Degradation of normal and proliferated peroxisomes in rat hepatocytes: Regulation of peroxisomes quantity in cells

2003 ◽  
Vol 61 (2) ◽  
pp. 151-160 ◽  
Author(s):  
Sadaki Yokota
Keyword(s):  
Rat Hepatocytes ◽  
In Cells

scholarly journals Glucose induces the translocation of glycogen synthase to the cell cortex in rat hepatocytes

1997 ◽  
Vol 321 (1) ◽  
pp. 227-231 ◽  
Author(s):  
Josep M. FERNÁNDEZ-NOVELL ◽  
David BELLIDO ◽  
Senen VILARÓ ◽  
Joan J. GUINOVART
Keyword(s):  
Laser Scanning ◽  
Glycogen Synthase ◽  
Protein A ◽  
Rat Hepatocytes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Glycogen Deposition ◽  
In Cells

After incubation with glucose a dramatic change in the intracellular distribution of glycogen synthase was observed in rat hepatocytes. Confocal laser scanning microscopy showed that glycogen synthase existed diffusely in the cytosol of control cells, whereas in cells incubated with glucose it accumulated at the cell periphery. Colocalization analysis between glycogen synthase immunostaining and actin filaments showed that the change in glycogen synthase distribution induced by glucose correlated with a marked increase in the co-distribution of the two proteins, indicating that, in response to glucose, glycogen synthase moves to the actin-rich area close to the membrane. When glycogen synthase was immunostained with rabbit anti-(glycogen synthase) and Protein A–colloidal gold, few particles were observed close to the membrane in control cells. In contrast, in cells incubated with glucose most of the gold particles were found near the membrane, confirming that glycogen synthase had moved to the cell cortex. Furthermore, in agreement with the glycogen synthase distribution, glycogen deposition appeared to be more active at the periphery of the cell.

scholarly journals Evaluation of oxidative status in acetaminophen treated rat hepatocytes in culture

2009 ◽  
pp. 239-246
Author(s):  
T Roušar ◽  
O Kučera ◽  
P Křiváková ◽  
H Lotková ◽  
R Kanďár ◽  
...  
Keyword(s):  
Functional Capacity ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Oxidative Status ◽  
Ros Production ◽  
Cellular Viability ◽  
Time Intervals ◽  
Leakage Test ◽  
Cultured Rat Hepatocytes ◽  
In Cells

The present study describes the estimation of acetaminophen (AAP) toxicity in cultured rat hepatocytes. We used different concentrations of AAP – 1, 2.5, 5, 10 and 20 mM, to test influence of AAP on cellular viability, functional capacity and oxidative status at given time intervals. WST-1 test showed decrease of dehydrogenase activity in 5, 10 and 20 mM AAP to 75 % of control values after 1 hour of incubation. At 12 h of treatment, all AAP concentrations decreased WST-1 signal; no enzyme activity was found since 18 h in cells treated with 20 mM AAP according to LDH leakage test performed at 24 h of incubation. Functional capacity was tested by albumin assay where the decrease was strictly related to AAP dose. Intracellular oxidative status was assessed by analysis of GSH/GSSG levels and time course of ROS production and glutathione reductase (GR) activity. Increased ROS production was found already after 3 h of incubation in 2.5, 5, 10 and 20 mM AAP, respectively. The highest ROS production was measured after 12 h treatment. GR activity was decreased already after 3 h of incubation and remained also decreased in cells treated with 2.5, 5, 10 and 20 mM AAP during further incubation.

Inhibition of GSH efflux from rat liver by methionine: effects of GSH synthesis in cells and perfused organ

1990 ◽  
Vol 258 (6) ◽  
pp. G967-G973 ◽  
Author(s):  
J. C. Fernandez-Checa ◽  
T. Maddatu ◽  
M. Ookhtens ◽  
N. Kaplowitz
Keyword(s):  
Rat Liver ◽  
Reduced Glutathione ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
Perfused Liver ◽  
Isolated Rat Hepatocytes ◽  
Dependent Inhibition ◽  
Krebs Henseleit Buffer ◽  
Isolated Rat ◽  
In Cells

The inhibition of efflux of intracellular reduced glutathione (GSH) by methionine was determined in isolated rat hepatocytes suspended either in Krebs-Henseleit buffer or in modified Fisher's medium. Methionine (1 mM) added to Krebs-Henseleit suspensions of isolated rat hepatocytes inhibited GSH efflux, with greater retention of GSH in the cells compared with control. Results were similar with methionine and 0.3 mM propargylglycine cystathionase inhibitor), suggesting no net synthesis of GSH from methionine. In Fisher's medium, the inhibitory effect of methionine on GSH efflux was masked due to increasing cellular GSH; however, the inhibitory effect of methionine was unmasked by propargylglycine, which prevented the utilization of methionine for GSH synthesis. The addition of serine (0.1 mM) to methionine in Krebs-Henseleit buffer raised cellular GSH, overcoming the inhibition of GSH efflux. In the perfused liver, infusion of 1 and 5 mM methionine initially inhibited GSH efflux, but the inhibition was reversed with continued methionine infusion. After removal of methionine, GSH efflux increased immediately. The reversal and rebound were blocked by propargylglycine, revealing concentration-dependent inhibition of sinusoidal GSH efflux by methionine. Thus, when methionine is utilized to promote GSH synthesis, its inhibitory effect on GSH efflux tends to be overcome.

scholarly journals Maintenance of glutathione content is isolated hepatocyctes

1978 ◽  
Vol 170 (3) ◽  
pp. 627-630 ◽  
Author(s):  
J Viña ◽  
R Hems ◽  
H A Krebs
Keyword(s):  
Reduced Glutathione ◽  
Standard Procedure ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Perfusion Medium ◽  
In Cells

1. During the standard procedure for the preparation of rat hepatocytes, about half of the cellular GSH (reduced glutathione) is lost. 2. This loss is prevented by the addition of 0.1 mM-EGTA (but no EDTA) to the perfusion medium. 3. On incubation with and without EGTA, isolated hepatocytes prepared in the presence of EGTA lose GSH. This loss is prevented by near-physiological concentrations of methionine or homocysteine, but not of cysteine. 4. Cysteine, at concentrations above 0.2 mM, causes a loss of GSH probably by non-enzymic formation of a mixed disulphide. 5. Serine together with methionine or homocystein increases GSH above the value in cells from starved rats in vivo. This is taken to suggest that cystathionine may be a cysteine donor in the synthesis of gamma-glutamylcysteine, the precursor of GSH.

The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver

2004 ◽  
Vol 286 (3) ◽  
pp. E439-E448 ◽  
Author(s):  
Martha H. Stipanuk ◽  
Lawrence L. Hirschberger ◽  
Monica P. Londono ◽  
Carrie L. Cresenzi ◽  
Anthony F. Yu
Keyword(s):  
Proteasome Inhibitor ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Cysteine Dioxygenase ◽  
Ubiquitin Proteasome ◽  
Excess Sulfur ◽  
Cells Cultured ◽  
In Cells

Hepatic cysteine dioxygenase (CDO) activity is a critical regulator of cellular cysteine concentration and availability of cysteine for anabolic processes and is markedly higher in animals fed diets containing excess sulfur amino acids compared with those fed levels at or below the requirement. Rat hepatocytes responded to a deficiency or excess of cysteine in the culture medium with a decrease or increase in CDO level but no change in CDO mRNA level. The cysteine analog, cysteamine, but not cysteine metabolites or thiol reagents, was also effective in increasing CDO. Inhibitors of the 26S proteasome blocked CDO degradation in cysteine-deficient cells but had little or no effect on CDO concentration in hepatocytes cultured with excess cysteine. High-molecular-mass CDO-ubiquitin conjugates were observed in cells cultured in cysteine-deficient medium, whether or not proteasome inhibitor was present, but these CDO-ubiquitin conjugates were not observed in cells cultured in cysteine-supplemented medium with or without proteasome inhibitor. Similar results were observed for degradation of recombinant CDO expressed in human heptocarcinoma cells cultured in cysteine-deficient or cysteine-supplemented medium. CDO is an example of a mammalian enzyme that is robustly regulated via its substrate, with the presence of substrate blocking the ubiquitination of CDO and, hence, the targeting of CDO for proteasomal degradation. This regulation occurs in primary hepatocytes in a manner that corresponds with changes observed in intact animals.

Activity and secretion of sialyltransferase in primary monolayer cultures of rat hepatocytes cultured with and without dexamethasone

1986 ◽  
Vol 64 (2) ◽  
pp. 79-84 ◽  
Author(s):  
Willem van Dijk ◽  
Willem Boers ◽  
Mieke Sala ◽  
Anne-Marie Lasthuis ◽  
Sailen Mookerjea
Keyword(s):  
Enzyme Activity ◽  
Culture Media ◽  
Rat Hepatocytes ◽  
Progressive Increase ◽  
Isolated Rat Hepatocytes ◽  
Sialyltransferase Activity ◽  
The Media ◽  
Primary Monolayer ◽  
Triton X ◽  
In Cells

Monolayers of hepatocytes attached on collagen-coated dishes were cultured for 20–24 h and were found suitable to study the activity and secretion of CMP-N-acetylneuraminate:asialo-α1-acid glycoprotein sialyltransferase. A progressive increase of sialyltransferase activity in the culture medium was observed during incubation of the hepatocytes. After 24 h 34–48% of the total sialyltransferase activity of the hepatocyte incubation system was present in the medium. The enzyme activity present in the medium was soluble in nature and could not be stimulated by Triton X-100. The secretion of the enzyme was stimulated about twofold by dexamethasone. The activity of sialyltransferase in the hepatocytes was also increased by dexamethanasone. The Km of either hepatocyte or medium sialyltransferase for CMP-sialic acid was only slightly changed by dexamethasone, whereas the Vmax was increased about twofold. The secretion of sialyltransferase could be inhibited partially by the anti-microtubular agent colchicine. The dexamethasone-induced increase of the sialyltransferase activity in cells and media could be eliminated by inclusion of α-amanitin in the culture media at 0 h. The inhibiting effect of α-amanitin was only partially expressed when the drug was added 4 h after the addition of dexamethasone to the media. The results suggest that isolated rat hepatocytes actively secrete sialyltransferase and that the increase in the sialyltransferase activity in cells and media owing to the synthetic glucocorticosteroid dexamethasone results from increased synthesis of the enzyme molecule. It is supposed that in the intact rat the increased levels of the enzyme activity in serum observed in inflammation may originate from an induction of the synthesis of sialyltransferase in the hepatocytes of rat liver by the increased levels of circulating corticosteroids.

scholarly journals Development of a Rat Sandwich-Cultured Hepatocytes Model Expressing Functional Human Organic Anion Transporting Polypeptide (OATP) 1B3: A Potential Screening Tool for Liver-Targeting Compounds

2021 ◽  
Vol 24 ◽  
pp. 475-483
Author(s):  
Taleah Farasyn ◽  
Chao Xu ◽  
Wei Yue
Keyword(s):  
Biliary Excretion ◽  
In Vitro Model ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Drug Uptake ◽  
Liver Targeting ◽  
Organic Anion Transporting Polypeptide ◽  
Vitro Model ◽  
In Cells

Purpose: Organic anion transporting polypeptide (OATP) 1B3 transports many clinically important drugs, including statins, from blood into the liver. It exclusively expresses in human liver under normal physiological conditions. There is no rodent ortholog of human OATP1B3. Tissue targeting of therapeutic molecules mediated by transporters, including liver-targeting via liver-specific OATPs, is an emerging area in drug development. Sandwich-cultured primary hepatocytes (SCH) are a well characterized in vitro model for assessment of hepatic drug uptake and biliary excretion. The current study was designed to develop a novel rat SCH model expressing human OATP1B3 to study the hepatic disposition of OATP1B3 substrates. Methods: Primary rat hepatocytes transduced with adenoviral vectors expressing FLAG-tagged OATP1B3 (Ad-OATP1B3), a control vector Ad-LacZ, or that were non-transduced were cultured in a sandwich configuration. FLAG immunoblot and immunofluorescence-staining determined expression and localization of OATP1B3. Uptake of [3H]-cholecystokinin octapeptide (CCK-8), a specific OATP1B3 substrate, was determined. Taurocholate (TC) is a substrate routinely used in SCH to assess biliary excretion via bile canaliculi (BC) and is also a substrate of OATP1B3. [3H]-TC accumulation in cells+BC, cells, biliary excretion index (BEI) and in vitro Clbiliary were determined using B-CLEAR® technology. Results: OATP1B3 protein was extensively expressed and primarily localized on the plasma membrane in day 4 Ad-OATP1B3-transduced rat SCH. [3H]-CCK-8 accumulation in cells+BC was significantly greater (~5-13 folds, p<0.001) in day 4 SCH with vs. without Ad-OATP1B3-transduction. Expressing OATP1B3 in rat SCH significantly increased [3H]-TC accumulation in cells+BC and cells, without affecting BEI and in vitro Clbiliary. Conclusions: Rat SCH expressing human OATP1B3-is a novel in vitro model allowing simultaneous assessment of hepatic uptake, hepatocellular accumulation and biliary excretion process of a human OATP1B3 substrate. This model could be a potential tool for screening for liver-targeting compounds mediated by OATP1B3.

scholarly journals The turnover of L-type pyruvate kinase in cultured rat hepatocytes

1982 ◽  
Vol 204 (1) ◽  
pp. 89-95 ◽  
Author(s):  
Graeme P. Poole ◽  
David P. Bloxham
Keyword(s):  
Pyruvate Kinase ◽  
Rat Hepatocytes ◽  
Cell Protein ◽  
Rapid Phase ◽  
First Order Kinetics ◽  
Enzyme Degradation ◽  
Fructose 1,6 Bisphosphatase ◽  
Cultured Rat Hepatocytes ◽  
In Cells

Hepatocytes were isolated by collagenase perfusion of livers from rats that had been allowed access to a carbohydrate-rich diet or laboratory chow or had been deprived of food 48h before use. By incubation with l-[4,5-3H]leucine and precipitation with anti-(L-type pyruvate kinase) sera the rates of synthesis and degradation of L-type pyruvate kinase were measured in freshly prepared cells and hepatocytes maintained in monolayer culture for up to 5 days. Hepatocytes from carbohydrate-rich-diet-fed rats synthesized more L-type pyruvate kinase than did cells from chow-fed animals, which in turn synthesized more than cells from 48h-starved rats. Hepatocytes maintained in culture for up to 5 days synthesized L-type pyruvate kinase at similar rates to freshly prepared cells. The degradation of [3H]leucine-labelled L-type pyruvate kinase was shown to be biphasic. A phase with t½ (half-time) 4.9h and a duration of 8–10h was followed by a phase with t½ 79.2h. Cells from chow-fed and carbohydrate-rich-diet-fed rats showed similar patterns of degradation of L-type pyruvate kinase. The addition of 2mm-fructose and 0.1μm-insulin to the culture medium increased the t½ of the rapid phase to 12h in cells isolated from carbohydrate-rich-diet-fed rats, but not in cells from chow-fed rats. The secondary, slower, phase of degradation remained unaffected. The degradation of fructose 1,6-bisphosphatase and total cell protein followed first-order kinetics. The half-life of fructose 1,6-bisphosphatase was 41.0h in cells from chow-fed animals and 48.5h in cells from carbohydrate-rich-diet-fed donors. Fructose and insulin did not affect the rate of enzyme degradation. We propose that there is a role for protein catabolism in the short-term and long-term control of L-type pyruvate kinase concentration.

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