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Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor alpha-induced apoptosis
(2003)
BACKGROUND/AIMS: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFalpha-induced apoptosis in rat hepatocytes. METHODS: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFalpha/ actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFalpha receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [125I]-TNFalpha. RESULTS: IL-6 treatment doubled TNFalpha/actinomycin D- induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [125I]-TNFalpha 3-fold. The latter and TNFalpha-mediated caspase activation was attenuated by prostaglandin E(2). CONCLUSIONS: IL-6 - in contrast to its anti-apoptotic modulation of the Fas-induced pathway - exerted a pro-apoptotic effect on the TNFalpha/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes.
The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.
Eight heptahelical receptors have been characterized for prostaglandin (PG) D(2), PGE(2), PGF(2alpha), prostacyclin and thromboxane A(2). They share a sequence identity of 40%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3beta-subtype PGE(2) receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3beta receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE(2) of the EP3alpha receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells.
Non-alcoholic fatty liver diseases (NAFLD) including the severe form with steatohepatitis (NASH) are highly prevalent ailments to which no approved pharmacological treatment exists. Dietary intervention aiming at 10% weight reduction is efficient but fails due to low compliance. Increase in physical activity is an alternative that improved NAFLD even in the absence of weight reduction. The underlying mechanisms are unclear and cannot be studied in humans. Here, a rat NAFLD model was developed that reproduces many facets of the diet-induced NAFLD in humans. The impact of endurance exercise was studied in this model. Male Wistar rats received control chow or a NASH-inducing diet rich in fat, cholesterol, and fructose. Both diet groups were subdivided into a sedentary and an endurance exercise group. Animals receiving the NASH-inducing diet gained more body weight, got glucose intolerant and developed a liver pathology with steatosis, hepatocyte hypertrophy, inflammation and fibrosis typical of NAFLD or NASH. Contrary to expectations, endurance exercise did not improve the NASH activity score and even enhanced hepatic inflammation. However, endurance exercise attenuated the hepatic cholesterol overload and the ensuing severe oxidative stress. In addition, exercise improved glucose tolerance possibly in part by induction of hepatic FGF21 production.
Non-alcoholic fatty liver diseases (NAFLD) including the severe form with steatohepatitis (NASH) are highly prevalent ailments to which no approved pharmacological treatment exists. Dietary intervention aiming at 10% weight reduction is efficient but fails due to low compliance. Increase in physical activity is an alternative that improved NAFLD even in the absence of weight reduction. The underlying mechanisms are unclear and cannot be studied in humans. Here, a rat NAFLD model was developed that reproduces many facets of the diet-induced NAFLD in humans. The impact of endurance exercise was studied in this model. Male Wistar rats received control chow or a NASH-inducing diet rich in fat, cholesterol, and fructose. Both diet groups were subdivided into a sedentary and an endurance exercise group. Animals receiving the NASH-inducing diet gained more body weight, got glucose intolerant and developed a liver pathology with steatosis, hepatocyte hypertrophy, inflammation and fibrosis typical of NAFLD or NASH. Contrary to expectations, endurance exercise did not improve the NASH activity score and even enhanced hepatic inflammation. However, endurance exercise attenuated the hepatic cholesterol overload and the ensuing severe oxidative stress. In addition, exercise improved glucose tolerance possibly in part by induction of hepatic FGF21 production.
Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors
(2020)
Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity.
A tight hormonal control of energy homeostasis is of pivotal relevance for animals. Recent evidence suggests an involvement of the nuclear receptor NR1i3 (CAR). Fasting induces CAR by largely unknown mechanisms and CAR-deficient mice are defective in fasting adaptation. In rat hepatocytes CAR was induced by WY14643, a PPARalpha-agonist. A DR1 motif in the CAR promoter was necessary and sufficient for this control. The PPARalpha-dependent increase in CAR potentiated the phenobarbital-induced transcription of the prototypical CAR-dependent gene CYP2B1. Since free fatty acids are natural ligands for PPARalpha, a fasting-induced increase in free fatty acids might induce CAR. In accordance with this hypothesis, CAR induction by fasting was abrogated in PPARalpha-deficient mice.
For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.