Intermediate Steroids Research Articles

Identification of a novel class of cortisol biosynthesis inhibitors and its implications in a therapeutic strategy for hypercortisolism

AimsDysregulation of adrenocortical steroid (corticosteroids) biosynthesis leads to pathological conditions such as Cushing's syndrome. Although several classes of steroid biosynthesis inhibitors have been developed to treat cortisol overproduction, limitations such as insufficient efficacy, adverse effects, and/or tolerability still remain. The present study aimed to develop a new class of small molecules that inhibit cortisol production, and investigated their putative modes of action. Main methodsWe screened an in-house chemical library with drug-like chemical scaffolds using human adrenocortical NCI-H295R cells. We then evaluated and validated the effects of the selected compounds at multiple regulatory steps of the adrenal steroidogenic pathway. Finally, genome-wide RNA expression analysis coupled with gene enrichment analysis was conducted to infer possible action mechanisms. Key findingsA subset of benzimidazolylurea derivatives, including a representative compound (designated as CJ28), inhibited both basal and stimulated production of cortisol and related intermediate steroids. CJ28 attenuated the mRNA expression of multiple genes involved in steroidogenesis and cholesterol biosynthesis. Furthermore, CJ28 significantly attenuated de novo cholesterol biosynthesis, which contributed to its suppression of cortisol production. SignificanceWe identified a novel chemical scaffold that exerts inhibitory effects on cortisol and cholesterol biosynthesis via coordinated transcriptional silencing of gene expression networks. Our findings also reveal an additional adrenal-directed pharmacological strategy for hypercortisolism involving a combination of inhibitors targeting steroidogenesis and de novo cholesterol biosynthesis.

MicroRNA-150 regulates steroidogenesis of mouse testicular Leydig cells by targeting STAR.

Leydig cells are essential for male reproductive development throughout life. Production of androgens as well as intermediate steroids is tightly regulated. Although microRNAs (miRNAs) are suggested to play important roles in spermatogenesis, little is currently known regarding the regulation of steroidogenesis by miRNAs in Leydig cells. Here, we found that miR-150 was predominantly expressed in Leydig cells within mouse testis. Therefore, we determined steroidogenesis of the Leydig cells in which miR-150 was knocked down or overexpressed using miR-150 antagomir and agomir, respectively. Compared with negative control group, a significant increase of STAR expression was observed in miR-150 antagomir-treated Leydig cells. Conversely, STAR expression was significantly reduced in miR-150 agomir-transfected Leydig cells. Production of sex-steroid precursors and testosterone of Leydig cells was also negatively controlled by miR-150. We further identified Star as a target of miR-150 using luciferase reporter assay. Finally, we confirmed that miR-150 was necessary for steroidogenesis and spermatogenesis in vivo via intratesticular injection of miR-150 antagomir or agomir. Taken together, our studies suggest that miR-150 negatively regulates the expression of STAR and steroidogenesis of Leydig cells in mice.

Degradation of phytosterols in tobacco waste extract by a novel Paenibacillus sp.

Phytosterols have been demonstrated to be precursors of polycyclic aromatic hydrocarbons (PAHs) formed during biomass pyrolysis. Here, a novel Paenibacillus sp. was evaluated for its ability to degrade phytosterols in tobacco waste extract (TWE). The optimal conditions for cell growth and stigmasterol (a representative of phytosterols) degradation were 37 °C, pH 7.0, 1.0g/L yeast extract, and 6.0g/L glucose. Paenibacillus sp. could degrade stigmasterol under high concentrations of glucose (up to 130g/L) and tolerate wide pH (5.0-9.0) and temperature (25-42 °C) ranges. The new strain could degrade stigmasterol completely into CO2 and H2 O, and no intermediate steroids were detected during the degradation process. Phytosterol degradation in TWE was demonstrated by high-performance liquid chromatography-tandem mass spectrometry. Under optimal conditions (37 °C, pH 7.0, with the exponential-phase cells), the total degradation ratio of phytosterols reached 38.5% in TWE, including 45.2% of stigmasterol, 37.4% of β-sitosterol, 27.3% of campesterol, and 28.7% of cholesterol. These results showed that Paenibacillus sp. is a candidate for phytosterol degradation in TWE and other biomass and is potentially useful in reducing the PAHs generated from biomass pyrolysis.

MEF2 and NR2F2 cooperate to regulate Akr1c14 gene expression in mouse MA‐10 Leydig cells

Leydig cells are essential for male reproductive development and health throughout life. Production of androgens [testosterone, dihydrotestosterone (DHT)] as well as intermediate steroids [progesterone, dihydroprogesterone (DHP)] is tightly regulated. In the mouse, the 3α-hydroxysteroid dehydrogenase enzyme (3α-HSD, AKR1C14) catalyses the interconversion of DHP and DHT into less potent steroids. Despite its importance, nothing is currently known regarding the regulation of Akr1c14 expression in Leydig cells. Recently, the transcription factors MEF2 and NR2F2 were identified in the mouse testis including in Leydig cells where they were found to regulate expression of genes involved in steroidogenesis. Analyses of transcriptomic data from MEF2- or NR2F2-deficient MA-10 Leydig cells revealed a significant decrease in Akr1c14 mRNA levels. Using qPCR, we confirmed that Akr1c14 mRNA levels were decreased in MEF2- and in NR2F2-deficient conditions. Conversely, overexpression of MEF2A or/and NR2F2 in MA-10 Leydig cells led to an increase in endogenous Akr1c14 mRNA levels. Recruitment of MEF2 and NR2F2 to the Akr1c14 promoter was confirmed by ChIP while DNA precipitation assays revealed direct binding of MEF2 but not NR2F2 to this region. In functional promoter studies, NR2F2 was found to activate the Akr1c14 promoter while MEF2A on its own had no effect. Combination of both NR2F2 and MEF2A led to a cooperative activation of the Akr1c14 promoter and this required intact MEF2 and NR2F2 elements. Finally, co-immunoprecipitation experiments showed that MEF2 and NR2F2 are present in the same protein complex. In conclusion, our results identify a novel cooperation between MEF2 factors and NR2F2 in the expression of the Akr1c14 gene involved in the regulation of DHP/DHT levels.

Simultaneous quantification of individual intermediate steroids in silkworm ecdysone biosynthesis by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring

The concentration changes of endogenous ecdysteroids are closely related to the regulation of insect growth and development. Although they are frequently measured by immunoassays with anti-steroid antibodies, the separate estimations of the individual concentrations of ecdysone and other ecdysteroids with similar chemical structures are quite difficult to accomplish. In this study, an efficient method for the simultaneous, individual quantification of intermediate steroids in ecdysone biosynthesis was developed, using LC–MS/MS. By employing multiple reaction monitoring (MRM) in the MS detection, the selectivity and sensitivity of the method were greatly enhanced, allowing the estimation of trace amounts of steroids in biological samples from silkworm prothoracic glands and hemolymph.

Convenient Labeling of Sterols with Eight Deuterium Atoms :

For quantitative and metabolic examination of sitosterol in plants, deuterio-labeled sitosterol and related steroids were synthesized from stigmasterol. The base-catalyzed enolization of a 3, 6-diketone group and reduction of the diketone with lithium aluminum deuteride were conducted as key reactions for the introduction of deuterium atoms into the steroidal A/B-ring. [2, 2, 3, 4, 4, 6, 7, 7-2H8] Sitosterol was obtained at 89% isotopic purity. The present labeling method for introducing eight deuterium atoms would appear, based on the present results, to be applicable to other sterols. Possible biosynthetic intermediate steroids from sitosterol to sitostanol were also prepared in deuterio-labeled form.

Immature adrenal steroidogenesis in preterm infants

Screening for congenital adrenal hyperplasia is associated with a high rate of false positive results in preterm infants. Some RIA studies have detected elevated levels of 17 alpha-hydroxy-progesterone in severely ill preterm infants. However, because the antisera used with RIAs have a high degree of cross-reactivity, RIA results may not be reliable. Adrenal steroidogenesis was evaluated in 33 preterm infants born at less than 32 weeks' gestation using reversed-phase high performance liquid chromatography. 17 alpha-hydroxyprogesterone, 11-deoxycortisol, and/or corticosterone were detectable temporarily at about 29 weeks of equivalent gestational age in 27 of 33 preterm infants. Levels of cortisol and cortisone were markedly decreased in 4 of 16 infants whose mothers had received steroids. These findings suggest that the activities of 21-hydroxylase, 11 beta-hydroxylase, and 18-hydroxylase are decreased at about 29 weeks of equivalent gestational age. Steroid levels should be carefully monitored in preterm infants with elevated intermediate steroids and preterm infants whose mothers receive steroids before delivery using reversed-phase high performance liquid chromatography.

Biological Activities of Biosynthetically-related Congeners of Brassinolide

Biological activity of intermediate steroids in the biosynthesis of brassinolide was examined by the rice-lamina inclination test and wheat-leaf unrolling test. In both bioassays, biological activity of intermediates increased according to the order in the biosynthetic pathway. The introduction of a hydroxyl group in the side chain (C-22), the step of 6-oxocampestanol to cathasterone, strikingly enhanced the activity. The activities of teasterone, 3-dehydroteasterone, and typhasterol were almost the same. Relative activities in the rice-lamina inclination test were as follows: 6-oxocampestanol (0.002), cathasterone (1), teasterone (20), castasterone (240), and brassinolide (1200).

Peroxisomal cholesterol synthesis in vivo: Accumulation of 4-methyl intermediate sterols after aminotriazole inhibition of cholesterol synthesis

To clarify the importance and pathway of peroxisomal cholesterol synthesis in vivo, we have examined whether or not 4,4-dimethyl-5α-cholest-8-en-3β-ol and 4α-methyl-5α-cholest-7-en-3β-olare accumulated in hepatic peroxisomes of aminotriα-zole-treated rats (we have shown that these intermediate steroids accumulate in rat liver when cholesterol synthesis is inhibited by aminotriazole: Hashimoto, F. and Hayashi, H. (1991) Biochim. Biophys. Acta 1086, 115). Differential centrifugation and Nycodenz gradient centrifugation showed that these intermediate steroids were localized in peroxisomes and microsomes. Cholestyramine (3-hydroxy-3-methylglutaryl-CoA reductase activator) pretreatment of aminotriazole-treated rats increased the contents of the intermediate steroids in both peroxisomes and microsomes. In peroxisomes, both 4α-methyl-5α-cholest-7-en-3β-ol and 4,4-dimethyl-5α-cholest-8-en-3β-ol were increased to about 3 times the control (aminotriazole-treated rat), and they were predominantly (about 70%) recovered in the membrane fraction after treatment with 0.05% deoxycholate or 100 mM Na 2CO 3. Gemfibrozil (peroxisomal proliferator) pretreatment enhanced the contents of 4α-methyl-5α-cholest-7-en-3β-ol and 4,4-di-methyl-5α-cholest-8-en- of peroxisomes to 4.5 times and 37 times the control, respectively. The effects of aminotriazole, cholestyramine and gemfibrozil on the intermediate contents were different between peroxisomes and microsomes. We suggest that peroxisomes in addition to microsomes participate in cholesterol synthesis in vivo, and the biosynthetic pathway includes 4α-methyl-5α-cholest-7-en-3β-ol and 4,4-dimethyl-5α-cholest-8-en-3β-ol.

Maturation of the normal pituitary-testicular axis, as assessed by gonadotropin-releasing hormone agonist challenge.

Pilot studies in boys with abnormalities of puberty have suggested that both gonadotropin and sex steroid responses to GnRH agonist undergo characteristic maturational changes. However, it is not known how the pattern of hormonal secretory responses to GnRH agonist changes during normal puberty. Therefore, we have assessed the responses of normal boys at various stages of pubertal development to a single dose of the GnRH agonist nafarelin. Baseline LH, FSH, testosterone (T), estradiol (E2), and steroid intermediates, including 17-hydroxyprogesterone, increased during puberty. LH responses peaked at 3 h in prepubertal (P1; n = 8) and midpubertal (P2; n = 4) boys, at 12 h in late pubertal (P3; n = 8) boys, but earlier (0.5-1 h) in adults (P4; n = 10). LH levels remained high for 24 h in P3-P4. Indexes of both the readily releasable and reserve pools of pituitary LH increased significantly with advancing pubertal stage (P < 0.003). FSH responses differed among groups only at 24 h. Nafarelin stimulated T significantly in groups P2 < P3 < P4. The maximal responses of T were 0.54 +/- 0.25 (+/- SEM), 8.7 +/- 2.9, 18.5 +/- 1.0, and 15.3 +/- 1.4 nmol/L in these respective groups (by analysis of variance, F = 40.5; P < 0.001). However, nafarelin did not stimulate E2 significantly until late puberty; the maximal E2 responses to nafarelin treatment were 17.9 +/- 5.9, 26.7 +/- 8.7, 318 +/- 71.3, and 329 +/- 17.4 pmol/L in P1, P2, P3, and P4 (F = 18.3; P < 0.001). In contrast to E2, most intermediate steroids measured increased significantly in response to nafarelin in stages P2-P4. We conclude that a single dose of the GnRH agonist nafarelin stimulates gonadotropin secretion in normal prepubertal and pubertal males; it also stimulates gonadal steroid production in pubertal males. Pubertal maturation of gonadotrope function appears to involve mainly increases in both the readily releasable and reserve pools of LH. The findings that late pubertal boys had LH responses similar to those of men, but greater T and lesser 17-hydroxyprogesterone outputs are compatible with their pituitary-testicular axis operating at a less down-regulated state than that of adults. The apparently isolated failure of E2 to increase in response to nafarelin in early puberty suggests that maturation of aromatase activity normally does not become appreciable until late puberty. These findings suggest that a single dose of GnRH agonist may provide a simple means of assessing the maturation of the neuroendocrine-testicular axis.

Early Time Sequence in Pregnenolone Metabolism to Testosterone in Homogenates of Human and Rat Testis

The time sequence of the metabolism of [4-14C] pregnenolone to testosterone in homogenates of human and rat testis was studied with special emphasis on the chain of events in the early 15 min of incubation. The incubations were performed at 32 C in the presence of NAD and a NADPH-generating system. The various intermediate steroids were separated by means of HPLC using a silica aliphatic diol column. Correction for procedural losses was performed by dual labeling. The present study confirms earlier reported results which showed that in the rat metabolism of pregnenolone to testosterone proceeds via the delta 4 pathway. However, this discloses for the first time that the conversion of pregnenolone proceeds very fast: progesterone, 17 alpha-hydroxyprogesterone, and 17 alpha-hydroxypregnenolone as the only important delta 5 intermediate, peak and decline again to almost undetectable levels within the first 15 min of incubation. Androstenedione and testosterone start to accumulate from 1 min on under the conditions used. In contrast, in the human testis, homogenates metabolism of pregnenolone to testosterone proceeds comparatively slowly and almost exclusively via the delta 5 intermediates dehydroepiandrosterone and androstenediol. Testosterone makes its appearance only after about 8 min of incubation. The data illustrate the importance of short-term incubations in evaluating the metabolism of steroids.

Separation of the major adrenal steroids by reversed-phase high-performance liquid chromatography

The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3β-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17α-hydroxyprogesterone, 17α-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.