Adenine Nucleotide Translocase Activity Research Articles

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

ObjectiveEnhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. MethodNAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). ResultNAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. ConclusionNAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications.

High doses of butyrate induce a reversible body temperature drop through transient proton leak in mitochondria of brain neurons

AimsSodium butyrate (SB) is a major product of gut microbiota with signaling activity in the human body. It has become a dietary supplement in the treatment of intestinal disorders. However, the toxic effect of overdosed SB and treatment strategy remain unknown. The two issues are addressed in current study. Materials and methodsSB (0.3–2.5 g/kg) was administrated through a single peritoneal injection in mice. The core body temperature and mitochondrial function in the brown adipose tissue and brain were monitored. Pharmacodynamics, targeted metabolomics, electron microscope, oxygen consumption rate and gene knockdown were employed to dissect the mechanism for the toxic effect. Key findingsThe temperature was reduced by SB (1.2–2.5 g/kg) in a dose-dependent manner in mice for 2–4 h. In the brain, the effect was associated with SB elevation and neurotransmitter reduction. Metabolites changes were seen in the glycolysis, TCA cycle and pentose phosphate pathways. Adenine nucleotide translocase (ANT) was activated by butyrate for proton transportation leading to a transient potential collapse through proton leak. The SB activity was attenuated by ANT inhibition from gene knockdown or pharmacological blocker. ROS was elevated by SB for the increased ANT activity in proton leak in Neuro-2a. SignificanceExcessive SB generated an immediate and reversible toxic effect for inhibition of body temperature through transient mitochondrial dysfunction in the brain. The mechanism was quick activation of ANT proteins for potential collapse in mitochondria. ROS may be a factor in the ANT activation by SB.

NF-κB in mitochondria regulates PC12 cell apoptosis following lipopolysaccharide-induced injury

To determine the relationship between nuclear transcription factor κB (NF-κB) expression in mitochondria and neuronal cell apoptosis after lipopolysaccharide (LPS)-induced injury. The effect of drug administration on PC12 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Hoechst 33342 staining. The morphology and function of mitochondria were investigated with electron microscopy and rhodamine 123, respectively. The activity of adenine nucleotide translocase 1 (ANT1), lipid peroxide, and antiperoxidase enzymes was measured by enzyme-linked immunosorbent assay (ELISA). Relative expression levels of NF-κB were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Pyrrolidine dithiocarbamate (PDTC, an NF-κB inhibitor) and nerve growth factor (NGF) not only reduced apoptosis but also had a protective effect on mitochondrial structure and function in a PC12 cell LPS damage model. The subcellular distribution of NF-κB demonstrated that NGF, diterpene acid atractyloside (ATR, an ANT1 antagonist), and PDTC alleviated increases in mitochondrial NF-κB after LPS-induced injury. (1) NF-κB is activated in mitochondria via uptake of ANT1 during apoptosis following LPS-induced injury in neuronal cells; (2) NGF not only decreases the activity of NF-κB but also reduces ANT1 activity, which in turn decreases NF-κB levels in mitochondria and suppresses mitochondria-mediated apoptosis.

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes

Ecto-F1-ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F1-ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y13-mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F1-ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F1-ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F1-ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes.This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis.

Spermine cycling in mitochondria is mediated by adenine nucleotide translocase activity: mechanism and pathophysiological implications.

Spermine, besides to be transported in mitochondria by an energy dependent electrophoretic mechanism, can be also released by two different mechanisms. The first one is induced in deenergizing conditions by FCCP or antimycin A and it is mediated by an electroneutral exchange spermine protons. The second one takes place in energizing conditions during the activity of the adenine nucleotide translocase and is mediated by an electroneutral symport mechanism involving the efflux in co-transport of spermine and phosphate and the exchange of exogenous ADP with endogenous ATP. The triggering of this mechanism permits an alternating cycling of spermine across the mitochondrial membrane, that is spermine is transported or released by energized mitochondria in the absence or presence of ATP synthesis, respectively. The physiological implications of this cycling of spermine are related to the induction or prevention of mitochondrial permeability transition and, consequently, on apoptosis or its prevention.

Transgenic overexpression of adenine nucleotide translocase 1 protects ischemic hearts against oxidative stress.

Ischemia impairs the adenine nucleotide translocase (ANT), which transports ADP and ATP across the inner mitochondrial membrane. We investigated whether ANT1 overexpression has protective effects on ischemic hearts. Myocardial infarction was induced in wild-type (WT) and heart-specific ANT1-transgenic (ANT1-TG) rats, and hypoxia was set in isolated cardiomyocytes. ANT1 overexpression reduced the myocardial infarct area and increased the survival rate of infarcted rats. Reduced ANT1 expression and increased 4-hydroxynonenal modification of ANT paralleled to impaired ANT function in infarcted WT hearts. ANT1 overexpression improved ANT expression and function. This was accompanied by reduced mitochondrial cytochrome C release and caspase-3 activation. ANT1-TG hearts suffered less from oxidative stress, as shown by lower protein carbonylation and 4-hydroxynonenal modification of ANT. ANT1 overexpression also increased cell survival of hypoxic cardiomyocytes and attenuated reactive oxygen species (ROS) production. This was linked to higher stability of mitochondrial membrane potential and lower activity of ROS detoxifying catalase. ANT1-TG cardiomyocytes also showed higher resistance against H2O2 treatment, which was independent of catalase activity. In conclusion, ANT1 overexpression compensates impaired ANT activity under oxygen-restricted conditions. It reduces ROS production and oxidative stress, stabilizes mitochondrial integrity, and increases survival, making ANT1 a component in ROS management and heart protection during ischemia. ANT1 overexpression reduces infarct size and increases survival after infarction. ANT1 overexpression compensates restricted ANT expression and function in infarcted hearts. Increased ANT1 expression enhances mitochondrial integrity. ANT1-overexpressing hearts reduce oxidative stress by decreasing ROS generation. ANT1 is a component in ROS management and heart protection.

Human Adenine Nucleotide Translocase (ANT) Modulators Identified by High-Throughput Screening of Transgenic Yeast

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast “knock-out/knock-in” screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.

Atractylosides, chlorogenic acids and antioxidant capacity of raw Arabica green coffee beans (AGCB) used by regular and spontaneous dieters to supports body weight loss and slimming

Using DPPH, ABTS and Folin-Ciocalteu procedures [1] the antioxidant capacity was characterized of fresh infusions of the raw AGCB distributed in Poland and originating from Central and South America. Next, UHPLC-TOF-MS/MS analyses of all infusions and MeOH extracts of raw AGCB were made using a Waters ACQUITY UPLC TabMode system interfaced with a Bruker microOTOF-Q mass spectrometer with an ESI source operating in negative mode at 150 °C and 4.5 kV with N2 nebulisation at 1.2 bar and a dry gas flow at 6 mL/min. An Agilent Poroshell 120EC-C18 column (2.1 × 150, 2.7 µm) was used operating at 0.6 mL/min flow rate with a mobile phase consisted of a linear gradient of 0.1% formic acid in water (A) and 0.1% formic acid in AcN (B), mixed by increasing eluate B in the range 5 – 90% from 0 – 20 min and followed by a split of column effluent flow 3:2 before the ESI ion source was used. MSMS spectra were analyzed with a Bruker Compass Data Analyzer v.4. The atractyligenin derivatives, i.e. 2-O-beta-D-glucopyranosyl-carboxyatractyligenin (m/z 525) (1), 2-O-(2'-O-isovaleryl-beta-D-glucopyranosyl)-carboxyatractyligenin (m/z 608) (2) and 3'-O-beta-D-glucopyranosyl-2'-O-isovaleryl-2-beta-(2-desoxy-carboxyatractyligenin)-beta-D-glucopyranoside (m/z 771) (3), were identified in the MeOH extracts of the raw but not milled AGCB from Colombia. Only compound 1 with a high inhibitory activity of adenine nucleotide translocase (ANT) [2] was confirmed by MSMS in the infusions of this AGCB sample. This indicates that some atractylosides in raw AGCB were hydrolyzed by hydrothermal treatment [3]. Compounds 1 and 3 with, respectively, significant and non-significant ANT inhibition were recently detected in the AGCB [2], but glucoside 2 was identified here for the first time.

Palmitoyl-carnitine increases RyR2 oxidation and sarcoplasmic reticulum Ca2 + leak in cardiomyocytes: Role of adenine nucleotide translocase

Long chain fatty acids bind to carnitine and form long chain acyl carnitine (LCAC), to enter into the mitochondria. They are oxidized in the mitochondrial matrix. LCAC accumulates rapidly under metabolic disorders, such as acute cardiac ischemia, chronic heart failure or diabetic cardiomyopathy. LCAC accumulation is associated with severe cardiac arrhythmia including ventricular tachycardia or fibrillation. We thus hypothesized that palmitoyl-carnitine (PC), alters mitochondrial function leading to Ca2+ dependent-arrhythmia. In isolated cardiac mitochondria from C57Bl/6 mice, application of 10μM PC decreased adenine nucleotide translocase (ANT) activity without affecting mitochondrial permeability transition pore (mPTP) opening. Mitochondrial reactive oxygen species (ROS) production, measured with MitoSOX Red dye in isolated ventricular cardiomyocytes, increased significantly under PC application. Inhibition of ANT by bongkrekic acid (20μM) prevented PC-induced mitochondrial ROS production. In addition, PC increased type 2 ryanodine receptor (RyR2) oxidation, S-nitrosylation and dissociation of FKBP12.6 from RyR2, and therefore increased sarcoplasmic reticulum (SR) Ca2+ leak. ANT inhibition or anti-oxidant strategy (N-acetylcysteine) prevented SR Ca2+ leak, FKBP12.6 depletion and RyR2 oxidation/S-nitrosylation induced by PC. Finally, both bongkrekic acid and NAC significantly reduced spontaneous Ca2+ wave occurrences under PC. Altogether, these results suggest that an elevation of PC disturbs ANT activity and alters Ca2+ handling in a ROS-dependent pathway, demonstrating a new pathway whereby altered FA metabolism may contribute to the development of ventricular arrhythmia in pathophysiological conditions.

Acute and Long-Term Treatments with an Herbal Formula V-Vital Capsule Increase Exercise Endurance Capacity in Weight-Loaded Swimming Mice

Fatigue is a self-limiting response arising from physical and/or mental weariness, with a consequent personal and economic morbidity on work performance and social relationships. Anti-fatigue intervention is therefore urgently sought. “Qi-invigorating” Chinese tonic herbs, which can improve the energy status in the body according to the theory of traditional Chinese medicine, may produce beneficial effects in fatigue individuals. The herbal formula V-Vital capsule (VVC), which comprises 3 “Qi-invigorating” herbs, namely the root of Rhodiola rosea, Eleutherococcus senticosus and Panax quinquefolium, may produce anti-fatigue effect. In the present study, we investigated the effect of acute/long-term VVC treatment (acute: 0.75, 0.2 and 3.75 kg/day × 1 dose; long-term: 0.075 and 0.25 g/kg/day × 14 doses) on weight-loaded swimming female ICR mice. The weight-loaded swimming time until exhaustion, indicative of exercise endurance capacity, was recorded. Plasma levels of glucose, non-esterified fatty acid (NEFA), lactate and reactive oxygen metabolites (ROM) were measured in the exhausted mice. Glycogen levels in skeletal muscle and liver tissues were also measured. Mitochondrial function status [such as adenine nucleotide translocase (ANT) activity and coupling efficiency] was assayed. Results showed that acute VVC treatment increased the exercise endurance capacity in weight-loaded swimming mice. The ability of acute VVC treatment to enhance the exercise endurance was associated with increases in plasma glucose levels as well as glycogen levels in skeletal muscles and liver tissues, presumably due to the utilization of plasma lactate for gluconeogenesis and/or glycogen synthesis in the liver. While acute VVC treatment reduced the plasma ROM level in weight-loaded swimming mice, it increased the ANT activity. In this regard, the enhancement in exercise endurance afforded by acute VVC treatment might be due to an increase in the glucose supply to the skeletal muscle, the amelioration of systemic oxidative stress and the improvement in mitochondrial function of skeletal muscle. Consistent with the results obtained in acute VVC treatment experiment, the long-term VVC treatment enhances the exercise endurance in weight-loaded swimming mice. The ensemble of results suggests that VVC may offer a promising prospect for enhancing the exercise endurance and alleviating fatigue in humans.

2-O-β-d-Glucopyranosyl-carboxyatractyligenin from Coffea L. inhibits adenine nucleotide translocase in isolated mitochondria but is quantitatively degraded during coffee roasting

Atractyloside (1) and carboxyatractyloside (2) are well-known inhibitors of the adenine nucleotide translocase (ANT) in mitochondria, thus effectively blocking oxidative phosphorylation. Structurally related derivatives atractyligenin (3), 2-O-β-D-glucopyranosyl-atractyligenin (4), 3'-O-β-D-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-atractyligenin)-β-D-glucopyranoside (5), and 2-O-β-D-glucopyranosyl-carboxyatractyligenin (6) were isolated from raw beans of Coffea L. and the impact of 1-6 on ANT activity was evaluated in isolated mitochondria. Among the coffee components, 6 significantly inhibited ANT activity leading to reduced respiration. Quantitative analysis in commercial coffees, experimental roastings of coffee, and model experiments using purified compound 6 consistently revealed a complete degradation during thermal treatment. In comparison, raw coffee extracts were found to contain high levels of 6, which are therefore expected to be present in food products enriched with raw coffee extracts. This implies the necessity of analytically controlling the levels of 6 in raw coffee extracts when used as additives for food products.

The Adenine Nucleotide Translocase 2, a Mitochondrial Target for Anticancer Biotherapy

Apoptosis or programmed cell death is one of the most important signaling pathways, which controls the cell fate and is frequently impaired in cancer cells. The major consequences of apoptosis inhibition are the accumulation of mutated cells and their enhanced resistance to chemotherapeutic agents. More generally, intrinsic or acquired apoptosis resistance may favor tumor growth and dissemination of mutated cells, and this resistance can be responsible of treatment failure. Mitochondria are central organelles in the signaling pathway of apoptosis and have been proposed as favorite candidates for anticancer biotherapy because they accommodate potential biological targets. Indeed, although cancer cells are highly glycolytic and become energetically independent of oxidative phosphorylation. Mitochondrial proteins involved in the so-called mitochondrial membrane permeabilization (MMP), such as the adenine nucleotide translocase (ANT) can be instrumental to elicit cancer cell death. Thus, multiple pharmacological and molecular studies revealed ANT could be a promising therapeutic target for the following reasons: (i) ANT is a bi-functional protein, it mediates the vital exchange of cytosolic ADP and mitochondrial ATP and participates to MMP via its capacity to become a lethal pore in the mitochondrial inner membrane; (ii) both ANT functions are under the control of the (anti)-oncogenes from the Bax/Bcl-2 family, (iii) several chemotherapeutic agents directly modulate the pore-forming activity of ANT and (iv) ANT2 isoform, which is anti-apoptotic, can be overexpressed in human cancers and its invalidation sensitize cells to apoptosis. In this review, we will introduce the knowledge of the role of ANT in MMP, illustrate the modulation of ANT by several strategies and propose the possibility to target preferentially the ANT2 isoform for induction of cancer cell apoptosis.

RETRACTION: Protective Effect of PGC-1 on Lipid Overload-induced Apoptosis in Vascular Endothelial Cell

Background: Fatty acids contribute to endothelial cell dysfunction and apoptosis by inducing accumulation of long chain fatty acyl CoA (LCAC), which increases oxidative stress in vascular endothelial cells. Forced expression of PGC-1 was shown to induce mitochondrial biogenesis and to control expression of mitochondrial enzymes involved in fatty acid oxidation. This study was undertaken to test the hypothesis that PGC-1 overexpression could prevent endothelial cell apoptosis by enhancing fatty acid oxidation and relieving oxidative stress in vascular endothelium. Methods: Adenoviruses containing human PGC-1 (Ad-PGC-1) and -galactosidase (Ad--gal) were transfected to confluent human aortic endothelial cells (HAECs). To investigate the effect of adenoviral PGC-1 gene transfer on apoptosis, combined treatment of linoleic acid (LA), an unsaturated fatty acid, was performed. Results: PGC-1 overexpression inhibited the increase in ROS production and apoptosis of HAECs induced by LA. Also, PGC-1 led to a significant increase in fatty acid oxidation and decrease in triglyceride content in HAECs. LA caused the decrease of adenine nucleotide translocase (ANT) activity and transient mitochondrial hyperpolarization, which was followed by depolarization. PGC-1 overexpression prevented these processes. Conclusion: In summary, PGC-1 overexpression inhibited mitochondrial dysfunction and apoptosis by facilitating fatty acid oxidation and protecting against the damage from oxidative stress in HAECs. The data collectively suggest that the regulation of intracellular PGC-1 expression might play a critical role in preventing atherosclerosis.

Accelerated Mitochondrial Adenosine Diphosphate/Adenosine Triphosphate Transport Improves Hypertension-Induced Heart Disease

Strong evidence suggests that mitochondrial malfunction, which leads to disturbed energy metabolism and stimulated apoptosis, is a linchpin in the induction and manifestation of cardiac failure. An adequate exchange of ATP and ADP over the inner mitochondrial membrane by the adenine nucleotide translocase (ANT) is thereby essential to guarantee the cellular energy supply. To explore the effect of an ameliorated mitochondrial ATP/ADP transportation on cardiac dysfunction, we generated transgenic rats overexpressing ANT1 in the heart (ANT rats) and crossed them with renin-overexpressing rats (REN rats) suffering from hypertension-induced cardiac insufficiency. Cardiac-specific ANT1 overexpression resulted in a higher ATP/ADP transportation and elevated activities of respiratory chain complexes. Increased ANT activity in double-transgenic (ANT/REN) animals did not influence excessive hypertension seen in REN rats. Hypertension-induced cardiac hypertrophy in the REN rats was prevented by parallel ANT1 overexpression, however, and left ventricular function remarkably improved. The ANT1 overexpression led to a reduction in fibrosis and an improvement in cardiac tissue architecture. Consequently, the survival rate of ANT/REN rats was enhanced. Further investigations into the cardioprotective mechanism of ANT1 overexpression revealed improved mitochondrial structure and function and significantly reduced apoptosis in ANT/REN rats, shown by lowered cytosolic/mitochondrial cytochrome c ratio, reduced caspase 3 level, and prevented DNA degradation. Myocardial ANT1 overexpression protects against hypertension-induced cardiac pathology. Thus, the improvement in mitochondrial function may be a basic principle for new strategies in treating heart disease.

The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H2O2), hydroxyl radical (HO•) or superoxide anion () on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO• slightly decreased F1F0-ATPase activity and HO• and partially inhibited the activity of adenine nucleotide translocase; H2O2 failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for and alpha-ketoglutarate dehydrogenase for H2O2 and HO•. Our results show that , H2O2 and HO• act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.

Changes in mitochondrial oxidative capacities during thermal acclimation of rainbow trout Oncorhynchus mykiss: roles of membrane proteins,phospholipids and their fatty acid compositions

Changes in the properties of mitochondria from oxidative muscle of rainbow trout Oncorhynchus mykiss were examined during warm (5 degrees C to 15 degrees C) acclimation. Trout were studied shortly after the initial thermal change and after 8 weeks acclimation to 15 degrees C. To identify potential mechanisms by which oxidative capacities change, the modifications of phospholipid composition, membrane proteins and functional capacities of red muscle mitochondria were examined. Marked functional changes of isolated muscle mitochondria during warm acclimation of rainbow trout were reflected by a host of modifications in phospholipid composition, but by few shifts in protein components. Shortly after transfer of trout from 5 degrees C to 15 degrees C, the maximal oxidative capacity of mitochondria measured at 15 degrees C increased slightly, but rates at both assay temperatures (5 degrees C and 15 degrees C) decreased markedly after warm acclimation. The increase in capacity in short-term warm exposed trout was most pronounced when rates at 15 degrees C were expressed relative to cytochrome a and c(1) levels. Non-phosphorylating (State 4) rates of oxygen uptake increased with short-term warm exposure before returning to initial levels after warm acclimation. Cytochrome c oxidase (CCO) activity in the mitochondrial preparations decreased with warm acclimation. The thermal sensitivity of the ADP affinity was markedly modified during short-term warm exposure, when the ADP/O ratio increased, but warm acclimation returned these values to those observed initially. ADP affinity increased after warm acclimation. Changes in the mitochondrial content of cytochromes and adenine nucleotide translocase (ANT) could not explain these patterns. On the other hand, changes in the proportions of the lipid classes and in the acyl chain composition of certain phospholipid classes mirror the modifications in functional properties. Short-term exposure to 15 degrees C decreased the ratio of diacylphosphatidylethanolamine/diacylphosphatidylcholine (diacylPE/diacylPC), whereas warm acclimation led to restructuring of fatty acids (FA) and to increases of plasmalogen forms of PE and PC. Modification of overall membrane unsaturation did not appear to be the primary aim of restructuring membrane FA during warm acclimation, as total mitochondrial phospholipids and the major phospholipid classes only showed slight shifts of their acyl composition with warm acclimation. On the other hand, natural lysophosphatidylcholine (LysoPC) showed dramatic changes in FA content, as 16:0 and 18:1n-9 doubled whereas 22:6n-3 decreased from around 50% to 32% in warm acclimated trout. Similarly, in cardiolipin (CL), the levels of 16:0 and 18:1n-7 halved while 18:2n-6 increased to over 20% of the FA with warm acclimation. Given the central role of CL in modulating the activity of CCO, F(0)F(1)-ATPase and ANT, these changes suggest that specific compositional changes in CL are important modulators of mitochondrial capacities. The many structural changes in membrane lipids contrast with the limited modifications of the membrane protein components examined and support the concept of lipid structure modulating mitochondrial capacities.

Necrosis Through RIP

If caspase activation is blocked, cells cannot undergo apoptosis but may still die through a process of necrotic cell death. Temkin et al. investigated the mechanism by which cell death occurred in response to activation of the death receptors in the presence of the nonspecific caspase inhibitor zVADfmk. Cell death caused by exposure of the cells to tumor necrosis factor-α (TNF-α) and zVADfmk was inhibited if receptor-interacting protein (RIP) abundance was diminished by geldanamycin or RNA interference (RNAi). In TNF-treated cells, RIP colocalized with mitochondria both in isolated mitochondria and by microscopic techniques. In cells exposed to both TNF-α and zVADfmk, the activity of mitochondrial adenine nucleotide translocase (ANT) was inhibited and adenosine triphosphate (ATP) concentrations dropped. Although reactive oxygen species were also produced, addition of antioxidants did not prevent death induced by TNF-α together with zVADfmk. ANT appeared to be a direct target of zVADfmk, because biotinylated zVADfmk was associated with ANT in the TNF-α-treated cells but not in cells in which RIP was decreased. Addition of zVADfmk to mitochondria isolated from TNF-α-treated cells inhibited the activity of ANT in two different assays. Cyclophilin D coimmunoprecipitated with ANT in control cells; however, TNF-α alone disrupted this interaction. Overexpression of cyclophilin D protected the cells from death induced by TNF-α+zVADfmk, and reduction of cyclophilin D by RNAi sensitized the cells to death induced by zVADfmk in the absence of TNF-α. Cyclophilin D and zVADfmk appeared to compete for the same interaction site on ANT. Thus, the authors propose a model whereby activation of a surface death receptor stimulates the translocation of the kinase RIP to the mitochondria, which results in displacement of cyclophilin D from ANT through an as-yet-undefined mechanism. In the presence of zVADfmk, the translocase activity of ANT is inhibited and ATP concentration falls, leading to necrotic cell death. These results have important implications for interpreting experiments using the inhibitor zVADfmk, because it not only inhibits apoptotic cell death through inhibition of caspases but also can promote necrotic cell death in the presence of death receptor signaling through inhibition of ANT. V. Temkin, Q. Huang, H. Liu, H. Osada, R. M. Pope, Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol. Cell. Biol. 26 , 2215-2225 (2006). [Abstract] [Full Text]

Inhibition of ADP/ATP Exchange in Receptor-Interacting Protein-Mediated Necrosis

Receptor-interacting protein (RIP) has been implicated in the induction of death receptor-mediated, nonapoptotic cell death. However, the mechanisms remain to be elucidated. Here we show that tumor necrosis factor alpha induced RIP-dependent inhibition of adenine nucleotide translocase (ANT)-conducted transport of ADP into mitochondria, which resulted in reduced ATP and necrotic cell death. The inhibition of ADP/ATP exchange coincided with the loss of interaction between ANT and cyclophilin D and the inability of ANT to adopt the cytosolic conformational state, which prevented cytochrome c release. Neither overexpression of Bcl-xL nor inhibition of reactive oxygen species prevented necrosis. In contrast, the ectopic expression of ANT or cyclophilin D was effective at preventing cell death. These observations demonstrate a novel mechanism initiated through death receptor ligation and mediated by RIP that results in the suppression of ANT activity and necrosis.

Protective Effect of PGC-1 on Lipid Overload-induced Apoptosis in Vascular Endothelial Cell

Background: Fatty acids contribute to endothelial cell dysfunction and apoptosis by inducing accumulation of long chain fatty acyl CoA (LCAC), which increases oxidative stress in vascular endothelial cells. Forced expression of PGC-1 was shown to induce mitochondrial biogenesis and to control expression of mitochondrial enzymes involved in fatty acid oxidation. This study was undertaken to test the hypothesis that PGC-1 overexpression could prevent endothelial cell apoptosis by enhancing fatty acid oxidation and relieving oxidative stress in vascular endothelium. Methods: Adenoviruses containing human PGC-1 (Ad-PGC-1) and -galactosidase (Ad--gal) were transfected to confluent human aortic endothelial cells (HAECs). To investigate the effect of adenoviral PGC-1 gene transfer on apoptosis, combined treatment of linoleic acid (LA), an unsaturated fatty acid, was performed. Results: PGC-1 overexpression inhibited the increase in ROS production and apoptosis of HAECs induced by LA. Also, PGC-1 led to a significant increase in fatty acid oxidation and decrease in triglyceride content in HAECs. LA caused the decrease of adenine nucleotide translocase (ANT) activity and transient mitochondrial hyperpolarization, which was followed by depolarization. PGC-1 overexpression prevented these processes. Conclusion: In summary, PGC-1 overexpression inhibited mitochondrial dysfunction and apoptosis by facilitating fatty acid oxidation and protecting against the damage from oxidative stress in HAECs. The data collectively suggest that the regulation of intracellular PGC-1 expression might play a critical role in preventing atherosclerosis.

Accumulation of nonesterified fatty acids causes the sustained energetic deficit in kidney proximal tubules after hypoxia-reoxygenation

Kidney proximal tubules exhibit decreased ATP and reduced, but not absent, mitochondrial membrane potential (Deltapsi(m)) during reoxygenation after severe hypoxia. This energetic deficit, which plays a pivotal role in overall cellular recovery, cannot be explained by loss of mitochondrial membrane integrity, decreased electron transport, or compromised F1F0-ATPase and adenine nucleotide translocase activities. Addition of oleate to permeabilized tubules produced concentration-dependent decreases of Deltapsi(m) measured by safranin O uptake (threshold for oleate = 0.25 microM, 1.6 nmol/mg protein; maximal effect = 4 microM, 26 nmol/mg) that were reversed by delipidated BSA (dBSA). Cell nonesterified fatty acid (NEFA) levels increased from <1 to 17.4 nmol/mg protein during 60- min hypoxia and remained elevated at 7.6 nmol/mg after 60 min reoxygenation, at which time ATP had recovered to only 10% of control values. Safranin O uptake in reoxygenated tubules, which was decreased 85% after 60-min hypoxia, was normalized by dBSA, which improved ATP synthesis as well. dBSA also almost completely normalized Deltapsi(m) when the duration of hypoxia was increased to 120 min. In intact tubules, the protective substrate combination of alpha-ketoglutarate + malate (alpha-KG/MAL) increased ATP three- to fourfold, limited NEFA accumulation during hypoxia by 50%, and lowered NEFA during reoxygenation. Notably, dBSA also improved ATP recovery when added to intact tubules during reoxygenation and was additive to the effect of alpha-KG/MAL. We conclude that NEFA overload is the primary cause of energetic failure of reoxygenated proximal tubules and lowering NEFA substantially contributes to the benefit from supplementation with alpha-KG/MAL.