Glucose suppresses superoxide generation in metabolically responsive pancreatic beta cells

High rates of glucose metabolism and mitochondrial electron transport have been associated with increased mitochondrial production of reactive oxygen species (ROS). This mechanism was also proposed as a possible cause for dysfunction and death of pancreatic beta cells exposed to high glucose levels. We examined whether high rates of glucose metabolism increase ROS production in purified rat beta cells. Glucose up to 20 mm did not stimulate H(2)O(2) or superoxide production, whereas it dose-dependently increased cellular NAD(P)H and FADH(2) levels with an EC(50) around 8 mm. On the contrary, glucose concentration-dependently suppressed H(2)O(2) and superoxide formation, with a major effect between 0 and 5 mm, parallel to an increase in cellular NAD(P)H levels. This suppressive effect was more marked in beta cells with higher NAD(P)H responsiveness to glucose; it was not observed in glucagon-containing alpha cells, which lacked a glucose-induced increase in NAD(P)H. Suppression was also induced by the mitochondrial substrates leucine and succinate. Experiments with electron transport chain inhibitors indicate a role of respiratory complex I in ROS production at low mitochondrial activity and low NADH levels. Superoxide production at low glucose is potentially cytotoxic, because scavenging by the superoxide dismutase mimetic agent manganese(III)tetrakis(4-benzoic acid)porphyrin was found to reduce the rate of beta cell apoptosis. Analysis of islets cultured at 20 mm glucose confirmed that this condition does not induce ROS production in beta cells as a result of their increased rates of glucose metabolism. Our study indicates the need of beta cells for basal nutrients maintaining mitochondrial NADH production at levels that suppress ROS accumulation from an inadequate respiratory complex I activity and thus inhibit a potential apoptotic pathway.     

Silibinin activates AMP-activated protein kinase to protect neuronal cells from oxygen and glucose deprivation-re-oxygenation

In this study, we explored the cytoprotective potential of silibinin against oxygen–glucose deprivation (OGD)-induced neuronal cell damages, and studied underling mechanisms. In vitro model of ischemic stroke was created by keeping neuronal cells (SH-SY5Y cells and primary mouse cortical neurons) in an OGD condition followed by re-oxygenation. Pre-treatment of silibinin significantly inhibited OGD/re-oxygenation-induced necrosis and apoptosis of neuronal cells. OGD/re-oxygenation-induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) reduction were also inhibited by silibinin. At the molecular level, silibinin treatment in SH-SY5Y cells and primary cortical neurons led to significant AMP-activated protein kinase (AMPK) signaling activation, detected by phosphorylations of AMPKα1, its upstream kinase liver kinase B1 (LKB1) and the downstream target acetyl-CoA Carboxylase (ACC). Pharmacological inhibition or genetic depletion of AMPK alleviated the neuroprotective ability of silibinin against OGD/re-oxygenation. Further, ROS scavenging ability by silibinin was abolished with AMPK inhibition or silencing. While A-769662, the AMPK activator, mimicked silibinin actions and suppressed ROS production and neuronal cell death following OGD/re-oxygenation. Together, these results show that silibinin-mediated neuroprotection requires activation of AMPK signaling.     

Mitochondrial complex I impairment in leukocytes from type 2 diabetic patients

Diabetes is associated with oxidative stress. This study evaluated the rates of oxidative stress and mitochondrial impairment in type 2 diabetes patients. The study population consisted of 182 diabetic patients and 50 body-composition- and age-matched controls. We assessed anthropometric and metabolic parameters and mitochondrial function by evaluating mitochondrial oxygen (O2) consumption, reactive oxygen species (ROS) production, glutathione (GSH) levels, GSH/GSSG ratio, mitochondrial membrane potential, and mitochondrial complex I activity in polymorphonuclear cells from diabetes type 2 patients. We found an increase in waist circumference and augmented serum levels of triglycerides, proinflammatory cytokines (IL-6 and TNF-α), homocysteine, glycated hemoglobin, ultrasensitive C-reactive protein, glucose, insulin, and homeostasis model assessment of insulin resistance score in diabetic patients versus controls. There was an impairment of mitochondrial function in diabetic patients, evidenced by a decrease in mitochondrial O2 consumption, an increase in ROS production, decreased GSH/GSSG ratio, a drop in GSH levels, and an undermining of the mitochondrial membrane potential. Furthermore, an impairment of mitochondrial complex I was detected. This study supports the hypothesis of an association of type 2 diabetes and the rate of impaired mitochondrial function. We also propose that one of the targets of oxidative stress responsible for diabetes is mitochondrial complex I.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Abnormalities of mitochondrial functioning can partly explain the metabolic disorders encountered in sarcopenic gastrocnemius

Aging triggers several abnormalities in muscle glycolytic fibers including increased proteolysis, reactive oxygen species (ROS) production and apoptosis. Since the mitochondria are the main site of substrate oxidation, ROS production and programmed cell death, we tried to know whether the cellular disorders encountered in sarcopenia are due to abnormal mitochondrial functioning. Gastrocnemius mitochondria were extracted from adult (6 months) and aged (21 months) male Wistar rats. Respiration parameters, opening of the permeability transition pore and ROS production, with either glutamate (amino acid metabolism) or pyruvate (glucose metabolism) as a respiration substrate, were evaluated at different matrix calcium concentrations. Pyruvate dehydrogenase and respiratory complex activities as well as their contents measured by Western blotting analysis were determined. Furthermore, the fatty acid profile of mitochondrial phospholipids was also measured. At physiological calcium concentration, state III respiration rate was lowered by aging in pyruvate conditions (-22%), but not with glutamate. The reduction of pyruvate oxidation resulted from a calcium-dependent inactivation of the pyruvate dehydrogenase system and could provide for the well-known proteolysis encountered during sarcopenia. Matrix calcium loading and aging increased ROS production. They also reduced the oxidative phosphorylation. This was associated with lower calcium retention capacities, suggesting that sarcopenic fibers are more prone to programmed cell death. Aging was also associated with a reduced mitochondrial superoxide dismutase activity, which does not intervene in toxic ROS overproduction but could explain the lower calcium retention capacities. Despite a lower content, cytochrome c oxidase displayed an increased activity associated with an increased n-6/n-3 polyunsaturated fatty acid ratio of mitochondrial phospholipids. In conclusion, we propose that mitochondria obtained from aged muscle fibers display several functional abnormalities explaining the increased proteolysis, ROS overproduction and vulnerability to apoptosis exhibited by sarcopenic muscle. These changes appear to be related to modifications of the fatty acid profile of mitochondrial lipids.     

P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK

Silibinin is an active constituent extracted from blessed milk thistle (Silybum marianum). Our previous study demonstrated that silibinin induced autophagy and apoptosis via reactive oxygen species (ROS) generation in HeLa cells. In this study, we investigated whether the autophagy- and apoptosis-associated molecules also involved in ROS generation. Silibinin promoted the expression phosphorylated-p53 (p-p53) in a dose-dependent manner. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced ROS production and reversed silibinin's growth-inhibitory effect. The ROS scavenger N-acetyl cysteine (NAC) attenuated silibinin-induced up-regulation of p-p53 expression, suggesting that p53 might be regulated by ROS and forms a positive feedback loop with ROS. On the other hand, silibinin dose-dependently promoted the expression of phosphorylated-c-Jun N-terminal kinase (p-JNK). Inhibition of JNK by SP600125 decreased ROS generation. NAC down-regulated the expression of p-JNK, indicating that JNK could be activated by ROS. Activation of p53 was suppressed by SP600125 and expression of p-JNK was inhibited by PFT-α, therefore silibinin might activate a ROS-JNK-p53 cycle to induce cell death. Silibinin up-regulated the PUMA and Bax expressions and down-regulated the mitochondrial membrane potential (MMP) level. PFT-α reduced the expression of PUMA and Bax. These results showed that p53 could interfere with mitochondrial functions such as MMP via PUMA pathways, thus resulting in ROS generation. In order to elucidate the functions of p53 in silibinin induced ROS generation, we have chosen the A431 cells (human epithelial carcinoma) because they lack p53 activity (p53His273 mutation). Interestingly, silibinin did not up-regulate the ROS level in A431 cells but lower the ROS level. PFT-α had no influence on ROS level in A431 cells. p53 activation plays a crucial role in silibinin induced ROS generation.     

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I

There is substantial evidence that Reactive Oxygen Species (ROS) play a major part in cell functioning. Although their harmfulness through oxidative stress is well documented, their role in signaling and sensing as an oxidative signal still needs to be investigated. In most cells, the mitochondrial Electron Transport Chain (ETC) is the primary source of ROS. The production of ROS by reverse electron transfer through complex I has been demonstrated both in an experimental context but also in many pathophysiological situations. Therefore, understanding the mechanisms that regulate this ROS production is of great interest to control its harmful effects. We used nigericin, Pi and valinomycin as tools to modulate the pH gradient (?pH) and the membrane potential (?Ψ) of the protonmotive force (?p) in liver and muscle mitochondria to accurately determine how these parameters control the ROS production. We show that a high ?Ψ is the “sine qua none” condition for ROS production from the reverse electron transfer (RET) through the complex I. However, a high ?Ψ is not the only condition governing ROS production. Indeed, using tools that modulate the mitochondrial NADH level, we also demonstrate that ROS production is directly related to the mitochondrial redox potential when the membrane potential is almost stable.     

Mitochondria in exercise-induced oxidative stress

In recent years it has been suggested that reactive oxygen species (ROS) are involved in the damage to muscle and other tissues induced by acute exercise. Despite the small availability of direct evidence for ROS production during exercise, there is an abundance of literature providing indirect support that oxidative stress occurs during exercise. The electron transport associated with the mitochondrial respiratory chain is considered the major process leading to ROS production at rest and during exercise. It is widely assumed that during exercise the increased electron flow through the mitochondrial electron transport chain leads to an increased rate of ROS production. On the other hand, results obtained by in vitro experiments indicate that mitochondrial ROS production is lower in state 3 (ADP-stimulated) than in state 4 (basal) respiration. It is possible, however, that factors, such as temperature, that are modified in vivo during intense physical activity induce changes (uncoupling associated with loss of cytochrome oxidase activity) leading to increased ROS production. The mitochondrial respiratory chain could also be a potential source of ROS in tissues, such as liver, kidney and nonworking muscles, that during exercise undergo partial ischemia because of reduced blood supply. Sufficient oxygen is available to interact with the increasingly reduced respiratory chain and enhance the ROS generation. At the cessation of exercise, blood flow to hypoxic tissues resumes leading to their reoxygenation. This mimics the ischemia-reperfusion phenomenon, which is known to cause excessive production of free radicals. Apart from a theoretical rise in ROS, there is little evidence that exercise-induced oxidative stress is due to its increased mitochondrial generation. On the other hand, if mitochondrial production of ROS supplies a remarkable contribution to exercise-induced oxidative stress, mitochondria should be a primary target of oxidative damage. Unfortunately, there are controversial reports concerning the exercise effects on structural and functional characteristics of mitochondria. However, the isolation of mitochondrial fractions by differential centrifugation has shown that the amount of damaged mitochondria, recovered in the lightest fraction, is remarkably increased by long-lasting exercise.     

Life-history Constraints on the Mechanisms that Control the Rate of ROS Production

The quest to understand why and how we age has led to numerous lines of investigation that have gradually converged to consider mitochondrial metabolism as a major player. During mitochondrial respiration a small and variable amount of the consumed oxygen is converted to reactive species of oxygen (ROS). For many years, these ROS have been perceived as harmful by-products of respiration. However, evidence from recent years indicates that ROS fulfill important roles as cellular messengers. Results obtained using model organisms suggest that ROS-dependent signalling may even activate beneficial cellular stress responses, which eventually may lead to increased lifespan. Nevertheless, when an overload of ROS cannot be properly disposed of, its accumulation generates oxidative stress, which plays a major part in the ageing process. Comparative studies about the rates of ROS production and oxidative damage accumulation, have led to the idea that the lower rate of mitochondrial oxygen radical generation of long-lived animals with respect to that of their short-lived counterpart, could be a primary cause of their slow ageing rate. A hitherto largely under-appreciated alternative view is that such lower rate of ROS production, rather than a cause may be a consequence of the metabolic constraints imposed for the large body sizes that accompany high lifespans. To help understanding the logical underpinning of this rather heterodox view, herein I review the current literature regarding the mechanisms of ROS formation, with particular emphasis on evolutionary aspects.     

Cancer cells metabolically “fertilize” the tumor microenvironment with hydrogen peroxide,driving the Warburg effect: Implications for PET imaging of human tumors

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.Key words: tumor stroma, microenvironment, hydrogen peroxide, aerobic glycolysis, mitochondrial oxidative phosphorylation, glucose uptake, oxidative stress, reactive oxygen species (ROS), cancer associated fibroblasts, PET imaging, the field effect, caveolin-1     

Mitochondrial ABC transporters function: the role of ABCB10 (ABC-me) as a novel player in cellular handling of reactive oxygen species

Mitochondria are one of the major sources of reactive oxygen species (ROS) in the cell. When exceeding the capacity of antioxidant mechanisms, ROS production may lead to different pathologies, such as ischemia-reperfusion injury, neurodegeneration, anemia and ageing. As a consequence of the endosymbiotic origin of mitochondria, eukaryotic cells have developed different transport mechanisms that coordinate mitochondrial function with other cellular compartments. Four mitochondrial ATP-binding cassette (ABC) transporters have been described to date in mammals: ABCB6, ABCB8, ABCB7 and ABCB10. ABCB10 is located in the inner mitochondrial membrane forming homodimers, with the ATP binding domain facing the mitochondrial matrix. ABCB10 expression is highly induced during erythroid differentiation and its overexpression increases hemoglobin synthesis in erythroid cells. However, ABCB10 is also expressed in nonerythroid tissues, suggesting a role not directly related to hemoglobin synthesis. Recent evidence points toward ABCB10 as an important player in the protection from oxidative stress in mammals. In this regard, ABCB10 is required for normal erythropoiesis and cardiac recovery after ischemia-reperfusion, processes intimately related to mitochondrial ROS generation. Here, we review the current knowledge on mitochondrial ABC transporters and ABCB10 and discuss the potential mechanisms by which ABCB10 and its transport activity may regulate oxidative stress. We discuss ABCB10 as a potential therapeutic target for diseases in which increased mitochondrial ROS production and oxidative stress play a major role.     

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide,driving the Warburg effect

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Metabolic effects of silibinin in the rat liver

The flavonolignan silibinin, which is a mixture of two diastereoisomers, silybin A and silybin B, is a component of the extract obtained from the fruit and seeds of the variegated milk thistle (Silybum marianum (L.) Gaertn. (Asteraceae)), known as silymarin. Among the therapeutic properties credited to silibinin, its antihyperglycaemic action has been extensively explored. Silibinin is structurally related to the flavonoids quercetin and fisetin, which have been previously demonstrated to be very active on liver metabolic processes related to glycaemic regulation. The aim of the present work was to investigate the effects of silibinin on metabolic pathways responsible for the maintenance of glycaemia, particularly glycogenolysis and gluconeogenesis, in the perfused rat liver. The activities of some key enzymes in these pathways and on parameters of energy metabolism in isolated mitochondria were also examined. At a concentration range of 50-300μM, silibinin inhibited gluconeogenesis in the fasted condition and inhibited glycogenolysis and glycolysis in the fed condition. The mechanisms by which silibinin exerted these actions were multiple and complex. It inhibited the activity of glucose 6-phosphatase, inhibited the pyruvate carrier, and reduced the efficiency of mitochondrial energy transduction. It can also act by reducing the supply of NADH for gluconeogenesis and mitochondria through its pro-oxidative actions. In general, the effects and the potency of silibinin were similar to those of quercetin and fisetin. However, silibinin exerted some distinct effects such as the inhibitory effect on oxygen consumption in the fed condition and a change in the energy status of the perfused livers. It can be concluded that the effects of silibinin on liver glucose metabolism may explain its antihyperglycaemic property. However, this effect was, in part, secondary to impairment in cellular energy metabolism, a finding that should be considered in its therapeutic usage.     

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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) 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.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.