol (100 µM) or oxypurinol (30 µM) for 30 min and then treated with 5 mM of 2-DG for 10 min.
Mentions: Mitochondria, NAD(P)H oxidase, and xanthine oxidase [37] can generate ROS in both physiological and pathological conditions. We sought to identify the major sources of ROS synthesis in 2-DG-treated endothelial cells. Mito-Tempol is a synthetic Tempol derivate that preferentially scavenges O2− from mitochondria. Pre-treatment of cells with 10 µM mito-Tempol for 30 min dramatically decreased 2-DG-induced phosphorylation of AMPK (Fig. 7A). Further, overexpression of MnSOD, a SOD isoform located in the mitochondrial matrix (Figure S3 in Text S1), attenuated the 2-DG-induced phosphorylation of AMPK and ACC (Fig. 7B).

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