Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 mu M), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However. LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn`t inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana. (C) 2010 Elsevier Ltd. All rights reserved.

Mitochondrial bioenergetics and redox state are unaltered in Trypanosoma cruzi isolates with compromised mitochondrial complex I subunit genes

In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.

Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ions and Small Molecules as Modulators of F1FO-ATPase, Mitochondrial Bioenergetics and Cell Metabolism

The properties of the mitochondrial F1FO-ATPase activated by the natural cofactor Mg2+ or by Ca2+, were studied, mainly on heart mitochondria from swine, widely used in translational medicine. The Ca2+ driven conformational changes in the F1FO-ATPase form the mitochondrial permeability transition pore (mPTP), which triggers regulated cell death and is involved in severe pathologies. The Ca2+-activated F1FO-ATPase hydrolyzes ATP with kinetics slightly different from those of the Mg2+-ATPase. Known F1-ATPase inhibitors inhibit both the Ca2+-activated F1FO-ATPase and the mPTP formation strengthening the molecular link between them. The different Gd3+ effects on the Ca2+- and Mg2+-activated F1FO-ATPases confirm their difference as also phenylglyoxal which preferentially inhibits the Ca2+-activated F1FO-ATPase. The effects of phenylarsine and dibromobimane, which interact with differently distant Cys thiols, show that mPTP opening is ruled by nearby or distant dithiols. Bergamot polyphenols and melatonin inhibit the mPTP and ROS formation. H2S, a known cardiovascular protector, unaffects the F1FO-ATPase, but inhibits Ca2+ absorption and indirectly the mPTP, both in swine heart and mussel midgut gland mitochondria. New generation triazoles inhibit the Ca2+-activated F1FO-ATPase and the mPTP, but unaffect the Mg2+-activated F1FOATPase. In parallel, the energy metabolism was investigated in mammalian cells. In boar sperm ATP is mainly produced by mitochondrial oxidative phosphorylation (OXPHOS), even if it decreases over time because of less active mitochondria. Insufficient ATP may induce sperm dysfunction. Also, canine mesenchymal stem cells rely on OXPHOS; those from umbilical cord which produce more ATP than those from adipose tissue, seem preferable for transplant studies. The intestinal porcine enterocyte cell line IPEC-J2, used for human gut research, responds to different fetal bovine serum concentrations by remodeling OXPHOS without altering the bioenergetic parameters. The IPEC-J2 bioenergetics is modulated by Vitamin K vitamers. These data shoulder cell bioenergetics as precious tool for medical research.

Overexpression Of Ucp1 In Tobacco Induces Mitochondrial Biogenesis And Amplifies A Broad Stress Response.

Uncoupling protein one (UCP1) is a mitochondrial inner membrane protein capable of uncoupling the electrochemical gradient from adenosine-5'-triphosphate (ATP) synthesis, dissipating energy as heat. UCP1 plays a central role in nonshivering thermogenesis in the brown adipose tissue (BAT) of hibernating animals and small rodents. A UCP1 ortholog also occurs in plants, and aside from its role in uncoupling respiration from ATP synthesis, thereby wasting energy, it plays a beneficial role in the plant response to several abiotic stresses, possibly by decreasing the production of reactive oxygen species (ROS) and regulating cellular redox homeostasis. However, the molecular mechanisms by which UCP1 is associated with stress tolerance remain unknown. Here, we report that the overexpression of UCP1 increases mitochondrial biogenesis, increases the uncoupled respiration of isolated mitochondria, and decreases cellular ATP concentration. We observed that the overexpression of UCP1 alters mitochondrial bioenergetics and modulates mitochondrial-nuclear communication, inducing the upregulation of hundreds of nuclear- and mitochondrial-encoded mitochondrial proteins. Electron microscopy analysis showed that these metabolic changes were associated with alterations in mitochondrial number, area and morphology. Surprisingly, UCP1 overexpression also induces the upregulation of hundreds of stress-responsive genes, including some involved in the antioxidant defense system, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glutathione-S-transferase (GST). As a consequence of the increased UCP1 activity and increased expression of oxidative stress-responsive genes, the UCP1-overexpressing plants showed reduced ROS accumulation. These beneficial metabolic effects may be responsible for the better performance of UCP1-overexpressing lines in low pH, high salt, high osmolarity, low temperature, and oxidative stress conditions. Overexpression of UCP1 in the mitochondrial inner membrane induced increased uncoupling respiration, decreased ROS accumulation under abiotic stresses, and diminished cellular ATP content. These events may have triggered the expression of mitochondrial and stress-responsive genes in a coordinated manner. Because these metabolic alterations did not impair plant growth and development, UCP1 overexpression can potentially be used to create crops better adapted to abiotic stress conditions.

The cyclophilin inhibitor alisporivir prevents hepatitis C virus-mediated mitochondrial dysfunction.

Alisporivir (Debio-025) is an analogue of cyclosporine A and represents the prototype of a new class of non-immunosuppressive cyclophilin inhibitors. In vitro and in vivo studies have shown that alisporivir inhibits hepatitis C virus (HCV) replication, and ongoing clinical trials are exploring its therapeutic potential in patients with chronic hepatitis C. Recent data suggest that the antiviral effect is mediated by inhibition of cyclophilin A, which is an essential host factor in the HCV life cycle. However, alisporivir also inhibits mitochondrial permeability transition by binding to cyclophilin D. Because HCV is known to affect mitochondrial function, we explored the effect of alisporivir on HCV protein-mediated mitochondrial dysfunction. Through the use of inducible cell lines, which allow to investigate the effects of HCV polyprotein expression independent from viral RNA replication and which recapitulate the major alterations of mitochondrial bioenergetics observed in infectious cell systems, we show that alisporivir prevents HCV protein-mediated decrease of cell respiration, collapse of mitochondrial membrane potential, overproduction of reactive oxygen species and mitochondrial calcium overload. Strikingly, some of the HCV-mediated mitochondrial dysfunctions could even be rescued by alisporivir. Conclusion: These observations provide new insights into the pathogenesis of HCV-related liver disease and reveal an additional mechanism of action of alisporivir that is likely beneficial in the treatment of chronic hepatitis C. (HEPATOLOGY 2012).

Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

Overexpression of UCP1 in tobacco induces mitochondrial biogenesis and amplifies a broad stress response

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

FIPRONIL AND IMIDACLOPRID REDUCE HONEYBEE MITOCHONDRIAL ACTIVITY

Bees have a crucial role in pollination; therefore, it is important to determine the causes of their recent decline. Fipronil and imidacloprid are insecticides used worldwide to eliminate or control insect pests. Because they are broad-spectrum insecticides, they can also affect honeybees. Many researchers have studied the lethal and sublethal effects of these and other insecticides on honeybees, and some of these studies have demonstrated a correlation between the insecticides and colony collapse disorder in bees. The authors investigated the effects of fipronil and imidacloprid on the bioenergetic functioning of mitochondria isolated from the heads and thoraces of Africanized honeybees. Fipronil caused dose-dependent inhibition of adenosine 5'-diphosphate-stimulated (state 3) respiration in mitochondria energized by either pyruvate or succinate, albeit with different potentials, in thoracic mitochondria; inhibition was strongest when respiring with complex I substrate. Fipronil affected adenosine 5'-triphosphate (ATP) production in a dose-dependent manner in both tissues and substrates, though with different sensitivities. Imidacloprid also affected state-3 respiration in both the thorax and head, being more potent in head pyruvate-energized mitochondria; it also inhibited ATP production. Fipronil and imidacloprid had no effect on mitochondrial state-4 respiration. The authors concluded that fipronil and imidacloprid are inhibitors of mitochondrial bioenergetics, resulting in depleted ATP. This action can explain the toxicity of these compounds to honeybees. (c) 2014 SETAC

Effect of hypoxia and hyperglycemia on cell bioenergetics

Mitochondria have a central role in energy supply in cells, ROS production and apoptosis and have been implicated in several human disease and mitochondrial dysfunctions in hypoxia have been related with disorders like Type II Diabetes, Alzheimer Disease, inflammation, cancer and ischemia/reperfusion in heart. When oxygen availability becomes limiting in cells, mitochondrial functions are modulated to allow biologic adaptation. Cells exposed to a reduced oxygen concentration readily respond by adaptive mechanisms to maintain the physiological ATP/ADP ratio, essential for their functions and survival. In the beginning, the AMP-activated protein kinase (AMPK) pathway is activated, but the responsiveness to prolonged hypoxia requires the stimulation of hypoxia-inducible factors (HIFs). In this work we report a study of the mitochondrial bioenergetics of primary cells exposed to a prolonged hypoxic period . To shine light on this issue we examined the bioenergetics of fibroblast mitochondria cultured in hypoxic atmospheres (1% O2) for 72 hours. Here we report on the mitochondrial organization in cells and on their contribution to the cellular energy state. Our results indicate that prolonged hypoxia cause a significant reduction of mitochondrial mass and of the quantity of the oxidative phosphorylation complexes. Hypoxia is also responsible to damage mitochondrial complexes as shown after normalization versus citrate synthase activity. HIF-1α plays a pivotal role in wound healing, and its expression in the multistage process of normal wound healing has been well characterized, it is necessary for cell motility, expression of angiogenic growth factor and recruitment of endothelial progenitor cells. We studied hypoxia in the pathological status of diabetes and complications of diabetes and we evaluated the combined effect of hyperglycemia and hypoxia on human dermal fibroblasts (HDFs) and human dermal micro-vascular endothelial cells (HDMECs) that were grown in high glucose, low glucose concentrations and mannitol as control for the osmotic challenge.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K(+) channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.

Efeitos de lantadenos sobre a bioenergética em mitocôndrias isoladas de fígado de rato

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mecanismos da infertilidade causada por Gossipol em ratos e efeito protetor da vitamina E

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Comparative effects of fipronil and its metabolites sulfone and desulfinyl on the isolated rat liver mitochondria

Fipronil is an insecticide extensively used to control pests in crops and animals. There are relates of poisoning due to exposure of fipronil in mammals and the liver has been suggested as potential target. In this study, we evaluated the effects of fipronil and its metabolites sulfone and desulfinyl on the bioenergetics, reactive oxygen species (ROS) production and calcium efflux from mitochondria isolated from rat liver. Fipronil (5-25 μM) inhibited state-3 respiration in mitochondria energized with glutamate plus malate, substrates of complex I of the respiratory chain and decreased the mitochondrial membrane potential resulting in inhibition of ATP synthesis. Fipronil also caused uncoupling in succinate-energized mitochondria and calcium efflux. The metabolites sulfone and desulfinyl also acted as mitochondrial inhibitors and uncouplers and caused calcium efflux, but with different potencies, being the sulfone the more potent one. These effects of fipronil and its metabolites on mitochondrial bioenergetics and calcium homeostasis may be related to toxic effects of the insecticide in the liver.

Mecanismos de toxicidade dos metabólitos do fipronil, dessulfinil e sulfona, em mitocôndrias isoladas de fígado de rato

Pós-graduação em Ciência e Tecnologia Animal - FEIS