Palmitate induces apoptosis via a direct effect on mitochondria

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Apoptosis 1999; 4: 81–87 ° C 1999 Kluwer Academic Publishers

Palmitate induces apoptosis via a direct effect on mitochondria M. A. de Pablo, S. A. Susin, E. Jacotot, N. Larochette, P. Costantini, L. Ravagnan, N. Zamzami and G. Kroemer Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 420, 19 rue Guy Moquet, ˆ F-94801 Villejuif, France

The fatty acid palmitate can induce apoptosis. Here we show that the palmitate-induced dissipation of the mitochondrial transmembrane potential (∆Ψm ), which precedes nuclear apoptosis, is not prevented by inhibitors of mRNA synthesis, protein synthesis, caspases, or pro-apoptotic ceramide signaling. However, the mitochondrial and nuclear effects of palmitate are inhibited by overexpression of anti-apoptotic proto-oncogene product Bcl-2 and exacerbated by 2-bromo-palmitate as well as by carnitine. The cytoprotective actions of Bcl-2, respectively, is not antagonized by etomoxir, an inhibitor of carnitine palmitoyl transferase 1 (CPT1), suggesting that the recently described physical interaction between CPT1 and Bcl-2 is irrelevant to Bcl-2-mediated inhibition of palmitate-induce apoptosis. When added to purified mitochondria, palmitate causes the release of soluble factors capable of stimulating the apoptosis of isolated nuclei in a cell-free system. Mitochondria purified from Bcl-2 overexpressing cells are protected against the palmitatestimulated release of such factors. These data suggest that palmitate causes apoptosis via a direct effect on mitochondria.

Keywords: Bcl-2; carnitine; fatty acids; mitochondrial megachannel; permeability transition pore.

(Received 17 November 1998; accepted 24 December 1998)

Introduction For long, it has been assumed that the activation of endonucleases and specific proteases (caspases) would not only constitute a distinctive feature of apoptosis but also determine the decisive step (the ‘decision to die’ or ‘commitment point’) which distinguishes living from dying cells. It is now clear that, in most models of apoptosis, inhibition of nucleases and caspases does not prevent cytolysis, indicating that the ‘decision to die’ is taken before Correspondence to: G. Kroemer, 19 rue Guy Moquet, ˆ B.P. 8, F-94801 Villejuif, France. Tel: 33-1-49 58 35 13; Fax: 33-1-49 58 35 09; email: [email protected]

catabolic enzymes are activated and that the activation of such enzymes is a by-product of the cell death process rather than a regulatory event.1−3 Recently, it has become clear that mitochondria are major players in the cell death decision process of mammalian cells. Four major arguments suggest the involvement of mitochondria in apoptosis. First, kinetic studies indicate that mitochondria undergo major changes in membrane integrity before classical signs of apoptosis become manifest. Thus, in several models of cell death, a dissipation of the mitochondrial transmembrane potential (19m ) and/or a disruption of outer mitochondrial membrane barrier function with release of soluble intermembrane proteins4−12 precedes other manifestations of apoptosis, including caspase activation,13 subtle changes in plasma membrane lipid orientation,14 redox potentials,15,16 and ion homeostasis.17,18 Second, cell free systems demonstrate that mitochondrial intermembrane proteins are rate limiting for the activation of caspases and endonucleases in cell extracts.4−6,8,9,19 Third, pharmacological data indicate cyclosporin A and bongkrekic acid, two drugs which inhibit the mitochondrial permeability transition (PT) pore can prevent the mitochondrial and post-mitochondrial manifestations of apoptosis.4,20−24 Fourth, extensive analysis of the proto-oncogene product Bcl-2 and of its apoptosis-inhibitory or apoptosis-inducing homologues has revealed that they regulate apoptosis via an effect on mitochondria and that they can influence PT pore opening.4,6,8−10,25−28 Within the PT pore complex, Bax interacts with the adenine nucleotide translocator (ANT), a member of the mitochondrial carrier protein family, to regulate mitochondrial membrane permeability and cell death.24 Palmitate and similar saturated long-chain acyl fatty acids have been previously reported to bind to the ANT,29,30 to uncouple the respiratory chain30−33 and to induce opening of the PT pore.34,35 Palmitate is also known to induce apoptosis via a mechanism which is not completely elucidated.36 It has been reported that palmitate can enhance the generation of the pro-apoptotic Apoptosis · Vol 4 · No 2 · 1999


M. A. de Pablo et al.

second messenger ceramide via a reaction that is stimulated by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1),36 another member of the mitochondria carrier protein family. This enzyme catbreak alyzes a reaction which is rate-limiting for the import of CoA-conjugated fatty acids into the mitochondrial matrix. Intrigued by this finding, as well as by the observation that CPT1 physically interacts with Bcl-2,37 we decided to reexamine the mechanism via which palmitate induces apoptosis. Here, we show that palmitate-induced apoptosis is probably not mediated via ceramide but rather involves a direct, Bcl-2-inhibited effect on mitochondria.

2 mM NaCl, 2.5 mM PO4 H2 K, 0.5 mM EGTA, 2 mM Cl2 Mg, 5 mM pyruvate, 0.1 mM phenylmethyl sulfonylfluoride, 1 mM dithiotreitol, 10 mM HEPES-NaOH, pH 7.4; reagents from Sigma). Osmotic shock of mitochondria leading to the disruption of the outer but not of the inner membrane was performed according to standard methods.41 AIF activity in the supernatant of mitochondria was tested on HeLa cell nuclei, as described.42 Briefly, AIF-containing supernatants of mitochondria were added to purified HeLa (90 min, 37◦ C), which were stained with propidium iodide (PI) and analyzed in an Elite II cytofluorometer (Coulter) to determine the frequency of hypoploid nuclei.

Materials and methods Induction and inhibition of apoptosis. 2B4.11 T cell hybridoma cell lines stably transfected with a SFFV.neo vector containing the human bcl-2 gene or the neomycin resistance gene (Neo) only38 were kindly provided by Jonathan Ashwell (Bethesda, NIH). Cells were cultured in RPMI 1640 medium supplemented with 10% FCS, L-glutamine, HEPES and antibiotics, in the presence of the indicated concentrations of palmitate (Sigma, St. Louis, MO; diluted as described 36 ) to induce apoptosis. C2 ceramide (Sigma, 50 µM), A23187, ionomycin, and carbonyl cyanide m-chlorophenylhydrazone (CCCP; 50 µM, Sigma) served as positive controls. Cells were cultured with these reagents, alone or in combination with cycloheximide (35 µM, Sigma), actinomycin D (3 µM, Sigma), the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk; 100 µM, Enzyme Systems, Dublin, CA, USA), 2-bromo-palmitate (100 µM, Sigma), (+)-2- [6-(4chlorophenoxy)hexyl]-oxiranecarboxylate (generic name: etomoxir; 100 µM; Research Biochemicals International, Natick, MA, USA), sphingosine-1-phosphate (5 µM) or L-carnitine (1 mM, Sigma-Tau). Cytofluorometric detection of apoptosis-associated changes. To evaluate the mitochondrial transmembrane potential (19m ) and superoxide anion generation, cells (5 × 105/ml) were incubated with 3,30 -dihexyloxacarbocyanine iodide (DiOC6 (3); Molecular Probes; 40 nM in PBS)15 and hydroethidine (HE; Molecular Probes; 2 µM)16 for 15 min at 37◦ C, followed by analysis on an Epics cytofluorometer (Coulter, Miami, FL). The frequency of cells having undergone chromatinolysis (subdiploid cells) was determined by ethanol permeabilization, followed by propidium iodine (PI) staining.39 Cell-free system of nuclear apoptosis. Mitochondria were purified by Percoll density centrifugation as described40 and were stored on ice in B buffer (400 mM mannitol; 10 mM PO4 H2 K, 5 mg/ml BSA, 50 mM TRIS-HCl, pH 7.2) for up to 2 h. Mitochondria were spun down and washed twice in CFS buffer (220 mM mannitol; 68 mM sucrose, 82 Apoptosis · Vol 4 · No 2 · 1999

Results and discussion Bcl-2 inhibits palmitate-induced apoptosis. As other inducers of apoptosis, palmitate caused a dose-dependent reduction of the 19m (detected by the potential-sensitive dye DiOC6 (3), green fluorescence), followed by an increase in the production of reactive oxygen species capable of oxidizing the non-fluorescent compound HE to the red fluorescent dye Eth (Figure 1a). Both these effects, as well as the palmitate-driven induction of nuclear apoptosis (Figure 1b; quantified as a reduction of DNA content measured with the DNA-intercalating dye PI) were inhibited in cells overexpressing the anti-apoptotic proto-oncogene Bcl-2 (Figure 1, a–c). Kinetic experiments indicate that the 19m collapse precedes nuclear apoptosis induced by palmitate (Figure 1d). These data indicate that palmitate induces bona fide apoptosis and confirm the strong correlation between nuclear and mitochondrial parameters of apoptosis observed in other models of apoptosis induction. Palmitate affects mitochondrial function in a protein-synthesis and caspase-independent fashion. As demonstrated in Figure 2, the inhibition of RNA transcription and protein synthesis by CHX and actinomycin D (which by itself has some toxic effects) did not inhibit palmitateinduced apoptosis, neither at the level of 19m loss (Figure 2b) nor at the level of nuclear DNA loss (Figure 2a). In contrast, it appears that the broad-spectrum caspase inhibitor Z-VAD.fmk prevents palmitate-induced nuclear apoptosis (Figure 2a), in line with the notion that apoptotic DNA fragmentation is a strictly caspase-dependent process.2,43−45 However, Z-VAD.fmk did not prevent the palmitate-induced 19m dissipation and only partially reduced ROS generation (Figure 2b), indicating that the 19m loss indicative of palmitate-induced cell damage does not rely on the activation of Z-VAD.fmk-inhibitible caspases. Putative relationship between palmitate, carnitine palmitoyltransferase I, and ceramide. In accord with previously published data,36 we observed that the non-hydrolyzable

Mitochondrial site of action for palmitate Figure 1. Effects of palmitate on apoptosis-associated parameters. 2B4.11 T cell hybridoma cell lines stably transfected with a SFFV.neo vector containing the human bcl-2 gene or the neomycin resistance gene (Neo) only were treated with 300 µM palmitate for 6 h, followed by cytofluorometric detection of the 19m , ROS generation (a), and nuclear DNA content (b). The effects of palmitate, as measured after 6 h, are dose dependent (c). Results are representative of five independent determinations. The kinetics of the effect of lonidamine on Neo cells have been measured for 19m loss, ROS generation, and nuclear DNA content (d).

palmitate derivative 2-bromo-palmitate (which by itself has no apoptosis-inducing effect) enhanced the apoptogenic effect of palmitate (Figure 3). In contrast, etomoxir, a specific inhibitor of CPT1, enhanced the 19m loss induced by palmitate (Figure 3b), yet had no effect on palmitate-triggered nuclear apoptosis (Figure 3a). While 2-bromo-palmitate reverses the apoptosis-inhibitory effects of Bcl-2, no such effect was observed for etomoxir (Figure 3). Thus, specific CPT1 inhibition does not interfere with the apoptosis-inhibitory effect of Bcl-2. Interestingly, L-carnitine can increase the cytotoxic effect of palmitate, both in Neo control cells and in Bcl-2overexpressing cells where it neutralizes the death antagonistic effect of Bcl-2. Moreover, we found that sphingosine-1-phosphate, a potent antagonist of ceramideinduced apoptosis,46,47 failed to antagonize-palmitateinduced cell death in conditions in which it effectively

Figure 2. Inhibitors of caspases or macromolecule synthesis fail to inhibit the 19m -dissipating effect of palmitate. Neo control cells were stimulated for 6 h in the presence or absence of 300 µM palmitate, 100 µM Z-VAD.fmk, 1 µM actinomycin D (Act. D), and/or 35 µM cycloheximide (CHX), followed by ethanol permeabilization, PI staining, and determination of nuclear apoptosis (a) or alternatively, staining with DiOC6 (3) and HE (b), as indicated in Materials and Methods. Shaded areas indicate the presence of the corresponding substance. One out of three experiments yielding similar results is shown.

blocked C2-ceramide induced mitochondrial and nuclear signs of apoptosis (Figure 4). This observation indicates that ceramide, which is overproduced after stimulation of cells with palmitate (Ref. 36 and data not shown), is not responsible for the lethal effect of palmitate. Bcl-2 prevents the palmitate-induced release of apoptogenic factors from isolated mitochondria. As a result of the above experiments, it appears that the palmitate-mediated mitochondrial effects are independent of macromolecule synthesis, caspase activation, and ceramide generation, yet are inhibited by Bcl-2. Given the ever increasing evidence suggesting that Bcl-2 acts on mitochondria to inhibit apoptosis,28 we speculated that palmitate might directly act on mitochondria to trigger the apoptotic process. We therefore incubated isolated mitochondria from Neo control Apoptosis · Vol 4 · No 2 · 1999


M. A. de Pablo et al. Figure 3. Effects of 2-bromopalmitate, etomoxir, or L-carnitine on palmitate induced apoptosis. Neo- or Bcl-2-transfected cells were cultured with palmitate (100 or 300 µM), 2-bromopalmitate (100 µM), etomoxir (100 µM), and/or L-carnitine (1 mM added each hour starting from beginning of the culture) during 6 h. Nuclear apoptosis (a) and the loss of the 19m (b) were measured. This experiment has been repeated three times with comparable results.

cells with palmitate and monitored their supernatants for the presence of soluble factor(s) capable of inducing nuclear apoptosis (measured with PI) in a cell-free system. Indeed, palmitate itself does not provoke nuclear apoptosis in such a system (Figure 5). However, the supernatant of palmitate-stimulated mitochondria (but not control mitochondria) contains such an apoptosis-inducing factor (AIF) activity (Figure 5). This effect was observed only for mitochondria from vector-only-transfected control cells. In strict contrast, mitochondria from Bcl-2 transfected cells, which contain an elevated amount of Bcl-2 in the outer membrane,6 fail to release AIF in response to palmitate, although they do contain detectable AIF activity when subjected to osmotic lysis (Figure 5). These data underscore that palmitate has a direct pro-apoptotic effect on mitochondria and that this effect is alleviated by the local action of Bcl-2. Bcl-2 partially inhibits ionophore-induced apoptosis. Palmitate has been previously shown to act on several different proteins from the mitochondrial transport protein family to cause uncoupling.29−35,48,49 If this uncoupling effect was responsible for the apoptogenic effect of palmitate, which is partially antagonized by Bcl-2 (Figure 1), then Bcl-2 should attenuate ionophore-induced apoptosis. 84 Apoptosis · Vol 4 · No 2 · 1999

Figure 4. Sphingosin-1-phosphate interferes with ceramide- but not with palmitate-induced apoptosis. Neo control cells were cultured for 6 h with 300 µM palmitate, 25 µM C2 ceramide and/or 5 µM sphingosin-1-phosphate, as indicated, and the frequency of subdiploid (a), DiOC6 (3)low and/or He->Ethhigh (b) cells was determined. Results are representative of three different experiments.

Indeed, we observed that Bcl-2 reduces nuclear apoptosis triggered by low doses of the protonophore CCCP, the K+ -specific ionophore valinomycin, and the Ca2+ specific ionophore A23187 (Figure 6). These data are compatible with a putative ionophore-like uncoupling action of palmitate accounting for its pro-apoptotic effect.

Conclusions The data contained in this paper are compatible with the hypothesis that palmitate triggers apoptosis via a specific effect on mitochondria. First, palmitate induces an early reduction of the 19m (Figure 1). Second, a series of agents that interfere with mRNA or protein synthesis, caspase activation or pro-apoptotic ceramide signaling fail to reduce the effect of palmitate on mitochondria (Figures 2–4). Third, the only manipulation which effectively reduces the palmitate effect on mitochondria is transfection-enforced expression of Bcl-2, an

Mitochondrial site of action for palmitate Figure 5. Palmitate induces the mitochondrial release of a factor causing nuclear apoptosis in vitro. Mitochondria were purified from Neo or Bcl-2 cells and were kept in the absence (Co.) or presence of palmitate (200 µM) for 60 min, followed by centrifugation and recovery of the supernatant. These supernatants were added undiluted to isolated HeLa nuclei, followed by another step of incubation (60 min), and the determination of DNA content using PI staining. Alternatively mitochondria were subjected to osmotic shock in order to determine the presence of apoptogenic factors. This result has been confirmed in an independent experiments for several doses of palmitate (200, 300 and 400 µM).

Figure 6. Bcl-2-mediated inhibition of ionophore-induced apoptosis. Cells were cultured for 18 h in the presence of the indicated dose of CCCP, valinomycin, or A23187, followed by quantitation of cells with a subdiploid DNA content.

Palmitate and other lipids can be generated locally in mitochondria by a phospholipase. A mitochondrial phospholipase A2 is activated in at least some pathways of apoptosis, including in Ca2+ 50 and tumor necrosis factorα-triggered cell death.51 Free fatty acids accumulate in mitochondria in some circumstances, e.g., following oxidative stress or exposure to Ca2+ ,52 and may limit the PT pore inhibitory effect of cyclosporin A.52 Cyclosporin A is a transient inhibitor of PT pore opening in vitro, in isolated mitochondria, and can prevent the 19m dissipation accompanying early apoptosis.53 The probable physiological relevance of phospholipase A2 activation for cell death is underlined by the observation that cyclosporin A and phospholipase A2 inhibitors such a trifluoperazine or aristolochic acid have a synergistic, stable inhibitory effect on both PT pore opening and apoptosis induced by oxidative stress,54 tumor necrosis factor-α 22 and overexpression of the Bcl-2-antagonist Bax.23 As shown here, Bcl-2, which also inhibits PT pore opening, can prevent mitochondrial effects of palmitate, both in cells (Figure 1) and in isolated mitochondria (Figure 5). This may explain why Bcl-2, at difference with cyclosporin A, has a long-term inhibitory effect on the PT pore and can provide true cytoprotection, including in response to continuous pro-apoptotic signals. Irrespective of these theoretical possibilities, it appears clear that palmitate-like fatty acids may act as an endogenous stress signals on mitochondria and that the apoptosis-inhibitory agent Bcl-2 can prevent their local action.


apoptosis-inhibitory protein whose mitochondrial effects are well documented. Indeed, it appears that the local presence of Bcl-2 also prevents the release of apoptogenic factors from isolated mitochondria treated with palmitate (Figure 5). It has previously been documented that palmitate can uncouple mitochondria, an effect that may involve a direct action on the ANT29,30 and perhaps other proteins from the mitochondrial transport family including the aspartate/glutamate antiporter48,49 and the dicarboxylate carrier.33 As shown here, specific inhibition of a further member of the mitochondrial transport family, CPT1, by etomoxir does not abolish the Bc-2-mediated inhibition of apoptosis, at least in the T cell hybridoma cell lines employed in this study. In strict contrast, we found that 2-bromo-palmitate and carnitine can enhance the toxic effect of palmitate, including in Bcl-2 overexpressing cells (Figure 3). The mechanism accounting for this effect is elusive and requires further investigation.

Supported by ANRS, ARC, CNRS, FRM, LNC, INSERM, and a grant from Sigma Tau. Manuel A. de Pablo was on leave of absence of the University of Ja´en, Spain. Santos A. Susin, Etienne Jacotot and Paola Costantini received fellowships from the European Commission, Sidaction, and ARC, respectively.

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