Treatment of the mouse fibrosarcoma cell line L929 with tumor necrosis factor (TNF) induces necrotic cell death. A crucial step in the cytotoxic action mechanism of TNF involves perturbation of mitochondrial functions leading to the formation of reactive oxygen intermediates (ROI). L929 cells have energy requirements adapted to a high proliferation rate. Glutamine (Gln) is utilized as a major energy source and drives mitochondrial ATP formation, while glucose is mainly converted to lactate

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... gh glycolysis. We investigated the role of the bioenergetic pathways involved in substrate utilization on the cytotoxic action of TNF and established a link between Gln oxidation and TNF-induced mitochondrial distress. Omission of Gln from the medium desensitizes the cells to TNF cytotoxicity, while the lack of glucose in the medium does not alter the TNF response. Sudden depletion of Gln from the culture medium results in a sharp decline in mitochondrial respiration in the cells, which might explain the decreased TNF responsiveness. However, when L929 cells are adapted to long term growth under conditions without Gln, these so-called L929/Gln ؊ cells have restored respiration, but they still display a decreased sensitivity to TNF cytotoxicity. Thus the TNF responsiveness of L929 cells depends on bioenergetic reactions that are specifically involved in the oxidation of Gln. This is further confirmed by the desensitizing effect of specific inhibitors of these Glnlinked enzyme reactions on TNF cytotoxicity in the parental cells, but not in the L929/Gln ؊ cells. Analysis of the induction of mitochondrial ROI formation by TNF in parental and L929/Gln ؊ cells suggests that the effect of Gln on the sensitivity to TNF cytotoxicity involves a mechanism that renders the mitochondria more susceptible to TNF-induced mediators, resulting in enhanced ROI production and accelerated cytotoxicity. Tumor necrosis factor (TNF) 1 is a pleiotropic cytokine mainly produced by activated macrophages. Besides its role in the host defense against microorganisms and bacterial pathogens, TNF is involved in the pathology of various diseases, such as the systemic inflammatory response syndrome (1-3). Furthermore, TNF is specifically cytotoxic for many types of transformed cells, especially in the presence of interferon. Most TNF-mediated activities, including cytotoxicity, are initiated by ligandinduced cross-linking of the p55 TNF receptor; only in T-lymphocytes has a role of the p75 TNF receptor in cell proliferation and in cytotoxicity been unambiguously demonstrated (4). Cross-linking of the receptors initiates signal transduction, possibly through association of the intracellular death domain of the p55 receptor (5). In the mouse L929 fibrosarcoma cell line, TNF signaling leads to necrotic cell death (6, 7). A major step in the cytotoxic mechanism is the formation of ROI in the mitochondria. Their crucial role was demonstrated by the interference of specific inhibitors of the electron transport chain with necrotic cell response (8) and by the correlation between sensitivity to TNF cytotoxicity and mitochondrial activity in the cell (9). More recently, we followed directly the TNF-induced mitochondrial ROI and correlated it with cytotoxicity (10). Hence the change from a sensitive to a resistant phenotype observed under hypoxic growth conditions (11, 12) can be explained by the inability to generate ROI in the mitochondria. Besides oxygen, another important parameter that affects mitochondrial functionality is the availability of energy substrates. Normal and malignant cells often exhibit different metabolic requirements (13-17); the exuberant proliferation rate of transformed cells demands an adapted energy metabolism. Therefore, the amino acid Gln, instead of the Glc-derived pyruvate, is used preferentially as a substrate for ATP production by oxidative phosphorylation. The underlying adaptations of the intermediary metabolism include (i) an increased activity of the mitochondrial matrix enzyme glutaminase, converting Gln to glutamate, (ii) formation in the mitochondria of the citric acid cycle intermediate ␣-ketoglutarate via transamination of glutamate, and (iii) the presence of malic enzyme, generating intramitochondrial pyruvate (14, 18, 19) . Here we describe the result of the above changes in mitochondrial enzyme composition and substrate utilization to TNF cytotoxicity. We show that L929 cells use Gln and not Glc as the major energy substrate and that this particular energy metabolism promotes the cytotoxic response of the cells to TNF. Our results also demonstrate that the dependence of TNF cytotoxicity on Gln is not due to the overall rate of mitochondrial respiration per se. Enzymatic pathways specifically utilized in mitochondrial oxidation of Gln appear to sensitize the mitochondria to TNF-induced perturbation of their activity and thereby amplify the resulting production of cytocidal ROI. MATERIALS AND METHODS Cell Culture-L929, a murine fibrosarcoma cell line, was grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-in-*