Regulation of Tumor Necrosis Factor-Induced, Mitochondria- and Reactive Oxygen Species-Dependent Cell Death by the Electron Flux through the Electron Transport Chain Complex I

Abstract

Tumor necrosis factor (TNF) induces a caspase-independent but mitochondria-dependent cell death process in the mouse fibrosarcoma cell line L929. Mitochondria actively participate in this TNF-induced necrotic cell death by the generation of mitochondrial reactive oxygen species (ROS). The aim of this study was to identify the mitochondrial components involved in TNF-induced production of ROS and their regulation by bioenergetic pathways. Therefore, we analyzed the bioenergetic characteristics in two metabolic L929 variants that exhibit different sensitivities to TNF. L929^gln cells use glutamine as respiratory substrate and are far more susceptible to TNF-induced ROS generation and cell death as L929^glc cells that use glucose as respiratory substrate. We show that the higher levels of reducing NAD(P)H equivalents, detected in the desensitized L929^glc cells, do not cause diminished ROS generation. To the contrary, TNF increases the levels of NAD(P)H, probably altering complex I activity. A multiparameter analysis of electron flux through the mitochondrial electron transport chain, TNF-induced ROS levels, and cell death convincingly demonstrates a dependence of TNF signaling on complex I activity. Also, the sensitizing effect of glutamine metabolism correlates with an enhanced contribution of complex I to the overall electron flux. This participation of complex I activity in TNF-induced cell death is regulated by substrate availability rather than by a direct modification of complex I proteins. From the results presented in this paper we conclude that TNF-induced ROS generation and cell death are strongly regulated by bioenergetic pathways that define electron flux through complex I of the electron transport chain.