Effect of estragole on glucocorticoid induction of tyrosine aminotransferase and tryprophan oxygenase in the rat and mouse liver

A single intraperitoneal injection of Estragole (300 mg/kg) to female ICR mice 19 hours prior to Dexamethasone induction decreased induced activities of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) nearly to 50% of the control values. In these mice, activities of the marker enzymes of liver damage: alanine aminotransferase (ALAT) and aspartate aminotransferase (AAT) increased in the blood 1.7-2.3-fold as compared with the untreated controls. By contrast, carbon tetrachloride (100 mg/kg) increased the blood AIAT and AsAT activities 135- and 30-fold as compared with the control, but inhibited the TAT and TO induction much less than Estragole did. Estragole seems to inhibit the glucocorticoid induction of these hepatic enzymes not via the unspecific toxic damage of the liver.     

Tissue-specific DNaseI hypersensitive sites in the 5''-flanking sequences of the tryptophan oxygenase and the tyrosine aminotransferase genes.

The genes for tryptophan oxygenase (TO) and tyrosine aminotransferase (TAT) are expressed in a tissue- and development-specific manner and are regulated by glucocorticoids (TO and TAT) and glucagon or its intracellular mediator cAMP (TAT) in rat liver. We have analyzed the chromatin structure of these genes in the vicinity of the 5' ends with regard to DNaseI hypersensitivity and have found DNaseI hypersensitive sites upstream of each of the promoters. Mapping of this region reveals three closely spaced cleavage sites near the TO promoter and a doublet of sites near the TAT promoter. In both genes additional cleavage sites are found further upstream. All hypersensitive sites of both genes are absent in kidney nuclei and therefore appear to be specific for the tissue expressing the genes. A correlation of expression and modified chromatin structure was also observed in a hepatoma cell line expressing TAT but not TO: hypersensitive sites are present in TAT but not in TO chromatin. Upon glucocorticoid induction an additional hypersensitive site is detected approximately 2 kb upstream of the TAT promoter in liver and hepatoma cells.     

Dehydroepiandrosterone regulation of the hepatic glucocorticoid receptor in the zucker rat. The obesity research program

Dehydroepiandrosterone (DHEA) decreases the activity of hepatic tyrosine aminotransferase (TAT), a glucocorticoid-inducible enzyme, in the obese, hypercorticosteronemic Zucker rat. To investigate the mechanism of this antiglucocorticoid action, the effect of exogenous DHEA on hepatic glucocorticoid receptor (GC) number and affinity was quantitated. Food supplementation with DHEA (0.6% w/w) for 1 or 7 days had no effect on either receptor number or affinity in obese Zucker rats. After 28 days, however, DHEA treatment resulted in a nearly 40% decrease in cytosolic hepatic receptor content (B_max; fmol/mg cytosolic protein) without any change in affinity (K_d) in both lean and obese rats. DHEA treatment for 28 days also resulted in an increased liver size and cytosolic protein content. When the hepatic GC receptor content was normalized based on the change in liver size and protein content, the apparent number of GC binding sites per liver was not affected by DHEA treatment. This observation suggests that DHEA's effect on GC receptor content may not be a specific action and that downregulation of the GC receptor is not the mechanism of DHEA action on GC induced TAT activity. This is supported by the effect of DHEA on obese rat TAT activity in the same experiment where the greatest inhibition occurred after only 1 day of treatment. From these experiments it is concluded that although long-term DHEA treatment may decrease the relative concentration of GC receptors in rat liver, this change is not the mechanism through which DHEA mediates its acute antiglucocorticoid action.     

Duration of antagonizing effect of RU486 on the agonist induction of tyrosine aminotransferase via glucocorticoid receptor

The duration of the antagonizing activity of RU486 on tyrosine aminotransferase (TAT) induction and the glucocorticoid receptor in rat liver was studied. A single dose of RU486 (10 mg/kg) caused occupation of the cytosol glucocorticoid receptor in rat liver at 1h. During this time no nuclear binding of [³H]dexamethasone ([³H]Dex) receptor complex was recorded, and TAT induction was completely blocked. TAT inducibility recovery parallelled receptor binding in both the cytosol and the nuclei, reaching maximum at 12 h. In contrast, nuclear binding recovered in 24 h, and [³H]Dex receptor binding in cytosol 48 h after RU486 application. It is concluded that the inhibitory effect of a single dose of RU486 on TAT induction is of rather short duration. At concomitant presence of agonist and antagonist in vivo, no direct correlation between agonist receptor occupancy and TAT induction could be observed.     

Studies on the nuclear binding of steroid hormone-receptor complex; binding of liver and thymus dexamethasone-receptor complex and prostate dihydrotestosterone-receptor complex to nuclei from various tissues.

To examine the binding specificity of steroid hormone-cytoplasmic receptor complexes to nuclei, binding of 3H-dexamethasone (Dex)-liver, 3H-Dex-thymus and 3H-dihydrotestosterone (DHT)-prostate receptor complexes to nuclei from liver, prostate, thymus, spleen and kidney was studied. It was observed that a significant amount of steroid-receptor complexes was bound to any nuclei used in the present study and the extent of the binding of receptor complexes to nuclei from homologous tissues was not always greater than that to nuclei from heterogenous tissues. However, a significant portion of the 3H-Dex-liver and 3H-DHT-prostate receptor complexes was not absorbed by nuclei from kidney, spleem, and thymus, and the unabsorbed complexes were efficiently bound to liver and prostate nuclei. The results obtained indicate that two types of receptor complex with regard to nuclear binding were present in cytosols of liver and prostate; one binds to nuclei from kidney, spleen, thymus, liver and prostate and the other does not bind to nuclei from kidney, spleen and thymus but does bind to nuclei of liver and prostate. The latter type of receptor complex was not observed in the cytosol from the thymus.     

Reduced cell sensitivity to glucocorticoid hormones in hypercholesterolemia

The number of 3H-dexamethasone binding sites in lymphocytes of subjects with hypercholesterolemia (HCS) was found to be decreased as compared to the receptor level in normolipidemic patients (N). In HCS-lymphocytes, the dexamethasone-induced inhibition of 3H-thymidine and 14C-acetate incorporation was less pronounced (by 20% and 22%, respectively) than in control cells, which is suggestive of the decreased sensitivity of HCS-lymphocytes to the hormone. An addition of 5-25% HCS blood sera to human skin fibroblast cultures caused a 10-50% decrease in the number of 3H-dexamethasone binding sites and diminished the Kd values 2-3 times. Lipid-depleted HCS-sera had no effect on the glucocorticoid reception in fibroblasts, whereas very low (VLDL) and low (LDL) density lipoproteins inhibited the 3H-dexamethasone binding to the cells. The most potent inhibiting effect was exerted by VLDL (both N-VLDL and HCS-VLDL). HCS-VLDL were more effective than N-LDL. HCS-HDL and N-HDL did not effect the 3H-dexamethasone binding to fibroblasts. In cells preincubated with VLDL dexamethasone inhibited the incorporation of 3H-thymidine and 14C-acetate less intensively (by 27% and 20%, respectively) than in control fibroblasts. The experimental results are suggestive of a decreased sensitivity of peripheral HCS-cells to glucocorticoids, which may shed some light on the mechanism of hypercholesterolemia realization into coronary heart disease and atherosclerosis.     

Sphingosine inhibition of tyrosine aminotransferase and tryptophan oxygenase induction by dexamethasone in primary culture of rat hepatocytes

The effect of sphingosine, a known selective inhibitor of protein kinase C, on the induction of tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) by dexamethasone was studied in the primary culture of rat hepatocytes to determine the possible involvement of protein kinase C in the expression of glucocorticoid action. Sphingosine inhibits the induction of TAT by dexamethasone in a concentration- and time-dependent manner in primary culture of rat hepatocytes. It does not inhibit the induction of TAT by Bt2cAMP. Sphingosine inhibits also the induction of TO by dexamethasone in a manner similar to TAT inhibition. It has no effect on the activity of lactate dehydrogenase, a cytosolic marker enzyme and on the protein content of the cultured hepatocytes. These findings indicate that endogenous modulator of protein kinase C, such as sphingosine, may influence the expression of glucocorticoid action in rat hepatocytes.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Description and analysis of differential sensitivity to glucocorticoids in Fao cells

This study shows that the derived hepatoma cell line Fao displays different sensitivities for glucocorticoid induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and gamma-glutamyltransferase (GGT). This was seen in the different behaviors of nine steroids with respect to these three effects: (1) in the presence of full agonists (dexamethasone or deacylcortivazol), half-maximal induction of GGT occurred at approx 5- to 6-fold higher agonist concentrations than those required for half-maximal induction of AAT and TAT; (2) in the presence of full antagonists (RU 486, R5020, or progesterone) the GGT response induced by an equal agonist concentration was inhibited at concentrations approx 4- to 5-fold lower than those required for an equivalent inhibition of TAT response; (3) in the presence of cortexolone, deoxycorticosterone, 11 beta-hydroxyprogesterone and dexamethasone-3'-oxetanone, there was a partial agonistic effect (30-50%) on TAT and AAT responses, whereas there was a mainly antagonistic effect (very weak agonistic effect: 0-10%) on GGT response; (4) regardless of the steroid or its full or partial agonist activity, a given TAT induction level (50%, for example) always corresponded to the same AAT and GGT induction levels (50 and 10% respectively). We provide evidence showing that the three above-mentioned biological responses are mediated via the same type of glucocorticoid receptor binding site. Consequently, this differential behavior probably originates from a phenomenon occurring after the common steps (activation, translocation) that follow the formation of the steroid-receptor complex. This leads us to propose a model in which this phenomenon is assumed to originate from a difference in the affinities of the activated receptor for the nuclear acceptor sites of the TAT and GGT genes.     

Insulin inhibition of tyrosine aminotransferase induction by dexamethasone is species-related

Dexamethasone induces TAT in the embryonic chick liver, but this effect is not blocked by prior administration of insulin at a dose sufficient to increase the hepatic protein/DNA ratio. Similarly, insulin does not block dexamethasone induction of TAT in chick embryo hepatocytes in vitro. In contrast, insulin reduces TAT induction by dexamethasone in fetal rat hepatocytes by 35-40%. No significant differences in insulin binding were noted between chick and rat hepatocytes.     

Cell-cycle regulation of insulin-stimulated tyrosine aminotransferase activity in rat hepatoma cells

Amongst the proteins that are subjected to variation during the cell division cycle few are under hormonal regulation. The variation in amount of tyrosine aminotransferase (TAT) in the hepatic tissue is under the control of glucagon, glucocorticoids and insulin. It has been reported that the inducibility of TAT activity by dexamethasone in rat hepatoma (HTC) is limited to the late G1 and the S portions of the cell cycle. Evidence is presented in this report that in the rat hepatoma Fao, insulin (which has the capability to promote both cell growth and hormonal effects via its own receptors) modulates the TAT activity during the cell cycle. The maximal insulin-stimulated induction of TAT activity was observed at the end of the G1 phase and then decreased as cells progressed through their mitotic cycle. The number of insulin binding sites per cell was decreased by only 30% during the same period of time. Furthermore, the extent of receptor autophosphorylation decreased in the same proportion, suggesting that insulin receptors remained functional through the whole cell cycle. In fact, another insulin-stimulated cellular function, neutral amino-acid transport, was not modified as cells progressed into the S phase. Hydroxyurea, which is known to prevent cell progression into the S phase, stabilized the insulin-induced TAT activity at its maximal level for several hours. Reciprocally, removal of hydroxyurea resulted in a concomitant decrease in TAT activity and reinitiation of DNA synthesis.     

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.     

Autogenic regulation of the sensitivity of liver cells to glucocorticoids during prolonged hormonal stimulation

The mechanisms of reversible decrease of hormone-dependent induction of tyrosine aminotransferase (TAT) by rat liver cells after prolonged administration of the glucocorticoid was studied. It was shown that the main links of the glucocorticoid action mechanism (i.e., the formation of a cytoplasmic hormone-receptor complex and the hormone accumulation in the nuclei) do not change under these conditions. It was found also that one of the necessary prerequisites for the decrease of the hormone-dependent induction of TAT is the constant production by liver cells of large amounts of TAT irrespective of whether this process is induced by the glucocorticoid or by a non-hormonal inducer, e.g., tryptophan. Using the dot-hybridization technique, it was demonstrated that the inhibition of hormone-dependent induction of TAT is correlated with the reduction of mRNA TAT. It was supposed that the main links in the mechanism of inhibition of the hormone-dependent induction are the formation of a large excess of the inducible protein--TAT--in the cells as well as the accumulation of end products of the TAT-catalyzed transamination reaction which cause a feed-back repression of the de novo synthesis of TAT. Studies with cell cultures of Morris hepatoma which is known to be sensitive to glucocorticoids revealed the ability of glucose, the end product of gluconeogenesis reactions, to provide for selective inhibition of the hormone-induced accumulation of mRNA TAT in hepatoma cells.     

Mechanism of a decrease of inducibility of tyrosine aminotransferase by carcinogenic aminoazo dyes. Absence of direct influence of o-aminoazotoluene on the enzyme molecules

The effect of o-aminoazotoluene (OAT) on the activity of tyrosine aminotransferase (TAT) from mouse liver cytosol under its incubation in the presence of the systems providing for the metabolic activation of the cancerogen (liver microsomes and NADPH2) and dephosphorylation of TAT molecules (light mitochondria and ATP) was studied. It was shown that OAT has neither direct nor indirect (via the phsophorylation--dephosphorylation systems) effect on the activity of TAT. It was concluded that the decrease of TAT induction by hydrocortisone in vivo resulting from injection of OAT to the mice is not due to the direct influence of the cancerogen on the enzyme molecules.     

Studies on the nuclear binding of steroid hormone-receptor complex; characteristics of binding of nuclei from fetal rat liver to 3H-dexamethasone-liver cytoplasmic receptor complex

Binding of 3H-dexamethasone (Dex)-rat liver cytoplasmic receptor complex to nuclei from fetal rat livers in vitro exhibited a high-affinity and saturable nature (Kd=1.5 X 10- M, maximal binding sites=470 fmole/mg DNA), and the binding was inhibited competitively by prior injection of Dex in vivo. While binding of 3H-Dex-receptor complex to nuclei from adult rat liver was in low affinity and unsaturable, and injection of Dex prior to the sacrifice of animals did not influence the nuclear binding to 3H-Dex-receptor complex in vitro. Differential salt-extraction with KCl solution of the nuclear bound 3H-Dex receptor complex revealed the presence of salt-extractable and residual forms of bound receptors. The amount of the fraction extracted with 0.3 M KCl reached its maximum at 10 min after the start of incubation, while the 1.0 M KCl-extractable and residual fractions reached their maximum plateaus after 30 min of the incubation. Scatchard analysis revealed that the binding of the receptor complex to the 0.3M and 1.0M KCl fractions was saturable, while the residual fraction did not show any tendency of saturation under the experimental conditions employed in the present study. The results obtained in this work were compared to those which have been reported by other investigators.     

Involvement of transforming growth factor-alpha secreted by macrophages in metallothionein induction by endotoxin

The mechanism of metallothionein (MT) induction of the liver by endotoxin, which is mediated by a factor secreted by endotoxin-stimulated macrophages, was studied in vitro. MT induction of the liver cells by the endotoxin-stimulated macrophage conditioned medium was inhibited by a monoclonal antiepidermal growth factor (EGF) / transforming growth factor-alpha (TGF-alpha) receptor antibody, which acts as an antagonist of EGF and TGF-alpha. MT was induced by the substance, which was adsorbed by polyclonal antibody to TGF-alpha, but not by a monoclonal antibody to EGF, in the conditioned medium of endotoxin-stimulated macrophages. These results suggest that TGF-alpha secreted by macrophages is involved in MT induction by endotoxin.     

A potential mechanism in medroxyprogesterone acetate teratogenesis.

MPA (medroxyprogeste)rone acetate) has been shown to be te)ratogenic in rabbits but not in rats or mice (Andrew and Staples 1977). Since normal steroid action appears to be mediated, in large part, through interaction with specific steroid receptors, it was postulated that the species difference in teratogenicity might be due to a difference in the interaction of MPA with target cells. A primary event in steroid-cell interaction is the binding of a steroid to intracellular receptors. Studies were initiated to measure the specific nature of MPA binding to glucocorticoid and progestin receptors in appropriate rat and rabbit target tissues. The competition of MPA with 3H-dexamethasone binding in liver cytosol (glucocorticoid receptor) and with 3H-progesterone binding in uterine cytosol (progesterone receptor) was determined. In rabbit liver cytosol, MPA was as effective at competing for specific dexamethasone binding as the natural glucocorticoids and considerably more effective than the nonspecific steroids. In rat liver cytosol MPA was only 10% as effective as the natural glucocorticoids and the competition could not be distinguished from that of nonspecific steroids. A similar species difference was not seen in uterine cytosol; MPA competed with progesterone in a similar fashion in both rat and rabbit. These data demonstrate a distinct species difference in the competitive nature of MPA for the glucocorticoid receptor but not for the progestin receptor. The results suggest that MPA, or possibly a metabolite, may be teratogenic in rabbits by binding with specific glucocorticoid receptors to inhibit or alter normal steroidal function in embryo-fetal development.     

Induction of tyrosine aminotransferase of primary cultured rat hepatocytes depends on the organization of microtubules

We investigated the relationship between the expression of tyrosine aminotransferase (TAT) and cytoskeletal systems of cultured rat hepatocytes by using serum-free culture conditions and changing three factors: (1) the concentration of calcium, (2) the dish-coating material, and (3) the cell-plating density. In hepatocytes in low-calcium medium, induction of TAT by dexamethasone and glucagon was maintained, although cell-cell adhesion was lost. Hepatocytes on Matrigel formed a nonspreading, spherical shape that provided them with the full extent of TAT activity without cell-cell adhesion. Hepatocytes plated on collagen at low cell density spread and changed shape, and the induction of TAT activity was markedly reduced. By using confocal laser-scanning microscopy, we analyzed the three-dimensional organization of cytoplasmic microtubules of hepatocytes maintaining the ability of TAT induction. Hepatocytes plated on collagen at low cell density possessed the radial filamentous structure of cytoplasmic microtubules. When the spherical shape of hepatocytes was maintained by cultivating cells on Matrigel, a ring-like structure of cytoplasmic micotubules beneath the plasma membrane was dominant. Moreover, the induction of TAT activity of hepatocytes in a standard culture system was strongly inhibited by the addition of 1 μM colchicine. These studies suggest that the organization of cytoplasmic microtubules may participate in the shape-related regulation of cell function. J. Cell. Physiol. 175:41–49, 1998. © 1998 Wiley-Liss, Inc.