Regulation of tyrosine aminotransferase messenger ribonucleic acid in rat liver. effect of cycloheximide on messenger ribonucleic acid turnover

Tyrosine aminotransferase messenger ribonucleic acid (mRNA) activity in rat liver was rapidly increased 3-6-fold following in vivo administration of hydrocortisone acetate, dibutyryladenosine cyclic 3',5'-phosphate, or the protein synthesis inhibitor cycloheximide. Treatment with the steroid hormone or cyclic nucleotide in combination with cycloheximide resulted in levels of tyrosine aminotransferase mRNA 10-20-fold greater than control values. These changes in mRNA activity were not accompanied by changes in albumin mRNA or total liver template activity. The rapid decline in tyrosine aminotransferase mRNA activity following cordycepin inhibition of de novo RNA synthesis was prevented by cycloheximide treatment. This protection was not observed when pactamycin was substituted for cycloheximide, demonstrating that the inhibition of protein synthesis per se was not responsible for the stabilization of tyrosine aminotransferase mRNA. Based upon the effects of cycloheximide and pactamycin on rat liver polysome structure, it is concluded that the cycloheximide-mediated increase in tyrosine aminotransferase mRNA activity is the result of stabilization of the mRNA molecule which renders the message less susceptible to inactivation and degradation in the cytoplasm. The action of cycloheximide is very specific for tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, and probably several other mRNAs that code for minor liver proteins that turn over rapidly in response to hormonal or metabolic stimuli.     

Regulation of hepatic tyrosine aminotransferase in genetically obese rats

The activities of hepatic tyrosine aminotransferase, tryptophan oxygenase and serine dehydratase were increased in obese rats shortly after weaning. Immunotitration experiments showed that the increase in tyrosine aminotransferase activity resulted from an increase in enzyme protein in obese rats. No increase in hepatic tyrosine aminotransferase was observed in suckling pre-obese rats. The post-weaning increase in hepatic tyrosine aminotransferase of obese rats was only observed during the light phase of the diurnal cycle, but was prevented by pair-feeding and by starvation. Tryptophan increased hepatic tyrosine aminotransferase of lean rats to obese levels but had no effect in obese rats until tyrosine aminotransferase levels were reduced by starvation or adrenalectomy. Adrenalectomy abolished the increase in hepatic tyrosine aminotransferase activity in obese rats although serum corticosterone was normal in these animals. Hepatic and brain tyrosine concentrations were decreased in obese rats but normalized after adrenalectomy. The results suggest that the corticosteroid-dependent increase in food and tryptophan intake may be the primary cause of the increased hepatic amino acid catabolism of obese rats.     

Dibutyryl cyclic AMP increases the amount of functional messenger RNA coding for tyrosine aminotransferase in rat liver.

The administration of N6,O2-dibutyryl cyclic AMP and theophylline to adrenalectomized rats results in an increase in the amount of functional mRNA coding for tyrosine aminotransferase that can be isolated from liver. The induction of this specific mRNA, as quantitated in a mRNA-dependent reticulocyte lysate system, and using poly(A)+ mRNA extracted from total tissue and polysomes, is very rapid. Within an hour after the intraperitoneal injection of the cyclic AMP derivative there is a 5- to 7-fold elevation of functional mRNA coding for tyrosine aminotransferase (mRNATAT), and by 3 h this has returned to basal levels. In contrast, the 4- to 5-fold induction of tyrosine aminotransferase catalytic activity is maximal at 2 h and is still significantly greater than the basal level at 5 h. In the basal state, tyrosine aminotransferase mRNA codes for 0.019 +/- 0.003% of the protein synthesized in the in vitro system, whereas after cyclic nucleotide treatment this value 0.115 +/- 0.015%, hence the increase in mRNATAT activity is relatively specific. Cordycepin, at a concentration which prevents the accumulation in cytoplasm of poly(A)+ mRNA, completely blocks the increase in both the catalytic and mRNA activity of this enzyme. The marked increase in functional mRNA, the requirement for continued synthesis of poly(A)+ RNA, and the rapid induction and deinduction suggest that the cyclic nucleotide is enhancing specific mRNA synthesis and/or, processing, however an effect on mRNA degradation cannot be excluded.     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

Evidence for a dual effect of dibutyryl cyclic AMP on the synthesis of tyrosine aminotransferase in rat liver

A single injection of dibutyryl cyclic AMP (Bt2cAMP) into adrenalectomized rats results in rapid and proportionate increases in hepatic tyrosine aminotransferase catalytic activity and in the amount of functional mRNA coding for this enzyme. This effect is transient in that mRNATAT peaks at 0.065% of total poly(A)+RNA activity at 1 h and is back to the basal level of 0.012% in 2.5 h. Enzyme activity peaks at 2.5 h and is back to the basal level by 5 h. If Bt2cAMP is repeatedly injected (0, 1, 2.5, and 4 h), enzyme activity remains at maximal levels for 4 to 5 h, whereas changes in mRNATAT activity are identical with those observed in the single injected rats. The rate of tyrosine aminotransferase synthesis at 5.5 h in the multiply injected rats, a time when mRNATAT has already returned to the basal level, is 3 to 4 times greater than that in either control or singly injected rats at the same time (0.3% of total protein versus 0.07%) and is equivalent to the maximal rate seen 1 h after the initial injection of the cyclic nucleotide. Since the rate of synthesis is increased in proportion to the increase in enzyme catalytic activity, stabilization of the enzyme against degradation is excluded as an induction mechanism at this late time point. These responses are not due to differences in the metabolism of Bt2cAMP, and the effect depends on the presence of metabolically active derivatives of this nucleotide. It thus appears that Bt2cAMP induces the synthesis of tyrosine aminotransferase in rat liver in two distinct ways. One is pretranslational and involves a transient and rapid increase in mRNATAT activity. The second appears to involve a delayed but sustained increase in translation of a basal level of mRNATAT.     

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.     

Regulation of the synthesis of tyrosine aminotransferase: the relationship to mRNATAT

The activity of the hepatic enzyme tyrosine aminotransferase (TAT) is the sum of many diverse regulatory factors. These include the developmental stage of the animal, the hormonal and nutritional environment of the animal (or tissue culture cell), other extrinsic and intrinsic regulatory cycles and factors (including cytoplasmic substances), and chromatin structure. Although TAT is subject to a number of post-translational modifications, alterations in catalytic activity always parallel changes in enzyme amount. In a few instances this is due to a selective change in TAT degradation, but most are due to changes in the rate of aminotransferase synthesis. Recent studies have shown that TAT synthesis is generally directly correlated with the activity, and presumably amount, of the mRNA that codes for tyrosine aminotransferase.     

Effect of high- and low-protein diets on the regulation of rat liver tyrosine aminotransferase and tryptophan oxygenase activities by l-tyrosine and l-tryptophan

Induction of rat liver tyrosine aminotransferase by l-tyrosine and tryptophan oxygenase by l-tryptophan was studied in groups of rats fed on diets containing 18 or 5% protein. The basal activity of hepatic tyrosine aminotransferase of rats receiving 5% protein gradually increased with the age of the animals but that of rats receiving 18% protein did not. l-Tyrosine induced hepatic tyrosine aminotransferase in rats receiving 18% protein when tested at ages from 4 to 20 weeks. When induction by l-tyrosine was carried out in rats receiving the 5% protein diet, significant induction of tyrosine aminotransferase occurred only in 4- or 6-week-old rats. Induction by l-tryptophan of tryptophan oxygenase in liver or the basal activity of this enzyme in liver did not differ between the groups fed on 5 and 18% protein. On changing the diet from 0 to 18% protein, the above-mentioned effects on the induction of hepatic tyrosine aminotransferase were reversed.     

Translation of mRNA from rat-liver polysomes into tyrosine aminotransferase and tryptophan oxygenase in a protein-synthesizing system from wheat germ. Effects of cortisol on the translatable levels of mRNA for these two enzymes.

Messenger RNA was isolated from rat liver polysomes by phenol/chloroform extraction and subsequent oligo(dT)-cellulose chromatography. The mRNA was translated in a protein-synthesizing system in vitro derived from wheat germ. The system was optimized in respect to Mg2+ and K+. The presence of spermidine or spermine is necessary for the synthesis of polypeptides having molecular weights of over 20 000. In the absence of the bases only small molecular weight products are formed. The amount of protein synthesized is linearly dependent on the amount of mRNA added up to concentrations of 80 mug mRNA/ml. The synthesis of tyrosine aminotransferase and tryptophan oxygenase in the system in vitro has been demonstrated by specific immunoprecipitation and sodium-dodecylsulfate polyacrylamide gel electrophoresis of the precipitate with enzyme proteins as marker. The amount of specific product formed is linearly dependent on the amount of mRNA present. The amount of translatable tyrosine aminotransferase mRNA and tryptophan oxygenase mRNA increases after administration of hydrocortisone to adrenalectomized rats. At low doses of hormone (2 mg/100 g body weight) maximal values are observed at 4 h, control levels being reached at 6-8 h after hormone application. With higher doses of hydrocortisone (20 mg/100 g body weight) maximal values are attained at 6 h, tending to control levels 14 h after treatment. The enzyme activity curves are parallel to the mRNA curves, the peak of enzyme activity occurring 2 h after the peak of mRNA activity.     

The effect of stress or glucose feeding on hepatic tyrosine aminotransferase activity and liver and plasma tyrosine level of intact and adrenalectomized rats.

The increase of hepatic tyrosine aminotransferase and the fall of plasma tyrosine in rats subjected to immobilization is reconfirmed. Moreover, the same effects three hrs after exposuing the animals to 400 revolutions in Noble-Collip drums are described. However, in bilaterally adrenalectomized rats both hepatic tyrosine aminotransferase and plasma tyrosine remain unchanged after injury and the liver tyrosine level increase. Finally, in animals fed overnight exclusively with 15% glucose solution the well-known decrease of hepatic tyrosine aminotransferase was found paralleled by increased plasma tyrosine levels. A regulatory role of tyrosine aminotransferase in establishing the level of tyrosine in plasma is suggested.     

Catalytic subunit of cAMP-dependent protein kinase is essential for cAMP-mediated mammalian gene expression

Cyclic AMP-stimulated mRNA levels in cultured rat hepatocytes were inhibited by three different inhibitors of cAMP-dependent protein kinase activity: (i) Rp-cAMPS, a cAMP analog with a sulfur substitution at the equatorial oxygen of the cyclic monophosphate; (ii) H8, an isoquinoline sulfonamide derivative; and (iii) PKI, a 20-amino acid synthetic peptide of the Walsh protein kinase inhibitor. These inhibitors specifically blocked the cAMP-stimulated increase in mRNA for tyrosine aminotransferase and phosphoenolpyruvate carboxykinase; they had no effect on the level of albumin mRNA which is not cAMP regulated. These results provide functional evidence that kinase activity involving protein phosphorylation is required in cAMP-mediated gene expression in mammalian cells.     

Effect of cortisol on the content of mRNA coding inducible tyrosine aminotransferase isoenzyme in rat liver]

In vitro estimation of synthesis of inducible tyrosine aminotransferase isoenzyme, directed by poly-A-containing RNA from liver of intact and corticol treated rats, is carried out. Total poly-A-containing RNA from liver polyribosomes of intact and induced rats was translated in cell-free system from wheat germs. Two antibodies immunoprecipitation was used to identify the translocation product (tyrosine aminotransferase). It was found that a synthesis of a specific protein product, precipitated by antibodies to tyrosine aminotransferase, takes place in cell-free system under translation of polysomic poly-A-containing liver RNA. The amount of immunoprecipitated product indicates, that the content of individual poly-A-containing mRNA for inducible tyrosine aminotransferase isoenzyme in liver of cortisol-induced rats is considerably higher than in intact animals.     

Pretranslational control of tyrosine aminotransferase synthesis by 8-bromo-cyclic AMP in H-4 rat hepatoma cells

The H-4 rat hepatoma cell line grown in tissue culture was used as a model system to investigate the action of cAMP in tyrosine aminotransferase induction. An immunoprecipitation technique was used to quantitate the amount and the rate of synthesis of tyrosine aminotransferase; the level of mRNA coding for tyrosine aminotransferase was determined by in vitro translation of poly(A)+ RNA isolated from hepatoma cells. Our results demonstrated that 8-bromo-cAMP gave time-dependent and proportionate increases in the tyrosine aminotransferase activity, the amount of immunoprecipitable tyrosine aminotransferase, the rate of synthesis of tyrosine aminotransferase, and the level of mRNATAT in H-4 hepatoma cells. The time course of increase in mRNATAT preceded the increase in synthesis of tyrosine aminotransferase and was dependent on the continuous production of poly(A)+ RNA. Pretreatment of the cells with cordycepin completely abolished the 8-bromo-cAMP-evoked increase in mRNATAT activity. These results provided evidence that the primary action of cAMP in tyrosine aminotransferase induction is the increase of functional mRNATAT and that this increase can completely account for the increase in tyrosine aminotransferase activity.     

Involvement of tyrosine kinase and cAMP in growth hormone-induced vitellogenin synthesis in the anuran, Rana esculenta

In the frog Rana esculenta, a multihormonal control of vitellogenin synthesis was previously demonstrated. Now in this study, the identity of intracellular second messengers that mediate the GH effects on hepatic VTG synthesis are described. The results clearly indicate that the effect of GH on frog hepatocytes, in vitro, works through a local production of IGF I; in fact, IGF I affects VTG synthesis and its action occurs faster with respect to that of GH. The effect of IGF I was abolished by the anti-estrogen tamoxifen, indicating the involvement of estrogen receptor in VTG induction by IGF I. Furthermore, in vitro treatment of frog hepatocytes with GH resulted in an increase of cAMP with maximum levels after 20 min of treatment. Besides the increase of cAMP, GH induced the appearance of a new phosphotyrosine protein at 20 min, suggesting the occurrence of tyrosine kinase activation. Addition of adenylate cyclase or protein tyrosine kinase inhibitors completely abolished the induction of VTG synthesis, indicating the involvement of cAMP and of a phosphotyrosine protein in VTG synthesis stimulated by both GH and IGF I.