Critical Role of H₂O₂ Generated by NOX4 during Cellular Response under Glucose Deprivation

<div><p>Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.</p> </div

Induction of AKT phosphorylation under glucose deprivation is mediated by ROS generated by NOX4.

<p>(A) siRNA-treated HepG2 cells were subjected to reverse-transcriptase PCR (RT-PCR) to confirm NOX4 knockdown. (B) NOX4 knockdown HepG2 Cells were stained with 5 µM of BES-H₂O₂ in the absence or presence of 5.5 mM of glucose for 0.5 h. ROS production was measured using flow cytometry. (C) Immunoblotting analyses of HepG2 cells transfected with a non-targeting siRNA or two separate NOX4 siRNAs in the absence or presence of 5.5 mM of glucose or treatment with exogenous H₂O₂ for 0.5 h.</p

AKT phosphorylation was induced under glucose deprivation.

<p>(A) Immunoblotting analyses after incubation of HepG2 cells in the absence or presence of 5.5 mM of glucose and absence or presence of 30 µM of LY294002 for the indicated times. (B) HepG2 cells treated or not treated with various concentrations of glucose for 0.5 h were subjected to immunoblotting. (C) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of galactose, or 5.5 mM of fructose for 0.5 h. (D) Immunoblotting analyses of HepG2 cells treated or not treated with 5.5 mM of glucose, 5.5 mM of 2-DG, or 5.5 mM of glucose plus 5.5 mM of 2-DG for 0.5 h.</p

ROS mediates AKT phosphorylation under glucose deprivation.

<p>(A)(B)(D) HepG2 cells were cultured in either glucose-containing medium or glucose-deprived medium in the absence or presence of 12.5 mM of NAC for 0.5 h. ROS production was measured using flow cytometry. Cells were stained with (A) 5 µM of DCFDA or (B) 5 µM of BES-H₂O₂. Cells were gated within a range contained in the upper 5% of the total cell count under the glucose replete condition. (D) The AKT phosphorylation level was evaluated by immunoblotting. (C) Addition of H₂O₂ to media containing 5.5 mM of glucose in the absence or presence of 30 µM of LY294002 for 0.5 h, followed by immunoblotting.</p

SRC and OSSA are indispensable for the AKT phosphorylation induced by glucose deprivation.

<p>(A) Immunoblotting analyses of HepG2 cells in the absence or presence of 5.5 mM of glucose <u>in the</u> and absence or presence of 20 µM of PP2 for the indicated times. (B) Addition of H₂O₂ to the culture medium containing 5.5 mM glucosein the absence or presence of 20 µM of PP2 for 0.5 h, followed by immunoblotting. (C) HepG2 cells were cultured in medium containing or not containing (glucose-deprived) 5.5 mM of glucose in the absence or presence of 30 µM of LY204002 or 20 µM of PP2 for 0.5 h. The cells were stained with 5 µM of BES-H₂O₂. ROS production was measured using flow cytometry. (D) siRNA-treated HepG2 cells were subjected to immunoblotting analyses using OSSA antibody. (E) Immunoblotting analyses of HepG2 cells transfected with a non-targeting siRNA or two separate OSSA siRNAs in the absence or presence of 5.5 mM of glucose for the indicated times. (F) Addition of H₂O₂ to the medium of OSSA-knockdown cells containing 5.5 mM glucose for 0.5 h, followed by immunoblotting.</p