Metabolic control mechanisms in mammalian systems. Hormonal regulation of phosphofructokinase in the rat prostate and seminal vesicles

1. The hormonal regulation of phosphofructokinase was investigated in the accessory reproductive organs of the orchidectomized rat. 2. Phosphofructokinase activities declined to 51% and 47% in the prostate and 9% and 6% of the normal values in seminal vesicles 4 and 8 weeks after castration respectively. Administration of testosterone (100mug./100g. body wt.) for 3 days reversed substantially the effects of orchidectomy, and phosphofructokinase activity increased to 173% in the prostate and 536% in seminal vesicles as compared with the values of castrated controls. 3. Time-course studies demonstrated that after a single injection of testosterone (5mg./100g. body wt.) phosphofructokinase activity was maximally elevated to 236% in the prostate and 342% in seminal vesicles at 24hr. 4. Dose-response studies revealed that 2.5mg. of testosterone propionate/100g. body wt. was the minimal amount necessary to induce significant increases in enzyme activity in both accessory sex organs; maximal increases were obtained with a dose of 5mg./100g. body wt. 5. The observed enzyme increases induced by testosterone were inhibited by the simultaneous administration of oestradiol-17beta, and phosphofructokinase activity in this group of rats remained at 97% in the prostate and 137% of the control values in seminal vesicles. Oestradiol-17beta by itself failed to produce any significant effect on enzyme activity in either of these secondary sexual tissues. 6. The nature of the testosterone-induced increases in phosphofructokinase activity was studied by using a variety of inhibitors of RNA and protein synthesis. Cycloheximide, 5-fluorouracil and ethionine largely blocked the androgen-stimulated rise in enzyme activity observed 24hr. after steroid injection. The inhibitory effect of ethionine was completely reversed by the simultaneous administration of methionine. 7. Actinomycin, which is known to inhibit the synthesis of messenger RNA as well as the synthesis of other cellular RNA fractions, when given simultaneously with the hormone, also inhibited the testosterone-induced increases in prostatic and seminal-vesicular phosphofructokinase. However, when the antibiotic was given 6 or 12hr. after injection of the steroid, practically no inhibition of phosphofructokinase induction was obtained. This indicates that, once the enzyme-forming machinery is turned on and allowed to operate for a few hours, actinomycin is incapable of reversing the hormone-induced enzyme responses. 8. The results presented suggest that new RNA and protein synthesis may be involved in the observed androgen-induced increases in phosphofructokinase activity in the prostate and seminal vesicles of the orchidectomized rat.     

The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

Variations in the concentrations of androstenedione in peripheral plasma of women the day and during the menstrual cycle

The target tissues (e.g., hypothalamus, pituitary, uterus and vagina) of mature female ovariectomized rats show selective uptake of radioactivity in one hour after the injection of 6,7, ³H-estradiol-17β in a dose of 0.1 μg per 100 g body weight. Injection of 100 μg norethindrone or norgestrel per 100 g body weight 15 min before or 15 min after the administration of tritiated estradiol reduced the radioactivity in most target tissues, and also in the non-target tissues to a lesser extent. The uptake of radioactivity in the pituitary and uterus is reduced more by norethindrone than by norgestrel treatment when these Steroids were injected 15 min after estradiol-17β injection. It appears that there exists a competitive inhibition of estradiol-17β by these contraceptive Steroids in the rat. It is speculated that such competition with estradiol-17β may be an inherent property of the 17-substituted 19-nortestosterone group of Steroids.     

The in vivo metabolism of 7 beta, 17-dimethyltestosterone-6,7-3H.

7 beta, 17-Dimethyltestosterone (17 beta-hydroxy-7 beta, 17-dimethyl-4-androsten-3-one) (I) was given to three subjects in oral doses of 400 mg per day for ten days. The initial dose contained the steroid tritiated in the 6 and 7 positions. Plasma levels and urinary excretion patterns were followed in all three subjects. Isolations were done on the urine, plasma, and stools of one patient. From the urine 7 beta, 17-dimethyl- 5 alpha-androstane-3 beta,17 beta-diol (VI) was isolated from the nonhydrolyzed fractions. Unchanged (I), 7 beta,17-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (III) and 7 beta, 17-dimethyl-5 beta-androstane-3 beta,17 beta-diol (IV) were isolated from the nonhydrolyzed and enzyme-hydrolyzed fractions. 7 beta,17-dimethyl-5 alpha-androstane-3 alpha,17 beta-diol (V) was isolated from the enzymatic fractions. From the stools were isolated unchanged (I), (III), (IV), (V), and (VI). Unchanged (I) and its 5 alpha-dihydro derivative (17 beta-hydroxy-7 beta,17-dimethyl-5 alpha-androstan-3-one) (II) were identified in the plasma. The total recovery of radioactivity in the one patient on whom the isolations were done was 57%; 40% from the urine and 17% from the stools.     

Dose–response effect of Red Maca (Lepidium meyenii) on benign prostatic hyperplasia induced by testosterone enanthate

The main goal of this study was to determine the effect of a freeze-dried aqueous extract of the red variety of Lepidium meyenii (Red Maca) on testosterone-induced benign prostatic hyperplasia (BPH) in adult rats of the Holtzman strain. Rats were treated with freeze-dried aqueous extract of Red Maca at doses of 0, 0.01, 0.05, 0.1, and 0.5 g/kg body wt. A positive control group received Finasteride (0.6 mg/kg body wt.). After treatment, the animals were sacrificed, and the ventral prostate was extracted, and weighed. HPLC was used to determine the presence of glucosinolates in Red Maca. The prostate weight diminished in a dose-dependent fashion in rats treated with Red Maca. The effect of Red Maca was better than that observed with Finasteride. Finasteride, but not Red Maca, reduced seminal vesicles weight. Analysis of the HPLC indicated the presence of benzyl glucosinolate (Glucotropaeolin) with a content of 0.639%. Serum testosterone levels were not affected by Red Maca. Moreover, serum testosterone levels were not related to prostate or seminal vesicles weight in rats treated with vehicle and Red Maca. In conclusion, Red Maca administered orally in rats seems to exert an inhibitory effect at a level post DHT conversion, on the BPH-induced experimentally, although a direct measure of reductase action would still be required.     

Effect of hypothalamic surgery on prolactin release induced by 5-hydroxytryptophan (5-HTP) in rats.

Intravenous injections of varying doses of 5-HTP (1, 3 and 5 mg/100 g body wt), a precursor of serotonin, caused a significant and dose-related increase in plasma prolactin concentrations in urethane-anesthetized rats. Increases in plasma prolactin concentrations caused by 5-HTP (1 mg/100 g body wt iv) were abolished by the concomitant administration of L-DOPA (2 mg/100 g body wt iv). Plasma prolactin levels were also significantly elevated following the injection of 5-HTP in rats with complete hypothalamic deafferentation, whereas 5-HTP had no significant effect on plasma prolactin levels in rats with extensive hypothalamic ablation. These results suggest that 5-HTP causes prolactin secretion by stimulating the serotoninergic mechanism in the hypothalamus.     

Developmental pattern of Δ5 3β-hydroxysteroid dehydrogenase activity in isolated rat Leydig cells

A direct method for determination of Δ⁵ 3β-hydroxysteroid dehydrogenase (3β-HSD) activity was employed in isolated Leydig cells (LC) derived from rats on fetal day 19 (F₁₉) and postnatal (N) days 1,12,24, 34 and 45 and adults. The activity of 3β-HSD in the adult LC was 1.15 ± 0.02 (μmole/μg DNA/hr, mean ± SEM, n = 73). Activities in the other groups, expressed as a percentage of the respective adult control, were: F₁₉-38%; N₁-39%; N₁₂-8%; N₂₄-89%; N₃₄-166%; and N₄₅-118%. A good correlation was found between histochemical staining for 3β-HSD and the quantitive method employed. Using (³H)-DHA as a substrate, LC isolated from F₁₉, n₁ and N₁₂ produced testosterone in appreciable amounts (41%, 55% and 20% of the toal products respectively) whereas at advanced stages of development (N₂₄ to adulthood) the major product was androstenedione (93 ± 1%). These findings may be explained by the observed decrease in 17β-hydroxysteroid dehydrogenase (17β-HSD) activity, due to an insufficient supply of NADPH, in the older vs. earlier stages of development. This study indicates the presence of steroidogenic enzymatic activity in LC throughout development in the rat. It also provides a relatively simple in vitro model for studies of testicular regulation during development.     

Androgen regulation of androgen receptor content and distribution in the ventral and dorsolateral prostates of aging AXC rats

Total androgen receptor content of ventral or dorsolateral prostate of intact, aged (730–740 day old) rats is decreased 50% when compared to intact, young mature (150–170 day old) rats. Treatment with exogenous testosterone increased ventral and dorsolateral prostate androgen receptor content per cell in aged rats to values identical to those of prostates of young mature rats. The increase in prostate receptor content was not attributable to testosterone mediated cellular hypertrophy or hyperplasia. At 24 hr post-orchiectomy ventral prostate cytoplasmic androgen receptors are depleted of endogenous androgen, without any decrease in number of receptors per cell, and nuclear androgen receptors are undetectable. During 30 to 60 min after a single 200 μg testosterone injection, ventral prostate nuclear receptor content increased to the level of intact control rats without producing any reduction in total cytoplasmic androgen receptor content. Although dorsolateral prostate is devoid of cytoplasmic androgen receptor, the effects of orchiectomy and testosterone treatment upon nuclear androgen receptor are comparable to those seen in ventral prostate. These effects of orchiectomy and testosterone injection upon prostatic receptor content and distribution were identical in prostates of young and aged rats. Our studies show that receptor processing in prostates of young and aged rats does not involve a process by which nuclear receptor is derived by depletion of cytoplasmic receptor. Moreover, our studies of the effect of short-term (48 hr) exogenous testosterone treatment upon androgen receptor content in prostates of aged rats are the first demonstration that androgen receptor content may be enhanced independent of generalized androgen mediated anabolic effects in prostate.     

Simultaneous radioimmunoassay of testosterone,dihydrotestosterone, 5α-androstane-3α, 17β-diol and 5α-androstane-3β, 17β-diol in the plasma of adult male rats

A single thin layer chromatography and three antibodies were used for the specific radioimmunoassay of four androgens in pooled rat plasma (Sprague-Dawley adult males). The following values were found (pg/ml ± SD). Testosterone : 3, 138 ± 173; dihydrotestosterone : 374 ± 20; 5α-androstane-3α 17β-diol : 284 ± 24; 5α-androstane-3β, 17β-diol : 223 ± 11.     

A simple radioimmunoassay for the measurement of testosterone glucosiduronate in unextracted urine

A simple, reliable and rapid radioimmunoassay (RIA) for the determination of testosterone glucosiduronate (TG) in crude urine is described. Two protein-TG complexes were investigated in raising antibodies: a) Bovine serum albumin (BSA)-TG and b) human plasma Cohn's fraction IV-4 (CF)-TG. In rabbits, high liters of antibodies were obtained after the injection of CF-TG. The specificity of the antiserum was sufficiently high (cross reaction with free testosterone 27%, with 5α-dihydrotestosterone-glucosiduronate 20%). TG was estimated in small aliquots of male and female urine after evaporation overnight at 50° C in order to eliminate interfering material. The intraassay coefficient of varation (CV) was found to be 6% and the interassay CV 11%. TG has been determined in 40 samples of urine simultaneously by “direct” RIA and by a “classical” RIA following hydrolysis with β-glucuronidase. The coefficient of correlation was found to be 0.89. The mean excretion of TG in the urine of 26 healthy men amounted to 164 ± 51 μg/24 hours with a range from 97 to 546 μg/24 hours. In a group of 16 women a mean urinary excretion of TG of 24 ± 10 μg/24 hours was determined. The method allows a technician to assay 40 samples per day.     

The effect of ACTH administration on plasma testosterone, dihydrotestosterone and serum LH concentrations in normal men

Plasma cortisol (F), testosterone (T), dihydrotestosterone (DHT) and serum luteinizing hormone (LH) concentrations were measured in 8 normal young men at 8 AM on two control days. Exogenous adrenocorticotrophin (ACTH) 20 U.S.P. units per m² of body surface area every 12 or 6 hours was administered intramuscularly for 4 days. Twenty-four hours after starting ACTH administration, the plasma T and DHT concentrations were significantly lower than those of the control days on a paired t test. No significant change in serum LH concentration could be demonstrated. Similar results were observed after 48, 72 and 96 hours of ACTH stimulation.     

A radioimmunoassay for the measurement of 5-androstene-3β,17β-diol in plasma

A reliable radioimmunoassay for the determination of 5-androstene-3β, 17β-diol in plasma is described. Antisera were obtained by immunization of rabbits with 3β,17β-dihydroxy-5-androsten-16-one coupled to bovine serum albumin in position 16. The antiserum was characterized by titer, affinity, and specificity. Only dehydroepi-androsterone (24.3 %) and pregnenolone (2.7 %) showed a small crossreactivity. The assay method consisted of extraction with ether, thin-layer chromatography and endpoint determination.The reliability of the method was studied. The interassay variability was 7.5 % at a concentration of 1.22 μg/l. The limit of detection was 0.068 μg/l. Specificity was achieved by Chromatographic separation of the crossreacting steroids. Mass recovery experiments with 250 and 500 pg were performed, in which 99.0 ± 4.6 % of the smaller and 97.6 ± 11.3 % of the greater mass were recovered. In 45 healthy adult males plasma concentrations between 0.44 and 1.80 μg/l were found. The median was 1.06 μg/l. Stimulation of the Leydig cells with human chorionic gonadotropin (HCG) increased plasma concentrations by 93 % (average in 12 males). Therapeutic castration in 8 men caused an average decrease of 55.4 % in plasma values.     

Vitamin D3 sulfoconjugate in pregnant and lactating mother rats after dosing with 3H vitamin D3

Twenty four hours after dosing of pregnant rats with 3H vitamin D3 i.v. the sulfoconjugate was detected only in the kidney. In contrast, 24 or 48 hours after 3H vitamin D3 i.v. dosing the vitamin D3 sulfoconjugate was detected in the plasma, liver, kidney and mammary glands of lactating mother rats.     

Discovery of 2-methyl-1-{1-[(5-methyl-1H-indol-2-yl)carbonyl]piperidin-4-yl}propan-2-ol: A novel,potent and selective type 5 17β-hydroxysteroid dehydrogenase inhibitor

Type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5), also known as aldo-keto reductase 1C3 (AKR1C3), is a member of the aldo-keto reductase superfamily of enzymes and is expressed in the human prostate. One of the main functions of 17β-HSD5 is to catalyze the conversion of the weak androgen, androstenedione, to the potent androgen, testosterone. The concentration of intraprostatic 5α-dihydrotestosterone (DHT) in patients following chemical or surgical castration has been reported to remain as high as 39% of that of healthy men, with 17β-HSD5 shown to be involved in this androgen synthesis. Inhibition of 17β-HSD5 therefore represents a promising target for the treatment of castration-resistant prostate cancer (CRPC). To investigate this, we conducted high-throughput screening (HTS) and identified compound 2, which displayed a structure distinct from known 17β-HSD5 inhibitors. To optimize the inhibitory activity of compound 2, we first introduced a primary alcohol group. We then converted the primary alcohol group to a tertiary alcohol, which further enhanced the inhibitory activity, improved metabolic stability, and led to the identification of compound 17. Oral administration of compound 17 to castrated nude mice bearing the CWR22R xenograft resulted in the suppression of androstenedione (AD)-induced intratumoral testosterone production. Compound 17 also demonstrated good isoform selectivity, minimal inhibitory activity against either CYP or hERG, and enhanced pharmacokinetic and physicochemical properties.     

The stimulatory effect of testosterone propionate and 17β-estradiol on 5′-methylthioadenosine phosphorylase activity in rat target tissues

The effect of castration and subsequent administration of 17β-estradiol and testosterone propionate on 5′-methylthioadenosine phosphorylase activity in rat target tissues was studied. Castration 34 days earlier resulted in a 95 reduction in ventral prostate 5′-methylthioadenosine phosphorylase activity and 16 days earlier in a 67% reduction in uterine 5′-methylthiodenosine phosphorylase activity. Four days of testosterone propionate administration stimulated ventral prostate 5′-methylhioadenosine phosphorylase activity 32% above castrate levels, which represented more than 50% of the intact control levels. 17β-Estradiol on the other hand stimulated uterine 5′-methylthioadenosine phosphorylase activity of 35% above castrate controls within 24h and with 3 days of continuous hormone treatment to within 97% of the intact control levels. However, castration and subsequent 17β-estradiol administration did not affect 5′-methylthioadenosine phosphorylase activity in rat liver and lung. Both prostate and uterine 5′-methylthioadenosine phosphorylase were shown to metabolize 5′-methylthioadenosine to 5′-methylthioribose through a 5′-methythiribose 1-phosphate intermediate. The data suggest that 5′-methylthioadenosine is not allowed to accumulate in rat target tissues even under conditions which are known to stimulate polyamine synthesis.     

Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo

Aiming at the development of new drugs for the treatment of prostate cancer, the effects of steroidal compounds and one non-steroidal substance on androgen biosynthesis were evaluated in vitro and in vivo. Sa 40 [17-(5-pyrimidyl)androsta-5,16-diene-3beta-ol], its 3-acetyl derivate Sa 41 and BW 19 [3,4-dihydro-2-(4-imidazolylmethyl)-6-methoxy-1-methyl-naphthalene] are compounds from our group, which have been developed as inhibitors of CYP 17 (17alpha-hydroxylase-C17, 20-lyase, the key enzyme in androgen biosynthesis). They have been compared with CB 7598 [abiraterone: 17-(3-pyridyl)androsta-5,16-diene-3beta-ol], its 3-acetyl compound CB 7630 and ketoconazole, compounds which already have been used clinically. The most potent compound toward human CYP 17 (testicular microsomes) was Sa 40 (IC(50) value of 24 nM), followed by Sa 41, CB 7598, BW 19, CB 7630 and ketoconazole. Sa 40 shows a type II difference spectrum and a non-competitive type of inhibition (K(i) value of 16 nM). No recovery of enzyme activity was observed after preincubation of CYP 17 with Sa 40 and subsequent charcoal treatment. In Escherichia coli cells coexpressing human CYP 17 and NADPH-P450 reductase, Sa 40 was more active than CB 7598 and BW 19, whereas the acetyl compounds were not active. The latter three compounds were equally active towards rat CYP 17. Male Sprague-Dawley (SD) rats were administered daily for 14 days BW 19 and the acetyl derivatives Sa 41 and CB 7630 as prodrugs (0.1 mmol/kg intraperitoneally). The test compounds strongly reduced plasma testosterone concentration, as well as prostate and seminal vesicles weights. They showed moderate inhibitory effects on the weights of levator ani, bulbocavernosus and testes, whereas they led to an increase in adrenal and pituitary weights. The only exception was BW 19 which did not change pituitary weights. Based on its superiority on the human enzyme, it was concluded that Sa 40 in its 3beta-acetate form (Sa 41) could be a promising candidate for clinical evaluation.     

Isolation of non-heparin-binding and heparin-binding proteins of boar prostate

Proteins of boar prostate secretion were separated by affinity chromatography on heparin-polyacrylamide to non-heparin-binding (H) and heparin-binding (H+) protein fractions. H- and H+ fractions were then subjected to RP HPLC. Elution profiles of H-and H+ fractions of prostate secretion were compared with those of seminal plasma and the amounts of corresponding proteins were compared. Besides, the isolated proteins were characterized by SDS-PAGE. In the H- fraction of prostate secretion, PSP I and PSP II spermadhesins and in the H+ fraction AQN 2 and AWN 1 spermadhesins were found in substantially lower amounts than in seminal plasma. On the contrary, beta-microseminoprotein was identified in abundant amounts both in H- and H+ fractions of boar prostate secretion. AQN 2 and AWN 1 spermadhesins were proved by their antibodies. Some seminal plasma proteins originating mainly in seminal vesicles could also be secreted by the prostatic gland. beta-Microseminoprotein was found to be produced mainly by the prostate.     

Conversion of orally administered 2-n.pentylaminoacetamide into glycinamide and glycine in the rat brain

Male Sprague Dawley albino rats were treated orally with 2-n.pentylaminoacetamide (10 to 100 mg/kg b.wt). This oral administration provoked a dose-related and time-dependent accumulation of glycinamide in forebrain, cerebellum, and medulla, and to increased levels of glycine in the three brain areas, and of serine in medulla. In kidney, liver and plasma, the accumulation of glycinamide was lower and there was no increase in glycine and serine levels. With a dose of 100 mg/kg b.wt, 28% of the drug were eliminated unchanged and 16% as glycinamide, in urines collected for 24 h. In all tissues examined, 2-n.pentylaminoacetamide and glycinamide levels peaked at 1 h and were nil again after 24 h, the ratio of 2-n.pentylaminoacetamide over glycinamide decreasing more rapidly in brain than in kidney and liver. Contrasting with the effects of 2-n.pentylaminoacetamide, the oral administration of glycinamide (66 mg/b.wt) led, 2 hours later, to similar low rises of glycinamide in plasma and brain. In another control experiment, the intraperitoneal injection of a large dose of glycine (450 mg/kg b.wt) provoked, 30 min later, modest rises of glycine levels in the central nervous system that merely reflected a contamination by plasma glycine.     

Estrogen effects on NADH oxidase and superoxide dismutase in prepubertal female rats

Thirty-four day old, ovariectomised rats were treated with increasing doses of estradiol, 2-hydroxyestradiol 2,3-dimethyl ether (23E₂), 4-hydroxyestradiol 3,4-dimethyl ether (34E₂) and 4-methoxy-estradiol (4ME₂) for five days by subcutaneous injection. Superoxide dismutase, phenol activated NADH oxidase and uterine dry weights were determined. Only estradiol was found to be uterotrophic and increased NADH oxidase activity in these experiments. Both 23E₂ and 34E₂ treatment reduced the enzyme activity significantly. Though 4ME₂ showed a decrease in NADH oxidase at 0.05μg/100gm body weight there was no further decrease at higher dose (5μg/100gm). The Superoxide dismutase (SOD) in uterus and liver was unaffected by estradiol, while 23E₂, 34E₂, and 4ME₂ significantly reduced SOD in both liver and uterus. These results indicate that 23E₂, 34E₂ and 4ME₂, in spite of their non-uterotrophic property, affect uterine metabolism. Furthermore, in view of the reports indicating the importance of SOD levels in various tumors and since catecholestrogens are observed to reduce SOD levels in liver and uterus, it is suggestive that catecholestrogens may play an important role in the pathophysiology of certain tumors.     

Epitestosterone--an endogenous antiandrogen?

Epitestosterone (17 alpha-hydroxy-4-androsten-3-one), a normal constituent of human plasma and urine, prevents the testosterone induced changes in body weight and in organ weights of seminal vesicles and kidney of castrated male mice. It competes with methyltrienolone in the binding to rat prostate cytosol (Ki = 29.8 nmol.l-1. It inhibits also the activity of 5 alpha-reductase from rat prostate pellet (Ki = 1.2 mumols.l-1). Epitestosterone can be considered as a weak antiandrogen in the term of displacement of androgen from receptor binding and as an efficient inhibitor of 5 alpha-reductase.