Pyruvate-induced Long-term Maintenance of Glutathione S-Transferase in Rat Hepatocyte Cultures

2001 ◽  
Vol 29 (3) ◽  
pp. 335-346 ◽  
Author(s):  
Tamara Vanhaecke ◽  
André Foriers ◽  
Albert Geerts ◽  
Elizabeth A. Shephard ◽  
Antoine Vercruysse ◽  
...  
Keyword(s):  
Mrna Level ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Strong Increase ◽  
Mrna Levels ◽  
Glutathione S Transferase ◽  
Albumin Secretion ◽  
Rat Hepatocyte ◽  
Hepatocyte Cultures ◽  
Transmission Electron

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, μ and α class GST activities were well maintained, whereas GST π activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: μ class proteins remained relatively constant, whereas a decrease in the a class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, π class GST activity was increased, but total, μ, and α class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.

scholarly journals Differential regulation of γ-glutamylcysteine synthetase heavy and light subunit gene expression

1997 ◽  
Vol 326 (1) ◽  
pp. 167-172 ◽  
Author(s):  
Jiaxin CAI ◽  
Zong-Zhi HUANG ◽  
Shelly C. LU
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Steady State ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Mrna Levels ◽  
Subunit Gene ◽  
Subunit Mrna ◽  
Glutamylcysteine Synthetase ◽  
Steady State Mrna Level

γ-Glutamylcysteine synthetase (GCS) is the rate-limiting enzyme in the biosynthesis of glutathione and is composed of a heavy and a light subunit. Although the heavy subunit is enzymically active alone, the light subunit plays an important regulatory role by making the holoenzyme function more efficiently. In the current study we examined whether conditions which are known to influence gene expression of the heavy subunit also influence that of the light subunit, and the mechanisms involved. Treatment of cultured rat hepatocytes with hormones such as insulin and hydrocortisone, or plating hepatocytes under low cell density increased the steady-state mRNA level of the heavy subunit only. Treatment with diethyl maleate (DEM), buthionine sulphoximine (BSO) and t-butylhydroquinone (TBH) increased the steady state mRNA level and gene transcription rates of both subunits. These treatments share in common their ability to induce oxidative stress and activate nuclear factor κB (NF-κB). Treatment with protease inhibitors 7-amino-1-chloro-3-tosylamido-2-heptanone (TLCK) or L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) had no influence on the basal NF-κB and GCS subunit mRNA levels, but blocked the activation of NF-κB by DEM, BSO and TBH, and the increase in GCS heavy subunit mRNA level by BSO and TBH. On the other hand, the DEM-, BSO- and TBH-induced increase in GCS light-subunit mRNA level was unaffected by TLCK and TPCK. Thus only the heavy subunit is hormonally regulated and growth sensitive, whereas both subunits are regulated by oxidative stress. Signalling through NF-κB is involved only in the oxidative-stress-mediated changes in the heavy subunit gene expression.

Physiological oxygen tensions modulate expression of the mdr1b multidrug-resistance gene in primary rat hepatocyte cultures

2000 ◽  
Vol 350 (2) ◽  
pp. 443-451 ◽  
Author(s):  
Karen I. HIRSCH-ERNST ◽  
Thomas KIETZMANN ◽  
Christina ZIEMANN ◽  
Kurt JUNGERMANN ◽  
Georg F. KAHL
Keyword(s):  
Multidrug Resistance ◽  
Mrna Expression ◽  
Rat Hepatocytes ◽  
Mrna Levels ◽  
Multidrug Resistance Gene ◽  
Hepatocyte Cultures ◽  
Dependent Signal ◽  
Oxygen Tensions ◽  
Arterial Po2 ◽  
Haem Proteins

P-Glycoprotein transporters encoded by mdr1 (multidrug resistance) genes mediate extrusion of an array of lipophilic xenobiotics from the cell. In rat liver, mdr transcripts have been shown to be expressed mainly in hepatocytes of the periportal region. Since gradients in oxygen tension (pO2) may contribute towards zonated gene expression, the influence of arterial and venous pO2 on mRNA expression of the mdr1b isoform was examined in primary rat hepatocytes cultured for up to 3 days. Maximal mdr1b mRNA levels (100%) were observed under arterial pO2 after 72h, whereas less than half-maximal mRNA levels (40%) were attained under venous pO2. Accordingly, expression of mdr protein and extrusion of the mdr1 substrate rhodamine 123 were maximal under arterial pO2 and reduced under venous pO2. Oxygen-dependent modulation of mdr1b mRNA expression was prevented by actinomycin D, indicating transcriptional regulation. Inhibition of haem synthesis by 25µM CoCl2 blocked mdr1b mRNA expression under both oxygen tensions, whereas 80µM desferrioxamine abolished modulation by O2. Haem (10µM) increased mdr1b mRNA levels under arterial and venous pO2. In hepatocytes treated with 50µM H2O2, mdr1b mRNA expression was elevated by about 1.6-fold at venous pO2 and 1.5-fold at arterial pO2. These results support the conclusion that haem proteins are crucial for modulation of mdr1b mRNA expression by O2 in hepatocyte cultures and that reactive oxygen species may participate in O2-dependent signal transduction. Furthermore, the present study suggests that oxygen might be a critical modulator for zonated secretion of mdr1 substrates into the bile.

Modulation of cytokeratin and actin gene expression and fibrillar organization in cultured rat hepatocytes

1992 ◽  
Vol 70 (10-11) ◽  
pp. 1238-1248 ◽  
Author(s):  
Normand Marceau ◽  
Andrée Grenier ◽  
Micheline Noel ◽  
Donald Mailhot ◽  
Anne Loranger
Keyword(s):  
Gene Expression ◽  
Dimethyl Sulfoxide ◽  
Intermediate Filaments ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Culture Conditions ◽  
Actin Gene ◽  
Mrna Levels ◽  
Cultured Rat Hepatocytes ◽  
Hepatocyte Spheroids

Intermediate filaments of rat hepatocytes are composed of cytokeratins 8 and 18 (CK8 and CK18, respectively). Recent work from our laboratory has indicated a close relationship between the synthesis of these cytokeratins, their organization into intermediate filaments, and the promotion of growth and differentiation of cultured rat hepatocytes by insulin, epidermal growth factor, and dexamethasone. In the present study, we examined the mRNA expression, level of protein synthesis, and fibrillar distribution of cytokeratins 8 and 18 and actin in hepatocytes, isolated from normal and dexamethasone-injected rats and cultured as monolayers or spheroids in the presence of insulin, or from normal rat hepatocytes, cultured as monolayers in the presence of dexamethasone, insulin, and dimethyl sulfoxide. The CK8 mRNA level was lower in hepatocytes isolated from noninjected rats and cultured as either monolayers or spheroids, than in those from dexamethasone-injected rats. However, the CK18 mRNA level varied in a manner that was different from that of CK8 mRNA, showing that the modes of expression of the two genes were independent. The various changes in hepatocyte culture conditions led to variations in albumin mRNA levels that largely followed those observed in CK8 mRNA levels. In the case of actin, the amount of mRNAs varied from relatively high levels in hepatocyte monolayers to extremely low levels in hepatocyte spheroids, even though in both cases the cells were isolated from dexamethasone-injected rats. These changes in mRNA levels did not necessarily correlate with changes in the synthesis of cytokeratins 8 and 18, and actin. Changes in culture conditions induced a major reorganization in the distribution of cytokeratin intermediate filaments and actin filament between the region near the surface membrane and the cytoplasm. The most divergent patterns in cytokeratin intermediate filaments and actin filament distributions were observed between hepatocytes cultured as spheroidal aggregates and as monolayers in the presence of dimethyl sulfoxide. The former condition resulted in patterns of cytokeratin and actin gene expression and fibrillar organization that best matched those in situ. In the latter condition, inappropriate patterns were obtained, in spite of the fact that dimethyl sulfoxide treated hepatocytes are known to exhibit survival and functional activities equivalent to that of hepatocyte spheroids. These results demonstrate for the first time that the survival and functional activity (i.e., albumin production) of rat hepatocytes in vitro is not necessarily correlated with a particular pattern of cytokeratin and actin gene expression and fibrillar arrangement.Key words: gene expression, cytokeratins, intermediate filaments, cytoskeleton, hepatocytes.

scholarly journals Anti-Inflammatory Effect of Charantadiol A, Isolated from Wild Bitter Melon Leaf, on Heat-Inactivated Porphyromonas gingivalis-Stimulated THP-1 Monocytes and a Periodontitis Mouse Model

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5651
Author(s):  
Tzung-Hsun Tsai ◽  
Chi-I Chang ◽  
Ya-Ling Hung ◽  
Wen-Cheng Huang ◽  
Hsiang Chang ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Mrna Level ◽  
Ethanolic Extract ◽  
Inhibitory Effect ◽  
Mrna Levels ◽  
Bitter Melon ◽  
Separation And Purification ◽  
Periodontal Pathogens ◽  
Periodontal Inflammation ◽  
Anti Inflammatory

Porphyromonas gingivalis has been identified as one of the major periodontal pathogens. Activity-directed fractionation and purification processes were employed to identify bioactive compounds from bitter melon leaf. Ethanolic extract of bitter melon leaf was separated into five subfractions by open column chromatography. Subfraction-5-3 significantly inhibited P. gingivalis-induced interleukin (IL)-8 and IL-6 productions in human monocytic THP-1 cells and then was subjected to separation and purification by using different chromatographic methods. Consequently, 5β,19-epoxycucurbita-6,23(E),25(26)-triene-3β,19(R)-diol (charantadiol A) was identified and isolated from the subfraction-5-3. Charantadiol A effectively reduced P. gingivalis-induced IL-6 and IL-8 productions and triggered receptors expressed on myeloid cells (TREM)-1 mRNA level of THP-1 cells. In a separate study, charantadiol A significantly suppressed P. gingivalis-stimulated IL-6 and tumor necrosis factor-α mRNA levels in gingival tissues of mice, confirming the inhibitory effect against P. gingivalis-induced periodontal inflammation. Thus, charantadiol A is a potential anti-inflammatory agent for modulating P. gingivalis-induced inflammation.

Regulation of hepatic glucose metabolism by leptin in pig and rat primary hepatocyte cultures

2004 ◽  
Vol 286 (1) ◽  
pp. R206-R216 ◽  
Author(s):  
Priya Raman ◽  
Shawn S. Donkin ◽  
Michael E. Spurlock
Keyword(s):  
Glucose Metabolism ◽  
Leptin Receptor ◽  
Rat Hepatocytes ◽  
Underlying Mechanism ◽  
Acute Effect ◽  
Model Systems ◽  
Mrna Levels ◽  
Hepatocyte Cultures ◽  
Hepatic Glucose Metabolism ◽  
Hepatic Glucose

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phospho enolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% ( P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease ( P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.

Regulation of Heme Oxygenase-1 Gene Expression by Anoxia and Reoxygenation in Primary Rat Hepatocyte Cultures

2003 ◽  
Vol 228 (5) ◽  
pp. 584-589 ◽  
Author(s):  
Andreas Ohlmann ◽  
Susanne Giffhorn-Katz ◽  
Ivonne Becker ◽  
Norbert Katz ◽  
Stephan Immenschuh
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Heme Oxygenase ◽  
Heme Oxygenase 1 ◽  
Protein Synthesis Inhibitor ◽  
Mrna Levels ◽  
Rat Hepatocyte ◽  
Hsp70 Mrna ◽  
Hepatocyte Cultures ◽  
Antioxidant Agents

Heme oxygenase (HO) catalyzes the rate-limiting enzymatic step of heme degradation and regulates the cellular heme content. Gene expression of the inducible isoform of HO, HO-1, is upregulated in response to various oxidative stress stimuli. To investigate the regulatory role of anoxia and reoxygenation (A/R) on hepatic HO-1 gene expression, primary cultures of rat hepatocytes were exposed after an anoxia of 4 hr to normal oxygen tension for various lengths of time. For comparison, gene expression of the noninducible HO isoform, HO-2, and that of the heat-shock protein 70 (HSP70) were determined. During reoxygenation, a marked increase of HO-1 and HSP70 steady-state mRNA levels was observed, whereas no alteration of HO-2 mRNA levels occurred. Corresponding to HO-1 mRNA, an increase of HO-1 protein expression was determined by Western blot analysis. The anoxia-dependent induction of HO-1 was prevented by pretreatment with the transcription inhibitor, actinomycin D, but not by the protein synthesis inhibitor, cycloheximide, suggesting a transcriptional regulatory mechanism. After exposure of hepatocytes to anoxia, the relative levels of oxidized glutathione increased within the first 40 min of reoxygenation. Pretreament of cell cultures with the antioxidant agents, β-carotene and allopurinol, before exposure to A/R led to a marked decrease of HO-1 and HSP70 mRNA expression during reoxygenation. An even more pronounced reduction of mRNA expression was observed after exposure to desferrioxamine. Taken together, the data demonstrate that HO-1 gene expression in rat hepatocyte cultures after A/R is upregulated by a transcriptional mechanism that may be, in part, mediated via the generation of ROS and the glutathione system.

Effect of hepatocyte swelling on microtubule stability and tubulin mRNA levels

1994 ◽  
Vol 72 (1-2) ◽  
pp. 12-19 ◽  
Author(s):  
Dieter Häussinger ◽  
Barbara Stoll ◽  
Stephan vom Dahl ◽  
Panayiotis A. Theodoropoulos ◽  
Emmanuel Markogiannakis ◽  
...  
Keyword(s):  
Cell Function ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Inhibitory Effect ◽  
Cell Swelling ◽  
Mrna Levels ◽  
Microtubule Network ◽  
Isolated Rat Hepatocytes ◽  
Tubulin Mrna ◽  
Induced Changes

Incubation of isolated rat hepatocytes under conditions known to induce cell swelling caused several alterations in microtubule physiology. As shown by immunofluorescence microscopy experiments in the absence and presence of triethyllead or colchicine (two well-established microtubule inhibitors), an apparent stabilization of the microtubule network became evident in hepatocytes exposed to hypotonic (190 mosmol/L) conditions. A similar stabilizing effect was also observed upon cell swelling induced by addition of insulin (100 nmol/L) or glutamine (10 mmol/L). The differential microtubule stabilities were not attributed to a differential incorporation of the antimicrotubular agents into hepatocytes as shown by [3H]colchicine-uptake experiments. The swelling-induced alterations of microtubules may contribute to the swelling-induced changes of liver cell function: in perfused rat liver it was found that the established inhibitory effect of hypotonic cell swelling on hepatic proteolysis was largely abolished in presence of colchicine. Tubulin mRNA levels increased by 1.9-, 2.1- and 2.7-fold in isolated hepatocytes being exposed for 120 min to hypotonic medium, insulin, or glutamine, respectively. The results suggest an involvement of microtubular structures in the regulation of liver metabolism in response to alterations of the cellular hydration state.Key words: microtubules, cell swelling, glutamine, gene expression, proteolysis.

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