Metyrapone modulation of tyrosine aminotransferase induction by dexamethasone in cultured hepatocytes

Metyrapone, an inhibitor of cytochrome P-450-dependent monooxygenases, enhanced the induction of tyrosine aminotransferase by dexamethasone in primary cultures of hepatocytes, while it had no effect on the basal level of the enzyme activity in the absence of the hormone. The amplification of the hormonal induction of tyrosine aminotransferase activity was strictly correlated with the concentration and with the inhibitory action of the compound on cytochrome P-450. The phenomenon occurred even at the maximally effective concentrations of dexamethasone, thus showing that metyrapone is a 'Glucocorticoid Potency Amplifier'. The dexamethasone activity amplification by metyrapone could be the consequence of a modulation of the glucocorticoid biotransformations due to the cytochrome P-450 inhibitor.     

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.     

Insulin-mediated inhibition of the induction of tyrosine aminotransferase by dexamethasone.

Insulin-mediated regulation of glucocorticoid-induced expression of the liver-specific gene tyrosine aminotransferase was studied in a clone of the Reuber rat hepatoma cells. Insulin inhibited dexamethasone-induced chloramphenicol acetyltransferase expression from approximately 4 kb of TAT 5' flanking sequence. The degree of this inhibition was comparable to the response of the endogenous gene. A construct of approximately 3 kbp of 5' flanking sequence exhibited no significant basal expression but retained sensitivity to glucocorticoids and to insulin inhibition of the glucocorticoid response. Results of further analysis of the insulin response in deletion constructs and constructs containing glucocorticoid responsive elements ligated to a heterologous promoter suggest that in addition to the glucocorticoid response elements a region close to the start site in the TAT promoter is necessary for insulin to inhibit glucocorticoid-mediated induction of expression.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.     

Epidermal growth factor as a new regulator of induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids

Epidermal growth factor (EGF) dose-dependently enhanced the induction of tyrosine aminotransferase and tryptophan oxygenase by glucocorticoids in primary cultures of adult rat hepatocytes without itself having any effect on these enzymes in the absence of glucocorticoids. The amplifications were observed even with dexamethasone at high concentrations (10(-6) M-10(-5) M) that had a maximal effect. EGF had no effect on induction of tyrosine aminotransferase by glucagon or Bt2cAMP. The effect of EGF was also observed in adrenal-ectomized and submaxillary gland-ectomized rats. These results suggest that EGF is an endogenous amplifier of the action of glucocorticoids.     

Cytoplasmic binding of dexamethasone and induction of tyrosine aminotransferase in neonatal rat liver

Dexamethasone administration markedly increases the activity of tyrosine aminotransferase in postnatal rat liver. The glucocorticoid fails to induce the enzyme in foetal rats when administered in utero. Dexamethasone binding activity of rat liver cytoplasm is low or absent in foetal animals but increases to adult levels 1–2 days after birth. In vitro experiments with isolated nuclei indicate that foetal nuclei have the capacity to accumulate dexamethasone but only when presented with cytosol-bound glucocorticoid.     

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.     

Inhibition of hormonal induction of tyrosine aminotransferase by polyamines in freshly isolated rat hepatocytes.

We have analysed the effects of natural aliphatic polyamines on hormonal induction of tyrosine aminotransferase (TAT) in suspensions of hepatocytes isolated from adult fed rats. Glucagon or cyclic AMP derivatives (dibutyryl and 8-bromo) used alone caused a 4-5 fold increase in enzyme activity within 4h. This effect was independent of glucocorticoids, which also increased TAT activity (2.5-fold); when combined, the effects of the two inducers were additive. Spermine and putrescine totally inhibited the hormonally-mediated increase in enzyme activity when added at the onset of incubation with the inducers. Furthermore, polyamines could block the hormonal effect at any time during the course of TAT induction, with, however, a 30 min lag period, suggesting that they must enter the cells. Hepatocytes were indeed shown to take up spermine. At low external concentrations (less than 50 microM), an Na+-dependent, saturable and concentrative mechanism was predominant; at high concentrations (greater than 0.5 mM) transport occurred mainly through a non-saturable, Na+-independent mechanism, building up intracellular concentrations slightly lower than those in the medium. Dose-dependence analysis of the polyamine effect on enzyme induction indicated that half-maximal and maximal inhibition occurred with 0.75 mM- and 2.5 mM-spermine respectively, whereas 2.5mM- and 7.5 mM-putrescine were required respectively to obtain similar effects. Spermidine was much less effective and cadaverine had virtually no effect. None of the polyamines affected the rate of decay of TAT, nor did they directly or indirectly cause enzyme inactivation, indicating that a post-translational modification was unlikely to account for the polyamine effects. Similarly, these effects could not be ascribed to a non-specific inhibition of overall protein synthesis. We conclude that, in hepatocytes, polyamines (or their metabolites) directly interfere with one or several steps controlled by hormones in the synthesis of tyrosine aminotransferase.     

Hormonal control of the development of tryptophan oxygenase in primary cultures of young rat hepatocytes

Developmental increase of tryptophan oxygenase (L--tryptophan: oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11) was studied using hepatocytes of neonatal rats in primary culture. Hepatocytes from rats of 2–30-days-old were isolated and cultured for 2 days. In cultured hepatocytes of 2-day-old rats, tryptophan (2.5 mM), dexamethasone (1.10^?5 M) and glucagon (1.10^?7 M) did not cause the appearance of tryptophan oxygenase. But the enzyme activity became detectable, when heptocytes from 5-day-old rats were incubated wiht tryptophan, the oxygenase could be induced precociously by dexamethasone, but not by glucagon. The effect of glucagon was first seen 2 weeks after birth. However, in hepatocytes of 9-day-old rats glucagon stimulated formation of cyclic AMP and protein kinase activity (EC 2.7.1.37) and also induced tyrosine aminotransferase (EC 2.6.1.5). When heptocytes of 9-day-old rats were cultured for 4 days, their tryptophan oxygenase became inducible by glucagon. Insulin almost completely inhibited precocious appearance of the enzyme activity evoked by tryptophan plus dexamethasone in hepatocytes of 9-day-old rats. These results suggest that the appearance of tryptophan oxygenase in rat liver during development is due to first the onset of gene coding for tryptophan oxygenase and then stimulation by the sequential of glucocorticoid and glucagon.