Alterations in bile acid metabolizing gut microbiota and specific bile acid genes as a precision medicine to subclassify NAFLD.

Multiple mechanisms for the gut microbiome contributing to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) have been implicated. Here, we aim to investigate the contribution and potential application for altered bile acids (BA) metabolizing microbes in NAFLD by post hoc analysis of whole metagenome sequencing (WMS) data. The discovery cohort consisted of 86 well-characterized patients with biopsy-proven NAFLD and 38 healthy controls. Assembly-based analysis was performed to identify BA-metabolizing microbes. Statistical tests, feature selection, and microbial coabundance analysis were integrated to identify microbial alterations and markers in NAFLD. An independent validation cohort was subjected to similar analyses. NAFLD microbiota exhibited decreased diversity and microbial associations. We established a classifier model with 53 differential species exhibiting a robust diagnostic accuracy [area under the receiver-operator curve (AUC) = 0.97] for detecting NAFLD. Next, eight important differential pathway markers including secondary BA biosynthesis were identified. Specifically, increased abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH), 3α-hydroxysteroid dehydrogenase (baiA), and bile acid-coenzyme A ligase (baiB) was detected in NAFLD. Furthermore, 10 of 50 BA-metabolizing metagenome-assembled genomes (MAGs) from Bacteroides ovatus and Eubacterium biforme were dominant in NAFLD and interplayed as a synergetic ecological guild. Importantly, two subtypes of patients with NAFLD were observed according to secondary BA metabolism potentials. Elevated capability for secondary BA biosynthesis was also observed in the validation cohort. These bacterial BA-metabolizing genes and microbes identified in this study may serve as disease markers. Microbial differences in BA-metabolism and strain-specific differences among patients highlight the potential for precision medicine in NAFLD treatment.

Combined MD and QM/MM Investigations of Hydride Reduction of 5α-Dihydrotestosterone Catalyzed by Human 3α-Hydroxysteroid Dehydrogenase Type 3: Importance of Noncovalent Interactions.

3α-Hydroxysteroid dehydrogenase (3α-HSD) is an enzyme that is essential in the regulation of the concentration of 5α-dihydrotestosterone (5α-DHT) in the prostate. It catalyzes the hydride reduction of 5α-DHT to 3α-androstanediol, which activates androgen receptors. Elucidating details about the hydride reduction of 5α-DHT by 3α-HSD and the environment around the active site of the enzyme could lead to the development of effective drugs for the treatment of prostate cancer. In this study, the X-ray crystal structure of human 3α-HSD type 3 was comprehensively evaluated. Moreover, molecular dynamics (MD) simulations and hybrid ONIOM-type quantum mechanics/molecular mechanics (QM/MM) calculations were performed using a large QM region (maximum 232 atoms). It was determined that the reaction proceeded in a single step without the formation of an alkoxide ion owing to the direct hydride reduction of the substrate by nicotinamide adenine dinucleotide phosphate (NADPH) and concerted proton transfer by Tyr55 and Lys84. Noncovalent interaction (NCI) analysis highlighted the roles of Tyr216 and Trp227 in 3α-HSD. Specifically, Tyr216 assisted the reaction by π/π interactions with the neighboring nicotinamide ring of NADP(H), whereas Trp227 played an important role in recognition of the size of the substrate by CH/π interactions.

Enzymatic methods may underestimate the total serum bile acid concentration.

Enzymatic assays based on bacterial 3α-hydroxysteroid dehydrogenase are the method of choice for quantification of total bile acids (BAs) in serum. Although non-specific, it is generally considered precise and robust. The aim of this study was to investigate how changes in the BA spectrum might affect the reliability of the method. We measured standard solutions of twenty-three human and murine BAs using a commercial enzymatic assay and compared the measured vs. expected concentrations. Additionally, total BA concentrations in rat and human cholestatic samples with an abnormal BA spectrum were measured using an enzymatic assay, and a more specific LC-MS/MS method. We observed a great variability in the response of individual BAs in the enzymatic assay. Relative signal intensities ranged from 100% in glycocholic acid (reference) to only 20% in α-muricholic acid. The enzymatic assay markedly underestimated the BA concentrations in both human and rat cholestatic sera when compared to the LC-MS/MS assay. Our study indicated that the performance of an enzymatic assay largely depends on the BA spectrum, and the total concentration of BAs can be markedly underestimated. Samples with an atypical BA spectrum (viz. in rodents) should preferably be measured by other methods.

Role of human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C3) in the extrahepatic metabolism of the steroidal aromatase inactivator Formestane.

The clinical use of the steroidal aromatase inhibitor Formestane (4-hydroxandrostenedione, 4-OHA) in the treatment of advanced ER+ breast cancer has been discontinued, and therefore, interest in this remarkable drug has vanished. As a C-19 sterol, 4-OHA can undergo extensive intracellular metabolism depending on the expression of specific enzymes in the corresponding cells. We used the metabolites 4ß-hydroxyandrosterone, 4ß-hydroxyepiandrosterone and its 17ß-reduced derivative as standards for the proof of catalytic activity present in the cell culture medium and expressed by the isolated enzymes. All of the aldo-keto reductases AKR1C1, AKR1C2, AKR1C3 and AKR1C4 catalysed the reduction of the 3-keto-group and the Δ4,5 double bond of 4-OHA at the same time. Molecular docking experiments using microscale thermophoresis and the examination of the kinetic behaviour of the isolated enzymes with the substrate 4-OHA proved that AKR1C3 had the highest affinity for the substrate, whereas AKR1C1 was the most efficient enzyme. Both enzymes (AKR1C1and AKR1C3) are highly expressed in adipose tissue and lungs, exhibiting 3ß-HSD activity. The possibility that 4-OHA generates biologically active derivatives such as the androgen 4-hydroxytestosterone or some 17ß-hydroxy derivatives of the 5α-reduced metabolites may reawaken interest in Formestane, provided that a suitable method of administration can be developed, avoiding oral or intramuscular depot-injection administration.

Sex differences in the brain expression of steroidogenic molecules under basal conditions and after gonadectomy.

The brain is a steroidogenic tissue. It expresses key molecules involved in the synthesis and metabolism of neuroactive steroids, such as steroidogenic acute regulatory protein (StAR), translocator protein 18 kDa (TSPO), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), 3ß-hydroxysteroid dehydrogenases (3ß-HSD), 5α-reductases (5α-R) and 3α-hydroxysteroid oxidoreductases (3α-HSOR). Previous studies have shown that the levels of brain steroids are different in male and female rats under basal conditions and after gonadectomy. In the present study, we assessed gene expression of key neurosteroidogenic molecules in the cerebral cortex and cerebellum of gonadally intact and gonadectomised adult male and female rats. In the cerebellum, the basal mRNA levels of StAR and 3α-HSOR were significantly higher in females than in males. By contrast, the mRNA levels of TSPO and 5α-R were significantly higher in males. In the cerebral cortex, all neurosteroidogenic molecules analysed showed similar mRNA levels in males and females. Gonadectomy increased the expression of 5α-R in the brain of both sexes, although it affected the brain expression of StAR, TSPO, P450scc and 3α-HSOR in females only and with regional differences. Although protein levels were not investigated in the present study, our findings indicate that mRNA expression of steroidogenic molecules in the adult rat brain is sexually dimorphic and presents regional specificity, both under basal conditions and after gonadectomy. Thus, local steroidogenesis may contribute to the reported sex and regional differences in the levels of brain neuroactive steroids and may be involved in the generation of sex differences in the adult brain function.

Site-specific observation of the conformational change of a protein with 15N-labeled Tyr residues using NMR.

One of the reasons it is difficult to analyze protein structural dynamics at atomic resolution using NMR is the molecular size of the protein. The selective amino acid labeling method is one of the effective methods that can solve this problem. In this study, to determine the site-specific conformational change in 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD), which forms a dimer composed of two 26 kDa subunits, we expressed and purified 15N-Tyr labeled Ps3αHSD and its mutants, and analyzed the conformational change upon NADH binding. Using the Tyr substituted mutants, we first assigned the respective signals of four Tyr residues. In the titration experiments with NADH, the four Tyr signals changed uniquely; changes in chemical shift and signal broadening were observed. The NADH binding affinity, determined from the plots of the 1H and 15N chemical shift changes, was comparable to those reported previously. Together with the crystal structure information for Ps3αHSD in the NADH-free and -bound states, site-specific conformational changes including environmental changes could be deduced.

The impact of sensory and motor enrichment on the epigenetic control of steroidogenic-related genes in rat hippocampus.

In the present study, we analyzed the effects of a short-term environmental enrichment on the mRNA expression and DNA methylation of steroidogenic enzymes in the hippocampus. Thus, young adult (80-day-old) and middle-aged (350-day-old) Wistar female rats were exposed to sensory (SE) or motor (ME) enrichment during 10 days and compared to animals housed under standard conditions. SE was provided by an assortment of objects that included plastic tubes and toys; for ME, rodent wheels were provided. In young adult animals, SE and ME increased the mRNA expression of cytochrome P450 17α-hydroxylase/c17,20-lyase, steroid 5α-reductase type 1 (5αR-1) and 3α-hydroxysteroid dehydrogenase and decreased the methylation levels of 5αR-1 gene. In middle-aged rats, ME and SE upregulated the gene expression of aldosterone synthase and decreased the methylation state of its promoter. These results propose that SE and ME differentially regulate the transcription of neurosteroidogenic enzymes through epigenetic mechanisms in young and aged rats.

Targeted Synthesis and Characterization of a Gene Cluster Encoding NAD(P)H-Dependent 3α-, 3ß-, and 12α-Hydroxysteroid Dehydrogenases from Eggerthella CAG:298, a Gut Metagenomic Sequence.

Gut metagenomic sequences provide a rich source of microbial genes, the majority of which are annotated by homology or unknown. Genes and gene pathways that encode enzymes catalyzing biotransformation of host bile acids are important to identify in gut metagenomic sequences due to the importance of bile acids in gut microbiome structure and host physiology. Hydroxysteroid dehydrogenases (HSDHs) are pyridine nucleotide-dependent enzymes with stereospecificity and regiospecificity for bile acid and steroid hydroxyl groups. HSDHs have been identified in several protein families, including medium-chain and short-chain dehydrogenase/reductase families as well as the aldo-keto reductase family. These protein families are large and contain diverse functionalities, making prediction of HSDH-encoding genes difficult and necessitating biochemical characterization. We located a gene cluster in Eggerthella sp. CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474, and CDD59475) and synthesized the genes for heterologous expression in Escherichia coli We then screened bile acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-HSDH, and we determined that CDD59474 (3α-HSDH) and CDD59473 (3ß-HSDH) constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these HSDHs with other gut bacterial HSDHs and closest homologues in the database revealed predictable clustering of HSDHs by function and identified several likely HSDH sequences from bacteria isolated or sequenced from diverse mammalian and avian gut samples.IMPORTANCE Bacterial HSDHs have the potential to significantly alter the physicochemical properties of bile acids, with implications for increased/decreased toxicity for gut bacteria and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved in glucocorticoid metabolism and may alter signaling through nuclear receptors such as farnesoid X receptor and G-protein-coupled receptor TGR5. Biochemical or similar approaches are required to fill in many gaps in our ability to link a particular enzymatic function with a nucleic acid or amino acid sequence. In this regard, we have identified a novel 12α-HSDH and a novel set of genes encoding an iso-bile acid pathway (3α-HSDH and 3ß-HSDH) involved in epimerization and detoxification of harmful secondary bile acids.

Indirect electrochemical detection for total bile acids in human serum.

Bile acids level in serum is a useful index for screening and diagnosis of hepatobiliary diseases. As bile acids concentration is closely related to the degree of hepatobiliary diseases, detecting it is a vital factor to understand the stage of the diseases. The prevalent determination for bile acids is the enzymatic cycling method which has low sensitivity while reagent-consuming. It is desirable to develop a new method with lower cost and higher sensitivity. An indirect electrochemical detection (IED) for bile acids in human serum was established using the screen printed carbon electrode (SPCE). Since bile acids do not show electrochemical signals, they were converted to 3-ketosteroids by 3-α-hydroxysteroid dehydrogenase (3α-HSD) in the presence of nicotinamide adenine dinucleotide (NAD(+)), which was reduced to NADH. NADH could then be oxidized on the surface of SPCE, generating a signal that was used to calculate the total bile acids (TBA) concentration. A good linear calibration for TBA was obtained at the concentration range from 5.00µM to 400µM in human serum. Both the precisions and recoveries were sufficient to be used in a clinical setting. The TBA concentrations in 35 human serum samples by our IED method didn't show significant difference with the result by enzymatic cycling method, using the paired t-test. Moreover, our IED method is reagent-saving, sensitive and cost-effective.

Human 3α-hydroxysteroid dehydrogenase type 3: structural clues of 5α-DHT reverse binding and enzyme down-regulation decreasing MCF7 cell growth.

Human 3α-HSD3 (3α-hydroxysteroid dehydrogenase type 3) plays an essential role in the inactivation of the most potent androgen 5α-DHT (5α-dihydrotestosterone). The present study attempts to obtain the important structure of 3α-HSD3 in complex with 5α-DHT and to investigate the role of 3α-HSD3 in breast cancer cells. We report the crystal structure of human 3α-HSD3·NADP(+)·A-dione (5α-androstane-3,17-dione)/epi-ADT (epiandrosterone) complex, which was obtained by co-crystallization with 5α-DHT in the presence of NADP(+) Although 5α-DHT was introduced during the crystallization, oxidoreduction of 5α-DHT occurred. The locations of A-dione and epi-ADT were identified in the steroid-binding sites of two 3α-HSD3 molecules per crystal asymmetric unit. An overlay showed that A-dione and epi-ADT were oriented upside-down and flipped relative to each other, providing structural clues for 5α-DHT reverse binding in the enzyme with the generation of different products. Moreover, we report the crystal structure of the 3α-HSD3·NADP(+)·4-dione (4-androstene-3,17-dione) complex. When a specific siRNA (100 nM) was used to suppress 3α-HSD3 expression without interfering with 3α-HSD4, which shares a highly homologous active site, the 5α-DHT concentration increased, whereas MCF7 cell growth was suppressed. The present study provides structural clues for 5α-DHT reverse binding within 3α-HSD3, and demonstrates for the first time that down-regulation of 3α-HSD3 decreases MCF7 breast cancer cell growth.

Koumine enhances spinal cord 3α-hydroxysteroid oxidoreductase expression and activity in a rat model of neuropathic pain.

BACKGROUND: Koumine is an alkaloid monomer found abundantly in Gelsemium plants. It has been shown to reverse thermal hyperalgesia and mechanical allodynia induced by sciatic nerve chronic constriction injury (CCI) in rats in a dose-dependent manner. Interestingly, this effect is mediated by elevated allopregnanolone levels in the spinal cord (SC). Since 3α-hydroxysteroid oxidoreductase (3α-HSOR), the key synthetase of allopregnanolone, is responsible for allopregnanolone upregulation in the SC, the objective of the present study was to investigate the role of its expression in the SC in koumine-induced analgesia using a rat model of neuropathic pain following peripheral nerve injury. RESULTS: Time-course investigations of immunohistochemistry and real-time polymerase chain reaction revealed that the immunoreactivity and mRNA expression of 3α-HSOR markedly increased in a time-dependent manner in the SC of koumine-treated CCI rats. Furthermore, 3α-HSOR activity in the SC of koumine-treated CCI rats increased by 15.8% compared to the activity in untreated CCI rats. Intrathecal injection of medroxyprogesterone acetate, a selective 3α-HSOR inhibitor, reversed the analgesic effect of koumine on CCI-induced mechanical pain perception. Our results confirm that koumine alleviates neuropathic pain in rats with CCI by enhancing 3α-HSOR mRNA expression and bioactivity in the SC. CONCLUSION: This study demonstrates that 3α-HSOR is an important molecular target of koumine for alleviating neuropathic pain. Koumine may prove a promising compound for the development of novel analgesic agents effective against intractable neuropathic pain.

A biosynthetic pathway for a prominent class of microbiota-derived bile acids.

The gut bile acid pool is millimolar in concentration, varies widely in composition among individuals and is linked to metabolic disease and cancer. Although these molecules are derived almost exclusively from the microbiota, remarkably little is known about which bacterial species and genes are responsible for their biosynthesis. Here we report a biosynthetic pathway for the second most abundant class in the gut, 3ß-hydroxy(iso)-bile acids, whose levels exceed 300 µM in some humans and are absent in others. We show, for the first time, that iso-bile acids are produced by Ruminococcus gnavus, a far more abundant commensal than previously known producers, and that the iso-bile acid pathway detoxifies deoxycholic acid and thus favors the growth of the keystone genus Bacteroides. By revealing the biosynthetic genes for an abundant class of bile acids, our work sets the stage for predicting and rationally altering the composition of the bile acid pool.

Expression, purification and functional characterization of a novel 3α-hydroxysteroid dehydrogenase from Pseudomonas aeruginosa.

3α-Hydroxysteroid dehydrogenase (3α-HSD) catalyzes the oxidation of the 3-hydroxyl group of steroids. The enzymatic conversion is a critical step in the enzymatic assay of urinary sulfated bile acids (SBAs), which is a valuable diagnosis index of hepatobiliary diseases. However, the source of 3α-HSD for clinical applications is limited. In this study, an open reading frame (ORF) encoding a novel 3α-HSD was successfully cloned from Pseudomonas aeruginosa and expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified by immobilized metal ion affinity chromatography. Enzyme characterization studies revealed that the protein has 3α-HSD activity and the Km value for sodium cholate is 1.06 mmol L(-1). More than 60% relative enzyme activity was observed in a wide range of pH and temperature, with an optimum pH at 8.0 and an optimum temperature at 30°C. The enzyme's good thermostability under 40°C would be favorable in clinical applications. Ion interference experiments indicated that Zn(2+) was an activating cofactor which increased the enzyme activity 1.75-fold. With the favorable characteristics mentioned above, the new 3α-HSD is a promising enzyme for clinical applications. More importantly, the present work is the first report on a 3α-HSD from P. aeruginosa.

Environmental enrichment attenuates the age-related decline in the mRNA expression of steroidogenic enzymes and reduces the methylation state of the steroid 5α-reductase type 1 gene in the rat hippocampus.

We analyzed the effects of aging and environmental enrichment on the mRNA expression and DNA methylation state of steroidogenic enzymes in the hippocampus. The effects of aging were evaluated by comparing young adult (90-day-old) and middle-aged (450-day-old) female Wistar rats. To elucidate the effects of environmental enrichment, a subgroup of middle-aged rats exposed to sensory and social stimulation for 105 days was compared to rats housed under standard laboratory conditions. Aging decreased the transcription of neurosteroidogenic-related genes and increased the promoter methylation state of cytochrome P450 side chain cleavage, 3α-hydroxysteroid dehydrogenase (3α-HSD) and 5α-reductase-1. Exposure of middle-aged rats to environmental enrichment increased mRNA levels of 5α-reductase-1, 3α-HSD and cytochrome P450 17α-hydroxylase/c17,20-lyase and decreased the methylation state of the 5α-reductase-1 gene. Thus, sensory and social stimulation attenuate the age-related decline in the mRNA expression of hippocampal steroidogenic enzymes. Epigenetic mechanisms associated with differential promoter methylation could be involved.

[Effects of AKR1C3 on kidney damage of preeclamptic rats].

OBJECTIVE: To explore the effects of AKR1C3 on kidney damage of preeclamptic rats and elucidate the possible therapeutic mechanism of glyburide. METHODS: The rat model of preeclampsia was established by an intraperitoneal injection of Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME). A total of 40 Wistar rats were randomly divided into 4 groups of preeclampsia (pregnant rats and L-NAME), treatment (pregnant rats, L-NAME and glyburide), non-pregnancy (L-NAME) and control (pregnant rats and NS) (n = 10 each). The rats in treatment group received an intragastric dose of glyburide. Successful modeling was confirmed by measuring blood pressure and 24 h urine protein content and observing the structure of kidney under transmission electron microscopy (TEM). The methods of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were employed to detect the expression levels of AKR1C3, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA). RESULTS: The blood pressures of all rats had no significant difference prior to modeling. After modeling, the blood pressure of preeclampsia group was significantly higher than those of control and treatment groups [(143.83 ± 9.62), (120.83 ± 4.31), (129.43 ± 14.4) mmHg, both P < 0.05]. The 24 h urine protein content of all rats had no significant difference prior to modeling. After modeling, the 24 h urine protein content of preeclampsia group was higher than those of control and treatment groups. The TEM observation of kidney slices verified the success of modeling. In preeclampsia group, the expression levels of AKR1C3 in protein and mRNA were significantly lower than control group [(0.48 ± 0.09) vs (0.98 ± 0.27), (0.05 ± 0.02) vs (0.87 ± 0.45), both P < 0.05]. Compared with preeclampsia group, the expression levels of AKR1C3 in protein and mRNA significantly increased in treatment group [(0.48 ± 0.09) vs (1.05 ± 0.20), (0.05 ± 0.02) vs (0.22 ± 0.06), both P < 0.05]. As compared with control group, the levels of SOD, CAT and GSH-Px were significantly lower while MDA was higher. Compared with preeclampsia group, the levels of SOD, CAT and GSH-Px in treatment group were significantly higher while MDA was lower. CONCLUSION: The expression level of AKR1C3 decreases in kidney of preeclamptic rats and the mechanism is related with oxidative stress. Glyburide increases the expression of AKR1C3 and have therapeutic effect for preeclampsia.

Developmentally regulated neurosteroid synthesis enhances GABAergic neurotransmission in mouse thalamocortical neurones.

KEY POINTS: During neuronal development synaptic events mediated by GABAA receptors are progressively reduced in their duration, allowing for rapid and precise network function. Here we focused on ventrobasal thalamocortical neurones, which contribute to behaviourally relevant oscillations between thalamus and cortex. We demonstrate that the developmental decrease in the duration of inhibitory phasic events results predominantly from a precisely timed loss of locally produced neurosteroids, which act as positive allosteric modulators of the GABAA receptor. The mature thalamus retains the ability to synthesise neurosteroids, thus preserving the capacity to enhance both phasic and tonic inhibition, mediated by synaptic and extrasynaptic GABAA receptors, respectively, in physiological and pathophysiological scenarios associated with perturbed neurosteroid levels. Our data establish a potent, endogenous mechanism to locally regulate the GABAA receptor function and thereby influence thalamocortical activity. During brain development the duration of miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABAA receptors (GABAA Rs) progressively reduces, to accommodate the temporal demands required for precise network activity. Conventionally, this synaptic plasticity results from GABAA R subunit reorganisation. In particular, in certain developing neurones synaptic α2-GABAA Rs are replaced by α1-GABAA Rs. However, in thalamocortical neurones of the mouse ventrobasal (VB) thalamus, the major alteration to mIPSC kinetics occurs on postnatal (P) day 10, some days prior to the GABAA R isoform change. Here, whole-cell voltage-clamp recordings from VB neurones of mouse thalamic slices revealed that early in postnatal development (P7-P8), the mIPSC duration is prolonged by local neurosteroids acting in a paracrine or autocrine manner to enhance GABAA R function. However, by P10, this neurosteroid 'tone' rapidly dissipates, thereby producing brief mIPSCs. This plasticity results from a lack of steroid substrate as pre-treatment of mature thalamic slices (P20-24) with the GABAA R-inactive precursor 5α-dihydroprogesterone (5α-DHP) resulted in markedly prolonged mIPSCs and a greatly enhanced tonic conductance, mediated by synaptic and extrasynaptic GABAA Rs, respectively. In summary, endogenous neurosteroids profoundly influence GABAergic neurotransmission in developing VB neurones and govern a transition from slow to fast phasic synaptic events. Furthermore, the retained capacity for steroidogenesis in the mature thalamus raises the prospect that certain physiological or pathophysiological conditions may trigger neurosteroid neosynthesis, thereby providing a local mechanism for fine-tuning neuronal excitability.

5α-Reduced neurosteroids sex-dependently reverse central prenatal programming of neuroendocrine stress responses in rats.

Maternal social stress during late pregnancy programs hypothalamo-pituitary-adrenal (HPA) axis hyper-responsiveness to stressors, such that adult prenatally stressed (PNS) offspring display exaggerated HPA axis responses to a physical stressor (systemic interleukin-1ß; IL-1ß) in adulthood, compared with controls. IL-1ß acts via a noradrenergic relay from the nucleus tractus solitarii (NTS) to corticotropin releasing hormone neurons in the paraventricular nucleus (PVN). Neurosteroids can reduce HPA axis responses, so allopregnanolone and 3ß-androstanediol (3ß-diol; 5α-reduced metabolites of progesterone and testosterone, respectively) were given subacutely (over 24 h) to PNS rats to seek reversal of the "programmed" hyper-responsive HPA phenotype. Allopregnanolone attenuated ACTH responses to IL-1ß (500 ng/kg, i.v.) in PNS females, but not in PNS males. However, 3ß-diol normalized HPA axis responses to IL-1ß in PNS males. Impaired testosterone and progesterone metabolism or increased secretion in PNS rats was indicated by greater plasma testosterone and progesterone concentrations in male and female PNS rats, respectively. Deficits in central neurosteroid production were indicated by reduced 5α-reductase mRNA levels in both male and female PNS offspring in the NTS, and in the PVN in males. In PNS females, adenovirus-mediated gene transfer was used to upregulate expression of 5α-reductase and 3α-hydroxysteroid dehydrogenase mRNAs in the NTS, and this normalized hyperactive HPA axis responses to IL-1ß. Thus, downregulation of neurosteroid production in the brain may underlie HPA axis hyper-responsiveness in prenatally programmed offspring, and administration of 5α-reduced steroids acutely to PNS rats overrides programming of hyperactive HPA axis responses to immune challenge in a sex-dependent manner.

Expression levels of mRNA for neurosteroidogenic enzymes 17ß-HSD, 5α-reductase, 3α-HSD and cytochrome P450 aromatase in the fetal wild type and SF-1 knockout mouse brain.

The presence of steroidogenic enzymes in the brain suggests de novo synthesis of steroid hormones in the brain. The current study was designed to determine the developmental profiles of cytochrome p450 aromatase (cyp19), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), 5α-reductase type I and 3α-hydroxysteroid dehydrogenase (3α-HSD) mRNA expression levels in the fetal mouse brain and potential influence of peripheral steroids, and the steroidogenic factor 1 (SF-1) gene on their expression. Brains were collected from WT and SF-1 knockout male and female fetuses at embryonic (E) days E12, E14, E16, and E18. Quantitative PCR analyses revealed age related increases in the expression levels of 17ß-HSD and 5α-reductase. Differences between genotypes in the expression levels of 17ß-HSD and 5α-reductase were detected on E14, with reduced levels of expression in SF-1 KO males and females for 17ß-HSD and only between females for 5α-reductase. Expression of 3α-HSD mRNA did not differ significantly between sexes, age groups or genotypes with the exception of SF-1 KO males, which had an unexplained increase in mRNA for this enzyme on day E18. Expression of cyp19 was at the limit of detection and could not be analyzed effectively. There were no sex differences and, with the exception of small difference on E14 for 17ß-HSD and 5α-reductase, no differences between genotypes. The results suggest that gonadal steroids do not influence the production of neurosteroids in the fetal brain, nor does SF-1 play a major role in the regulation of steroidogenic enzyme expression in the brain.

Fluoxetine elevates allopregnanolone in female rat brain but inhibits a steroid microsomal dehydrogenase rather than activating an aldo-keto reductase.

BACKGROUND AND PURPOSE: Fluoxetine, a selective serotonin reuptake inhibitor, elevates brain concentrations of the neuroactive progesterone metabolite allopregnanolone, an effect suggested to underlie its use in the treatment of premenstrual dysphoria. One report showed fluoxetine to activate the aldo-keto reductase (AKR) component of 3α-hydroxysteroid dehydrogenase (3α-HSD), which catalyses production of allopregnanolone from 5α-dihydroprogesterone. However, this action was not observed by others. The present study sought to clarify the site of action for fluoxetine in elevating brain allopregnanolone. EXPERIMENTAL APPROACH: Adult male rats and female rats in dioestrus were treated with fluoxetine and their brains assayed for allopregnanolone and its precursors, progesterone and 5α-dihydroprogesterone. Subcellular fractions of rat brain were also used to investigate the actions of fluoxetine on 3α-HSD activity in both the reductive direction, producing allopregnanolone from 5α-dihydroprogesterone, and the reverse oxidative direction. Fluoxetine was also tested on these recombinant enzyme activities expressed in HEK cells. KEY RESULTS: Short-term treatment with fluoxetine increased brain allopregnanolone concentrations in female, but not male, rats. Enzyme assays on native rat brain fractions and on activities expressed in HEK cells showed fluoxetine did not affect the AKR producing allopregnanolone from 5α-dihydroprogesterone but did inhibit the microsomal dehydrogenase oxidizing allopregnanolone to 5α-dihydroprogesterone. CONCLUSIONS AND IMPLICATIONS: Fluoxetine elevated allopregnanolone in female rat brain by inhibiting its oxidation to 5α-dihydroprogesterone by a microsomal dehydrogenase. This is a novel site of action for fluoxetine, with implications for the development of new agents and/or dosing regimens to raise brain allopregnanolone.

Allopregnanolone and puberty: modulatory effect on glutamate and GABA release and expression of 3α-hydroxysteroid oxidoreductase in the hypothalamus of female rats.

The hypothalamic release of glutamate and GABA regulates neurosecretory functions that may control the onset of puberty. This release may be influenced by neurosteroids such as allopregnanolone. Using superfusion experiments we examined the role of allopregnanolone on the K(+)-evoked and basal [(3)H]-glutamate and [(3)H]-GABA release from mediobasal hypothalamus and anterior preoptic area in prepubertal, vaginal opening and pubertal (P) rats and evaluated its modulatory effect on GABAA and NMDA (N-methyl-d-aspartic acid) receptors. Also, we examined the hypothalamic activity and mRNA expression of 3α-hydroxysteroid oxidoreductase (3α-HSOR) - enzyme that synthesizes allopregnanolone - using a spectrophotometric method and RT-PCR, respectively. Allopregnanolone increased both the K(+)-evoked [(3)H]-glutamate and [(3)H]-GABA release in P rats, being the former effect mediated by the modulation of NMDA receptors - as was reverted by Mg(2+) and by the NMDA receptor antagonist AP-7 and the latter by the modulation of NMDA and GABAA receptors - as was reverted by Mg(2+) and the GABAA receptor antagonist bicuculline. The neurosteroid also increased the basal release of [(3)H]-glutamate in VO rats in an effect that was dependent on the modulation of NMDA receptors as was reverted by Mg(2+). On the other hand we show that allopregnanolone reduced the basal release of [(3)H]-GABA in P rats although we cannot elucidate the precise mechanism by which the neurosteroid exerted this latter effect. The enzymatic activity and the mRNA expression of 3α-HSOR were both increased in P rats regarding the other two studied stages of sexual development. These results suggest an important physiological function of allopregnanolone in the hypothalamus of the P rat where it might be involved in the 'fine tuning' of neurosecretory functions related to the biology of reproduction of the female rats.