Western Diet Decreases the Liver Mitochondrial Oxidative Flux of Succinate: Insight from a Murine NAFLD Model.

Mitochondria play an essential role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Previously, we found that succinate-activated respiration was the most affected mitochondrial parameter in mice with mild NAFLD. In this study, we focused on the role of succinate dehydrogenase (SDH) in NAFLD pathogenesis. To induce the progression of NAFLD to nonalcoholic steatohepatitis (NASH), C57BL/6J mice were fed a Western-style diet (WD) or control diet for 30 weeks. NAFLD severity was evaluated histologically and the expression of selected proteins and genes was assessed. Mitochondrial respiration was measured by high-resolution respirometry. Liver redox status was assessed using glutathione, malondialdehyde, and mitochondrial production of reactive oxygen species (ROS). Metabolomic analysis was performed by GC/MS. WD consumption for 30 weeks led to reduced succinate-activated respiration. We also observed decreased SDH activity, decreased expression of the SDH activator sirtuin 3, decreased gene expression of SDH subunits, and increased levels of hepatic succinate, an important signaling molecule. Succinate receptor 1 (SUCNR1) gene and protein expression were reduced in the livers of WD-fed mice. We did not observe signs of oxidative damage compared to the control group. The changes observed in WD-fed mice appear to be adaptive to prevent mitochondrial respiratory chain overload and massive ROS production.

Adaptation of Mitochondrial Substrate Flux in a Mouse Model of Nonalcoholic Fatty Liver Disease.

Maladaptation of mitochondrial oxidative flux seems to be a considerable feature of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to induce NAFLD in mice fed a Western-style diet (WD) and to evaluate liver mitochondrial functions. Experiments were performed on male C57BL/6J mice fed with a control diet or a WD for 24 weeks. Histological changes in liver and adipose tissue as well as hepatic expression of fibrotic and inflammatory genes and proteins were evaluated. The mitochondrial respiration was assessed by high-resolution respirometry. Oxidative stress was evaluated by measuring lipoperoxidation, glutathione, and reactive oxygen species level. Feeding mice a WD induced adipose tissue inflammation and massive liver steatosis accompanied by mild inflammation and fibrosis. We found decreased succinate-activated mitochondrial respiration and decreased succinate dehydrogenase (SDH) activity in the mice fed a WD. The oxidative flux with other substrates was not affected. We observed increased ketogenic capacity, but no impact on the capacity for fatty acid oxidation. We did not confirm the presence of oxidative stress. Mitochondria in this stage of the disease are adapted to increased substrate flux. However, inhibition of SDH can lead to the accumulation of succinate, an important signaling molecule associated with inflammation, fibrosis, and carcinogenesis.

Does Simple Steatosis Affect Liver Regeneration after Partial Hepatectomy in Rats?

AIM: The aim of our study was to assess whether simple steatosis impairs liver regeneration after partial hepatectomy (PHx) in rats. METHODS: Male Sprague-Dawley rats were fed a standard diet (ST-1, 10% kcal fat) and high-fat diet (HFD, 71% kcal fat) for 6 weeks. Then the rats were submitted to 2/3 PHx and animals were sacrificed 24, 48 or 72 h after PHx. Serum biochemistry, respiration of mitochondria in liver homogenate, hepatic oxidative stress markers, selected cytokines and DNA content were measured, and histopathological samples were prepared. Liver regeneration was evaluated by incorporation of bromodeoxyuridine (BrdU) to hepatocyte DNA. RESULTS: HFD induced simple microvesicular liver steatosis. PHx caused elevation of serum markers of liver injury in both groups; however, an increase in these parameters was delayed in HFD group. Hepatic content of reduced glutathione was significantly increased in both groups after PHx. There were no significant changes in activities of respiratory complexes I and II (state 3). Relative and absolute liver weights, total DNA content, and DNA synthesis exerted very similar changes in both ST-1 and HFD groups after PHx. CONCLUSION: PHx-induced regeneration of the rat liver with simple steatosis was not significantly affected when compared to the lean liver.

Comparison of HepaRG and HepG2 cell lines to model mitochondrial respiratory adaptations in non­alcoholic fatty liver disease.

Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non­alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non­alcoholic fatty liver disease (NAFLD) with similar findings. Despite being the most commonly used immortalized cell line for in vitro models of NAFLD, HepG2 cells exposed to free fatty acids (FFAs) exhibit a decreased mitochondrial respiration. On the other hand, the use of HepaRG cells to study mitochondrial respiratory changes following exposure to FFAs has not yet been fully explored. Therefore, the present study aimed to assess cellular energy metabolism, particularly mitochondrial respiration, and lipotoxicity in FFA­treated HepaRG and HepG2 cells. HepaRG and HepG2 cells were exposed to FFAs, followed by comparative analyses that examained cellular metabolism, mitochondrial respiratory enzyme activities, mitochondrial morphology, lipotoxicity, the mRNA expression of selected genes and triacylglycerol (TAG) accumulation. FFAs stimulated mitochondrial respiration and glycolysis in HepaRG cells, but not in HepG2 cells. Stimulated complex I, II­driven respiration and ß­oxidation were linked to increased complex I and II activities in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA­treated HepaRG cells than in FFA­treated HepG2 cells. TAG accumulation was less prominent in HepaRG cells than in HepG2 cells. On the whole, the present study demonstrates that stimulated mitochondrial respiration is associated with lipotoxicity in FFA­treated HepaRG cells, but not in FFA­treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early­stage NASH.

Chronic anthracycline cardiotoxicity: molecular and functional analysis with focus on nuclear factor erythroid 2-related factor 2 and mitochondrial biogenesis pathways.

Anthracycline anticancer drugs (e.g., doxorubicin or daunorubicin) can induce chronic cardiotoxicity and heart failure (HF), both of which are believed to be based on oxidative injury and mitochondrial damage. In this study, molecular and functional changes induced by chronic anthracycline treatment with progression into HF in post-treatment follow-up were analyzed with special emphasis on nuclear factor erythroid 2-related factor 2 (Nrf2) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) pathways. Chronic cardiotoxicity was induced in rabbits with daunorubicin (3 mg/kg, weekly for 10 weeks), and the animals were followed for another 10 weeks. Echocardiography revealed a significant drop in left ventricular (LV) systolic function during the treatment with marked progression to LV dilation and congestive HF in the follow-up. Although daunorubicin-induced LV lipoperoxidation was found, it was only loosely associated with cardiac performance. Furthermore, although LV oxidized glutathione content was increased, the oxidized-to-reduced glutathione ratio itself remained unchanged. Neither Nrf2, the master regulator of antioxidant response, nor the majority of its target genes showed up-regulation in the study. However, down-regulation of manganese superoxide dismutase and NAD(P)H dehydrogenase [quinone] 1 were observed together with heme oxygenase 1 up-regulation. Although marked perturbations in mitochondrial functions were found, no induction of PGC1α-controlled mitochondrial biogenesis pathway was revealed. Instead, especially in the post-treatment period, an impaired regulation of this pathway was observed along with down-regulation of the expression of mitochondrial genes. These results imply that global oxidative stress need not be a factor responsible for the development of anthracycline-induced HF, whereas suppression of mitochondrial biogenesis might be involved.

Characterization of calcium, phosphate and peroxide interactions in activation of mitochondrial swelling using derivative of the swelling curves.

We describe a new method for the analysis of mitochondrial swelling curves. Using classical swelling curves, only the maximum extent of the swelling can be calculated in a numerical form. However, taking the derivative of the classical swelling curves enables the evaluation of two additional parameters of the swelling process in a numerical form, namely, the maximum swelling rate after the addition of the swelling inducer (as dA520/10 s) and the time (in sec) at which the maximum swelling rate after the addition of the swelling inducer is obtained. The use of these three parameters enables the better characterization of the swelling process as demonstrated by the evaluation of calcium and phosphate interactions in the opening of the mitochondrial permeability transition pore and by the characterization of the peroxide potentiating action.

Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen.

BACKGROUND AND AIM: Acetaminophen overdose is the most frequent cause of acute liver failure. Non-alcoholic fatty liver disease is the most common chronic condition of the liver. The aim was to assess whether non-alcoholic steatosis sensitizes rat liver to acute toxic effect of acetaminophen. METHODS: Male Sprague-Dawley rats were fed a standard diet (ST-1, 10% kcal fat) and high-fat gelled diet (HFGD, 71% kcal fat) for 6 weeks and then acetaminophen was applied in a single dose (1 g/kg body weight). Animals were killed 24, 48 and 72 h after acetaminophen administration. Serum biochemistry, activities of mitochondrial complexes, hepatic malondialdehyde, reduced and oxidized glutathione, triacylglycerol and cholesterol contents, and concentrations of serum and liver cytokines (TNF-α, TGF-ß1) were measured and histopathological samples were prepared. RESULTS: The degree of liver inflammation and hepatocellular necrosis were significantly higher in HFGD fed animals after acetaminophen administration. Serum markers of liver injury were elevated only in acetaminophen treated HFGD fed animals. Concentration of hepatic reduced glutathione and ratio of reduced/oxidized glutathione were decreased in both ST-1 and HFGD groups at 24 h after acetaminophen application. Mild oxidative stress induced by acetaminophen was confirmed by measurement of malondialdehyde. Liver content of TNF-α was not significantly altered, but hepatic TGF-ß1 was elevated in acetaminophen treated HFGD rats. We did not observe acetaminophen-induced changes in activities of respiratory complexes I, II, and IV and activity of caspase-3. CONCLUSION: Liver from rats fed HFGD is more susceptible to acute toxic effect of acetaminophen, compared to non-steatotic liver.

Comparison of two anti-diabetic monoestolides regarding effects on intact murine liver tissue.

CONTEXT: Monoestolides belonging to the fatty acid-hydroxy fatty acid (FAHFA) family have recently emerged as promising insulin sensitizers. OBJECTIVE: To investigate and compare impact of two selected FAHFA isomers, namely 9-hexadecanoyloxy-octadecanoic acid [9-PAHSA] and 9-(9Z-octadecenoyloxy)-octadecanoic acid [9-OAHSA], on intact livers in C57BL/6J mice. MATERIALS AND METHODS: Short-term in vivo study with intragastric gavage of 13 mg/kg of substances. Morphological, biochemical and high-resolution respirometric assessment of plasma and liver tissue or homogenates thereof. RESULTS: The 9-OAHSA-gavaged mice had the highest final total body weight, the lowest free fatty acid circulating levels and the highest plasma activities of both ALT and AST. No significant changes of ambient glycaemia were found, however 9-PAHSA-gavaged mice tended to have lower glycaemia than other animals. Respirometry proved no substance-dependent differences. DISCUSSION AND CONCLUSION: 9-PAHSA was more metabolically beneficial and less hepatotoxic than 9-OAHSA. Bioenergetic machinery of liver homogenates seemed unaffected at our FAHFA dose.

The comparison of biological effects of bacterial and synthetic melanins in neuroblastoma cells.

Melanins belong to a group of pigments of different structure and origin. They can be produced synthetically or isolated from living organisms. A number of studies have reported testing of various melanins in neurological studies providing different outcomes. Because the structure of melanins can have an effect on obtained results in cell toxicity studies, we present here our original study which aimed to compare the biological effects of bacterial melanin (biotechnologically obtained from B. thuringiensis) with that of synthetic melanin in neuroblastoma cells. Both melanins were structurally characterized in detail. After melanin treatment (0-200 µg/mL), cell viability, glutathione levels, cell morphology and respiration were assessed in SH-SY5Y cells. The structural analysis showed that bacterial melanin is more hydrophilic according to the presence of larger number of -OH moieties. After melanin treatment, we found that synthetic melanin at similar dosage caused always larger cell impairment compared to bacterial melanin. In addition, more severe toxic effect of synthetic melanin was found in mitochondria. In general, we conclude that more hydrophilic, bacterial melanin induced lower toxicity in neuroblastoma cells in comparison to synthetic melanin. Our findings can be useable for neuroscientific studies estimating the potential use for study of neuroprotection, neuromodulation or neurotoxicity.

Is rat liver affected by non-alcoholic steatosis more susceptible to the acute toxic effect of thioacetamide?

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic condition of the liver in the western world. There is only little evidence about altered sensitivity of steatotic liver to acute toxic injury. The aim of this project was to test whether hepatic steatosis sensitizes rat liver to acute toxic injury induced by thioacetamide (TAA). Male Sprague-Dawley rats were fed ad libitum a standard pelleted diet (ST-1, 10% energy fat) and high-fat gelled diet (HFGD, 71% energy fat) for 6 weeks and then TAA was applied intraperitoneally in one dose of 100 mg/kg. Animals were sacrificed in 24-, 48- and 72-h interval after TAA administration. We assessed the serum biochemistry, the hepatic reduced glutathione, thiobarbituric acid reactive substances, cytokine concentration, the respiration of isolated liver mitochondria and histopathological samples (H+E, Sudan III, bromodeoxyuridine [BrdU] incorporation). Activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase and concentration of serum bilirubin were significantly higher in HFGD groups after application of TAA, compared to ST-1. There were no differences in activities of respiratory complexes I and II. Serum tumour necrosis factor alpha at 24 and 48 h, liver tissue interleukin-6 at 72 h and transforming growth factor ß1 at 24 and 48 h were elevated in TAA-administrated rats fed with HFGD, but not ST-1. TAA-induced centrilobular necrosis and subsequent regenerative response of the liver were higher in HFGD-fed rats in comparison with ST-1. Liver affected by NAFLD, compared to non-steatotic liver, is more sensitive to toxic effect of TAA.

Deteriorating effect of fluvastatin on the cholestatic liver injury induced by bile duct ligation in rats.

Antiinflammatory effect of statins mediated by the reduction of cytokine IL-6 in hepatocytes have been reported. Contrary to beneficial effect, statins can increase susceptibility to mitochondrial dysfunction. Extrahepatic biliary obstruction is associated with oxidative stress, pro-inflammatory response and hepatocyte mitochondrial dysfunction. The aim of our study was to verify the effect of fluvastatin on cholestatic liver injury. Cholestasis was induced in Wistar rats by bile duct ligation. Fluvastatin (1 or 5 mg/kg) was administered after surgery and then daily for 7 days. The dose of 5 mg/kg led to the deterioration of hepatocellular injury. Despite lower production of IL-6, decrease in GSH content, rise of TGFß and inhibition of respiratory complex I in mitochondria were determined. The mRNA expressions of canalicular transporter Mdr1b and basolateral transporter Mrp3 increased in cholestatic liver. Fluvastatin administration then led to the attenuation of this change. Analogously, mRNA expression of conjugative enzyme Ugt1a1 was diminished by fluvastatin administration to cholestatic rats. We can conclude that decrease in the antioxidative status and mitochondrial dysfunction could at least in part participate on the deteriorating effect of fluvastatin. Whether these processes can be a consequence of the alteration in metabolism and transport of potentially toxic substances remains to verify.

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells.

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

Dose-dependent regulation of mitochondrial function and cell death pathway by sorafenib in liver cancer cells.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth most frequent cause of cancer-related death worldwide. Sorafenib is the first line recommended therapy for patients with locally advanced/metastatic HCC. The low response rate is attributed to intrinsic resistance of HCC cells to Sorafenib. The potential resistance to Sorafenib-induced cell death is multifactorial and involves all hallmarks of cancer. However, the presence of sub-therapeutic dose can negatively influence the antitumoral properties of the drug. In this sense, the present study showed that the sub-optimal Sorafenib concentration (10 nM) was associated with activation of caspase-9, AMP-activated protein kinase (AMPK), sustained autophagy, peroxisome proliferator-activated receptor-coactivator 1α (PGC-1α) and mitochondrial function in HepG2 cells. The increased mitochondrial respiration by Sorafenib (10 nM) was also observed in permeabilized HepG2 cells, but not in isolated rat mitochondria, which suggests the involvement of an upstream component in this regulatory mechanism. The basal glycolysis was dose dependently increased at early time point studied (6 h). Interestingly, Sorafenib increased nitric oxide (NO) generation that played an inhibitory role in mitochondrial respiration in sub-therapeutic dose of Sorafenib. The administration of sustained therapeutic dose of Sorafenib (10 µM, 24 h) induced mitochondrial dysfunction and dropped basal glycolysis derived acidification, as well as increased oxidative stress and apoptosis in HepG2. In conclusion, the accurate control of the administered dose of Sorafenib is relevant for the potential prosurvival or proapoptotic properties induced by the drug in liver cancer cells.

The Reducing Hospital Costs through Horizontal Integration.

BACKGROUND: This paper investigated the impact of hospitals' horizontal integration in the Czech Republic on the cost behavior. The aim of the research was to examined the hospitals costs in specific environment of region hospitals at NUTS 3 level (Nomenclature of Units for Territorial Statistics) - Administrative Regions. METHODS: The survey was conducted in the period from April to August 2016 in the Czech Republic. The research was divided into two parts. The first part was based on data obtained from the Institute of Health Information and Statistics of the Czech Republic. We used Statgraphics Centurion XVII for the descriptive statistics and data visualization. Second part of the results was obtained through a survey research focused on managers of the horizontal integrated hospitals and their experiences with the cost behavior. RESULTS: The results from statistical survey showed that up to 80 percent of the observed region hospitals at NUTS 3 level, the cost of treatment for a patient per day has decreased after integration into an association. Based on primary survey, 73% hospital managers confirm these results and see one of the advantages that it is possible to reduce costs through integration of hospitals. The largest savings, according to hospital managers, are due to central purchasing and investments, together and they have a better negotiation position with suppliers. CONCLUSION: We can confirm that horizontal integration of hospitals can lead to reduction of costs and higher efficiency, in the specific environment of region hospitals at NUTS 3 level.

The toxic effect of thioacetamide on rat liver in vitro.

Thioacetamide (TAA) is a hepatotoxin frequently used for experimental purposes which produces centrilobular necrosis after a single dose administration. In spite of the fact that oxidative stress seems to play a very important role in the mechanism of TAA-induced injury, the effect of TAA on hepatocytes in primary culture with respect to the influence on mitochondria has yet to be verified. Hepatocytes were incubated for 24h in a medium containing TAA (0-70 mmol/l). Glutathione content (GSH/GSSG), reactive oxygen species and malondialdehyde formation were assessed as markers of cell redox state. Toxicity was determined by lactate dehydrogenase leakage and WST-1 assay. The functional capacity of hepatocytes was evaluated from albumin and urea production. Mitochondrial metabolism was assessed by measuring mitochondrial membrane potential and oxygen consumption. Our results show that a profound decrease in the GSH level in hepatocytes precedes a sharp rise in endogenous ROS production. ROS production correlates with an increase in lipoperoxidation. Mitochondria are affected by TAA secondarily as a consequence of oxidative stress. Oxidation of the NADH-dependent substrates of respiratory Complex I is significantly more sensitive to the toxic action of TAA than oxidation of the flavoprotein-dependent substrate of Complex II. Mitochondria can also maintain their membrane potential better when they utilize succinate as a respiratory substrate. It appears that GSH should be depleted below a certain critical level in order to cause a marked increase in lipid peroxidation. Mitochondrial injury can then occur and cell death develops.