The effects of O-acetylsterigmatocystin and related compounds on rat liver and cultured chicken embryonal liver cells.

Fine structural nucleolar changes induced in rat liver and primary tissue culture cells from 10-day-old chicken embryonal liver by O-acetylsterigmatocystin (AcO-stg), related compounds and aflatoxin B1 were compared. (1) Male Wistar rats were given a single i.p. injection of sterigmatocystin (stg), AcO-stg, and aflatoxin B1. 3 days after the injection of 15 mg/kg of stg, sporadic single cell necrosis was observed in rat liver, whereas rats treated with 8 mg/kg AcO-stg or more, and 3 mg/kg of aflatoxin B1 showed massive liver necrosis. Acetylation resulted in a marked increase in solubility in polar organic solvents. This increased solubility could play an important role in determining toxicity. (II) Treatment with the compounds with an unsaturateddelta1,2-furobenzofuranring system, such as AcO-stg, demethyl-diacetyl-stg (deMe-diAc-stg), and aflatoxin B1, resulted in nucleolar segregation and fragmentation of primary culture cells. Both parenchymal and mesenchymal cells in culture were susceptible to AcO-stg and deMe-diAc-stg, while the mesenchymal cells were more resistant to aflatoxin B1 than the hepatocytes. The inhibition of RNA synthesis in both cell types as determined in radioautography was in accordance with the electron-microscopic observations. Acetyldihydrosterigmatocystin (AcO-dihyd-stg), a saturated delta1,2-furobenzofuranring compound, was less toxic to primary tissue culture cells.     

The effect of polycyclic aromatic hydrocarbons on choline kinase activity in mouse hepatoma cells

Choline kinase catalyzes the first rate-limiting step in the pathway of biosynthesis of phosphatidylcholine. This enzyme was shown previously to be induced in liver by treatment of rats with polycyclic aromatic hydrocarbons (Ishidate et al. (1980) Biochem. Biophys. Res. Commun. 96, 946-952). The present study was undertaken to determine whether choline kinase in the murine hepatoma cell line, Hepa 1c1c7, is inducible by aromatic hydrocarbons and, if so, whether this induction is mediated by the aromatic hydrocarbon receptor. Treatment of Hepa 1c1c7 cells with 10 microM beta-naphthoflavone resulted in a 1.6-fold increase of choline kinase activity, but no response was seen when the cells were exposed to either 5.0 microM benzo[a]pyrene or 1.0 nM 2.3,7,8-tetrachlorodibenzo-p-doxin, both potent inducers of aryl hydrocarbon hydroxylase. Cell line variants with either deficient or elevated aromatic hydrocarbon receptors showed no increase in choline kinase activity following treatment with any of the polycyclic aromatic hydrocarbons. These results are not consistent with a role for the aromatic hydrocarbon receptor in increased choline kinase activity in Hepa 1c1c7 cells.     

Lipid-induced insulin resistance in cultured hepatoma cells is associated with a decreased insulin receptor tyrosine kinase activity.

We have shown previously that experimental modifications of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (Zajdela Hepatoma Culture, ZHC) affect both binding and biological actions of insulin. Discrepancies between insulin binding and actions implied a postbinding defect, responsible for the observed insulin resistance in lipid-treated cells. To elucidate the mechanism for this defect, we have studied insulin binding and insulin receptor kinase activity in partially purified receptor preparations from ZHC cells grown either in normal medium or in medium supplemented with linoleic acid or 25-hydroxycholesterol. Insulin binding to the lectin-purified insulin receptor showed only a small alteration in receptor affinity for the preparations from lipid-treated cells. Insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor, as well as insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr)4:1, was significantly decreased in the preparations from lipid-modified cells. Although differences in basal levels were observed, the magnitude of the insulin-stimulated kinase activity was significantly decreased in receptor preparations from lipid-treated cells. These findings indicate that experimental modification of the lipids of cultured hepatoma cells can produce in insulin receptor kinase activity changes that are proportional to the reduced insulin action observed in these cells.     

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.     

Purification and characterization of choline/ethanolamine kinase from rat liver

Choline kinase, the first enzyme in the CDP-choline pathway for phosphatidylcholine biosynthesis, was purified 26,000-fold from rat liver to a specific activity of 143,000 nmol.min-1.mg-1 protein. The subunit molecular mass was 47 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the apparent native molecular mass was 160 kDa by size exclusion chromatography, suggesting a tetrameric structure. Two peaks of choline kinase activity were obtained by chromatofocusing. These isoforms eluted at pH 4.7 (CKI) and 4.5 (CKII). CKII appeared to be homogeneous by sodium dodecyl sulfate gel electrophoresis. Peptide mapping of two isoforms indicated a high degree of similarity, although there were peptides not common to both. Ethanolamine kinase activity copurified with both isoforms. The ratio of choline to ethanolamine kinase activity was 3.7 +/- 0.7 throughout the purification procedure. Choline and ethanolamine were mutually competitive inhibitors. The respective Km values, 0.013 and 1.2 mM, were similar to the Ki values of 0.014 and 2.2 mM. An antibody raised against CKII immunoprecipitated both choline and ethanolamine kinase activities from liver cytosol at the same titer. These data suggest that both activities reside on the same protein and occur at the same active site. Similarly, both activities were immunoprecipitated from brain, lung, and kidney cytosols. Western blot analysis showed both purified liver isoforms, as well as brain, lung and kidney enzymes, to have a molecular mass of 47 kDa.     

Ontogeny of insulin-like growth factor I and insulin receptor kinase activity in rat liver.

IGF-I and insulin receptors possess tyrosine-kinase enzymatic activity considered to be essential for signal transduction and thereby mediating the putative effects of these hormones on fetal growth and development. We investigated the ontogeny of IGF-I and insulin receptor tyrosine-kinase activity in at least 3 separate membrane preparations from liver of rats at 21 day of embryonic life (21ED), 1 and 5 day of postnatal life (1PD and 5PD respectively) and adult. Receptors purified by wheat germ agglutinin chromatography (WGA) were exposed to graded concentrations of IGF-I or insulin, and tyrosine-kinase activity was measured by quantifying incorporation of 32P into the exogenous substrate poly[Glu,Tyr; 4:1]. IGF-I stimulated tyrosine-kinase solely at 1 PD as documented by a maximal increase of 346 +/- 167% over basal kinase activity with 6.6 nmol/L IGF-I. While the lack of response in adult animals could be explained by a striking decrease in receptors at that age, 125I-IGF-I binding and affinity labelling of the WGA preparations indicated substantial IGF-I receptors were present in the liver at each of the perinatal ages. Furthermore, this dissociation between IGF-I binding and the tyrosine-kinase activity of these IGF-I receptors could not be attributed to the presence/absence of IGF-I binding proteins as judged by affinity labelling. In contrast, insulin-stimulated tyrosine-kinase activity was observed at all ages tested although it appeared greatest at 1PD. We conclude that (i) expression of IGF-I tyrosine-kinase activity is linked to developmental events and differs from that found for the insulin receptor tyrosine-kinase activity, (ii) during the perinatal period there is an apparent dissociation between ligand binding by the IGF-I receptor and receptor tyrosine-kinase activity. These observations suggest modulation of IGF-I receptor tyrosine-kinase activity may be an important regulator of IGF-I action during the perinatal period.     

Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells.

Rat liver epithelial cells in culture (WIRL-3C) have the enzymes that synthesize serine from 3-phophoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine-phosphate (serine-P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL-3C cells, nor was it common for PGAD activity in other cultured cells. At time of subculture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone-phosphate (Dx-P) or dibutyryl cyclic AMP(cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL-3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.     

Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

We examined the effects of dexamethasone on creatine kinase (CK) activity and insulin-like growth factor I (IGF-I) binding in two skeletal muscle-derived cell lines (mouse, C2C12; rat, L6) and in one cardiac muscle-derived cell line (rat, H9c2). Dexamethasone treatment during differentiation of cultured cells caused a dose-dependent increase in CK activity as well as an increase in the degree of myotube formation in C2C12 and L6, whereas H9c2 cells did not exhibit significant CK activities during culture or dexamethasone treatment. Dexamethasone treatment of C2C12 did not stimulate proliferation in differentiating cultures, but a dose-dependent increase in the number of nuclei was observed for L6 concomitant with increased CK activity. In L6 the increased CK activity may therefore reflect a dose-dependent increase in proliferation. Short-term (48 hr) treatment of C2C12 with dexamethasone (20 nM) did not appear to alter myoblast fusion but reversibly increased CK activity. In C2C12 the observed increase in CK, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities with dexamethasone treatment suggest modulation of protein expression and/or turnover. Although the data for dexamethasone effects on CK activities varied in each of the cell lines, consistent behavior was observed in all three cell lines when IGF-I binding was examined. IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities. Affinity cross linking and autoradiography indicated that the increase in IGF-I binding was the result of dexamethasone up-regulation of type I IGF receptors. Our data for all three muscle cell lines suggest that similar heterologous hormone receptor modulation of type I IGF receptor sites occurs with dexamethasone treatment.     

Actinomycin D-sensitive induction of choline kinase by carbon tetrachloride intoxication in rat liver

A single intraperitoneal dose(1 ml/kg body weight) of carbon tetrachloride (CCl4) caused a rapid and drastic induction of choline kinase activity in rat liver cytosol. The administration of either cycloheximide or actinomycin D completely blocked the CCl4-mediated induction of choline kinase activity, indicating that the elevated activity could be due to the change in the enzyme level. The pretreatment of rats with phenobarbital did not cause any significant effect on hepatic choline kinase induction by CCl4, suggesting that the induction may not be directly related to the metabolic rate of CCl4. A considerable part of induced form(s) of choline kinase appeared not to be a form present in the liver of untreated rats. The contribution of adrenals to the CCl4-mediated hepatic choline kinase induction could be ruled out.     

Retinol esterification in cultured rat liver cells.

Retinol esterification was examined in cultured hepatocytes and stellate cells from the rat. Esterification of [3H]retinol was linear for 2 h in both cell types. By increasing the concentration of retinol in the medium, there was a marked increase in retinol esterification in both cell types. The capacity for esterification of retinol was in the same order of magnitude in the two cell types at 3.5 microM-retinol in the medium. This represents a rate of retinol esterification which far exceeds that required to esterify the amount of retinol absorbed in the intestine. It was demonstrated in particulate homogenates from cultured hepatocytes that the esterification of retinol was dependent on acyl-CoA. Addition of 25-hydroxycholesterol or mevalonolactone promoted an increase in cholesterol esterification, whereas retinol esterification was unaffected, suggesting that cholesterol and retinol are esterified by two different enzymes. Some 80% of vitamin A in cultured hepatocytes is retinyl esters, mostly retinyl palmitate. By adding 87 microM-retinol in the medium the cells accumulated 100-fold free retinol and 2.5-3.0-fold retinyl esters within 1 h. When retinol-loaded cells were incubated without retinol, there was a marked decrease especially in free but also in esterified retinol. In the presence of 1 mM-oleic acid in the medium the amount of retinyl oleate was twice that in control cells.     

Modulation of rat brain insulin receptor kinase activity in diabetes

Insulin receptors from rat brain regions were studied for insulin binding and receptor associated kinase activity, in alloxan induced short-term and long-term diabetes, and insulin induced hypoglycemia. Insulin receptor activity was assessed by [¹²³I]insulin binding, and basal as well as insulin stimulated kinase activity of the receptor, expressed as phosphorylation of the synthetic peptide poly (Glu-Tyr (4:1)). Regional distribution pattern elicited the highest binding and kinase activity in the olfactory bulb. Diabetes caused a significant increase in the kinase activity. The data suggests that brain insulin receptor kinase is regulated differently compared to peripheral tissues and supports the concept of an active brain insulin receptor in vivo.