Glycogen synthase turnover and phosphorylation in rat H4IIE hepatoma cells

Glycogen synthase was isolated from rat H4IIE hepatoma cells by the use of specific antibodies. Immunoprecipitates from cells grown in the presence of [35S]methionine contained two 35S-labeled polypeptides, designated GS1 and GS2, separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Labeling of both species was half-maximal after 3 h and remained constant up to 48 h. When cells were incubated with [32P]-phosphate, 32P was incorporated into both species with similar kinetics, half-maximal labeling occurring after 2-3 h. The steady-state ratio 32P/35S was significantly higher for the lower mobility GS2 polypeptide. Pulse-chase experiments showed that the two subunits followed similar kinetics with respect to 35S-labeling. However, the turnover of 32P on the GS2 subunit was significantly faster (t1/2 approximately 30 min) than that on the GS1 subunit (t1/2 approximately 2 h). We suggest that the two polypeptides represent different phosphorylation states of the glycogen synthase subunit and are rapidly interconverted.     

Osmotic regulation of betaine homocysteine-S-methyltransferase expression in H4IIE rat hepatoma cells

Cell hydration changes critically affect liver metabolism and gene expression. In the course of gene expression studies using nylon cDNA-arrays we found that hyperosmolarity (405 mosmol/l) suppressed the betaine-homocysteine methyltransferase (Bhmt) mRNA expression in H4IIE rat hepatoma cells. This was confirmed by Northern blot and real-time quantitative RT-PCR analysis, which in addition unraveled a pronounced induction of Bhmt mRNA expression by hypoosmotic (205 mosmol/l) swelling. Osmotic regulation of Bhmt mRNA expression was largely paralleled at the levels of Bhmt protein and enzymatic activity. Like hyperosmotic NaCl, hyperosmotic raffinose but not hyperosmotic urea suppressed Bhmt mRNA expression, suggesting that cell shrinkage rather than increased ionic strength or hyperosmolarity per se is the trigger. Hypoosmolarity increased the expression of a reporter gene driven by the entire human BHMT promoter, whereas destabilization of BHMT mRNA was observed under hyperosmotic conditions. Osmosensitivity of Bhmt mRNA expression was impaired by inhibitors of tyrosine kinases and cyclic nucleotide-dependent kinases. The osmotic regulation of BHMT may be part of a cell volume-regulatory response and additionally lead to metabolic alterations that depend on the availability of betaine-derived methyl groups.     

Detection of DNA damage in workers exposed to JP-8 jet fuel

The genotoxicity of jet propulsion fuel 8 (JP-8) was assessed in the leukocytes of archived blood specimens from U.S. Air Force personnel using the comet assay. No differences in mean comet assay measurements were found between low, moderate, and high exposure groups before or after a 4h work shift. Before the work shift, mean tail DNA and mean tail (Olive) moment increased as the concentration of benzene measured in end-exhaled breath increased, indicating that prior environmental or work-related exposures to benzene produced DNA damage. The number of cells with highly damaged DNA decreased as the pre-shift benzene concentration in breath increased. It is not clear why the decrease is occurring. Mean tail DNA and mean tail (Olive) moment decreased as the concentrations of benzene and naphthalene measured in breath immediately after the work shift increased. These inverse relationships may reflect a slower rate of absorption or a faster rate of expiration of benzene in the lung. The number of cells with highly damaged DNA increased as the concentration of urinary (2-methoxyethoxy)acetic acid (MEAA) increased. This relationship was not seen in urinary MEAA adjusted for creatinine. MEAA is a metabolite of the deicing agent 2-(2-methoxyethoxy)ethanol contained in JP-8. MEAA or a component of JP-8 correlated with MEAA may have a toxic effect on DNA.     

Hypoosmolarity influences the activity of transcription factor NF-kappaB in rat H4IIE hepatoma cells

The influence of anisoosmolarity on NF-kappaB binding activity was studied in H4IIE rat hepatoma cells. Hypoosmolarity induced a sustained NF-kappaB binding activity whereas the hyperosmotic NF-kappaB response was only minor. Hypoosmotic NF-kappaB activation was accompanied by degradation of the inhibitory IkappaB-alpha. Protein kinase C, PI(3)-kinase, reactive oxygen intermediates and the proteasome apparently participate in mediating the hypoosmotic effect on NF-kappaB. Hypoosmolarity plus PMA induced, amplified and prolonged IkappaB-alpha degradation and NF-kappaB binding activity. Transforming growth factor beta-induced apoptosis was diminished by hypoosmolarity. However, this anti-apoptotic effect was probably not related to NF-kappaB activation.     

Osmotic regulation of the heat shock response in H4IIE rat hepatoma cells.

The influence of cell hydration on the heat shock response was investigated in H4IIE hepatoma cells at the levels of HSP70 expression, MAP kinase activation, induction of c-jun and the MAP kinase phosphatase MKP-1, heat resistance, and development of tolerance/sensitization to arsenite after a priming heat treatment. Induction of HSP70, MKP-1, and c-jun by heat was delayed, but more pronounced or sustained, under hyperosmotic conditions compared with normo- and hypo-osmotically exposed cells. Anisosmolarity per se was ineffective to induce HSP70; some expression of the mRNAs for MKP-1 and c-jun in response to hyperosmolarity was found, but was small compared with the response to heat. Heat-induced activation of JNK-1 was increased under hyperosmotic conditions and more sustained than the JNK-activity induced by hyperosmolarity at 37 degrees C. A prominent Erk-2 activation was found immediately after heat shock under hypo- and normo-osmotic conditions, but Erk-2 activation was weak in hyperosmolarity-exposed cells. Despite anisosmotic alterations of the heat shock response at the molecular level, the heat resistance of H4IIE cells toward heat shock was not affected by ambient osmolarity. However, an osmolarity-dependent sensitization to arsenite was induced by a priming heat shock. The osmodependence of the H4IIE cell response to heat differs from that recently found in primary rat hepatocytes. The data are discussed in terms of cellular adaption mechanisms and their physiological relevance.     

DNA damage induced by nitropyrenes in primary mouse hepatocytes and in rat H4-II-E hepatoma cells

The capacity of nitropyrenes to cause DNA damage in primary mouse hepatocytes (C57BL/6N mice) and rat H4-II-E hepatoma cells was studied by estimating single-strand breaks using the alkaline elution technique. 1-Nitropyrene (10-200 microM) caused clear dose-dependent increases in DNA strand breaks in both cell types, whereas no increase in DNA strand breaks was observed in hepatocytes treated with 1.3-, 1,6-, 1,8-dinitropyrene, 1,3,6-trinitropyrene and 1,3,6,8-tetranitropyrene under standard assay conditions (5-20 microM 30-min incubation). However, 1,8-dinitropyrene (1,8-DNP) caused dose-dependent increases in DNA strand breaks when incubated with the H4-II-E cells for 48 h, while no single-strand breaks were observed following treatment with 1,6-dinitropyrene (1,6-DNP) under the same conditions. Neither 1,6-DNP nor 1,8-DNAP induced DNA crosslinks in the H4-II-E cells. These data indicate that substrate specificity exists in the metabolic activation of nitropyrenes in murine liver.     

Hepatic mitochondrial transport of glutathione: studies in isolated rat liver mitochondria and H4IIE rat hepatoma cells

Glutathione (GSH) is transported into renal mitochondria by the dicarboxylate (DIC; Slc25a10) and 2-oxoglutarate carriers (OGC; Slc25a11). To determine whether these carriers function similarly in liver mitochondria, we assessed the effect of competition with specific substrates or inhibitors on GSH uptake in isolated rat liver mitochondria. GSH uptake was uniphasic, independent of ATP hydrolysis, and exhibited K_m and V_max values of 4.08 mM and 3.06 nmol/min per mg protein, respectively. Incubation with butylmalonate and phenylsuccinate inhibited GSH uptake by 45-50%, although the individual inhibitors had no effect, suggesting in rat liver mitochondria, the DIC and OGC are only partially responsible for GSH uptake. H4IIE cells, a rat hepatoma cell line, were stably transfected with the cDNA for the OGC, and exhibited increased uptake of GSH and 2-oxoglutarate and were protected from cytotoxicity induced by H₂O₂, methyl vinyl ketone, or cisplatin, demonstrating the protective function of increased mitochondrial GSH transport in the liver.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Osmotic regulation of MG-132-induced MAP-kinase phosphatase MKP-1 expression in H4IIE rat hepatoma cells.

BACKGROUND/AIMS: Proteasome inhibitors such as MG-132 are considered as potential therapeutical tools in different clinical settings. The dual specificity MAP-kinase phosphatase MKP-1 plays a role in balancing signals mediating cell death or survival. Here the effect of cell hydration on MG-132-induced MKP-1 expression was investigated in H4IIE rat hepatoma cells. RESULTS: Hyperosmolarity (405mosmol/l) increased MKP-1 expression by MG-132, which was accompanied by an induction of c-Fos, c-Jun, cJun Ser73 phosphorylation, and AP-1 DNA binding. MKP-1 induction by MG-132 plus hyperosmolarity was sensitive to inhibition of p38(MAPK) and c-Jun-N-terminal kinases (JNKs) but not extracellular signal-regulated kinases Erk-1/Erk-2, and was accompanied by a decline of MAP-kinase activities. Although hyperosmolarity increased overall protein ubiquitination in presence of MG-132, ubiquitination of MKP-1 was found under normo-, but not hyperosmotic conditions. Hyperosmolarity also enabled MG-132 to induce poly-ADP-ribose polymerase (PARP) cleavage which was sensitive to inhibition of p38(MAPK) and JNKs but not Erk-1/Erk-2. PARP cleavage and caspase-3 activation in H4IIE cells treated with hyperosmolarity plus MG-132 was further increased by vanadate, consistent with a contribution of MKP-1 to counterbalance proapoptotic MAP-kinase signals. CONCLUSION: The findings suggest that among other factors cell hydration critically determines the cellular response to proteasome inhibitors.     

Oxidative stress induces phosphoenolpyruvate carboxykinase expression in H4IIE cells

Oxidative stress is closely associated with diabetes and is a major cause of insulin resistance. Impairment of hepatic insulin action is thought to be responsible for perturbations in hepatic glucose metabolism. In this study, we found that oxidative stress is involved in the dysregulation of gene expression of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, by a mechanism independent of insulin. Elevation of oxidative stress by injection of ferric nitrilotriacetate in rats increased the expression of hepatic PEPCK mRNA. To examine the direct action of oxidative stress on PEPCK expression, we treated H4IIE hepatoma cells with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO increased intracellular oxidative stress and the expression of PEPCK mRNA. Inhibition of p38 mitogen-activated protein kinase (p38 MAP kinase), which mediates responses to oxidative stress, suppressed the induction of PEPCK mRNA by BSO. These results suggest that oxidative stress dysregulates hepatic PEPCK expression by an insulin-independent mechanism.     

Control of angiotensinogen production by H4 rat hepatoma cells in serum-free culture

Summary A serum-free, hormone and attachment factor supplemented culture for rat H4 hepatoma cells was established. In the defined medium (Dulbecco's Modified Eagle's +Ham's F12+insulin, transferrin, fibronectin liver cell growth factor, and sodium selenite), H4 cells grew equally well as in 10% fetal bovine serum supplemented medium. H4 cells in either defined or serum-containing culture conditions produce transferrin but not albumin or alpha-fetoprotein. In this paper we have studied the effect of various hormones and pressor peptides on the production of angiotensinogen by H4 cells cultured in defined conditions. Only glucocorticoid hormone had a significant effect on the production of angiotensinogen, whereas other hormones previously reported to exert their effect on angiotensinogen production had little or no effect. This work was supported by grant P01 CA37589 from the National Institutes of Health, Bethesda, MD.     

Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.     

Analysis of rat testicular protein expression following 91-day exposure to JP-8 jet fuel vapor

We analyzed protein expression in preparations from whole testis in adult male Sprague-Dawley rats exposed for 6 h/d for 91 consecutive days to jet propulsion fuel-8 (JP-8) in the vapor phase (0, 250, 500, or 1000 mg/m(3) +/- 10%), simulating a range of possible human occupational exposures. Whole body inhalation exposures were carefully controlled to eliminate aerosol phase, and subjects were sacrificed within 48 h postexposure. Organ fractions were solubilized and separated via large-scale, high resolution two-dimensional electrophoresis, and gel patterns scanned, digitized and processed for statistical analysis. Seventy-six different testis proteins were significantly increased or decreased in abundance in vapor-exposed groups, compared to controls, and dose-response profiles were often nonlinear. A number of the proteins were identified by peptide mass fingerprinting and related to histopathological or physiological deficits shown in previously published studies to occur with repeated exposure to hydrocarbon fuels or solvents. These results demonstrate a significant effect of JP-8 exposure on protein expression, particularly in protein expression in the rodent testis, and suggest that a 91 d exposure to jet fuel vapor induces changes of equal or greater magnitude to those reported previously for shorter duration JP-8 aerosol exposures.