Overexpression of acyl-CoA synthetase-1 increases lipid deposition in hepatic (HepG2) cells and rodent liver in vivo

2006 ◽  
Vol 291 (4) ◽  
pp. E737-E744 ◽  
Author(s):  
Heidi A. Parkes ◽  
Elaine Preston ◽  
Donna Wilks ◽  
Mercedes Ballesteros ◽  
Lee Carpenter ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Hepg2 Cells ◽  
Hepatoma Cell ◽  
Acid Oxidation ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Chain Acyl

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 ± 3 vs. 67 ± 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 ± 6 vs. 100 ± 6 nmol/g protein in controls, P < 0.05) and increased [14C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 ± 4 vs. 20 ± 2 μmol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-14C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.

scholarly journals Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

2020 ◽  
Vol 22 (1) ◽  
pp. 202
Author(s):  
Josephin Glück ◽  
Julia Waizenegger ◽  
Albert Braeuning ◽  
Stefanie Hessel-Pras
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Pyrrolizidine Alkaloids ◽  
Defense Mechanism ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Dependent Manner ◽  
Human Hepatoma Cell

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

Abstract The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

scholarly journals A Combination of Caffeine, Arginine, Soy Isoflavones, and l-Carnitine Enhances Both Lipolysis and Fatty Acid Oxidation in 3T3-L1 and HepG2 Cells in Vitro and in KK Mice in Vivo

2007 ◽  
Vol 137 (10) ◽  
pp. 2252-2257 ◽  
Author(s):  
Shinji Murosaki ◽  
Tae Ryong Lee ◽  
Koutarou Muroyama ◽  
Eui Seok Shin ◽  
Si Young Cho ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Hepg2 Cells ◽  
Soy Isoflavones ◽  
Acid Oxidation ◽  
Kk Mice

scholarly journals Role of hydrogen peroxide in hypoxia-induced erythropoietin production

1994 ◽  
Vol 303 (2) ◽  
pp. 507-510 ◽  
Author(s):  
J Fandrey ◽  
S Frede ◽  
W Jelkmann
Keyword(s):  
Hepg2 Cells ◽  
Hepatoma Cell ◽  
Inhibitory Effect ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Endogenous Production ◽  
Hepatoma Cell Line Hepg2 ◽  
Haem Protein ◽  
Exogenous H2o2

The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.

PEGylation potentiates hepatoma cell targeted liposome-mediated in vitro gene delivery via the asialoglycoprotein receptor

2017 ◽  
Vol 72 (7-8) ◽  
pp. 293-301 ◽  
Author(s):  
Nkosiyethu K. Mkhwanazi ◽  
Charles B. de Koning ◽  
Willem A.L. van Otterlo ◽  
Mario Ariatti ◽  
Moganavelli Singh
Keyword(s):  
Gene Delivery ◽  
Hepatoma Cell ◽  
Asialoglycoprotein Receptor ◽  
Sub Saharan Africa ◽  
Treatment Modalities ◽  
Condensed State ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell

AbstractHepatocellular carcinoma is a burgeoning health issue in sub-Saharan Africa and East Asia where it is most prevalent. The search for gene medicine treatment modalities for this condition represents a novel departure from current treatment options and is gaining momentum. Here we report on nonPEGylated and on sterically stabilized PEGylated cationic liposomes decorated with D-galacto moieties linked to 24.1 Å spacers for asialoglycoprotein receptor (ASGP-R)-targeted vehiculation of pCMV-luc plasmid DNA. Cargo DNA is fully liposome associated at N/P ratio=3:1 and is partially protected from the effects of serum nucleases. Moreover, at this ratio, lipoplex dimensions (89–97 nm) are compatible with the requirements for extravasation in vivo. Ethidium displacement assays show that the reporter DNA is in a less condensed state when bound to PEGylated liposomes than with nonPEGylated liposomes. PEGylated lipoplexes were well tolerated by both HEK293 (ASGP-R-negative) and HepG2 (ASGP-R-positive) cell lines and delivered DNA to the human hepatoma cell line HepG2 by ASGP-R mediation at levels three-fold greater than nonPEGylated lipoplexes. PEGylated ASGP-R-targeted liposomes reported in this study possess the required characteristics for hepatotropic gene delivery and may be considered for further application in vivo.

scholarly journals In Vivo and In Vitro Effect of a Nutrient Mixture on Human Hepatoma Cell Line SK‐Hep‐1

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
M. Waheed Roomi ◽  
Vadim Ivanov ◽  
Aleksandra Niedzwiecki ◽  
Matthias Rath
Keyword(s):  
Cell Line ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
Vitro Effect

Microscopic Analysis of Oxidative Stress in Cultured Cells. I. Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 634-635
Author(s):  
Beverly E. Maleeff ◽  
Tracy L. Gales ◽  
Padma K. Narayanan ◽  
Mark A. Tirmenstein ◽  
Timothy K. Hart
Keyword(s):  
Oxidative Stress ◽  
Hepg2 Cells ◽  
Cultured Cells ◽  
Cell Model ◽  
Hepatoma Cell ◽  
Common Mechanism ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Drug Induced

Cellular oxidative stress, a common mechanism of drug-induced toxicity, is a result of the formation of reactive oxygen species (ROS) in response to chemical stimuli. An endpoint of ROS production is lipid peroxidation, which can in turn lead to disruption of cellular membranes, loss of mitochondrial function, protein oxidation and DNA damage. This toxicity can be organ-specific due to the varying capacities of tissues to handle oxidative events. Liver is particularly sensitive to the effects of oxidative stress, and hepatic toxicity is seen clinically. HepG2 cells are an immortalized human hepatoma cell line used as an in vitro model for mammalian hepatotoxicity studies. The purpose of this study was to characterize the effects of chemically induced oxidative stress in this cultured cell model.HepG2 cells were grown to subconfluence in poly-L-lysine coated 4-well LabTek™ II chambered coverglasses (Nalge Nunc International).

scholarly journals In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 617-623 ◽  
Author(s):  
J Fandrey ◽  
HF Bunn
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Human Hepatoma Cell ◽  
Competitive Polymerase Chain Reaction ◽  
Polymerase Chain

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.

Abstract 418: Bifunctional Role of Autophagy in Intracellular ApoB Degradation and Lipid Droplet Mobilization

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Patricia Christian ◽  
Mostafa Zamani ◽  
Wei Qui ◽  
Rianna Zhang ◽  
Khosrow Adeli
Keyword(s):  
Cell Line ◽  
Hepg2 Cells ◽  
Ex Vivo ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Human Hepatoma ◽  
Human Hepatoma Cell ◽  
Human Hepatoma Cell Line ◽  
Vldl Assembly

Introduction: As apolipoprotein B (apoB) is translated and translocated into the ER, lipids from cytoplasmic lipid droplets (LDs) are added to promote folding and to initiate very-low-density-lipoprotein (VLDL) assembly. However, without sufficient lipid availability, apoB is misfolded and subject to proteasomal degradation. Evidence now shows that apoB can also be degraded through autophagy under certain conditions, and that LDs are also subject to autophagic degradation, a process referred to as lipophagy. We postulate that apoB autophagy and LD lipophagy integrate to regulate hepatic lipid export and VLDL production. Methods/Results: Studies were conducted in vitro using the human hepatoma cell line, HepG2 and ex vivo using primary Syrian Golden hamster hepatocytes. Cells were fat loaded with/without 0.4 mM OA for 4 hours and simultaneously treated with an autophagy inhibitor 3-methyadenine (3-MA; 100uM) or an autophagy inducer, Torin 1 (250nM). HepG2 cells were transfected with 10 nM of atg12 siRNA, an essential autophagy related gene, for a total of 72 hours. In freshly isolated primary hamster hepatocytes, inhibition of autophagosome formation, through treatment with 3-MA, significantly increased cellular levels of newly synthesized apoB, without a significant increase in apoB secreted into the media. Interestingly, treating these cells with Torin 1 to promote autophagy also significantly increased apoB recovery. However, modulation of autophagy activity also affected the average number of LDs per cell, indicating that lipophagy activity had also been modified, potentially affecting VLDL formation. Conversely, while inhibition and induction of autophagy in HepG2 cells, a human hepatoma cell line, reduced and increased apoB co-localization with autophagosomes respectively, siRNA knockdown of atg12 as well as 3-MA treatment decreased apoB recovery. However, this did not appear to be due to reduction in LD breakdown through autophagy. The data obtained with primary hamster hepatocytes suggest that autophagy may play a dual role in VLDL assembly in vivo by regulating both degradation of apoB and lipidation of VLDL particles through mobilization of lipid from LDs. Defects in these pathways can induce hepatic LD accumulation and steatosis.

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