Mitochondrial Cholesterol Side-chain Cleavage Research Articles

1420P The pharmacokinetics and the pharmacodynamic effect of ODM-208, an inhibitor of cholesterol side-chain cleavage enzyme (CYP11A1)

ODM-208 is a first-in-class, oral, non-steroidal small molecule that selectively and completely inhibits the mitochondrial cholesterol side-chain cleavage cytochrome P450 (CYP11A1) enzyme, the first and rate limiting step in steroidogenesis, leading to deficiency of endogenous steroid hormones. The pharmacokinetics (PK) of ODM-208 and the pharmacodynamics (PD) of ODM-208 by measuring serum testosterone levels were studied in the Cypides phase 1 trial in males with progressive mCRPC, who had received at least one prior line of both 2nd generation AR pathway inhibitor (ARPI) and taxane chemotherapy.

Autism-Like Behavior in the Offspring of CYP11A1-Overexpressing Pregnant Rats.

Autism spectrum disorders (ASD) represent a complex group of neurodevelopmental disorders that are characterized by impaired social behavior and communication as well as repetitive behavior and restricted interests. Prenatal exposure to high levels of testosterone and preeclampsia are thought to be risk factors of ASD. We had previously reported that overexpression of the mitochondrial cholesterol side-chain cleavage enzyme (CYP11A1) could lead to both preeclampsia-like symptoms and increased testosterone levels in pregnant rats. In this study, we investigated the association between high CYP11A1 levels in pregnant rats and autism-like behavior in their offspring. Timed-pregnant Sprague-Dawley (SD) rats were injected with CYP11A1 gene-carrying adenoviruses on gestational day 8.5, and their offspring were then compared with those from timed-pregnant control SD rats. Compared with their control counterparts, the offspring of the CYP11A1-ovexpressing dams displayed more symptoms of anxiety and spent less time in social interactions and more time in self-grooming and rearing, all indicators of autism-like behavior. Sequencing of the transcriptome in primary microglia from the offspring of CYP11A1-overexpressing dams revealed that immune pathways were highly activated, and the gamma-aminobutyric acid type A (GABAA) receptor genes were among the top differentially expressed genes. Using primary microglia cultures generated from neonatal rats, tumor necrosis factor-alpha expression was found to be elevated in the cells transfected with CYP11A1-carrying adenoviruses. Additionally, the offspring of CYP11A1-overexpressing dams displayed dysregulated GABAA receptor expression. Taken together, these results suggest that abnormal CYP11A1 gene expression in pregnant rats could lead to microglial immune activation and dysregulated GABAA receptor expression in their offspring and thereby anxiety and autism-related behavior. Our study suggests that the pathways regulated by CYP11A1 could be promising preventative and therapeutic targets for ASD.

De novo steroid biosynthesis in human prostate cell lines and biopsies

Intratumoral androgen formation may be a factor in the development of prostate cancer (PCa), particularly castration-resistant prostate cancer (CRPC). To evaluate the ability of the human prostate to synthesize de novo steroids, we examined the expression of key enzymes and proteins involved in steroid biosynthesis and metabolism. Using TissueScan™ Cancer qPCR Arrays and quantitative RT-PCR, we performed comparative gene expression analyses between various prostate cell lines and biopsies, including normal, hyperplastic, cancerous, and androgen-deprived prostate cells lines, as well as normal, benign prostate hyperplasia (BPH), PCa, and CRPC human specimens. These studies were complemented with steroid biosynthesis studies in normal and BPH cells. Normal human prostate WPMY-1 and WPE1-NA22, benign prostate hyperplasia BPH-1, and cancer PC-3, LNCaP, and VCaP cell lines, as well as normal, BPH, PCa, and CRPC specimens, were used. Although all cell lines express mRNA encoding for hydroxymethylglutaryl-CoA reductase (HMGCR), the mitochondrial translocator protein TSPO and cholesterol side chain cleavage enzyme CYP11A1 were only observed in WPMY-1, BPH-1, and LNCaP cells. HSD3B1, HSD3B2, and CYP17A1 are involved in androgen formation and were not found in most cell lines. WPE1-NA22 and BPH-1 cells were unable to synthesize de novo steroids from mevalonate. Moreover, androgen-deprived cells did not have alterations in the expression of enzymes that could lead to de novo steroid formation. All prostate specimens expressed TSPO and CYP11A1. HSD3B1/2, CYP17A1, HSD17B5, and CYP19A1 mRNA expression was distinct to the profile observed in cells lines. The majority of BPH (90.9%) and PCa (83.1%) specimens contained CYP17A1, compared to control (normal) specimens (46.7%). BPH (82%), PCa (59%), normal (40%), and CRPC (34%) specimens expressed the four key enzymes that metabolize cholesterol to androgens. These studies question the use of prostate cell lines to study steroid biosynthesis and demonstrate that human prostate samples contain transcripts encoding for key steroidogenic enzymes and proteins indicating that they have the potential to synthesize de novo steroids. We propose CYP17A1 as a candidate enzyme that can be used for patient stratification and treatment in BPH and PCa.

The stimulation of rat Leydig cell steroidogenesis by human ovarian steroidogenesis-inducing protein (SIP) may not require endogenous cAMP.

Recently a protein from ovarian follicular fluid was isolated which stimulates steroid production in different cells (Khan et al., 1990). The present study was performed to further characterize the short term effects of this steroidogensis-inducing protein (SIP) on steroid production in isolated rat Leydig cells and to compare the effects with LH/hCG and LHRH. SIP stimulated testosterone production in a dose-dependent manner. In Leydig cells isolated from adult rats the degree of stimulation was much higher than that obtained with hCG, dibutyryl cAMP (db cAMP) or LHRH. Moreover, the stimulated steroid production in the presence of hCG or db cAMP was further enhanced by SIP. The time courses of hCG and SIP action on testosterone production were comparable and maximal stimulation of steroid production was obtained within one hour. In contrast to hCG, SIP did not stimulate cAMP production. An antagonist of LHRH action was unable to block the effects of SIP on Leydig cells indicating that SIP does not act via LHRH receptors. Neither SIP nor LH could further stimulate the steroid production in the presence of 22-R-OH-cholesterol, illustrating that both stimulators control the availability of cholesterol as substrate. An inhibitor of mitochondrial cholesterol side chain cleavage (CSCC), aminoglutethemide, completely blocked the stimulatory effects of SIP and LH/hCG. Thus the effects of SIP on steroid production are not the result of conversion of contaminating steroids in the SIP-preparation. SIP and LH/hCG actions were also blocked when the cells were incubated in the presence of cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Contact sites from human placental mitochondria: Characterization and role in progesterone synthesis

To understand the functional compartmentalization of human placental mitochondria, we analyzed the composition and steroidogenic activity of contact sites. Several fractions containing contact sites were isolated using osmotic shock treatment and sucrose gradient centrifugation. These fractions contained various proteins and marker enzymes associated with mitochondrial membranes. The fractions containing the cytochrome P450 side chain cleavage system, cholesterol, nicotinamide adenine dinucleotide phosphate–isocitrate dehydrogenase, porin, and adenosine 5 ′-triphosphate–diphosphohydrolase activity showed the capacity to synthesize progesterone. Our observations indicate that all necessary elements and enzymes for steroidogenesis are present and functional in placental mitochondrial contact sites. This organization may facilitate the metabolism of cholesterol delivered to the outer mitochondrial membrane into steroid hormones by the inner mitochondrial membrane cholesterol side chain cleavage system.

Formation and functioning of fused cholesterol side-chain cleavage enzymes.

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.

MLN64 Mediates Mobilization of Lysosomal Cholesterol to Steroidogenic Mitochondria

This study demonstrates that the steroidogenic acute regulatory protein-related lipid transfer (START) domain-containing protein, MLN64, participates in intracellular cholesterol trafficking. Analysis of the intracellular itinerary of MLN64 and MLN64 mutants tagged with green fluorescent protein showed that the N-terminal transmembrane domains mediate endocytosis of MLN64 from the plasma membrane to late endocytic compartments. MLN64 constitutively traffics via dynamic NPC1-containing late endosomal tubules in normal cells; this dynamic movement was inhibited in cholesterol-loaded cells, and MLN64 is trapped at the periphery of cholesterol-laden lysosomes. The MLN64 START domain stimulated free cholesterol transfer from donor to acceptor mitochondrial membranes and enhanced steroidogenesis by placental mitochondria. Expression of a truncated form of MLN64 (DeltaSTART-MLN64), which contains N-terminal transmembrane domains but lacks the START domain, caused free cholesterol accumulation in lysosomes and inhibited late endocytic dynamics. The DeltaSTART-MLN64 dominant negative protein was located at the surface of the cholesterol-laden lysosomes. This dominant negative mutant suppressed steroidogenesis in COS cells expressing the mitochondrial cholesterol side chain cleavage system. We conclude that MLN64 participates in mobilization and utilization of lysosomal cholesterol by virtue of the START domain's role in cholesterol transport.

Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3β-Hydroxysteroid Dehydrogenase

Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.

Biphasic effects of octylphenol on testosterone biosynthesis by cultured Leydig cells from neonatal rats

The present studies evaluated the suitability of using cultured dispersed testicular cells from neonatal rats as a source for fetal Leydig cells and the use of these cells to examine direct toxic effects of environmental/occupational chemicals on androgen biosynthesis. For the current studies, the direct actions of octylphenol (OP), a surfactant additive widely used in the manufacture of various detergents, on testosterone biosynthesis by cultured rat neonatal Leydig cells were examined. Octylphenol is considered a xenoestrogen and has been reported to mimic the actions of estrogen in many cellular systems. Following exposure of cultured cells for 24 h to varying concentrations of OP (1 to 2000 nM) together with 10 mIU/mL human chorionic gonadotropin (hCG), the lower concentrations of OP (1 and 10 nM) consistently enhanced testosterone levels (approximately 10 to 70% above control), whereas higher OP concentrations (100 to 2000 nM) progressively decreased testosterone from peak levels to approximately 40 to 80% below control at the highest OP concentration. Interestingly, increasing concentrations of 17β-estradiol (1 to 1000 nM) were without effect on testosterone biosynthesis under the same conditions, and the biphasic pattern of testosterone biosynthesis elicited by increasing OP concentrations was unaffected by concomitant treatment with 10 or 100 nM ICI 182,780, which is considered a pure estrogen antagonist. Therefore, the actions of OP on testosterone biosynthesis by cultured neonatal Leydig cells do not appear to be mediated through the classic estrogen receptor α or β pathway. Although the increase in testosterone levels after exposure to lower OP concentrations and to 0.1 and 1.0 mM 8-Br-cAMP was attenuated, suggesting that lower OP concentrations may alter cellular cAMP levels, because hCG-stimulated cAMP levels were unaffected by any of the OP concentrations evaluated, it appears that its main site(s) of action occurs after the generation of cAMP. In addition, because pretreatment of cells with increasing OP concentrations and hCG had no effect on the conversion of steroid precursors (22(R)-hydroxycholesterol, pregnenolone, progesterone, or androstenedione) to testosterone, it seems that the main actions of OP under the present conditions occur before the mitochondrial cholesterol side-chain cleavage step. Furthermore, because concomitant treatment of cells with various antioxidants (α-tocopherol, butylated hydroxyanisole, or ascorbic acid) did not alter the biphasic pattern of testosterone response to increasing concentrations of OP and hCG, it seems that OP is not acting as an anti- or pro-oxidant in producing these effects. It will be important to determine whether this dose-sensitive response to OP is observed in vivo, and whether the maturational status of Leydig cells influences their pattern of response to OP and similar chemicals.

Regulation of follicular luteinization by a gonadotropin‐releasing hormone agonist: Relationship between steroidogenesis and apoptosis

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol(E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins. Mol. Reprod. Dev. 51:287–294, 1998. © 1998 Wiley-Liss, Inc.

Regulation of follicular luteinization by a gonadotropin-releasing hormone agonist: relationship between steroidogenesis and apoptosis.

The purpose of this study was to evaluate the effects of GnRH-analog (Leuprolide acetate, LA) administration on follicular luteinization in equine chorionic gonadotropin plus human chorionic gonadotropin (eCG + hCG)-superovulated prepubertal treated rats. Results indicate that LA treatment decreases circulating levels of progesterone (P) and P accumulation in collagenase-dispersed ovarian cell cultures, though estradiol (E2) production is increased. These data suggest that cells from the LA group may be less luteinized following gonadotropin treatment. Studies performed on histological ovarian sections after different times of eCG administration showed that LA injections produce lower amounts of corpora lutea and antral follicles, and a greater number of atretic and preantral follicles. The basal and LH-stimulated P and progestagen accumulations are decreased in incubations of corpora lutea isolated from the LA group. In addition, the mitochondrial cholesterol side-chain cleavage (P450SCC) levels in corpora lutea from LA-treated rats are reduced, indicating that the decrease in P production observed is due in part to an alteration in the steroidogenic luteal capability. Immunocytochemical localization of nuclei exhibiting DNA fragmentation by the technique of terminal deoxynucleotidyl transferase end-labeling showed that LA treatment causes an increase in the number of apoptotic cells in preantral and antral follicles at all times studied (1, 2, 4, or 7 days of LA administration). A similar effect, though less pronounced, was observed in corpora lutea. It is concluded that LA treatment produces a failure in the steroidogenic luteal capability and an increase of apoptotic mechanisms in the ovary, producing as a consequence an interference in the follicular recruitment, growth, and luteinization induced by gonadotropins.

Inhibition of Testicular Steroid Metabolism by Administration of 1-Aminobenzotriazole to Rats

Effects of 1-aminobenzotriazole (ABT) on testicular steroid metabolism were evaluated in rats. Administration of ABT to adult male rats caused dose-dependent decreases in testicular microsomal and mitochondrial cytochrome P450 concentrations. Significant losses of P450 occurred within 8 h of ABT treatment. Accompanying the declines in testicular P450 content were decreases in microsomal 17α-hydroxylase and mitochondrial cholesterol sidechain cleavage activities. Incubation of testicular microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on rates of steroid metabolism. By contrast, incubation of hepatic microsomes with ABT under the same conditions decreased P450 levels and xenobiotic-metabolizing activity. The results indicate that ABT in vivo causes inactivation of steroidogenic P450 isozymes in the testis, but the mechanism of inactivation differs from that on xenobiotic-metabolizing isozymes.

Interleukin-1 beta stimulates sphingomyelin hydrolysis in cultured granulosa cells: evidence for a regulatory role of ceramide on progesterone and prostaglandin biosynthesis.

In granulosa cells labeled to isotopic steady-state with [3H]serine, addition of interleukin-1 beta (IL1 beta) or bacterial sphingomyelinase (SMase) induced a rapid decrease (approximately 60% by 10 min) in cellular [3H]Sphingomyelin content and a prolonged generation (up to 60 min) of [3H]ceramide, the immediate lipid-moiety generated in response to sphingomyelin hydrolysis. In FSH-treated cells, IL1 beta (0.3-30 ng/ml) inhibited progesterone biosynthesis in a dose-dependent manner, an effect that was also observed in cells exposed to increasing concentrations of bacterial SMase (0.003-0.3 U/ml) or the membrane-permeable ceramide analogue N-hexanoylsphingosine (C6-cer:0.1-10 microM). Abrogation of progesterone biosynthesis was not a sole consequence of inadequate cAMP biosynthesis because cyclic nucleotide levels remained elevated (3- to 4-fold over untreated cultures) after addition of IL1 beta, SMase, or two different cell permeable ceramide analogues (C2-cer and C6-cer) to gonadotropin-stimulated granulosa cells. Moreover, taken into account that exogenous SMase or C6-cer partially abolished progesterone biosynthesis induced by But2cAMP (0.5 mM) or cholera toxin (CTX: 1 microgram/ml), the above mentioned results support the notion that activation of the sphingomyelin pathway exerts its inhibitory effects on granulosa cell steroidogenic activity at site(s) of action both proximal and distal to cAMP generation. As determined by RT-PCR analysis, the inhibitory effect of IL1 beta, SMase, or C6-cer on gonadotropin-stimulated steroidogenesis was accompanied by arrested transcription of the mitochondrial cholesterol side chain cleavage enzyme (P450scc) and 3 beta-hydroxysteroid dehydrogenase/delta 5-4isomerase, the two FSH-inducible steps involved in progesterone biosynthesis. Although bacterial SMase or the ceramide analogue C6-cer alone did not exactly reproduce the effect of IL1 beta on granulosa cell prostaglandin E2 (PGE2) biosynthesis, both agents augmented net PGE2 production and messenger RNA levels of the inducible prostaglandin endoperoxide synthase/cyclooxygenase (PGHS-2) in cytokine-treated cells. Although the effect on PGHS-2 messenger RNA may account for the facilitatory role of ceramide on IL1 beta-induced PGE2 biosynthesis, neither SMase nor the membrane-permeant ceramide analogue were able to augment prostaglandin accumulation in the presence of exogenously added arachidonate precursor. Collectively, whereas these results show that ceramide triggers a negative-effector pathway that is both necessary and sufficient to reproduce the inhibitory effect of IL1 beta on FSH-stimulated granulosa cell steroidogenesis, they also support the notion that sphingomyelin hydrolysis may be important for cytokine-induced PGHS-2 expression but not sufficient to reproduce IL1 beta-stimulated PGE2 biosynthesis.

Alteration of the adrenal antioxidant defense system during aging in rats.

The goal of this study was to determine to what extent aging affects the antioxidant defense system of the rat adrenal and to evaluate the impact of any change in this system on the recognized age-related decline in steroidogenic capacity of adrenocortical cells. The studies were conducted on young (2-5 mo) and aging (12-27 mo) Sprague-Dawley rats and involved procedures measuring steroidogenesis; oxidative damage to tissue; non enzymatic antioxidants such as vitamin C, E, and glutathione; and tissue antioxidant enzyme (Mn and CuZn superoxide dismutases, catalase, and glutathione peroxidase) activity and expression (mRNA, protein mass, and location). Some measurements were made also on rats maintained on vitamin E-deficient diets. The data show that adrenals from young animals are especially well protected against oxidative events; i.e., these adrenals show the least endogenous lipid peroxidation and the highest level of resistance to prooxidant-induced damage (of various tissues measured) and show exceedingly high levels of tissue antioxidants. Aging, on the other hand, results in oxidative changes in adrenal tissue that are generally linked in time to a reduction in efficiency of the normally protective antioxidant defense system and to the decline in corticosterone production. We speculate that these events are causally related, i.e., that the age-related reduction in oxidative mechanisms in adrenal tissues leads to oxidative damage of membrane or cytosolic factors important to cholesterol transport, and, as a consequence of this damage, cholesterol cannot reach appropriate mitochondrial cholesterol side chain cleavage sites, and corticosterone production fails.

Inactivation of adrenal cytochromes P450 by 1-aminobenzotriazole: Divergence of in vivo and in vitro actions

Recent investigations demonstrated that administration of 1-aminobenzotriazole (ABT) to rats caused adrenal gland enlargement. Studies were done to pursue the mechanism(s) involved. Preliminary experiments revealed that the adrenal enlargement caused by ABT was associated with a decline in plasma corticosterone concentrations, suggesting inhibition of adrenal steroidogenesis. Indeed, a single injection of ABT (25 or 50 mg/kg body weight) to rats caused concentration-dependent declines (60–80%) in adrenal mitochondrial and microsomal cytochrome P450 (P450) concentrations. The decreases in adrenal P450 levels exceeded those in hepatic microsomes. Accompanying the declines in adrenal P450 concentrations were decreases in steroid hydroxylase activities. Mitochondrial 11β-hydroxylase and cholesterol side-chain cleavage activities and microsomal 21-hydroxylase activity were diminished markedly (60–90%) by ABT treatment. In contrast, activity of adrenal 3β-hydroxysteroid dehydrogenase-isomerase was not affected by ABT, indicating specificity for P450-dependent reactions. Incubation of adrenal microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on steroid hydroxylase activities. Similar incubations with hepatic microsomes caused declines in P450 levels and in the rates of P450-mediated xenobiotic metabolism. The results demonstrate that ABT is a potent inhibitor of adrenal steroid hydroxylases in vivo, but the in vitro studies indicate that the mechanism of action differs from that on other P450 isozymes. The absence of inhibitor effects in vitro suggests that an extra-adrenal metabolite of ABT is responsible for the in vivo inactivation of steroidogenic enzymes.

Routes and regulation of NADPH production in steroidogenic mitochondria.

The first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system that is dependent on NADPH. The pathways of NADPH generation in steroidogenic mitochondria include three major routes catalyzed by: 1. NADP-linked malic enzyme, 2. NADP-linked isocitrate dehydrogenase, and 3. nicotinamide nucleotide transhydrogenase. The main route may differ among cell types and across species. Generally operation of alternative routes, with different substrates is not excluded. The oxidation of NADPH by the mitochondrial P450 systems is not tightly coupled with substrate metabolism, as these systems can reduce O2 by a single electron to produce harmful superoxide radical. To minimize such futile NADPH oxidation, NADPH generation may be regulated by two types of mechanisms: 1. Feedback mechanisms that maintain the ratio of NADPH/NADP+ at a steady-state level by enhancing the rate of NADPH production to keep up with its rate of oxidation, e.g., allosteric regulation of enzymes involved in NADPH production. 2. Hormonal signals that enhance the level of NADPH production in coordination with steroidogenesis. One major hypothesis with experimental evidence is that stimulation of mitochondrial NAD(P)H synthesis is mediated by Ca++ as a second messenger of tropic factors. Tropic stimulation of cells increases the levels of Ca++ in the cytosol and then in the mitochondrial matrix, and the rise in Ca++ activates enzymes involved in NAD(P)H synthesis. These regulatory mechanisms most probably operate in concert adjusted to the steroidogenic activity of the cell.

Peroxisome proliferators and retinoids affect JEG-3 choriocarcinoma cell function.

To examine the hypothesis that nutritional signals regulate trophoblast cell function, JEG-3 choriocarcinoma cells were treated with drugs that stimulate peroxisome proliferator-activated receptors (PPARs). These receptors are thought to mediate in part the effects of lipidic nutrients on gene expression. Because PPARs are modulated by interactions with retinoid-X receptors, we also examined the actions of the peroxisome proliferators in the presence of retinoids. Clofibric acid, a known peroxisome proliferator, suppressed JEG-3 cell growth in association with increases in the tumor suppressor p53 protein and its messenger RNA (mRNA). It reduced CG secretion and CG alpha and CG beta mRNAs in growing cells. However, clofibric acid did not induce peroxisome proliferation in the JEG-3 cells, as assessed by electron microscopy and immunostaining for catalase, a peroxisomal enzyme, or alter levels of mRNAs for peroxisomal proteins, sterol carrier protein-X/sterol carrier protein-2 and acyl-Coenzyme-A oxidase. The mitochondrial cholesterol side-chain cleavage enzyme, cytochrome P450scc, was modestly increased in some experiments. All-trans-retinoic acid and 9-cis-retinoic acid increased CG secretion and CG alpha and CG beta mRNAs, but clofibric acid blunted these stimulatory effects. WY 14,643, another peroxisome proliferator, also reduced CG gene expression without increasing mRNAs encoding peroxisomal proteins or altering P450scc mRNA. The mRNA for a human PPAR, NUC1, was demonstrated in JEG-3 cells, and NUC1 mRNA was shown to be upregulated by 8-bromo-cAMP. We conclude 1) that JEG-3 cells express a PPAR and are subject to regulation by PPAR stimulators; 2) that PPAR stimulation in JEG-3 cells does not promote peroxisome proliferation; and 3) that peroxisome proliferators and retinoids differentially regulate JEG-3 cell endocrine activities. We suggest from these findings that JEG-3 cells possess mechanisms to respond to nutrient cues.

Peroxisome proliferators and retinoids affect JEG-3 choriocarcinoma cell function

To examine the hypothesis that nutritional signals regulate trophoblast cell function, JEG-3 choriocarcinoma cells were treated with drugs that stimulate peroxisome proliferator-activated receptors (PPARs). These receptors are thought to mediate in part the effects of lipidic nutrients on gene expression. Because PPARs are modulated by interactions with retinoid-X receptors, we also examined the actions of the peroxisome proliferators in the presence of retinoids. Clofibric acid, a known peroxisome proliferator, suppressed JEG-3 cell growth in association with increases in the tumor suppressor p53 protein and its messenger RNA (mRNA). It reduced CG secretion and CG alpha and CG beta mRNAs in growing cells. However, clofibric acid did not induce peroxisome proliferation in the JEG-3 cells, as assessed by electron microscopy and immunostaining for catalase, a peroxisomal enzyme, or alter levels of mRNAs for peroxisomal proteins, sterol carrier protein-X/sterol carrier protein-2 and acyl-Coenzyme-A oxidase. The mitochondrial cholesterol side-chain cleavage enzyme, cytochrome P450scc, was modestly increased in some experiments. All-trans-retinoic acid and 9-cis-retinoic acid increased CG secretion and CG alpha and CG beta mRNAs, but clofibric acid blunted these stimulatory effects. WY 14,643, another peroxisome proliferator, also reduced CG gene expression without increasing mRNAs encoding peroxisomal proteins or altering P450scc mRNA. The mRNA for a human PPAR, NUC1, was demonstrated in JEG-3 cells, and NUC1 mRNA was shown to be upregulated by 8-bromo-cAMP. We conclude 1) that JEG-3 cells express a PPAR and are subject to regulation by PPAR stimulators; 2) that PPAR stimulation in JEG-3 cells does not promote peroxisome proliferation; and 3) that peroxisome proliferators and retinoids differentially regulate JEG-3 cell endocrine activities. We suggest from these findings that JEG-3 cells possess mechanisms to respond to nutrient cues.

Mechanisms of regulation of ovarian sterol metabolism by insulin-like growth factor type II: in vitro studies with swine granulosa cells.

The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 +/- 1.2 ng/ml (0.61 +/- 0.16 nM) between 0-48 h of culture and 27 +/- 5.7 ng/ml (3.6 +/- 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 microgram/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Maturational changes in steroidogenesis in the inner and outer zones of the guinea pig adrenal cortex

Studies were done to determine the effects of age on steroidogenesis in the inner (zona reticularis) and outer (zona fasciculta plus glomerulosa) zones of the guinea pig adrenal cortex. In 35-day-old animals, cortisol production by adrenal outer zone cells was approximately twice as great as that by inner zone cells. With aging, cortisol secretion by inner zone cells decreased to very low levels, but there was no detectable change in the capacity for cortisol production by the outer zone. However, the outer zone comprised a progressively decreasing fraction of the total adrenal mass in older animals. To determine the basis for the decline in cortisol production by inner zone cells with aging, the activities of several steroidogenic enzymes were determined. Microsomal 21-hydroxylase activity was greater in the inner than outer zone but was not significantly affected by age. By contrast, 17α-hydroxylase activity was greater in the outer zone at all ages, and decreased with aging in the inner but not the outer zone. Mitochondrial cholesterol sidechain cleavage and 11β-hydroxylase activities were also higher in the outer than inner zone and declined in the zone only in older animals. The decrease in inner zone cholesterol sidechain cleavage activity with aging was proportionately greater than the age-dependent changes in other enzyme activities. The results indicate that the effects of aging on steroidogenesis are both zone- and enzyme-specific. The overall decline in cortisol secretion by the guinea pig adrenal cortex with aging is attributable to both a decrease in cortisol production by the cells of the zone reticularis and a disproportionate increase in the mass of the gland comprised by this zone. The decrease in cortisol secretion correlates closely with a decline in cholesterol sidechain cleavage activity in the zona reticularis, and may be causally related.