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.     

Quantification of the importance of individual steps in the control of aromatic amino acid metabolism.

The quantitative importance of the individual steps of aromatic amino acid metabolism in rat liver was determined by calculation of the respective Control Coefficients (Strengths). The Control Coefficient of tryptophan 2,3-dioxygenase for tryptophan degradation was determined in a variety of physiological conditions and with a range of activities of tryptophan 2,3-dioxygenase. The Control Coefficient varied from 0.75 with basal enzyme activity to 0.25 after maximal induction of the enzyme by dexamethasone. The remainder of the control for tryptophan degradation was associated with the transport of the amino acid across the plasma membrane, with only very small contributions from kynureninase and kynurenine hydroxylase. The Control Coefficients of tyrosine aminotransferase for tyrosine degradation were approx. 0.70 and 0.20 with basal and dexamethasone-induced tyrosine aminotransferase activities respectively; the Control Coefficients of the transport of the amino acid into the cell were 0.22 and 0.58 respectively. Phenylalanine hydroxylase was found to have a Control Coefficient for the degradation of phenylalanine of approx. 0.50 under conditions of basal enzyme activity; after maximal activation by glucagon, the Control Coefficient decreased to 0.12. The transport of phenylalanine was responsible for the remaining control in the pathway. These results have important implications, directly for the regulation of aromatic amino acid metabolism in the liver, and indirectly for the regulation of neuroamine synthesis in the brain.     

Regulation of the expression of the serine dehydratase gene in the kidney and liver of the rat

Serine dehydratase was induced in the kidneys of normal rats by the administration of either glucagon or dexamethasone. The increase in enzyme activity was associated with an increase in both enzyme protein and its mRNA, which were determined respectively by Western blot and RNA blot analysis. No apparent differences were observed between kidney and liver in the molecular weights of serine dehydratase proteins and the sizes of their mRNAs. Although kidney serine dehydratase was dramatically induced by either glucagon or dexamethasone, the liver enzyme was induced by glucagon but not by dexamethasone alone in the intact rat. On the other hand, liver serine dehydratase was induced in starvation, diabetes mellitus, and a high-protein diet. The kidney enzyme could not be induced under any of these conditions.     

The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.     

Induction of tryptophan 2,3-dioxygenase in the mouse endometrium during implantation

Indoleamine 2,3-dioxygenase (IDO) is expressed in trophoblasts and defends the conceptus against rejection by reducing the tryptophan level and suppressing the T cell activity. We isolated a cDNA for tryptophan 2,3-dioxygenase (TDO), another key catabolizing enzyme of tryptophan, from a mouse uterus cDNA library enriched with pregnancy-induced genes. Northern blot and in situ hybridization analyses demonstrated that the TDO mRNA was induced in the decidualized stromal cells around the implanted embryo at the time of implantation. The expression was then upregulated and primarily localized at the mesometrial decidua. TDO mRNA was induced by deciduoma formation as well as embryo transfer but not by ovarian steroid hormones. These findings demonstrated that TDO is induced in the endometrial stromal cells concomitant with decidualization and suggested its involvement in the implantation process by regulating the tryptophan level at the implantation site.     

Induction of cytosolic aspartate aminotransferase by glucagon in primary cultured rat hepatocytes

The activity and the mRNA content of cytosolic aspartate aminotransferase (EC 2.6.1.1) were examined in cultured rat hepatocytes. Addition of glucagon (1 x 10(-7) M) in the presence of dexamethasone (1 x 10(-7) M) caused about 2-fold increase in the activity and mRNA content. Dibutyryl cAMP (1 x 10(-4) M) could replace glucagon for this effect. Maximal induction of cytosolic aspartate aminotransferase mRNA was observed 8 h after their additions. Insulin (1 x 10(-7) M) did not inhibit the enzyme induction by glucagon or dibutyryl cAMP. These results suggest that the cytosolic aspartate aminotransferase gene is regulated by cAMP, and not by insulin.     

Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells

Acute hormonal effects on the synthesis rate of the cytosolic form of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (GTP), were investigated using rat hepatocytes maintained in short-term suspension culture. Cells were pulse-labeled with [3H]leucine or [35S]methionine and the rate of synthesis of phosphoenolpyruvate carboxykinase was estimated after immunoprecipitation of cell extracts with specific antibodies or following high-resolution two-dimensional gel electrophoresis of cell proteins. Total RNA was also extracted from cultured cells and subsequently translated in a wheat germ cell-free protein-synthesis system, in order to quantify the level of functional mRNA coding for phosphoenolpyruvate carboxykinase. Glucagon, the single most effective inducer, causes a 15--20-fold increase in the level of specific mRNA in 2 h, accompanied by a similar increase in enzyme synthesis rate. The extent of induction is further amplified about threefold when dexamethasone is added to the culture medium. The synergistic action of dexamethasone does not require pre-exposure of the cells to the glucocorticoid, but on the contrary occurs without lag upon simultaneous addition of glucagon and dexamethasone. The induction of phosphoenolpyruvate carboxykinase mRNA by glucagon is markedly depressed in hepatocytes inhibited for protein synthesis by cycloheximide. Cycloheximide-inhibited cells, however, display a considerable induction of the message after joint stimulation with dexamethasone and glucagon. Thus, the synergistic action of dexamethasone does not require concomitant protein synthesis. These data provide indirect evidence for a primary effect of the glucocorticoids on the expression of the phosphoenolpyruvate carboxykinase gene. Besides glucagon and dexamethasone, the thyroid hormones are shown to influence the rate of phosphoenolpyruvate carboxykinase synthesis in isolated liver cells. The stimulatory effect of 3,5,3'-triiodothyronine (T3) is best demonstrated as a twofold increase in relative rate of enzyme synthesis in cells supplied with T3 plus glucagon, as compared to cells challenged with glucagon alone. The effect of T3 relies on a pretranslational mechanism, as shown by a commensurate increase in functional mRNA coding for phosphoenolpyruvate carboxykinase. Dose-response experiments with T3 as well as dexamethasone demonstrate effects at very low hormone levels, consistent with a role for these hormones as physiological modulators of phosphoenolpyruvate carboxykinase expression.     

Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice

Indoleamine 2,3-dioxygenase activity was found to be ubiquitously distributed in various tissues of mice, such as brain, lung, stomach, intestine, and epididymis. The highest enzyme activity was detected in the alimentary canal and the epididymis. Developmental and daily rhythmic changes of indoleamine 2,3-dioxygenase activity and the effects of various regulatory factors were studied with the supernatant fractions derived from the small intestine and the epididymis. The enzyme activity in these two tissues was absent during the first 2 weeks (the weaning period). From the third week, there was a rapid increase in activities and a maximum was reached when the mice were 8 to 10 weeks of age (adolescence). The enzyme activity in the small intestine then gradually diminished to zero level at 30 weeks of age (prime) or later, while that in the epididymis remained at the high level throughout 69 weeks of age (senescence). The enzyme activity of the small intestine from mice fed during the hours 9:00–13:00 showed daily rhythmic changes; high in the daytime and low at night. Under night feeding (21:00–1:00), the enzyme activity was high at night and low in the daytime. The epididymal enzyme activity showed no daily fluctuations by either feeding schedule. With regard to the developmental and daily rhythmic changes, indoleamine 2,3-dioxygenase activity in the small intestine was similar to that of hepatic tryptophan 2,3-dioxygenase. However, in contrast to the hepatic tryptophan 2,3-dioxygenase activity, indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis was not affected by adrenalectomy or intraperitoneal administration of adrenal steroid or tryptophan.     

The stimulating effect of cyclic AMP, glucagon and insulin on guanidine acetate-N-methyltransferase activity in rat liver and pancreas]

N6,O2'-dibutyrylcyclo-3',5'-AMP injected to intact rats alone or in combination with theophylline increases the activity of guanidine acetate methyltransferase (GAMT) in liver and pancreas. Cyclic 3',5'-AMP and its dibutyryl analog administered immediately or two hours after the suturing of common bile duct (SCBD) stimulate the increase of pancreatic GAMT activity 2-3 fold. Glucagon, injected intraabdominally simultaneously with SCBD and administration of theophylline, dramatically increases the theophylline effect on the GAMT activity. The freezing of rat pancreas pretreated witn secretin, a hormone structurally similar to glucagon, results in a 1.5-2-fold increase of creatine synthesis from S-adenosylmethionine and guanidinacetic acid. An hour after glucagon administration to intact rats the GAMT activity of liver increases 9 times. The effect of glucagon is enhanced by insulin. Cycloheximide inhibits the increase of GAMT activity, induced by glucagon or a combination of glucagon and insulin. Experiments on tissue homogenates demonstrate that 3',5'-AMP in concentrations of 10(-8) --10(-2) M does not affect the GAMT activity or to some extent inhibits the enzyme. The homogenate incubation in a medium containing 10(-5) M epinephrine or 10(-7) M caffeine and 5 mM Mg2+ leads to an increase in the GAMT activity. Oligomycin removes the stimulating effects of caffeine and Mg2+ on the enzyme activation. This is probably due to the presence of 3',5'-AMP-dependent protein kinase in the mechanism of GAMT activation by cyclic AMP.     

Expression of carbamoyl-phosphate synthetase I mRNA in Reuber hepatoma H-35 cells. Regulation by glucocorticoid and insulin

Reuber hepatoma H-35 cells actively synthesize the urea cycle enzyme, carbamoyl-phosphate synthetase I. Treatment of H-35 cells with dexamethasone (0.14 microM), however, enhanced synthesis of the enzyme (as measured by incorporation of [35S]methionine) by 4-5-fold. Insulin (0.18 microM) completely inhibited dexamethasone-dependent stimulation of enzyme synthesis. In vitro translation and cDNA hybridization assays were employed to measure effects of dexamethasone plus or minus insulin on levels of mRNA encoding the biosynthetic precursor of carbamoyl-phosphate synthetase I (pCPS) in Reuber H-35 cells. Both measurements yielded similar results: dexamethasone increased pCPS mRNA levels by 4-5-fold and insulin suppressed this response, but only by 50%. Specific cDNA hybridization assays also demonstrated that Reuber H-35 cells, even after hormone treatments, contain only very low levels of albumin mRNA, and no detectable ornithine carbamoyl-transferase mRNA.