Carbonyl reductase 1 catalyzes 20ß-reduction of glucocorticoids, modulating receptor activation and metabolic complications of obesity.

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20ß-dihydrocortisol (20ß-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20ß-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20ß-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.

Evolution of hormone signaling in elasmobranchs by exploitation of promiscuous receptors.

Specific interactions among proteins, nucleic acids, and metabolites drive virtually all cellular functions and underlie phenotypic complexity and diversity. Despite the fundamental importance of interactions, the mechanisms and dynamics by which they evolve are poorly understood. Here we describe novel interactions between a lineage-specific hormone and its receptors in elasmobranchs, a subclass of cartilaginous fishes, and infer how these associations evolved using phylogenetic and protein structural analyses. The hormone 1alpha-hydroxycorticosterone (1alpha-B) is a physiologically important steroid synthesized only in elasmobranchs. We show that 1alpha-B modulates gene expression in vitro by activating two paralogous intracellular transcription factors, the mineralocorticoid receptor (MR) and glucocorticoid receptor (GR), in the little skate Leucoraja erinacea; MR serves as a high-sensitivity and GR as a low-sensitivity receptor. Using functional analysis of extant and resurrected ancestral proteins, we show that receptor sensitivity to 1alpha-B evolved millions of years before the hormone itself evolved. The 1alpha-B differs from more ancient corticosteroids only by the addition of a hydroxyl group; the three-dimensional structure of the ancestral receptor shows that the ligand pocket contained ample unoccupied space to accommodate this moiety. Our findings indicate that the interactions between 1alpha-B and elasmobranch GR and MR proteins evolved by molecular exploitation: a novel hormone recruited into new functional partnerships two ancient receptors that had previously interacted with other ligands. The ancestral receptor's promiscuous capacity to fortuitously bind compounds that are slight structural variants of its original ligands set the stage for the evolution of this new interaction.

Abnormal steroidogenesis in three patients with Antley-Bixler syndrome: apparent decreased activity of 17alpha-hydroxylase, 17,20-lyase and 21-hydroxylase.

BACKGROUND: Antley-Bixler syndrome (ABS) is characterized mainly by abnormal skeletal morphogenesis such as craniosynostosis and radiohumeral synostosis, and by ambiguous genitalia in some cases. The mechanisms resulting in these deformities have not been determined. METHODS: The adrenal and gonadal function of three Japanese ABS patients were evaluated. Patient 1 (17-year-old-male) had bilateral cryptoorchidism, delayed puberty and symptoms of glucocorticoid deficiency. Patient 2 (14-year-old male) and patient 3 (4-year-old female) presented with emaciation. Additionally, patient 3 had partial labial fusion and common urogenital sinus. In each patient, blood sampling for steroid analysis before and after rapid adrenocorticotropic hormone (ACTH) stimulation was carried out. Additionally, urinary steroids were quantified. Molecular analysis of CYP17 and CYP21A2 were also performed. RESULTS: All patients showed elevated basal 17alpha-deoxysteroid levels. Although the 17alpha-deoxysteroid levels further increased after rapid ACTH stimulation, 17alpha-hydroxysteroids including cortisol did not respond, suggesting impaired 17alpha-hydroxylation. Patient 1 and patient 2 showed low adrenal androgen blood levels both before and after rapid ACTH stimulation. Patient 3 showed lower than normal excretions of urinary androgens. Additionally, a prolonged ACTH stimulation in patient 3 failed to elicit significant increase of adrenal androgens. These findings suggested impaired 17,20-lyase activity. In contrast to attenuated 17alpha-hydroxycorticosteroids, notably cortisol, elevated 17alpha-hydroxyprogesterone (17OHP) levels were observed, not only in pubertal patients (1 and 2) but also in prepubertal patient 3, indicating impaired 21-hydroxylation. This assumption was supported by increased urinary 21-deoxycortisol metabolite excretion in patients 2 and 3. With the exception of a heterozygous mutation of CYP17 in one of the patients, other mutations of this gene or CYP21A2 were identified in any of the patients. CONCLUSION: Combined decreased 17alpha-hydroxylation, 17,20-lyase activity and 21-hydroxylation was detected in three ABS patients. Considering that the enzymes responsible are all cytochrome P450 enzymes and that another cytochrome P450 enzyme, lanosterol 14alpha-demethylase, has recently been shown to be impaired in an ABS patient, we speculate that dysfunction of a system which commonly regulates cytochrome P 450 activity may be responsible for the ABS phenotype.

Microbial hydroxylation of progesterone with Acremonium strictum.

Microbial hydroxylation of progesterone occurred in the culture of Acremonium strictum PTCC 5282 to produce two hydroxylated pregnene-like steroids. The metabolites were purified and characterized using spectroscopic methods and identified as 15alpha-hydroxyprogesterone and 15alpha-hydroxydeoxycorticosterone.

Effects of beta-endorphin and naloxone on corticosterone and cortisol release in the newt (Triturus carnifex): studies in vivo and in vitro.

The effects of beta-endorphin and its receptor antagonist, naloxone, on corticosterone and cortisol production in male and female Triturus carnifex were studied in vivo and in vitro. In the in-vivo experiment, the animals were injected s.c. with beta-endorphin and/or naloxone, and killed after 15, 30, 90 and 360 min. In the in-vitro experiment, interrenal tissues, with and without added pituitary, were incubated with beta-endorphin and/or naloxone for 15, 30, 60 and 120 min. The data obtained in vivo and in vitro from males and females were in agreement. Treatment with beta-endorphin caused a significant decrease in corticosterone and cortisol release, while naloxone induced an increase in the two corticosteroids at the same times as the decrease caused by beta-endorphin. The combined beta-endorphin plus naloxone treatment did not change corticosterone and cortisol levels. These results suggest that, in Triturus carnifex, opioids are involved in the regulation of the hypothalamo-pituitary-interrenal axis. In particular, the in-vitro results indicate a direct effect of opioids on interrenal steroidogenesis.

Splanchnic nerve stimulation modulates steroid secretion in hypophysectomized dogs.

To test whether or not splanchnic neural input to the adrenal gland affects secretion of steroids from the adrenal cortex, the thoracic splanchnic nerve was electrically stimulated in pentobarbital-anesthetized dogs after hypophysectomy and replacement with physiological concentrations of ACTH. An adrenal vein cannula was placed to permit measurement of cortisol, corticosterone, 11-deoxycortisol, epinephrine and norepinephrine secretion rates and adrenal blood flow. Plasma ACTH was measured and the presentation rate of ACTH was calculated as the product of plasma ACTH concentration and adrenal plasma flow. Dogs were infused initially with ACTH for 60 min at 2 ng/min followed by infusion for 60 min at 10 ng/min. Within each infusion period, the distal end of the nerve was stimulated (20 V; 0.5-ms pulse duration) at 4 and at 20 Hz for 10 min each. Nerve stimulation resulted in a frequency-dependent increase in mean arterial pressure, in epinephrine and norepinephrine secretion and in adrenal blood flow. Arterial ACTH remained constant during nerve stimulation; however, increased adrenal blood flow resulted in increased presentation rate of ACTH to the adrenal. Cortisol secretion increased in response to nerve stimulation at 4 and 20 Hz during infusion of 2 and 10 ng/min ACTH and occurred prior to changes in presentation rate of ACTH. Corticosterone secretion also increased after stimulation at both frequencies, but the response was observed only during infusion of 10 ng/min ACTH. In contrast, 11-deoxycortisol decreased after nerve stimulation at 4 Hz but showed no response after stimulation at 20 Hz during infusion of 2 and 10 ng/min ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of the cytoskeleton in the steroidogenic response of frog adrenal glands to angiotensin II, acetylcholine and serotonin.

In order to determine the role of the cytoskeleton in adrenal steroidogenesis, we have studied the effect of cytochalasin B (a microfilament-disrupting agent) and vinblastine (an antimicrotubular drug) on corticosteroid secretion by frog interrenal tissue in vitro. Perifusion of interrenal fragments with cytochalasin B (50 mumol/l) induced a marked inhibition of basal corticosteroid output. In addition, stimulation of corticosteroidogenesis by all corticotrophic factors was also inhibited by cytochalasin B. Using an immunohistochemical technique and specific anti-tubulin antiserum, we verified that vinblastine (10 mumol/l) was responsible for the disappearance of the microtubular network in adrenocortical cells. Administration of vinblastine (10 mumol/l) did not affect the spontaneous secretion of corticosterone and aldosterone and had no effect on the steroidogenic response of interrenal glands to angiotensin II and acetylcholine. In contrast, vinblastine was responsible for a marked decrease in serotonin-induced stimulation of corticosteroid production. On the other hand, data from high-performance liquid chromatography showed that infusion of cytochalasin B or vinblastine was not associated with the production of any new steroid which could interfere in the radioimmunoassays. Taken together, these data suggest that microfilaments are involved in a late and common step of corticosteroidogenesis while microtubules are only required for the coupling of the secretory response to certain corticotrophic factors such as ACTH and serotonin.

Steroid sequestration within the rat adrenal zona glomerulosa.

Accumulated data from in-vitro experiments have suggested that 18-hydroxysteroids may be stored within the intact rat adrenal zona glomerulosa. The phenomenon was further investigated by comparing the amount of steroid remaining in the zona glomerulosa tissue with that secreted into the media during incubation in vitro. The results showed that 18-hydroxydeoxycorticosterone (18-OH-DOC) and 18-hydroxycorticosterone (18-OH-B) were retained within the tissue against a considerable concentration gradient, with smaller amounts of aldosterone and corticosterone. Lysis of the intact zona glomerulosa, by preincubation in distilled water, yielded an enriched plasma membrane preparation. After subsequent incubation in Krebs-Ringer bicarbonate this preparation contained significantly more 18-OH-DOC than did the intact tissue, suggesting that tissue-sequestered 18-OH-DOC is normally metabolized to other products. These may include 18-OH-B and aldosterone. Fractionation of homogenized intact zona glomerulosa and the enriched plasma membrane preparation by density gradient centrifugation showed that tissue 18-OH-DOC banded in fractions of density 1.063-1.21 g/ml and that its distribution was highly correlated with protein. Corticosterone, 18-OH-B and aldosterone banded like added free [3H]18-OH-DOC in fractions of density less than 1.006 g/ml. The results suggest that 18-OH-DOC is the major sequestered steroid within the rat adrenal zona glomerulosa and that this sequestration is attributable to the association of 18-OH-DOC with a high-density component of the plasma membrane.

Effects of ACTH on steroidogenesis in bovine adrenocortical cells in primary culture--increased secretion of 17 alpha-hydroxylated steroids associated with a refractoriness in total steroid output.

The long-term effects of ACTH on steroidogenesis in bovine adrenocortical cells maintained in primary culture have been investigated. Cells in monolayer culture were incubated in the presence or absence of ACTH for up to 72 h, and the steroid content of the incubation medium was assayed at 12 h intervals. During the first 12 h, adrenocortical cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) produced substantially more cortisol and corticosterone than did cells incubated in the absence of ACTH. The production of steroidogenic intermediates such as pregnenolone, progesterone, and 17 alpha-hydroxypregnenolone, as well as 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11-deoxycorticosterone also was increased by short-term (12 h) treatment with ACTH. Thereafter, corticosteroid production by cells incubated in the continued presence of ACTH decreased in a time and concentration dependent fashion. The maximal rate of cortisol production by cells incubated in the presence of ACTH (10(-9) M and 10(-6) M) for 72 h was only one third that of cells incubated in the presence of ACTH for 12 h. More dramatically, by 36 h, corticosterone secretion by cells incubated in the presence of ACTH (10(-6) M) declined to less than 20% of that of nontreated cells, and the production of 11-deoxycorticosterone was no longer detectable. ACTH also induced refractoriness in the production of other C21-steroids (pregnenolone, progesterone, 17 alpha-hydroxypregnenolone, 17 alpha-hydroxyprogesterone, and 11-deoxycortisol) as well as of C19-steroids (dehydroepiandrosterone, androstenedione, and 11 beta-hydroxyandrostenedione). The ACTH-induced refractoriness in the production of C21-steroids lacking a 17 alpha-hydroxyl group occurred earlier than that of 17-hydroxylated C21-steroids. Despite the decline in total corticosteroid production, the long term effect of ACTH was to enhance the relative secretion of 17 alpha-hydroxylated steroids and C19-steroids. Adrenocortical cells incubated for 72 h in the presence of ACTH continued to secrete cortisol, 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and 11 beta-hydroxyandrostenedione in increased amounts. In fact, 11-deoxycortisol became a major secretory product of the ACTH-refractory adrenocortical cell. These results are indicative that ACTH acts in diverse manners on the bovine adrenocortical cell to affect corticosteroid secretion. The initial stimulation of corticosteroid production appears to be reflective of an increase in overall substrate (cholesterol) utilization and probably is mediated, in part, by an increase in cholesterol side chain cleavage activity. The secretion of 17 alpha-hydroxysteroids and C19-steroids is enhanced further by an action of ACTH to increase 17 alpha-hydroxylase activity and possibly also 17,20-lyase activity. The ACTH-induced refractoriness in corticosteroid production, on the other hand, appears to result primarily from a decline in precursor (cholesterol) utilization.

Aldose and aldehyde reductase exhibit isocorticosteroid reductase activity.

In this paper we describe the reduction of corticosteroid metabolites containing the 17 beta-aldol side chain (isocorticosteroids) by aldose and aldehyde reductase from human tissues. Aldose reductase catalyzed the reduction of the aldehydes derived from cortisol and corticosterone at about the same rate, whereas aldehyde reductase preferentially acted on the aldehydes derived from 17-deoxycorticosteroids. At comparable rates of reduction the Michaelis constants for the best steroid aldehydes were one order of magnitude lower than for the hitherto best substrates. We propose that aldose and aldehyde reductase participate in the conversion of the corticosteroid ketol side chain to the glycol side chain via an aldol intermediate by the 'long loop' pathway proposed by Monder and Bradlow [(1977) J. Steroid Biochem. 8, 897-908].

Effect of metoclopramide on the secretion of aldosterone and other adrenocortical steroids.

The aim of this study was to determine the effect of metoclopramide, a dopamine antagonist, on the secretion of aldosterone and other adrenocortical steroids in normal subjects. An i.v. bolus injection of 10 mg of metoclopramide significantly increased the plasma PRL, plasma aldosterone and 18-hydroxycorticosterone, but the plasma renin activity, plasma deoxycorticosterone, corticosterone and cortisol remained unchanged. The changes in plasma aldosterone induced by metoclopramide were significantly correlated with the basal levels of plasma aldosterone and renin activity. These results suggest that the response of plasma aldosterone to metoclopramide in normal subjects is influenced by the basal activity of renin-angiotensin-aldosterone system and the late step of aldosterone synthesis is stimulated by metoclopramide.

Quantification of polar glucocorticosteroids in the urine of pregnant and nonpregnant women: a comparison with 6 alpha-hydroxylated metabolites of cortisol in neonatal urine and amniotic fluid.

6 alpha-Hydroxy metabolites of cortisol were determined in the urine of pregnant (36-40 weeks of gestation) and nonpregnant women and in amniotic fluid from nearly fullterm pregnant women because relatively large amounts of these compounds are excreted in the urine of 2-day-old infants (> 200 micrograms/day). The corticosteroids analyzed by high pressure liquid chromatography and gas chromatography-mass spectrometry were 6 alpha-hydroxy derivatives of (allo)tetrahydrocortisone (3 alpha, 17 alpha, 21-trihydroxy-5 epsilon-pregnan-11,20-dione), (allo)tetrahydrocortisol (3 alpha, 11 beta, 17 alpha, 21-tetrahydroxy-5 epsilon-pregnan-20-one), and alpha- and beta-cortolone (3 alpha, 17 alpha, 20 epsilon, 21-tetrahydroxy-5 beta-pregnan-11-one). All of these compounds were found in the urine samples from both groups of women and in the amniotic fluid samples in contrast to those found in the urine samples from the neonates where 6 alpha-hydroxy compounds of (allo)tetrahydrocortisol and allotetrahydrocortisone were not positively identified because of insufficient yields. The pregnant women excreted significantly larger amounts of 6 alpha-hydroxy metabolites of cortisol (approximately 600 micrograms/day) than the control women (approximately 90 micrograms/day), and the rate of urinary excretion of these 6 alpha-hydroxy compounds was 7.82 and 1.30 micrograms/kg . day, respectively, for these groups of women compared to 54.3 micrograms/kg . day for the neonates. The precursors of these metabolites within the fetal body originated largely from the maternal circulation, and, therefore, the 6 alpha-hydroxy metabolites of cortisol excreted by the mother refer mainly to fetal metabolism and to a lesser extent, to the fetal secretion of cortisol.

[Use of diazepam for medication of feed for pigs]. / Pouzitelnost diazepamu k medikaci krmiva u prasat.

Diazepam was given to weaned piglets intramuscularly (0.25 and 1.0 mg . kg-1), individually per os (2 mg . kg-1), or in feed mixture (5, 10, 15 mg . kg-1 of feed) either alone or in combination with phenobarbital (100 mg . kg-1 of feed). The highest tranquillizing effect of diazepam was obtained after the single administration of the intramuscular dose of 1 mg . kg-1, oral dose of 2 mg . kg-1, and after the intake of 0.65 mg together with 6.5 mg of phenobarbital per kg of body weight in feed during 22 hours. Adrenocortical activity, increased in the controls as a result of the stress from handling, was also blocked in these cases. The piglets which had consumed a dose of 0.66 mg of diazepam with feed during 14 days after weaning, or 0.26 and 0.34 mg of diazepam together with 5.2 and 3.4 mg phenobarbital per kg b. w., showed a manifest trend to a lower level of plasma corticoids. At repeated administration, the effect on body weight gains was not explicit; the addition of phenobarbital had no effect. No symptoms of intoxication or impairment to the metabolism of the main nutrients and vitamin A were observed. The suitability of diazepam for the tranquilization of piglets was demonstrated and the practical advantages of its administration in feed mixture were emphasized, the best time of administration being a day prior to handling which is expected to exert an adverse effect on pig production and to render human work difficult.