Metformin, a drug widely used in the treatment of Type II diabetes, has recently received attention owing to new findings regarding its mitochondrial and cellular effects. In the present study, the effects of metformin on respiration, complex 1 activity, mitochondrial permeability transition, cytochrome c release and cell death were investigated in cultured cells from a human carcinoma-derived cell line (KB cells). Metformin significantly decreased respiration both in intact cells and after permeabilization. This was due to a mild and specific inhibition of the respiratory chain complex 1. In addition, metformin prevented to a significant extent mitochondrial permeability transition both in permeabilized cells, as induced by calcium, and in intact cells, as induced by the glutathione-oxidizing agent t-butyl hydroperoxide. This effect was equivalent to that of cyclosporin A, the reference inhibitor. Finally, metformin impaired the t-butyl hydroperoxide-induced cell death, as judged by Trypan Blue exclusion, propidium iodide staining and cytochrome c release. We propose that metformin prevents the permeability transition-related commitment to cell death in relation to its mild inhibitory effect on complex 1, which is responsible for a decreased probability of mitochondrial permeability transition.

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca 2؉ -sensitive channel that plays a key role in different models of cell death. Because functional links between the PTP and the respiratory chain complex I have been reported, we have investigated the effects of rotenone on PTP regulation in U937 and KB cells. We show that rotenone was more potent than cyclosporin A at inhibiting Ca 2؉ -induced PTP opening in digitonin-permeabilized cells energized with succinate. Consistent with PTP regulation by electron flux through complex I, the effect of rotenone persisted after oxidation of pyridine nucleotides by duroquinone. tert-Butyl hydroperoxide induced PTP opening in intact cells (as shown by mitochondrial permeabilization to calcein and cobalt), as well as cytochrome c release and cell death. All these events were prevented by rotenone or cyclosporin A. These data demonstrate that respiratory chain complex I plays a key role in PTP regulation in vivo and confirm the importance of PTP opening in the commitment to cell death.Studies over the past few years have led to the recognition that, in addition to their established role in energy metabolism, mitochondria are main actors of cell death (see Refs. 1 and 2 for recent reviews). Mitochondrial proapoptotic factors such as cytochrome c, apoptosis-inducing factor, and smac/diablo are normally confined to the mitochondrial intermembrane space. Once released into the cytosol, cytochrome c binds to Apaf-1, which prompts activation of caspases in the presence of ATP or dATP (3, 4), and smac/diablo counteracts the inhibitory effect of inhibitor of apoptotic proteins on caspases activity (5, 6), whereas apoptosis-inducing factor activates nuclear endonucleases (7,8). The mechanisms by which the outer mitochondrial membrane becomes permeable to these proapoptotic proteins are not totally understood, but evidence suggests that two non-mutually exclusive pathways may be concerned (1, 2). One relies on outer membrane channel(s) involving Bcl-2 family proteins, whereas the other is due to outer membrane rupture secondary to mitochondrial swelling and implicates an inner membrane channel, the permeability transition pore (PTP). 1 PTP opening in vitro leads to collapse of the proton-motive force, disruption of ionic homeostasis, mitochondrial swelling, and release of intermembrane space proteins, especially cytochrome c (9 -11). Involvement of the PTP in the commitment to cell death is supported by a large body of evidence based on the protective effect of two PTP inhibitors, namely cyclosporin A (CsA) (reviewed in Refs. 12-15) and bongkrekic acid (BA) (e.g. see , in several models of cell death. PTP opening in vivo is commonly appreciated as a CsA-sensitive mitochondrial depolarization but can also be visualized directly by fluorescence imaging using CsA-sensitive calcein diffusion through the mitochondrial inner membrane (22)(23)(24)(25).PTP regulation has been studied extensively in rat liver mitochondria. Although it is generally assumed that there is ...

Inhibition of the mitochondrial permeability transition pore (PTP) has proved to be an effective strategy for preventing oxidative stress-induced cell death, and the pore represents a viable cellular target for drugs. Here, we report that inhibition of complex I by rotenone is more effective at PTP inhibition than cyclosporin A in tissues that express low levels of the cyclosporin A mitochondrial target, cyclophilin D; and, conversely, that tissues in which rotenone does not affect the PTP are characterized by high levels of expression of cyclophilin D and sensitivity to cyclosporin A. Consistent with a regulatory role of complex I in the PTP-inhibiting effects of rotenone, the concentrations of the latter required for PTP inhibition precisely match those required to inhibit respiration; and a similar effect is seen with the antidiabetic drug metformin, which partially inhibits complex I. Remarkably (i) genetic ablation of cyclophilin D or its displacement with cyclosporin A restored PTP inhibition by rotenone in tissues that are otherwise resistant to its effects; and (ii) rotenone did not inhibit the PTP unless phosphate was present, in striking analogy with the phosphate requirement for the inhibitory effects of cyclosporin A [Basso et al. (2008) J. Biol. Chem. 283, 26307-26311]. These results indicate that inhibition of complex I by rotenone or metformin and displacement of cyclophilin D by cyclosporin A affect the PTP through a common mechanism; and that cells can modulate their PTP response to complex I inhibition by modifying the expression of cyclophilin D, a finding that has major implications for pore modulation in vivo.