Mitochondria-targeted dodecyltriphenylphosphonium (C12TPP) combats high-fat-diet-induced obesity in mice.

BACKGROUND: A membrane-penetrating cation, dodecyltriphenylphosphonium (C12TPP), facilitates the recycling of fatty acids in the artificial lipid membrane and mitochondria. C12TPP can dissipate mitochondrial membrane potential and may affect total energy expenditure and body weight in animals and humans. METHODS: We investigated the metabolic effects of C12TPP in isolated brown-fat mitochondria, brown adipocyte cultures and mice in vivo. Experimental approaches included the measurement of oxygen consumption, carbon dioxide production, western blotting, magnetic resonance imaging and bomb calorimetry. RESULTS: In mice, C12TPP (50 µmol per (day•kg body weight)) in the drinking water significantly reduced body weight (12%, P<0.001) and body fat mass (24%, P<0.001) during the first 7 days of treatment. C12TPP did not affect water palatability and intake or the energy and lipid content in feces. The addition of C12TPP to isolated brown-fat mitochondria resulted in increased oxygen consumption. Three hours of pretreatment with C12TPP also increased oligomycin-insensitive oxygen consumption in brown adipocyte cultures (P<0.01). The effects of C12TPP on mitochondria, cells and mice were independent of uncoupling protein 1 (UCP1). However, C12TPP treatment increased the mitochondrial protein levels in the brown adipose tissue of both wild-type and UCP1-knockout mice. Pair-feeding revealed that one-third of the body weight loss in C12TPP-treated mice was due to reduced food intake. C12TPP treatment elevated the resting metabolic rate (RMR) by up to 18% (P<0.05) compared with pair-fed animals. C12TPP reduced the respiratory exchange ratio, indicating enhanced fatty acid oxidation in mice. CONCLUSIONS: C12TPP combats diet-induced obesity by reducing food intake, increasing the RMR and enhancing fatty acid oxidation.

Novel Mitochondrial Cationic Uncoupler C4R1 Is an Effective Treatment for Combating Obesity in Mice.

Obesity is associated with premature mortality, impaired quality of life, and large healthcare costs. However, treatment options remain quite limited. Here we studied potential anti-obesity effects of a novel cationic mitochondrial uncoupler, C4R1 (derivative of rhodamine 19) in C57Bl/6 mice. Obesity was induced by long-term (eight weeks) high fat diet feeding at thermoneutrality. The treated group of mice received consecutively two doses of C4R1 in drinking water (30 and 12-14 µmol/kg daily) during 30 days. Effects of C4R1 were dose-dependent. After six days of C4R1 treatment at dose 30 µmol/kg daily, food intake was reduced by 68%, body weight by 19%, and fat mass by 21%. Body weight decrease was explained partly by reduced food intake and partly by increased metabolism, likely resulting from uncoupling. Body fat reduction upon C4R1 treatment was associated with improved lipid utilization estimated from decrease in respiratory quotient to the minimal level (0.7). Interestingly, the classical uncoupler 2,4-dinitrophenol at similar dose (27 µmol/kg daily) did not have any effect. Our results are relevant to the search for substances causing mild uncoupling of mitochondria that could be a promising therapeutic strategy to treat obesity.

Age-associated murine cardiac lesions are attenuated by the mitochondria-targeted antioxidant SkQ1.

Age-related changes in mammalian hearts often result in cardiac hypertrophy and fibrosis that are preceded by inflammatory infiltration. In this paper, we show that lifelong treatment of BALB/c and C57BL/6 mice with the mitochondria-targeted antioxidant SkQ1 retards senescence-associated myocardial disease (cardiomyopathy), cardiac hypertrophy, and diffuse myocardial fibrosis. To investigate the molecular basis of the action of SkQ1, we have applied DNA microarray analysis. The global gene expression profile in heart tissues was not significantly affected by administration of SkQ1. However, we found some small but statistically significant modifications of the pathways related to cell-to-cell contact, adhesion, and leukocyte infiltration. Probably, SkQ1-induced decrease in leukocyte and mesenchymal cell adhesion and/or infiltration lead to a reduction in age-related inflammation and subsequent fibrosis. The data indicate a causative role of mitochondrial reactive oxygen species in cardiovascular aging and imply that SkQ1 has potential as a drug against age-related cardiac dysfunction.

Reduced content of α-synuclein in peripheral blood leukocytes of patients with LRRK2-associated Parkinson's disease.

Measurement of α-synuclein level in the peripheral blood was proposed as a diagnostic test for Parkinson's disease. However, the results of these studies remain contradictory, probably because the examined samples included patients with different etiology of Parkinson's disease. To verify this assumption we studied the levels of α-synuclein in peripheral blood leukocytes of patients with Parkinson's disease associated with mutations in the gene of leucine-rich kinase 2 (LRRK2). The mean α-synuclein level was significantly lower in patients with LRRK2-associated Parkinson's disease (N=8) than in patients with sporadic form of the disease (N=33; p<0.02) and in controls (N=18; p<0.05). On the other hand, we found no differences in the level of α-synuclein level between patients with sporadic form of the disease and controls. We hypothesize that the level of α-synuclein in the peripheral blood largely depends on the etiology of the disease and cannot be used as a universal diagnostic test for Parkinson's disease.

Generation of reactive oxygen species by mitochondria in senescence-accelerated OXYS rats.

Chemiluminescence assay showed that oxygen reduction and production of superoxide anion and hydrogen peroxide by liver mitochondria in OXYS rats highly sensitive to oxidative stress were less intensive than in Wistar rats. Experiments with cytochrome c oxidase inhibitors showed that decreased O2-. generation in mitochondria of OXYS rats is probably associated with changes in complex III of the electron transport chain.

Dynamics of structural and functional changes in hepatocyte mitochondria of senescence-accelerated OXYS rats.

Electron microscopy showed signs of degenerative dystrophic changes in hepatocytes and structural decompensation of mitochondria in senescence-accelerated OXYS rats in comparison with Wistar rats. These signs were detected in the presence of liver mitochondria dysfunction in OXYS rats: reduced oxygen consumption rate in all metabolic states, respiratory control volume, ADP/O ratio. transmembrane potential, and phosphorylation rate. The revealed disorders in mitochondrial structure and function are the key factors in the pathogenesis of accelerated aging in OXYS rats and visceropathies characteristic of these animals.

[Use of mirtilene forte and adrusen zinco for correction of oxidative lesions in mitochondria in rats OXYS with inherited hyperproduction of free radicals]. / Primenenie mirtilene forte i adruzena tsinco dlia korrektsii okislitel'nykh povrezhdenii mitokhondrii krys OXYS s vrozhdennoi sposobnost'iu k radikaloobrazovaniiu.

A long-term uptake of a diet supplemented with Mirtilene Forte (Vaccinum myrtillus extract) or Adrusen Zinco (a vitamin E complex with zinc, copper, selenium and omega-3 polyunsturated fatty acid) improved the functional state of liver mitochondria in OXYS rat strain. Adrusen Zinco sharply decreases the level of protein and lipid oxidation products. Possible mechanisms of the drug action are considered in relationship with their antioxidant properties.

Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid.

We have previously shown that one of the most potent rodent hepatocarcinogens, perfluorooctanoic acid (PFOA), induces apoptosis in human HepG2 cells in a dose- and time-dependent manner. In this study we have investigated the involvement of reactive oxygen species (ROS), mitochondria, and caspase-9 in PFOA-induced apoptosis. Treatment with 200 and 400 microM PFOA was found to cause a dramatic increase in the cellular content of superoxide anions and hydrogen peroxide after 3 h. Measurement of the mitochondrial transmembrane potential (Delta Psi(m)) after PFOA treatment showed a dissipation of Delta Psi(m) at 3 h. Caspase-9 activation was seen at 5 h after treatment with 200 microM PFOA. In order to evaluate the importance of these events in PFOA-induced apoptosis, cells were cotreated with PFOA and N-acetylcysteine (NAC), a precursor of glutathione, or Cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MPT pore). NAC reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced ROS. In addition, CsA also reduced Delta Psi(m) dissipation, caspase 9 activation, and apoptosis, indicating a role for PFOA-induced opening of the MPT pore. In summary, we have delineated a ROS and mitochondria-mediated pathway for induction of apoptosis by PFOA.

Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells.

The effects of perfluorooctanoic acid (PFOA), a potent hepatocarcinogen and peroxisome proliferator in rodents, on human cells have not yet been examined. In the present study we demonstrate that treatment of human hepatoblastoma HepG2 cells with PFOA induces apoptosis, as well as perturbs the cell cycle. This apoptosis was characterized by electron microscopy, which revealed typical nucleosomal fragmentation (also observed as a 'DNA ladder' upon electrophoresis on agarose) and was quantitated using propidium iodide staining of cellular DNA and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. This process was dose- and time-dependent: apoptosis became manifest with 200 microM and maximal (45% of the cells) upon exposure to 450 microM PFOA for 24 h. Electrophoresis of the DNA from HepG2 cells exposed to 500 microM PFOA for 24 h or to 400 microM PFOA for 48 h revealed a smear typical of non-specific degradation. These findings indicate that in the presence of high concentrations of PFOA for long times, HepG2 cells undergo primary and secondary necrosis. Quantitation of trypan blue exclusion supported this conclusion. Flow cytometric analysis revealed that the cell cycle of HepG2 cells was perturbed by exposure to 50-150 microM PFOA. A 50 microM concentration resulted in a significant increase in the proportion of G(2)/M cells and, simultaneously, a decrease in the number of cells in the S phase, whereas treatment with 100 or 150 microM PFOA increased the proportion of cells in the G(0)/G(1) phase and decreased the number of cells in the G(2)/M and S phases. Simultaneous flow cytometric analysis of apoptosis-associated DNA strand breaks using the TUNEL procedure and of propidium iodide staining of cellular DNA revealed DNA breaks in HepG2 cells exposed to 150 microM PFOA, prior to nuclear fragmentation.

[Oxidative phosphorylation activity, F0F1-ATPase and level of liver mitochondrial cytochromes in rats with congenitally increased ability for free radical formation]. / Aktivnost' okislitel'nogo fosforilirovaniia, F0F1-ATPazy i soderzhanie tsitokhromov mitokhondrii pecheni krys s vrozhdennym povysheniem sposobnosti radikaloobrazovaniia.

The rates of oxygen consumption in liver mitochondria of strain S rats (2-3- and 10-12-months-old) with inherited free radical hyperproduction during oxidation of three types of substrates (glutamate+maleate, succinate+rotenone and ascorbate+TMPD) as well as cytochrome a level in the respiratory chain are lowered in comparison with Wistar controls. In liver mitochondria of 10-12-months-old rats these changes are more pronounced and manifest themselves as decreased ADP/O, phosphorylation rates and F0F1-ATPase activity. The phosphorylation potential, ATP content and adenine nucleotide pool in rat liver are also reduced. The experimental results give a better insight into the pathogenetic mechanisms of morbid states (e.g., degenerative diseases) in human beings and shed additional light on the origin of short lifespan and premature in strain S rats.

[The role of the adenine nucleotide carrier in regulating energy and ion permeability of rat liver mitochondria upon cold exposure]. / Rol' perenoschika adeninnukleotidov v reguliatsii énergizatsii i ionnoi provodimosti mitokhondrii pecheni pri vozdeistvii kholoda na krys.

The energy state of rat liver mitochondria on day 10 of cold acclimation, when the body temperature decreases significantly concomitantly with an increase in the content of long-chain acyl-CoAs in the liver, has been studied. State 4 and uncoupling respiration rates increase in parallel; however, the integral potentials of the adenine nucleotide system in the liver diminish. In the presence of oligomycin, ADP (20 and 50 microM) decreases the II(+)-permeability and increases the Ca(2+)-capacity of mitochondria in both control and, in a lesser degree, cold-exposed rats. At 90 microM ADP has the same effect on mitochondria of both groups of rats. Carboxyatractylate abolishes the ADP effect on the mitochondria. In EGTA-containing media carboxyatractylate decreases the respiration rate in oligomycin-treated mitochondria. Palmitoyl-CoA increases the II(+)-permeability of the mitochondrial membrane and decreases the Ca(2+)-capacity of mitochondria. ADP abolished the competitive effects of this long-chain acyl-CoA. Possible involvement of the ATP/ADP antiporter in the thermoregulatory response of liver mitochondria in cold-acclimated rats and the role of fatty acids and long-chain acyl-CoAs in this process are discussed.

[Energy status of rat liver during the dynamics of cold adaptation]. / Energeticheskoe sostoianie pecheni krys v dinamike adaptatsii k kholodu.

The energy state of rat liver at various times of cold acclimation has been studied. On day 10 of cold exposure, the energy state of the liver is characterized by a low level of ATP and the adenine nucleotide pool as well as by low values of the phosphorylation potential and the "energy charge". The respiratory control is decreased, while the low permeability of the inner mitochondrial membrane is increased. It is supposed that on days 10-15 after cold exposure the liver becomes actively involved in thermal stabilization. Cold acclimation on days 35-42 leads to an increase in State 3 respiration rate, respiratory control and energization of liver mitochondria. This allows to maintain the ATP level and the total adenine nucleotide pool in liver tissues at the control level. However, this does not seem to compensate completely for the energy expenditure because the phosphorylation potential and the "energy charge" of the liver are thereby decreased.

Impairment of respiratory functions in mitochondria of rats with an inherited hyperproduction of free radicals.

The functional characteristics of liver mitochondria and physical-chemical properties of mitochondrial membranes were studied in S rats with congenitally enhanced capacity to free radical generation in comparison to those in Wistar rats. It was shown previously that intense lipid peroxidation, numerous DNA rearrangements, protein oxidation, morbid states resembling human degenerative diseases and short life-span are characteristic of S rats. In present study we have demonstrated that in S rats at the age of 2-3 months the respiratory rate of the mitochondria in active metabolic state is lower than in Wistar rats, and so are the values for the respiratory control ratio, oxidative phosphorylation and membrane potential. By 10-12 months of age the decrease of the respiration rate and oxidative phosphorylation in S rat mitochondria become even more dramatic. These changes are associated with a decrease in the extent of dip of proteins into the membrane lipid layer or with the increase in the amount of protein aggregates. The results add to the understanding of the nature of morbid conditions developed under the effect of intensive free radical generation and to the comprehension of their role in aging.

[Control of the energizing of liver mitochondria at the adenine nucleotide carrier level under hypotonic conditions]. / Kontrol' énergizatsii mitokhondrii pecheni na urovne perenoschika adeninnukleotidov v usloviiakh nizkoi tonichenosti sredy inkubatsii.

The effects of ADP, carboxyatractyloside (CAT) and the local anaesthetic nupercaine on the energy-dependent Ca2+ uptake by rat liver mitochondria oxidizing succinate in the presence of oligomycin were compared, using incubation media of 320 mosM and 120 mosM tonicities. In hypotonic media the mitochondrial Ca2+ capacity was increased by 50%, and the mitochondria were more stable to the damaging effects of Ca + Pi. In the presence of ADP the Ca2+ capacities of mitochondria increased both in normotonic and hypotonic media; however, the absolute amounts of calcium consumed were levelled off. CAT abolished the effect of ADP on the mitochondrial Ca2+ uptake and equalized the Ca2+ capacities of rat liver mitochondria in the both media. The local anaesthetic nupercaine also increased the Ca2+ capacity of mitochondria. The effects of nupercaine and ADP were additive. CAT abolished the effect of ADP but not that of nupercaine. Measurements of the intramitochondrial contents of adenine nucleotides showed that in 120 mosM media there was a significant increase in the intramitochondrial content of ATP and the total pool of adenine nucleotides. It was concluded that in hypotonic media the mitochondrial adenine nucleotide carrier exists predominantly in the m-conformation thus facilitating the energization of mitochondria.