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

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.葡萄糖是最高效的能量来源，各类癌细胞依赖糖酵解（glycolysis）获取能量。为维持存活与增殖，癌细胞需精准调控葡萄糖感知（glucose sensing），并适应营养匮乏的环境。尽管酵母菌中的葡萄糖感知机制已得到充分研究，但哺乳动物细胞内葡萄糖感知的分子机制仍有待阐明。我们曾报道，葡萄糖剥夺可通过磷脂酰肌醇3-激酶（PI3K）激活，快速诱导蛋白激酶B（AKT）磷酸化。我们推测AKT的调控与葡萄糖感知密切相关，并进一步探究了其潜在分子机制。 本研究中，半乳糖与果糖可抑制葡萄糖剥夺诱导的AKT磷酸化，而2-脱氧葡萄糖（2-deoxyglucose，2-DG）则可诱导该磷酸化过程。研究发现，2-DG处理与葡萄糖剥夺均可诱导HepG2细胞中的AKT磷酸化。上述结果提示，葡萄糖转运蛋白可能不参与葡萄糖感知及AKT磷酸化的诱导，而下游代谢事件或发挥关键调控作用。 据报道，多种代谢应激可诱导活性氧（reactive oxygen species，ROS）的产生。本研究中，葡萄糖剥夺可诱导HepG2细胞内过氧化氢（hydrogen peroxide，H₂O₂）生成。抗氧化试剂N-乙酰半胱氨酸（N-acetylcysteine，NAC）可同时降低细胞内H₂O₂水平的升高，以及葡萄糖剥夺诱导的AKT磷酸化。上述结果强烈提示，葡萄糖剥夺诱导的细胞内H₂O₂升高介导了AKT的磷酸化过程。 针对NOX4的RNA干扰（RNA interference）可完全抑制葡萄糖剥夺诱导的AKT磷酸化与细胞内ROS水平升高，而针对NOX5的RNA干扰则无此效应；不过，在NOX4敲低的细胞中，外源性H₂O₂仍可诱导AKT磷酸化。 本研究证实，由NOX4产生的ROS通过感知代谢通量参与了细胞内的适应性应答。