[Cloning and functional analysis of 3ß-hydroxysteroid dehydrogenase gene involved in biosynthesis of digitoxin].

Digitoxin, an important secondary metabolite of Digitalis purpurea, is a commonly used cardiotonic in clinical practice. 3ß-Hydroxysteroid dehydrogenase(3ßHSD) is a key enzyme involved in the biosynthesis of digitoxin. It belongs to the short-chain dehydrogenase/reductase(SDR) family, playing a role in the biosynthesis of cardiac glycosides by oxidizing and isomerizing the precursor sterol. In this study, two 3ßHSD genes were cloned from D. purpurea. The results showed that the open reading frame(ORF) of Dp3ßHSD1 was 780 bp, encoding 259 amino acid residues. The ORF of Dp3ßHSD2 was 774 bp and encoded 257 residues. Dp3ßHSD1/2 had the cofactor binding site TGxxxA/GxG and the catalytic site YxxxK. In vitro experiments confirmed that Dp3ßHSD1/2 catalyzed the generation of progesterone from pregnenolone, and Dp3ßHSD1 had stronger catalytic capacity than Dp3ßHSD2. The expression level of Dp3ßHSD1 was much higher than that of Dp3ßHSD2 in leaves, and digitoxin was only accumulated in leaves. The results implied that Dp3ßHSD1 played a role in the dehydrogenation of pregnenolone to produce progesterone in the biosynthesis of digitoxin. This study provides a reference for further exploring the biosynthetic pathway of cardiac glycosides in D. purpurea.

The hypothalamic steroidogenic pathway mediates susceptibility to inflammation-evoked depression in female mice.

BACKGROUND: Depression is two-to-three times more frequent among women. The hypothalamus, a sexually dimorphic area, has been implicated in the pathophysiology of depression. Neuroinflammation-induced hypothalamic dysfunction underlies behaviors associated with depression. The lipopolysaccharide (LPS)-induced mouse model of depression has been well-validated in numerous laboratories, including our own, and is widely used to investigate the relationship between neuroinflammation and depression. However, the sex-specific differences in metabolic alterations underlying depression-associated hypothalamic neuroinflammation remain unknown. METHODS: Here, we employed the LPS-induced mouse model of depression to investigate hypothalamic metabolic changes in both male and female mice using a metabolomics approach. Through bioinformatics analysis, we confirmed the molecular pathways and biological processes associated with the identified metabolites. Furthermore, we employed quantitative real-time PCR, enzyme-linked immunosorbent assay, western blotting, and pharmacological interventions to further elucidate the underlying mechanisms. RESULTS: A total of 124 and 61 differential metabolites (DMs) were detected in male and female mice with depressive-like behavior, respectively, compared to their respective sex-matched control groups. Moreover, a comparison between female and male model mice identified 37 DMs. We capitalized on biochemical clustering and functional enrichment analyses to define the major metabolic changes in these DMs. More than 55% of the DMs clustered into lipids and lipid-like molecules, and an imbalance in lipids metabolism was presented in the hypothalamus. Furthermore, steroidogenic pathway was confirmed as a potential sex-specific pathway in the hypothalamus of female mice with depression. Pregnenolone, an upstream component of the steroid hormone biosynthesis pathway, was downregulated in female mice with depressive-like phenotypes but not in males and had considerable relevance to depressive-like behaviors in females. Moreover, exogenous pregnenolone infusion reversed depressive-like behaviors in female mice with depression. The 5α-reductase type I (SRD5A1), a steroidogenic hub enzyme involved in pregnenolone metabolism, was increased in the hypothalamus of female mice with depression. Its inhibition increased hypothalamic pregnenolone levels and ameliorated depressive-like behaviors in female mice with depression. CONCLUSIONS: Our study findings demonstrate a marked sexual dimorphism at the metabolic level in depression, particularly in hypothalamic steroidogenic metabolism, identifying a potential sex-specific pathway in female mice with depressive-like behaviors.

Hypothalamic pregnenolone mediates recognition memory in the context of metabolic disorders.

Obesity and type 2 diabetes are associated with cognitive dysfunction. Because the hypothalamus is implicated in energy balance control and memory disorders, we hypothesized that specific neurons in this brain region are at the interface of metabolism and cognition. Acute obesogenic diet administration in mice impaired recognition memory due to defective production of the neurosteroid precursor pregnenolone in the hypothalamus. Genetic interference with pregnenolone synthesis by Star deletion in hypothalamic POMC, but not AgRP neurons, deteriorated recognition memory independently of metabolic disturbances. Our data suggest that pregnenolone's effects on cognitive function were mediated via an autocrine mechanism on POMC neurons, influencing hippocampal long-term potentiation. The relevance of central pregnenolone on cognition was also confirmed in metabolically unhealthy patients with obesity. Our data reveal an unsuspected role for POMC neuron-derived neurosteroids in cognition. These results provide the basis for a framework to investigate new facets of POMC neuron biology with implications for cognitive disorders.

Stress and drug abuse-related disorders: The promising therapeutic value of neurosteroids focus on pregnenolone-progesterone-allopregnanolone pathway.

The pregnenolone-progesterone-allopregnanolone pathway is receiving increasing attention in research on the role of neurosteroids in pathophysiology, particularly in stress-related and drug use disorders. These disorders involve an allostatic change that may result from deficiencies in allostasis or adaptive responses, and may be downregulated by adjustments in neurotransmission by neurosteroids. The following is an overview of findings that assess how pregnenolone and/or allopregnanolone concentrations are altered in animal models of stress and after consumption of alcohol or cannabis-type drugs, as well as in patients with depression, anxiety, post-traumatic stress disorder or psychosis and/or in those diagnosed with alcohol or cannabis use disorders. Preclinical and clinical evidence shows that pregnenolone and allopregnanolone, operating according to a different or common pharmacological profile involving GABAergic and/or endocannabinoid system, may be relevant biomarkers of psychiatric disorders for therapeutic purposes. Hence, ongoing clinical trials implicate synthetic analogs of pregnenolone or allopregnanolone, and also modulators of neurosteroidogenesis.

Steroid profiles in quail brain and serum: Sex and regional differences and effects of castration with steroid replacement.

Both systemic and local production contribute to the concentration of steroids measured in the brain. This idea was originally based on rodent studies and was later extended to other species, including humans and birds. In quail, a widely used model in behavioural neuroendocrinology, it was demonstrated that all enzymes needed to produce sex steroids from cholesterol are expressed and active in the brain, although the actual concentrations of steroids produced were never investigated. We carried out a steroid profiling in multiple brain regions and serum of sexually mature male and female quail by gas chromatography coupled with mass spectrometry. The concentrations of some steroids (eg, corticosterone, progesterone and testosterone) were in equilibrium between the brain and periphery, whereas other steroids (eg, pregnenolone (PREG), 5α/ß-dihydroprogesterone and oestrogens) were more concentrated in the brain. In the brain regions investigated, PREG sulphate, progesterone and oestrogen concentrations were higher in the hypothalamus-preoptic area. Progesterone and its metabolites were more concentrated in the female than the male brain, whereas testosterone, its metabolites and dehydroepiandrosterone were more concentrated in males, suggesting that sex steroids present in quail brain mainly depend on their specific steroidogenic pathways in the ovaries and testes. However, the results of castration experiments suggested that sex steroids could also be produced in the brain independently of the peripheral source. Treatment with testosterone or oestradiol restored the concentrations of most androgens or oestrogens, respectively, although penetration of oestradiol in the brain appeared to be more limited. These studies illustrate the complex interaction between local brain synthesis and the supply from the periphery for the steroids present in the brain that are either directly active or represent the substrate of centrally located enzymes.

Polycistronic expression of the mitochondrial steroidogenic P450scc system in the HEK293T cell line.

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3ß-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.

Structural insights into the function of steroidogenic cytochrome P450 17A1.

Cytochrome P450 17A1 (CYP17A1) operates at the core of human steroidogenesis, directing precursors into mineralocorticoids, glucocorticoids, or sex steroids. Although the 17α-hydroxylase and 17,20-lyase activities of this dual function enzyme have been investigated extensively, until recently no CYP17A1 structures were available to inform our understanding. Structures of CYP17A1 with a range of steroidal inhibitors and substrates are now available. This review relates functional knowledge of this enzyme to structural features defining the selective differentiation between its various substrates. While both hydroxylase and lyase substrates have similar orientations with respect to the heme, subtle differences in hydrogen bonding between CYP17A1 and the C3 substituent at the opposite end of ligands appear to correlate with differential substrate utilization and product formation. Complementary structural information from solution NMR supports cytochrome b5 allosteric modulation of the lyase reaction, implicating regions involved in ligand access to the otherwise buried active site.

Gonadotropin-releasing hormone stimulates the biosynthesis of pregnenolone sulfate and dehydroepiandrosterone sulfate in the hypothalamus.

The sulfated neurosteroids pregnenolone sulfate (Δ(5)PS) 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 Δ(5)PS 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 hybridization histochemistry indicated that GnRHR-1 and GnRHR-3 mRNAs are particularly abundant in preoptic area and magnocellular nucleus whereas the concentration of GnRHR-2 mRNA in these 2 nuclei is much lower. Pulse-chase experiments using tritiated Δ(5)P and DHEA as steroid precursors, and 3'-phosphoadenosine 5'-phosphosulfate as a sulfonate moiety donor, showed that GnRH1 stimulates, in a dose-dependent manner, the biosynthesis of Δ(5)PS and DHEAS in frog diencephalic explants. Because Δ(5)PS and DHEAS, like GnRH, stimulate sexual activity, our data strongly suggest that some of the behavioral effects of GnRH could be mediated via the modulation of sulfated neurosteroid production.

In vitro effects of genistein and daidzein on the activity of adrenocortical steroidogenic enzymes in mature female pigs.

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), 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17α-hydroxylase/C17-20 lyase (P450c17) or 21-hydroxylase (P450c21) and 11ß-hydroxylase (P45011ß), 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 3ß-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 3ß-HSD level.

Inhibitory effect of protopanaxatriol ginseng metabolite M4 on the production of corticosteroids in ACTH-stimulated bovine adrenal fasciculata cells.

AIMS: We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. MAIN METHODS: The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography-mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. KEY FINDINGS: An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. SIGNIFICANCE: Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.

Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and clinical evidence.

Emerging preclinical and clinical evidence suggests that pregnenolone may be a promising novel therapeutic candidate in schizophrenia. Pregnenolone is a neurosteroid with pleiotropic actions in rodents that include the enhancement of learning and memory, neuritic outgrowth, and myelination. Further, pregnenolone administration results in elevations in downstream neurosteroids such as allopregnanolone, a molecule with neuroprotective effects that also increases neurogenesis, decreases apoptosis and inflammation, modulates the hypothalamic-pituitary-adrenal axis, and markedly increases GABA(A) receptor responses. In addition, pregnenolone administration elevates pregnenolone sulfate, a neurosteroid that positively modulates NMDA receptors. There are thus multiple mechanistic possibilities for pregnenolone as a potential therapeutic agent in schizophrenia, including the amelioration of NMDA receptor hypofunction (via metabolism to pregnenolone sulfate) and the mitigation of GABA dysregulation (via metabolism to allopregnanolone). Additional evidence consistent with a therapeutic role for pregnenolone in schizophrenia includes neurosteroid changes following administration of certain antipsychotics in rodent models. For example, clozapine elevates pregnenolone levels in rat hippocampus, and these increases may potentially contribute to its superior antipsychotic efficacy [Marx et al. (2006a) Pharmacol Biochem Behav 84:598-608]. Further, pregnenolone levels appear to be altered in postmortem brain tissue from patients with schizophrenia compared to control subjects [Marx et al. (2006c) Neuropsychopharmacology 31:1249-1263], suggesting that neurosteroid changes may play a role in the neurobiology of this disorder and/or its treatment. Although clinical trial data utilizing pregnenolone as a therapeutic agent in schizophrenia are currently limited, initial findings are encouraging. Treatment with adjunctive pregnenolone significantly decreased negative symptoms in patients with schizophrenia or schizoaffective disorder in a pilot proof-of-concept randomized controlled trial, and elevations in pregnenolone and allopregnanolone post-treatment with this intervention were correlated with cognitive improvements [Marx et al. (2009) Neuropsychopharmacology 34:1885-1903]. Another pilot randomized controlled trial recently presented at a scientific meeting demonstrated significant improvements in negative symptoms, verbal memory, and attention following treatment with adjunctive pregnenolone, in addition to enduring effects in a small subset of patients receiving pregnenolone longer-term [Savitz (2010) Society of Biological Psychiatry Annual Meeting New Orleans, LA]. A third pilot clinical trial reported significantly decreased positive symptoms and extrapyramidal side effects following adjunctive pregnenolone, in addition to increased attention and working memory performance [Ritsner et al. (2010) J Clin Psychiatry 71:1351-1362]. Future efforts in larger cohorts will be required to investigate pregnenolone as a possible therapeutic candidate in schizophrenia, but early efforts are promising and merit further investigation. This article is part of a Special Issue entitled: Neuroactive Steroids: Focus on Human Brain.

Chronic administration of the anabolic androgenic steroid nandrolone alters neurosteroid action at the sigma-1 receptor but not at the sigma-2 or NMDA receptors.

Studies have shown that anabolic androgenic steroids (AASs) can induce profound changes to mental health. Commonly reported psychiatric side effects among AAS users include aggression, anxiety, depression, drug abuse and cognitive disabilities. In experimental animals, many of these effects have been associated with alterations in a number of neurotransmitter systems. We have observed that chronic administration of the AAS nandrolone (nandrolone decanoate) can affect excitatory amino acids as well as monoaminergic and peptidergic pathways in a way that is compatible with nandrolone-induced behavioural changes. The aim of the present work was to further explore the mechanisms underlying nandrolone-induced effects, with a particular focus on components known to be involved in aggression and cognitive function. Male rats were given daily injections of nandrolone decanoate for 14 days and the effects on neurosteroid interactions with sites on the N-methyl-D-aspartyl (NMDA) and sigma receptors were examined. These receptors were chosen because of their involvement in aggressive and cognitive behaviors and the hypothesis that nandrolone might affect the brain via interaction with neurosteroids. Radiolabelled [³H]ifenprodil was used in the binding studies because of its significant affinity for the NMDA and sigma receptors. The results indicated that [³H]ifenprodil binds to both sigma-1 and sigma-2 sites and can be displaced to a certain extent from both sites by the neurosteroids pregnenolone sulphate (PS), pregnanolone sulphate (3α5ßS) and dehydroepiandrosterone sulphate (DHEAS). The remainder of the [³H]ifenprodil was displaced from the sigma-1 site by the sigma-1 receptor-selective ligand (+)-SKF 10,047. Chronic nandrolone treatment changed the sigma-1 receptor target for the neurosteroids but not for ifenprodil. The sigma-2 receptor site was unaltered by treatment with nandrolone decanoate. The results also indicated that the neurosteroid-induced allosteric modulation of the NMDA receptor subunit NR2B was not affected by nandrolone treatment. We conclude that chronic treatment with nandrolone changes the affinity of the neurosteroids PS, 3α5ßS and DHEAS at the sigma-1 site but not at the sites on the sigma-2 receptor or the NMDA receptor subunit NR2B.

Anxiolytic properties of a 2-phenylindolglyoxylamide TSPO ligand: Stimulation of in vitro neurosteroid production affecting GABAA receptor activity.

A number of neurosteroids have been demonstrated to exert anxiolytic properties by means of a positive modulation of inhibitory GABAergic neurotransmission. The observation that neurosteroid synthesis can be pharmacologically regulated by ligands to the mitochondrial translocator protein (TSPO) has prompted the search for new, more selective TSPO ligands able to stimulate steroidogenesis with great efficacy. In the present study, the potential anxiolytic activity of a selective TSPO ligand, N,N-di-n-propyl-2-(4-methylphenyl)indol-3-ylglyoxylamide (MPIGA), was tested by means of the elevated plus maze paradigm. Moreover, the in vitro effects on synaptoneurosomal GABA(A) receptor (GABA(A)R) activity exerted by the conditioned salt medium from MPIGA-treated ADF human glial cells were investigated. MPIGA (30mg/kg) was found to affect rats' performance in the elevated plus maze test significantly, leading to an increase in both entries and time spent in the open arms. This same dose of MPIGA had no effect on rats' spontaneous exploratory activity. The conditioned salt medium from MPIGA-treated ADF cells potentiated the (36)Cl(-) uptake into cerebral cortical synaptoneurosomes. The exposure of ADF cells to MPIGA stimulated the production of pregnelonone derivatives including allopregnanolone, one of the major positive GABA(A)R allosteric modulator. In conclusion, the TSPO ligand MPIGA is a promising anxiolytic drug. The mechanism of action by which MPIGA exerts its anxiolytic activity was identified in the stimulation of endogenous neurosteroid production, which in turn determined a positive modulation of GABA(A)R activity, thus opening the way to the potential use of this TSPO ligand in anxiety and depressive disorders.

Brain distribution and behavioral effects of progesterone and pregnenolone after intranasal or intravenous administration.

Neurosteroids hold great promise for the treatment of diseases of the central nervous system (CNS). We compared the uptake by 11 brain regions and appearance in blood of tritium-labeled pregnenolone and progesterone after intranasal and intravenous (IV) injection. Both neurosteroids appeared in blood and brain after either method of administration, but with important differences in uptake. Bioavailability based on appearance in arterial serum showed that about 23% and 14% of the intranasal administered doses of pregnenolone and progesterone, respectively, entered the blood. Brain levels were about two fold lower after intranasal administration for the two neurosteroids. With intranasal administration, brain levels of the two steroids did not vary over time (2-120 min), whereas brain levels were higher early (10 min or less) after i.v. administration. With i.v. administration, uptake by brain regions did not vary, whereas the olfactory bulb, hippocampus, and hypothalamus had high uptake rates after intranasal administration. Intranasal administration of prenenolone improved memory, whereas progesterone decreased anxiety, thus demonstrating that therapeutic levels of neurosteroids can be delivered to the brain by intranasal administration. The neurosteroids were rapidly degraded after i.v. or intranasal delivery, but pregnenolone was more resistant to degradation in the brain after intranasal administration and in serum after i.v. administration. These results show that either the i.v. or intranasal routes of administration can deliver neurosteroids to blood and brain, but that the two routes have significant differences with intranasal administration favoring some brain regions.

Testosterone synthesized in cultured human SZ95 sebocytes derives mainly from dehydroepiandrosterone.

Human sebaceous gland possesses all the steroidogenic enzymes required for androgen synthesis. It remains unclear whether the testosterone produced in situ mainly derives from circulating dehydroepiandrosterone (DHEA) or from de novo synthesis utilizing serum cholesterol. Using testosterone radioimmunoassay, we found that testosterone was barely detectable in the supernatant of cultured human SZ95 sebocytes when cholesterol was added alone, indicating a low basal expression of steroidogenic acute regulatory protein (StAR) in SZ95 cells. Human chorionic gonadotropin and fibroblast growth factor-9 were as potent as forskolin in activating StAR to enhance testosterone production, while interleukin-1 beta, dexamethasone, insulin and insulin-like growth factor-1 showed no stimulatory effect. A two-fold increase of testosterone production was observed in supplementation of DHEA as compared to pregnenolone, progesterone or 17 alpha-hydroxyprogesterone. Based on our findings, testosterone synthesized in cultured sebocytes derived mainly from DHEA and inhibition of 3beta-hydroxysteroid dehydrogenase and 17beta-hydroxysteroid dehydrogenase may be a new target of androgen suppression for acne treatment.

Pregnenolone, dehydroepiandrosterone, and schizophrenia: alterations and clinical trials.

Neurosteroids, such as pregnenolone (PREG), dehydroepiandrosterone (DHEA), and their sulfates (PREGS and DHEAS) are reported to have a modulatory effect on neuronal excitability and synaptic plasticity. They also have many other functions associated with neuroprotection, response to stress, mood regulation, and cognitive performance. Furthermore, these neurosteroids have been linked to, and their levels are altered in, neuropsychiatric disorders. This review highlights what is currently known about the metabolism and mode of action of PREG and DHEA, as well as about alterations of these neurosteroids in schizophrenia. This review also provides substantial information about clinical trials with DHEA and PREG augmentation with of antipsychotic agents in schizophrenia.

Effects of evodiamine and rutaecarpine on the secretion of corticosterone by zona fasciculata-reticularis cells in male rats.

Evodiamine (EVO) and rutaecarpine (RUT) are two bioactive alkaloid isolated from Chinese herb named Wu-Chu-Yu. Previous studies have shown that EVO and RUT possess thermoregulation, vascular regulation, anti-allergic, anti-nociceptive and anti-inflammatory activities. The mechanisms of EVO and RUT effect on steroidogenesis are not clear. The goal of this study was to characterize the mechanism by which EVO and RUT affect corticosterone production in rat zona fasciculata-reticularis (ZFR) cells. ZFR cells were isolated from adrenal glands of male rats and incubated with adrenalcorticotropin (ACTH, 10(-9) M), forskolin (an adenylyl cyclase activator, 10(-5) M), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP, a permeable cAMP analog, 10(-4) M), or steroidogenic precursors including 25-hydroxycholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10(-5) M each, in the presence or absence of EVO and RUT respectively (0-10(-3) M) at 37 degrees C for 1 h. The concentrations of corticosterone, pregnenolone and progesterone in the media were measured by radioimmunoassay. After administration of ZFR cells with EVO or RUT (10(-4) M) for 60 and 120 min, Western blot analysis was employed to explore the influence of EVO and RUT on the expression of cytochrome P450 side chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). EVO and RUT reduced both basal and ACTH-, forskolin-, as well as 8-Br-cAMP-stimulated corticosterone production in rat ZFR cells. The enhanced corticosterone production caused by the administration of four steroidogenic precursors was decreased following EVO or RUT challenge. These results suggest that EVO and RUT inhibit corticosterone production in rat ZFR cells via a mechanism involving: (1) a decreased activity of cAMP-related pathways; (2) a decreased activity of the steroidogenic enzymes, that is, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 11beta-hydroxylase (P450c11), during steroidogenesis; and (3) an inhibition of StAR protein expression.

Umbelliferone and esculetin: inhibitors or substrates for polyphenol oxidases?

Recently, an interesting debate arose about the nature (substrate versus inhibitor) of esculetin, a coumarin derivative, for mushroom polyphenol oxidase (PPO). The present study examined the behavior of PPOs preparations from fungal and plant origin towards esculetin as a substrate. Both enzymes were able to oxidize esculetin though at a slow rate. A higher sensitivity was reached when the assay was performed in the presence of 3-methyl-2-benzothiazolinone hydrazone (MBTH) even with a lower amount of PPO. These observations unambiguously confirmed that esculetin has to be considered a substrate for mushroom polyphenol oxidase. The oxidation of esculetin was also demonstrated for the first time by a fungal laccase. This should be taken into account because some mushroom PPO preparations could exert contaminant laccase activity. In addition, a PPO preparation from Ferula communis was demonstrated to use esculetin as a substrate. Umbelliferone, the monophenolic precursor of esculetin along the phenylpropanoid pathway, behaved as a competitive inhibitor for the monophenolase activity of mushroom PPO with a K(i) value=0.014 mM. This is worth a mention because only a few couples of mono- and corresponding o-diphenol show such opposite behavior towards PPO. A possible role of PPO in the esculetin fate along biosynthesis pathway of coumarin derivatives is also discussed.

The influence of Sutherlandia frutescens on adrenal steroidogenic cytochrome P450 enzymes.

AIM OF THE STUDY: The aim of this study was to investigate whether Sutherlandia frutescens, subsp. microphylla (family: Fabaceae/Leguminosa), which is traditionally used to treat symptoms of chronic stress generally associated with increased circulating glucocorticoids, influences the biosynthesis of these glucocorticoids. METHODS: We investigated the interaction of Sutherlandia frutescens with cytochrome P450 enzymes, CYP17 and CYP21, which catalyse key reactions in glucocorticoid biosynthesis. The binding of progesterone and pregnenolone to these enzymes and their metabolism were assayed in the presence of extracts and the bioactive compounds, l-canavanine, pinitol, GABA, flavonoids and triterpenoid glucosides present in the shrub. RESULTS: While the aqueous and methanol extracts inhibited the type I progesterone-induced difference spectrum (p<0.05), inhibition of pregnenolone binding (p=0.25) was negligible, with the aqueous extract exhibiting greater inhibition. The triterpenoid fraction inhibited both the type I pregnenolone- and progesterone-induced difference spectra and elicited a type II difference spectrum in the absence of substrate. Both pregnenolone and progesterone metabolism were inhibited by the aqueous extract, the inhibition of CYP21 being greater than that of CYP17, influencing the flux through glucocorticoid precursor pathways. CONCLUSION: This attenuation of adrenal P450 enzymes may thus demonstrate a possible mechanism by which Sutherlandia frutescens reduces glucocorticoid levels and alleviates symptoms associated with stress.

Steroid pregnenolone sulfate enhances NMDA-receptor-independent long-term potentiation at hippocampal CA1 synapses: role for L-type calcium channels and sigma-receptors.

Severe stress elevates plasma and CNS levels of endogenous neuroactive steroids that can contribute to the influence of stress on memory formation. Among the neuroactive steroids, pregnenolone sulfate (PREGS) reportedly strengthens memories and is readily available as a memory-enhancing supplement. PREGS actions on memory may reflect its ability to produce changes in memory-related neuronal circuits, such as long-term potentiation (LTP) of excitatory transmission in hippocampus. Here, we report a previously undiscovered pathway by which PREGS exposure promotes activity-dependent LTP of field excitatory postsynaptic potentials at CA1 synapses in hippocampal slices. Thus, application of PREGS, but not the phosphated conjugate of the steroid, selectively facilitates the induction of a slow-developing LTP in response to high-frequency (100 Hz) afferent stimulation, which is not induced in the absence of the steroid. The slow-developing LTP is independent of NMDA-receptor function (i.e., dAP5 insensitive) but dependent on functional L-type voltage-gated calcium channels (VGCC) and sigma-receptors. By contrast, PREGS at the highest concentration tested produces a depression in NMDA-receptor-dependent LTP, which is evident when sigma-receptor function is compromised by the presence of a sigma-receptor antagonist. We found that at early times during the induction phase of L-type VGCC-dependent LTP, PREGS via sigma-receptors transiently enhances presynaptic function. As well, during the maintenance phase of L-type VGCC-dependent LTP, PREGS promotes a further increase in presynaptic function downstream of LTP induction, as evidenced by a decrease in paired-pulse facilitation. The identification of complex regulatory actions of PREGS on LTP, involving sigma-receptors, L-type VGCCs, NMDA-receptors, and inhibitory circuits will aid future research endeavors aimed at understanding the precise mechanisms by which this stress-associated steroid may engage multiple LTP-signaling pathways that alter synaptic transmission at memory-related synapses.