An inhibitor of 11-ß hydroxysteroid dehydrogenase type 1 (PF915275) alleviates nonylphenol-induced hyperadrenalism and adiposity in rat and human cells.

BACKGROUND: Nonylphenol (NP) is an environmental endocrine-disrupting chemical (EDC) detected in human cord blood and milk. NP exposure in developmental periods results in hyperadrenalism and increasing 11ß-hydroxysteroid dehydrogenase I (11ß-HSD1) activity in an adult rat model. Alleviating 11ß-HSD1 activity is therefore a logical and common way to treat hyperadrenalism. PF915275 (PF; 4'-cyano-biphenyl-4-sulfonic acid (6-amino-pyridin-2-yl)-amide) is a selective inhibitor for 11ß-HSD1. This study aimed to determine whether PF915275 could alleviate the hyperadrenalism induced by NP. In addition to a rat model, the effects of NP and PF915275 were measured in human preadipocytes. METHODS: For the in vivo rat model, female adult rats exposed to NP during the developmental period were divided into two treatment groups, with one receiving oral DMSO solution and the other receiving PF915275 once per day for 4 weeks. After the final treatment, the rats from each group were sacrificed for analysis. For the in vitro human model, human preadipocytes received 2 regimens of NP treatment. One treatment regimen occurred before differentiation (to mimic the sensitive developmental period; P exposure), and the other included continuous exposure from preadipocytes to fully differentiated adipocytes (to mimic the growing and adult periods, respectively; C exposure). Protein and RNA were extracted from rat tissues and the preadipocytes for western blot and real-time PCR analysis. RESULTS: In the rat model, PF915275 alleviated NP-induced effects by interfering with adipogenesis pathways, including enhancing PPARα expression, decreasing PPARγ expression, and reducing both 11ß-HSD1 protein and mRNA expression levels. Additionally, PF915275 reduced the effects of the adrenal corticoid synthesis pathway by reducing StAR expression and 11ß-hydroxylase and aldosterone synthase activities. With short-term exposure, NP enhanced PPARγ and FASN mRNA expression levels and reduced PPARα expression, whereas PF915275 alleviated these effects. With C exposure, the NP-induced accumulation of intracellular lipids was reduced by PF915275 treatment, which was mediated by decreased PPARγ mRNA and protein expression levels and increased PPARα protein expression. CONCLUSIONS: The effects of NP and PF915275 treatment in both rat and human cell models are similar. Rats may be an appropriate model to study the effects of NP in humans, especially during the developmental period.

Effects of nonylphenol on aldosterone release from rat zona glomerulosa cells.

Alkylphenol ethoxylate, which consists of approximately 80% nonylphenol ethoxylate (NPE), is a major nonionic surfactant. Nonylphenol (NP), the primary degradation product of NPE, has been reported to interfere with reproduction in fish, reptiles, and mammals by inducing cell death in the gonads and by affecting other reproductive parameters. However, the effects of NP on rat adrenal zona glomerulosa cells (ZG) and the underlying mechanisms remain unclear. In this study, we explored the effects of NP on aldosterone release. ZG cells were incubated with NP in the presence or absence of the secretagogues angiotensin II (ANG II), potassium, 8-Br-cAMP, 25-OH-cholesterol, corticosterone or cyclopiazonic acid (CPA). After performing radioimmunoassay (RIA) and Western blot analysis, we found that (1) NP stimulated aldosterone release in cells induced by ANG II, KCl, 8-Br-cAMP, 25-OH-cholesterol, corticosterone, and CPA; (2) NP triggered the release of higher amounts of pregnenolone in cells treated with vehicle and 25-OH-cholesterol+trilostane than in cells treated with other compounds; and (3) the stimulatory effect of NP seemed to be mediated through steroidogenic acute regulatory protein (StAR) and aldosterone synthase activity. These observations suggest that the effects of NP are mediated via increased free Ca(2+) in the cytoplasm.

Effects of dehydroepiandrosterone on aldosterone release in rat zona glomerulosa cells.

The present study was to investigate the effects and action mechanisms of dehydroepiandrosterone (DHEA) on steroidogenesis in rat adrenal zona glomerulosa cells (ZG). ZG cells were incubated with DHEA in the presence or absence of angiotensin II (AngII), a high concentration of potassium, 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone, deoxycorticosterone, corticosterone, A23187, or cyclopiazonic acid (CPA) at 37 degrees C for 1 h. The concentration of aldosterone or pregnenolone in the culture medium was then measured by radioimmunoassay (RIA). The cells were used to determine the cellular cAMP content. The data demonstrated that: (1) DHEA inhibited AngII-, high concentration of KCl-, forskolin-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone-, deoxycorticosterone-, corticosterone-, A23187-, or CPA-stimulated aldosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA noncompetitively inhibited aldosterone synthase but showed uncompetitive inhibition of P450scc. These results suggest that DHEA acts directly on rat ZG cells to diminish aldosterone secretion by inhibition of a post-cAMP pathway or by acting on intracellular Ca2+ mobilization. In addition it affects the function of post-P450scc steroidogenic enzymes.

Mechanisms of inhibition of dehydroepiandrosterone upon corticosterone release from rat zona fasciculata-reticularis cells.

We have demonstrated that dehydroepiandrosterone (DHEA) acts directly on rat zona fasciculata-reticularis (ZFR) cells to diminish corticosterone secretion by an inhibition of post-cAMP pathway, and decreases functions of steroidogenic enzymes after P450(scc) as well as steroidogenic acute regulatory (StAR) protein expression. However, the mechanisms by which DHEA engages with environmental messenger signals which translate into interfering StAR protein expression are still unclear. This study explored the effects of DHEA on the phosphorylation/activation of extracellular signal-regulated kinases (ERKs). ERK activation resulted in enhancing phosphorylation of steroidogenic factor-1 (SF-1) and increased StAR protein expression. ZFR cells were incubated in the presence or absence of adrenocorticotropin (ACTH), forskolin (FSK), 25-OH-cholesterol, U0126, and H89 at 37 degrees C. The concentration of corticosterone released into the media was measured by radioimmunoassay (RIA). The cells were used to extract protein for Western blot analysis of ERKs or StAR protein expression or immunoprecipitation of SF-1 analysis. The results suggested that (1) ERK pathway of rat ZFR cells might be PKA dependent, (2) ERK activity was required for SF-1 phosphorylation to upregulate steroidogenesis in rat ZFR cells, and (3) DHEA did not affect ERK phosphorylation, however, it attenuated forskolin-stimulated SF-1 phosphorylation to affect StAR protein expression.

Effects of S-petasin on cyclic AMP production and enzyme activity of P450scc in rat zona fasciculata-reticularis cells.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of P. hybridus, which has been used to relieve gastrointestinal pain, lung disease, and spasms of urogenital tract. We have demonstrated that S-petasin inhibited corticosterone release from rat zona fasiculata-reticularis cells. However, the mechanism and molecular effects of S-petasin on zona fasiculata-reticularis cells are still unclear. This study explored the effects of S-petasin on cellular adenosine 3':5'-cyclic monophosphate (cAMP) production, the functions of steroidogenic enzymes including cytochrome P450 side-chain cleavage enzyme (P450scc), 11beta-hydroxylase, and the expression levels of steroidogenic acute regulatory protein or P450scc. In this experiment, zona fasciculata-reticularis cells were incubated with S-petasin in the presence or absence of adrenocorticotropin (ACTH), 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), forskolin, 25-OH-cholesterol, deoxycorticosterone at 37 degrees C for 0.5, 1 or 3 h. The media were used to measure the concentration of corticosterone or pregnenolone by radioimmunoassay. The cells were used to measure the content of cAMP by radioimmunoassay and extracted protein for Western blot or messenger RNA (mRNA) for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Our data demonstrated that (1) S-petasin inhibits ACTH- or forskolin-stimulated cellular cAMP production, (2) S-petasin increased the Michaelis constants of P450scc and 11beta-hydroxylase and (3) S-petasin decreased the expression levels and mRNA of steroidogenic acute regulatory protein. In summary, the actions of S-petasin mediate the inhibition of cAMP formation, decrease the activities of key enzymes P450scc and 11beta-hydroxylase, and reduce mRNA of steroidogenic acute regulatory protein and expression of steroidogenic acute regulatory protein.

Effects of dehydroepiandrosterone on corticosterone release in rat zona fasciculata-reticularis cells.

The decline of plasma dehydroepiandrosterone (DHEA) and maintenance of glucocorticoid levels with increasing age contribute to excess body fat accumulation, hyperglycaemia, hyperlipidaemia, hyperinsulinaemia and cancer. Although opposing actions of DHEA and corticosterone have been proposed in a rat model, the effects and action mechanisms of DHEA on rat adrenal zona fasciculata-reticularis (ZFR) cells are still unclear. This study addressed the effects of DHEA on corticosterone release, cellular cAMP production, the functions of steroidogenic enzymes and the expression levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc). ZFR cells were incubated with DHEA in the presence or absence of adrenocorticotropin (ACTH), 8-Br-cAMP, forskolin, 25-OH-cholesterol, pregnenolone, progesterone or deoxycorticosterone at 37 degrees C for 30 min, 1 h or 5 h and the concentration of corticosterone or pregnenolone measured subsequently in the media by RIA. The cells were used to measure the content of cAMP by RIA and to extract protein for Western blot or mRNA for RT-PCR analysis. The data demonstrated that (1) DHEA inhibited ACTH-, 8-Br-cAMP-, 25-OH-cholesterol-, pregnenolone-, progesterone- or deoxycorticosterone-stimulated corticosterone release; (2) DHEA increased 25-OH-cholesterol-stimulated pregnenolone release but not when 25-OH-cholesterol was combined with trilostane; (3) DHEA increased the K(m) of 11beta-hydroxylase but not P450scc; (4) DHEA affected the expression levels of StAR protein but not of P450scc. These results suggest that DHEA acts directly on rat ZFR cells to diminish corticosterone secretion by inhibition within the post-cAMP pathway, by inhibiting steroidogenic enzymes downstream from P450scc and by inhibiting StAR expression.

Effects of fasting on corticosterone production by zona fasciculata-reticularis cells in ovariectomized rats.

BACKGROUND: To investigate the function and mechanism of fasting on the production of corticosterone in vitro by zona fasciculata-reticularis (ZFR) cells from ovariectomized (OVX) rats. METHODS: Female rats were OVX for 4 days before decapitation. Rats were fed or fasted for 1 day before experiment. ZFR cells from fed and fasted rats were incubated with adrenocorticotropic hormone (ACTH), forskolin, 8-bromo-3',5'-cyclic adenosine monophosphate, SQ22536, nifedipine, chelerythrine chloride, trilostane or steroidogenic precursors at 37 degrees C for either 60 or 30 minutes. Corticosterone, pregnenolone concentrations in spent media, and the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration were determined by radioimmunoassay. The effects of fasting in response to ACTH on the protein expressions of steroidogenic acute regulatory protein (StAR) or cytochrome P450 side-chain cleavage enzyme (P450scc) in ZFR cells were determined by Western blot analysis. RESULTS: The concentration of plasma corticosterone in fasted rats was significantly higher than that in fed rats (P<0.01). One-day fasting significantly increased the responsiveness of ZFR cells to ACTH, forskolin, and precursor-stimulated corticosterone productions and to forskolin-stimulated cAMP accumulation. The corticosterone production was reduced in fasted group when adenylyl cyclase was inhibited by SQ22536. The fasting-enhanced level of corticosterone production in ZFR cells was decreased by the administration of nifedipine but not altered by that of chelerythrine chloride. Fasting significantly increased trilostane-stimulated production of pregnenolone in ZFR cells. The activities of enzymes which converting cholesterol, pregnenolone, progesterone, and deoxycorticosterone to corticosterone and the expressions of StAR in ZFR cells were greater in fasted rats than in fed rats. CONCLUSIONS: This study demonstrated that fasting increased the release of corticosterone and the accumulation of cAMP by rat ZFR cells. The action mediated through enhancing the responsiveness to ACTH stimulation, cAMP cascades and the activity of L-type calcium channels. The activities of steroidogenic enzymes including P450scc, 3beta-hydroxysteroid dehydrogenase, 21-hydroxylase, and 11beta-hydroxylase were all enhanced by the fasting treatment.

Effects of S-petasin on corticosterone release in rats.

Petasites hybridus is used in Chinese herbal medicine. S-petasin is a bioactive compound isolated from leaves or roots of Petasites hybridus. S-petasin has been used to relieve gastrointestinal pain, lung disease, and spasms of the urogenital tract. However, the side effect of S-petasin on endocrine systems are still not clear. This study explored the effects of S-petasin on the release of corticosterone in vivo and in vitro. An intravenous injection of S-petasin (10 microg/kg) decreased both basal and adrenocorticotropin (ACTH)-induced plasma corticosterone concentration in male rats. In vitro, S-petasin (3 x 10(-6) - 10(-4) M) caused a significant reduction of basal and ACTH-stimulated release of corticosterone from the enzymatically dispersed rat zona fasciculata-reticularis (ZFR) cells in a dose-dependent manner. In order to study possible mechanisms, ZFR cells were incubated with S-petasin (10(-5) M) in the presence or absence of forskolin (adenylate cyclase activator, 10(-6) - 10(-4) M), 8-Br-cAMP (a cAMP analogue, 10(-6) 10(-4) M), 25-OH-cholesterol (pregnenolone biosynthesis precursor, 10(-5) M) combined with trilostane (a blocker of 3beta-hydroxysteriod dehydrogenase, 3beta-HSD, 10(-6) M) and deoxycorticosterone (corticosterone biosynthesis precursor, 10(-9) - 10(-6) M) at 37 degrees C for 1h. The concentration of pregnenolone and corticosterone in media were measured by radioimmunoassay. The stimulatory effects of corticosterone secretion induced by forskolin (10(-5) - 10(-4) M), 8-Br-cAMP (10(-5) - 10(-4) M) and deoxycorticosterone (10(-7) - 10(-6) M) were reduced by S-petasin at 10(-5) M. The stimulatory effects of pregnenolone secretion induced by 25-OH-cholesterol combined with or without trilostane was reduced by S-petasin at 10(-5) M. These results suggest that S-petasin inhibits the production of corticosterone from rat ZFR cells in part through decreasing the activities of adenylyl cyclase, P450scc and 11beta-hydroxylase.