Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

Inhibitory effect of metformin on oxidation of NADH-dependent substrates in rat liver homogenate.

Metformin is widely used in the treatment of Type 2 diabetes, however, mechanisms of its antihyperglycemic effect were not yet fully elucidated. Complex I of mitochondrial respiration chain is considered as one of the possible targets of metformin action. In this paper, we present data indicating that the inhibitory effect of metformin can be tested also in liver homogenate. Contrary to previous findings on hepatocytes or mitochondria under our experimental conditions, lower metformin concentrations and shorter time of preincubation give significant inhibitory effects. These conditions enable to study the mechanism of the inhibitory effect of metformin in small samples of biological material (50-100 mg wet weight) and compare more experimental groups of animals because isolation of mitochonria is unnecessary.

A novel deficiency of mitochondrial ATPase of nuclear origin.

We report a new type of fatal mitochondrial disorder caused by selective deficiency of mitochondrial ATP synthase (ATPase). A hypotrophic newborn from a consanguineous marriage presented severe lactic acidosis, cardiomegaly and hepatomegaly and died from heart failure after 2 days. The activity of oligomycin-sensitive ATPase was only 31-34% of the control, both in muscle and heart, but the activities of cytochrome c oxidase, citrate synthase and pyruvate dehydrogenase were normal. Electrophoretic and western blot analysis revealed selective reduction of ATPase complex but normal levels of the respiratory chain complexes I, III and IV. The same selective deficiency of ATPase was found in cultured skin fibroblasts which showed similar decreases in ATPase content, ATPase hydrolytic activity and level of substrate-dependent ATP synthesis (20-25, 18 and 29-33% of the control, respectively). Pulse-chase labelling of patient fibroblasts revealed low incorporation of [(35)S]methionine into assembled ATPase complexes, but increased incorporation into immunoprecipitated ATPase subunit beta, which had a very short half-life. In contrast, no difference was found in the size and subunit composition of the assembled and newly produced ATPase complex. Transmitochondrial cybrids prepared from enucleated fibroblasts of the patient and rho degrees cells derived from 143B. TK(-)human osteosarcoma cells fully restored the ATPase activity, ATP synthesis and ATPase content, when compared with control cybrids. Likewise, the pattern of [(35)S]methionine labelling of ATPase was found to be normal in patient cybrids. We conclude that the generalized deficiency of mitochondrial ATPase described is of nuclear origin and is caused by altered biosynthesis of the enzyme.

Defective kinetics of cytochrome c oxidase and alteration of mitochondrial membrane potential in fibroblasts and cytoplasmic hybrid cells with the mutation for myoclonus epilepsy with ragged-red fibres ('MERRF') at position 8344 nt.

We have investigated pathogenic effects of the tRNA(Lys) A8344G mutation associated with the syndrome myoclonus epilepsy with ragged-red fibres (MERRF) by using fibroblasts and fibroblast-derived cytoplasmic hybrid cells harbouring different percentages of mutated mitochondrial DNA (mtDNA). The activity of cytochrome c oxidase (COX) in patient fibroblasts with 89% mutated mtDNA was decreased to 20% of the control levels. COX exhibited altered kinetics, with a decreased V(max) for both the low-affinity and high-affinity phases; however, the K(m) values were not significantly changed. The substrate-dependent synthesis of ATP was decreased to 50% of the control. Analysis of the mitochondrial membrane potential, DeltaPsi, in digitonin-treated cells with tetramethylrhodamine methyl ester (TMRM) with the use of flow cytometry showed a 80% decrease in DeltaPsi at state 4 and an increased sensitivity of DeltaPsi to an uncoupler in fibroblasts from the patient. The investigation of transmitochondrial cytoplasmic hybrid clones derived from the patient's fibroblasts enabled us to characterize the relationship between heteroplasmy of the MERRF mutation, COX activity and DeltaPsi. Within the range of 87-73% mutated mtDNA, COX activity was decreased to 5-35% and DeltaPsi was decreased to 6-78%. These results demonstrate that the MERRF mutation affects COX activity and DeltaPsi in different proportions with regard to mutation heteroplasmy and indicate that the biochemical manifestation of the MERRF mutation exerts a very steep threshold of DeltaPsi inhibition.

Effect of triiodothyronine administration on the recovery of liver oxidative capacity after partial hepatectomy.

The aim of the present work was to investigate the role of triiodothyronine on liver regeneration after partial hepatectomy in the rat. During the first 3 days of liver regeneration, total protein, total DNA content and total cytochrome c oxidase activity of the residual tissue increased from 30% immediately after partial hepatectomy to 70% of the preoperative values in control rats. In triiodothyronine-treated rats, the increase in total protein was the same (70%), however, the increase in total liver DNA content and total cytochrome c oxidase acitivity was 100 and 135%, respectively during the first 3 days of regeneration. Triiodothyronine administration also increased significantly the activity of mitochondrial ATPase in regenerating liver.

Hypothyroidism leads to a decreased expression of mitochondrial F0F1-ATP synthase in rat liver.

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F0F1-ATP synthase are accompanied by a decrease in the amount of immunodetected beta-F1, F0 1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for beta-F1 subunit in liver of hypothyroid rats. Administration of 3,5,3'-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F0F1-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F0F1-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.

The effect of triiodothyronine on cell oxidative capacity in regenerating rat liver.

The recovery of total DNA content and recovery of total cytochrome c oxidase activity in the rat liver after partial hepatectomy is accelerated by triiodothyronine applied in three doses, two before and one immediately after liver resection. Triiodothyronine-treated animals already have higher cytochrome c oxidase activity before resection. The recovery of the tissue oxidative capacity after partial hepatectomy is more rapid in triiodothyronine-treated animals. These data indicate that hormonal activation of the liver regeneration process is involved.

Inhibition of mitochondrial cytochrome C oxidase by dicarbanonaborates.

Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitochondrial ATPase or glycerol phosphate dehydrogenase, inhibition of cytochrome c oxidase was not competitive and the residual, drug-insensitive activity was higher. These results indicate that dicarbanonaborates inhibit various mitochondrial membrane-bound enzymes through different mechanisms.

Respiratory control index of mitochondria isolated from regenerating rat liver.

Mitochondria were isolated from regenerating rat liver 12, 24 and 48 h after partial hepatectomy. The "State 3" and "State 4" respiration were measured in the presence of succinate. The P/O quotient and respiratory control index (RCI) were calculated. The experimental data showed that the partial uncoupling of oxidative phosphorylation in regenerating liver mitochondria occurring in the early period of regeneration is partly due to free fatty acids.

The role of mitochondria in aging.

Aging is a process drawing attention of many researchers, and at present many theories exists, which try to explain this chain of inevitable events leading to death of organism. In this article we focused our attention on a theory explaining the degenerative changes occurring during aging by the effect of oxygen free radicals. These highly reactive radicals are produced during oxidative phosphorylation in mitochondria. All cellular components appear to be sensitive to oxygen-radical damage. Lipids, proteins and nucleic acids are probably the most susceptible to this injury. Lipoperoxidation of lipids together with cross-linking of proteins with phospholipids and nucleic acids caused changes in membrane fluidity. Mitochondrial DNA coding several subunits of respiratory chain enzymes can be also damaged by these radicals. All these changes together have negative impact on mitochondrial metabolism resulting progressive decrease of the efficiency of oxidative phosphorylation and thus of the whole organism.

Dicarbanonaborates--new inhibitors of mitochondrial L-glycerol-3-phosphate dehydrogenase.

The inhibitory effect of mercapto- and chlorodicarbanonaborates on mitochondrial, FAD-linked, L-glycerol-3-phosphate dehydrogenase activity was tested. The mercaptodicarbanonaborate was found to be a more potent inhibitor than the chloro derivative. The Ki of mercapto- and chlorodicarbanonaborates were 0.8 M and 480 M, respectively. Whereas the mercaptodicarbanonaborate inhibited the enzyme activity competitively, the inhibitory effect of chloro derivative was noncompetitive. The inhibitory effect of mercaptodicarbanonaborate was completely prevented by addition of bovine serum albumin.

The effect of lipid peroxidation on the activity of various membrane-bound ATPases in rat kidney.

Peroxidation of membrane phospholipids is accompanied by alteration of the structural and functional characteristics of membranes. Lipid peroxidation changes the activities of various enzymes. The present study evaluates the effect of lipid peroxidation on the activity of various ATPases localized on kidney membranes. Our experiments were performed on crude preparation of rat kidney membranes which were exposed to Fe2+.ADP/NADPH-induced lipid peroxidation. The extent of peroxidation was estimated by measuring the thiobarbituric acid-reactive substances. Simultaneously the activities of different ATPases were determined and divided according to their ouabain sensitivity and Mg2+ dependency. We found that 10 min incubation of isolated rat kidney membranes at 37 degrees C with inductors of lipid peroxidation increased the production of thiobarbituric acid-reactive substances from 1.10 +/- 0.26 to 7.72 +/- 2.55 nmol malondialdehyde/mg prot. (+/- SD, n = 4). Under these conditions total ATPase activity was decreased from 681 +/- 77 to 507 +/- 82, ouabain-sensitive Mg(2+)-dependent ATPase (Na+,K(+)-ATPase) activity from 249 +/- 54 to 81 +/- 21 and ouabain-insensitive Mg(2+)-dependent activity from 287 +/- 48 to 173 +/- 58 whereas apparently Mg(2+)-independent ATPase activity was increased from 145 +/- 37 to 253 +/- 42 nmoles P/min/mg prot. (+/- SD, n = 4). The study indicates different mechanisms by which lipoperoxides affect the function of membrane-bound ATPases activities. It is concluded that the ATPases activities are changed during lipid peroxide formation.

Inhibition of mitochondrial ATPase by dicarbopolyborate, a new enzyme inhibitor.

Polyborate anions were found to inhibit mitochondrial ATPase. Mercapto and chloro derivatives of dicarbononaborates showed full inhibition of the enzyme activity at 0.5-0.8 mM. The inhibitory effect of dodecaborates was lower. The inhibition was of competitive type with respect to ATP. The inhibition of soluble F1-ATPase indicates a direct interaction of the polyborate anion with the catalytic part of the enzyme molecule.

Age dependent changes in mitochondrial FoF1 ATP synthase in regenerating rat-liver.

The effect of aging on rat liver regeneration and on the FoF1-ATP synthase complex of isolated liver mitochondria was followed after partial (70%) hepatectomy. ATP hydrolase activity in submitochondrial particles prepared from regenerating liver was first depressed; the time needed to reach the lowest activity was age dependent. This decrease was accompanied by parallel decrease of i) the respiratory rate of succinate supplemented mitochondria in state III; ii) the respiratory control index; iii) the rate of synthesis of ATP in succinate supplemented submitochondrial particles. This first phase of liver regeneration, characterized at all ages by a lag phase in the growth, was followed by a second phase in which the tissue mass was restored and the enzyme activities normalized. Immunoblot analysis showed that the changes in the catalytic activities of the FoF1-ATP synthase observed during liver regeneration were accompanied by parallel changes in the amount of subunits of both the catalytic (F1) and the membrane (Fo) sector of the complex.

The effect of phospholipase A2 on mitochondrial glycerol-3-phosphate oxidation.

Glycerol-3-phosphate oxidation in brown adipose tissue mitochondria of cold-adapted hamster is strongly inhibited by phospholipase A2 (PLA2). Our data show that the glycerol-3-phosphate branch of the respiratory chain is sensitive to PLA2 action more than the succinate branch and that the transfer of reducing equivalents from the glycerol-3-phosphate dehydrogenase to artificial electron acceptor is especially sensitive to the PLA2 action.

Lipid peroxidation in isolated membranes of cerebral cortex, heart and kidney.

The extent of ADP.Fe/NADPH-induced lipid peroxidation measured as production of thiobarbituric acid-reactive substances (TBARS) was determined in isolated membranes from cerebral cortex, heart and kidney of 21-days-old rats. The time course of lipid peroxidation showed higher production of TBARS in cerebral cortex than in heart and kidney. Our data indicate that high level of TBARS production is not due to high activity of NADPH oxidoreductase but due to high content of endogenous lipids in cerebral cortex membranes that could be modified. Higher production of TBARS in cerebral cortex is the result of higher content of lipids in cerebral cortex membranes because NADPH cytochrome c reductase activity in membranes of cerebral cortex is lower than that of heart and kidney.

Age-dependent changes in the mitochondrial F0F1 ATP synthase.

The age dependence of ATP hydrolase activity and oligomycin sensitive passive proton conduction in sonicated submitochondrial particles of rat brain and rat heart has been investigated. The results show an increase of Vmax of the ATP hydrolase activity and decrease of oligomycin sensitive passive proton conduction with increase of the age of rats from 3 to 6 months. Decrease of ATPase activity and increase of oligomycin sensitive proton conduction occur with further aging to 24 months. Immunoblot analysis shows that both the F(1) and F(0) contents of mitochondria vary with the age of rats, the former exhibiting relatively larger changes.

The effect of lysophosphatidylcholine on the activity of various mitochondrial enzymes.

The influence of lysophosphatidylcholine (LPC) on H(+)-ATPase, cytochrome oxidase (COX), glycerolphosphate dehydrogenase (GPDH) and malate dehydrogenase (MDH) was followed. The activities of H(+)-ATPase and COX increased with increasing LPC concentration up to 0.5 mg/mg protein when maxima were achieved. This activatory effect is LPC-specific, because Lubrol-treated or frozen-thawed mitochondria showed lower activities of these enzymes. H(+)-ATPase was not influenced by higher concentration of LPC, while COX activity decreased with increasing amount of LPC. The activity of GPDH decreased at very low concentration of LPC and was not further modified at higher LPC concentration. In an attempt to find the concentration of LPC necessary for a complete permeabilization of inner mitochondrial membrane we followed the influence of lysolipid on the release of MDH activity from the mitochondrial matrix. The full activity of this enzyme was obtained with a concentration 0.75 mg LPC/mg protein indicating that mitochondria were completely broken. Our data indicate that LPC significantly affects activity of enzymes connected with mitochondrial membrane and can be useful for evaluation of the importance of phospholipid microenvironment for the enzyme function.

Metabolic differentiation of bloodstream forms of Trypanosoma brucei brucei into procyclic forms in hemin-depleted medium and in the presence of respiratory inhibitors.

Bloodforms of Trypanosoma brucei brucei (STIB 247) differentiated in vitro into procyclic forms as described in the accompanying paper (Markos et al. 1989). The importance of the respiratory chain for the process was tested by the inhibition of its development (omission of hemin from the medium) or function (respiratory inhibitors). In the absence of hemin, all enzyme markers of the procyclic state, except for hemoproteins, developed to 50-70 per cent of control values. The presence of hemin is therefore not essential for the onset of differentiation, although the process cannot be completed under hemin limitation. Addition of 1 mumole.dm-3 KCN, 10 mumole.dm-3 antimycin A, or 100 mumole.dm-3 salicyl hydroxamate (SHAM) did not block the differentiation, although it proceeded at a slower rate. The development of the inner mitochondrial membrane markers--succinate: cytochrome c reductase, and NADH: cytochrome c reductase--was strongly inhibited by KCN or antimycin. None of these inhibitors had a significant effect on the activity of procyclic state marker--glycosomal malate dehydrogenase.