Various strategies have been employed to improve the reliability of 2D, 3D, and co-culture in vitro models of nonalcoholic fatty liver disease, including using extracellular matrix proteins such as collagen I to promote cell adhesion. While studies have demonstrated the significant benefits of culturing cells on collagen I, its effects on the HepG2 cell line after exposure to palmitate (PA) have not been investigated. Therefore, this study aimed to assess the effects of PA-induced lipotoxicity in HepG2 cultured in the absence or presence of collagen I. HepG2 cultured in the absence or presence of collagen I was exposed to PA, followed by analyses that assessed cell proliferation, viability, adhesion, cell death, mitochondrial respiration, reactive oxygen species production, gene and protein expression, and triacylglycerol accumulation. Culturing HepG2 on collagen I was associated with increased cell proliferation, adhesion, and expression of integrin receptors, and improved cellular spreading compared to culturing them in the absence of collagen I. However, PA-induced lipotoxicity was greater in collagen I-cultured HepG2 than in those cultured in the absence of collagen I and was associated with increased α2β1 receptors. In summary, the present study demonstrated for the first time that collagen I-cultured HepG2 exhibited exacerbated cell death following exposure to PA through integrin-mediated death. The findings from this study may serve as a caution to those using 2D models or 3D scaffold-based models of HepG2 in the presence of collagen I.
- MeSH
- buněčná adheze * účinky léků MeSH
- buněčná smrt účinky léků MeSH
- buňky Hep G2 MeSH
- integrin alfa2beta1 metabolismus MeSH
- integriny metabolismus genetika MeSH
- kolagen typu I * metabolismus genetika MeSH
- lidé MeSH
- nealkoholová steatóza jater metabolismus patologie MeSH
- palmitany toxicita farmakologie MeSH
- proliferace buněk * účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- viabilita buněk * účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Although some clinical studies have reported increased mitochondrial respiration in patients with fatty liver and early non‐alcoholic steatohepatitis (NASH), there is a lack of in vitro models of non‐alcoholic 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 FFA‐treated 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, II‐driven respiration and β‐oxidation were linked to increased complex I and II activities in FFA‐treated HepaRG cells, but not in FFA‐treated HepG2 cells. Exposure to FFAs disrupted mitochondrial morphology in both HepaRG and HepG2 cells. Lipotoxicity was induced to a greater extent in FFA‐treated HepaRG cells than in FFA‐treated 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 FFA‐treated HepaRG cells, but not in FFA‐treated HepG2 cells. These findings suggest that HepaRG cells are more suitable for assessing mitochondrial respiratory adaptations in the developed in vitro model of early‐stage NASH.
- MeSH
- buněčné linie MeSH
- buňky Hep G2 MeSH
- dýchání MeSH
- kyseliny mastné neesterifikované MeSH
- lidé MeSH
- mitochondrie MeSH
- nealkoholová steatóza jater * MeSH
- triglyceridy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- dieta s vysokým obsahem tuků škodlivé účinky MeSH
- glutathion metabolismus MeSH
- jaterní mitochondrie metabolismus MeSH
- játra metabolismus patologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nealkoholová steatóza jater etiologie metabolismus MeSH
- peroxidace lipidů * MeSH
- sukcinátdehydrogenasa metabolismus MeSH
- tuková tkáň metabolismus patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
CONTEXT: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. OBJECTIVES: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. MATERIALS AND METHODS: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24 h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). RESULTS: APAP from concentrations of 2.5 and 0.75 mmol/L induced a decrease in viability of rat (p < 0.001) and mouse (p < 0.001) hepatocytes (WST-1), respectively. In contrast to rat hepatocytes, there was no activation of caspase-3 in mouse hepatocytes after APAP treatment. Earlier damage to plasma membrane and faster depletion of reduced glutathione were detected in mouse hepatocytes. Mouse hepatocytes showed increased glutamate + malate-driven respiration in state 4 and higher susceptibility of the outer mitochondrial membrane (OMM) to APAP-induced injury. CONCLUSION: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.
- MeSH
- biologické markery metabolismus MeSH
- buněčná membrána účinky léků metabolismus MeSH
- druhová specificita MeSH
- glutathion metabolismus MeSH
- hepatocyty cytologie účinky léků metabolismus MeSH
- jaterní mitochondrie účinky léků enzymologie metabolismus MeSH
- kultivované buňky MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondriální membrány účinky léků metabolismus MeSH
- myši inbrední C57BL MeSH
- neopioidní analgetika škodlivé účinky MeSH
- oxidace-redukce MeSH
- oxidační stres účinky léků MeSH
- paracetamol škodlivé účinky MeSH
- peroxidace lipidů účinky léků MeSH
- potkani Wistar MeSH
- reaktivní formy kyslíku agonisté metabolismus MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- Publikační typ
- abstrakt z konference MeSH
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.
- MeSH
- hepatektomie * MeSH
- jaterní mitochondrie patologie fyziologie MeSH
- jaterní testy MeSH
- játra patologie patofyziologie MeSH
- krysa rodu rattus MeSH
- potkani Sprague-Dawley MeSH
- regenerace jater fyziologie MeSH
- ztučnělá játra patologie patofyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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.
- MeSH
- apoptóza účinky léků MeSH
- biologické markery metabolismus MeSH
- galaktosamin farmakologie MeSH
- hepatocyty účinky léků metabolismus patologie MeSH
- jaterní mitochondrie účinky léků metabolismus patologie MeSH
- kultivované buňky MeSH
- L-laktátdehydrogenasa metabolismus MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- modely nemocí na zvířatech MeSH
- nealkoholová steatóza jater metabolismus patologie MeSH
- nekróza MeSH
- peroxidace lipidů účinky léků MeSH
- potkani Wistar MeSH
- primární buněčná kultura MeSH
- reaktivní formy kyslíku metabolismus MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
