Anna Aronis

Potentiation of Fas-mediated apoptosis by attenuated production of mitochondria-derived reactive oxygen species

AbstractThe role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill-defined. Here, we show that ROS levels play a role in moderating Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or (mitochondrial uncoupler) and Fas-activating antibody (CH11) facilitated rapid cell death that was not associated with decreased ATP production or increased DEVDase activity and cytochrome c release. However, a decrease in cellular ROS production was associated with CH11 treatment, and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS production. Thus, decreased ROS production is correlated with enhanced cell death. A transition from state 3 to state 4 mitochondrial respiration accounted for the attenuated ROS production and membrane potential. Similar observations were demonstrated in isolated rat liver mitochondria. These data show that ROS production is important in receptor-mediated apoptosis, playing a pivotal role in cell survival.

Selenite sensitizes mitochondrial permeability transition pore opening in vitro and in vivo: a possible mechanism for chemo-protection.

There is a known connection between selenium supplementation and chemo-protective anti-cancer activity. This biological phenomenon may be due to the ability of selenium to instigate cellular apoptosis. However, the mechanism by which selenium promotes cellular apoptosis is still obscure. The present study shows that sodium selenite, a common dietary form of selenium, promotes the mitochondrial permeability transition (MPT) in isolated rat liver mitochondria both in vitro and following in vivo supplementation. A low selenium concentration (0.1-10 microM) strongly induced cyclosporin A-sensitive mitochondrial swelling. Selenium also promoted both calcium release from the matrix of isolated mitochondria and uncoupled respiration. The MPT-inducing effect of selenium provoked the release of cytochrome c, a pro-apoptotic factor, into the incubation medium. Selenium did not increase intra-mitochondrial peroxide production, but did consume endogenous mitochondrial glutathione. Moreover, the effect of MPT induction was greatly potentiated in the presence of thiol-bearing antioxidants, e.g. N -acetylcysteine and lipoamide. During MPT progression, selenium induced NADH oxidation via electron acceptance from complex I. Supplementation for 20 days with 16 p.p.m. selenium in the drinking water of rats increased the propensity of mitochondria to undergo the MPT. More marked mitochondrial swelling in response to calcium and lower calcium-uptake capacity were observed, in the absence of liver damage or the intensive oxidation of reduced glutathione. Therefore selenite facilitates MPT pore opening via its thiol- and NADH/complex I-dependent reduction, and thereby may provide chemo-protection by potentiation of the capacity of the mitochondria to regulate programmed cell death. Data from the present study suggest that selenium can regulate important mitochondrial functions both in vivo and in vitro.

Membrane depolarization of isolated rat liver mitochondria attenuates permeability transition pore opening and oxidant production.

It has been suggested that one key feature of mitochondrial permeability transition (PT) regulation is its control by the proton electrochemical gradient and that depolarization favors pore opening, swelling, and reactive oxygen species (ROS) production. Moreover, ROS have been suggested to facilitate the process of mitochondrial PT pore opening. The aim of this study was to show that collapsing the mitochondrial membrane potential with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), at concentrations of up to 10 microM, does not induce mitochondrial swelling and, in fact, stabilizes mitochondria exposed to oxidant, protecting them from tert-butyl hydroperoxide (TBH)-induced high-amplitude swelling. FCCP decreased polyethylene glycol-induced mitochondrial contraction following exposure to TBH, indicating closing of the PT mega-channel. In the presence of the calcium uniporter inhibitor ruthenium red, FCCP induced PT due to suppression of calcium efflux. Under PT-favorable conditions, ROS production was evaluated in mitochondria following treatments with TBH, inorganic phosphate, or FCCP (with or without ruthenium red). FCCP alone and in combination with ruthenium red attenuated mitochondria-derived ROS production. FCCP also decreased the augmented ROS production induced by inorganic phosphate. It is concluded that mitochondrial depolarization protects and prevents high-amplitude swelling and PT-derived ROS production.

Lipotoxic effects of triacylglycerols in J774.2 macrophages

OBJECTIVE Triacylglycerols (TGs) are being considered as an independent risk factor in atherosclerosis and metabolic syndrome, acting by dysregulation of the TG/high-density lipoprotein axis. Accumulation of lipids in subendothelial space attracts macrophages, leading to atherosclerotic plaque formation and increased plaque instability due to formation of foam cells and macrophage death. The aim of this study was to evaluate lipotoxic effects in macrophages caused by TG uptake. METHODS J774.2 macrophages were exposed to soybean or olive oil-based lipid emulsions as a source of TGs (1 mg/mL) in a presence or absence of lipase inhibitor paraoxon (20 microM) or to bovine serum albumin-complexed palmitic (150 microM), linoleic (600 microM), and oleic (600 microM) fatty acids. RESULTS The results demonstrated accumulation of TGs, G1/S arrest, and cell death with necrotic morphologic features after exposure to TG emulsions. These effects were prevented by treatment with an antioxidant N-acetyl-cysteine (0.5 mM). Paraoxon inhibited intracellular TG degradation but did not prevent lipotoxicity and cell death. Olive oil TG triggered macrophage death in a manner similar to soybean oil. Treatment of the macrophages with free fatty acid, mainly with palmitic acid, showed a reactive oxygen species-independent cell death pathway, which was different from that of TG and was not prevented by N-acetyl-cysteine. CONCLUSION This study shows a direct lipotoxic pathway for TG molecules in macrophages, which is not associated with degradation of TG molecule to free fatty acids. This study for the first time can explain at a cellular level how TGs as an independent risk factor aggravate atherosclerotic outcomes.

Mitochondrion-mediated apoptosis is enhanced in long-lived αMUPA transgenic mice and calorically restricted wild-type mice

Caloric restriction (CR) can extend the life-span of multiple species and is the only intervention known to attenuate aging in mammals. Mechanisms mediating the CR influence are as yet unclear. To get insight into these mechanisms we took advantage of alphaMUPA transgenic mice that have previously been reported to spontaneously eat less and live longer compared with their wild-type (WT) control. Here we report that mitochondria isolated from young adult alphaMUPA livers showed increased susceptibility to calcium-induced high-amplitude swelling, increased cytochrome c release and enhanced glutathione levels. Furthermore, young adult alphaMUPA mice showed significantly enhanced caspase-3 activity in liver homogenates, increased fraction of apoptotic hepatocytes, and a lower level of serum IGF-1. In addition, alphaMUPA mice showed a decreased rate of spontaneously occurring lung tumors at an old age. Short-term (8 weeks) calorically restricted WT mice also showed an increase of mitochondrial swelling and caspase-3 activity compared with ad libitum (AL) fed WT mice. These results provide the first indication that CR can enhance mitochondrion-mediated apoptotic capacity. Collectively, the results are consistent with the possibility that long lasting, moderately increased apoptotic capacity, possibly linked in part to IGF-1 and GSH modulation, could play a role in the CR-induced anti-aging influence in mice.

Mitochondrial state 3 to 4 respiration transition during Fas-mediated apoptosis controls cellular redox balance and rate of cell death.

The role of reactive oxygen species (ROS) production in death receptor-mediated apoptosis is ill defined. We show that ROS levels play a novel role in moderating the rate of cell death in Fas-dependent apoptosis. Treatment of Jurkat T cells with oligomycin (ATP-synthase inhibitor) or FCCP (mitochondrial uncoupler) and Fas activating antibody (CH11), facilitated rapid cell death. ATP levels, DEVDase activity and cytochrome c release were not account for the synergistic killing effect. However, a decrease in cellular ROS production was associated with CH11 treatment and combinations of CH11 with oligomycin or FCCP further inhibited cellular ROS levels. Thus, decreased ROS production is correlated with accelerated cell death. A transition from state 3 to state 4 mitochondrial respiration following apoptotic stimuli accounted for an attenuated membrane potential and as a results mitochondria-derived ROS production capacity diminished. Similar observations were demonstrated in isolated rat liver mitochondria. Transfection with mitochondrial targeted catalase inhibited mitochondrial ROS production and potentiated cell death. These data show that ROS production is important in receptor-mediated apoptosis and may play a pivotal role in cell survival.

Triacylglycerol-induced impairment in mitochondrial biogenesis and function in J774.2 and mouse peritoneal macrophage foam cells.

The aim of this study was to detect mitochondrial alterations in J774.2 macrophages and mouse peritoneal macrophages (MPM) foam cells. J774.2 and MPM cells were exposed to triacylglycerol (TG) emulsion (1 mg/ml) for induction of fat accumulation. Impairment of mitochondrial function was reflected by reduced cellular ATP production and decreased expression of subunits of mitochondrial complexes I and III. The expression of subunit IV of complex IV remained unchanged, however, the content of its precursor in cells increased. Inhibitors of mitochondrial complexes, rotenone (0.1 microM) and myxothiazol (25 nM), protected the viability in TG-loaded macrophages. The exposure to TG caused downregulation of PPARgamma coactivator (PGC)-1alpha and nuclear respiratory factor (NRF)-1. Activation of peroxisome proliferator-activated receptors attenuated reactive oxygen species production in the foam cells. Treatment with antioxidant N-acetylcysteine (NAC) prevented lipid-mediated mitochondrial and cellular damage. In conclusion, this study demonstrates the important role of mitochondrial biogenesis dysfunction in TG-induced lipotoxicity in macrophages.