In vivo activation of the constitutive androstane receptor beta (CARbeta) by treatment with dehydroepiandrosterone (DHEA) or DHEA sulfate (DHEA-S)

We investigated whether dehydroepiandrosterone (DHEA) or DHEA-sulfate (S) affected the activities of nuclear receptors, with special reference to constitutive androstane receptor beta (CARbeta). Administration of DHEA or DHEA-S enhanced the DNA binding of hepatic nuclear extracts to responsive elements for the retinoic acid receptor, the retinoic acid receptor beta 2 and the peroxisome proliferator activated receptor. The bound complexes were shown to be the CARbeta-RXR heterodimer by antibody-supershift assays. The expression of a target gene of CARbeta, Cyp2b10, was increased in liver by DHEA or DHEA-S treatment, suggesting that DHEA or DHEA-S actually activated CARbeta in vivo. It was suggested that the metabolic conversion of DHEA, DHEA-S to CARbeta ligands could occur in vivo and the metabolites could regulate the expression of CARbeta target gene expression. Our results provide new insights into the in vivo relationship between DHEA/DHEA-S and CARbeta activation.     

Dehydroepiandrosterone-sulfate inhibits thrombin-induced platelet aggregation

Dehydroepiandrosterone (DHEA) and its sulfated form, DHEA-S, are the most abundant steroids circulating in human blood. DHEA stimulates endothelial cells to release high amounts of nitric oxide in the circulation. Nitric oxide activates guanylyl cyclase in platelets thus decreasing the responsiveness of these cells to physiological agonists. However, the impact of DHEA-S and DHEA on platelet function and their possible role in modulating the response of human platelets to physiological agonists were not yet investigated. Here, DHEA-S, but not DHEA, inhibited in vitro thrombin-dependent platelet aggregation in a dose-dependent manner. DHEA-S exerted this effect by decreasing thrombin-dependent dense granule secretion, and so impairing the positive feed-back loop provided by ADP. Furthermore, DHEA-S inhibited thrombin-dependent activation of Akt, ERK1/2, and p38 MAP kinase. Although both DHEA-S and DHEA directly activated in platelets the inhibitory cGMP/PGK/VASP pathway, these events were not responsible for the inhibitory action of DHEA-S in platelets. In addition DHEA-S acted in synergism with nitric oxide in inhibiting platelet aggregation. In conclusion DHEA-S inhibited platelet activation caused by a mild stimulus without completely hampering platelet functionality and thus DHEA-S may participate in the physiological mechanisms that maintain circulating platelets in a resting state. The role played by DHEA-S could be relevant mainly when the functionality of the vascular endothelium is compromised.     

Prevention of diabetes,hepatic injury,and colon cancer with dehydroepiandrosterone

The levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) peak in human in their twenties, then decrease gradually with age. The physiological importance of DHEA was not clear until recent research reports showing that DHEA has beneficial effects on preventing diabetes, malignancy, inflammation, osteoporosis, and collagen disease. We summarize our results concerning diabetes, hepatitis, and colon cancer.

In 1982, Coleman et al. [Diabetes 31 (1982) 830] reported that DHEA decreased hyperglycemia in diabetic db/db mice, which become insulin resistant. We measured hepatic gluconeogenic enzymes in an attempt to elucidate the mechanical mechanism of DHEA action. The activity and gene expression of hepatic gluconeogenic enzyme such as glucose-6-phosphatase (G6Pase) was increased in db/db mice despite hyperinsulinemia compared to control db/+m mice. DHEA, like troglitazone, decreased these levels in db/db mice. We also showed that DHEA improved the insulin resistance caused by aging or obesity using the glucose clamp technique in another animal model. In humans, the serum DHEA concentration was shown to be associated with hyperinsulinemia in diabetes. It also became clear that DHEA increased insulin secretion in old-aged db/db mice. DHEA increases not only insulin sensitivity due to the effects in the liver and muscle, but also insulin secretion.

As an effect of DHEA on T-cell mediated hepatitis induced by concanavalin A (ConA), DHEA reduced hepatic injury by inhibiting several inflammatory mediators and apoptosis. As an effect of DHEA on carcinogenesis, DHEA would be a potential chemopreventative agent against colon cancer because it decreases the number of azoxymethane (AOM) induced aberrant crypt foci, which is a possible precursor to adenoma and cancer in a murine model.

Thus, since DHEA has many beneficial effects experimentally, we should consider administration of DHEA in the future, and common mechanisms among these actions of DHEA should be elucidated in further studies.     

Dehydroepiandrosterone and Body Fat

Dehydroepiandrosterone sulfate (DHEA-S) is the most abundant circulating adrenal steroid in man, yet its physiologic role and that of its parent compound DHEA are unknown. Age-related decreases in DHEA in association with increases in obesity, insulin resistance, and atherosclerosis are well known. Recent investigations in lower mammals (which do not secrete DHEA) have suggested that DHEA (or its metabolites) may function as an antiobesity agent in these models of obesity independent of food intake. Proposed mechanisms for the decrease in fat mass and lower weight gain when DHEA is given orally include increases in futile cycling and peroxisomal β-oxidation and decreases in de novo lipogenesis. Alterations in the availability of reducing equivalents for lipid synthesis do not appear to explain this decrease. Changes in pancreatic insulin secretion or insulin sensitivity may also be responsible for some of these effects. Studies in humans have failed to demonstrate a beneficial effect of DHEA on body composition or energy expenditure at either pharmacologic or physiologic replacement doses for 1–3 months. Administration of DHEA to men or women has also not been shown to alter insulin sensitivity as measured by the minimal model or the euglycemic clamp technique. The effect of DHEA on peroxisomal β-oxidation and de novo lipogenesis is not known. We conclude that a significant role for DHEA in the pharmacologic treatment of human obesity is unlikely.     

Specific binding and effects of dehydroepiandrosterone sulfate (DHEA-S) on skeletal muscle cells: possible implication for DHEA-S replacement therapy in patients with myotonic dystrophy.

Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroidal products and major circulating steroids in humans. The serum concentrations of DHEA-S are lower in patients with myotonic dystrophy (DM) than normal controls, and possible improvement of myotonia and muscle weakness was recently reported following DHEA-S replacement therapy. However, the molecular mechanism of action of DHEA-S remains unknown. To understand the reported anti-DM action of DHEA-S, we investigated DHEA-S binding in skeletal muscle cells in vitro. We identified two populations of DHEA-S binding sites (Kd = 5-9 microM and 35-40 microM) in C2C12 myocytes. Similar binding sites were also identified in human skeletal muscles. The Kd value of the high-affinity site was within the range of serum concentrations of DHEA-S in adult humans. Our results suggest that DHEA-S might act directly on skeletal muscles under normal physiological conditions in humans.     

Adrenal C19-5-ene steroids induce full estrogenic responses in rat pituitary gonadotrophs

Previous studies have shown that the C19 adrenal steroid 5-androstene-3 beta, 17 beta-diol (5-ene-diol), a metabolite of dehydroepiandrosterone (DHEA), can stimulate typical estrogenic responses in target tissues. Since estrogens are known to cause a specific stimulatory effect on LHRH-induced LH release in rat anterior pituitary cells in culture, we have taken advantage of the precision of this system to study the effect of 5-ene-diol or DHEA on this precise estrogen-sensitive parameter. Pretreatment for 48 h with 17 beta-estradiol (E2), 5-ene-diol or DHEA induces a 2.4-, 2.7- and 2.6-fold stimulation of LH release induced by 0.3 nM LHRH, the effect being exerted at respective 50% maximally effective concentrations (ED50 values) of 0.015, 45 and 115 nM. Following a 48-h preincubation with 10 nM E2, 1 microM 5-ene-diol or 1 microM DHEA, the maximal LH and FSH responses to LHRH are increased by approx 50% above control. On the other hand, the sensitivities of the LH and FSH responses to LHRH as assessed by ED50 values of LHRH action are increased by 3.3- to 7.5-fold. As further proof of the estrogenic nature of the effect of 5-ene-diol and DHEA, the effects of E2, 5-ene-diol and DHEA are inhibited competitively by simultaneous incubation with the antiestrogen LY156758 (keoxifene). The 2-fold stimulation of LHRH-induced LH release caused by DHEA-S, at concentrations within the range found in the plasma of women, is also completely blocked by 120 nM LY156758. In direct binding studies, 5-ene-diol and DHEA or DHEA-S have approx 85- and greater than 10,000 lower affinities than E2, respectively, for the estrogen receptor in rat anterior pituitary homogenate and human breast carcinoma cytosol. The present data clearly show that 5-ene-diol, DHEA and DHEA-S can exert full estrogenic activity in rat gonadotrophs, thus supporting the potential estrogenic role of these C19 adrenal steroids in estrogen-dependent processes, especially breast cancer.     

Dehydroepiandrosterone protects human keratinocytes against apoptosis through membrane binding sites

Although the epidermis is importantly affected by steroid hormones, little is known about the effects of dehydroepiandrosterone (DHEA) on human keratinocytes, in spite of its abundance in human serum. Here, we demonstrate for the first time a protective role of DHEA against apoptosis in keratinocytes, using non-cancerous immortalized human HaCaT cells. We show that DHEA transmits its signal via specific G protein-coupled, membrane binding sites and inhibits apoptosis, through prevention of mitochondrial disruption and altered balance of Bcl-2 proteins. DHEA conjugated to the membrane impermeable molecule BSA, as well as DHEA-S, the most abundant form of DHEA in human serum exhibit similar anti-apoptotic effect. Our data provide new insights in the treatment of the epidermis with steroid hormones in apoptosis-related conditions.     

Perceived Stress at Work Is Associated with Lower Levels of DHEA-S

Background

It is known that long-term psychosocial stress may cause or contribute to different diseases and symptoms and accelerate aging. One of the consequences of prolonged psychosocial stress may be a negative effect on the levels of dehydroepiandrosterone (DHEA) and its sulphated metabolite dehydroepiandrosterone sulphate (DHEA-S). The aim of this study is to investigate whether levels of DHEA and DHEA-S differ in individuals who report perceived stress at work compared to individuals who report no perceived stress at work.

Methods

Morning fasting DHEA-S and DHEA levels were measured in serum in a non-stressed group (n = 40) and a stressed group (n = 41). DHEA and DHEA-S levels were compared between the groups using ANCOVA, controlling for age.

Results

The mean DHEA-S levels were 23% lower in the subjects who reported stress at work compared to the non-stressed group. Statistical analysis (ANCOVA) showed a significant difference in DHEA-S levels between the groups (p = 0.010). There was no difference in DHEA level between the groups.

Conclusions

This study indicates that stressed individual have markedly lower levels of DHEA-S. Given the important and beneficial functions of DHEA and DHEA-S, lower levels of DHEA-S may constitute one link between psychosocial stress, ill health and accelerated ageing.     

Pharmacokinetics of oral dehydroepiandrosterone (DHEA) in the ovariectomised cynomolgus monkey

Humans and primates are unique in having adrenals that secrete large amounts of DHEA and DHEA-S in the circulation. These steroids act as precursors of active androgens and estrogen's in a long series of peripheral target intracrine tissues. The marked decline of serum DHEA and DHEA-S concentrations with age in men and women has been incriminated in the development of various pathologies. This study provides detailed information on the effect of a single 50mg oral dose of DHEA on circulating estrogen's as well as androgens and their metabolites over 10h in adult ovariectomised (OVX) Cynomolgus monkeys. Serum DHEA, DHEA-S, testosterone (Testo) and androstenedione (4-dione) concentrations increased rapidly with a maximal value at approximately 1h after DHEA administration followed by a 60-80% decrease during the next 2-6h. An important sulfatation of DHEA occurs through first hepatic pass, thus, leading to a marked increase in serum DHEA-S. Serum androst-5-ene-3beta,17beta-diol and androsterone glucuronide (ADT-G) levels remained elevated on a plateau for 6h. Androstan-3alpha,17beta-diol-glucuronide, estradiol and estrone levels remained unchanged. The present data indicate the predominant transformation of the adrenal precursor DHEA into active androgens in peripheral tissues and support the importance of measurement of circulating glucuronide derivatives as index of peripheral or intracrine androgen formation and action.     

Differential gene expression profile of glucocorticoids, testosterone, and dehydroepiandrosterone in human cells.

Glucocorticoids are the major immunomodulating hormones in the human body. Recently, increasing interest in androgens as immunomodulators has emerged. In particular, Dehydroepiandrosterone (DHEA) has been suggested as beneficial in the treatment of some autoimmune disorders. However, the action and role of testicular and adrenal androgens on human immune cells remains unclear. This is the first study to provide large-scale gene expression data on the action of different steroids (DHEA, glucocorticoids, and testosterone) on human peripheral blood mononuclear cells using the recently developed genomic-scale technology of microarrays. Novel computational tools and techniques such as Principal Component Analysis (PCA) were used for analysis, clustering and visualization. We have demonstrated that each steroid has its distinct gene expression profile, although DHEA and testosterone co-regulated most genes in a similar direction while glucocorticoids frequently regulated the same genes in an opposite direction. Our data suggest an important and a complex regulatory role for androgens on human immune cells that should be considered in androgen replacement or treatment strategies.     

DHEA-sulfate causes a phase-dependent increase in melatonin secretion: a study of perifused rat pineal glands

Steroid hormones affect various metabolic activities, including melatonin synthesis, in mammals and nonmammals. We report here the effects of dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), two steroids with weak androgen potency, on the levels of isoproterenol-stimulated melatonin released by perifused rat pineal glands removed in the middle of the light and dark spans [7 and 19 Hours After Light Onset (HALO), respectively] in a L/D 12:12 regimen. DHEA-S but not DHEA was found to have a direct action on beta-adrenergic-stimulated melatonin release. DHEA-S increased melatonin secretion (by 50-80%) dose-dependently in pineals obtained during the light span. This effect depended on the circadian stage, because at night (19 HALO), only the highest concentration (10(-3) M) of DHEA-S increased melatonin secretion (by 25%). In contrast, DHEA had no effect on melatonin release in pineals obtained during the light span. This work shows that DHEA-S but not DHEA was able to stimulate melatonin secretion by adrenergic-stimulated pineals removed during the light phase. It also suggests that the effects observed, or their intensity, or both depend on the circadian stage.     

Dehydroepiandrosterone up-regulates the Adrenoleukodystrophy-related gene (ABCD2) independently of PPARalpha in rodents

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disease caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC transporter, ALDP, supposed to participate in the transport of very long chain fatty acids (VLCFA). The adrenoleukodystrophy-related protein (ALDRP), which is encoded by the ABCD2 gene, is the closest homolog of ALDP and is considered as a potential therapeutic target since functional redundancy has been demonstrated between the two proteins. Pharmacological induction of Abcd2 by fibrates through the activation of PPARalpha has been demonstrated in rodent liver. DHEA, the most abundant steroid in human, is described as a PPARalpha activator and also as a prohormone able to mediate induction of several genes. Here, we explored the in vitro and in vivo effects of DHEA on the expression of peroxisomal ABC transporters. We show that Abcd2 and Abcd3 but not Abcd4 are induced in primary culture of rat hepatocytes by DHEA-S. We also demonstrate that Abcd2 and Abcd3 but not Abcd4 are inducible by an 11-day treatment with DHEA in the liver of male rodents but not in brain, testes and adrenals. Finally and contrary to Abcd3, we show that the mechanism of induction of Abcd2 is independent of PPARalpha.     

Chemiluminescence enzyme immunoassay of dehydroepiandrosterone and its sulfate using peroxidase as label

We report a highly sensitive enzyme immunoassay for dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) using horseradish peroxidase as the label enzyme. Separation of free and bound DHEA-peroxidase conjugate was by insolubilized antibody, prepared by coupling purified IgG of goat anti-rabbit IgG serum with Sepharose 4B or a polystyrene tube. The enzyme activity was measured by the chemiluminescence reaction using luminol and hydrogen peroxide as substrate. The faint chemiluminescence was measured by a photon counter. The sensitivity was 25 pg/assay tube for DHEA and 100 pg/assay tube for DHEA-S. Upon comparison, results obtained by radioimmunoassay and this method showed good agreement; r = 0.86 for free DHEA, r = 0.92 for acid-hydrolyzed DHEA-S and r = 0.91 for solvolyzed DHEA-S. The present method is applicable in the routine determination of DHEA and DHEA-S in biological fluid.     

Ex vivo studies on the acute and chronic effects of DHEA and DHEA-sulfate on melatonin synthesis in young- and old-rat pineal glands

This study investigates the effects of acute and chronic injections of the neurosteroid dehydroepiandrosterone (DHEA) and its sulfate DHEA-S on pineal gland melatonin synthesis. Pineal melatonin production and plasma melatonin levels were investigated in young (9-week-old) and old (27-month-old) male Wistar rats. DHEA or DHEA-S have been administered acutely in a single intraperitoneal injection at a dosage of 50, 250, or 500 microg per animal, or on a long-term basis, i.e., for 8 days at a dosage of 100 microg per animal, 1 h before the onset of darkness. DHEA, at a dose of 50, 250, or 500 microg per animal, administered acutely to rats had no significant effects on pineal melatonin production whatever the age of the animals. In contrast, 500 microg DHEA-S induced a significant increase in the pineal melatonin content (15% in young animals and 35% in old animals) and the activity of N-acetyltransferase, the rate-limiting enzyme for melatonin synthesis in the pineal gland, (40% in young animals and 20% in old animals), without altering the activity of hydroxyindole-O-methyltransferase whatever the age of the animals. At lower concentrations (50 or 250 microg) DHEA-S had no effect on pineal melatonin production regardless of the age of the rats. Chronic injection of DHEA or DHEA-S at a dose of 100 microg had no effect on pineal melatonin or NAT and HIOMT activities in the two age groups. This work shows that DHEA-S (and not DHEA) is able, at pharmacological concentrations, to stimulate melatonin production by rat pineal glands regardless of the age of the animals.     

Ovine prolactin potentiates the action of adrenocorticotropic hormone on the secretion of dehydroepiandrosterone sulfate and dehydroepiandrosterone from cultured bovine adrenal cells

To determine the direct effect of prolactin on adrenal androgen secretion, the daily secretions of dehydroepiandrosterone sulfate (DHEA-S), dehydroepiandrosterone (DHEA), androstenedione and cortisol were determined in monolayer culture of bovine adrenal cells in the presence or absence of adrenocorticotropic hormone (ACTH) and/or prolactin. In the absence of ACTH ovine prolactin alone had no effect on steroid secretion during seven-day culture. Ovine prolactin, when administered in combination with ACTH, significantly potentiated the stimulatory effect of ACTH on DHEA-S and DHEA but not androstenedione secretion on the seventh day in culture. On the first day in culture prolactin showed no synergistic effect with ACTH on DHEA and DHEA-S secretion, although ACTH significantly increased DHEA and cortisol secretion. DHEA-S secretion increased as a function of prolactin concentration in the presence of ACTH. These results indicated that long-term treatment by ovine prolactin with ACTH caused the increase in adrenal androgen secretion from bovine adrenal cells. The site of action of prolactin was suggested to be the partial inhibition of adrenal 3 beta-hydroxysteroid dehydrogenase by the result of increases in DHEA-S and DHEA but not androstenedione secretion.     

Effect of prolactin on the secretion of dehydroepiandrosterone (DHEA), its sulfate (DHEA-S), and cortisol by the human fetal adrenal in vitro

The effect of prolactin on the secretions of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) as well as that of cortisol were studied in vitro in order to investigate the possible regulatory role of prolactin on steroidogenesis of the human fetal adrenal at mid-gestational age. The addition of 0.5 microgram/ml of human prolactin to the incubation medium produced a significant (P less than 0.05) increase in DHEA, DHEA-S, and cortisol secretion. These results indicate that prolactin has a regulatory role in steroidogenesis in the human fetal adrenal at mid-gestation.     

Effects of short-term dehydroepiandrosterone supplementation on body composition in young athletes

Dehydroepiandrosterone (DHEA) is often promoted as a slimming and weight/fat loss agent and ingestion of DHEA may have hypolipidemic and anti-obesity properties. The main aim of this study was to examine the effects of acute DHEA intake on body composition and serum steroid hormones in young athletes. Twenty young (19 to 22 years) male soccer players were allocated into two randomly assigned trials in double-blind design by ingesting 100-mg daily oral DHEA or as placebo (PLA) for 28 days. Body mass was not affected by 4 weeks of DHEA supplementation (P > 0.05). No significant changes in body mass index (BMI), waist-to-hip ratio (WHR) and body fat or total muscle mass for the two groups were detected at the end of the trial (P > 0.05). There was no within- or between-group difference in arm fat index (AFI) and corrected mid-upper-arm muscle area (cAMA) (P > 0.05). Treatment with DHEA resulted in a significant increase of total testosterone, estradiol and DHEA-S levels in treated subjects versus the placebo group (P < 0.05). Results of this study suggest that DHEA supplementation has no beneficial effects on body composition in young competitive athletes.