2-Deoxy-D-Glucose Treatment of Endothelial Cells Induces Autophagy by Reactive Oxygen Species-Mediated Activation of the AMP-Activated Protein Kinase

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 H2O2 (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.

Hexokinase II integrates energy metabolism and cellular protection: Akting on mitochondria and TORCing to autophagy

“…Interaction of hexokinase II and Akt/mTOR pathway DJ Roberts and S Miyamoto with 5-25 mM commonly used [164][165][166] ) inhibited autophagy development in the absence of glucose in cardiomyocytes. This observation implied that HK-II acts to stimulate autophagy in the absence of its substrate.…”

High glucose modulates IL‐6 mediated immune homeostasis through impeding neutrophil extracellular trap formation

“…It has been demonstrated that wortmannin and diphenylene iodinium, the inhibitors of PI3K/ autophagy and NADPH oxidase respectively, abrogated phorbol ester (PMA) induced NETosis [21]. 2-DG has been shown to induce autophagy, NADPH oxidase activity and reactive oxygen species in endothelial cells [22]. We hypothesize that 2-DG enhances NADPH oxidase activity impaired by high glucose thereby resensitizing NETs formation in cells derived from diabetic subjects.…”

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

“…Acidic pH e was reported to inhibit glycolysis via decreased glucose uptake in Ehrlich ascites tumor cells (72) and our data indicate that melanoma cells exposed to acidic pH show signs of glycolysis inhibition because they reduced glucose uptake and lactate release. Similarly, glycolysis inhibition by 2-deoxy-D-glucose was reported to induce cytoprotective autophagy in endothelial and melanoma cells (73,74). Because it is known that active mTOR stimulates glucose uptake via increased membrane expression of glucose transporters (75) it is likely that inhibition of mTOR by acidic pH may be the upstream event leading to reduced glycolysis and autophagy induction.…”