Antonio Galina

Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation ANTIOXIDANT ROLE OF MITOCHONDRIAL KINASE-DEPENDENT ADP RE-CYCLING ACTIVITY

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846–39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H2O2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H2O2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H2O2 production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ΔΨm and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate1

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

Sepsis induces brain mitochondrial dysfunction.

Objective:Mitochondrial dysfunctions have been associated with the pathogenesis of sepsis. A systematic survey of mitochondrial function in brain tissues during sepsis is lacking. In the present work, we investigate brain mitochondrial function in a septic mouse model. Design:Prospective animal study. Setting:University research laboratory. Subjects:Male Swiss mice, aged 6–8 wks. Interventions:Mice were subjected to cecal ligation and perforation (sepsis group) with saline resuscitation or to sham operation (control group). Measurements and Main Results:Oxygen consumption was measured polarographically in an oximeter. Brain homogenates from septic animals presented higher oxygen consumption in the absence of adenosine 5′-diphosphate (state 4) compared with control animals. The increase in state 4 respiration in animals in the cecal ligation and perforation group resulted in a drastic decrease in both respiratory control and adenosine 5′-diphosphate/oxygen ratios, indicating a reduction in the oxidative phosphorylation efficiency. Septic animals presented a significant increase in the recovery time of mitochondrial membrane potential on adenosine 5′-diphosphate addition compared with control animals, suggesting a proton leak through the inner mitochondrial membrane. The septic group presented a general reduction in the content of cytochromes. Moreover, the activity of cytochrome c oxidase was specifically and significantly decreased in the brain during sepsis. Hydrogen peroxide generation by brain mitochondria from septic mice did not respond to substrates of electron transport chain or to adenosine 5′-diphosphate, showing that mitochondrial function may be compromised in a critical level in the brain during sepsis. Conclusions:The mitochondrial dysfunctions demonstrated here indicate that uncoupling of oxidative phosphorylation takes place in the brain of septic mice, compromising tissue bioenergetic efficiency.

Reactive oxygen species generation is modulated by mitochondrial kinases: Correlation with mitochondrial antioxidant peroxidases in rat tissues

Mitochondrial hexokinase (mt-HK) and creatine kinase (mt-CK) activities have been recently proposed to reduce the rate of mitochondrial ROS generation through an ADP re-cycling mechanism. Here, we determined the role of mt-HK and mt-CK activities in regulate mitochondrial ROS generation in rat brain, kidney, heart and liver, relating them to the levels of classical antioxidant enzymes. The activities of both kinases were significantly higher in the brain than in other tissues, whereas the activities of catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) were higher in both liver and kidney mitochondria. In contrast, manganese superoxide dismutase (Mn-SOD) activity was not significantly different among these tissues. Activation of mitochondrial kinases by addition of their substrates increased the ADP re-cycling and thus the respiration by enhancing the oxidative phosphorylation. Succinate induced hydrogen peroxide (H(2)O(2)) generation was higher in brain than in kidney and heart mitochondria, and the lowest in liver mitochondria. Mitochondrial membrane potential (DeltaPsi(m)) and H(2)O(2) production, decreased with additions of 2-DOG or Cr to respiring brain and kidney mitochondria but not to liver. The inhibition of H(2)O(2) production by 2-DOG and Cr correspond to almost 100% in rat brain and about 70% in kidney mitochondria. Together our data suggest that mitochondrial kinases activities are potent preventive antioxidant mechanism in mitochondria with low peroxidase activities, complementing the classical antioxidant enzymes against oxidative stress.

Amyloid-β Triggers the Release of Neuronal Hexokinase 1 from Mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimers disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.

Reactive Oxygen Species Production by Potato Tuber Mitochondria Is Modulated by Mitochondrially Bound Hexokinase Activity

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (KMGlc = 140 μm versus KMFrc = 1,375 μm). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H2O2) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H2O2 release. The blockage of H2O2 release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F0F1ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H2O2 release. Inhibition in H2O2 release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H2O2 release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H2O2 release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.

Cold Tolerance in Hypothyroid Rabbits: Role of Skeletal Muscle Mitochondria and Sarcoplasmic Reticulum Ca2+ ATPase Isoform 1 Heat Production

Brown adipose tissue (BAT) is involved in rat and mice thermoregulation, and heat produced by BAT depends on the concerted action of thyroid hormones and catecholamines. Little is known about cold-induced thermogenesis in mammals that have little or no BAT, such as rabbits. In these animals, thermogenesis primarily occurs in skeletal muscle. In this work, we have studied the effect of cold acclimation (4 C for 10 d) in normal and hypothyroid rabbits. It is known that hypothyroid rats die after a few hours of cold exposure. We now show that, different from rats, hypothyroid rabbits sustain their body temperature and survive after 10 d cold exposure. When compared with rabbits kept at room temperature, the muscles of cold-exposed rabbits showed a dark red color characteristic of oxidative muscle fibers. According to this pattern, we observed that in both normal and hypothyroid rabbits, cold exposure promotes an increase in oxygen consumption by skeletal muscle mitochondria. Moreover, in red muscle, cold acclimation induces an increase in the expression and activity of sarcoplasmic reticulum Ca(2+) ATPase isoform 1 (SERCA1), one of the muscle enzymes involved in heat production. We conclude that rabbit cold tolerance is probably related to increased muscle oxidative metabolism and heat production by SERCA1 and that these changes are not completely dependent on normal thyroid function.

Succinate dehydrogenase (mitochondrial complex II) is a source of reactive oxygen species in plants and regulates development and stress responses.

Reactive oxygen species (ROS) are signaling molecules that regulate plant development and responses to stresses. Mitochondria are the source of most ROS in heterotrophic cells, and mitochondrial complex I and complex III are regarded as the main sites of ROS production in plant mitochondria. Recent studies have demonstrated that succinate dehydrogenase (SDH) also contributes to mitochondrial ROS production. However, the ability of SDH to generate ROS in plants is unclear. The aim of this study was to evaluate the role of SDH in mitochondrial ROS production. Our results demonstrated that SDH is a direct source of ROS in Arabidopsis thaliana and Oryza sativa, and the induction of ROS production by specific SDH inhibitors impaired plant growth. In addition, this effect was accompanied by the down-regulation of cell cycle genes and the up-regulation of stress-related genes. However, the partial inhibition of SDH by a competitive inhibitor decreased ROS production, which was associated with increased shoot and root growth, and prevented the down-regulation of cell cycle genes and the induction of stress-related genes by noncompetitive inhibitors. In conclusion, SDH plays an important role in ROS production, being a direct source of ROS in plant mitochondria and regulating plant development and stress responses.

Role of estrogen on skeletal muscle mitochondrial function in ovariectomized rats: a time course study in different fiber types

Postmenopausal women are prone to develop obesity and insulin resistance, which might be related to skeletal muscle mitochondrial dysfunction. In a rat model of ovariectomy (OVX), skeletal muscle mitochondrial function was examined at short- and long-term periods after castration. Mitochondrial parameters in the soleus and white gastrocnemius muscle fibers were analyzed. Three weeks after surgery, there were no differences in coupled mitochondrial respiration (ATP synthesis) with pyruvate, malate, and succinate; proton leak respiration; or mitochondrial reactive oxygen species production. However, after 3 wk of OVX, the soleus and white gastrocnemius muscles of the OVX animals showed a lower use of palmitoyl-carnitine and glycerol-phosphate substrates, respectively, and decreased peroxisome proliferator-activated receptor-γ coactivator-1α expression. Estrogen replacement reverted all of these phenotypes. Eight weeks after OVX, ATP synthesis was lower in the soleus and white gastrocnemius muscles of the OVX animals than in the sham-operated and estrogen-treated animals; however, when normalized by citrate synthase activity, these differences disappeared, indicating a lower muscle mitochondria content. No differences were observed in the proton leak parameter. Mitochondrial alterations did not impair the treadmill exercise capacity of the OVX animals. However, blood lactate levels in the OVX animals were higher after the physical test, indicating a compensatory extramitochondrial ATP synthesis system, but this phenotype was reverted by estrogen replacement. These results suggest early mitochondrial dysfunction related to lipid substrate use, which could be associated with the development of the overweight phenotype of ovariectomized animals.