Cortisol-treated Rats Research Articles

The neonatal glucocorticoid treatment-produced long-term changes of the pituitary-adrenal function and brain corticosteroid receptors in rats.

Two distinct periods of sensitivity to elevated glucocorticoid hormone levels during postnatal development of the pituitary-adrenal axis were studied. Wistar rats were injected subcutaneously (s.c.) with cortisol (1 mg/kg) on postnatal days 1-5 or 14-18. The steroid treatment during the first postnatal week resulted in a decrease of the morning basal and stress-induced plasma corticosterone levels in 30 day-old male rats, as well as in rats that were injected with cortisol on the third postnatal week. Stress-induced corticosterone levels in 90-day old cortisol-treated rats were determined in blood samples drawn from the tail vein before the restraint stress, immediately after the 20-min long stress, then 60 and 180 min afterwards. Only the rats treated with cortisol during the third week showed a prolonged stress-induced corticosterone secretion, with the highest corticosterone level in 180 min after the restraint stress. The early neonatal cortisol treatment had no effect on (3)H-corticosterone binding in all studied brain areas of the 90-day old rats. The rats treated with cortisol at the 14-17th postnatal days showed a significantly lower (3)H-corticosterone binding in the frontal cortex, hippocampus, and hypothalamus. These findings suggest that the third week of life in rats is more sensitive to elevated levels of corticosterone than the first one. The high level of glucocorticoids at this period has long-term effects on the efficiency of the negative feedback mechanisms provided by hypothalamus-pituitary-adrenal axis.

Binding and action of glucagon in isolated adipocytes from cortisol-treated rats

Evidence for pre-receptor, receptor and post-receptor glucagon defects was investigated in adipocytes from cortisol-treated rats. A decrease in glucagon binding due to a decreased number of receptors was observed. No changes in receptor affinity were detected. Both, the lipolytic response of glucagon and the ability of glucagon to increase basal and theophylline-stimulated cAMP accumulation remained unaltered. Moreover, a hyperglucagonemia accompanied by an increase in glucagon degradation in the serum of cortisol-treated rats was observed. Such alterations could represent a new mechanism by which glucocorticoids exert their biological actions.

Decrease in the response to ADH of the rat kidney as a result of early postnatal treatment with cortisol

Wistar rats were injected just once, intraperitoneally with cortisol (1 microgram/g) or saline at the age of 5 days. The cortisol-treated rats did not differ significantly in the (U/P)osm ratio from the saline-treated controls before 15 days of life. Their response to ADH was distinct but weaker than in the saline controls aged 30 days. This reduced response persisted to 60 days of life. In the collecting tubule fragments, (3H)AVP specific binding was lower in the cortisol-treated rats than in the controls at the age of 20 and 60 days. There was no (3H)AVP specific binding in the proximal convoluted tubules in the cortisol- and saline-injected rats of both ages. The ontogenetic patterns of cAMP specific binding in the papillary cytosolic fraction were different: the early increase in cAMP binding was protracted in the cortisol-treated rats, and no peak appeared at the age of 25 days. Cytosolic protein kinase activity was lower, no peak appeared at 30 days, no activation of protein kinase occurred to the end of weaning in the cortisol-treated rats. The difference between the cortisol and saline groups was abolished by day 30. The interference of cortisol with the ontogenetic changes in AVP binding capacity and cAMP-dependent protein kinase appears to be a plausible cause of the altered development of the response to ADH.

Sex dimorphism and hormonal regulation of rat tissue kallikrein mRNA

The regulation of rat tissue kallikrein mRNA levels was investigated by RNA filter hybridization using a kallikrein cDNA probe and by in vitro translation of isolated RNA. The synthesis of a 37 kDa polypeptide has been hybrid-arrested by the tissue kallikrein cDNA in cell-free translation assays directed by submandibular mRNA and the quantitation of this protein was used as a measure of translational activity of kallikrein mRNA. In Northern blot analyses, relative kallikrein mRNA levels were found to be 60-fold higher in submandibular gland than in pancreas and 20-fold higher than in kidney. In addition, it was noted that kallikrein mRNA is differentially regulated in tissues from male and female animals. Rat submandibular gland kallikrein mRNA is 2-times more abundant in male animals as in females. This trend is reversed in kidney, where the kallikrein mRNA in females is twice that in male animals. Relative submandibular gland kallikrein mRNA levels were found to be responsive to hormonal manipulation as measured by both Northern blotting and cell-free translation assays. Castration of male animals resulted in a decrease of kallikrein mRNA which could be partially restored by treatment with thyroxine and almost completely restored by testosterone treatment. The submandibular gland kallikrein mRNA in normal female rats was increased up to 30% by administration of testosterone or thyroxine. Adrenalectomized female rats showed a decrease in submandibular kallikrein mRNA which was significantly increased by testosterone, thyoxine, aldosterone and cortisol, with the largest effect being a 2- to 2.4-fold increase in cortisol-treated rats. These results indicate that hormones regulate tissue kallikrein synthesis in the rat submandibular gland, at least in part, by altering kallikrein mRNA accumulation.

Metabolism of amino acids by the atrophied soleus of tail-casted, suspended rats

Amino acid metabolism was investigated in atrophied soleus muscle from rats subjected to six days of tail-cast, hindlimb suspension. The fresh-frozen unloaded muscle showed higher concentrations of tyrosine and glutamate but lower amounts of aspartate, glutamine, ammonia, and a lower ratio of glutamine to glutamate than normal muscle. The atrophied muscle also showed faster in vitro production of alanine and tyrosine, and slower utilization of glutamate and aspartate. Despite a greater activity of glutamine synthetase, synthesis of glutamine was slower in the soleus muscle of suspended rats than in control muscle. Provision of ammonium chloride and/or glutamate showed that this slower synthesis of glutamine in the atrophied soleus probably was due to limiting amounts of free ammonia and not of glutamate. Flux through AMP deaminase was probably slower as demonstrated by the maintenance of a greater pool of total adenine nucleotides and by the slower release of nucleosides by the incubated soleus muscle of suspended v control rats. The extensor digitorum longus muscles of suspended animals showed greater glutamine production, glutamine synthetase activity, and aspartate utilization than control muscles. Data from muscles of intact, adrenalectomized and adrenalectomized, cortisol-treated rats suggested that the greater glutamine synthetase activity was mediated possibly by higher circulating glucocorticoid hormones and a greater response of the soleus muscle to these hormones. Glutamine synthesis in skeletal muscle may be regulated primarily by the availability of ammonia, which is associated with the degradation of adenine nucleotides, and secondarily by the amount of glutamine synthetase and glutamate in the tissue.

Influence of hormones and undernutrition on brain development in newborn rats.

High L-thyroxine (T4) and cortisol doses given to rats during the first 8 days of life and on the first day only respectively, produce decrease of body and brain weight and perturbances to brain energy substrates, i.e. glucose and ketone bodies. The same alterations are found in the undernourished rats from the prenatal period. The pituitary GH and TSH is decreased in the T4- and cortisol-injected animals. The plasma ACTH is decreased in the treated cortisol animals at 8, 12 and 22 days of life. The pituitary TSH content is reduced with respect to controls in the undernourished animals from 14 to 70 days of life. From the exposed experiments we suggest an alteration in the hypothalamic-pituitary complex in T4- and cortisol-treated rats, and describe the similarities between the three experimental models.

Precursor supply and hepatic enzyme activities as regulators of triacylglycerol synthesis in isolated hepatocytes and perfused liver

The relative significance of alterations in precursor supply and enzyme activities for the rate of triacylglycerol synthesis was studied in isolated hepatocytes and perfused livers. Precursor availability was varied in vitro by changing the fatty acid concentration in the incubation medium or adding ethanol to the perfusion medium in order to increase the cellular glycerol 3-phosphate concentration. The rate of glycerolipid synthesis in hepatocytes, measured in terms of the label incorporated into the various lipid classes from tritiated glycerol, was strongly dependent on the fatty acid concentration up to 2 m m of oleate (fatty acid/albumin molar ratio 7 1 ). Ethanol in vitro increased the incorporation of labeled oleate into phosphatidic acid and diacylglycerol in the isolated perfused liver, but its effect on the incorporation into triacylglycerol was small. Ethanol in vitro increased the label incorporation into both diacylglycerol and triacylglycerol in the livers from cortisol-treated rats. Although cortisol treatment increased the soluble phosphatidate phosphohydrolase activity 4.4-fold in the hepatocytes, it had no effect on the rate of triacylglycerol synthesis, whereas fasting increased this rate about 3-fold, although only a moderate concomitant increase in soluble phosphatidate phosphohydrolase activity was observed. Neither cortisol treatment nor fasting affected the microsomal glycerol-3-phoshate acyltransferase activity. The results demonstrate that substrate availability can override enzyme modulations in the regulation of triacylglycerol synthesis and that phosphatidate phosphohydrolase is not the main regulator of triacylglycerol synthesis.

Modification of pituitary-adrenal feedback sensitivity in young rats by neonatal treatment with cortisol.

Neonatal exposure of rats to cortisol acetate was found to alter pituitary-adrenal feedback regulation at 20-25 days of age. Plasma levels of adrenocorticotrophin (ACTH) after ether stress were reduced in cortisol-treated rats pre-treated with 100 microgram corticosterone/100 g body weight, while rats given vehicle neonatally did not show suppression of the ACTH response below levels in animals given saline only or not injected as pre-treatments. Neonatal cortisol increased sensitivity to dexamethasone in inhibition of the stress response; cortisol-treated animals had a reduced plasma corticosterone response to stress 3 h after pre-treatment with 1.25, 2.5, 25, or 250 microgram dexamethasone/100 g body weight, while the stress response in animals given vehicle neonatally was not inhibited by the lowest dosage of dexamethasone. Neonatal cortisol treatment did not affect corticosteroid-binding globulin (CBG) binding capacity in plasma of 25-day-old animals. Thus, neonatal treatment with cortisol appears to increase feedback sensitivity to circulating corticosteroids at 20-25 days of age.

Glucocorticoid-induced recovery from streptozotocin diabetes in the adult rat.

The effect of a large dose of cortisol (5 mg/kg), injected 24h and 3h before or 3h and 24h after streptozotocin (SZ), on the course of SZ-induced diabetes has been studied in the rat. Glucose tolerance tests, performed 1 or 2, 8, and 30 days after the SZ injection (35 mg/kg), gave the same results in cortisol-treated rats regardless of the time of cortisol injection [before SZ (C.SZ) or after it (SZ.C)]. During the first test, elevated plasma glucose, decreased glucose tolerance, and decreased glucose-induced insulin secretion were present in SZ, C.SZ, and SZ.C rats. During the second and the third tests, glucose tolerance and insulin secretion were significantly improved in both groups of cortisol-treated rats. On the contrary, rats receiving only SZ showed a worsening of the diabetic state. It should be noted that cortisol-induced recovery of diabetes was not obtained with larger doses of SZ (50 mg/kg). These findings indicate that an improvement of diabetes can be obtained by cortisol treatment after the onset of chemically induced diabetes. Whether this effect is obtained through an effect on pancreatic regeneration and/or through the anti-inflammatory properties of glucocorticoids is unknown at the present time.

The effects of cortisol, corticotropin and thyroxine on the synthesis of glycerolipids and on the phosphatidate phosphohydrolase activity in rat liver

1. Male rats were injected daily for 5 days with 0.15m-NaCl, corticotropin, cortisol or l-thyroxine and the rates of glycerolipid synthesis were measured in the livers after intraportal injection of [(14)C]palmitate and [(3)H]glycerol. 2. Injection of all three hormones decreased the rates of body-weight gain. 3. Cortisol treatment increased the weight of the liver relative to body weight. 4. Thyroxine treatment increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol and decreased the relative accumulation of (3)H and (14)C in diacylglycerol. It did not significantly alter the accumulation of these isotopes in phosphatidate nor the activity of the soluble phosphatidate phosphohydrolase in the total liver. However, this activity increased by 1.5-fold when expressed relative to the soluble protein of the liver. The increased triacylglycerol synthesis appears to be related to a general increase in the turnover of fatty acids in the liver. 5. Treatment with cortisol and corticotropin increased the relative rate of triacylglycerol synthesis from [(3)H]glycerol, decreased the accumulation of (3)H in phosphatidate and increased the flux of both isotopes from phosphatidate to diacylglycerol. This appeared to be caused by the increased activity of the soluble phosphatidate phosphohydrolase that was observed in the livers of the cortisol-treated rats. 6. It is proposed that cortisol could be directly or indirectly involved in increasing the activity of hepatic phosphatidate phosphohydrolase in starvation, diabetes, laparotomy, subtotal hepatectomy, liver damage, ethanol feeding and in obesity. This enzyme adaptation could contribute to the potential of the liver to increase its synthesis and accumulation of triacylglycerols or to secrete very-low-density lipoproteins.

Cycloheximide causes increased accumulation of translatable mRNA for tyrosine aminotransferase and tryptophan oxygenase in livers of cortisol-treated rats.

Messenger RNA activities for two cortisol-inducible enzymes, tyrosine aminotransferase and tryptophan oxygenase, have been determined by translation in a wheat germ system. The effects of cycloheximide on the two mRNA activities have been evaluated. Cortisol leads to an increase of the translatable mRNAs for tyrosine aminotransferase and tryptophan oxygenase with a maximum at approximately 6 h. Cycloheximide was administered 4 h after treatment with cortisol; 2 h later, the activities of tyrosine aminotransferase and tryptophan oxygenase mRNA had increased five-fold and two-fold, respectively, compared to the activities reached with cortisol alone. Thereafter the amount of the two translatable mRNAs declined, though 14 h after cortisol administration the mRNA activities were still several fold higher than in control animals. Application of alpha-amanitin together with cycloheximide did not prevent an increased accumulation of specific translatable mRNAs. The increase in tyrosine aminotransferase and tryptophan oxygenase activity by cortisol was immediately blocked by cycloheximide. Whereas tryptophan oxygenase activity rapidly declined after cycloheximide application, tyrosine aminotransferase activity remained at the same level. Approximately 4 h thereafter, both enzyme activities increased again.

Postnatal development of adrenocortical circadian rhythm in neonatally cortisol- and estriol-treated rats (author's transl)

The effect of a single neonatal administration of cortisol and estriol on the development of the adrenocortical circadian rhythm was investigated in immature rats. In normal control rats of both sexes, the plasma corticosterone rhythm was discernible on day 21. In male rats the adult pattern was observed on day 28, but in females as the plasma corticosterone level elevated progressively during puberty, the rhythm increased in amplitude and the adult pattern was established at 8 weeks of age. In neonatally cortisol-treated rats, the plasma corticosterone rhythm appeared at 4 weeks of age, but the plasma level was significantly lower than that of the controls until 6 weeks and attained a level similar to the controls at 8 weeks. Neonatally estriol-treated rats exhibited the rhythm as early as 2 weeks of age, but this was temporary. It disappeared at 3 weeks and reappeared a 4 weeks. Although 5-week-old males showed normal rhythm as seen in the controls, in females the plasma corticosterone level remained low for weeks and approached the control level at 8 weeks of age. The development of circadian periodicity of adrenal corticosterone content almost paralleled that of plasma corticosterone not only in the controls but also in steroid-treated groups, but the rhythm was occasionally equivocal. In neonatally estriol-treated rats, stress response of plasma and adrenal corticosterone to ether was similar to that of the controls, but the response to exposure to a strange environment was significantly small.

Histochemistry of 11-beta-hydroxysteroid dehydrogenase in rat submandibular gland. Effect of cortisol stimulation.

The activity pattern of NAD/NADP-linked 11β-hydroxysteroid dehydrogenase (HSD) in the submandibular gland of the rat was re-evaluated using several control experiments. The incubation time needed for the initial appearance of red and blue formazans was used to investigate the activity of NAD-dependent 11β-HSD in control and cortisol-treated rats. The following results were obtained. (1) Prefixation of small tissue blocks with 1% w/v methanol-free formaldehyde (pH 7.2) for up to 20 min preserved morphological integrity and maximal enzyme activity. The substantivity of formazans was enhanced. (2) The substantivity of Nitro BT was highly variable. The implication of this forin situ localization of enzymes was analysed. (3) Pretreatment with acetone and application of phenanthroline was necessary to avoid a false positive reaction caused by alcohol dehydrogenase. (4) No diffusion of 11β-HSD was noticed within 30 min of incubation, nor was rediffusion of reduced intermediates seen. (5) With either NAD or NADP as coenzyme, 11β-HSD was localized in the striated, intralobular, interlobular, interlobar, and main duct. (6) 11β-HSD was found to be primarily NAD-dependent. (7) With DMF or DMSO as solvent, the rate of utilization of substrates was as follows: Cortisol=11β-hydroxyandrostendione>11β-hydroxyprogesterone. Aldosterone was utilized very poorly, if at all. (8) After injection (i.p.) of a single pharmacological dose of cortisol, the activity of NAD-linked 11β-HSD was significantly increased 24 h later. (9) NADH-tetrazolium reductase was not inhibited by levamisole. (10) Distinct NADPH-tetrazolium reductase activity was localized in the apical part of cells (or cell membranes) of the interlobar ducts and the main duct.

Influence of cortisol on tryptophan-mediated stimulation of hepatic protein synthesis in well-fed adrenalectomized rats

Administration of either l-tryptophan or cortisol to well-fed adrenalectomized rats resulted in a marked enhancement in [ 3H]leucine incorporation into plasma albumin, fibrinogen, and liver ferritin in vivo, as compared to water-treated controls. In a cortisol-pretreated adrenalectomized rat, the magnitude of increment, over that of the water-treated control, of [ 3H]leucine incorporation into albumin and fibrinogen by tryptophan was less than that observed in the noncortisol-treated rats. Tryptophan administration to cortisol-treated rats also lowered the cortisol-mediated stimulation of [ 3H]leucine incorporation into albumin, fibrinogen, and ferritin.

Effect of graded doses of cortisol on total body calcium in rats

Male rats with an average body weight of 250 g were injected (sc) daily for 4 wk with 0.05, 0.20, 0.75, or 3.00 mg of cortisol acetate. Intact and adrenalectomized control animals were injected daily with 0.1 ml of vehicle (corn oil). Total body calcium (TBCa) was measured weekly in each rat by in vivo neutron activation analysis. The gain is body weight of rats treated with 0.75 mg cortisol was significantly less than controls, and the animals treated with 3.00 mg cortisol lost weight. In spite of these differences in body weight, the TBCa of all rats increased to an equal degree from an average of 1.93 g to 2.81 g in 4 wk. In addition, there were no significant differences in tibial ash calcium. However, calcium (mg) per unit length (mm) of tibia was increased in rats treated with the higher doses of cortisol; thus bone density was increased. These results demonstrate that the TBCa increases even when rats are subjected to cortisol. This is explained in part by the normal rate of intestinal calcium absorption in cortisol-treated rats.

Effect of adrenal steroids on bone resorption in rats.

Rats labeled with strontium-85 (85Sr) were rejected with adrenocortical steroids for 2 wk. The urinary-to-tibial (U/T) 85Sr ratio was used as an index of bone resorption. The glucocorticoids caused an inhibition of skeletal resorption, as judged by the 50% reduction in the U/T ratio, and decreased excretion of hydroxyproline. Thyroidal calcitonin levels were slightly elevated in glucocorticoid-treated animals, suggestive of a possible retardation of calcitonin release. The U/T ratios of thyroparathyroidectomized (TPTX) rats injected with corticosteroids were 50% of control values. The results indicate that glucocorticoids inhibit bone resorption independent of the action of calcitonin. Cortisol treatment increased the tibial density as measured by a radiographic technique. However, bone density was decreased and the U/T ratio increased in steroid-treated rats fed a low-calcium diet. In TPTX cortisol-treated rats, parathyroid extract (PTE) increased the U/T ratio and serum calcium but not to the degree observed in TPTX PTE-injected control animals. These experiments indicate that in rats glucocorticoids inhibit the rate of bone resorption but this effect can be overcome in part by PTE.

Nucleolar RNA synthesis in the liver of partially hepatectomized and cortisol-treated rats

RNA synthesis by isolated nucleoli from rat liver is significantly enhanced 12–14 h after partial hepatectomy and 4 h after cortisol administration. The increased RNA synthetic capacity is demonstrable also in the respective high salt nucleolar extracts and in Biogel A-1.5 filtration fractions of the nucleolar extracts. DNA saturation experiments using nucleoli and Biogel fractions from control and treated animals as RNA polymerase source, have demonstrated, that independent of the extent of RNA synthesis, saturation of transcription is reached at the same concentration of exogenous template. We conclude that the activity and not the amount of nucleolar RNA polymerase is increased as a result of partial hepatectomy or cortisol administration. Parallel to the effects on RNA polymerase, the activity of RNA-degrading enzymes present in nucleoli is also enhanced by the same treatment.

Eosinophils in the bone marrow of normal and cortisol-treated rats. Quantitative and autoradiographic studies.

Absolute counts of the eosinophil cells of the blood and bone marrow were carried out in a haemocytometer and combined with autoradiographic studies of smears of blood and bone‐marrow suspensions from normal rats and cortisol‐treated rats. A certain parallelism was found between the number of circulating eosinophil leucocytes and the eosinophils of the bone marrow. Calculations showed that the bone marrow contains more than 50 times as many eosinophils as the blood. Single injections of Cortisol (5 mg intraperitoneally) as well as repeated injections of Cortisol (5 mg every 6 hours for up to 3 days) induced blood and bone‐marrow eosinopenia. The decrease in the number of eosinophils in the bone marrow appears to be due primarily to a decrease of mature (non‐prolifera ting) eosinophils. During repeated injections there was continued proliferation of eosinophil cells in the bone marrow, but mitotic activity was reduced. Appearances like those in the bone marrow have previously been observed in the spleen (Bro‐Rasmussen 1972 and 1973).

Differential effects of cortisol on nucleolar and extranucleolar RNA synthesis of rat thymocytes

Nucleoli were prepared from thymocytes of cortisoltreated rats as well as from thymus cells treated in vitro with the hormone. Cortisol decreases very significantly the synthesis of nucleolar RNA and increases synthesis of RNA by the extranucleolar fraction. The RNA stimulated by cortisol is resistant to the action of α-amanitin.

Studies on ribonucleic acid turnover in rat organs: effects of cortisol, actinomycin D, and cycloheximide.

Effects of cortisol administration to adrenalectomized rats on the half-life of RNA in liver, kidney and spleen were examined. The effect of administration of Actinomycin D and cycloheximide was also studied. A significant increase in the half-life of RNA was observed in liver and kidney from cortisol-treated rats but the rate of RNA degradation in spleen was not significantly influenced. Administration of Actinomycin D at a dose which caused 30-50% inhibition of 3H-orotate incorporation into the liver, spleen and kidney also decreased the degradation of labeled RNA in liver and spleen but not in kidney, while cycloheximide, a dose which evoked approximately 40% inhibition of 3H-leucine incorporation into the tissues, exhibited no significant effect on the turnover rate of RNA in all tissues examined.