Dimethylbiguanide inhibits cell respiration via an indirect effect targeted on the respiratory chain complex I

We report here a new mitochondrial regulation occurring only in intact cells. We have investigated the effects of dimethylbiguanide on isolated rat hepatocytes, permeabilized hepatocytes, and isolated liver mitochondria. Addition of dimethylbiguanide decreased oxygen consumption and mitochondrial membrane potential only in intact cells but not in permeabilized hepatocytes or isolated mitochondria. Permeabilized hepatocytes after dimethylbiguanide exposure and mitochondria isolated from dimethylbiguanide pretreated livers or animals were characterized by a significant inhibition of oxygen consumption with complex I substrates (glutamate and malate) but not with complex II (succinate) or complex IV (N,N,N',N'-tetramethyl-1, 4-phenylenediamine dihydrochloride (TMPD)/ascorbate) substrates. Studies using functionally isolated complex I obtained from mitochondria isolated from dimethylbiguanide-pretreated livers or rats further confirmed that dimethylbiguanide action was located on the respiratory chain complex I. The dimethylbiguanide effect was temperature-dependent, oxygen consumption decreasing by 50, 20, and 0% at 37, 25, and 15 degrees C, respectively. This effect was not affected by insulin-signaling pathway inhibitors, nitric oxide precursor or inhibitors, oxygen radical scavengers, ceramide synthesis inhibitors, or chelation of intra- or extracellular Ca(2+). Because it is established that dimethylbiguanide is not metabolized, these results suggest the existence of a new cell-signaling pathway targeted to the respiratory chain complex I with a persistent effect after cessation of the signaling process.     

Inhibition of respiration in mitochondria and in digitonin-treated rat hepatocytes by podophyllotoxin

Summary The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain.     

Effects of ionophores and metabolic inhibitors on the mitochondrial membrane potential within isolated hepatocytes as measured with the safranine method.

A difference spectrum with a peak of absorbance at 526nm appears slowly upon addition of valinomycin or KCN in combination with oligomycin to a hepatocyte suspension in the presence of safranine. When the cells are incubated at 37 degrees C in a medium containing safranine, a slow decrease in the absorbance occurs at the wavelength pair 524-484 nm. The change in absorbance is completed within 20-30 min after additions of cells to a medium containing safranine. At this time the safranine concentration of the outer medium is considerably decreased. The safranine signal is completely reversed by valinomycin, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or KCN in combination with oligomycin. None of these treatments have any immediate effect on cellular ATP concentrations or the 36Cl- equilibrium potential across the plasma membrane. In the presence of iodoacetate a slow reversal of the trace can be induced upon addition of KCN, but not of oligomycin alone. Rotenone, in combination with oligomycin, does not reverse the safranine signal except when both KF and iodoacetate are present, in which case a slow reversal is seen. A subsequent addition of duroquinone brings back the signal to the same level as in the presence of rotenone alone. The results indicate that the spectral response of safranine in the presence of isolated hepatocytes is a result of a slow penetration of safranine into intracellular mitochondria, where aggregation of safranine molecules occurs as a response to the mitochondrial membrane potential.     

Adenine nucleotide compartmentation in foetal rat hepatocytes. Effects of atractyloside, oligomycin, calcium ionophore and adrenergic agonists

In the presence of lactate plus pyruvate, or glucose or alanine as substrates, ATP/ADP ratios in the cytosol were higher than in mitochondria in isolated rat foetal hepatocytes. The cytosolic ATP/ADP ratios were dependent on substrate (lactate + pyruvate greater than glucose greater than alanine). Oleate increased the cytosolic ATP/ADP ratios in the presence of the other substrates studied. Atractyloside decreased the cytosolic ATP/ADP ratios, oligomycin decreasing these values in both compartments. Isoproterenol, phenylephrine and Ca2+ ionophore decreased the cytosolic ATP/ADP ratio, without altering this value in mitochondria.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Biochemical mechanism of GSH depletion induced by 1,2-dibromoethane in isolated rat liver mitochondria. Evidence of a GSH conjugation process

HPLC measurements of GSH and GSSG levels in isolated rat liver mitochondria, on addition of 1,2-dibromoethane (DBE), revealed the presence of a glutathione (GSH)-conjugating pathway of DBE. This process required the structural integrity of the mitochondrial matrix and inner membrane complex and was inhibited by the uncouplers of oxidative phosphorylation, particularly 2,4-dinitrophenol. On the other hand it was not affected by the energetic state of the mitochondria, since other mitochondrial inhibitors like KCN and oligomycin did not have any effect on it. This process also did not require the involvement of mitochondrial inner membrane transport systems, based on the measurement of the mitochondrial transmembrane potential. The involvement of mitochondrial GSH-S-transferases, located either in the matrix or in the intermembrane space, is discussed.     

Respiratory activity of isolated hepatocytes

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.     

Isolation,Purification, and Characterization of Mitochondria from Chlamydomonas reinhardtii

Mitochondria were isolated from autotrophically grown Chlamydomonas reinhardtii cell-wall-less mutant CW 92. The cells were broken by vortexing with glass beads, and the mitochondria were collected by differential centrifugation and purified on a Percoll gradient. The isolated mitochondria oxidized malate, pyruvate, succinate, NADH, and [alpha]-ketoglutarate. Respiratory control was obtained with malate (2.0) and pyruvate (2.2) but not with the other substrates. From experiments with KCN and salicylhydroxamic acid, it was estimated that the capacity of the cytochrome pathway was at least 100 nmol O2 mg-1 protein min-1 and the capacity of the alternative oxidase was at least 50 nmol O2 mg-1 protein min-1. A low sensitivity to oligomycin indicates some difference in the properties of the mitochondrial ATPase from Chlamydomonas as compared to higher plants.     

Topology of superoxide production from different sites in the mitochondrial electron transport chain

We measured production of reactive oxygen species by intact mitochondria from rat skeletal muscle, heart, and liver under various experimental conditions. By using different substrates and inhibitors, we determined the sites of production (which complexes in the electron transport chain produced superoxide). By measuring hydrogen peroxide production in the absence and presence of exogenous superoxide dismutase, we established the topology of superoxide production (on which side of the mitochondrial inner membrane superoxide was produced). Mitochondria did not release measurable amounts of superoxide or hydrogen peroxide when respiring on complex I or complex II substrates. Mitochondria from skeletal muscle or heart generated significant amounts of superoxide from complex I when respiring on palmitoyl carnitine. They produced superoxide at considerable rates in the presence of various inhibitors of the electron transport chain. Complex I (and perhaps the fatty acid oxidation electron transfer flavoprotein and its oxidoreductase) released superoxide on the matrix side of the inner membrane, whereas center o of complex III released superoxide on the cytoplasmic side. These results do not support the idea that mitochondria produce considerable amounts of reactive oxygen species under physiological conditions. Our upper estimate of the proportion of electron flow giving rise to hydrogen peroxide with palmitoyl carnitine as substrate (0.15%) is more than an order of magnitude lower than commonly cited values. We observed no difference in the rate of hydrogen peroxide production between rat and pigeon heart mitochondria respiring on complex I substrates. However, when complex I was fully reduced using rotenone, rat mitochondria released significantly more hydrogen peroxide than pigeon mitochondria. This difference was solely due to an elevated concentration of complex I in rat compared with pigeon heart mitochondria.     

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.     

水生动物和大鼠线粒体的呼吸链比较

用陆生哺乳动物线粒体呼吸链与水生动物线粒体呼吸链相比较的研究方法,探讨了呼吸链的功能与环境相适应的关系。研究了淡水中生活的草鱼肝丝线粒体,观察到琥珀酸脱氢酶的活性非常低,而ＮＡＤＨ脱氢酶和泛醌细胞色素Ｃ还原酶的活性较高。但海洋生物海绵的线粒体ＮＡＤＨ脱氢酶和琥垢酸脱氢酶的活性都非常低。     

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum)

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.     

Import of rat liver mitochondrial transhydrogenase.

The biosynthesis of pyridine dinucleotide transhydrogenase has been studied in isolated rat hepatocytes and in a rabbit reticulocyte-lysate translation system supplemented with either intact isolated rat liver mitochondria or the soluble matrix fraction from isolated mitochondria. In intact hepatocytes, the transhydrogenase precursor was short-lived in the cytosol and was efficiently imported into the membranous fraction. When the cell-free translation mixture was incubated with intact mitochondria, the transhydrogenase precursor was processed to the mature form, to an extent that depended on the amount of added mitochondria. Incubation of the translation mixture with the soluble mitochondria matrix fraction converted the precursor to a mature-sized protein with 75% efficiency, this being blocked by various proteinase inhibitors such as EDTA, 1,10-phenanthroline and leupeptin.     

A role of ubiquinone in energy conservation in mitochondria

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.     

Mitophagy is not induced by mitochondrial damage but plays a role in the regulation of cellular autophagic activity

It was postulated that mitophagy removes damaged mitochondria, which is critical for proper cellular homeostasis; dysfunctional mitochondria can generate excess reactive oxygen species (ROS) that can further damage the organelle as well as other cellular components. Although proper cell physiology requires the maintenance of a healthy pool of mitochondria, little is known about the mechanism underlying the recognition and selection of damaged organelles. We investigated the cellular fate of mitochondria damaged by the action of oxidative phosphorylation inhibitors (antimycin A, myxothiazol, KCN, oligomycin, CCCP). Only antimycin A and KCN effectively induce nonspecific autophagy, but not mitophagy, in a wild-type strain; however, low or no autophagic activity was measured in strains deficient in genes, including ATG32, ATG11 and BCK1, encoding proteins that are involved in mitophagy. These results provide evidence for a major role of specific mitophagy factors in the control of a general autophagic cellular response induced by mitochondrial alteration. Moreover, significant reduction of cytochrome b, one of the components of the respiratory chain, could be the first signal of this induction pathway.     

The action of anoxia and cyanide on glycogen breakdown in the liver of the gsd/gsd rat

Perfusion of normal rat livers under anoxic conditions or the addition of KCN to aerobic perfusions activated phosphorylase and stimulated glycogen breakdown and glucose output. Livers from rats with a deficiency of liver phosphorylase kinase (gsd/gsd) showed a much smaller activation of phosphorylase with anoxia or KCN and produced glucose at about half the rate of normal livers. The increase in phosphorylase a in gsd/gsd livers was insufficient to account for the increase in glucose output. The addition of KCN to normal hepatocytes, activated phosphorylase and stimulated glucose output almost as effectively as glucagon. Hepatocytes from gsd/gsd rats showed only a very small increase in phosphorylase a on the addition of KCN, and glucose output did not increase. We conclude that in the perfused liver, anoxia and KCN stimulate glycogen breakdown and glucose output, at least in part, by a mechanism that does not involve conversion of phosphorylase b to phosphorylase a. In isolated hepatocytes KCN stimulates glucose output only by increasing the content of phosphorylase a.     

Free-radical metabolism of carbon tetrachloride in rat liver mitochondria. A study of the mechanism of activation.

Alterations in liver mitochondria as consequence of rat poisoning with carbon tetrachloride (CCl4) have been reported over many years, but the mechanisms responsible for causing such damage are still largely unknown. Isolated rat liver mitochondria incubated under hypoxic conditions with succinate and ADP were found able to activate CCl4 to a free-radical species identified as trichloromethyl free radical (CCl3) by e.s.r. spectroscopy coupled with the spin-trapping technique. The incubation of mitochondria in air decreased free-radical production, indicating that a reductive reaction was involved in the activation of CCl4. However, in contrast with liver microsomes (microsomal fractions), mitochondria did not require the presence of NADPH, and the process was not significantly influenced by inhibitors of cytochrome P-450. The addition of inhibitors of the respiratory chain such as antimycin A and KCN decreased free-radical formation by only 30%, whereas rotenone displayed a greater effect (approx. 84% inhibition), but only when preincubated for 15 min with mitochondria not supplemented with succinate. These findings suggest that the mitochondrial electron-transport chain is responsible for the activation of CCl4. A conjugated-diene band was observed in the lipids extracted from mitochondria incubated with CCl4 under anaerobic conditions, indicating that stimulation of lipid peroxidation was occurring as a result of the formation of free-radical species.     

Evidence for participation of the inner membrane in the import of sulfite oxidase into the intermembrane space of liver mitochondria

Sulfite oxidase, a soluble enzyme in mitochondrial intermembrane space, was synthesized as a precursor protein larger than the authentic enzyme when rat liver RNA was translated $\text{in}\text{vitro}$ using reticulocyte lysate. When the $\text{in}\text{vitro}$ translation products were incubated with isolated rat liver mitochondria, the precursor of sulfite oxidase was converted to the size of the mature enzyme. The $\text{in}\text{vitro}$ processed mature enzyme was no longer susceptible to externally added proteases and was extractable by a hypotonic treatment of the mitochondria, suggesting its location in the intermembrane space. When mitochondria were subfractionated, most of the processing activity was recovered in the mitoplast fraction. The import-processing activity of mitochondria was inhibited by CCCP, oligomycin, or atractyloside in the presence of KCN. These results suggest that the import of sulfite oxidase into mitochondrial intermembrane space requires the participation of inner membrane.     

Balance between Cyanide-Sensitive and -Insensitive Respiration in Wheat Root Mitochondria, as Influenced by Salt Concentration in the Plant Growth Medium

Mitochondria were isolated from 7-day-old wheat roots (Triticum vulgare Vill. cv. Svenno Spring Wheat) grown in either a full-strength culture medium (100%) or in the same medium diluted 100 times (1%). Outer membrane integrity was assayed using the cytochrome c reduction assay. This indicated about 20% damage. Using an oxygen electrode the respiration of the mitochondria was measured with either malate or succinate as the substrate (both 40 mM). KCN (3 mM) and salicylhydroxamic acid (SHAM, 1 mM) were used as inhibitors. The properties of the isolated mitochondria (STATE 3 rate, ADP/O ratio, and KCN-sensitivity) depend upon the ionic concentration of the growth medium of the roots. In the mitochondria isolated from roots grown in the 1% medium (1% mitochondria) there is a synergistic effect of KCN and SHAM. This means that electrons can be shifted from one pathway to the other when only one of the inhibitors is added. This flexibility between the electron pathways is almost nil in the mitochondria isolated from roots grown in the 100% medium (100% mitochondria). The maximal capacity of the alternative electron pathway (= rate in the presence of KCN) is higher in 1% (40 nmol O₂ min^?1 (mg protein)^?1) than in 100% mitochondria (20 nmol O₂ min^?1 (mg protein)^?1. In 100% mitochondria the alternative pathway seems to be operating at maximal capacity in the absence of KCN with both substrates and in both STATES 3 and 4. In 1% mitochondria the alternative pathway functions at >50% of its capacity in the absence of KCN.     

On the mechanism of the so-called uncoupling effect of medium- and short-chain fatty acids

Octanoate applied to rat liver mitochondria respiring with glutamate plus malate or succinate (plus rotenone) under resting-state (State 4) conditions stimulates oxygen uptake and decreases the membrane potential, both effects being sensitive to oligomycin but not to carboxyatractyloside. Octanoate also decreases the rate of pyruvate carboxylation under the same conditions, this effect being correlated with the decrease of intramitochondrial content of ATP and increase of AMP. The decrease of pyruvate carboxylation and the change of mitochondrial adenine nucleotides are both reversed by 2-oxoglutarate. Fatty acids of shorter chain length have similar effects, though at higher concentrations. Addition of octanoate in the presence of fluoride (inhibitor of pyrophosphatase) produces intramitochondrial accumulation of pyrophosphate, even under conditions when oxidation of octanoate is prevented by rotenone. In isolated hepatocytes incubated with lactate plus pyruvate, octanoate also increases oxygen uptake and produces a shift in the profile of adenine nucleotides similar to that observed in isolated mitochondria. It decreases the ‘efficiency’ of gluconeogenesis, as expressed by the ratio between an increase of glucose production and an increase of oxygen uptake upon addition of gluconeogenic substrates (lactate plus pyruvate), and increases the reduction state of mitochondrial NAD. These effects taken together are not compatible with uncoupling, but point to intramitochondrial hydrolysis of octanoyl-CoA and probably also shorter chain-length acyl-CoAs. This mechanism probably functions as a ‘safety valve’ preventing a drastic decrease of intramitochondrial free CoA under a large supply of medium- and short-chain fatty acids.