Undesirable effects of chemical inhibitors of NAD(P)+ transhydrogenase on mitochondrial respiratory function.

NAD(P)+ transhydrogenase (NNT) is located in the inner mitochondrial membrane and catalyzes a reversible hydride transfer between NAD(H) and NADP(H) that is coupled to proton translocation between the intermembrane space and mitochondrial matrix. NNT activity has an essential role in maintaining the NADPH supply for antioxidant defense and biosynthetic pathways. In the present report, we evaluated the effects of chemical compounds used as inhibitors of NNT over the last five decades, namely, 4-chloro-7-nitrobenzofurazan (NBD-Cl), N,N'-dicyclohexylcarbodiimide (DCC), palmitoyl-CoA, palmitoyl-l-carnitine, and rhein, on NNT activity and mitochondrial respiratory function. Concentrations of these compounds that partially inhibited the forward and reverse NNT reactions in detergent-solubilized mouse liver mitochondria significantly impaired mitochondrial respiratory function, as estimated by ADP-stimulated and nonphosphorylating respiration. Among the tested compounds, NBD-Cl showed the best relationship between NNT inhibition and low impact on respiratory function. Despite this, NBD-Cl concentrations that partially inhibited NNT activity impaired mitochondrial respiratory function and significantly decreased the viability of cultured Nnt-/- mouse astrocytes. We conclude that even though the tested compounds indeed presented inhibitory effects on NNT activity, at effective concentrations, they cause important undesirable effects on mitochondrial respiratory function and cell viability.

Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity.

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p 50). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

Effects of partial inhibition of respiratory complex I on H2O 2 production by isolated brain mitochondria in different respiratory states.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Proliferating Astrocytes in Primary Culture Do Not Depend upon Mitochondrial Respiratory Complex I Activity or Oxidative Phosphorylation.

Understanding the role of astrocytes in the development of the nervous system and neurodegenerative disorders implies a necessary knowledge of the oxidative metabolism of proliferating astrocytes. The electron flux through mitochondrial respiratory complexes and oxidative phosphorylation may impact the growth and viability of these astrocytes. Here, we aimed at assessing to which extent mitochondrial oxidative metabolism is required for astrocyte survival and proliferation. Primary astrocytes from the neonatal mouse cortex were cultured in a physiologically relevant medium with the addition of piericidin A or oligomycin at concentrations that fully inhibit complex I-linked respiration and ATP synthase, respectively. The presence of these mitochondrial inhibitors for up to 6 days in a culture medium elicited only minor effects on astrocyte growth. Moreover, neither the morphology nor the proportion of glial fibrillary acidic protein-positive astrocytes in culture was affected by piericidin A or oligomycin. Metabolic characterization of the astrocytes showed a relevant glycolytic metabolism under basal conditions, despite functional oxidative phosphorylation and large spare respiratory capacity. Our data suggest that astrocytes in primary culture can sustainably proliferate when their energy metabolism relies only on aerobic glycolysis since their growth and survival do not require electron flux through respiratory complex I or oxidative phosphorylation.

A dataset describing glycolytic inhibitors overcoming the underestimation of maximal mitochondrial oxygen consumption rate in oligomycin-treated cells.

Determination of oxygen consumption is one of the most valuable methodologies to evaluate mitochondrial (dys)function. Previous studies demonstrated that a widely used protocol, consisting of adding the ATP synthase inhibitor oligomycin before mitochondrial respiratory uncoupling by sequential addition of a protonophore (e.g., carbonyl cyanide 3-chlorophenyl hydrazone [CCCP]), may lead to underestimation of maximal oxygen consumption rate (OCRmax) and spare respiratory capacity (SRC) parameters in highly glycolytic tumor cell lines. In this dataset, we report the effects of the glycolytic inhibitors 2-deoxy-D-glucose, iodoacetic acid, and lonidamine on overcoming the underestimation of OCRmax and SRC in oligomycin-treated cells. We propose a protocol in which 2-deoxy-D-glucose is added after oligomycin and just before the sequential addition of CCCP to avoid underestimation of OCRmax and SRC parameters in A549, C2C12, and T98G cells. The oxygen consumption rates were determined in intact suspended cell lines using a high-resolution oxygraph device. The data can be used in several fields of research that require characterization of mitochondrial respiratory parameters in intact cells.

High glycolytic activity of tumor cells leads to underestimation of electron transport system capacity when mitochondrial ATP synthase is inhibited.

This study sought to elucidate how oligomycin, an ATP synthase blocker, leads to underestimation of maximal oxygen consumption rate (maxOCR) and spare respiratory capacity (SRC) in tumor cells. T98G and U-87MG glioma cells were titrated with the protonophore CCCP to induce maxOCR. The presence of oligomycin (0.3-3.0 µg/mL) led to underestimation of maxOCR and a consequent decrease in SRC values of between 25% and 40% in medium containing 5.5 or 11 mM glucose. The inhibitory effect of oligomycin on CCCP-induced maxOCR did not occur when glutamine was the metabolic substrate or when the glycolytic inhibitor 2-deoxyglucose was present. ATP levels were reduced and ADP/ATP ratios increased in cells treated with CCCP, but these changes were minimized when oligomycin was used to inhibit reverse activity of ATP synthase. Exposing digitonin-permeabilized cells to exogenous ATP, but not ADP, resulted in partial inhibition of CCCP-induced maxOCR. We conclude that underestimation of maxOCR and SRC in tumor cells when ATP synthase is inhibited is associated with high glycolytic activity and that the glycolytic ATP yield may have an inhibitory effect on the metabolism of respiratory substrates and cytochrome c oxidase activity. Under CCCP-induced maxOCR, oligomycin preserves intracellular ATP by inhibiting ATP synthase reverse activity.

Underestimation of the Maximal Capacity of the Mitochondrial Electron Transport System in Oligomycin-Treated Cells.

The maximal capacity of the mitochondrial electron transport system (ETS) in intact cells is frequently estimated by promoting protonophore-induced maximal oxygen consumption preceded by inhibition of oxidative phosphorylation by oligomycin. In the present study, human glioma (T98G and U-87MG) and prostate cancer (PC-3) cells were titrated with different concentrations of the protonophore CCCP to induce maximal oxygen consumption rate (OCR) within respirometers in a conventional growth medium. The results demonstrate that the presence of oligomycin or its A-isomer leads to underestimation of maximal ETS capacity. In the presence of oligomycin, the spare respiratory capacity (SRC), i.e., the difference between the maximal and basal cellular OCR, was underestimated by 25 to 45%. The inhibitory effect of oligomycin on SRC was more pronounced in T98G cells and was observed in both suspended and attached cells. Underestimation of SRC also occurred when oxidative phosphorylation was fully inhibited by the ATP synthase inhibitor citreoviridin. Further experiments indicated that oligomycin cannot be replaced by the adenine nucleotide translocase inhibitors bongkrekic acid or carboxyatractyloside because, although these compounds have effects in permeabilized cells, they do not inhibit oxidative phosphorylation in intact cells. We replaced CCCP by FCCP, another potent protonophore and similar results were observed. Lower maximal OCR and SRC values were obtained with the weaker protonophore 2,4-dinitrophenol, and these parameters were not affected by the presence of oligomycin. In permeabilized cells or isolated brain mitochondria incubated with respiratory substrates, only a minor inhibitory effect of oligomycin on CCCP-induced maximal OCR was observed. We conclude that unless a previously validated protocol is employed, maximal ETS capacity in intact cells should be estimated without oligomycin. The inhibitory effect of an ATP synthase blocker on potent protonophore-induced maximal OCR may be associated with impaired metabolism of mitochondrial respiratory substrates.

Rotenone exerts similar stimulatory effects on H2O2 production by isolated brain mitochondria from young-adult and old rats.

Chronic and systemic treatment of rodents with rotenone, a classical inhibitor of mitochondrial respiratory complex I, results in neurochemical, behavioral, and neuropathological features of Parkinson's disease. The aim of the present study was to evaluate whether brain mitochondria from old rats (24 months old) would be more susceptible to rotenone-induced inhibition of oxygen consumption and increased generation of H2O2 than mitochondria from young-adult rats (3-4 months old). Isolated brain mitochondria were incubated in the presence of different rotenone concentrations (5, 10, and 100nM), and oxygen consumption and H2O2 production were measured during respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration). Respiratory state 3 and citrate synthase activity were significantly lower in mitochondria from old rats. Mitochondria from young-adult and old rats showed similar sensitivity to rotenone-induced inhibition of oxygen consumption. Similarly, H2O2 production rates by both types of mitochondria were dose-dependently stimulated to the same extent by increasing concentrations of rotenone. We conclude that rotenone exerts similar effects on oxygen consumption and H2O2 production by isolated brain mitochondria from young-adult and old rats. Therefore, aging does not increase the mitochondrial H2O2 generation in response to complex I inhibition.

Identification of compounds from high-fat and extra virgin olive oil-supplemented diets in whole mouse liver extracts and isolated mitochondria using mass spectrometry.

Nonalcoholic steatohepatitis (NASH) is a fatty liver disorder that could be improved with extra virgin olive oil (EVOO) supplementation in diet. We propose the monitoring, in whole mouse liver extracts and in isolated mitochondria, of the absorption of compounds from three different diets: standard (CT), high-fat (HFD) and high-fat supplemented with EVOO (HFSO). Male mice were submitted to one of the following three diets: CT or HFD for 16 weeks or HFD for 8 weeks followed by additional 8 weeks with HFSO. Following this period, liver was extracted for histological evaluation, mitochondria isolation and mass spectrometry analyses. Diets, liver extracts and Percoll-purified mitochondria were analyzed using ESI-MS and the lipidomics approach. Morphological, histological and spectrometric results indicated a decrease in NASH severity with EVOO supplementation in comparison with animals maintained with HFD. Spectrometric data also demonstrated that some compounds presented on the diets are absorbed by the mitochondria. EVOO was shown to be a potential therapeutic alternative in food for NASH. Our results are in accordance with the proposition that the major factor that influences different responses to diets is their composition - and not only calories - especially when it comes to studies on obesity.