Loss of oxidative phosphorylation in mitochondria isolated from kidneys of mercury poisoned rats

Mercurials at low concentrations suppress oxidative phosphorylation in isolated rat kidney mitochondria. The kidneys of rats administered toxic doses of HgCl₂ have been found to accumulate mercury selectively compared e.g. to liver. Mitochondria isolated from such kidneys are defective in coupled ATP synthesis. Providing the time subsequent to the injection of a toxic dose of HgCl₂ does not exceed a critical time, the defect in the mitochondria isolated from the kidneys of such poisoned rats is correctable by addition of Mg++ to the assay medium. Acute mercury toxicity in rats appears to be referable to the impairment of kidney function in turn caused by impairment of mitochondrial function.     

Synthesis of adenosine triphosphate in isolated nuclei and intact cells

1. It has previously been demonstrated that nuclei isolated from normal and neoplastic lymphoid cells are capable of oxygen-dependent ATP synthesis. In this paper it is shown that also the corresponding intact cells can synthesize ATP under those conditions in which nuclei can synthesize ATP. 2. In nuclei isolated from liver, kidney, rhabdomyosarcoma and osteosarcoma, oxygen-dependent ATP synthesis could not be demonstrated. The cells isolated from these tissues or tumours could not synthesize ATP either. The alternatives that such nuclei lost their ability for oxidative phosphorylation during the isolation procedure or that the process does not occur in these nuclei were explored. 3. Janus Green B, a vital stain for mitochondria, was used as a differential inhibitor of mitochondrial and nuclear ATP synthesis in intact cells. 4. Oxidative phosphorylation in mitochondria isolated from cells that had been incubated with various concentrations of Janus Green B (1–10μm) was seriously uncoupled, whereas at these concentrations oxygen-dependent ATP synthesis in isolated nuclei and in isolated cells were only inhibited to a small extent. 5. The results suggest that oxygen-dependent ATP synthesis in isolated cells measured under `nuclear' conditions and in the presence of Janus Green B and Ca²⁺ is mainly due to nuclear oxygen-dependent ATP synthesis. The stimulation of cellular ATP synthesis by glucose was completely inhibited by Janus Green B. 6. It is tentatively concluded that the stimulation of ATP synthesis in isolated cells by glucose, which is not found in isolated nuclei, represents mitochondrial ATP synthesis, and nuclear and mitochondrial ATP synthesis can then be studied differentially in the intact cell. The possibility is considered that oxygen-dependent nuclear ATP synthesis is not a general property of cell nuclei.     

Fatty liver and insulin resistance in obese Zucker rats: no role for mitochondrial dysfunction

The relationship between insulin resistance and mitochondrial function is of increasing interest. Studies looking for such interactions are usually made in muscle and only a few studies have been done in liver, which is known to be a crucial partner in whole body insulin action. Recent studies have revealed a similar mechanism to that of muscle for fat-induced insulin resistance in liver. However, the exact mechanism of lipid metabolites accumulation in liver leading to insulin resistance is far from being elucidated. One of the hypothetical mechanisms for liver steatosis development is an impairment of mitochondrial function. We examined mitochondrial function in fatty liver and insulin resistance state using isolated mitochondria from obese Zucker rats. We determined the relationship between ATP synthesis and oxygen consumption as well as the relationship between mitochondrial membrane potential and oxygen consumption. In order to evaluate the quantity of mitochondria and the oxidative capacity we measured citrate synthase and cytochrome c oxidase activities. Results showed that despite significant fatty liver and hyperinsulinemia, isolated liver mitochondria from obese Zucker rats display no difference in oxygen consumption, ATP synthesis, and membrane potential compared with lean Zucker rats. There was no difference in citrate synthase and cytochrome c oxidase activities between obese and lean Zucker rats in isolated mitochondria as well as in liver homogenate, indicating a similar relative amount of hepatic mitochondria and a similar oxidative capacity. Adiponectin, which is involved in bioenergetic homeostasis, was increased two-fold in obese Zucker rats despite insulin resistance. In conclusion, isolated liver mitochondria from lean and obese insulin-resistant Zucker rats showed strictly the same mitochondrial function. It remains to be elucidated whether adiponectin increase is involved in these results.     

Competition between extramitochondrial and intramitochondrial ATP-consuming processes.

The relationship between intra- and extramitochondrial ATP utilization was investigated in liver mitochondria isolated from normally fed, starved and high-protein fed rats. ATP export was provoked by adding a hexokinase-glucose-trap and intramitochondrial ATP consumption by adding ammonia, bicarbonate and ornithine in order to stimulate citrulline synthesis. Both processes compete for ATP produced via oxidative phosphorylation; the rate of citrulline formation declines as the extramitochondrial [ATP]/[ADP] ratio decreases. It is concluded that ATP for adenine nucleotide translocation and that for carbamoyl phosphate synthesis are delivered from a common intramitochondrial pool of adenine nucleotides. In mitochondria from rats with a high-protein diet, citrulline synthesis greatly stimulates the rate of oxidative phosphorylation (about two thirds of state 3 respiration). Under these conditions the intramitochondrial [ATP]/[ADP] ratio is significantly reduced. The intramitochondrial [ATP]/[ADP] ratio is not in thermodynamic equilibrium with the extramitochondrial one.     

The role of mitochondrial calcium transport during inflammation and the effect of anti-inflammatory drugs

The effects of inflammation induced by the inoculation of rats with Freund's adjuvant on calcium transport by isolated rat liver mitochondria and on mitochondrial in vivo protein synthesis were investigated. Mitochondria isolated from the liver of inflamed rats exhibited (i) a reduction in 45Ca2+ uptake and, (ii) a reduction in protein synthesis. Addition of ATP to the calcium uptake medium stimulate the uptake in inflamed rat liver mitochondria. After inflammation was controlled by treatment with a mixture of Clerodendron inerme flavonoidal glycosides and indomethacin, rat liver mitochondria showed (i) an increase in 45Ca2+ uptake and, (ii) an increase in mitochondrial in vivo protein synthesis. The mechanism of mitochondrial calcium transport and the mitochondrial protein metabolism during inflammation and after treatment with anti-inflammatory drugs were discussed.     

Failure of dietary erucic acid to impair oxidative capacity or APT production of rat heart mitochondria isolated under controlled conditions.

1. Male, 8-week old rats were fed Purina Rat Chow for semisynthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days. 2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglyerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed. 3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rats of oxidation of different substrates and of ATP synthesis by these mitochondria were compared. 4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets. 5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals. 6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.     

Failure of dietary erucic acid to impair oxidative capacity or ATP production of rat heart mitochondria isolated under controlled conditions

1. Male, 8-week old rats were fed Purina Rat Chow or semi-synthetic diets containing 20% by weight of rapeseed oil or corn oil for 3 days.2. The hearts from the animals fed the three diets were analyzed for total lipid, phospholipid, free fatty acids, cholesterol esters, tri-, di- and monoacylglycerols. There was a seven-fold increase in the levels of triacylglycerols in the hearts of rats fed rapeseed oil diet compared to the levels in the hearts of animals fed the other two diets. Smaller increases in the content of other neutral lipid fractions were also observed.3. Heart mitochondria from the three groups of animals were isolated under controlled conditions in the presence or absence of heparin. The rates of oxidation of different substrates and of ATP synthesis by these mitochondria were compared.4. Mitochondria isolated in the absence of heparin from rapeseed oil-fed rats had much lower rates of oxidation and ATP synthesis than mitochondria isolated similarly from rats fed the other two diets.5. With mitochondria freshly isolated in the presence of heparin, no significant differences in rates of oxidation or ATP synthesis were found among the three groups of animals.6. It is concluded that, when properly isolated, mitochondria from rapeseed oil-fed rats are functionally intact with respect to oxidation and energy-coupling capacity.     

Cardiolipin content is involved in liver mitochondrial energy wasting associated with cancer-induced cachexia without the involvement of adenine nucleotide translocase

Cancer-induced cachexia describes the progressive skeletal muscle wasting associated with many cancers leading to shortened survival time in cancer patients. We previously reported that cardiolipin content and energy-wasting processes were both increased in liver mitochondria in a rat model of peritoneal carcinosis (PC)-induced cachexia. To increase the understanding of the cellular biology of cancer cachexia, we investigated the involvement of adenine nucleotide translocator (ANT) in mitochondrial energy-wasting processes in liver mitochondria of PC and pair-fed control rats and its interactions with cardiolipin in isolated liver mitochondria from healthy rats exposed to cardiolipin-enriched liposomes. We showed in this study that functional ANT content was decreased in liver mitochondria from PC rats but without any effects on the efficiency of ATP synthesis. Moreover, non-phosphorylating energy wasting was not affected by saturating concentrations of carboxyatractylate (CAT), a potent inhibitor of ANT, in liver mitochondria from PC rats. Decreased efficiency of ATP synthesis was found in normal liver mitochondria exposed to cardiolipin-enriched liposomes, with increased non-phosphorylating energy wasting, thus mimicking mitochondria from PC rats. However, the functional ANT content in these cardiolipin-enriched mitochondria was unchanged, although non-phosphorylating energy wasting was reduced by CAT-induced inhibition of ANT. Finally, non-phosphorylating energy wasting was increased in cardiolipin-enriched mitochondria with substrates for complexes 1 and 2, but not for complex 4. In conclusion, increased energy wasting measured in liver mitochondria from rats with cancer cachexia is dependent on cardiolipin but independent of ANT. Interactions between ANT and cardiolipin are modified when cancer cachexia occurs.     

Control of adenine nucleotide metabolism in hepatic mitochondria from rats with ethanol-induced fatty liver

Male rats developed fatty liver after being fed on an ethanol-containing diet for 31 days. Liver mitochondria from these animals catalysed ATP synthesis at a slower rate when compared with mitochondria from pair-fed control rats (control mitochondria), and demonstrated lowered respiratory control with succinate as substrate, owing to a decrease in the State-3 respiratory rate. Respiration in the presence of uncoupler was comparable in mitochondria from both groups of rats. Translocation of both ATP and ADP was decreased in mitochondria from ethanol-fed rats, with ADP uptake being lowered more dramatically by ethanol feeding. Parameters influencing adenine nucleotide translocation were investigated in mitochondria from ethanol-fed rats. Experiments performed suggested that lowered adenine nucleotide translocation in these mitochondria is not the result of inhibition of the translocase by either long-chain acyl-CoA derivatives or unesterified fatty acids. Analysis of endogenous adenine nucleotides in these mitochondria revealed lowered ATP concentrations, but no decrease in total adenine nucleotides. In experiments where the endogenous ATP in these mitochondria was shifted to higher concentrations by incubation with oxidizable substrates or defatted bovine serum albumin, the rate of ADP translocation was increased, with a linear correlation being observed between endogenous ATP concentrations and the rate of ADP translocation. The depressed ATP concentration in mitochondria from ethanol-fed rats suggests that the ATP synthetase complex is replenishing endogenous ATP at a slower rate. The lowered ATPase activity of the ATP synthetase observed in submitochondrial particles from ethanol-fed animals suggests a decrease in the function of the synthetase complex. A decrease in the rate of ATP synthesis in mitochondria from ethanol-fed rats is sufficient to explain the decreased ADP translocation and State-3 respiration.     

Stimulation of mitochondrial functions by glucagon treatment, starvation and by treatment of isolated mitochondria with glycogen-bound enzymes

Liver mitochondria isolated from rats starved overnight, or fed rats injected with glucagon, exhibited a similar increase of the respiration rate with succinate (by 30-40%) and glutamate plus malate (by 20-30%), as compared to mitochondria from control fed animals. The content of mitochondrial adenine nucleotides was elevated by 30-45% by glucagon treatment or starvation. Mitochondrial respiration and citrulline synthesis were stimulated by 30-40% when mitochondria isolated from fed rats were briefly preincubated with the extract from liver glycogen granules, ATP and MgCl2. This effect was abolished by heating the extract at 100 degrees C.     

Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.

The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.     

Optimal conditions for studies of amino acid incorporation in vitro by isolated skeletal muscle mitochondria

Optimal conditions for amino acid incorporation into protein in vitro by isolated skeletal muscle mitochondria were established. Maximum incorporation rates were obtained when atractylate and glutamate were added to the incubation medium in the absence of any exogenous adenine nucleotides. Under these conditions, the rate of amino acid incorporation was more than 5-fold greater than that observed with glutamate and ADP and nearly 12-fold greater than that observed with ATP and an ATP-regenerating system consisting of phosphoenolpyruvate and pyruvate kinase. The optimal concentrations of adenine nucleotides, glutamate, cofactors and the substrate leucine were determined for all three energy-providing systems. The inhibitors of protein synthesis, puromycin and chloramphenicol, completely blocked amino acid incorporation by isolated skeletal muscle in mitochondria, while cycloheximide had no effect. Analysis of the labeled mitochondrial proteins by sodium dodecylsulfate polyacrylamide gel electrophoresis revealed five labeled bands of molecular weights ranging from 38,000 to 10,000.Amino acid incorporation by skeletal muscle mitochondria isolated from diabetic rats was decreased over 60% as compared to mitochondria from controls when measured in the presence of glutamate and atractylate, ADP and glutamate or the ATP regenerating system. By contrast, amino acid incorporation by liver mitochondria isolated from diabetic rats did not differ significantly from control values when measured with four different energy sources.     

Regulation of pyruvate oxidation in mitochondria isolated from fetal and adult rat liver

The effect of ATP on the velocity of oxygen uptake during the oxidation of pyruvate plus malate, in the presence of oligomycin, 2,4-dinitrophenol and fluorocitrate, was studied in mitochondria, isolated from the livers of adult and fetal rats.It was found that the addition of ATP caused an inhibition in the rate of oxygen uptake of 21 ± 6% in mitochondria from adult rat liver and 49 ± 8% in mitochondria from fetal rat liver. Measurements of the velocity of oxygen uptake during the oxidation of pyruvate plus malate and of palmitoylcarnitine in adult rat liver mitochondria in the presence of ATP showed that the activity of pyruvate dehydrogenase was lower than the activity of citrate synthase.In fetal mitochondria, addition of ATP resulted in an increase in the CoASH/acetyl-CoA ratio, indicating that pyruvate dehydrogenase was rate limiting here as well.It is concluded that ATP inhibited pyruvate oxidation by phosphorylation of the pyruvate dehydrogenase complex, rather than by inhibiting citrate synthase under these conditions.     

Effect of single exposure to the carcinogen 4-dimethylaminoazobenzene on several properties of rat liver mitochondria]

The hepatospecific carcinogen 4-dimethylaminoazobenzene (DAB), applied as a single interperitoneal injection, induced no changes in the activity of mitochondrial and cytoplasmic malate-dehydrogenase in the rat liver. The same carcinogen and non-carcinogenic isomer 4-diethylaminoazobenzene brought about decreased activity of cytochromeoxidase in isolated rat liver mitochondria. During 18 days after a single interperitoneal injection of DAB the swelling-contraction properties of isolated liver mitochondria were seen altered in the presence of succinate and ATP whereas DAB exerted no influence on mitochondria from the kidney, which organ is not a target-tissue for carcinogenic action.     

Ischemia-induced alterations of mitochondrial structure and function in brain, liver, and heart muscle of young and senescent rats

Young and senescent rats (3 and 28-30 months old) were subjected to complete ischemia at 37 degrees C in order to study function and structure of mitochondria isolated from liver, heart muscle, and brain. The rates of energy-coupled respiration and ATP synthesis were found to decrease progressively in relation to time of ischemia. The respiratory rates in the absence of ADP (state 4 respiration) did not increase after exposure to ischemia, suggesting that ischemia primarily affects electron transport rather than the energy coupling system. Mitochondria of heart muscle were more affected by ischemia than mitochondria of brain and liver. Liver and heart muscle mitochondria obtained from young rats were found to be slightly more sensitive to short periods of ischemia than those isolated from senescent animals.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Mitochondria alterations in Cd2+-treated rats: general regression of inner membrane cristae and electron transport impairment

Young adult rats absorbed 50 p.p.m. Cd2+ added to drinking water. After 6 weeks, 3, 6 and 9 months of treatment, the ultrastructural condition of liver, kidney and muscle was observed by electron microscopy. The choice of these tissues was determined by their differences in the capacity to accumulate Cd2+: the liver is able to concentrate a considerable amount of metal, but redistributes it throughout the entire organism, while the kidney collects it in view of its elimination. Muscle contains the least Cd2+. A general regression in mitochondria cristae accompanied by a vesiculation and a fragmentation of endoplasmic reticulum appeared simultaneously in the three tissues, at as early as 6 weeks of treatment, and extended progressively with its continuation supporting evidence of a general attack of the intracellular membrane systems. Cd2+ stimulation of membrane-degrading enzymes such as phospholipases and proteases was suggested. A concomitant diminution in glycogen stores was noted. Active synthesis of neutral lipids, especially cholesterol esters, took place in liver mitochondria of treated rats in collaboration with rough endoplasmic reticulum, and progressively generated a multiplication of electron-transparent inclusions in cytoplasm. Isolated mitochondria from liver, kidney and muscle of Cd2+-treated rats maintained partial energy coupling, but displayed a rapid early fall in cytochrome oxidase followed by a partial restoration after 6 months of treatment, and a progressively slackening of succinate dehydrogenase. Isolated vesicles of liver mitochondria inner membrane of treated rats behaved as intact mitochondria, indicating changes inside the membrane itself. Addition in vitro of the metal ion to mitochondria and also to inner membrane vesicles isolated from control rats revealed that Cd2+ was able to stop completely succinate dehydrogenase, but was totally ineffective on cytochrome oxidase. Membrane fixation of Cd2+ on the flavoprotein or SH associated with succinate dehydrogenase is proposed. Considering the close parallelism of the extensive depression of microsomal NADPH cytochrome c reductase and the rapid fall in mitochondrial cytochrome oxidase, it is suggested that an indirect inhibition process occurs, through Cd2+-induced diminution of a constituent common to all cytochromes in the cell.     

Studies on the relationship between ATP synthesis and transport and the proton electrochemical gradient in rat liver mitochondria

The effect of ATP synthesis on delta mu H in rat liver mitochondria has been analyzed by separating the steps of adenine nucleotide translocation and ATP synthesis in the matrix. Either exchange of ATP, synthesized by substrate level phosphorylation in the matrix of oligomycin-treated mitochondria, for external ADP, or activity of the membrane-bound ATP synthase complex results in delta mu H depression with respect to resting state levels. This depression appears to be more pronounced, under strictly comparable conditions, when arsenate is used to stimulate ATP synthase activity than when the ornithine-citrulline conversion reaction is used for the same purpose.     

Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.     

Stabilization of hsp70 mRNA on prolonged cell exposure to hypertonicity

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria.The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O(2) Clark electrodes.The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86+/-0.02 micromol/min g wet weight (min g ww)] than in control [1.44+/-0.18 micromol/min g ww]. However, mitochondrial respiration remained unchanged [2.20+/-0.13 micromol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33+/-0.035 (control) to 0.42+/-0.015 (ethanol).The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.