Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.     

Epoxyeicosatrienoic acids in cardioprotection: ischemic versus reperfusion injury

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels (K(ATP)); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 +/- 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 +/- 1.9%, 11,12-EET; and 8.4 +/- 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 +/- 2.1%, 11,12-EET; and 9.7 +/- 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 +/- 4.6%) by the K(ATP) channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 +/- 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12,12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP omega-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 +/- 1.2%, DDMS; and 22.2 +/- 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP omega-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP omega-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac K(ATP) channels.     

Site-mapping of in vitro S-nitrosation in cardiac mitochondria: implications for cardioprotection

S-nitrosation (SNO) of mitochondrial protein cysteines can be cardioprotective. Several targets have been implicated, yet the scope and identification of specific residues has not been fully assessed. To address this, a comprehensive assessment of mitochondrial SNO-modifiable cysteines was performed to determine nitric oxide (NO) susceptible pathways and identify novel mechanisms of oxidative cardioprotection. The biotin switch assay and mass spectrometry were used on rat cardiac mitochondrial lysates treated with the nitric oxide donor, S-nitrosoglutathione, and controls (n=3) to map 83 SNO-modified cysteine residues on 60 proteins. Of these, three sites have been reported, 30 sites are new to 21 proteins previously known to be S-nitrosated but which lacked site-specific information and 50 sites were found on 39 proteins not previously implicated in SNO pathways. The SNO-modifications occurred in only a subset of available cysteines, indicating a specific targeted effect. Functional annotation and site-specificity analysis revealed a twofold greater nitric oxide-susceptibility for proteins involved in transport; including regulators of mitochondrial permeability transition suggesting SNO-regulation and a possible protective mechanism. Additionally, we identified many novel SNO-modified proteins with cardioprotective potential involved in the electron transport chain, tricarboxylic acid cycle, oxidative stress defense, fatty acid and amino acid metabolism. These findings suggest that SNO-modification may represent a novel mechanism for the regulation of oxidative phosphorylation and/or cell death. S-nitrosation of mitochondrial permeability transition-associated proteins represents an intriguing potential link to cardioprotection.     

Injection of isolated mitochondria during early reperfusion for cardioprotection

Previously, we demonstrated that ischemia induces mitochondrial damage and dysfunction that persist throughout reperfusion and impact negatively on postischemic functional recovery and cellular viability. We hypothesized that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, would enhance postischemic functional recovery and limit infarct size. New Zealand White rabbits (n = 52) were subjected to 30 min of equilibrium and 30 min of regional ischemia (RI) induced by snaring the left anterior descending coronary artery. At 29 min of RI, the RI zone was injected with vehicle (sham control and RI vehicle) or vehicle containing mitochondria (7.7 x 10(6) +/- 1.5 x 10(6)/ml) isolated from donor rabbit left ventricular tissue (RI-Mito). The snare was released at 30 min of RI, and the hearts were reperfused for 120 min. Our results show that left ventricular peak developed pressure and systolic shortening in RI-Mito hearts were significantly enhanced (P < 0.05 vs. RI-vehicle) to 75% and 83% of equilibrium value, respectively, at 120 min of reperfusion compared with 57% and 62%, respectively, in RI-vehicle hearts. Creatine kinase-MB, cardiac troponin I, and infarct size relative to area at risk were significantly decreased in RI-Mito compared with RI-vehicle hearts (P < 0.05). Confocal microscopy showed that injected mitochondria were present and viable after 120 min of reperfusion and were distributed from the epicardium to the subendocardium. These results demonstrate that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, significantly enhance postischemic functional recovery and cellular viability.     

Ceramide and mitochondria in ischemic brain injury

Sphingolipids are essential structural components of cellular membranes, playing prominent roles in signal transduction that governs cell proliferation, differentiation and apoptosis. Ceramides, a family of distinct molecular species characterized by various acyl chains, are synthesized de novo at the cytosolic side of the endoplasmic reticulum serving as precursors for the biosynthesis of sphingolipids in the Golgi. Recently, mitochondria emerged as an important intracellular compartment of sphingolipid metabolism. Thus, several sphingolipid-metabolizing enzymes were found to be associated with mitochondria, including neutral ceramidase, novel neutral sphingomyelinase, and (dihydro) ceramide synthase, an important ceramide-generating enzyme in de novo ceramide synthesis and recycling pathway. Mitochondrial dysfunction appears to be essential in tissue damage after brain ischemia/reperfusion (IR). Mitochondria are known to be involved in both the necrosis and apoptosis detected in animal models of ischemic stroke, and treatments that ameliorate tissue infarction were associated with better recovery of mitochondrial function. Although mitochondrial injury in stroke has been extensively studied and key mitochondrial functions affected by IR are mainly characterized, the nature of the molecule that causes loss of mitochondrial integrity and function remains obscure. Emerging data indicate a deregulation of ceramide metabolism in mitochondria damaged by IR suggesting that ceramides could play critical roles in cerebral IR-induced mitochondrial damage. This review will examine the experimental evidence supporting the key role of ceramides in mitochondrial dysfunction in cerebral IR and highlight potential targets for development of novel therapeutic approaches for stroke treatment.     

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria.

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD⁺, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD⁺ was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.     

Effects of long-chain fatty acids on the inhibition by antimycin of respiration in hepatocytes and isolated mitochondria from rat liver

A previous study [Berry, M. N., Gregory, R. B., Grivell, A. R. & Wallace, P. G. (1983) Eur. J. Biochem. 131, 215-222] suggested that long-chain fatty acid (palmitate) oxidation by hepatocytes was less sensitive than short-chain fatty acid (hexanoate) oxidation to inhibition by a given concentration of antimycin. Re-examination of this phenomenon showed that palmitate oxidation by hepatocytes could be depressed by antimycin to the same degree as other NAD+-linked substrates, only if the concentration of the inhibitor was raised 2-4-fold. The presence of palmitate also reduced the sensitivity to antimycin of hepatocytes metabolizing lactate or pyruvate. Over the range of fatty acids tested, butyrate (C4) to stearate (C18), only long-chain (greater than C10) fatty acids endowed cells with decreased sensitivity towards antimycin. 2-Bromopalmitate, a non-metabolizable fatty acid, and inhibitor of fatty acid oxidation, also decreased the inhibitory effect of antimycin in cells, suggesting that long-chain fatty acids per se rather than their metabolites, reverse the inhibition by antimycin. Moreover, another inhibitor of fatty acid oxidation, 2-tetradecylglycidic acid, did not diminish the effects of palmitate. Succinate oxidation in isolated mitochondria that had been inhibited by a low concentration of antimycin could be restored by subsequent addition of palmitate or other long-chain fatty acids such as dodecanoate, tetradecanoate and oleate under conditions where fatty acid oxidation was prevented. 2-Bromopalmitate, likewise partially restored antimycin-depressed succinate oxidation. This amelioration of antimycin inhibition was counteracted by the addition of more antimycin and was not seen upon addition of defatted bovine serum albumin, palmitoylcarnitine or octanoate. The total amount of antimycin bound to mitochondria was not affected by the presence of palmitate. The data suggest that long-chain fatty acids are able to interact with the mitochondrial inner membrane in a manner which can relieve the inhibitory effect of antimycin, whether the antimycin is added to the cell or mitochondrial suspension before or after fatty acid addition.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

Incorporation of fatty acids into the outer and inner membranes of isolated rat liver mitochondria

Acyl-CoA: phospholipid acyl-transferase activity as well as phospholipase A activity were detected in inner and outer membrane preparations from rat liver mitochondria. Both enzyme systems have an optimum pH around 8 and act preferentially on phosphatidylethanolamine. While phospholipase A activity is much lower in the inner membrane than in the outer membrane of mitochondria the reverse is true for the incorporation of (14C)-oleic acid into endogenous phosphatidylethanolamine. These results bring an indirect evidence that the inner membrane per se possesses a phospholipase A activity.     

Effects of fatty acids on phosphatidylcholine biosynthesis in isolated hamster heart

The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.     

Activation and transport of fatty acids in ovarian mitochondria: effect of Lh

Enzymes of fatty acid activation and transport were studied in luteinized rat ovaries. Luteal mitochondria were found to contain high levels of palmitoyl-CoA synthetase and carnitine palmitoyl-transferase activities. In addition, studies on the effect of palmitate concentration on palmitoyl-CoA synthetase activity revealed the possible existence of two forms of the enzyme: Km values of 0.34 mM and 21.33 mM, with Vmax of 3.64 and 66.67 nmoles/min/mg mitochondrial protein respectively, were obtained for the two activities. Similar kinetic data for carnitine palmitoyl-transferase activity in intact mitochondria are a Km of 21 microM and a Vmax of 18.2 nmoles/min/mg mitochondrial protein. Only one activity of this enzyme could be detected in luteal mitochondria. It appears that the activities of both enzymes were not affected by prior administration of LH in vivo. The possibility that this negative finding was due to the experimental procedures employed, rather than a reflection of the situation in vivo, could not be discounted, although its more likely that these two enzymes are probably not locus of LH stimulation. The results indicate that fatty acid oxidation is an important metabolic capability of luteal mitochondria, and support the view regarding the lipid nature of the respiratory fuel of ovarian tissue.     

Omega-3 fatty acids in neurodegenerative diseases: Focus on mitochondria

Mitochondrial dysfunction represents a common early pathological event in brain aging and in neurodegenerative diseases, e.g., in Alzheimer’s (AD), Parkinson’s (PD), and Huntington’s disease (HD), as well as in ischemic stroke. In vivo and ex vivo experiments using animal models of aging and AD, PD, and HD mainly showed improvement of mitochondrial function after treatment with polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA). Thereby, PUFA are particular beneficial in animals treated with mitochondria targeting toxins. However, DHA showed adverse effects in a transgenic PD mouse model and it is not clear if a diet high or low in PUFA might provide neuroprotective effects in PD. Post-treatment with PUFA revealed conflicting results in ischemic animal models, but intravenous administered DHA provided neuroprotective efficacy after acute occlusion of the middle cerebral artery. In summary, the majority of preclinical data indicate beneficial effects of n-3 PUFA in neurodegenerative diseases, whereas most controlled clinical trials did not meet the expectations. Because of the high half-life of DHA in the human brain clinical studies may have to be initiated much earlier and have to last much longer to be more efficacious.     

Lipoxygenase- mediated cleavage of fatty acids in plant mitochondria

Incubation of cauliflower bud mitochondria in the presence of 5 mM CaCl2 results in a rapid hydrolysis of the main membrane phospholipsds. Under the action of phospholipase D, phosphatidic acid is produced and forms, within the membranes, a very labile complex with Ca2+ and HPO42-ions present in the incubation medium. With time, one observes a first step characterized by the formation of phosphatidic acid, followed by a second step linked to the breakdown of this phospholipid. The enzyme responsible for the disappearance of phosphalidic acid has been identified as lipoxygenase. In the presence of molecular oxygen, this enzyme acts on the polyun-saturated fatty acids of phosphatidic add (mainly C18:2 and C18:3) yielding small water-soluble molecules, one of them being identified as malondialdehyde (1, 3-propanedial). Experiments involving inhibitory conditions of the breakdown of phosphatidic acid indicate that lipoxygenase acts directly on membrane-bound phosphatidic acid without previous, involvement of a lipolytic acyl hydrolase activity. In addition, the lipoxygenase activity is fully sensitive to hydroxamate derivatives. It is proposed that the lipoxygenase activity may account for a part of the mitochondrial alternative electron pathway that is insensitive to cyanide.     

Pyruvate provides cardioprotection in the experimental model of myocardial ischemic reperfusion injury

The present study was designed to evaluate the cardioprotective potential of pyruvate and to characterize the mechanism underlying the protection. Wistar albino rats were randomly divided into three groups. Two groups were administered saline orally (sham, ischemia-reperfusion (I-R) control group) and animals of third group received pyruvate (500 mg/kg) for 4 weeks. On the 29th day, animals of the I-R control and pyruvate treated groups underwent 45 min of occlusion of the left anterior descending (LAD) coronary artery and were thereafter reperfused for 60 min. In the I-R control group, a significant cardiac necrosis, depressed mean arterial pressure (MAP) and heart rate (HR), decline in myocardial antioxidant status and elevation in lipid peroxidation were observed as compared to sham control. Pyruvate treatment restored the myocardial antioxidant status and favorably modulated the altered MAP as compared to I-R control. Furthermore, I/R-induced lipid peroxidation was significantly inhibited by pyruvate treatment. These beneficial cardioprotective effects translated into significant improvement in MAP. Histopathological examination and restored specific myocardial injury marker CK-MB isoenzyme activity further confirmed protective effects of pyruvate. In conclusion, our study has demonstrated that the beneficial effect of pyruvate likely results from improved MAP and suppression of oxidative stress.