Metabolic alterations produced in the liver by chronic ethanol administration. Comparison between the effects produced by ethanol and by thyroid hormones

1. Liver slices from rats treated with thyroxine show an increased rate of O₂ consumption. The extra consumption, but not the basal respiration, can be abolished by ouabain. 2. Dinitrophenol is not effective in increasing the rate of O₂ consumption of liver slices from thyroxine-treated animals but its effectiveness can be recovered in the presence of ouabain. 3. (Na⁺+K⁺)-stimulated adenosine triphosphatase activity of liver was increased by administration of thyroxine in vivo. No changes were found in total Mg²⁺-stimulated adenosine triphosphatase activity. 4. Mitochondrial α-glycerophosphate dehydrogenase and microsomal NADPH oxidase activity were increased by both thyroxine and chronic ethanol treatment. 5. Liver slices from animals chronically treated with ethanol synthesize urea at an increased rate. 6. Mitochondrial size (section area) is markedly increased in the liver of animals chronically treated with ethanol. 7. Acute administration of ethanol in doses of 4 and 6g/kg significantly increases the uptake of ¹³¹I-labelled thyroxine by the liver. 8. Work reported here, along with results from other investigators, indicates marked similarities between the effects produced in the liver by chronic administration of ethanol and by thyroid hormones.     

Metabolic alterations produced in the liver by chronic ethanol administration. Changes related to energetic parameters of the cell

1. Chronic ethanol administration to rats for 21–27 days increases the rate of O₂ consumption as measured in liver slices. The extra respiration can be abolished by inhibition of the active transport of Na⁺ and K⁺. Dinitrophenol activates the respiratory rate in the liver of the treated animals only in the presence of ouabain. 2. Active (ouabain-sensitive) transport of ⁸⁶Rb and (Na⁺+K⁺)-stimulated adenosine triphosphatase activity were increased in the livers of the ethanol-treated animals. 3. Chronic ethanol administration also led to a decrease in the phosphorylation potential ([ATP]/[ADP][P_i]) in the liver cell owing to a decrease in [ATP] and an increase in [P_i]. 4. It is suggested that an increased sodium pump activity is responsible for the increased oxidative capacity and for the insensitivity to dinitrophenol observed in the livers of ethanol-treated animals.     

Ethanol modulates calmodulin-dependent Ca2+-activated ATPase in synaptic plasma membranes

The effects of ethanol in vitro on calmodulin-dependent Ca²⁺-activated ATPase (CaM–Ca²⁺-ATPase) activity were studied in synaptic plasma membranes (SPM) prepared from the brain of normal and chronically ethanol-treated rats. In SPM from normal animals, ethanol at 50–200 mM inhibited the Ca²⁺-ATPase activity. Lineweaver-Burk analysis indicates that the inhibition was the result of a decreased affinity of the enzyme for calmodulin, whereas the maximum activity of the enzyme was not changed. Arrhenius analysis indicates that the enzyme activity was influenced by lipid transition of the membranes, and ethanol in vitro resulted in a shift of the transition temperature toward a lower value. From animals receiving chronic ethanol treatment (3 weeks), the SPM were resistant to the inhibitory effect of ethanol on the enzyme activity. The resistance to ethanol inhibition was correlated with a higher enzyme affinity for calmodulin and a higher transition temperature, as compared with normal SPM. Since the calmodulin-dependent Ca²⁺-ATPase in synaptic plasma membranes is believed to be the Ca²⁺ pump controlling free Ca²⁺ levels in synaptic terminals, its inhibition by ethanol could therefore lead to altered synaptic activity.Abbreviations used ATPase adenosine triphosphatase - CaM calmodulin - CaM–Ca²⁺-ATPase calmodulin-dependent Ca²⁺-activated ATPase - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - EtOH ethanol - Hepes N—2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - SPM synaptic plasma membranes - TFP trifluoperazine - Tris tris(hydroxymethyl)aminomethane - K_m Michaelis constant - T_d transition temperature - V_max maximum velocity     

The localization of adenosine triphosphatases in morphologically characterized subcellular fractions of guinea-pig brain

1. The distribution of adenosine triphosphatase was studied in morphologically characterized subcellular fractions of guinea-pig brain. The conditions of homogenization were selected so as to favour the survival of nerve endings as organized structures. 2. A fraction consisting mainly of the external membranes of nerve endings was rich in a ouabain-sensitive Na⁺–K⁺-stimulated adenosine triphosphatase which closely resembled that present in the classical microsomal fraction studied by other workers, but which showed a higher specific activity. 3. A dinitrophenol-stimulated adenosine triphosphatase was located in the nerve-ending mitochondria. 4. The synaptic-vesicle fraction contained a small amount of adenosine triphosphatase that differed in its response to several ions and other compounds from the membrane, myelin and mitochondrial fractions, indicating freedom from contamination by these elements.     

The cellular production of hydrogen peroxide

1. The enzyme–substrate complex of yeast cytochrome c peroxidase is used as a sensitive, specific and accurate spectrophotometric H₂O₂ indicator. 2. The cytochrome c peroxidase assay is suitable for use with subcellular fractions from tissue homogenates as well as with pure enzyme systems to measure H₂O₂ generation. 3. Mitochondrial substrates entering the respiratory chain on the substrate side of the antimycin A-sensitive site support the mitochondrial generation of H₂O₂. Succinate, the most effective substrate, yields H₂O₂ at a rate of 0.5nmol/min per mg of protein in state 4. H₂O₂ generation is decreased in the state 4→state 3 transition. 4. In the combined mitochondrial–peroxisomal fraction of rat liver the changes in the mitochondrial generation of H₂O₂ modulated by substrate, ADP and antimycin A are followed by parallel changes in the saturation of the intraperoxisomal catalase intermediate. 5. Peroxisomes supplemented with uric acid generate extraperoxisomal H₂O₂ at a rate (8.6–16.4nmol/min per mg of protein) that corresponds to 42–61% of the rate of uric acid oxidation. Addition of azide increases these H₂O₂ rates by a factor of 1.4–1.7. 6. The concentration of cytosolic uric acid is shown to vary during the isolation of the cellular fractions. 7. Microsomal fractions produce H₂O₂ (up to 1.7nmol/min per mg of protein) at a ratio of 0.71–0.86mol of H₂O₂/mol of NADP⁺ during the oxidation of NADPH. H₂O₂ is also generated (6–25%) during the microsomal oxidation of NADH (0.06–0.025mol of H₂O₂/mol of NAD⁺). 8. Estimation of the rates of production of H₂O₂ under physiological conditions can be made on the basis of the rates with the isolated fractions. The tentative value of 90nmol of H₂O₂/min per g of liver at 22°C serves as a crude approximation to evaluate the biochemical impact of H₂O₂ on cellular metabolism.     

Nanoparticle-mediated catalase delivery protects human neurons from oxidative stress

Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H₂O₂) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H₂O₂-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H₂O₂, NP-mediated catalase delivery successfully protected cultured neurons from H₂O₂-induced oxidative stress. Catalase-loaded NPs significantly reduced H₂O₂-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H₂O₂ pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.     

The cerium perhydroxide-diaminobenzidine (Ce-H2O2-DAB) procedure

Summary New light microscopic visualization methods were developed for the histochemical detection of non-specific alkaline and acid phosphatase, Mg-, Ca-and Na, K-dependent adenosine triphosphatase, myosin adenosine triphosphatase, glucose-6-phosphatase, 5-nucleotidase and thiamine pyrophosphatase with cerium ions as trapping agents in cryostat and plastic sections. The techniques are based on the conversion of cerium phosphate into cerium perhydroxide by H₂O₂ which decomposes at 55°–60° C into cerium hydroxide and oxygen radicals. These radicals are able to oxidize diaminobenzidine (DAB) to DAB brown. Addition of nickel ions to the DAB-H₂O₂ mixture generates bluish-black stained nickel-DAB complexes. Compared with the classical metal precipitation, azo, azoindoxyl and tetrazolium procedures the H₂O₂-DAB and especially the H₂O₂-DAB-nickel methods provided identical or superior results in catalytic phosphatase histochemistry and immunohistochemistry when using non-specific alkaline phosphatase as the enzyme label.     

Oxygen metabolism of mammalian spermatozoa. Generation of hydrogen peroxide by rabbit epididymal spermatozoa

Rabbit spermatozoa from the cauda epididymis produced 0.7–0.8nmol of H₂O₂/min per 10⁸ cells at cell concentrations below 10⁷ cells/ml with linear dependence on cell concentration. Above 2 × 10⁷ cells/ml, the rate again became linear with cell concentration but decreased to 0.1–0.2nmol/min per 10⁸ cells. Spermatozoa treated with amphotericin B, which makes the plasma membrane highly permeable to low-molecular-weight compounds, showed a similar dependence of H₂O₂ production rate on cell concentration; below 10⁷ cells/ml the rate was 0.3–0.4nmol/min per 10⁸ cells; above 2 × 10⁷ cells/ml, the rate was 0.1–0.2nmol/min per 10⁸ cells. Hypo-osmotically treated rabbit epididymal spermatozoa, a preparation useful for studying mitochondrial function in sperm [Keyhani & Storey (1973) Biochim. Biophys. Acta 305, 557–565] produced 0.1–0.2nmol/min per 10⁸ cells in the absence of added substrates. The dependence of rate on cell concentration was linear from 10⁷ to 2.2 × 10⁸ cells/ml. This endogenous rate was unaffected by rotenone, but stimulated 4-fold by antimycin A. Addition of the mitochondrial substrates lactate plus malate increased the rate of H₂O₂ production to 0.3nmol/min per 10⁸ cells. The decreased rate of H₂O₂ production observed with intact sperm at high cell concentrations is attributed to reaction of H₂O₂ with the cells, possibly with the plasma membrane, which is lost after hypo-osmotic treatment. Rabbit spermatozoa have glutathione peroxidase and glutathione reductase activities, but these seem to play little role in removal of H₂O₂ generated. The rate at low cell concentration is taken to be the unperturbed rate. The sources of H₂O₂ production in rabbit spermatozoa have been tentatively resolved into a low-molecular-weight component, lost after amphotericin treatment, a mitochondrial component and a rotenone-insensitive component that has not been identified.     

Ethanol-induced oxidative stress: basic knowledge

After a general introduction, the main pathways of ethanol metabolism (alcohol dehydrogenase, catalase, coupling of catalase with NADPH oxidase and microsomal ethanol-oxidizing system) are shortly reviewed. The cytochrome P₄₅₀ isoform (CYP2E1) specifically involved in ethanol oxidation is discussed. The acetaldehyde metabolism and the shift of the NAD/NADH ratio in the cellular environment (reductive stress) are stressed. The toxic effects of acetaldehyde are mentioned. The ethanol-induced oxidative stress: the increased MDA formation by incubated liver preparations, the absorption of conjugated dienes in mitochondrial and microsomal lipids and the decrease in the most unsaturated fatty acids in liver cell membranes are discussed. The formation of carbon-centered (1-hydroxyethyl) and oxygen-centered (hydroxyl) radicals during the metabolism of ethanol is considered: the generation of hydroxyethyl radicals, which occurs likely during the process of univalent reduction of dioxygen, is highlighted and is carried out by ferric cytochrome P₄₅₀ oxy-complex (P₄₅₀–Fe³⁺O₂^·−) formed during the reduction of heme-oxygen. The ethanol-induced lipid peroxidation has been evaluated, and it has been shown that plasma F₂-isoprostanes are increased in ethanol toxicity.     

Control of Interspecies Electron Flow during Anaerobic Digestion: Significance of Formate Transfer versus Hydrogen Transfer during Syntrophic Methanogenesis in Flocs

Microbial formate production and consumption during syntrophic conversion of ethanol or lactate to methane was examined in purified flocs and digestor contents obtained from a whey-processing digestor. Formate production by digestor contents or purified digestor flocs was dependent on CO₂ and either ethanol or lactate but not H₂ gas as an electron donor. During syntrophic methanogenesis, flocs were the primary site for formate production via ethanol-dependent CO₂ reduction, with a formate production rate and methanogenic turnover constant of 660 μM/h and 0.044/min, respectively. Floc preparations accumulated fourfold-higher levels of formate (40 μM) than digestor contents, and the free flora was the primary site for formate cleavage to CO₂ and H₂ (90 μM formate per h). Inhibition of methanogenesis by CHCl₃ resulted in formate accumulation and suppression of syntrophic ethanol oxidation. H₂ gas was an insignificant intermediary metabolite of syntrophic ethanol conversion by flocs, and its exogenous addition neither stimulated methanogenesis nor inhibited the initial rate of ethanol oxidation. These results demonstrated that >90% of the syntrophic ethanol conversion to methane by mixed cultures containing primarily Desulfovibrio vulgaris and Methanobacterium formicicum was mediated via interspecies formate transfer and that <10% was mediated via interspecies H₂ transfer. The results are discussed in relation to biochemical thermodynamics. A model is presented which describes the dynamics of a bicarbonate-formate electron shuttle mechanism for control of carbon and electron flow during syntrophic methanogenesis and provides a novel mechanism for energy conservation by syntrophic acetogens.     

Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide

Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O₂^·− and H₂O₂. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H₂O₂ but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (^·OH) as a consequence of the reaction of H₂O₂ with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H₂O₂ in the presence of an α-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)–ethanol spin-trapping system, the 4-POBN–^·CH(CH₃)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating ^·OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H₂O₂ in a similar manner, the magnitude of ^·OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of ^·OH spin trapped. Exogenous SOD failed to inhibit ^·OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in ^·OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H₂O₂-mediated killing seen in these mutants lacking SOD.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H₂O₂ was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H₂O₂ to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H₂O₂ on kinase activity. Similar to the results obtained with H₂O₂-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H₂O₂ are increased. These results demonstrate that physiologically relevant concentrations of H₂O₂ can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves

Leaf extracts of both Nicotiana tabacum and Nicotiana sylvestris contain multiple forms of catalase (H₂O₂:H₂O₂ oxidoreductase, EC 1.11.1.6) which are separable at different pH values by chromatofocusing columns. Marked changes in distribution of these catalases occur during seedling development and leaf maturation. The form of catalase eluting first (peak 1) was predominant during early seedling growth and present at all stages of development. Two more acidic forms (peaks 2 and 3) appeared later and comprised 29% of the total activity by 11 days postgermination. Mature leaves of N. tabacum contained peak 1 catalase, but peaks 2 and 3 represented 62% of the total activity. No interconversion of peaks 1, 2, and 3 was detected. The three forms of catalase differed in thermal stability with peak 1 > peak 2 peak 3. For N. sylvestris, t_½ at 55°C was 31.5 and 3.0 min for peaks 1 and 3, respectively, and for N. tabacum, t_½ was 41.5 and 3.2 min, respectively. All forms of catalase in tobacco show peroxidatic (measured as ethanol to acetaldehyde conversion) as well as catalatic activities. However, for both Nicotiana species the ratio peroxidatic/catalatic activity is at least 30-fold higher in peak 3 than in peaks 1 and 2. Chromatofocusing of extracts from spinach leaves separated at least four peaks of catalase activity, one of which had a 10-fold higher ratio of peroxidatic/catalatic activity than the others. Short-term growth (5 days) of tobacco seedlings under atmospheric conditions suppressing photorespiration (1% CO₂/21% O₂) reduced total catalase activity and caused a decline in peak 1 catalase and a substantial increase in the activity of peaks 2 and 3 relative to air-grown seedlings at the same stage.     

Phenotypic analysis of the ccp1Δ and ccp1Δ-ccp1 mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

The Molecular Mechanism of Amyloid β42 Peptide Toxicity: The Role of Sphingosine Kinase-1 and Mitochondrial Sirtuins

Our study focused on the relationship between amyloid β 1–42 (Aβ), sphingosine kinases (SphKs) and mitochondrial sirtuins in regulating cell fate. SphK1 is a key enzyme involved in maintaining sphingolipid rheostat in the brain. Deregulation of the sphingolipid metabolism may play a crucial role in the pathogenesis of Alzheimer’s disease (AD). Mitochondrial function and mitochondrial deacetylases, i.e. sirtuins (Sirt3,-4,-5), are also important for cell viability. In this study, we evaluated the interaction between Aβ_1–42, SphKs and Sirts in cell survival/death, and we examined several compounds to indicate possible target(s) for a strategy protecting against cytotoxicity of Aβ_1–42. PC12 cells were subjected to Aβ_1–42 oligomers and SphK inhibitor SKI II for 24–96 h. Our data indicated that Aβ_1–42 enhanced SphK1 expression and activity after 24 h, but down-regulated them after 96 h and had no effect on Sphk2. Aβ_1–42 and SKI II induced free radical formation, disturbed the balance between pro- and anti-apoptotic proteins and evoked cell death. Simultaneously, up-regulation of anti-oxidative enzymes catalase and superoxide dismutase 2 was observed. Moreover, the total protein level of glycogen synthase kinase-3β was decreased. Aβ_1–42 significantly increased the level of mitochondrial proteins: apoptosis-inducing factor AIF and Sirt3, -4, -5. By using several pharmacologically active compounds we showed that p53 protein plays a significant role at very early stages of Aβ_1–42 toxicity. However, during prolonged exposure to Aβ_1–42, the activation of caspases, MEK/ERK, and alterations in mitochondrial permeability transition pores were additional factors leading to cell death. Moreover, SphK product, sphingosine-1-phosphate (S1P), and Sirt activators and antioxidants, resveratrol and quercetin, significantly enhanced viability of cells subjected to Aβ_1–42. Our data indicated that p53 protein and inhibition of SphKs may be early key events responsible for cell death evoked by Aβ_1–42. We suggest that activation of S1P-dependent signalling and Sirts may offer a promising cytoprotective strategy.     

Flux through mitochondrial redox circuits linked to nicotinamide nucleotide transhydrogenase generates counterbalance changes in energy expenditure

Compensatory changes in energy expenditure occur in response to positive and negative energy balance, but the underlying mechanism remains unclear. Under low energy demand, the mitochondrial electron transport system is particularly sensitive to added energy supply (i.e. reductive stress), which exponentially increases the rate of H₂O₂ (JH₂O₂) production. H₂O₂ is reduced to H₂O by electrons supplied by NADPH. NADP⁺ is reduced back to NADPH by activation of mitochondrial membrane potential–dependent nicotinamide nucleotide transhydrogenase (NNT). The coupling of reductive stress-induced JH₂O₂ production to NNT-linked redox buffering circuits provides a potential means of integrating energy balance with energy expenditure. To test this hypothesis, energy supply was manipulated by varying flux rate through β-oxidation in muscle mitochondria minus/plus pharmacological or genetic inhibition of redox buffering circuits. Here we show during both non-ADP– and low-ADP–stimulated respiration that accelerating flux through β-oxidation generates a corresponding increase in mitochondrial JH₂O₂ production, that the majority (∼70–80%) of H₂O₂ produced is reduced to H₂O by electrons drawn from redox buffering circuits supplied by NADPH, and that the rate of electron flux through redox buffering circuits is directly linked to changes in oxygen consumption mediated by NNT. These findings provide evidence that redox reactions within β-oxidation and the electron transport system serve as a barometer of substrate flux relative to demand, continuously adjusting JH₂O₂ production and, in turn, the rate at which energy is expended via NNT-mediated proton conductance. This variable flux through redox circuits provides a potential compensatory mechanism for fine-tuning energy expenditure to energy balance in real time.     

Enhancement of hydrogen peroxide formation by protophores and ionophores in antimycin-supplemented mitochondria

Rat and pigeon heart mitochondria supplemented with antimycin produce 0.3–1.0nmol of H₂O₂/min per mg of protein. These rates are stimulated up to 13-fold by addition of protophores (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, carbonyl cyanide m-chloromethoxyphenylhydrazone and pentachlorophenol). Ionophores, such as valinomycin and gramicidin, and Ca²⁺ also markedly stimulated H₂O₂ production by rat heart mitochondria. The enhancement of H₂O₂ generation in antimycin-supplemented mitochondria and the increased O₂ uptake of the State 4-to-State 3 transition showed similar protophore, ionophore and Ca²⁺ concentration dependencies. Thenoyltrifluoroacetone and N-bromosuccinimide, which inhibit succinate–ubiquinone reductase activity, also decreased mitochondrial H₂O₂ production. Addition of cyanide to antimycin-supplemented beef heart submitochondrial particles inhibited the generation of O₂⁻, the precursor of mitochondrial H₂O₂. This effect was parallel to the increase in cytochrome c reduction and it is interpreted as indicating the necessity of cytochrome c₁³⁺ to oxidize ubiquinol to ubisemiquinone, whose autoxidation yields O₂⁻. The effect of protophores, ionophores and Ca²⁺ is analysed in relation to the propositions of a cyclic mechanism for the interaction of ubiquinone with succinate dehydrogenase and cytochromes b and c₁ [Wikstrom & Berden (1972) Biochim. Biophys. Acta 283, 403–420; Mitchell (1976) J. Theor. Biol. 62, 337–367]. A collapse in membrane potential, increasing the rate of ubisemiquinone formation and O₂⁻ production, is proposed as the molecular mechanism for the enhancement of H₂O₂ formation rates observed on addition of protophores, ionophores and Ca²⁺.     

Mitochondrial transmembrane potential is diminished in phorbol myristate acetate-stimulated peritoneal resident macrophages isolated from wild-type mice,but not in those from gp91-phox-deficient mice

Macrophages produce superoxide (O₂^–) during phagocytosis or upon stimulation with a variety of agents including phorbol myristate acetate (PMA) through the activation of NADPH oxidase, and the formed O₂^– is converted to other reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂). The aim of the present study was to elucidate the effect of the intracellularly produced ROS on mitochondrial transmembrane potential (MTP) in mouse (C57BL/6) peritoneal resident macrophages stimulated with PMA. Using a fluorescent dye, succinimidyl ester of dichlorodihydrofluorescein (H₂DCFDA), O₂^– was visualized in intracellular compartments in a certain subpopulation of macrophages isolated from wild-type mice. Cells deficient in gp91-phox, one of the membrane components of NADPH oxidase, were negative for the fluorescence. When cells were loaded with both H₂DCFDA and MitoCapture, a fluorescent dye for mitochondria, mitochondrial fluorescence was diminished in O₂^–-producing cells, but not in O₂^–-deficient cells. Flow cytometry also revealed the decrease of mitochondrial fluorescence in wild-type cells, but not in gp91-phox-deficient cells. The loss of mitochondrial fluorescence was prevented by microinjection of catalase into cells. The present findings demonstrate that MTP is diminished by ROS, including the H₂O₂ dismutated from O₂^–, produced intracellularly by activation of the NADPH oxidase in mouse peritoneal resident macrophages stimulated with PMA.