Age-Dependent Changes in the Function of Mitochondrial Membrane Permeability Transition Pore in Rat Liver Mitochondria.

Mitochondria play an important role in the cell aging process. Changes in calcium homeostasis and/or increased reactive oxygen species (ROS) production lead to the opening of mitochondrial permeability transition pore (MPTP), depolarization of the inner mitochondrial membrane, and decrease of ATP production. Our work aimed to monitor age-related changes in the Ca2+ ion effect on MPTP and the ability of isolated rat liver mitochondria to accumulate calcium. The mitochondrial calcium retention capacity (CRC) was found to be significantly affected by the age of rats. Measurement of CRC values of the rat liver mitochondria showed two periods when 3 to 17-week old rats were tested. 3-week and 17-week old rats showed lower CRC values than 7-week old animals. Similar changes were observed while testing calcium-induced swelling of rat liver mitochondria. These findings indicate that the mitochondrial energy production system is more resistant to calcium-induced MPTP opening accompanied by the damaging effect of ROS in adult rats than in young and aged animals.

Factors affecting the function of the mitochondrial membrane permeability transition pore and their role in evaluation of calcium retention capacity values.

Values of the calcium retention capacity (CRC) of rat liver mitochondria are highly dependent on the experimental conditions used. When increasing amounts of added calcium chloride are used (1.25-10 nmol), the values of the CRC increase 3-fold. When calcium is added in 75 s intervals, the CRC values increase by 30 % compared with 150 s interval additions. CRC values are not dependent on the calcium/protein ratio in the measured sample in our experimental design. We also show that a more detailed evaluation of the fluorescence curves can provide new information about mitochondrial permeability transition pore opening after calcium is added.

Modification of calcium retention capacity of rat liver mitochondria by phosphate and tert-butyl hydroperoxide.

By determining the calcium retention capacity (CRC) of rat liver mitochondria, we confirmed and extended previous observations describing the activation of mitochondrial swelling by phosphate and tert-butyl hydroperoxide (t-BHP). Using CRC measurements, we showed that both phosphate and t-BHP decrease the extent of calcium accumulation required for the full mitochondrial permeability transition pore (MPTP) opening to 35 % of control values and to only 15 % when both phosphate and t-BHP are present in the medium. When changes in fluorescence were evaluated at higher resolution, we observed that in the presence of cyclosporine A fluorescence values return after each Ca(2+) addition to basal values obtained before the Ca(2+) addition. This indicates that the MPTP remains closed. However, in the absence of cyclosporine A, the basal fluorescence after each Ca(2+) addition continuously increased. This increase was potentiated both by phosphate and t-BHP until the moment when the concentration of intramitochondrial calcium required for the full opening of the MPTP was reached. We conclude that in the absence of cyclosporine A, the MPTP is slowly opened after each Ca(2+) addition and that this rate of opening can be modified by various factors such as the composition of the media and the experimental protocol used.

Glucagon-like peptide-1 analogues exenatide and liraglutide exert inhibitory effect on the early phase of liver regeneration after partial hepatectomy in rats.

Glucagon-like peptide-1 (GLP-1) is an incretin known for proliferative and antiapoptotic effects on various tissues. Exenatide and Liraglutide are GLP-1 analogues used in clinical practice as antidiabetic drugs. Since GLP-1 and its analogues exert significant effect on liver metabolism and since changes in intermediary metabolism play an important role in the process of liver regeneration, we decided to determine the effect of Exenatide and Liraglutide on the early phase of liver regeneration and selected metabolic parameters in a model of 2/3 partial hepatectomy (PHx) in rats. Animals were submitted either to PHx or laparotomy and received 3 doses of either GLP-1 analogues (Exenatide - 42 microg/kg b.w., Liraglutide - 0.75 mg/kg b.w.) or saline intraperitoneally. We analyzed body and liver weight, liver bromodeoxyuridine incorporation, liver content of DNA, triacylglycerols and cholesterol and biochemical serum parameters. Bromodeoxyuridine labeling was significantly lower in hepatectomized rats receiving either type of GLP-1 analogues when compared to hepatectomized controls. This effect was more pronounced in the Liraglutide group compared to Exenatide (p<0.001). In addition, liver DNA content was lower in hepatectomized rats receiving Liraglutide than in hepatectomized control rats (p<0.001). In conclusion, GLP-1 analogues Exenatide and Liraglutide significantly inhibited an early phase of liver regeneration after PHx in rats. This inhibitory effect was more pronounced in rats receiving Liraglutide.

In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine.

Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca(2+)), phosphate (P(i)), and triiodothyronine (T(3)) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM P(i), 50 microM Ca(2+) and 25 microM T(3) when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca(2+) and P(i) are used as activating factors, the addition of T(3) doubled the rate of swelling. T(3) has also an activating effect on mitochondrial membrane potential. The T(3) activating effect was also found after in vivo application of T(3). Our data thus demonstrate that T(3) has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions.

The effect of alpha-tocopheryl succinate on succinate respiration in rat liver mitochondria.

We compared the effect of alpha-tocopheryl succinate (TOS) on succinate-dependent respiration in rat liver mitochondria, homogenate and permeabilized hepatocytes in both a coupled and uncoupled state. In isolated mitochondria, a significant inhibitory effect was observed at a concentration of 5 microM, in liver homogenate at 25 microM and in permeabilized hepatocytes at 50 microM. The inhibitory effect of TOS on succinate respiration in an uncoupled state was less pronounced than in a coupled state in all the experimental models tested. When the concentration dependence of the TOS inhibitory effect was tested, the most sensitive in both states were isolated mitochondria; the most resistant were permeabilized hepatocytes.

The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.

In vitro models serve as a tool for studies of steatosis. Palmitic and oleic acids can induce steatosis in cultured hepatocytes. The aim of our study was to verify steatogenic and cytotoxic effects of palmitic acid (PA), oleic acid (OA) and their combinations as well as their impact on functional capacity of rat primary hepatocytes. Hepatocytes were exposed to OA or PA (0.125-2 mmol/l) or their combination at ratios of 3:1, 2:1 or 1:1 at the final concentrations of 0.5-1 mmol/l. Both OA and PA caused a dose-dependent increase in triacylglycerol content in hepatocytes. PA was more steatogenic at 0.25 and 0.5 mmol/l while OA at 0.75 and 1 mmol/l. PA exhibited a dose-dependent cytotoxic effect associated with ROS production, present markers of apoptosis and necrosis and a decrease in albumin production. OA induced a damage of the cytoplasmic membrane from 1 mM concentration. Mixture of OA and PA induced lower cytotoxicity with less weakened functional capacity than did PA alone. Extent of steatosis was comparable to that after exposure to OA alone. In conclusion, OA or combination of OA with PA is more suitable for simulation of simple steatosis than PA alone.

The effect of D-galactosamine on lean and steatotic rat hepatocytes in primary culture.

The aim of our work was to compare the effect of D-galactosamine (GalN) on primary cultures of lean and steatotic rat hepatocytes isolated from intact and fatty liver, respectively. GalN caused more severe injury to steatotic hepatocytes than to lean cells as documented by lactate dehydrogenase leakage. Necrotic mode of cell death strongly prevails over apoptosis since we did not observe any significant increase in activities of caspase 3, 8 and 9 in any group of hepatocytes treated with GalN. Reactive oxygen species (ROS) formation and lipid peroxidation were elevated in a dose-dependent manner by GalN and were significantly more pronounced in fatty hepatocytes. A decrease in the percentage of hepatocytes with energized mitochondria was observed from 30 mM and 10 mM GalN in lean and steatotic hepatocytes, respectively. Our results undoubtedly indicate that steatotic hepatocytes exert higher sensitivity to the toxic effect of GalN. This sensitivity may be caused by more intensive GalN-induced ROS production and lipid peroxidation and by higher susceptibility of mitochondria to loss of mitochondrial membrane potential in steatotic hepatocytes. In our experimental arrangement, apoptosis does not seem to participate considerably on hepatotoxic action of GalN in either group of hepatocytes.

Comparison of acetaminophen toxicity in primary hepatocytes isolated from transgenic mice with different appolipoprotein E alleles.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor, important for combating electrophilic and oxidative stress in the liver and other organs. This encompasses detoxification of hepatotoxic drugs, including acetaminophen (APAP). Recently, an association between apolipoprotein E (ApoE) genotype and Nrf2 expression was described. We compared the toxicity of APAP on primary culture hepatocytes isolated from transgenic mice carrying two different human ApoE alleles and wild-type controls. The cells were exposed to APAP in concentrations from 0.5 to 4 mM for up to 24 hours. APAP led to a dose-dependent hepatotoxicity from 1 mM after 16 h exposure in all mice tested. The toxicity was higher in hepatocytes isolated from both transgenic strains than in wild-type controls and most pronounced in ApoE3 mice. Concurrently, there was a decline in mitochondrial membrane potential, especially in ApoE3 hepatocytes. The formation of reactive oxygen species was increased after 24 hours with 2.5 mM APAP in hepatocytes of all strains tested, with the highest increase being in the ApoE3 genotype. The activity of caspases 3 and 7 did not differ among groups and was minimal after 24 hour incubation with 4 mM APAP. We observed higher lipid accumulation in hepatocytes isolated from both transgenic strains than in wild-type controls. The expression of Nrf2-dependent genes was higher in ApoE3 than in ApoE4 hepatocytes and some of these genes were induced by APAP treatment. In conclusion, transgenic mice with ApoE4 and ApoE3 alleles displayed higher susceptibility to acute APAP toxicity in vitro than wild-type mice. Of the two transgenic genotypes tested, ApoE3 allele carriers were more prone to injury.

Impairment of mitochondrial function of rat hepatocytes by high fat diet and oxidative stress.

Fatty liver disease associated with obesity is an important medical problem and the mechanisms for lipid accumulation in hepatocytes are not fully elucidated yet. Recent findings indicate that mitochondria play an important role in this process. Our data on hepatocytes in which mitochondria are in contact with other cytosolic structures important for their function, extend observations obtained on isolated mitochondria and confirm inhibition of Complex I activity in hepatocytes isolated from rats fed by high fat diet (HFD) compared with controls fed by standard diet (STD). Furthermore we have found that HFD-hepatocytes are more sensitive to the peroxidative stress because under these conditions also Complex II activity is disturbed. Therefore in HFD animals decrease of Complex I activity cannot be compensated by Complex II substrates as in STD hepatocytes. Our data thus indicates that combination of HFD and peroxidative stress potentiates HFD damaging effect of mitochondria because both branches of the respiratory chain (NADH- and flavoprotein-dependent) are disturbed.

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.

[Effects of GLP-1 (glucagon-like peptide 1) on liver]. / Úcinky GLP-1 (glucagonlike peptide 1) na játra.

Effects of glucagonlike peptide 1 (GLP1) on liver cells are very intensively studied. In the metabolism of saccharides GLP1 stimulates synthesis of glycogen and reduces glucose production -  thus acting like insulin. In the lipid metabolism it enhances fatty acid oxidation and lipid transport from hepatocytes while reducing de novo lipogenesis -  effects more similar to glucagon action. Some studies suggest beneficial effects of GLP1 on oxidative stress, endoplasmic reticulum stress, production of inflammatory mediators and dysfunction of biliary secretion. Current results suggest that drugs affecting incretin system could be used in the treatment of certain liver diseases (e.g. NAFLD and NASH) in the future. In the following article we mention the known effects of GLP 1 on liver functions and liver metabolism and we point out its possible future therapeutic use in the treatment of liver diseases.

Purified acetaminophen-glutathione conjugate is able to induce oxidative stress in rat liver mitochondria.

Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104 ± 13 % and 130 ± 10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production.

Transcriptome temporal and functional analysis of liver regeneration termination.

Decades of liver regeneration studies still left the termination phase least elucidated. However regeneration ending mechanisms are clinicaly relevant. We aimed to analyse the timing and transcriptional control of the latest phase of liver regeneration, both controversial. Male Wistar rats were subjected to 2/3 partial hepatectomy with recovery lasting from 1 to 14 days. Time-series microarray data were assessed by innovative combination of hierarchical clustering and principal component analysis and validated by real-time RT-PCR. Hierarchical clustering and principal component analysis in agreement distinguished three temporal phases of liver regeneration. We found 359 genes specifically altered during late phase regeneration. Gene enrichment analysis and manual review of microarray data suggested five pathways worth further study: PPAR signalling pathway; lipid metabolism; complement, coagulation and fibrinolytic cascades; ECM remodelling and xenobiotic biotransformation. Microarray findings pertinent for termination phase were substantiated by real-time RT-PCR. In conclusion, transcriptional profiling mapped late phase of liver regeneration beyond 5(th) day of recovery and revealed 5 pathways specifically acting at this time. Inclusion of longer post-surgery intervals brought improved coverage of regeneration time dynamics and is advisable for further works. Investigation into the workings of suggested pathways might prove helpful in preventing and managing liver tumours.

Steatotic rat hepatocytes in primary culture are more susceptible to the acute toxic effect of acetaminophen.

Acetaminophen (APAP) overdose is the most common cause of acute liver failure in humans. Non-alcoholic fatty liver disease is the most frequent chronic liver disease in developed countries. The aim of our work was to compare the effect of APAP on intact rat hepatocytes and hepatocytes isolated from steatotic liver in primary cultures. Male Wistar rats were fed with standard diet (10 % energy from fat) and high-fat diet (71 % energy from fat) for 6 weeks and then hepatocytes were isolated. After cell attachment, APAP (1; 2.5; 3.75 and 5 mM) was added to culture media (William's E medium) and hepatocytes were cultured for up to 24 hours. APAP caused more severe dose-dependent damage of steatotic hepatocytes as documented by increased release of lactate dehydrogenase (LDH) and LDH leakage, decreased activity of cellular dehydrogenases (WST-1 test) and reduced albumin production. Intact steatotic hepatocytes contained lower amount of reduced glutathione (GSH). Treatment with APAP (1 and 2.5 mmol/l) caused more pronounced decrease in GSH in steatotic hepatocytes. ROS (reactive oxygen species) formation after 24-hour incubation was significantly higher in fatty hepatocytes using APAP at concentration of 3.75 and 5 mmol/l. Interleukin 6 (IL-6) production was elevated in 2.5 mM APAP-treated nonsteatotic and steatotic hepatocyte cultures at 8 hours, compared to appropriate controls. In conclusions, our results indicate that steatotic hepatocytes exert higher sensitivity to the toxic action of APAP. This sensitivity may be caused by lower content of GSH in intact steatotic hepatocytes and by more pronounced APAP-induced decrease in intracellular concentration of GSH.

Developmental changes of the sensitivity of cardiac and liver mitochondrial permeability transition pore to calcium load and oxidative stress.

Opening of the mitochondrial membrane permeability transition pore (MPTP) is an important factor in the activation of apoptotic and necrotic processes in mammalian cells. In a previous paper we have shown that cardiac mitochondria from neonatal rats are more resistant to calcium load than mitochondria from adult animals. In this study we have analyzed the ontogenetic development of this parameter both in heart and in liver mitochondria. We found that the high resistance of heart mitochondria decreases from day 14 to adulthood. On the other hand, we did not observe a similar age-dependent sensitivity in liver mitochondria, particularly in the neonatal period. Some significant but relatively smaller increase could be observed only after day 30. When compared with liver mitochondria cardiac mitochondria were more resistant also to the peroxide activating effect on calcium-induced mitochondrial swelling. These data thus indicate that the MPTP of heart mitochondria is better protected against damaging effects of the calcium load and oxidative stress. We can only speculate that the lower sensitivity to calcium-induced swelling may be related to the higher ischemic tolerance of the neonatal heart.

The effect of rat strain, diet composition and feeding period on the development of a nutritional model of non-alcoholic fatty liver disease in rats.

Non-alcoholic fatty liver disease (NAFLD) is an important cause of liver-related morbidity and mortality. The aim of this work was to establish and characterize a nutritional model of NAFLD in rats. Wistar or Sprague-Dawley male rats were fed ad libitum a standard diet (ST-1, 10 % kcal fat), a medium-fat gelled diet (MFGD, 35 % kcal fat) and a high-fat gelled diet (HFGD, 71 % kcal fat) for 3 or 6 weeks. We examined the serum biochemistry, the hepatic malondialdehyde, reduced glutathione (GSH) and cytokine concentration, the respiration of liver mitochondria, the expression of uncoupling protein-2 (UCP-2) mRNA in the liver and histopathological samples. Feeding with MFGD and HFGD in Wistar rats or HFGD in Sprague-Dawley rats induced small-droplet or mixed steatosis without focal inflammation or necrosis. Compared to the standard diet, there were no significant differences in serum biochemical parameters, except lower concentrations of triacylglycerols in HFGD and MFGD groups. Liver GSH was decreased in rats fed HFGD for 3 weeks in comparison with ST-1. Higher hepatic malondialdehyde was found in both strains of rats fed HFGD for 6 weeks and in Sprague-Dawley groups using MFGD or HFGD for 3 weeks vs. the standard diet. Expression of UCP-2 mRNA was increased in Wistar rats fed MFGD and HFGD for 6 weeks and in Sprague-Dawley rats using HFGD for 6 weeks compared to ST-1. The present study showed that male Wistar and Sprague-Dawley rats fed by HFGD developed comparable simple steatosis without signs of progression to non-alcoholic steatohepatitis under our experimental conditions.

Glutathione reductase is inhibited by acetaminophen-glutathione conjugate in vitro.

The aim of the present work was to investigate a new mechanism likely contributing to the toxic action of acetaminophen, especially to explore the possible inhibition of glutathione reductase through an acetaminophen-glutathione conjugate (APAP-SG). APAP-SG conjugate was synthesized by organic synthesis and purified by column chromatography. The inhibitory effect of the conjugate on two types of glutathione reductase (from yeasts and rat hepatocytes) was tested spectro-photometrically. We found that the enzyme activity was reduced similarly after the treatment with 2.96 mM acetaminophen-glutathione conjugate in both yeast and hepatocyte glutathione reductases (GR); the enzyme activity was inhibited to 52.7+/-1.5 % (2.4+/-0.3 mU/ml) in yeast GR (control activity was 5.6+/-0.3 mU/ml) and to 48.1+/-8.8 % (2.2+/-0.2 mU/ml) in rat hepatocytes lysate GR (control activity was 5.2+/-0.2 mU/ml). In addition, the enzyme activity (from hepatocytes lysate) was decreased to 79+/-7 %, 67+/-2 % and 39+/-7 %, in 0.37, 1.48 and 3.7 mM concentration of the conjugate, respectively. We found that glutathione reductase, the essential enzyme of the antioxidant system, was dose-dependently inhibited by the product of acetaminophen metabolism - the conjugate of acetaminophen and glutathione.

Peroxidative damage of mitochondrial respiration is substrate-dependent.

The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 microM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 microM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage.

Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture.

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP - 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST 1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.