Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation

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Prostaglandins, Leukotrienes and Essential Fatty Acids 78 (2008) 147–155 www.elsevier.com/locate/plefa

Palmitate-induced NO production has a dual action to reduce cell death through NO and accentuate cell death through peroxynitrite formation Simon W. Rabkin, Shaun S. Klassen University of British Columbia, 9th Floor, 2775 Laurel Street, Vancouver, BC, Canada V5Z 1M9 Received 27 April 2007; accepted 23 September 2007

Abstract The objective of this study was to determine the role of palmitate-induced stimulation of nitric oxide synthase (NOS) on palmitate-induced cell death, specifically distinguishing the effects of the subtype NOS2 from NOS3, defining the effect of NO on mitochondria death pathways, and determining whether palmitate induces peroxynitrite formation which may impact cardiomyocyte cell survival. Cardiomyocytes from embryonic chick hearts were treated with palmitate 300–500 mM. Cell death was assessed by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The ability of palmitate to induce NO production and its consequences were tested by using the NOS inhibitor 7-nitroindazole (7-N) and the peroxynitrite scavenger (5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) chloride) (FeTPPS). The effect of palmitate on the mitochondria was assessed by Western blotting for cytochrome c release into the cytosol, and assessment of mitochondrial transmembrane potential (DCm) by 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-benzimidazolyl-carbocyanine iodide staining and immunocytochemistry. The NOS inhibitor 7-N, which is selective for NOS2 and not for NOS3, significantly (po0.05) increased palmitate-induced cell death. In contrast, 7-N did not alter cell death produced by the combination of potassium cyanide and deoxyglucose, which, respectively, inhibit glycolysis and oxidative phosphorylation. The mitochondrial actions of palmitate, specifically palmitate-induced translocation of mitochondrial cytochrome c to cytosol and loss of mitochondrial transmembrane potential, were not altered by pretreatment with 7-N. FeTPPS, which isomerizes peroxynitrite to nitrate and thereby reduces the toxic effects of peroxynitrite, produced a significant reduction in palmitate-induced cell death. In summary, these data suggest that palmitate stimulates NO production, which has a dual action to protect against cell death or to induce cell death. Palmitate-induced cell death is mediated, in part, through NO generation, which leads to peroxynitrite formation. The protective effect of NO is operative through stimulation of NOS2 but not NOS3. The actions of NO on palmitate-induced cell death are independent of mitochondrial cell death pathways. r 2008 Elsevier Ltd. All rights reserved.

1. Introduction Fatty acid-induced cell death has recently generated considerable interest because it potentially plays a causative role in a spectrum of diseases—from diabetes mellitus to heart failure and renal disease [1–4]. While the saturated fatty acid palmitate (C16:0) is a common essential element of cellular structure, high concentrations induce cell death in many cell types, including the Corresponding author. Tel.: +1 604 875 5847; fax: +1 604 875 5849. E-mail address: [email protected] (S.W. Rabkin).

0952-3278/$ - see front matter r 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2007.09.003

cardiomyocyte element of the heart [5–9]. One important modulator of the action of palmitate on cell death is nitric oxide (NO). We have shown that in cardiomyocytes, palmitate induces a significant concentrationdependent increase in nitric oxide synthase (NOS) activity measured by the conversion of [3H]-arginine to [3H]-citrulline and with the resultant NO-modulating palmitate-induced cell death [10]. NOS, which facilitates NO generation via a five-electron oxidation of a terminal guanidinium nitrogen on the amino acid L-arginine, exists mainly as three different isoforms—NOS type I (NOS1 or nNOS), type II (NOS2 or iNOS) or type III (NOS3 or eNOS) [11,12]. In addition

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there may be a distinct mitochondrial NOS (mtNOS) [13,14] or the NOS in mitochondrial maybe a NOS1-a splice variant [15]. In the heart, the functional role of NO depends, in part, on the spatial proximity of the target to the active NOS subtype as well as the surrounding microenvironment. In cardiac myocytes, NOS1 is preferentially localized to the sarcoplasmic reticulum (SR), the organelle responsible for excitation–contraction coupling [16,17] and depresses both systolic and diastolic cardiac functions [17,18]. NOS2 is localized to contractile fibers, the plasma membrane T-tubules, nuclear envelope, mitochondria and Golgi complex [19]. NOS3 is preferentially localized to cell-membrane caveolae, organelles that link extracellular hormones with appropriate intracellular signalling pathways [17]. In cardiomyocytes, palmitate induces a significant increase in cellular NOS2 and NOS3, determined by immunocytochemistry and Western blotting [10]. Which of these two NOS isoenzymes is operative in palmitate-induced cell death is unknown. The dual action of NO on cell viability, namely the ability of the same molecule to both induce as well as prevent cell death, remains confusing and not completely explained [20,21]. The extent to which the different NOS isoenzymes contribute to the cell death/cell survival paradigm for fatty acid-induced cell death has received limited attention. In ischemia-reperfusion injury, NOS2 or NOS3 each have a cardioprotective action in myocardial cell death [22–25] and both also prevent myocardial dysfunction and mortality in murine models of septic shock [26,27]. There are some exceptions, chronic cardiac-specific up-regulation of NOS2 in mice causes heart block and death [28]. It has been proposed that the cellular effect of NO is dependent on the surrounding microenvironment, which includes, among other things, the local concentrations of metal ions and reactive oxygen species [29]. The formation of peroxynitrite from the reaction of NO and superoxide anion is an attractive mechanism to explain NO-induced cell death because of the cellular damage that peroxynitrite can produce [30]. Whether this mechanism is operative in palmitate-induced cell death, however, is not certain. The objectives of this study were to determine the effect of NO and peroxynitrite on cardiomyocyte viability in palmitateinduced cell death, to define the effect of NO on mitochondria, and to examine the role of NOS in palmitate-induced cell death, specifically separating the effect of NOS2 from NOS3. 2. Methodology 2.1. Cell culture Chick embryonic ventricular cells were cultured from 7-day chick embryos from white Leghorn eggs as previously described [8]. Myocytes were maintained in

culture in medium 818A [73% DBSK (NaCl 116 mM, MgSO4 0.8 mM, NaH2PO4 0.9 mM, dextrose 5.5 mM, CaCl2 1.8 mM, NaHCO3 26 mM), 20% M199, 6% fetal calf serum, and 1% antibiotic–antimycotic (10,000 mg/ml streptomycin sulfate, 10,000 U/ml penicillin G sodium and 25 mg/ml amphotericin B)] for 72 h prior to the experiment. The proportion of myocytes at this time was at least 90% as verified by the proportions of cells showing spontaneous contraction or displaying musclespecific markers on immunohistologic examination [8]. 2.2. Cell viability: MTT assay The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay serves as an index of cell viability [7]. Cardiomyocytes were grown in multiwell microtiter plate (Falcon ]3072, Becton Dickinson, Lincoln Park, NJ, USA) at 30,000 cells per well and incubated at 37 1C for 72 h. Cardiomyocytes were then treated with various concentrations of palmitate or its diluent (ethanol), with or without NOS inhibitors or their diluent, for 24 h. MTT dye, suspended in phosphate-buffered saline (pH 7.35) (Promega, Madison, WI, USA) was added to each well 4 h before the end of the 24-h incubation period. The reactions were stopped with the addition of solubilization reagent (Promega, Madison, WI, USA) and the absorbance was measured at a wavelength of 570 nm on a multiwell plate reader (BioRad, Mississauga, Canada) as previously described [8,10]. Background absorbance of medium, in the absence of cells, was subtracted from all the raw data. All experiments were done in duplicate and the mean results for each experiment were calculated. There is a highly significant linear relationship between cell number and absorbance [8,10]. 2.3. Western blot Cardiomyocytes were incubated for 72 h at 37 1C and 5% CO2 prior to treatments. For whole cell lysates, cells were lysed with RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8). For cytosol/mitochondria separations, samples were first centrifuged at 600g for 10 min at 4 1C, and then this supernatant further centrifuged at 15 000g for 5 min at 4 1C to separate the mitochondria (pellet) from the cytosol (supernatant). The mitochondrial pellets were resuspended in RIPA buffer. Equivalent amounts of protein were then loaded on to 12% PAGE gels for electrophoresis and Western blotting as previously described [10]. Briefly, the proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk overnight at 4 1C. The membrane was then washed with tris-buffered saline containing 0.3% Tween-20 (0.3% TBST) and incubated with 1:1000 dilution of anti-cytochrome c antibodies followed

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by 1:1000 dilution of horseradish peroxidase (HRP)linked secondary antibody. Signals were detected using chemiluminescence reagents (Amersham, Piscataway, NJ, USA) on a chemiluminescence film (Kodak, Rochester, NY, USA). Densitometric analysis was performed with the Scion Image program (Scion Corporation, Maryland, USA).

Culture media, fetal calf serum, antibiotic–antimycotic were obtained from Gibco (Burlington, Canada). The MTT assay kit was obtained from Promega (Madison, WI, USA). Chemiluminescent reagents were from Amersham Biosciences (Piscataway, NJ, USA). Anticytochrome c antibodies were from BD Pharmingen (San Jose, CA, USA). 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylTM benzimidazolyl-carbocyanine iodide (DePsipher ) stain was from Trevigen Inc. (Gaithersburg, MD, USA). 7-Nitroindazole (7-N) and FeTPPS (5,10,15,20-tetrakis (4-sulfonatophenyl)porphyrinato iron (III) chloride), were from Calbiochem (San Diego, CA, USA). Chemiluminescent film was from Kodak (Rochester, NY, USA). All other chemicals were from Sigma Chemical Co. (St. Louis, MO, USA).

3. Results 3.1. 7-Nitroindazole increases palmitate-induced cell death Palmitate induced a concentration-dependent increase in cell death in these cardiomyocytes (Fig. 1), consistent with our previous findings [6–8]. In order to test the hypothesis that selective inhibition of NOS alters palmitate-induced cell death, the NOS inhibitor 7-N was utilized. Based on the IC50 for NOS2 inhibition, 7-N, 0.1 mM, was selected to separate NOS2 from NOS3 isoenzymes inhibited at this low concentration. In combination with 300 mM palmitate for 24 h, 7-N, 0.1 mM, significantly (po0.05) increased cell death from 42.873.6% to 58.676.2%. With 500 mM palmitate, 7-N, 0.1 mM, significantly (po0.05) increased cell death from 64.374.4% to 80.977.0%. 7-N, 0.1 mM, did not alter cell death in the absence of palmitate. 3.2. 7-Nitroindazole does not prevent KCN plus DOG-induced cell death Next we sought to determine whether the effects of 7-N were generalizable to more than the modes by which palmitate-induced cell death. A mode of cell death that is also mediated at the mitochondrial level was chosen. Cardiomyocytes were treated with the combination of potassium cyanide (KCN) and deox-

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Palmitate Fig. 1. 7-Nitroindazole increases palmitate-induced cell death. Cardiomyocytes were maintained in culture for 72 h in multiwell plates, after which they were incubated with the indicated combinations of palmitate and/or 7-nitroindazole (7-N) (N ¼ 8), for 24 h. Cell death was examined by MTT assay. Absorbance was measured at 570 nm and is shown in the inset. Results are presented as percent change of cell death relative to control, and are expressed as mean7SEM. Analysis of variance compared palmitate (plus diluent-treated cell) with and without 7-N treatment (*po0.05).

yglucose (DOG), which, respectively, inhibit glycolysis and oxidative phosphorylation. 7-N did not significantly alter KCN plus DOG-induced cell death (Fig. 2). KCN 1 mM plus DOG 5 mM induced 41.0719.2% cell death alone, and in combination with 7-N, showed little change, with 42.3721.4%, 45.0718.8%, and 53.0714.9% cell death with 0.1, 1, and 10 mM, 7-N, respectively. 3.3. 7-Nitroindazole does not prevent palmitate-induced cytosolic cytochrome c accumulation We have previously demonstrated that palmitateinduced cell death is mediated, in part, through the release of cytochrome c from the mitochondria [7,8]. Thus we examined the effect of 7-N on palmitate-induced cytochrome c release into the cytosole, by Western blotting (Fig. 3). 7-N did not significantly alter palmitate-induced cytochrome c release. Palmitate, 300 mM, caused a 2.070.6 increase of cytosolic cytochrome c, whereas its combination with 7-N was associated with a similar 2.27.01 fold increase. 7-N alone was associated with a small non-significant increase of the amount of cytochrome c in the cytosolic cellular fraction. 3.4. 7-Nitroindazole partially restores palmitate-induced loss of mitochondrial transmembrane potential Palmitate induces a loss of mitochondrial transmembrane potential [31]. To determine whether 7-N could

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+ 1 mM KCN + 5 mM DOG Fig. 2. 7-Nitroindazole does not alter cell death induced by potassium cyanide (KCN) and deoxyglucose (DOG). Cardiomyocytes were maintained in culture for 72 h in multiwell plates. The amount of cell death following the combinations of KCN plus DOG and 7-nitroindazole (7-N) for 24 h was examined by MTT assay (control, 7-N 0 mM (N ¼ 9), 0.1 mM (N ¼ 7), 1.0 mM (N ¼ 8) and 10 mM (N ¼ 9). Absorbance was measured at 570 nm and is shown in the inset. Results are presented as percent change of cell death relative to control, and are expressed as mean7SEM.

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Fig. 3. 7-Nitroindazole does not prevent palmitate-induced cytosolic cytochrome c accumulation. Cardiomyocytes in culture for 72 h were treated with palmitate 300 mM, 7-nitroindazole 10 mM or their combination for 24 h (N ¼ 3). Cytosolic and mitochondrial fractions were obtained by differential centrifugation. Equal amounts of protein, assessed by the Bradford assay, were loaded on to 15% SDS–PAGE gels, transferred to nitrocellulose membranes, probed with cytochrome c-specific antibodies as outlined in the method. A representative Western blot is shown to illustrate the change in cytosolic cytochrome c. Densitometric analysis of cytochrome c in the cytosolic and mitochondrial fractions was performed and the ratio of the amount in the cytosolic and mitochondrial fractions was calculated. The data is shown relative to control.

alter this effect of palmitate, mitochondrial transmembrane potential was assessed using a fluorescent probe and immunocytochemistry to determines the overall state of mitochondrial DCm in the cell population [32]. At lower DCm less dye enters the mitochondria resulting in monomers that fluoresce green while at higher DCm the dye accumulates sufficiently in mitochondria to form aggregates that fluoresce red [33] There will always be some mitochondria with a lower potential (green monomers) even though the cell is healthy (predominantly red aggregates) [31]. In control cells, red aggregates indicating the presence of mitochondria with a high DCm were readily apparent (Fig. 4). There were no changes induced by 7-N. Palmitate produced a dramatic loss of red coloration, with the green monomeric form of the dye accumulating under conditions of reduced potential. 7-N did not modify (further accentuate) palmitate-induced loss of DCm. 3.5. The peroxynitrite isomerase FeTPPS reduces palmitate-induced cell death As palmitate induces NO generation [10], some of the NO may combine with oxidative free radicals, specifically superoxide anions, to produce peroxynitrite [34]. We sought to determine whether peroxynitrite was

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Fig. 4. 7-Nitroindazole does not restore palmitate-induced reduction of mitochondrial transmembrane potential. Cardiomyocytes were plated directly on to glass coverslips and maintained in culture for 72 h. Cells were treated with either palmitate 300 mM, 7-nitroindazole 10 mM, or the combination for 24 h. Coverslips were stained with 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethyl-benzimidazolyl-carbocyanine iodide to visualize mitochondrial membrane potential by fluorescent microscopy. Red/orange aggregates indicate healthy mitochondria; green monomers indicate reduced mitochondrial membrane potential. Representative cells are shown.

playing a role in palmitate-induced cell death. We chose the peroxynitrite isomerase FeTPPS to isomerize peroxynitrite to nitrate and thereby reduce the toxic effects of peroxynitrite. FeTPPS at concentrations from 10 to 50 mM were selected based on data that suggest cytoprotection near these concentrations [35] and toxic effects with higher ones [36]. FeTPPS reduced palmitateinduced cell death (Fig. 5). Notably, 20 mM FeTPPS reduced the cell death induced by 300 mM palmitate 19.077.1% or from 61.276.8% to 52.677.2, and 500 mM palmitate by 21.676.1% from 86.278.2% to 64.776.5%.

4. Discussion The present study expands the understanding of the pathogenesis of fatty acid (palmitate)-induced cell death. It presents the novel findings that palmitate-induced cell death is mediated in part through peroxynitrite formation, demonstrates the dual action of NO on cell viability applies to fatty acid-induced cell death as NO also has a protective role in this mode of cell death. It further suggests that the site of action of the protective effect of NO is independent of the mitochondrial death pathways, and suggests the responsible NOS isoenzymes stimulated by palmitate leading to cell survival. We demonstrate that low concentrations of the NOS inhibitor 7-N accentuated palmitate-induced cell death.

These findings confirm and extend our previous observation that high concentrations of L-NAME accentuated palmitate-induced cell death [10]. L-NAME is a relatively non-specific NOS inhibitor and the concentrations of 100 mM, which was previously used to evaluate palmitate-induced cell death, inhibits all three types of NOS [37]. We have previously suggested a role for NOS2 and NOS3 in palmitate-induced cell death because palmitate increases protein expression of both of these NOS isoenzymes as determined by immunocytochemistry and Western blotting [10]. Our current data suggest that palmitate-induced NO production does not occur through NOS2. It is unlikely that 7-N could have meaningfully inhibited NOS2 because the concentration of 7-N employed in this study was less than one tenth of the EC50 for NOS2 [38]. A role for NOS1 in palmitate-induced cell death cannot be excluded because the EC50 for NOS1 and NOS3 are, respectively, 0.6 and 0.8 mM [39]. Interestingly, the amount of palmitoylation of NOS regulates NOS3 activity subsequent NO release [40]. Consistent with this concept, NOS3 palmitoylation-deficient mutant cells release less NO [40]. Recognizing that in some other cell types and modes of cell death, for example clonal beta-cell HIT-T15, NOS inhibition limits cell death [41], we speculate that the beneficial effects of palmitate-induced NO production may operate through NO-induced inhibition of caspase activation. This can be supported by several

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Fig. 5. FeTPPS reduces palmitate-induced cell death. Cardiomyocytes were maintained in culture for 72 h in multiwell plates. The amount of cell death following the indicated combinations of palmitate and FeTPPS (N ¼ 4) for 24 h was examined by MTT assay. Absorbance was measured at 570 nm and the percent change of cell death relative to control was determined and shown in the inset. The effect of FeTPPS on cell death at each concentration of palmitate is shown. Data is expressed as mean7SEM (*po0.05, **po0.01).

lines of evidence. First, the data consistently demonstrate that caspases are activated by palmitate [42–44]. Palmitate-induced activation of caspase-3, -6, -7, and -9 is translated into apoptotic cell death [42,43,45,46]. Cleaved (active) caspase-3 accumulates in the cytosol within 9 h of palmitate treatment [46]. Second, NOinduced protein nitrosylation can play a major role in regulating cell death [47]. Third and most importantly, NO inhibits caspase activity through caspase nitration. The NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) inhibits caspase-3 activation induced by doxorubicin [48]. NO-induced reduction in caspase-3 activity is mediated in part through blockage of the proteolytic conversion of pro-caspase-3 to active caspase-3 [49]. SNAP-induced reductions in caspase-1, -2, -3, -4, -6, -7 and -8 activity was reversed by DTT indicating caspase inactivation occurred through S-nitrosylation [50]. Other NO donors antagonize the activation of caspase-3, -8 or -9 in various cell types through protein nitrosylation of these proteases [51–54]. NO-induced caspase nitrosylation antagonizes the effect of cytochrome c release [52,53,55,56]. Nitrosylation can affect caspases located in the cytosol or mitochondria [57]. Caspase activation in response to palmitate is relatively modest compared with other inducers of apoptosis [45,46]. Our data suggest a potential explanation for that observation. In addition to palmitateinduced caspase activation palmitate enhances NO production, which in turn reduces caspase activity. We have also identified that part of the mechanism of palmitate-induced cell death is mediated through NO

production that in turn combines with superoxide ions to generate peroxynitrite. The peroxynitrite scavenger FeTPPS is highly effective in catalyzing the isomerization of peroxynitrite to nitrate [35], thereby removing peroxynitrite from the cell and reducing the deleterious effects of peroxynitrite. Peroxynitrite at low concentrations and in some situations can be beneficial [55]. Peroxynitrite-induced nitration of M-CPT I occurs, can decrease CPT I activity [58] which would be anticipated to protect against further palmitate-induced cell death [8]. In the majority of circumstances, however, peroxynitrite is damaging to the cell [30]. Our findings are consistent with an adverse effect of peroxynitrite on cell viability. Whether palmitate directly stimulates superoxide formation is controversial and opposite results has been reported [9,45]. Cardiomyocytes, under usual conditions, however, generate superoxide anions that would combine with excess NO produced from palmitate to generate more peroxynitrite. Our data suggest that the effect of palmitate-induced NO generation to modulate cell death operates independent of mitochondrial death pathways based on several lines of evidence. First cytochrome c release is a fundamental part of palmitate-induced cell death [7,8] yet the NOS inhibitor 7-N did not alter cytochrome c release in response to palmitate treatment. Second, 7-N did not affect one of the deleterious effects of palmitate on the mitochondria namely palmitate-induced loss of mitochondrial transmembrane potential. Third, NO generated by NOS isoenzymes inhibited by 7-N did not alter cell death due to agents that directly inhibit

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mitochondrial function, namely KCN and DOG, which inhibit glycolysis and oxidative phosphorylation of mitochondria [59]. In contrast, other agents moderate KCN plus DOG-induced cell death [60]. 7-N has some selectivity for the NOS1 variant mitochondrial NOS [13,61]. Our data can also be interpreted to suggest that mitochondrial NOS is not operative in regulating palmitate-induced loss of cytochrome c from the mitochondria or loss of DCm. Thus our data suggest that palmitate-induced NO release is acting either upstream or downstream of mitochondrial glycolysis and oxidative phosphorylation, generation of DCm and cytochrome c release. There are several important considerations relevant to the limitations of the study. The avian and embryonic nature of the cardiomyocyte population needs to be considered in extrapolation of the data. However, the avian model possesses a high degree of homology with other vertebrates [62] which makes it a suitable system for exploring the mechanisms of cell death with potential cross-species application. Second, we did not measure peroxynitrite formation because it is difficult to do so as peroxynitrite is not stable and exists for only a short time period. Third, mitochondrial DCm was visualized by DePsipher staining, and the effects of NO were only qualitatively described. However, we have shown a close correlation between immunohistochemistry and quantitative assessment using the same dye in flow cytometry analysis [7,8]. Fourth, we did not study a particular mode of cell death such as apoptosis, because palmitate produces both apoptotic and oncotic cell death [8]. The MTT assay was the only cell death assay used in these experiments, because it reflects the sum of cell death from apoptosis plus oncosis and in these cardiomyocytes, and correlates highly with other indices of cell death such as trypan blue and FACS analysis with different stains for cell death [7,63]. We can now construct the following schema of palmitate-induced cell death (Fig. 6). Palmitate needs to enter the mitochondria in order to produce the major portion of cell death [7,8]. Palmitate reduces mitochondrial cardiolipin which in turn affects cytochrome c binding to the inner membrane with resultant higher levels of soluble cytochrome c in the mitochondrial intermembrane space [64]. Palmitate produces a loss of cytochrome c from the mitochondria into the cytosole [8]. Cytochrome c, once it is in the cytoplasm, activates cellular caspases leading to apoptotic cell death [65] and indeed palmitate induces activation of caspase-3, -6, -7, and -9, which is translated into apoptotic cell death [42,43,45,46,66]. Other mitochondrial constituents such as endonuclease G are also released from the mitochondria by saturated free fatty acids and lead to cell death [42]. Palmitate-induced NO is an essential component of palmitate-induced cell death, in part, through generation of peroxynitrite. Palmitate-induced NO production

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Fig. 6. Schema for the consequences of palmitate-induced NO generation on cardiomyocyte viability.

also acts as a negative regulator of palmitate-induced cell death perhaps through reducing the extent of caspase activation. References [1] O. Leonardi, G. Mints, M.A. Hussain, Beta-cell apoptosis in the pathogenesis of human type 2 diabetes mellitus, Eur. J. Endocrinol. 149 (2003) 99–102. [2] J.M. McGavock, R.G. Victor, R.H. Unger, L.S. Szczepaniak, Adiposity of the heart, revisited, Ann. Intern. Med. 144 (2006) 517–524. [3] E.P. Haber, J. Procopio, C.R.O. Carvalho, A.R. Carpinelli, P. Newsholme, R. Curi, New insights into fatty acid modulation of pancreatic beta-cell function, Int. Rev. Cytol. 248 (2006) 1–41. [4] J.M. Weinberg, Lipotoxicity, Kidney Int. 70 (2006) 1560–1566. [5] J.E. de Vries, M.M. Vork, T.H. Roemen, Y.F. de Jong, J.P. Cleutjens, G.J. van der Vusse, M. van Bilsen, Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes, J. Lipid Res. 38 (1997) 1384–1394. [6] S.W. Rabkin, M. Huber, G. Krystal, Modulation of palmitateinduced cardiomyocyte cell death by interventions that alter intracellular calcium, Prostaglandins Leukot. Essent. Fatty Acids 61 (1999) 195–201. [7] J.Y. Kong, S.W. Rabkin, Palmitate-induced apoptosis in cardiomyocytes is mediated through alterations in mitochondria: prevention by cyclosporin A, Biochim. Biophys. Acta 1485 (2000) 45–55. [8] J.Y. Kong, S.W. Rabkin, Palmitate-induced cardiac apoptosis is mediated through CPT-1 but not influenced by glucose and insulin, Am. J. Physiol.—Heart Circ. Physiol. 282 (2002) H717–H725. [9] D.L.M. Hickson-Bick, G.C. Sparagna, L.M. Buja, J.B. McMillin, Palmitate-induced apoptosis in neonatal cardiomyocytes is not dependent on the generation of ROS, Am. J. Physiol.—Heart Circ. Physiol. 282 (2002) H656–H664. [10] M.Y.C. Tsang, S.E. Cowie, S.W. Rabkin, Palmitate increases nitric oxide synthase activity that is involved in palmitate-induced cell death in cardiomyocytes, Nitric Oxide 10 (2004) 11–19. [11] S. Moncada, A. Higgs, The L-arginine-nitric oxide pathway, N. Engl. J. Med. 329 (1993) 2002–2012. [12] W.K. Alderton, C.E. Cooper, R.G. Knowles, Nitric oxide synthases: structure, function and inhibition, Biochem. J. 357 (2001) 593–615.

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