Mitochondrial Fatty Acid Composition Research Articles

Glutathione peroxidase 2 (Gpx2) preserves mitochondrial function and decreases ROS levels in chronologically aged yeast

Glutathione peroxidase 4 (Gpx4) counteracts mitochondrial lipid peroxidation in mammals. In yeast, Gpx2 is orthologous of Gpx4, is localized in mitochondria, and reduces both inorganic and organic peroxides. However, a phenotype of oxidative stress hypersensitivity has not been observed with gpx2 deletion. We hypothesized that the absence of polyunsaturated fatty acids (PUFA) in yeast membranes may mask an antioxidant role of Gpx2 in mitochondria. Thus, we tested the effects of PUFA on cell viability, mitochondrial function, ROS production, and mitochondrial fatty acid composition of a gpx2Δ mutant subjected to chronological aging. As expected, gpx2Δ mutation did not alter these parameters with respect to wild-type (WT) cells after 30 h growth, even in the presence of linolenic acid (C18:3), except for some activities of the electron transport chain (ETC) complexes. Conversely, aged gpx2Δ cells exhibited lower viability, impaired respiration, decreased ETC activities, and increased ROS generation in comparison to aged WT cells. These effects were exacerbated by C18:3, as gpx2Δ cells displayed residual respiration, full inhibition of ETC complexes, and a burst in ROS production on day 15 that decreased on day 30, although ROS remained several-fold higher than in WT cells. gpx2 was not involved in the preservation of PUFA levels, as no differences in mitochondrial C18:3 content were observed between WT and gpx2Δ cells. These results indicate that gpx2 is a late – acting antioxidant system that decreases mitochondrial ROS production and preserves ETC function, without being involved in the preservation of PUFA levels in mitochondria.

Antipsychotics inhibit the mitochondrial bioenergetics of pancreatic beta cells isolated from CD1 mice

Antipsychotics (APs) are widely used medications with reported diabetogenic side effects. This study investigated the effect of commonly used APs, namely chlorpromazine (CPZ), haloperidol (HAL) and clozapine, on the bioenergetics of male CD1 mice isolated pancreatic beta cells as an underlying mechanism of their diabetogenic effects. The effect of APs on Alamar blue reduction, adenosine triphosphate (ATP) production and glucose-stimulated insulin secretion (GSIS) of isolated beta cells was evaluated. Then, the effects of APs on the activities of mitochondrial complexes and their common coding genes expression, oxygen consumption rate (OCR), mitochondrial membrane potential (MMP) and lactate production were investigated. The effects of APs on the mitochondrial membrane fluidity (MMF) and mitochondrial membrane fatty acid composition were also examined. Results showed that the tested APs significantly decreased cellular ATP production and GSIS of the beta cells. The APs significantly inhibited the activities of mitochondrial complexes and their coding gene expression, MMP and OCR of the treated cells, with a parallel increase in lactate production to different extents with the different APs. CPZ and HAL showed increased MMF and mitochondrial membrane polyunsaturated fatty acid content. In conclusion, the tested APs-induced mitochondrial disruption can play a role in their diabetogenic side effect.

Chilling injury in pineapple fruit is related to mitochondrial antioxidative metabolism

Abstract Mitochondrial fatty acid composition and mitochondrial antioxidative metabolism were studied in relation to chilling injury (CI) in the fruit pulp of two pineapple (Ananas comosus) cultivars: Pattavia (a tolerant cultivar) and Trad-See-Thong (a susceptible cultivar) which were stored at 10 °C for 14 d. CI symptoms, mainly water soaking, were only found in the susceptible cultivar Trad-See-Thong. Only small differences in the ratio of saturated to unsaturated fatty acids were determined between mitochondria of the two cultivars. Mitochondrial superoxide dismutase, catalase, and ascorbate peroxidase activities were all lower in the susceptible cultivar. Mitochondrial total antioxidant capacity, determined by the FRAP and the ABTS methods, was lower in the susceptible cultivar and mitochondrial dysfunction, determined by the absence of an increase in respiration after ADP addition, was also more prominent in the susceptible cultivar. Mitochondria of Trad-See-Thong were also less tolerant to treatment with exogenous hydrogen peroxide. It is concluded that the impaired functioning of mitochondria in pulp cells of Trad-See-Thong fruit stored at 10 °C, and its higher sensitivity to damage by ROS, is likely due to its lower total antioxidant capacity and lower activities of antioxidative enzymes. A higher sensitivity to ROS-induced mitochondrial dysfunction was apparently the cause of the increased CI levels in the pulp of chilled Trad-See-Thong fruit.

Cardiac mitochondrial function, nitric oxide sensitivity and lipid composition following hypoxia acclimation in sablefish.

In fishes, the effect of O2 limitation on cardiac mitochondrial function remains largely unexplored. The sablefish (Anoplopoma fimbria) encounters considerable variations in environmental oxygen availability, and is an interesting model for studying the effects of hypoxia on fish cardiorespiratory function. We investigated how in vivo hypoxia acclimation (6 months at 40% then 3 weeks at 20% air saturation) and in vitro anoxia-reoxygenation affected sablefish cardiac mitochondrial respiration and reactive oxygen species (ROS) release rates using high-resolution fluorespirometry. Further, we investigated how hypoxia acclimation affected the sensitivity of mitochondrial respiration to nitric oxide (NO), and compared mitochondrial lipid and fatty acid (FA) composition between groups. Hypoxia acclimation did not alter mitochondrial coupled or uncoupled respiration, or respiratory control ratio, ROS release rates, P50 or superoxide dismutase activity. However, it increased citrate synthase activity (by ∼20%), increased the sensitivity of mitochondrial respiration to NO inhibition (i.e., the NO IC50 was 25% lower), and enhanced the recovery of respiration (by 21%) and reduced ROS release rates (by 25-30%) post-anoxia. In addition, hypoxia acclimation altered mitochondrial FA composition [increasing arachidonic acid (20:4ω6) and eicosapentaenoic acid (20:5ω3) proportions by 11 and 14%, respectively], and SIMPER analysis revealed that the phospholipid:sterol ratio was the largest contributor (24%) to the dissimilarity between treatments. Overall, these results suggest that hypoxia acclimation may protect sablefish cardiac bioenergetic function during or after periods of O2 limitation, and that this may be related to alterations in mitochondrial sensitivity to NO and to adaptive changes in membrane composition (fluidity).

Consequences of oxidative damage and mitochondrial dysfunction on the fatty acid profile of muscle of Indian Major Carps considering metal toxicity

Current study aims to find interrelation between mitochondrial enzyme function and fatty acid profile in fish muscle and role of antioxidant agents to maintain their balance in response to metal accumulation. Fishes (Labeo rohita, Catla catla, Cirrhinus cirrhosus) were collected from two sites (Nalban Bheri and Diamond Harbour, India). Concentrations of metals (lead, cadmium, copper, nickel, zinc), enzymatic and non-enzymatic antioxidant activity (malondialdehyde, superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione S-transferase), muscle enzyme activity (acetylcholinesterase, succinate dehydrogenase, lactate dehydrogenase, Ca2+ATPase, AMP-deaminase, lipoamide reductase, cytochrome C oxidase, aldolase) and fatty acid composition in muscle tissues were analyzed. Metal concentrations were significantly higher (P < 0.05) in fish muscles from Nalban compared to those in Diamond Harbour. Increased activity of antioxidant enzymes was noted with diminished mitochondrial enzymes activity and altered fatty acid composition in response to higher metal accumulation. Higher metal concentration in fish muscle of Nalban seems to significantly (P < 0.05) affect poly and monounsaturated fatty acid content, possibly due to oxidative damage and accumulation of hazardous reactive oxygen species (ROS) molecules. Changes in fatty acid contents following metal accumulation were observed to be species specific. Current study is the first correlative study to illuminate the level of oxidative damage and possible consequences on muscle cellular integrity, mitochondrial functionality and flesh quality against bioaccumulation of different metals in carps. Future studies are needed to quantify the relative contributions of enzymatic and low-molecular-mass antioxidants in protecting mitochondrial function and maintenance of proper fatty acid oxidation during acclimation to long term metal exposure.

Skeletal muscle overexpression of short isoform Sirt3 altered mitochondrial cardiolipin content and fatty acid composition.

Cardiolipin (CL) is a phospholipid at the heart of mitochondrial metabolism, which plays a key role in mitochondrial function and bioenergetics. Among mitochondrial activity regulators, SIRT3 plays a crucial role in controlling the acetylation status of many enzymes participating in the energy metabolism in particular concerning lipid metabolism and fatty acid oxidation. Data suggest that possible connection may exist between SIRT3 and CL status that has not been evaluated in skeletal muscle. In the present study, we have characterized skeletal muscle lipids as well as mitochondrial lipids composition in mice overexpressing long (SIRT3-M1) and short (SIRT3-M3) isoforms of SIRT3. Particular attention has been paid for CL. We reported no alteration in muscle lipids content and fatty acids composition between the two mice SIRT3 strains and the control mice. However, mitochondrial CL content was significantly decreased in SIRT3-M3 mice and associated to an upregulation of tafazzin gene expression. In addition, mitochondrial phospholipids and fatty acids composition was altered with an increase in the PC/PE ratio and arachidonic acid content and a reduction in the MUFA/SFA ratio. These modifications in mitochondrial membrane composition are associated with a reduction in the enzymatic activities of mitochondrial respiratory chain complexes I and IV. In spite of these mitochondrial enzymatic alterations, skeletal muscle mitochondrial respiration remained similar in SIRT3-M3 and control mice. Surprisingly, none of those metabolic alterations were detected in mitochondria from SIRT3-M1 mice. In conclusion, our data indicate a specific action of the shorter SIRT3 isoform on lipid mitochondrial membrane biosynthesis and functioning.

DHA but Not EPA Emulsions Preserve Neurological and Mitochondrial Function after Brain Hypoxia-Ischemia in Neonatal Mice

Background and PurposeTreatment with triglyceride emulsions of docosahexaenoic acid (tri-DHA) protected neonatal mice against hypoxia-ischemia (HI) brain injury. The mechanism of this neuroprotection remains unclear. We hypothesized that administration of tri-DHA enriches HI-brains with DHA/DHA metabolites. This reduces Ca2+-induced mitochondrial membrane permeabilization and attenuates brain injury.Methods10-day-old C57BL/6J mice following HI-brain injury received tri-DHA, tri-EPA or vehicle. At 4–5 hours of reperfusion, mitochondrial fatty acid composition and Ca2+ buffering capacity were analyzed. At 24 hours and at 8–9 weeks of recovery, oxidative injury, neurofunctional and neuropathological outcomes were evaluated. In vitro, hyperoxia-induced mitochondrial generation of reactive oxygen species (ROS) and Ca2+ buffering capacity were measured in the presence or absence of DHA or EPA.ResultsOnly post-treatment with tri-DHA reduced oxidative damage and improved short- and long-term neurological outcomes. This was associated with increased content of DHA in brain mitochondria and DHA-derived bioactive metabolites in cerebral tissue. After tri-DHA administration HI mitochondria were resistant to Ca2+-induced membrane permeabilization. In vitro, hyperoxia increased mitochondrial ROS production and reduced Ca2+ buffering capacity; DHA, but not EPA, significantly attenuated these effects of hyperoxia.ConclusionsPost-treatment with tri-DHA resulted in significant accumulation of DHA and DHA derived bioactive metabolites in the HI-brain. This was associated with improved mitochondrial tolerance to Ca2+-induced permeabilization, reduced oxidative brain injury and permanent neuroprotection. Interaction of DHA with mitochondria alters ROS release and improves Ca2+ buffering capacity. This may account for neuroprotective action of post-HI administration of tri-DHA.

Tissue‐Specific Seasonal Changes in Mitochondrial Membrane Composition and Respiratory Function in the Golden‐Mantled Ground Squirrel

Mammals that hibernate, such as the golden‐mantled ground squirrel (Callospermophilus lateralis; GMGS), cease to feed, reduce metabolic rate, and lower body temperature during the winter months, surviving almost exclusively on the oxidation of lipids from endogenous fat stores. Whether mitochondria, the cellular sites of oxidative metabolism, undergo adaptive changes in response to hypometabolism and decreasing ambient temperature to facilitate this remarkable phenotype is unclear. It has been postulated that changes in mitochondrial membrane composition reported in response to cold exposure in some species may facilitate maintenance of respiratory enzyme function with decreasing temperature. However, no studies to date have investigated the function and membrane fatty acid composition of mitochondria from different tissues from the warm summer months, to fall and winter in a hibernator. We hypothesize that tissue‐specific differences in mitochondrial respiration occur across seasons in the GMGS that may parallel distinct changes in mitochondrial membrane composition. We compared the respiratory capacity, oxidative enzyme activity, and membrane composition of GMGS mitochondria isolated from liver, heart, skeletal muscle, and brown adipose tissue (BAT) from summer, fall, and winter (torpid) seasons. Maximal mitochondrial oxidative phosphorylation (OXPHOS)‐supported respiration was determined by high‐resolution respirometry at 37°C, 25°C, and 15°C in the presence of saturating concentrations of ADP and respiratory substrates. Mitochondrial membrane fatty acid composition was determined by gas chromatography on phospholipid fractions obtained from isolated mitochondria. Maximal OXPHOS assayed at 37°C tended to increase from summer to winter in liver, heart and BAT, and decrease in skeletal muscle, with tissue‐ and season‐specific changes in pyruvate versus fatty acid (palmitoylcarnitine) oxidation capacity. OXPHOS capacity was uniformly suppressed by decreasing assay temperature in all tissue mitochondria from torpid GMGS in the winter, despite widely variable changes in mitochondrial membrane composition across tissues and seasons. Interestingly, a consistent relative decrease in mitochondrial membrane docosahexaenoic acid (DHA; 22:6n3) was seen from summer to winter in all tissues, the biological relevance of which merits further investigation. Taken together, these findings argue against a consistent relationship between changes in mitochondrial membrane composition and respiratory function across seasons in GMGS, but highlight distinct tissue‐ and season‐specific differences that may have important biological effects that remain to be elucidated.

Omega-3 fatty acids partially revert the metabolic gene expression profile induced by long-term calorie restriction

Calorie restriction (CR) consistently extends longevity and delays age-related diseases across several animal models. We have previously shown that different dietary fat sources can modulate life span and mitochondrial ultrastructure, function and membrane fatty acid composition in mice maintained on a 40% CR. In particular, animals consuming lard as the main fat source (CR-Lard) lived longer than CR mice consuming diets with soybean oil (CR-Soy) or fish oil (CR-Fish) as the predominant lipid source. In the present work, a transcriptomic analysis in the liver and skeletal muscle was performed in order to elucidate possible mechanisms underlying the changes in energy metabolism and longevity induced by dietary fat in CR mice. After 8months of CR, transcription downstream of several mediators of inflammation was inhibited in liver. In contrast, proinflammatory signaling was increased in the CR-Fish versus other CR groups. Dietary fish oil induced a gene expression pattern consistent with increased transcriptional regulation by several cytokines (TNF, GM-CSF, TGF-β) and sex hormones when compared to the other CR groups. The CR-Fish also had lower expression of genes involved in fatty acid biosynthesis and increased expression of mitochondrial and peroxisomal fatty acid β-oxidation genes than the other CR diet groups. Our data suggest that a diet high in n-3 PUFA, partially reverts CR-related changes in gene expression of key processes, such as inflammation and steroid hormone signaling, and this may mitigate life span extension with CR in mice consuming diets high in fish oil.

Lactation Affects Isolated Mitochondria and Its Fatty Acid Composition but Has No Effect on Tissue Protein Oxidation, Lipid Peroxidation or DNA-Damage in Laboratory Mice.

Linking peak energy metabolism to lifespan and aging remains a major question especially when focusing on lactation in females. We studied, if and how lactation affects in vitro mitochondrial oxygen consumption and mitochondrial fatty acid composition. In addition, we assessed DNA damage, lipid peroxidation and protein carbonyls to extrapolate on oxidative stress in mothers. As model system we used C57BL/6NCrl mice and exposed lactating females to two ambient temperatures (15 °C and 22 °C) while they nursed their offspring until weaning. We found that state II and state IV respiration rates of liver mitochondria were significantly higher in the lactating animals than in non-lactating mice. Fatty acid composition of isolated liver and heart mitochondria differed between lactating and non-lactating mice with higher n-6, and lower n-3 polyunsaturated fatty acids in the lactating females. Surprisingly, lactation did not affect protein carbonyls, lipid peroxidation and DNA damage, nor did moderate cold exposure of 15 °C. We conclude that lactation increases rates of mitochondrial uncoupling and alters mitochondrial fatty acid composition thus supporting the “uncoupling to survive” hypothesis. Regarding oxidative stress, we found no impact of lactation and lower ambient temperature and contribute to growing evidence that there is no linear relationship between oxidative damage and lactation.

Protective effects of dietary avocado oil on impaired electron transport chain function and exacerbated oxidative stress in liver mitochondria from diabetic rats.

Electron transport chain (ETC) dysfunction, excessive ROS generation and lipid peroxidation are hallmarks of mitochondrial injury in the diabetic liver, with these alterations also playing a role in the development of non-alcoholic fatty liver disease (NAFLD). Enhanced mitochondrial sensitivity to lipid peroxidation during diabetes has been also associated to augmented content of C22:6 in membrane phospholipids. Thus, we aimed to test whether avocado oil, a rich source of C18:1 and antioxidants, attenuates the deleterious effects of diabetes on oxidative status of liver mitochondria by decreasing unsaturation of acyl chains of membrane lipids and/or by improving ETC functionality and decreasing ROS generation. Streptozocin-induced diabetes elicited a noticeable increase in the content of C22:6, leading to augmented mitochondrial peroxidizability index and higher levels of lipid peroxidation. Mitochondrial respiration and complex I activity were impaired in diabetic rats with a concomitant increase in ROS generation using a complex I substrate. This was associated to a more oxidized state of glutathione, All these alterations were prevented by avocado oil except by the changes in mitochondrial fatty acid composition. Avocado oil did not prevented hyperglycemia and polyphagia although did normalized hyperlipidemia. Neither diabetes nor avocado oil induced steatosis. These results suggest that avocado oil improves mitochondrial ETC function by attenuating the deleterious effects of oxidative stress in the liver of diabetic rats independently of a hypoglycemic effect or by modifying the fatty acid composition of mitochondrial membranes. These findings might have also significant implications in the progression of NAFLD in experimental models of steatosis.

Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Eicosapentaenoic acid mediates mitochondrial fatty acid composition and fusion protein OPA-1 in associated with preservation of oxidative phosphorylation after myocardial infarction

Purpose: Eicosapentaenoic acid (EPA) is a first line drug in the management after myocardial infarction. Mitochondria are major contributors to energy metabolism and recent mounting evidence suggests that mitochondrial dynamics, such as fusion and fission, has a pivotal role in regulating mitochondrial function. This study was designed to determine whether oral EPA mediates mitochondrial fatty acid composition, dynamics, and oxidative phosphorylation, leading to the attenuation of cardiac remodeling after myocardial infarction (MI). Methods and results: Anterior MI was produced in male rats by ligating the left anterior descending coronary artery (MI group). In the EPA-treated group, EPA (1,000 mg/kg/day) was administrated for 12 weeks after coronary ligation (MI+EPA group). Myocardial infarct size and blood pressure were comparable between groups. At 12 weeks after MI, mitochondria were isolated in non-infarcted myocardium and mitochondrial fatty acid composition was determined using gas chromatography mass spectrometry. In EPA+MI group, mitochondrial EPA content was approximately 10 times higher than that in untreated-MI group. Cardiac function was assessed by echocardiography and 2F micro-manometer-tipped catheter at 12 weeks of MI. EPA significantly improved %fractional shortening, +dP/dt, and -dP/dt, and reduced left ventricular (LV) end-diastolic diameter and pressure. In addition, histological examination showed EPA significantly suppressed myocyte hypertrophy and interstitial fibrosis in non-infarcted myocardium by 15% and 30%, respectively. Levels of ATP in cardiac tissue were measured by high-performance liquid chromatography and mitochondrial oxidative phosphorylation was assessed by O2 consumption using isolated mitochondria. After 12 weeks after MI, ATP contents in non-infarcted myocardium were significantly decreased, and mitochondrial complex II, III, and IV activities were also impaired, while EPA treatment significantly preserved mitochondrial complex activities, as a consequence of an increment in myocardial ATP content. Furthermore, MI decreased optic atrophy-1 (OPA-1) protein, a mitochondrial fusion protein, without any effects on dynamin-related protein-1 (Drp-1) protein, a mitochondrial fission protein, leading to attenuation of mitochondrial damage. Conclusion: These results suggest that oral EPA administration mediates mitochondrial EPA composition, preserves mitochondrial fusion protein OPA-1 and function of oxidative phosphorylation, leading to the attenuation of left ventricular remodeling after MI.

3,5-Diiodo-L-Thyronine increases FoF1-ATP synthase activity and cardiolipin level in liver mitochondria of hypothyroid rats

Short-term effects of 3,5-L-diiodothyronine (T(2)) administration to hypothyroid rats on F(o)F(1)-ATP synthase activity were investigated in liver mitochondria. One hour after T(2) injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. F(o)F(1)-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T(2) administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of F(o)F(1)-ATP synthase was found, ruling out a T(2) genomic effect. In T(2)-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T(2) administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T(2)-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced F(o)F(1)-ATP synthase activity observed following injection of T(2) to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.

Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal muscle

Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.

Dietary supplementation with docosahexaenoic acid, but not eicosapentanoic acid, remodels cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Dietary supplementation with docosahexaenoic acid, but not eicosapentanoic acid, remodels cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca 2+-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca 2+-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca 2+-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca 2+ retention capacity and decreased Ca 2+-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca 2+-induced MPTP opening.

Liver Injury in Acute Fatty Liver of Pregnancy: Possible Link to Placental Mitochondrial Dysfunction and Oxidative Stress

Acute fatty liver of pregnancy (AFLP) is a rare disorder which is fatal if not recognized and treated early. Delivery of the feto-placental unit results in dramatic improvement in maternal liver function, suggesting a role for the placenta. However, the mechanisms by which defects in the fetus or placenta lead to maternal liver damage are not well understood and form the focus of this study. Placenta and serum were obtained at delivery from patients with AFLP, and placental mitochondria and peroxisomes were isolated. Placental mitochondrial function, oxidative stress, and fatty acid composition as well as serum antioxidants, oxidative and nitrosative stress markers, and fatty acid analysis were carried out. Hepatocytes in culture were used to evaluate cell death, mitochondrial function, and lipid accumulation on exposure to fatty acids. Oxidative stress was evident in placental mitochondria and peroxisomes of patients with AFLP, accompanied by compromised mitochondrial function. Increased levels of arachidonic acid were also seen in AFLP placenta when compared to control. Patients with AFLP also had a significant increase in oxidative and nitrosative stress markers in serum, along with decreased antioxidant levels and elevated levels of arachidonic acid. These levels of arachidonic acid were capable of inducing oxidative stress in hepatocyte mitochondria accompanied by induction of apoptosis. Exposure to arachidonic acid also resulted in increased lipid deposition in hepatocytes. Oxidative stress in placental mitochondria and peroxisomes is accompanied by accumulation of toxic mediators such as arachidonic acid, which may play a causative role in maternal liver damage seen in AFLP.

A Silybin-Phospholipid Complex Prevents Mitochondrial Dysfunction in a Rodent Model of Nonalcoholic Steatohepatitis

Mitochondrial dysfunction and oxidative stress are determinant events in the pathogenesis of nonalcoholic steatohepatitis. Silybin has shown antioxidant, anti-inflammatory, and antifibrotic effects in chronic liver disease. We aimed to study the effect of the silybin-phospholipid complex (SILIPHOS) on liver redox balance and mitochondrial function in a dietary model of nonalcoholic steatohepatitis. To accomplish this, glutathione oxidation, mitochondrial oxygen uptake, proton leak, ATP homeostasis, and H(2)O(2) production rate were evaluated in isolated liver mitochondria from rats fed a methionine- and choline-deficient (MCD) diet and the MCD diet plus SILIPHOS for 7 and 14 weeks. Oxidative proteins, hydroxynonenal (HNE)- and malondialdehyde (MDA)-protein adducts, and mitochondrial membrane lipid composition were also measured. Treatment with SILIPHOS limited glutathione depletion and mitochondrial H(2)O(2) production. Moreover, SILIPHOS preserved mitochondrial bioenergetics and prevented mitochondrial proton leak and ATP reduction. Finally, SILIPHOS limited the formation of HNE- and MDA-protein adducts. In conclusion, SILIPHOS is effective in preventing severe oxidative stress and preserving hepatic mitochondrial bioenergetics in nonalcoholic steatohepatitis induced by the MCD diet. The modifications of mitochondrial membrane fatty acid composition induced by the MCD diet are partially prevented by SILIPHOS, conferring anti-inflammatory and antifibrotic effects. The increased vulnerability of lipid membranes to oxidative damage is limited by SILIPHOS through preserved mitochondrial function.

Genetically Modified Arabidopsis thaliana Cells Reveal the Involvement of the Mitochondrial Fatty Acid Composition in Membrane Basal and Uncoupling Protein-Mediated Proton Leaks

We investigated the role of membrane fatty acids in basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild-type cells, cold-sensitive fad2 mutant cells, deficient in omega-6-oleate desaturase, and cold-tolerant FAD3(+) transformant cells, overexpressing omega-3-linoleate desaturase, we showed that basal proton leak in the non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in the fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3(+) mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3(+) and lower in fad2. UCP content was higher in both the fad2 mutant and FAD3(+) transgenic lines compared with wild-type cells and so was the UCP activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP-proton leak in the non-phosphorylating state (flux-force relationships between proton flux and membrane potential). The potential uncoupling capacity of the UCP was high enough to allow the loss of respiratory control in the three genotypes. Taken together, the data reported here suggest that the cold tolerance of FAD3(+) cells and the cold sensitivity of fad2 cells are associated with changes in their mitochondrial membrane basal proton leaks, whereas differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells.