Regulation of tyrosine aminotransferase in foetal rat liver.

A specific tyrosine aminotransferase, separate from the aspartate aminotransferases, is present in low concentration in foetal rat liver at the 21st day of gestation. Intraperitoneal injections of tyrosine methyl ester into the foetuses in utero increase the activity 2-fold, whereas glucose injections decrease it. Tyrosine, dexamethasone and dibutyryl cyclic AMP induce the enzyme activity in organ culture to the same extent as in adult rat liver in vivo.     

Synthesis and secretion of albumin and transferrin by foetal rat hepatocyte cultures

Hepatocytes derived from foetal rat liver synthesize and secrete albumin and transferrin when maintained in primary culture. These proteins are produced for at least seven days under the conditions of culture. Studies on hepatocyte cultures derived from 12, 13, 14, 15 and 19-day foetal rats show that the maximal cellular rate of secretion of both proteins increases about 50-fold over this period. The maximal rate of albumin secretion in all cultures is achieved after one day in culture and decreases in hepatocytes from early foetuses after the fourth to sixth day in culture. Transferrin secretion by hepatocytes from 12 to 15 day foetuses increases markedly during the second day of culture and is relatively constant thereafter. In contrast, secretion of transferrin by hepatocytes from 19-day foetuses is constant from the first day of culture. The results show that both albumin and transferrin are synthesized and secreted by the foetal liver as early as the twelfth day of gestation. The increase in the rate of transferrin secretion that occurs during culture of hepatocytes from 12 to 15 day foetuses may reflect the development of a secretory mechanism that is different from that for albumin.     

Glucagon-induced changes in fructose 2,6-bisphosphate and 6-phosphofructo-2-kinase in cultured rat foetal hepatocytes.

The sensitivity of 6-phosphofructo-2-kinase to glucagon and cyclic AMP was studied during the perinatal period. In liver homogenates from foetal and neonatal rats, incubation with cyclic AMP produced inactivation of 6-phosphofructo-2-kinase 3 h after birth. The maximal effect was obtained 12 h after birth. In primary cultures of hepatocytes from 22-day-old foetuses, glucogon induced an inhibition of 6-phosphofructo-2-kinase that required 45 min to reach the half-maximal effect. Cycloheximide prevented the glucagon-induced changes in this activity from cultured foetal hepatocytes. These results suggest that the adult form of 6-phosphofructo-2-kinase is rapidly induced after birth, probably by the hormonal changes that occur in this period.     

Hormonal inducibility of liver-specific enzymes in cultured rat embryos

In monolayer cultures, hepatocyte-specific enzymes are inducible by hormones as soon as hepatocytes differentiate from the embryonic foregut (15-somite stage). Though offering an excellent opportunity for quantitative studies, several features of a normal cell environment are lost in such a model system. To determine the inducibility of such tissue-specific enzymes in intact organisms, rat embryos were cultured in vitro for 48 h and exposed to the hormonal factors that had been found effective in monolayer culture, viz. dexamethasone, triiodothyronine and dibutyryl cyclic AMP. Normal development of the embryos during culture in vitro was assessed by general criteria reflecting growth, morphogenesis and cytodifferentiation. Development of external features, organogenesis, the distribution of cell divisions and the appearance of tissue-specific proteins such as alpha-fetoprotein and glutamate dehydrogenase served as parameters. Despite undisturbed development of the embryos as judged by these criteria, irrespective of whether the culture was started at day 10 or at day 11 of gestation (just before, respectively after the appearance of the liver primordium), induction of hepatocyte-specific enzymes like carbamoylphosphate synthetase by hormones could not be demonstrated immunohistochemically. However, induction of this enzyme by hormones could be demonstrated in monolayers of hepatocytes isolated from such embryos after 48 h of culture, providing yet another demonstration of the adequate culture conditions. In addition, an adequate uptake of hormones by the embryo during culture could be shown with radio-actively labeled dexamethasone and triiodothyronine and with a radioreceptor assay for cyclic AMP. Therefore, the presence of factors in young embryos that inhibit tissue-specific enzyme synthesis has to be postulated.     

Induction of alkaline phosphatase in choriocarcinoma cells by 1-beta-D-arabinofuranosyl-cytosine, mitomycin C, phleomycin, and cyclic nucleotides.

Alkaline phosphatase is induced in cultured human choriocarcinoma cells by three inhibitors of DNA synthesis which alter DNA structure: 1-β-D-arabinofuranosyl-cytosine, mitomycin C, and phleomycin. No induction is observed with the inhibitors, hydroxyurea and thymidine, which do not alter DNA structure. Cyclic AMP, analogs of cyclic nucleotides, and sodium butyrate also induce alkaline phosphatase in these cells. Among the cyclic nucleotides tested, dibutyryl cyclic AMP is the best inducer, whereas dibutyryl cyclic GMP is a poor inducer. Induction of alkaline phosphatase by inhibitors of DNA synthesis or by exposure to dibutyryl cyclic AMP appears to utilize different mechanisms. Maximum induction is observed after simultaneous addition of both types of inducers at the concentrations found to be optimal for each inducer alone. Under these conditions, the induced activity is equal to or greater than the sum of the activities induced by each inducer. RNA synthesis and protein synthesis are required for induction. Dibutyryl cyclic AMP added to cultures of choriocarcinoma cells is not degraded in the culture medium, but is extensively degraded in the cells. Nevertheless, significant amounts of dibutyryl and monobutyryl cyclic AMP are found intracellularly throughout the experiment. Since the cellular uptake of dibutyryl cyclic AMP is extremely slow, the amount of butyrate released by intracellular degradation cannot account for the observed induction. Neither the rate of uptake nor the stability of dibutyryl cyclic AMP are changed by the addition of 1-β-D-arabinofuranosyl-cytosine to the culture medium. Furthermore, 1-β-D-arabinofuranosyl-cytosine inhibits the induction by sodium butyrate. The results indicate that butyrate is not the major mediator of induction by dibutyryl cyclic AMP.     

Co-ordinate induction of hepatic mitochondrial and peroxisomal carnitine acyltransferase synthesis by diet and drugs.

The effects of pancreatic hormones and cyclic AMP on the induction of ketogenesis and long-chain fatty acid oxidation were studied in primary cultures of hepatocytes from fetal and newborn rabbits. Hepatocytes were cultivated during 4 days in the presence of glucagon (10(-6) M), forskolin (2 x 10(-5) M), dibutyryl cyclic AMP (10(-4) M), 8-bromo cyclic AMP (10(-4) M) or insulin (10(-7) M). Ketogenesis and fatty acid metabolism were measured using [1-14C]oleate (0.5 mM). In hepatocytes from fetuses at term, the rate of ketogenesis remained very low during the 4 days of culture. In hepatocytes from 24-h-old newborn, the rate of ketogenesis was high during the first 48 h of culture and then rapidly decreased to reach a low value similar to that measured in cultured hepatocytes from term fetuses. A 48 h exposure to glucagon, forskolin or cyclic AMP derivatives is necessary to induce ketone body production in cultured fetal hepatocytes at a rate similar to that found in cultured hepatocytes from newborn rabbits. In fetal liver cells, the induction of ketogenesis by glucagon or cyclic AMP results from changes in the partitioning of long-chain fatty acid from esterification towards oxidation. Indeed, glucagon, forskolin and cyclic AMP enhance oleate oxidation (basal, 12.7 +/- 1.6; glucagon, 50.0 +/- 5.5; forskolin, 70.6 +/- 5.4; cyclic AMP, 77.5 +/- 3.4% of oleate metabolized) at the expense of oleate esterification. In cultured fetal hepatocytes, the rate of fatty acid oxidation in the presence of cyclic AMP is similar to the rate of oleate oxidation present at the time of plating (85.1 +/- 2.6% of oleate metabolized) in newborn rabbit hepatocytes. In hepatocytes from term fetuses, the presence of insulin antagonizes in a dose-dependent fashion the glucagon-induced oleate oxidation. Neither glucagon nor cyclic AMP affect the activity of carnitine palmitoyltransferase I (CPT I). The malonyl-CoA concentration inducing 50% inhibition of CPT I (IC50) is 14-fold higher in mitochondria isolated from cultured newborn hepatocytes (0.95 microM) compared with fetal hepatocytes (0.07 microM), indicating that the sensitivity of CPT I decreases markedly in the first 24 h after birth. The addition of glucagon or cyclic AMP into cultured fetal hepatocytes decreased by 80% and 90% respectively the sensitivity of CPT I to malonyl-CoA inhibition. In the presence of cyclic AMP, the sensitivity of CPT I to malonyl-CoA inhibition in cultured fetal hepatocytes is very similar to that measured in cultured hepatocytes from 24-h-old newborns.     

Influence of glucagon, 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate and triamcinolone on the arginine synthetase system in perinatal rat liver

1. The administration of triamcinolone (19-190mug/animal) to postnatal rats increased the arginine synthetase system activity 1.2-2.5-fold above control values 24h after exposure to the hormone. Cortisol (hydrocortisone), however, increased the arginine synthetase system activity only when larger (190mug/animal) or repeated daily doses were given. Glucagon (100mug/animal) stimulated arginine synthetase system activity only after the second postnatal day. None of these agents increased the activity in 19.5-21.5-day foetuses after intrauterine administration. 2. The viability of foetal rat liver explants maintained in organ culture for up to 54h was validated both by ultramicroscopic examination and by incorporation of radioactive leucine and orotic acid. 3. In organ cultures of foetal rat liver explants (18.5 days to term), triamcinolone (20mug/ml of medium) evoked a 2.8-4.3-fold increase after 24h of incubation. This increase was completely inhibited by actinomycin D (25mug/ml) or cycloheximide (10mug/ml). Cortisol (5-50mug/ml) or glucagon (0.067-67mug/ml) also increased the arginine synthetase system activity above the respective control values, but there was no increase in activity with insulin (0.05-0.25i.u./ml). 4. Maximum concentrations of glucagon (67mug/ml), dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.1mm) and triamcinolone (20mug/ml) incubated for 24h with foetal rat liver explants each produced between a two-and three-fold increase in the activity of the arginine synthetase system. Combinations of maximum amounts of glucagon and the cyclic nucleotide did not produce a greater effect than either agent alone. However, the combination of dibutyryl cyclic AMP with triamcinolone appeared to produce somewhat less than additive effects. 5. The effects of the cyclic nucleotide and triamcinolone were evident after 12h of incubation and increased steadily throughout the 24h of observation. This time-course of increased enzyme activity is very much slower than that reported for the induction of other enzymes in explant cultures of foetal rat liver.     

Hormonal regulation of four urea cycle enzymes in postnatal rat liver in organ culture

The administration of hydrocortisone to 3- to 15-day-old rats increased the levels of hepatic argininosuccinate synthetase (ASS) and arginase. In 13-day-old rat liver explants maintained in organ culture, ornithine carbamoyltransferase (OTC), carbamoylphosphate synthetase (CPS) and arginase were stimulated by betamethasone. Actinomycin D prevented the responses of the latter two enzymes. Dibutyryl cyclic AMP raised OTC, CPS, ASS and arginase in vitro. The responses of the latter three enzymes were blocked by cycloheximide and puromycin and partially inhibited by actinomycin D. The simultaneous presence of betamethasone and dibutyryl cyclic AMP in the culture medium raised CPS and OTC in an additive manner. The sequential treatment of the cultures with betamethasone followed by dibutyryl cyclic AMP increased CPS and arginase synergistically and amplified the response of ASS to dibutyryl cyclic AMP.     

Changes in enzyme patterns produced by high potassium concentration and dibutyryl cyclic AMP in organ cultures of sympathetic ganglia

—Preliminary experiments had shown that acetylcholine, the putative mediator of trans-synaptic induction of tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) in vivo, did not lead to an increase in these enzyme activities in mouse superior cervical ganglia kept in organ culture. It was the aim of the present study to evaluate whether increases in tyrosine hydroxylase and dopamine β-hydroxylase evoked by other stimuli such as potassium or dibutyryl cyclic AMP in such an in vitro system are representative for in vivo trans-synaptic induction where changes in the levels of enzymes involved in norepinephrine synthesis or degradation are strictly confined to TH and DBH. In the presence of elevated concentrations of potassium or 5 mm dibutyryl cyclic AMP under organ culture conditions TH and DBH as well as DOPA decarboxylase and monoamine oxidase were significantly (P < 0.025) increased. The increase in total activities of TH and DBH were completely, those of DOPA decarboxylase and monoamine oxidase partially, inhibited by cycloheximide. In the presence of high concentrations of potassium, the total protein content of the ganglia was 28 per cent higher than in culture controls while dibutyryl cyclic AMP had no significant effect. Cycloheximide alone caused the protein content to fall to 70 per cent of that in control cultures. The loss of protein in the presence of cycloheximide was not accompanied by a simultaneous loss of TH, DOPA decarboxylase or monoamine oxidase, but DBH was decreased. Potassium was shown to increase the incorporation of [³H]leucine into TCA-insoluble protein during an early culture period but dibutyryl cyclic AMP showed no such effect. An increase in the rate of incorporation of [³H]leucine into protein was seen in both the control and elevated potassium cultures after 48 h. This increase did not occur in the presence of dbcAMP. The difference in enzyme patterns under conditions of elevated potassium and dibutyryl cyclic AMP and the fact that no changes in the levels of endogenous cyclic AMP were observed during exposure to 54 mm -potassium for a time period sufficient to initiate changes ultimately leading to elevated TH levels argues against the mediation of the potassium-induced enzyme increases by cAMP. Since changes in enzyme patterns caused by potassium and dbcAMP were not similar to patterns seen in vivo under conditions of trans-synaptic induction we conclude that use of this system as an in vitro model for in vivo trans-synaptic induction necessitates great caution.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

Occurrence of phenolsulfotransferase in primary glial culture cells of rat

Phenolsulfotransferase (PST) activity towards phenol and monoamines was determined in rat brain and in primary cultures of rat astrocytes. The pH requirement.K _m values and the proportion of PST activity with respect to phenol and dopamine as substrates were similar between PST from the glial cells and the rat cortex. The enzyme activity increased with age in the brain of older animals, and also with increasing incubation time in the primary culture of astroglia. The specific PST activity of the astroglia appeared to be higher than that of the brain enzyme. In glial cultures treated with 0.25 mM dibutyryl cyclic AMP in the same culture conditions, PST activity is suppressed to about 25% of its untreated counterpart, even though dibutyryl cyclic AMP at concentrations of 1 mM only slightly inhibited PST activity in vitro.     

Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine

We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells. Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes. Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity. Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values. Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value. After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen. In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250%. 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled. The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol. In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity. The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol. On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures. It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.     

Hepatocyte differentiation in culture. Appearance of tyrosine aminotransferase.

Liver of rat foetuses from 14 to 19 days of gestation and cultured hepatocytes derived from foetuses of 14 or 15 days gestation show a limited capacity to transaminate tyrosine. This low tyrosine transamination activity can be ascribed to aspartate aminotransferase. Definitive tyrosine aminotransferase can be demonstrated in 1-day-old cultures of hepatocytes taken from 19-day foetuses, but not from 15-day foetuses. However, after 3 days of culture hepatocytes from 15-day foetuses are able to synthesize tyrosine aminotransferase. Induction studies reveal that dexamethasone is capable of increasing tyrosine aminotransferase activity once it is detectable in culture.     

Regulation of ornithine decarboxylase activity by amino acids, cyclic AMP and luteinizing hormone in cultured mammalian cells

Any one of five amino acis (alanine, asparagine, glutamine, glycine, and serine) is an essential requirement for the induction of ornithine decarboxylase (EC 4.1.1.17) in cultured chinese hamster ovary (CHO) cells maintained with a salts/glucose, medium. Each of these amino acids induced a striking activation of ornithine decarboxylase in the presence of dibutyryl cyclic AMP and luteinizing hormone. The effect of the other amino acids was considerably less or negligible. The active amino acids at optimal concentrations (10 mM) induced only a 10-20 fold enhancement of enzyme activity alone, while in the presence of dibutyryl cyclic AMP, ornithine decarboxylase activity was increased 40-50 fold within 7-8 h. Of the hormones and drugs tested, luteinizing hormone resulted in the highest (300-500 fold) induction of ornithine decarboxylase with optimal concentrations of dibutyryl cyclic AMP and asparagnine. Omission of dibutyryl cyclic AMP reduced this maximal activation to one half while optimal levels of luteinizing hormone alone caused no enhancement of ornithine decarboxylase activity. The induction of ornithine decarboxylase elicited by dibutyryl cyclic AMP, amino acid and luteinizing hormone was diminished about 50% with inhibitors of RNA and protein synthesis. The specific amino acid requirements for ornithine decarboxylase induction in chinese hamster ovary cells was similar to the requirements for induction in two other transformed cell lines. Understanding the mechanism of enzyme induction requires an identification of the essential components of the regulatory system. The essential requirement for enzyme induction is one of five amino acids. The induction of ornithine decarboxylase by dibutyryl cyclic AMP and luteinizing hormone was additive in the presence of an active amino acid.     

Hormonal regulation of glycogen metabolism in neonatal rat liver

1. The development of active and inactive phosphorylase was determined in rat liver during the perinatal period. No inactive form could be found in tissues from animals less than 19 days gestation or older than the fifth postnatal day. 2. The regulation of phosphorylase in organ cultures of foetal rat liver was examined. None of the agents examined [glucagon, insulin or dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate)] changed the amount of phosphorylase activity. 3. Glycogen concentration in these explants were nevertheless decreased more than twofold by 4h of incubation with glucagon or dibutyryl cyclic AMP. Incubation with insulin for 4h increased the glycogen content twofold. 4. Glycogen synthetase activity was examined in these explants. I-form activity (without glucose 6-phosphate) was found to decrease by a factor of two after 4h of incubation with dibutyryl cyclic AMP, whereas I+D activity (with glucose 6-phosphate) remained nearly constant. Incubation for 4h with insulin increased I-form activity threefold, with only a slight increase in I+D activity. 5. When explants were incubated with insulin followed by addition of dibutyryl cyclic AMP, the effects of insulin on glycogen concentration and glycogen synthetase activity were reversed. 6. These results indicate that the regulation of glycogen synthesis may be the major factor in the hormonal control of glycogen metabolism in neonatal rat liver.     

Effect of dibutyryl cyclic AMP and dexamethasone on glutamine synthetase gene expression in rat astrocytes in culture

Astrocytes are the primary site of glutamate conversion to glutamine in the brain. We examined the effects of treatment with either dibutyryl cyclic AMP and/or the synthetic glucocorticoid dexamethasone on glutamine synthetase enzyme activity and steady-state mRNA levels in cultured neonatal rat astrocytes. Treatment of cultures with dibutyryl cyclic AMP alone (0.25 mM–1.0 mM) increased glutamine synthetase activity and steady state mRNA levels in a dose-dependent manner. Similarly, treatment with dexamethasone alone (10^–7–10^–5 M) increased glutamine synthetase mRNA levels and enzyme activity. When astrocytes were treated with both effectors, additive increases in glutamine synthetase activity and mRNA were obtained. However, the additive effects were observed only when the effect of dibutyryl cyclic AMP alone was not maximal. These findings suggest that the actions of these effectors are mediated at the level of mRNA accumulation. The induction of glutamine synthetase mRNA by dibutyryl cyclic AMP was dependent on protein synthesis while the dexamethasone effect was not. Glucocorticoids and cyclic AMP are known to exert their effects on gene expression by different molecular mechanisms. Possible crosstalk between these effector pathways may occur in regulation of astrocyte glutamine synthetase expression.Abbreviations used GS glutamine synthetase - dbcAMP dibutyryl cyclic AMP - MEM minimal essential medium - cyx cycloheximide - GRE glucocorticoid response element - CRE cyclic AMP response element