Effects of glucagon and an analogue of cAMP on tyrosine aminotransferase in isolated chick embryo hepatocytes.

Hepatocytes were isolated from 17-day-old chick embryos by the use of collagenase. Glucagon and dibutyryl cAMP (bt2cAMP), individually or in combination, stimulated tyrosine aminotransferase (TAT) activity and synthesis in the isolated hepatocytes; maximal stimulation occurred 4 h after exposure of hepatocytes to the inducers. The stimulatory effects produced by glucagon and bt2cAMP were abolished by treatment of hepatocytes with cordycepin or cycloheximide. The effects of the hormone and the cyclic nucleotide were not additive. The induction of the enzyme by glucagon suggests a physiological role for the hormone in the expression of TAT activity during chick embryonic development.     

Role of glucocorticoids and resident liver macrophages in induction of tyrosine aminotransferase

Administration of cortisol to an animal induces tyrosine aminotransferase (TAT) in the liver. A similar effect was observed after stimulation of resident liver macrophages (Kupffer cells) by dextran sulfate. Actinomycin D completely blocks enzyme induction both by cortisol and dextran sulfate, whereas their combined effect gives an additive result. In primary culture of hepatocytes, dextran sulfate inhibits TAT activity, but conditioned macrophage medium reliably increases enzyme activity in hepatocytes. However, incubation of isolated macrophages in the presence of dextran sulfate and such medium transfer into hepatocyte culture results in even more pronounced increase in TAT activity. In a combined culture of hepatocytes and non-parenchymal liver cells, reproducing intercellular interactions in vitro, cortisol and non-parenchymal cells exhibit an additive effect on TAT activity. These results show that liver macrophages release a factor of unknown nature launching the mechanism of TAT induction independently of cortisol, a classic TAT inducer.     

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.     

Differential sensitivity of HTC and Fu5-5 cells for induction of tyrosine aminotransferase by 3',5'-cyclic adenosine monophosphate

The two independently derived hepatoma cell lines (HTC and Fu5-5) have previously been shown to display different sensitivities for the induction of tyrosine aminotransferase (TAT) enzyme activity and mRNA levels by glucocorticoids with the enzyme being half-maximally induced at approximately 7-fold higher concentrations of dexamethasone in HTC cells than in Fu5-5 cells. In the present study we investigated the induction of TAT activity by cAMP in order to see whether the difference is limited to the steroidal induction. Using the stable cAMP derivative (8-(4-chlorophenylthio)-cAMP) as an inducer, we found that a 6-fold higher cAMP concentration was needed in HTC cells to achieve the same extent of enzyme induction as in Fu5-5 cells. The induction of TAT enzyme activity could be accounted for by an increased amount of TAT mRNA. Further experiments involving sequential addition of both inducers in general showed a synergism of steroids and cAMP for TAT induction in HTC cells only at submaximal concentrations of steroid; in Fu5-5 cells, the occurrence of synergism depended on the order of addition of inducers. The maximal response in HTC cells was limited to the value that could be achieved by induction with steroid alone. In Fu5-5 cells, however, the steroid response could be augmented when cAMP was added to cells already maximally induced by steroid. This demonstrates that the effect of a combination of cAMP and steroids depends on their concentration, the sequence of their addition, and the rat hepatoma cell line used. Collectively the data suggest that a common pretranslational event determines the differential sensitivity of TAT induction by glucocorticoids and by cAMP in HTC and Fu5-5 cells. Furthermore a second, or possibly the same, common event also regulates the maximum level of TAT induction that is obtainable under most conditions with glucocorticoids and/or cAMP.     

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.     

Tumor-promoting phorbol ester amplifies the inductions of tyrosine aminotransferase and ornithine decarboxylase by glucocorticoid

In adrenalectomized rats, the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the inductions of tyrosine aminotransferase (TAT) and ornithine decarboxylase by glucocorticoids, even with sufficient concentration of glucocorticoids to have a maximal effect, whereas it had no effect on TAT activity and increased ornithine decarboxylase activity only slightly in the absence of glucocorticoids. Phorbol derivatives and components of TPA such as 4 beta-phorbol, phorbol 12-tetradecanoate, phorbol 13-acetate, and 4-O-methylphorbol 12-tetradecanoate 13-acetate, which have no tumor-promoting activity or ability to activate protein kinase C, did not have any effect on TAT induction by glucocorticoid. TPA enhanced the induction of TAT by various glucocorticoids but had no effect on induction of TAT by glucagon or insulin and did not enhance the induction of glucose-6-phosphate dehydrogenase by 17 beta-estradiol. These results suggest that TPA specifically enhances the induction of TAT and ornithine decarboxylase by glucocorticoids. Similar effects of TPA on TAT induction by glucocorticoid were observed in primary cultures of adult rat hepatocytes. Another activator of protein kinase C, rac-1,2-dioctanoylglycerol, was also found to have similar effects on the cells.     

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.     

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.     

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.     

The increase in hepatic tyrosine aminotransferase activity by ethanol administration involves an acceleration of enzyme synthesis

The present study was conducted to examine the nature of the increase in tyrosine aminotransferase (TAT) activity by acute ethanol administration. A significant rise in aminotransferase activity was observed as early as 1 hr after intact rats were gavaged with ethanol. Ethanol administration also increased TAT activity in adrenalectomized rats. Inhibition of ethanol metabolism by pyrazole administration had no effect on the ethanol-induced increase in TAT activity. Immunochemical analyses revealed that the enhancement of TAT activity in ethanol-fed rats correlated with an increase in aminotransferase protein. Measurement of the rate of TAT synthesis showed that in ethanol-fed rats, [3H]leucine was incorporated into the aminotransferase protein at a higher rate than in controls by a factor which was similar to the enhancement in enzyme activity. Our findings indicate that an acceleration of TAT synthesis fully accounts for the increase in TAT activity during the early stage of enzyme induction. TAT induction by ethanol administration is not dependent upon an increase in adrenal corticosteroid production, nor does it require ethanol metabolism.     

The regulation of hepatic tyrosine aminotransferase

Tyrosine aminotransferase induction has been studied in hepatocytes from untreated, partially and fully glucocorticoid-induced rats: enzyme activities were initially 12.9 +/- 1.7 (n = 16), 41.4 +/- 3.2 (n = 6) and 117.9 +/- 10.5 (n = 7) munits/mg protein, respectively. Untreated or fully induced hepatocytes maintain initial levels, whereas partially induced hepatocytes increase their tyrosine aminotransferase activity even in the presence of actinomycin D. Fully induced hepatocytes possess a normal protein synthetizing machinery and the mechanisms to degrade selectively tyrosine aminotransferase. The effect of progesterone treatment is consistent with these cells retaining a high dexamethasone level. Glucagon induces tyrosine aminotransferase via its second messenger, cyclic AMP. This induction decreases dramatically with in vivo glucocorticoid treatment. Time courses and effects of inhibitors are consistent with these in vivo and in vitro treatments being alternative methods of inducing tyrosine aminotransferase by the same basic pretranslational step.     

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.     

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.     

Unlinked regulation of the sensitivity of primary glucocorticoid-inducible responses in mouse mammary tumor virus infected Fu5-5 rat hepatoma cells

The enzyme tyrosine aminotransferase (TAT) is induced by unusually low concentrations of glucocorticoids in Fu5-5 cells. We have isolated clones of Fu5-5 cells infected with mouse mammary tumor virus (MMTV) in order to simultaneously compare the glucocorticoid regulation of the host cell gene, TAT, with that of another primary inducible gene, MMTV. In the two clones that were examined in detail, MMTV RNA induction occurred at 4- to 11-fold higher concentrations of dexamethasone than those needed for induction of TAT mRNA. Furthermore, the amount of agonist activity displayed by the irreversible antiglucocorticoid dexamethasone 21-mesylate was greater for the induction of TAT mRNA than for MMTV RNA. These results extend our previous observations of unequal sensitivity of induction of TAT enzyme activity in two hepatoma cell lines and show that differential glucocorticoid regulation of gene induction within the same cell can occur at a pretranslational step. The present data also indicate that the unusual properties of TAT gene induction are not shared by all primary, glucocorticoid-inducible responses of the same cell and imply that additional factors mediating differential regulation of glucocorticoid-responsive genes are involved.     

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.     

Different responses of DD/He and CC57BR/Mv mice to fasting

Reaction to fasting of 2 mice strains differing in their sensitivity to spontaneous and induced hepatocarcinogenesis, has been investigated. It was shown that mice of both strains displayed similar stress reaction after 3-day fasting manifested in increase in blood corticosterone level; and decrease in testosterone level. At the same time, both strains demonstrated opposite changes at tissue- and enzyme levels in the liver. It was shown that DD/He mice, highly sensitive to induction of liver tumors, were characterized by significant increase in tyrosine aminotransferase (TAT) activity and reduction of lipid droplets in hepatocytes. CC57BR/Mv mice, demonstrating high frequency of spontaneous hepatomas and insensitive to induction of such tumors, were characterized by a decrease in the TAT activity and fatty infiltration of the liver.     

Enhancement by streptozotocin and N-methyl-N'-nitro-N-nitrosoguanidine of the tyrosine aminotransferase activity in cultured rat liver cells: role of dexamethasone and NAD

The activity of tyrosine aminotransferase (TAT) (EC 2.6.1.5) was enhanced 3-fold after a 5-h exposure of cultured rat liver cells (RLC) to streptozotocin (SZ) at concentrations higher than 100 microgram/ml (0.38 mM) in the presence of 10 nM dexamethasone, a potent glucocorticoid inducer for the enzyme. The structurally related carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) also enhanced the aminotransferase in the presence of the glucocorticoid, but its optimal concentration was at 100 ng/ml (0.68 microM). While the cellular NAD (NAD+ + NADH) concentration was reduced to 60% of the control levels, the rate of poly(ADP-ribose) formation in the isolated cell nuclei was unaffected by treating the cells with SZ. The enhancement of tyrosine aminotransferase by SZ and MNNG was effectively prevented by nicotinamide. Using nicotinamide and its derivatives such as 1-methyl-, N'-methyl- or 6-amino-derivatives it was found that the degree of enzyme induction is almost inversely proportional to the cellular NAD content, though the activity of nuclear poly(ADP-ribose)polymerase remains unchanged. The results indicate that SZ or MNNG, in combination with dexamethasone, stimulate the induction of tyrosine aminotransferase through their NAD lowering action.     

The influence of culture conditions on cell morphology and tyrosine aminotransferase levels in rat liver epithelial cell lines

Experimental conditions known to alter the shape, permeability and organization of cells were used to find out their effects on tyrosine aminotransferase (TAT) in rat liver epithelial cell lines. To produce a more spheroidal morphology for non-malignant cells than that obtainable on plastic, floating collagen and poly(2-hydroxyethylmethacrylate) (poly(HEMA)), were used as substrata. Under these experimental conditions the basal level of TAT activity increased 1.5–2.5-fold. When monolayer cultures were permeabilized by the use of a hypertonic salt solution, the basal activity increased 4–5-fold. TAT activity was also elevated in hepatoma cells cultured in anchorage-independent conditions. The enzyme was not inducible by dexamethasone (DEX) under any of these culture conditions, and the lack of induction was not due to the absence of receptors for this hormone. These studies have shown that the production of TAT, one of the characteristics of adult liver, has persisted in a number of rat epithelial cell lines derived from normal, malignant or regenerating liver and its activity was influenced by the different culture conditions employed.