Showing papers on "Pregnenolone published in 2013"
TL;DR: Mitochondria in the steroidogenic cells of the adrenal, gonad, placenta and brain contain the cholesterol side-chain cleavage enzyme, P450scc, and its two electron-transfer partners, ferredoxin reductase and ferred toxin, so that it serves as the chronic regulator of steroidogenesis.
321 citations
TL;DR: The skin can be defined as an independent steroidogenic organ, whose activity can affect its functions and the development of local or systemic inflammatory or autoimmune diseases.
289 citations
TL;DR: The levels of some neuroactive steroids in cerebrospinal fluid as well as in plasma may be valuable to predict their levels in the nervous system, and some steroids showed a full correlation with tissue levels.
111 citations
TL;DR: Age related neuropathological changes in AD brains were associated with modified levels of specific neuroactive steroids and particularly with changes in the levels of progesterone and testosterone metabolites, which might impact on the activation of neuroprotective signaling mediated by classic and nonclassic steroid receptors.
95 citations
TL;DR: This review will focus on molecular aspects of the neurosteroid, pregnenolone sulfate, its metabolism, concentrations in serum and tissues and last not least will summarize the functional data.
Abstract: Pregnenolone sulfate is a steroid metabolite with a plethora of actions and functions. As a neurosteroid, pregnenolone sulfate modulates a variety of ion channels, transporters, and enzymes. Interestingly, as a sulfated steroid, pregnenolone sulfate is not the final- or waste-product of pregnenolone being sulfated via a phase II metabolism reaction and renally excreted, as one would presume from the pharmacology textbook knowledge. Pregnenolone sulfate is also the source and thereby the starting point for subsequent steroid synthesis pathways. Most recently, pregnenolone sulfate has been functionally "upgraded" from modulator of ion channels to an activating ion channel ligand. This review will focus on molecular aspects of the neurosteroid, pregnenolone sulfate, its metabolism, concentrations in serum and tissues and last not least will summarize the functional data.
71 citations
TL;DR: Results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and D HEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species.
Abstract: Synthesis of the androgen dehydroepiandrosterone (DHEA) by the fetal adrenal gland is important for placental estrogen production and may also be important for modulating the effects of glucocorticoids on the developing brain. The presence of cortisol in spiny mouse (Acomys cahirinus) blood led us to determine whether the adrenal gland of this precocial rodent also synthesized DHEA. Cytochrome P450 enzyme 17α-hydroxylase/17,20-lyase (P450c17), cytochrome-b5 (Cytb5), and 3β-hydroxysteroid dehydrogenase (3βHSD) were detected in the adrenal gland from 30 days gestation (term = 39 days), and DHEA, cortisol, and aldosterone were detected in fetal plasma from this time. Plasma DHEA concentrations increased 4-fold, whereas cortisol concentrations decreased from day 30 of gestation until the day of birth. Explant culture of fetal adrenal tissue showed that DHEA was produced from exogenous pregnenolone, and thus, the DHEA in the fetal circulation is likely to be of fetal origin. Clear zonation of the fetal adrenal cortex was evident by 38 days gestation when expression of Cytb5 was present throughout the cortex, and coexpression of P450c17 and Cytb5 occurred in the zona reticularis and fasciculata. 3βHSD was expressed in the cortex from at least 30 days gestation and decreased as term approached, consistent with the fall of cortisol in late gestation in this species. These results show that the spiny mouse adrenal gland, like that of the human fetus, can synthesize and secrete DHEA from at least 30 days (relative gestation length, 30 days of a 39-day gestation, 0.76) of gestation, and DHEA may have important roles in placental biosynthesis of estrogens and in modulating the actions of glucocorticoids in the developing brain in this species.
53 citations
TL;DR: Although P 450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450Scc mutations may present later without disordered sexual development.
Abstract: Context: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously. Objective: We sought to expand clinical and genetic experience with P450scc deficiency. Patients and Methods: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc–Ferredoxin Reductase–Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel. Results: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe2...
50 citations
TL;DR: A novel process of delayed-onset potentiation whereby PRegS approximately doubles the cell’s response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS is described.
Abstract: N-methyl D-aspartate (NMDA) receptors (NMDARs) mediate fast excitatory synaptic transmission and play a critical role in synaptic plasticity associated with learning and memory. NMDAR hypoactivity has been implicated in the pathophysiology of schizophrenia, and clinical studies have revealed reduced negative symptoms of schizophrenia with a dose of pregnenolone that elevates serum levels of the neuroactive steroid pregnenolone sulfate (PregS). This report describes a novel process of delayed-onset potentiation whereby PregS approximately doubles the cell’s response to NMDA via a mechanism that is pharmacologically and kinetically distinct from rapid positive allosteric modulation by PregS. The number of functional cell-surface NMDARs in cortical neurons increases 60–100% within 10 minutes of exposure to PregS, as shown by surface biotinylation and affinity purification. Delayed-onset potentiation is reversible and selective for expressed receptors containing the NMDAR subunit subtype 2A (NR2A) or NR2B, but not the NR2C or NR2D, subunits. Moreover, substitution of NR2B J/K helices and M4 domain with the corresponding region of NR2D ablates rapid allosteric potentiation of the NMDA response by PregS but not delayed-onset potentiation. This demonstrates that the initial phase of rapid positive allosteric modulation is not a first step in NMDAR upregulation. Delayed-onset potentiation by PregS occurs via a noncanonical, pertussis toxin–sensitive, G protein–coupled, and Ca2+-dependent mechanism that is independent of NMDAR ion channel activation. Further investigation into the sequelae for PregS-stimulated trafficking of NMDARs to the neuronal cell surface may uncover a new target for the pharmacological treatment of disorders in which NMDAR hypofunction has been implicated.
38 citations
TL;DR: The first approval by FDA of an inhibitor of CYP17A1 for the treatment of castrate-resistant prostate cancer is approved, and ongoing clinical trials are now evaluating the agent in earlier stages of prostate cancer and even rare forms of androgen-dependent breast cancer.
Abstract: Cytochrome P450 17A1 (CYP17A1; also P450c17and P450sccII) is a critically important enzyme in humans that catalyzes the formation of all endogenous androgens. It is an atypical cytochrome P450 enzyme in that it catalyzes two distinct types of substrate oxidation. Through its hydroxylase activity, it catalyzes the 17α-hydroxylation of pregnenolone to 17α-OH pregnenolone. Subsequently, through its C17,20lyase activity, it can further convert 17α-OH pregnenolone to the androgen dehydroepiandrosterone, which is a precursor to androstenedione, testosterone, and dihydrotestosterone. The importance of androgens in diseases such as prostate cancer has been appreciated for decades and the discovery of extra-testicular formation of androgens has helped clarify the pathology of the disease, especially the castrate- resistant disease. Therefore, specific inhibition of CYP17A1 by therapeutic intervention has been an area of considerable effort in several research laboratories. This basic research has led to the discovery of several promising drug candidates followed by the conduct of several clinical trials. Recently, all these efforts have culminated in the first approval by FDA of an inhibitor of CYP17A1 for the treatment of castrate-resistant prostate cancer. Ongoing clinical trials are now evaluating the agent in earlier stages of prostate cancer and even rare forms of androgen-dependent breast cancer. Accordingly, this review focuses on the biochemistry, chemistry, and clinical inhibitors of CYP17A1.
38 citations
TL;DR: An accurate determination of tissue steroids was developed using LC-MS analysis and it showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver.
Abstract: The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography–electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.
34 citations
TL;DR: This is the first report showing that Aβ25-35, a main etiological factor of Alzheimer's disease, can alter the level and metabolism of neuroactive steroids in the prefrontal cortex and hippocampus, which are brain regions significantly involved in learning and memory.
TL;DR: The results indicate that thioxanthone derivatives can act as potential endocrine disruptors both at the level of nuclear receptor signaling and steroid hormone production.
TL;DR: Apigenin was more potent than other polyphenols in increasing the levels of pregnenolone and 17α-hydroxyprogesterone, suggesting that it inhibits cytochrome P450 (CYP) 17 and CYP21, as well as 3β-HSD, the first study to demonstrate the inhibitory effects of apigenin on CYP17 and CyP21.
Abstract: Modulating steroid hormone levels is a curative and preventive measure for Cushing's syndrome, aldosteronism, and various stress-triggered symptoms. Polyphenols have been reported to inhibit steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (3β-HSD) and aromatase. However, evidence for their inhibitory effects is fragmentary because it has been determined in studies with small groups of steroid hormones. To investigate the effects of steroids on complete steroidogenic pathways, comprehensive analysis of steroid hormones is necessary. Here we cultured forskolin-stimulated NCI-H295R, a human adrenocortical carcinoma cell line, in the presence of a polyphenol and employed GC-MS to simultaneously determine the levels of nine steroid hormones (pregnenolone, progesterone, deoxycorticosterone, aldosterone, 17α-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and estradiol) in cell culture supernatant. We found that daidzein, genistein, apigenin, hesperetin, naringenin, and eriodictyol significantly reduced deoxycorticosterone and androstenedione levels (p<0.05), suggesting inhibition of 3β-HSD by these polyphenols. Apigenin was more potent than other polyphenols in increasing the levels of pregnenolone and 17α-hydroxyprogesterone, suggesting that it inhibits cytochrome P450 (CYP) 17 and CYP21, as well as 3β-HSD. Real-time reverse transcription polymerase chain reaction showed that apigenin significantly downregulated the expression levels of 3β-HSD, CYP17, and CYP21 mRNA (p<0.05). This is the first study to demonstrate the inhibitory effects of apigenin on CYP17 and CYP21.
TL;DR: The results indicated that compounds 15, 17a, 18 and 20e among the tested compounds showed the highest cytotoxicity against the cancer cell lines.
TL;DR: Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T(H)2 differentiation.
Abstract: Background Cytochrome P450, family 11, subfamily A, polypeptide 1 (Cyp11a1), a cytochrome P450 enzyme, is the first and rate-limiting enzyme in the steroidogenic pathway, converting cholesterol to pregnenolone. Cyp11a1 expression is increased in activated T cells. Objectives We sought to determine the role of Cyp11a1 activation in the development of peanut allergy and T H cell functional differentiation. Methods A Cyp11a1 inhibitor, aminoglutethimide (AMG), was administered to peanut-sensitized and challenged mice. Clinical symptoms, intestinal inflammation, and Cyp11a1 levels were assessed. The effects of Cyp11a1 inhibition on T H 1, T H 2, and T H 17 differentiation were determined. Cyp11a1 gene silencing was performed with Cyp11a1-targeted short hairpin RNA. Results Peanut sensitization and challenge resulted in diarrhea, inflammation, and increased levels of Cyp11a1 , IL13 , and IL17A mRNA in the small intestine. Inhibition of Cyp11a1 with AMG prevented allergic diarrhea and inflammation. Levels of pregnenolone in serum were reduced in parallel. AMG treatment decreased IL13 and IL17A mRNA expression in the small intestine without affecting Cyp11a1 mRNA or protein levels. In vitro the inhibitor decreased IL13 and IL17A mRNA and protein levels in differentiated T H 2 and T H 17 CD4 T cells, respectively, without affecting GATA3 , retinoic acid–related orphan receptor γt (RORγt) , or T H 1 cells and IFNG and T-bet expression. Short hairpin RNA–mediated silencing of Cyp11a1 in polarized T H 2 CD4 T cells significantly decreased pregnenolone and IL13 mRNA and protein levels. Conclusion Cyp11a1 plays an important role in the development of peanut allergy, regulating peanut-induced allergic responses through effects on steroidogenesis, an essential pathway in T H 2 differentiation. Cyp11a1 thus serves as a novel target in the regulation and treatment of peanut allergy.
TL;DR: Concise data are presented on three of the most important biological targets for nitrogen-containing steroids: enzymes 17α-hydroxylase-17/20-lyase (CYP17), aromatase ( CYP19), and 24-sterol methyltransferase (SMT).
Abstract: Data published over the last 15 years on the chemical synthesis of biologically active steroids with nitrogen-containing substituents (mostly containing nitrogen heterocycles) in ring D are reviewed. Modern methods for the synthesis of nitrogen-containing derivatives from 17-keto steroids and 20-keto steroids and certain other methods for the synthesis of the target products are discussed. Concise data are also presented on three of the most important biological targets for nitrogen-containing steroids: enzymes 17α-hydroxylase-17/20-lyase (CYP17), aromatase (CYP19), and 24-sterol methyltransferase (SMT).
TL;DR: Assessment of whether chronic hyperammonemia alters the modulation by different neurosteroids of GABAA, NMDA, and/or sigma receptors and of the glutamate–NO–cGMP pathway in cerebellum found pregnanolone and pregnenolone sulfate could restore cognitive function in hyper ammonemia and encephalopathy.
Abstract: Several neurosteroids modulate the glutamate-nitric oxide (NO)-cGMP pathway in cerebellum through modulation of NMDA- GABAA - or sigma receptors. Hyperammonemia alters the concentration of several neurosteroids and impairs the glutamate-NO-cGMP pathway, leading to impaired learning ability. This work aimed to assess whether chronic hyperammonemia alters the modulation by different neurosteroids of GABAA, NMDA, and/or sigma receptors and of the glutamate-NO-cGMP pathway in cerebellum. Neurosteroids were administered through microdialysis probes, and extracellular cGMP and citrulline were measured. Then NMDA was administered to assess the effects on the glutamate-NO-cGMP pathway activation. Hyperammonemia completely modifies the effects of pregnanolone and pregnenolone. Pregnanolone acts as a GABAA receptor agonist in controls, but as an NMDA receptor antagonist in hyperammonemic rats. Pregnenolone does not induce any effect in controls, but acts as a sigma receptor agonist in hyperammonemic rats. Hyperammonemia potentiates the actions of tetrahydrodeoxy-corticosterone (THDOC) as a GABAA receptor agonist, allopregnanolone as an NMDA receptor antagonist, and pregnenolone sulfate as an NMDA receptor activation enhancer. Neurosteroids that reduce the pathway (pregnanolone, THDOC, allopregnanolone, DHEAS) may contribute to cognitive impairment in hyperammonemia and hepatic encephalopathy. Pregnenolone would impair cognitive function in hyperammonemia. Neurosteroids that restore the pathway in hyperammonemia (pregnenolone sulfate) could restore cognitive function in hyperammonemia and encephalopathy.
TL;DR: Investigation has shown that SPE may relatively easily extract and recover steroid hormones, potentially replacing LLE, in H295R studies.
TL;DR: This short review summarizes studies that have begun to provide insight into the role of the cytoskeleton, mitochondrial transport, and the physical interaction of the ER and mitochondria in the production of adrenocortical steroid hormones.
TL;DR: Docking simulations and experimental data indicate that 22-NBD-cholesterol, a common fluorescent sterol analog, binds into active sites of bovine cytochrome P450scc and microbial cholesterol dehydrogenases (CHDHs) and then undergoes regiospecific oxidations by these enzymes, indicating intricate character of the reaction.
TL;DR: In vertebrates, the biosynthesis of steroid hormones is initiated by cytochrome P450 CYP11A1 which converts cholesterol to pregnenolone, and it is demonstrated that P450 association with the lipid bilayer determines, in many cases, a drug's effect on enzyme activity.
Journal Article•
TL;DR: It is suggested that genistein and daidzein present in soy products may negatively affect glucocorticoid synthesis of mature gilts by disrupting adrenal steroidogenesis at the 3β-HSD level.
Abstract: Soy products, commonly used as a protein source in farm animals' diets, contain considerable quantities of non-nutrient constituents such as phytoestrogens. Genistein and daidzein are known to affect the reproductive processes in humans and animals. However, reports concerning phytoestrogens and porcine adrenal steroidogenesis are scarce, and the adrenal mechanism of phytoestrogen action in species other than humans and rodents is poorly recognized. The goal of the present paper was to examine the in vitro effects of genistein and daidzein on the activity of key enzymes for cortisol and corticosterone synthesis in porcine adrenocortical cells harvested during the luteal or follicular phase of the porcine estrous cycle. The cells were treated with genistein or daidzein (10 µM), with or without ACTH (5 nM), in the presence or absence of precursors (1 µM) of cortisol (pregnenolone, P5; progesterone, P4; 17-hydroxyprogesterone, 17OH-P4; or 11-deoxycortisol, 11d-cortisol) or corticosterone: (P5 or P4) synthesis. The supplementation of a medium with P5, P4, 17OH-P4 or 11d-cortisol enabled us to measure the activity of cholesterol side-chain cleavage enzyme (P450scc), 3bhydroxysteroid dehydrogenase (3b-HSD), 17a-hydroxylase/C17-20 lyase (P450c17) or 21-hydroxylase (P450c21) and 11b-hydroxylase (P45011b), respectively. We demonstrated that in sexually mature, cyclic pigs, regardless of the phase of the estrous cycle, phytoestrogens genistein and daidzein suppressed basal and ACTH-stimulated in vitro secretion of cortisol and corticosterone via progesterone synthesis inhibition. This indicates that phytoestrogens specifically inhibit the 3b-HSD activity in porcine adrenocortical cells. We suggest that genistein and daidzein present in soy products may negatively affect glucocorticoid synthesis of mature gilts by disrupting adrenal steroidogenesis at the 3b-HSD level.
TL;DR: The main neurobiological effects of both forms, DHEA and DHEAS, on the brain structures, include the neuroprotective effects, neurogenesis and survival of neurons, apoptosis, and the effect on the synthesis and secretion of catecholamines.
Abstract: In the past 30 years it has become clear that the brain tissue and the nervous system are steroidproducing structures. Steroids synthesized in the brain structures are called neurosteroids. This paper summarizes the results of studies on the biosynthesis and metabolism of dehydroepiandrosterone (DHEA), including its metabolism in the adipose tissue, where it serves as a substrate for intracellular formation of biologically active metabolites estradiol and testosterone. The role of sulfatase and sulfotransferase in mutual conversions of DHEA and DHEA sulfate (DHEAS) is described. Species-related differences in the synthesis of DHEA in the adrenal cortex are considered. The adrenal glands of primates (humans and monkeys, including the lower ones) produce large quantities of free and sulfated DHEA. Their synthesis proceeds by the Δ5 pathway: cholesterol → pregnenolone → 17-hydroxypregnenolone → DHEA. The adrenal glands of other species, including rats and mice, do not synthesize DHEA. Out point of view on the possible mechanisms of penetration of endogenous or exogenous DHEA sulfate into the brain structures is described: desulfurization of molecules to form free DHEA penetrating the blood-brain barrier and the possibility of penetration of the sulfate form into the hypothalamic structures, which are not protected by the blood-brain barrier. The results of studies of the use of DHEA as a neurosteroid in clinical practice and the analysis of its role in the development of Alzheimer’s disease, cognitive disorders, and other CNS disorders are also presented. The possible mechanisms underlying the effects of DHEA on the brain are considered. The main neurobiological effects of both forms, DHEA and DHEAS, on the brain structures, which were identified experimentally in various animal models, include the neuroprotective effects, neurogenesis and survival of neurons, apoptosis, and the effect on the synthesis and secretion of catecholamines. Neurosteroids also carry out antioxidative, antiinflammatory, and antiglucocorticoid activity.
TL;DR: The SNP located within the WT1 allele may have a potential application in marker-assisted selection of sheep exhibiting a greater release of cortisol from the adrenal gland in response to stressors.
Abstract: In commercial production systems, the full expression of the genetic potential of an animal is limited by its intrinsic and extrinsic environment. It is therefore necessary to include robustness as a breeding goal because robustness is defined as the ability of an animal to express a high production potential in a wide variety of environmental conditions. The ability of mammals to produce sufficient cortisol on stimulation of the hypothalamic-pituitary-adrenal (HPA) axis is vital in its adaptation to stress. The biosynthesis of cortisol is dependent on the enzymatic activity of the microsomal enzyme, cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17). Two isoforms for sheep (Ovis aries) CYP17, previously identified in 2 independent studies, differ by 2 nucleotides, resulting in 2 AA differences (Ser210Gly and Tyr464Asn). The present study investigates the effect of these differences on cortisol production as a function of the HPA axis activity by comparing the catalytic activities of these isoforms. The activities of the CYP17 isoforms were compared by expressing the enzymes in vitro. The kinetic constants, Vmax and Km, which were determined for pregnenolone and progesterone (in the absence of cytochrome b(5)), showed no significant difference (P > 0.05) between the CYP17 isoforms. In contrast, a time course of the metabolism of pregnenolone, 17-hydroxypregnenolone, and progesterone, assayed in the presence and absence of ovine cytochrome b(5) overexpression, showed significant differences (P < 0.05) between the isoforms. Wild-type 1 CYP17 (WT1, GenBank accession number L40335) yielded more cortisol precursors than wild-type 2 (WT2, GenBank accession number AF251388). Site-directed mutagenesis indicated that a tyrosine residue at position 464 of WT1 increased the 17α-hydroxylation of progesterone compared with an asparagine residue at that position of WT2. In a subsequent insulin-induced hypoglycemic stress test, the presence of WT1 resulted in a greater cortisol output from the sheep adrenal than the presence of WT2, as homozygous WT1/WT1 sheep produced more cortisol than heterozygous WT1/WT2 sheep. The SNP located within the WT1 allele may therefore have a potential application in marker-assisted selection of sheep exhibiting a greater release of cortisol from the adrenal gland in response to stressors.
TL;DR: 3β,14α-Dihydroxypregn-5-en-7,20-dione was identified as a new metabolite.
Abstract: In this work, incubations of testosterone, dehydroepiandrosterone and pregnenolone with Mucor hiemalis MRC 70325 have been reported. Incubation of testosterone afforded androst-4-en-3,17-dione (3%)...
TL;DR: Galeterone is a selective and potent CYP17 lyase inhibitor that also antagonizes and degrades the AR, and exhibits minimal evidence of deleterious steroid changes associated with ME, recapitulate phase I clinical experience where no ME was observed or prednisone required in CRPC patients.
Abstract: 184 Background: Because CYP17 has a central role in converting progestogens to androgens, inhibitors have been developed to treat CRPC. CYP17 has both hydroxylase and lyase catalytic functions. However, selective hydroxylase inhibition causes build-up of progestogens and mineralocorticoids, resulting in secondary mineralocorticoid excess (ME), edema, hypokalemia and hypertension (treated clinically with prednisone). We analyzed four inhibitors for their effects on CYP17 hydroxylase and lyase activities, and more globally on the steroidogenic pathway. Methods: Human CYP17 expressed in yeast microsomes was incubated with pregnenolone or 17α-hydroxypregnenolone and products quantitated using LC/MS/MS. Galeterone, abiraterone, orteronel or ketoconazole were added, and hydroxylase or lyase IC50 values calculated. In a separate experiment, H295R adenocarcinoma cells were incubated with a range of drug concentrations between 0.316 nM and 10 μM for 24 hours and media was analyzed for steroid production by LC/MS/M...
TL;DR: The study shows that the spatial separation of steroidogenic enzymes within the ER has a minimal effect on the rate of steroid synthesis and the compartmentalization of the enzymes into different organelles of a cell creates cellular steroid gradients and can affect the balance of the different steroids.
TL;DR: Pulse-chase experiments showed that GnRH1 stimulates, in a dose-dependent manner, the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants, which strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.
Abstract: The sulfated neurosteroids pregnenolone sulfate (Δ5PS) and dehydroepiandrosterone sulfate (DHEAS) are known to play a role in the control of reproductive behavior. In the frog Pelophylax ridibundus, the enzyme hydroxysteroid sulfotransferase (HST), responsible for the biosynthesis of Δ5PS and DHEAS, is expressed in the magnocellular nucleus and the anterior preoptic area, two hypothalamic regions that are richly innervated by GnRH1-containing fibers. This observation suggests that GnRH1 may regulate the formation of sulfated neurosteroids to control sexual activity. Double labeling of frog brain slices with HST and GnRH1 antibodies revealed that GnRH1-immunoreactive fibers are located in close vicinity of HST-positive neurons. The cDNAs encoding 3 GnRH receptors (designated riGnRHR-1, -2, and -3) were cloned from the frog brain. RT-PCR analyses revealed that riGnRHR-1 is strongly expressed in the hypothalamus and the pituitary whereas riGnRHR-2 and -3 are primarily expressed in the brain. In situ hybridiz...
TL;DR: A stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work and may open some perspectives for further generation of novel microbial biocatalysts.
Abstract: Mammalian cytochrome P450scc enzyme system catalyzes the initial step in steroid hormone biosynthesis—cholesterol hydroxylation followed by cleavage of the side-chain to yield pregnenolone. This system consists of three components—the cytochrome P450scc (CYP11A1), a flavoprotein (NADPH-adrenodoxin reductase, AdR) and an iron-sulfur protein (adrenodoxin, Adx). In this work, the three-component electron transport chain (AdR/Adx/CYP11A1) from bovine adrenal cortex has been implemented in Escherichia coli by co-expression of the corresponding coding sequences from a tricistronic plasmid. The cDNAs of AdR, Adx and CYP11A1 are situated in a single transcription unit and separated by ribosome binding sequences. The recombinant strain created was capable of synthesizing functional proteins identical to the bovine CYP11A1, AdR and Adx on molecular weights and immuno-specificity. The experiments in vivo showed pregnenolone production from cholesterol by the transformed bacteria. Maximal productivity of 0.42 ± 0.015 mg/l pregnenolone for 24 h has been reached for the induced cells in the presence of cholesterol solubilizing agent—methyl-β-cyclodextrin. Thus, a stable transgenic E. coli strain with the functional reconstructed bovine cholesterol side-chain cleavage system has been firstly generated in this work. The findings are of importance for studies of mammalian steroidogenic system features, and may open some perspectives for further generation of novel microbial biocatalysts.
TL;DR: The idea that the most upstream effect of mono-OH MXC on steroidogenesis is by reducing the availability of pregnenolone is supported, and that adding pregnanolone may not be sufficient to prevent inhibited follicle growth and survival is supported.