Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.     

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex LH-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not trptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortison which are not identical to any of the known metabolites.     

The effect of hydrocortisone on the alkaline proteinase activity in the target organs of rats]

The effect of hydrocortisone on the thymus and liver proteinases activity in the alkaline pH range was studied on rats. It is shown that the alkaline proteinases are activated in the thymus and inhibited in the liver of hormone-treated animals. Possible relationship between the effect of glucocorticoids on protein metabolism and alkaline proteinases activity is suggested.     

Effects of hydrocortisone and insulin on the electrokinetic potential of hepatocyte nuclei

The electrokinetic potential of liver cell nuclei of rats after the in vivo separate and joint injection of hydrocortisone and insulin was determined. It was shown that the steroid induces a significant elevation of the value of electrokinetic potential while insulin does not change this parameter. An insignificant elevation of the potential by the joint action of the hormones was found, which apparently is indicative of the antagonism between hydrocortisone and insulin.     

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H]hydrocortisone

After administration of [³H]hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [³H]hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5–60 min after administration of biologically active doses of hydrocortisone. 3ξ,11β,17α,20ξ, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in ronide on the basis of its chromatographic behavior. The biological significance of this conjugate of hydrocortisone as well as that of the extremely polar conjugate found in fraction DE-3 cannot be understood on the basis of the published data pertaining to biological functions and metabolism of glucocorticosteroids.Our finding that no ‘classical’ glucocorticosteroid receptor can be detected in rat liver cytosol raises again the question of the way in which hydrocortisone and its active metabolites enter the nucleus. On the basis of the published data, the possibility cannot be ruled out that glucocorticosteroids are transported via the endoplasmic reticulum. A transport by this way has been inferred for the uptake of sodium and inulin by liver nuclei [40–42].     

Effect of cold exposure on the concentration of triglyceride in the liver of the rat

The aim of the present study was to examine an effect of cold exposure on the concentration of triglycerides (TG) in the rat's liver. The rats were divided into the following groups: control, fed with oil, treated with hydrocortisone, fed with oil and treated with hydrocortisone, treated with noradrenaline. The rats exposed to cold were kept in wire cages (one rat in one cage) in the cold room at temperature +2 degrees C. They had free access to food (pellet diet for rodents) and water. In the control group the exposure to cold increased mildly (though significantly) the TG concentration after 1 and 3 h and had no effect after 2 and 24 h. It did not affect the concentration of plasma free fatty acids (FFA). At room temperature feeding with oil (2 ml/100 g of body weight) alone, and combined with hydrocortisone treatment (5 mg/100 g of body weight) as well as treatment with noradrenaline (0.1 mg/100 g of body weight) had no effect on the liver TG concentration, although the concentration of plasma FFA was increased. Exposure to cold for 3 h increased markedly the liver TG concentration in each of those groups. It is concluded that exposure to cold elicits a mechanism, which in the presence of elevated plasma FFA concentration induces accumulation of TG in the liver.     

Effect of insulin and hydrocortisone on various indices of energy metabolism in rats irradiated with 6.0 MeV fast neutrons

Neutron irradiation (1 Gy, 0.5 + 0.5 Gy) causes hypersecretion of glucocorticoids. The administration of hydrocortisone imitates, in intact rats, and increases, in irradiated rats, the radiation inhibition of energy metabolism. The increase of radical oxidation in irradiated tissues is a probable cause of radiation hypercorticism; glucocorticoids are the inhibitors of radical oxidation. Insulin injections to irradiated rats normalize the main indices of energy metabolism.     

Lipogenesis and gluconeogenesis in the liver of irradiated rats

The incorporation of 14C from [U-14C] glucose and 3H from 3H2O into the total lipids fatty acids and glycogen of the liver incorporation of 3H from 3H2O into blood glucose was studied in rats totally irradiated in a dose of 14.4 Gy. It is shown that in the liver of irradiated rats glucose is accumulated in considerable amounts as glycogen but it is slightly used as a source of carbon for lipid synthesis. The study of 3H incorporation shows that irradiation stimulates glucogenesis, glyconeogenesis and lipogenesis in the liver.     

Differential Regulation of Malate Dehydrogenase Isoenzymes by Hydrocortisone in the Liver and Brain of Aging Rats

The activities and induction patterns of the isoenzymes of malate dehydrogenase (MDH) of the liver and brain of male rats of various ages were studied. The activities of both the isoenzymes of MDH of the liver and brain show a gradual increase with increasing age of the rats. Adrenalectomy decreases and hydrocortisone treatment increases the activity of cytoplasmic MDH of the liver and brain of rats of all the ages except that of the brain isoenzyme of old rats. This hormone-mediated induction of the isoenzyme is actinomycin D-sensitive. Furthermore, adrenalectomy decreases and hydrocortisone treatment increases the activity of mitochondrial MDH of the liver of young and adult rats but not in old rats. However, these treatments do not show any significant effect on the activity of mitochondrial MDH of the brain of rats of all the ages.     

Age-dependent effects of sodium butyrate and hydrocortisone on acetylation of high mobility group proteins of rat liver

The in vitro acetylation of high mobility group (HMG) proteins and its modulation by sodium butyrate and hydrocortisone have been studied using liver slices of young (13-) and old (114-week-old) rats. Acetylation of total HMG proteins was significantly higher in young than old rats. HMG 1, in particular, showed greater acetylation than others. Whereas acetylation of HMG 1 and 2 decreased drastically, that of HMG 14 and 17 increased in old age. In young rats, sodium butyrate and hydrocortisone stimulated acetylation of HMG 14 and 17, and decreased that of HMG 2. Butyrate had no effect on HMG 1, but hydrocortisone decreased it. In old rats, butyrate and hydrocortisone decreased acetylation of all HMGs, except HMG 17, which was stimulated to a slight extent by butyrate.     

Effects of ubiquinone-9 on lipids of nuclei and chromatin of the rat liver under continuous irradiation]

The influence of continuous gamma irradiation on the lipids of nuclei and chromatin of rat liver at a dose-rate of 0,129 Gy/day for 155 days (a total dose of 20 Gy) and by feeding of ubiquinone-9 has been studied. The amount of phosphatidylcholine with phosphatidylserine and phosphatidyl-ethanolamine in liver nuclei of irradiated rats was found to increase. Ubiquinone-9 had a normalizing effect. A decrease of cardiolipin was observed in the liver chromatin of irradiated rats. The amount of free fatty acids had a tendency to decrease in homogenate, nuclei and liver chromatin of irradiated rats. Ubiquinone was found to increase the amount of free fatty acids up to the control level. The amount of cholesterol in nuclei was increased after irradiation and that in chromatin tended to rise. Ubiquinone-9 significantly decreased the amount of cholesterol in nuclei and chromatin of irradiated rats.     

Comparison of the properties of L-threonine dehydratase isolated from the livers of intact, adrenalectomized or cortisol-treated rats]

L-Threonine dehydratase preparations were isolated from liver of intact, treated with hydrocortisone and adrenalectomized rats. These preparations had different properties in stability, sensitivity to proteases and kinetic patterns. The preparations possessed also serine dehydratase activity, and the ratio threonine: serine activities was modified during the procedure of enzyme purification. It appears that the hormones affect not only the amount of enzyme proteins, but the qualitative properties of these proteins.     

Alterations in the activities of subcellular fractions marker enzymes in rat liver and brain by hydrocortisone and corticosterone treatment

The effect of subcutaneous injection of hydrocortisone and corticosterone on the activity values of some subcellular fractions marker enzymes from rat liver and brain was investigated and compared with controls (without treatment with hormones). The following enzymes were studied (subcellular fraction are shown between parentheses): N-acetyl-beta-D-glucosaminidase and beta-glucuronidase (lysosomes); succinate dehydrogenase = SDH (mitochondria); glucose-6-phosphatase (endoplasmic reticulum); 5'-nucleotidase and Na+-K+-Mg2+ ATPase (plasma membrane). The specific activity of lysosomal enzymes from liver showed no change when rats were injected either with hydrocortisone or corticosterone. The same enzymes from brain showed significant increases in their activities with both hydrocortisone or corticosterone except beta-glucuronidase; this enzyme gave activity values remaining between the control levels, after treatment with corticosterone. The activity of mitochondrial SDH was increased after corticosterone injection either in liver or brain. After hydrocortisone injection, its activity rises significantly in brain (72%), but it falls in liver compared to the control values. Glucose-6-phosphatase behaves similarly in brain or liver fractions; its activity increases always after corticosterone treatment and decreases by hydrocortisone. The plasma membrane marker enzymes did not change practically in brain fractions, excepted Na+-K+-Mg2+ ATPase which tends to rise its activity after hydrocortisone injection. In liver fractions, both 5'-nucleotidase and Na+-K+-Mg2+ ATPase activities increase either by corticosterone or hydrocortisone treatment, except 5'-nucleotidase which specific activity decreases in liver after hydrocortisone treatment.     

Possibility of hydrocortisone induction of intertissue recombination of DNA fragments

Incorporation of 3H-thymidine in DNA of different organs and blood of rats was studied in health and under the action of hydrocortisone. Based on the data obtained it may be suggested that there is a possibility of intertissular recombination of DNA fragments, which is increased under the action of hydrocortisone mainly in the liver.     

Inhibitory action of microwave radiation on gamma-glutamyl transpeptidase activity in liver of rats treated with hydrocortisone

The influence of microwave irradiation on the activity of gamma-glutamyl transpeptidase (GGT) induced by hydrocortisone (HC) in the liver of rats was investigated. Animals were subjected to microwave irradiation (frequency 53.57 GHz, power density 10 mW/cm2 and 1 mW/cm2) during and after hydrocortisone (HC) treatment (20 mg/kg for 60 days). The results indicate that microwave radiation may block an inducible effect of HC on GGT activity in the liver of rats. This effect depends on the power density of millimetre microwaves.     

Participation of the hypophyseal-adrenal cortex system in thrombin clearance during immobilization stress]

The examination carried out with thrombin marked by 131J resulted in a considerable increase of the thrombin clearance rate in healty male rats during the stress (caused by an immobilization lasting 30 minutes) and in an increase of thrombin deposits in the liver. A further increase of thrombin clearance occurred by the combination of immobilization and administration of ACTH. Contrary to ACTH the thrombin clearance is not stimulated in healthy animals by hydrocortisone. Thrombin clearance and thrombin deposits in the liver are lowered in adrenalectomized rats. In these animals the administration of ACTH does not result in an increase of thrombin clearance. The rate of thrombin clearance is normalized in adrenalectomized animals after administering hydrocortisone without as well as under conditions of stress. In adrenalectomized animals having received hydrocortisone as well as in healthy animals the administration of ACTH will results in an increase of thrombin clearance. From these experiments the conclusion can be drawn that ACTH will increase the intensity of thrombin clearance in stress and that hydrocortisone plays a transmitting part here.     

Effect of glucagon and hydrocortisone on aldolase turnover in the rat liver after total x-irradiation

A study was made of the effect of glucagon and hydrocortisone on aldolase metabolism in rat liver after total-body X-irradiation. The hormonal regulation of the enzyme metabolism in the exposed rats was shown to vary from that of intact animals.     

Antidiuretic hormone content in the neurohypophysis of adult white rats after hydrocortisone administration in the early postnatal period

The age-related time-course of changes in arginine-vasopressin (AVP) content in the pituitary gland was studied in adult intact Wistar rats. In 60-day-old rats, the hormone content was measured before and after 24 h of water deprivation. In adult rats treated with a single injection of hydrocortisone at different times after birth, the content of AVP remained high in rats injected with hydrocortisone on day 2 or day 5 after birth, exceeding significantly the content of AVP in intact rats. The animals injected with hydrocortisone on day 9 or 15 manifested a more noticeable reduction in the hormone content, as was the case in intact rats. It is suggested that the first five days after birth is a critical period in the formation of the central regulation of AVP secretion with high sensitivity to short-term changes in corticosteroid balance.     

Inhibition of proliferation of normal and neoplastic liver cells under conditions of prolonged hormonal stimulation

Single injections of rats with hydrocortisone led to the inhibition of regenerating liver cell proliferation and protooncogene++ Ha-ras mRNA synthesis within 48 hours of hormonal induction. Administration of hydrocortisone to rats daily for 10 days resulted in a persistent decrease of the liver cell capacity to proliferate in response to partial hepatectomy. This inhibiting effect was observed for at least 7 days after cessation of hormonal stimulation; the level of Ha-ras mRNA was thereby decreased. A marked inhibition of ascite hepatoma cell growth was demonstrated after injections of those cells to mice induced with hydrocortisone for 10 days. Such a persistent effect of hydrocortisone is thought to be due to the depletion of the hormone-dependent hepatotrophic factors. The effect of the glucocorticoid hormone in vivo can be supposed to involve both the direct and indirect regulation of target cell proliferation. The latter is mediated via the changes in the activity of exogenous factors which control cell growth and proliferation.     

Tissue-specific differential induction of aspartate transcarbamylase by hydrocortisone in rats of various ages

The activity and induction pattern of aspartate transcarbamylase (ATCase) of the liver (a mitotic tissue) and heart (a post-mitotic tissue) of rats of various ages were studied. The activity of the enzyme of both tissues was highest in the immature rat and decreased significantly thereafter with increasing age of the animal. Adrenalectomy and hydrocortisone treatments altered the activity of liver ATCase in rats of all the ages. However, these treatments altered the heart enzyme of the immature and adult rats, but not of senescent rat. The magnitude of induction of the enzyme by hydrocortisone was highest in the immature rat. Actinomycin D inhibited the hormone-mediated induction of ATCase in both the liver and heart.