Phorbol ester inhibition of hormonal induction of tyrosine aminotransferase

The liver specific enzyme, tyrosine aminotransferase, can be induced by glucocorticoids, cAMP analogs, or insulin. Each of these different inducing agents is believed to act through a separate pathway. The tumor promoting phorbol esters have been reported to stimulate phosphorylation of the insulin receptor and thereby decrease the ability of insulin to induce tyrosine aminotransferase. Our results demonstrate that TPA will not only inhibit the insulin stimulated increase in tyrosine aminotransferase, but will also inhibit induction of the enzyme by glucocorticoids or by cAMP.     

Regulation of expression of tyrosine aminotransferase in somatic cell hybrids between rat hepatoma cells and mouse fibroblasts

Somatic cell hybrids between rat hepatoma cells and mouse 3T3 fibroblasts fail to produce the liver-specific enzyme tyrosine aminotransferase. A novel approach using gamma-irradiation to induce chromosome loss from the non-expressing parent cell was applied to dissect genetically the factors in 3T3 cells that interact with the regulation of expression of tyrosine aminotransferase in these hybrids. Suppression of basal and steroid-inducible tyrosine aminotransferase activities was progressively relieved with increasing dose of radiation. The wide range in degree of reexpression suggests a complex of regulatory mechanisms. Suppression of steroid-inducibility was not linked to the mouse X-chromosome. Nor did the mouse genome affect the modulation of enzyme activity induced by insulin and by serum.     

The hormonal regulation of enzymes in prenatal and postnatal rat liver. Effects of adenosine 3′,5′-(cyclic)-monophosphate

1. The administration of glucagon, cAMP [adenosine 3',5'-(cyclic)-monophosphate], BcAMP [6-N-2'-O-dibutyryladenosine 3',5'-(cyclic)-monophosphate] or adrenaline to foetal rats during the last 2 days of gestation evoked the appearance of tyrosine aminotransferase and enhanced the accumulation of glucose 6-phosphatase in the liver. In foetuses 1-2 days younger only BcAMP was effective. After birth liver glucose 6-phosphatase no longer responds to glucagon or BcAMP. Tyrosine aminotransferase is still inducible by these agents in 2-day-old rats, but not in 50-day-old rats. After adrenalectomy of adults glucagon or BcAMP can enhance the induction of the enzyme by hydrocortisone. The results indicate that the ability to synthesize tyrosine aminotransferase and glucose 6-phosphatase when exposed to cAMP develops sooner than the ability to respond to glucagon with an increase in the concentration of cAMP; the responsiveness of enzymes to different hormones changes with age. A scheme illustrating the sequential development of competence in regulating the level of an enzyme is presented. 2. Actinomycin inhibited the effects of glucagon and BcAMP on liver tyrosine aminotransferase and glucose 6-phosphatase in foetal rats. Growth hormone, insulin and hydrocortisone did not enhance the formation of these enzymes. 3. The time-course of accumulation of glucose 6-phosphatase in the kidney is different from that in the liver. Hormones that increase the accumulation in foetal liver do not do so in the kidney of the same foetus or in the livers of postnatal rats.     

Stabilization of rat liver tyrosine aminotransferase in vivo by pyridoxine administration.

Administration of pyridoxine stabilizes rat liver tyrosine aminotransferase in vivo, whereas administration of cortisol, cyclic AMP, glucagon, insulin, tryptophan or tyrosine does not. The results of these and other experiments with pyridoxine are discussed in relation to the mechanisms of action of this vitamin on the activity of the enzyme.     

Immunological study of carbon tetrachloride-mediated induction of tyrosine aminotransferase in rat liver

Administration of CCl₄ (1.0 ml/kg) to rats resulted in a rise of liver tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) activity to a maximum of about 3.6 times the normal level 6 hr later. An immunological titration study proved that the phenomenon was due to increased enzyme content. Using an isotopic-immunochemical procedure the half-life of liver tyrosine aminotransferase at 3.5 hr after CCl₄ administration was shown to be 11.9 hr in contrast to 2.1 hr in the normal liver. Immunochemical analysis revealed that enzyme synthesis was decreased by CCl₄. Thus, in the early stage of CCl₄ poisoning, enzyme synthesis proceeded at a moderate rate while degradation was markedly impaired, resulting in the rise of tyrosine aminotransferase in the liver tissue.Several hours after administration of hydrocortisone to adrenalectomized rats, induced tyrosine aminotransferase reached its peak activity and then subsided to the basal level. At any time following hydrocortisone administration, administration of CCl₄ consistently caused an elevation of the enzyme activity above the level in controls not treated with CCl₄. Actinomycin D (5 mg/kg) also increased the enzyme at an early period of induction cycle but failed to do so at a later period.The CCl₄-mediated “superinduction” of hormonally preinduced tyrosine aminotransferase, like the induction of this enzyme by CCl₄ at a basal level, was found to be caused by the differential inhibitory effect of CCl₄ on the synthesis and degradation of this enzyme.     

Effects on induction of tyrosine aminotransferase in fetal mouse liver in vitro of prednisolone,insulin and thyroxine

The hormonal requirements for formation of tyrosine aminotransferase (EC 2.6.1.5) in fetal mouse liver were investigated in organ culture using chemically defined medium. The hormones tested were insulin, thyroxine and prednisolone. Prednisolone alone resulted in a two-fold increase in tyrosine amino-transferase activity in explanted liver in hormone-free medium on day 6, and its effect was dose dependent, but neither insulin nor thyroxine alone induced the enzyme. Addition of prednisolone plus thyroxine and prednisolone plus insulin increased the enzyme activity 1.4- and 1.3-fold, respectively, over that of explants with prednisolone alone. These three hormones together had the greatest effect, causing induction of 1.5-fold more activity than that with prednisolone plus insulin or plus thyroxine. The three hormones were not all needed continuously during the culture period: prednisolone and insulin were required during the early part of cultivation and thyroxine during the later part. The effects of these hormones were blocked by actinomycin D or puromycin, suggesting that these hormones increase de novo synthesis of tyrosine aminotransferase. Phase-contrast microscopy showed that prednisolone stimulated liver epithelial cell outgrowth, probably acting with insulin.     

L-tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo

The administration of l-tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l-tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo. When l-tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy-dl-tryptophan and indole administration in amounts equimolar to l-tryptophan also result in similar increases in activity whereas α-methyl-dl-tryptophan produces little or no increase.Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.     

Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: the role of enzyme synthesis and degradation

The pyrimidine analogs, 5-fluoroorotate and 5-azacytidine, have been shown to stimulate the basal level as well as the cortisone, tryptophan, and casein hydrolysate-induced levels of the rat liver enzyme, tyrosine aminotransferase. This stimulation was most marked in the case of dietary and hormonal induction when the analog was given 4–6 hr prior to the administration of the inducer. When tryptophan induced tyrosine aminotransferase, maximal stimulation by the analog occurred if it were given 2 hr prior to the administration of the amino acid. The optimal stimulatory dose of 5-azacytidine was 5 mg/kg body weight whereas 5-fluoroorotate gave its highest stimulation at a dose of 60 mg/kg. Of several orotic acid analogs tested, only the chloro-analog had an effect similar to the fluoro-congener.Utilizing quantitative immunochemical precipitation and pulse labeling in vivo, it was demonstrated that the administration of 5-fluoroorotate or 5-azacytidine at doses of 60 and 5 mg per kg, respectively, while causing a stimulation in the basal level of tyrosine aminotransferase, did not result in any change in the rate of enzyme synthesis. Furthermore, after cortisone induction of the enzyme, the delayed administration of these analogs caused either a further stimulation in the level of the enzyme or the maintenance of a high level while the enzyme activity decayed in animals not given the analogs. The rates of synthesis either showed no change or a decrease while the amount of enzyme was increasing. Prelabeling of the enzyme in vivo after induction with cortisone and followed by the administration of 5-fluoroorotate resulted in a marked decrease in the t$\text{1}\text{2}$ of the decay rate of the enzyme measured either by loss of radioactivity or by loss of enzyme activity. These studies suggest that these analogs act in some manner to prevent enzyme turnover by an inhibition of enzyme degradation.     

The role of messenger RNA in tyrosine aminotransferase superinduction: effects of camptothecin on hepatoma cells in culture

Camptothecin inhibited the hydrocortisone but not the insulin induction of tyrosine aminotransferase activity in hepatoma cells in culture. However, camptothecin did not cause “superinduction” of tyrosine aminotransferase activity even though it reportedly inhibits messenger RNA synthesis. In hydrocortisone pre-induced cultures, camptothecin treatment caused a rapid decline in tyrosine aminotransferase activity suggesting it did not block degradation of the enzyme. A comparison of actinomycin D with camptothecin indicated that some of the effects of actinomycin D on tyrosine aminotransferase activity may not be mediated through inhibition of messenger RNA synthesis.     

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.     

Influence of streptozotocin upon the induction of tyrosine aminotransferase, the content of NAD and of 5-methyl cytosine in rat liver

The antibiotic, streptozotocin, has carcinostatic, carcinogenic, and diabetogenic properties. Moreover, it is capable of inducing the enzyme tyrosine aminotransferase in a permanent line of rat liver cells. In the present publication, the effects of streptozotocin upon the induction of tyrosine aminotransferase, NAD synthesis, and methylation of DNA in different organs were analyzed in vivo. If administered alone, streptozotocin slightly induced tyrosine aminotransferase. The induction of tyrosine aminotransferase caused by tryptophan or nicotinamide was inhibited by streptozotocin. Streptozotocin reduced the NAD content of the liver. NAD synthesis induced by tryptophan was reduced by streptozotocin, while that induced by nicotinamide was enhanced. DNA methylation in the form of 5-methyl cytosine was not influenced by streptozotocin.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol L-serrine. Pyruvate aminotransferase by dibutyryl adenosine 3,5-monophosphate.

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutyryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine:pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have not effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serin aminotransferase diminishes with the age of animall. Increases in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyryl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Inhibitory effect of cortisone acetate on the stimulation of rat liver cytosol l-serine Pyruvate aminotransferase by dibutyryl adenosine 3′,5′-monophosphate

An injection of cortisone acetate at a dose of 5 mg/100 g body weight concomitant with dibutryl cyclic AMP prevents the increase in the activity of rat liver cytosol serine aminotransferase (L-serine: pyruvate aminotransferase, EC 2.6.1.51) elicited by the nucleotide with a lag of about 2 h. If the glucocorticoid is given 2 h prior to the nucleotide inducer, the lag disappears. The inhibitory effect of cortisone acetate gradually decays and is no longer detectable 12 h following its administration. Theophylline, insulin and glucose at doses which affect significantly the level of tyrosine aminotransferase, have no effect on the level of serine aminotransferase and on the cortisone inhibition. The inhibitory effect of the glucocorticoid on the dibutyryl cyclic AMP-mediated increase in serine aminotransferase diminishes with the age of animals. Increase in the enzyme activity by a single dose of glucagon can also be inhibited by cortisone acetate and actinomycin D as in the case with dibutyrl cyclic AMP as an inducer. The possibility of the existence of a specific inhibitory factor which is formed in response to cortisone acetate is discussed.     

Age-related changes of liver tyrosine aminotransferase in senescent rats

Tyrosine aminotransferase was induced in adult and senescent rat liver and its properties studied. We show the appearance of a 'cross-reacting material' for induced tyrosine aminotransferase of old rats compared to basal enzyme; this cross-reacting material can be provoked in adult rats after injection of cycloheximide, and suppressed in adult and old rats after injection of a serine protease inhibitor (tosylphenylalanine chloromethylketone). Other properties of induced tyrosine aminotransferase (thermostability, Km for tyrosine, isoelectrofocusing) are identical except for the proportion of the three forms and their sensitivity to trypsin in the absence of pyridoxal phosphate, which is increased in senescent animals. The suppression of cross-reacting material clearly indicates that it is not due to errors on old rat liver DNA but rather to post-translational modifications. This demonstrates also the role of serine proteases in tyrosine aminotransferase degradation. We suggest that induced enzyme of senescent rats would undergo a conformational change, possibly due to a release of pyridoxal phosphate from the enzymic molecules, which would thus become more susceptible to proteolytic attack than those of adult rats.     

Effects of insulin on messenger RNA activities in rat liver

Liver poly(A) RNA, isolated from adrenalectomized rats after insulin treatment, was translated in a nuclease-treated lysate of rabbit reticulocytes and quantitated for both total activity and the capacity to synthesize the insulin-inducible enzyme tyrosine aminotransferase. Analysis of the translated products from poly(A) RNA isolated 1 h after insulin treatment showed a 2.7-fold increase in activity of tyrosine aminotransferase mRNA. During the same interval, the capacity of poly(A) RNA to direct the synthesis of total protein in lysates also changed, showing a 30 to 40% increase in translational activity/unit of RNA. Increased translatability was apparent in all fractions of poly(A) RNA separated by centrifugation on sucrose gradients. Insulin thus appears to mediate a generalized change in mRNAs leading to increased capacity for translation; induction of tyrosine aminotransferase may reflect sensitivity to effect of the hormone.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Ontogeny of tyrosine aminotransferase in Xenopus laevis.

The development of tyrosine aminotransferase (TAT) activity in Xenopus laevis embryos was studied. Undivided eggs can transaminate tyrosine to some extent. The enzyme activity increases after hatching on the third day of development. In the early stages of development, the transamination of tyrosine is due to aspartate aminotransferase (ASAT, EC 2.6.1.1), both isoenzymes of which are present in the undivided egg. No specific TAT (EC 2.6.1.5) can be detected until the age of about 1 day, at which time neurulation is complete and the rapid development of the foregut and visceral pouches and arches has begun. The appearance of the enzyme is immediately preceded by a steep increase in the concentration of free tyrosine. Tyrosine aminotransferase is known to be induced by its substrate in the adult liver, and a similar effect may operate in the embryo.     

Development of tyrosine aminotransferase in perinatal rat liver: changes in functional messenger RNA and the role of inducing hormones

Expression of the hepatic enzyme tyrosine aminotransferase was analyzed in the perinatal period of development in the rat, when this expression undergoes significant changes associated with hepatocyte differentiation. In late prenatal liver both enzyme and functional mRNA gene products are present at levels 10- to 15-fold below those in the fully differentiated adult liver. This low level of expression in fetal liver is refractory to induction by glucocorticoids, but both gene products are increased to a limited extent by cyclic AMP. This induction by cyclic AMP (cAMP) does not confer glucocorticoid-responsiveness on expression. By 3 hr after birth both functional mRNA and enzyme levels are significantly increased, an increase which continues until a peak is reached at 12 hr that is appreciably above the adult levels. Both gene products then decline until adult levels are reached by 24 hr. The postnatal shift in aminotransferase expression is accompanied by acquisition of the capacity to respond to glucocorticoids. Treatment of newborns with an antiglucocorticoid steroid or with glucose suppresses the postnatal overshoot of expression, but neither treatment affects the increase from fetal to adult levels of expression. The results indicate that prior to birth, expression of the aminotransferase gene is partially repressed, a repression that is lifted essentially immediately upon birth. The hormones capable of inducing aminotransferase synthesis have no apparent necessary role in this process.