Mechanism underlying oxidative stress-mediated lipotoxicity: Exposure of J774.2 macrophages to triacylglycerols facilitates mitochondrial reactive oxygen species production and cellular necrosis

The aim of this study was to elucidate death pathways in macrophages resulting from exposure to triacylglycerols (TG), mechanisms which may be relevant to the development of atherosclerosis. A commercial TG emulsion (lipid emulsion, LE; 0.1-1.5 mg lipids/ml) was added to J774.2 cells in culture. Within the first 24 h after TG treatment, cellular reactive oxygen species (ROS) levels were strongly elevated and basal caspase-3 activity was attenuated. In contrast, after 48 h, ROS production was arrested. TG-mediated ROS production was demonstrated to be via mitochondrial complex 1 of the electron-transfer chain since the inhibitor of complex 1 rotenone significantly attenuated the cellular ROS levels in TG-treated cells. The TG effect culminated in cell death, with no caspase-3 activation. We therefore evaluated the effect of TG on apoptotic cells showing high caspase activity. TG induced elevated ROS levels and suppressed caspase-3 in apoptotic cells pretreated for 24 h with cycloheximide. Dual staining with propidium iodide and Annexin V followed by flow cytometric analysis showed that TG facilitated cell death with clear necrotic characteristics. To elucidate whether the necrotic cell death process is indeed oxidant dependent, antioxidant protection was studied. Treatment with N-acetylcysteine (NAC) (0.5 mM), ascorbic acid (0.5 mM), and resveratrol (0.2 mM) protected against the TG lipotoxic effect, while, surprisingly, lipophilic antioxidants did not. The combination of NAC, ascorbic acid, and resveratrol, each at much lower concentrations, had a synergistic protective effect. In conclusion, we show here for the first time that exposure to TG can directly regulate lipotoxicity in macrophages by inducing mitochondria-mediated prolonged oxidative stress; this, in turn, can inactivate the apoptotic caspase system, resulting in necrotic cell death which can be prevented by specific antioxidants.