ABSTRACT

The presence and production of 5-ene-steroid fatty acid esters (SFA) has been previously reported in bovine adrenals. A study was conducted, using a series of chromatographic procedures and radioimmunoassays, to determine the levels of SFA in adrenals from man, cattle, dog, rat and guinea-pig, and to assess, in both rats and guinea-pigs, the effect of ACTH on SFA production by adrenals and their subsequent secretion into the circulation. The effects of ACTH on plasma SFA and non-conjugated steroid levels were also investigated in human subjects. Our data indicated that adrenal pregnenolone fatty acid ester (PREG-FA) levels were below 40% of PREG levels in cattle, dog, rat and guinea-pig while, in man, PREG-FA levels were threefold those of PREG. A large proportion of dehydroepiandrosterone (DHEA) and 5-androstene-3β,17β-diol were present as fatty acid ester derivatives in the adrenals of all species, with the exception of cattle. In both rats and guineapigs, administration of ACTH caused a sharp increase in adrenal PREG of approximately threefold which lasted for 6 h, while the concentration of adrenal PREG-FA was slightly increased for a short time. In plasma, however, a marked rise in PREG-FA occurred, while the changes in PREG levels were much lower than those of its acylated counterpart. In man, PREG and DHEA concentrations were rapidly stimulated two-fold in the first 30 min following the administration of ACTH, while PREG-FA and DHEA-FA levels were increased by approximately 2·5-fold (P<0·01) at 120 and 180 min. Our data suggest that there is a rapid turnover of SFA in adrenals which causes accumulation of non-polar steroids in the plasma.

Journal of Endocrinology (1990) 127, 505–511

ABSTRACT

Recent reports have shown that RU 486, a synthetic glucocorticoid and progestin antagonist, has direct effects on tissues secreting steroids. In order to characterize the effects of RU 486 on steroidogenesis further, guinea-pig fasciculata-glomerulosa (FG) cells in primary culture were treated for 48 h with RU 486. RU 486 caused an alteration of basal as well as ACTH-stimulated steroid secretion. Corticosterone and cortisol secretion decreased by 50% while the secretion of 17-hydroxyprogesterone and C₁₉ steroids were increased. The activity of steroidogenic enzymes was measured using tritiated steroids. In RU 486-treated cells, the activity of 21-hydroxylase was dramatically inhibited while there was an increase in 17-hydroxylase and 17,20-desmolase activities. The effects of RU 486 on enzyme activities were dependent upon dose and time. The effects of the compound were not reversed by concomitant treatment of FG cells with R-5020 or dexamethasone, thus suggesting that RU 486 acted directly on steroidogenic enzymes to alter their activity.

Journal of Endocrinology (1991) 130, 71–78

ABSTRACT

The present study examined the effects of steroids on steroidogenic enzyme activity in adrenal glands. Guinea-pig fasciculata-glomerulosa (FG) cells maintained in primary culture were exposed to steroids for 48 h. Although the treatment with androstenedione alone had no effect on 3β-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3β-HSD), 17-hydroxylase and 17,20-lyase activities, there was inhibition of 11-hydroxylase and 21-hydroxylase activities. When FG cells were exposed to 10 nmol ACTH/l for the last 24 h of incubation, ACTH alone had no effect on steroidogenic enzymes but, while combined with androstenedione, it further decreased 21-hydroxylase activity and stimulated 17-hydroxylase and 17,20-lyase activities. Cortisol, corticosterone, oestradiol and 11β-hydroxy androstenedione had no effect on steroidogenic enzyme activities while the inhibitory effect on 21-hydroxylase activity was only observed with androstenedione, testosterone and dihydrotestosterone. Addition of hydroxyflutamide, a pure antiandrogen, did not block the inhibitory effect of androstenedione on 21-hydroxylase and 11-hydroxylase activities. The reduction in oxygen tension from 19 to 2% which was aimed at examining the oxygen-mediated effects on steroidogenic enzymes, revealed that the reduction in 21-hydroxylase activity induced by androstenedione could not be prevented by low oxygen tension. An interaction of C₁₉ steroids at the level of the enzymes is also suggested by our finding that androstenedione had no effect on basal and ACTH-stimulated steady-state 11-hydroxylase, 17-hydroxylase, 17,20-lyase and 21-hydroxylase mRNA levels. These results indicate that C₁₉ steroids alter the adrenal steroidogenic enzyme activities in such a manner that C₁₉ steroid synthesis is increased while glucocorticoid production is inhibited. The mechanism of action of C₁₉ steroids does not involve gene expression for steroidogenic enzymes but probably a direct interaction with steroidogenic enzymes, namely 21-hydroxylase, 17-hydroxylase and 17,20-lyase. Our data suggest that C₁₉ steroids may reduce the amount of 21-hydroxylase in the microsomal fraction which may have a major impact on the levels of microsomal P450 reductase available for 17-hydroxylase and 17,20-lyase activities.

Journal of Endocrinology (1992) 132, 269–276

Abstract

Dehydroepiandrosterone (DHEA) administered percutaneously by twice daily application for 7 days to the dorsal skin of the rat stimulates an increase in ventral prostate weight with approximately one third the potency of the compound given by subcutaneous injection. The doses required to achieve a 50% reversal of the inhibitory effect of orchiectomy are approximately 3 and 1 mg respectively. By the oral route, on the other hand, DHEA has only 10–15% of the activity of the compound given percutaneously. Taking the bioavailability obtained by the subcutaneous route as 100%, it is estimated that the potencies of DHEA by the percutaneous and oral routes are approximately 33 and 3% respectively. Similar ratios of activity were obtained when dorsal prostate and seminal vesicle weight were used as parameters of androgenic activity. When examined on an estrogen-sensitive parameter, namely uterine weight in ovariectomized rats, the stimulatory effect of DHEA was much less potent than its androgenic activity measured in the male animal, a 50% reversal of the inhibitory effect of ovariectomy on uterine weight being observed at the 3 and 30 mg doses of DHEA administered by the subcutaneous and percutaneous routes respectively. When measured on uterine weight, percutaneous DHEA thus shows a 10% potency compared with the subcutaneous route. The sulfate of DHEA (DHEA-S), on the other hand, was approximately 50% as potent as DHEA at increasing ventral prostate weight after subcutaneous or percutaneous administration. When the effect was measured on dorsal prostate and seminal vesicle weight, percutaneous DHEA-S had 10–25% of the activity of DHEA. DHEA decreased serum LH levels in ovariectomized animals, an effect which was completely reversed by treatment with the antiandrogen flutamide. On the other hand, flutamide had no significant effect on the increase in uterine weight caused by DHEA, thus suggesting a predominant estrogenic effect of DHEA at the level of the uterus and an androgenic effect on the feedback control of LH secretion. The present data show a relatively high bioavailability of percutaneous DHEA as measured by its androgenic and/or estrogenic biological activity in well-characterized peripheral target intracrine tissues in the rat.

Journal of Endocrinology (1996) 150, S107–S118

Abstract

Obesity is a heterogeneous condition and not every obese patient is at increased risk of cardiovascular diseases (CVD). It is now well established that the regional distribution of body fat is a critical correlate of the metabolic complications of obesity. Studies that have assessed adipose tissue distribution by imaging techniques such as computed tomography have demonstrated the importance of the intra-abdominal (visceral) fat depot as a marker of a cluster of metabolic abnormalities which include glucose intolerance, insulin resistance, hyperinsulinemia, hypertriglyceridemia, elevated number of apo B-carrying lipoproteins as well as hypoalphalipoproteinemia. Although the association between visceral obesity and metabolic complications can hardly be questioned, it has been suggested that it may not necessarily represent a causal relationship. For instance, concomitant alterations in sex steroid levels have been found in both men and women with abdominal (visceral) obesity which have also been reported to be significantly correlated with the insulin resistant-dyslipidemic state found in abdominal obese subjects. In women, abdominal obesity is associated with increased free testosterone concentrations and reduced sex hormone binding globulin (SHBG) levels, whereas in men this condition is associated with reduced testosterone and adrenal C₁₉ steroid (dehydroepiandrosterone, androstenedione, androstene-3β, 17β-diol) levels as well as decreased SHBG concentrations. These altered steroid and SHBG levels have been reported to be independent correlates of the metabolic complications of visceral obesity although they cannot solely account for the increased CVD risk found in these patients. In this regard, intervention studies are clearly warranted to better quantify the respective contribution of excess visceral adipose tissue and of the concomitant alterations in sex steroid levels as modulators of metabolic disturbances increasing CVD risk in obesity.

Journal of Endocrinology (1996) 150, S155–S164

Abstract

We have evaluated the effect of dehydroepiandrosterone (DHEA) replacement therapy in 60- to 70-year-old women (n=15) who received a single daily percutaneous application of a 10% DHEA cream for 12 months. While anthropometric measurements showed no change in body weight, we observed a 9·8% decrease in subcutaneous skinfold thickness at 12 months (P<0·05). This was confirmed by measurements of midthigh fat and muscle areas by computed tomography where a 3·8% decrease (P<0·05) in femoral fat and a 3·5% increase (P<0·05) in femoral muscular areas were observed at 12 months. There was no significant change in abdominal fat measurements but the waist-to-hip ratio was only 0·83 at the onset of treatment. These changes in body fat and muscular mass were associated with a 11% decrease (P<0·05) in fasting plasma glucose and a 17% decrease (P<0·05) in fasting insulin levels. Treatment with DHEA had no adverse effect on the lipid or lipoprotein profile. In fact, an overall trend towards a decrease in total cholesterol and its lipoprotein fractions was observed. Plasma triglycerides were not affected. Plasma high-density lipoprotein (HDL) cholesterol decreased by 8% but the ratio HDL/cholesterol was unchanged by DHEA treatment because of a parallel decrease in total cholesterol. The index of sebum secretion showed a 73% increase (P<0·05) during the 12 months of DHEA therapy followed by a return to pretreatment values 3 months after cessation of therapy. At the same time, sex hormone-binding globulin levels decreased (P<0·05) during treatment and returned to pretreatment values 3 months after the end of therapy. Serum gonadotropins were not changed by DHEA treatment. Although not significant, we observed a tendency towards an elevation in serum GH levels. Values of serum IGF-I remained unchanged while plasma IGF-binding protein-3 levels significantly decreased (P<0·05) during treatment and returned to pretreatment values after cessation of DHEA therapy. The present data clearly indicate the beneficial effects of DHEA therapy in postmenopausal women through its transformation into androgens and/or estrogens in specific intracrine tissues without any significant side effects.

Journal of Endocrinology (1996) 150, S43–S50

Abstract

In addition to dehydroepiandrosterone (DHEA) sulfate (S), the human plasma also contains a second form of DHEA ester: DHEA-fatty acid esters (DHEA-FA). In the human adult, the plasma concentrations of DHEA-FA, DHEA and DHEAS are in the range of 6, 12 and 2000 nm respectively. Although the adrenal is responsible for almost all production of DHEAS in the circulation, DHEA-FA is formed from DHEA by an enzyme present in the circulation. Our work has clearly demonstrated that lecithincholesterol acyltransferase, localized on high density lipoprotein, is responsible for DHEA-FA production. Once DHEA-FA is formed, it is subsequently transferred to very low density lipoprotein (VLDL) and low density lipoprotein (LDL), like cholesteryl esters. Plasma lipoproteins contain at least 90% of circulating DHEA-FA of which 40% are found in the LDL fraction. Analysis of the fatty acid composition of tritiated DHEA-FA-labelled LDL ([³H]DHEA-FA-LDL) indicated the prevalence of DHEA-linoleate/palmitoleate and DHEA-oleate. Treatment of [³ H]steroid-FA-LDL with charcoal does not remove radioactivity, thus suggesting that the non-polar steroid is incorporated into the central non-polar core of the lipoproteins. Incubation of [³H]DHEA-FA-LDL with ZR-75–1 breast cancer cells produced a time-dependent increase in labeled non-conjugated steroids in the cell culture medium, whereas the levels of tritiated DHEA-FA decreased. Lipoidal radioactivity in cells increased with time, but non-conjugated radioactivity associated with the cells showed no such increase. HPLC analysis of the culture medium indicated the presence of tritiated DHEA and androst-5-ene-3β, 17β-diol. Our study indicates that circulating DHEA-FA incorporated into lipoproteins may indeed act as a substrate for potent steroid formation following their entry into steroid target cells.

Journal of Endocrinology (1996) 150, S119–S124

The biosynthesis of steroid hormones in endocrine steroid-secreting glands results from a series of successive steps involving both cytochrome P450 enzymes, which are mixed-function oxidases, and steroid dehydrogenases. So far, the subcellular distribution of steroidogenic enzymes has been mostly studied following subcellular fractionation, performed in placenta and adrenal cortex. In order to determine in situ the intracellular distribution of some steroidogenic enzymes, we have investigated the ultrastructural localization of the three key enzymes: P450 side chain cleavage (scc) which converts cholesterol to pregnenolone; 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) which catalyzes the conversion of 3 beta-hydroxy-5-ene steroids to 3-oxo-4-ene steroids (progesterone and androstenedione); and P450(c17) which is responsible for the transformation of C(21) into C(19) steroids (dehydroepiandrosterone and androstenedione). Immunogold labeling was used to localize the enzymes in rat adrenal cortex and gonads. The tissues were fixed in 1% glutaraldehyde and 3% paraformaldehyde and included in LR gold resin. In the adrenal cortex, both P450(scc) and 3 beta-HSD immunoreactivities were detected in the reticular, fascicular and glomerular zones. P450(scc) was exclusively found in large mitochondria. In contrast, 3 beta-HSD antigenic sites were mostly observed in the endoplasmic reticulum (ER) with some gold particles overlying crista and outer membranes of the mitochondria. P450(c17) could not be detected in adrenocortical cells. In the testis, the three enzymes were only found in Leydig cells. Immunolabeling for P450(scc) and 3 beta-HSD was restricted to mitochondria, while P450(c17) immunoreactivity was exclusively observed in ER. In the ovary, P450(scc) and 3 beta-HSD immunoreactivities were found in granulosa, theca interna and corpus luteum cells. The subcellular localization of the two enzymes was very similar to that observed in adrenocortical cells. P450(c17) could also be detected in theca interna cells of large developing and mature follicles. As observed in Leydig cells, P450(c17) immunolabeling could only be found in the ER. These results indicate that in different endocrine steroid-secreting cells P450(scc), 3 beta-HSD and P450(c17) have the same association with cytoplasmic organelles (with the exception of 3 beta-HSD in Leydig cells), suggesting similar intracellular pathways for biosynthesis of steroid hormones.

Dehydroepiandrosterone (DHEA) is not a hormone but it is a very important prohormone secreted in large amounts by the adrenals in humans and other primates, but not in lower species. It is secreted in larger quantities than cortisol and is present in the blood at concentrations only second to cholesterol. All the enzymes required to transform DHEA into androgens and/or estrogens are expressed in a cell-specific manner in a large series of peripheral target tissues, thus permitting all androgen-sensitive and estrogen-sensitive tissues to make locally and control the intracellular levels of sex steroids according to local needs. This new field of endocrinology has been called intracrinology. In women, after menopause, all estrogens and almost all androgens are made locally in peripheral tissues from DHEA which indirectly exerts effects, among others, on bone formation, adiposity, muscle, insulin and glucose metabolism, skin, libido and well-being. In men, where the secretion of androgens by the testicles continues for life, the contribution of DHEA to androgens has been best evaluated in the prostate where about 50% of androgens are made locally from DHEA. Such knowledge has led to the development of combined androgen blockade (CAB), a treatment which adds a pure anti-androgen to medical (GnRH agonist) or surgical castration in order to block the access of the androgens made locally to the androgen receptor. In fact, CAB has been the first treatment demonstrated to prolong life in advanced prostate cancer while recent data indicate that it can permit long-term control and probably cure in at least 90% of cases of localized prostate cancer. The new field of intracrinology or local formation of sex steroids from DHEA in target tissues has permitted major advances in the treatment of the two most frequent cancers, namely breast and prostate cancer, while its potential use as a physiological HRT could well provide a physiological balance of androgens and estrogens, thus offering exciting possibilities for women’s health at menopause.