OBJECTIVE

To determine if insulin-like growth factor 1 (IGF-1) and androgen levels (1) correlate with the presence and severity of acne in adult men and women, and (2) correlate directly with each other and interact in affecting acne.

METHODS

Case-control study and single-center examination of hormone levels in a cohort of volunteers.

METHODS

Academic referral center.

METHODS

Thirty-four subjects (8 women and 8 men with clinical acne, 10 women and 8 men without clinical acne). Clinical acne is defined by a history of persistent acne (acne present on most days for several years), recent acne treatment, and the presence of 10 or more inflammatory acne lesions and 15 or more comedones.

METHODS

Single visit for serum sampling.

METHODS

Serum levels of IGF-1 and androgens were determined, adjusted for age, and compared based on the presence or absence of clinical acne using an analysis of covariance. Correlations between hormone levels and acne lesion counts were calculated within each subgroup. Correlations were also calculated between serum levels of IGF-1 and androgens. Further statistical testing was conducted to determine whether IGF-1 or androgens had a greater effect on acne lesion counts.

RESULTS

Dehydroepiandrosterone (DHEAS), dihydrotestosterone (DHT), and IGF-1 correlated positively with acne lesion counts in women. Androstenedione and DHEAS correlated with acne lesion counts in men. Although the age-adjusted mean serum levels of IGF-1 were higher in women with clinical acne than in women without clinical acne, this difference did not achieve statistical significance. No difference in IGF-1 level was noted in men based on the presence of clinical acne. In women with clinical acne, IGF-1 correlated with DHT. In men with clinical acne, IGF-1 correlated with DHEAS and androstenedione. In men and women with clinical acne, the effects of androgens on increased acne lesion counts were dependent on the influence of IGF-1.

CONCLUSIONS

Increased IGF-1 levels in addition to androgens may influence acne in adult men and women. While IGF-1 appears to have a stronger effect on acne in women, androgens may play a greater role in acne for men. However, in both men and women these hormones are interrelated, possibly owing to reciprocal effects on hormone production.

OBJECTIVE

Increasing evidence indicates that enhanced intratumoral androgen synthesis contributes to prostate cancer progression after androgen deprivation therapy. This phase II study was designed to assess responses to blocking multiple steps in androgen synthesis with inhibitors of CYP17A1 (ketoconazole) and type I and II 5alpha-reductases (dutasteride) in patients with castration-resistant prostate cancer (CRPC).

METHODS

Fifty-seven men with CRPC were continued on gonadal suppression and treated with ketoconazole (400 mg thrice daily), hydrocortisone (30 mg/AM, 10 mg/PM), and dutasteride (0.5 mg/d).

RESULTS

Prostate-specific antigen response rate >> or =50% decline) was 56% (32 of 57; 95% confidence interval, 42.4-69.3%); the median duration of response was 20 months. In patients with measurable disease, 6 of 20 (30%) responded by the Response Evaluation Criteria in Solid Tumors. Median duration of treatment was 8 months; 9 patients remained on therapy with treatment durations censored at 18 to 32 months. Median time to progression was 14.5 months. Grade 3 toxicities occurred in 32% with only one reported grade 4 (thrombosis) toxicity. Dehydroepiandrosterone sulfate declined by 89%, androstenedione by 56%, and testosterone by 66%, and dihydrotestosterone declined to below detectable levels compared with baseline levels with testicular suppression alone. Median baseline levels and declines in dehydroepiandrosterone sulfate, androstenedione, testosterone, and dihydrotestosterone were not statistically different in the responders versus nonresponders, and hormone levels were not significantly increased from nadir levels at relapse.

CONCLUSIONS

The response proportion to ketoconazole, hydrocortisone, and dutasteride was at least comparable with previous studies of ketoconazole alone, whereas time to progression was substantially longer. Combination therapies targeting multiple steps in androgen synthesis warrant further investigation.

It is a common practice to extract steroids from plasma, serum, or tissue samples prior to steroid measurement by radioimmunoassay (RIA) or enzyme immunoassay (EIA). Steroid extraction is critical because it can remove substances that interfere with the RIA or EIA. Steroid extraction is commonly achieved using organic solvents, such as diethyl ether or dichloromethane. However, organic solvent extractions can suffer from low recovery, imprecise recovery, or incomplete removal of assay interference. Here, we describe validations of a simple protocol to extract steroids (e.g., dehydroepiandrosterone, corticosterone, and estradiol) from avian plasma, serum, and brain tissue using solid phase extraction (SPE) with commercially available C18 columns. We compare various methods for (1) eluting steroids from columns, (2) drying eluates, and (3) resuspending dried eluates prior to RIA. The SPE method yields high and consistent recoveries. The SPE method also effectively separates steroids from interfering substances, even when extracting steroids from lipid-rich plasma and brain tissue. These data indicate that SPE is superior to organic solvent extraction on several measures. SPE should be broadly useful for extracting steroids from plasma or tissue samples.

OBJECTIVE

The relationships between adipocytokines, sex steroids and the GH/IGF-I axis is poorly studied and subject to controversy in healthy elderly male subjects. We investigated the association between both adiponectin and leptin, and the metabolic syndrome (MetS), lipid parameters, insulin sensitivity, sex steroids and IGF-I in healthy non-diabetic Lebanese men.

METHODS

In this cross-sectional study, a total of 153 healthy non-diabetic men aged 50 and above (mean age 59.3 +/- 7 years) had a detailed clinical and biological evaluation. Subjects were classified according to the National Cholesterol Education Program criteria of the MetS. Insulin sensitivity was determined by the Quantitative Insulin Sensitivity Check Index (QUICKI).

RESULTS

Subjects with the MetS had lower adiponectin and higher leptin levels (P < 0.0001 for both variables) compared with individuals without the MetS. Adiponectin was significantly correlated with waist size, triglycerides, high-density lipoprotein (HDL) cholesterol and QUICKI (r = -0.33, -0.26, 0.45 and 0.36 respectively, P < 0.0001 for all variables). The relation between adiponectin and HDL cholesterol, triglycerides and QUICKI remained significant after adjustment for age and body mass index (BMI). Also, leptin was strongly correlated with waist size and QUICKI (r = 0.63 and -0.63 respectively, P < 0.001 for both variables). However, its relation to the lipid profile was weak (for cholesterol r = 0.16, P < 0.05; for triglycerides r = 0.17, P < 0.05) and disappeared after adjustment for BMI. Adiponectin was positively correlated with sex hormone-binding globulin (SHBG) (r = 0.39, P < 0.001) and inversely correlated with free-androgen index (r = -0.24, P < 0.01), estradiol and dehydroepiandrosterone sulfate (r = -0.165, P < 0.05; r = -0.21, P < 0.01 respectively). This difference remained significant for SHBG after adjustment for age and BMI (r = 0.20, P < 0.005). Finally, leptin was inversely correlated with total testosterone and SHBG (r = -0.44, P < 0.001; r = -0.30, P < 0.001 respectively); the relation with testosterone remained significant after adjustment for BMI. No significant relationship of either adiponectin or leptin with GH or IGF-I values was observed. In a stepwise multiple regression analysis, the independent predictors of adiponectin were HDL cholesterol, QUICKI, age and BMI (P < 0.0001, P = 0.005, P = 0.002 and P = 0.047 respectively) while for leptin, it was QUICKI, waist size and testosterone (P < 0.0001, P < 0.0001 and P = 0.004 respectively). The adjusted R2 values were 0.34 and 0.55.

CONCLUSIONS

Our results show that in a healthy elderly male population, both adiponectin and leptin are related to insulin sensitivity, independent of age and BMI. While adiponectin is independently related to triglycerides and HDL cholesterol, the weak relationship of leptin to the lipid profile is completely mediated by BMI. In addition, and more interestingly, both adipocytokines are strongly associated with sex steroids. We speculate that SHBG is regulated by adiponectin and that there is an inhibitory effect of testosterone on the adiponectin gene. Further studies are needed to fully elucidate these relationships.

The concentrations of the adrenal steroids dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and delta 4-androstenedione (delta 4-A) have been measured by RIA before and after sexual maturation in plasma of rodents, domestic animals, and primates to determine whether these species exhibit and adrenarchal process comparable to man. The average concentrations of DHA and DHAS were less than 60 ng/dl and 5 microgram/dl, respectively, in plasma of sexually mature rodents and domestic animals, and a significant increase in the plasma DHA level after sexual maturation was seen only in the rabbit and dog. The concentrations of DHA, DHAS, and delta 4-A in 21 rhesus monekeys from 0-3 yr of age were 2021 +/- 235 ng/dl (mean +/- SE), 357 +/- 60 microgram/dl, and 107 +/- 9 ng/dl, respectively, and did not increase during sexual maturation. By contrast, DHA, DHAS, and delta 4-A levels in plasma of chimpanzees were 5.9-fold, 3.3-fold, and 4.8-fold greater, respectively, in 7- to 22-compared to 0- to 3-yr-old animals. Temporally, the increase in DHA levels in the chimpanzee is apparent at 5 yr and this precedes the increase in gonadal steroids, as is characteristic of human adrenarche. It is apparent that adrenal androgen levels and their developmental patterns differ markedly among species, and that among the species examined, only the chimpanzee exhibits an adrenarche comparable to that of man.

A great number of epidemiological studies have demonstrated that the frequency of the epsilon4 allele of the apolipoprotein E gene (APOE) is markedly higher in sporadic and in familial late onset Alzheimer disease (AD). In the frontal cortex of AD patients, oxidative damage is elevated. We address the hypothesis that the APOE genotype and reactive oxygen-mediated damage are linked in the frontal cortex of AD patients. We have related the APOE genotype to the levels of lipid oxidation (LPO) and to the antioxidant status, in frontal cortex tissues from age-matched control and AD cases with different APOE genotypes. LPO levels were significantly elevated in tissues from Alzheimer's cases which are homozygous for the epsilon4 allele of APOE, compared to AD epsilon3/epsilon3 cases and controls. Activities of enzymatic antioxidants, such as catalase and glutathione peroxidase (GSH-PX), were also higher in AD cases with at least one epsilon4 allele of APOE, while superoxide dismutase (SOD) activity was unchanged. In the frontal cortex, the concentration of apoE protein was not different between controls and AD cases, and was genotype independent. The Ginkgo biloba extract (EGb 761), the neurosteroid dehydroepiandrosterone (DHEA) and human recombinant apoE3 (hapoE3rec) were able to protect control, AD epsilon3/epsilon3 and epsilon3/epsilon4 cases against hydrogen peroxide/iron-induced LPO, while hapoE4rec was completely ineffective. Moreover, EGb 761 and DHEA had no effect in homozygous epsilon4 cases. These results demonstrate that oxidative stress-induced injury and protection by antioxidants in the frontal cortex of AD cases are related to the APOE genotype.

We investigated whether a low plasma testosterone level is related to endothelial dysfunction in men with coronary risk factors. One hundred and eighty-seven consecutive male outpatients (mean age+/-SD: 47+/-15 years) who underwent measurement of flow-mediated vasodilation (FMD) of the brachial artery using ultrasonography were enrolled. The relationship between plasma hormones and FMD was analyzed. Total and free testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were significantly correlated with %FMD (r=0.261, 0.354 and 0.295, respectively; p<0.001), while estradiol and cortisol were not. %FMD in the highest quartile of free testosterone was 1.7-fold higher than that in the lowest quartile. Multiple regression analysis revealed that total and free testosterone were related to %FMD independent of age, body mass index, hypertension, hyperlipidemia, diabetes mellitus and smoking (beta=0.198 and 0.247, respectively; p<0.01), and were independent of age, body mass index, systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, fasting plasma glucose, smoking and nitroglycerin-induced dilation (beta=0.196 and 0.227, respectively; p<0.01). DHEA-S was not significantly related to %FMD in multivariate analysis. In conclusion, a low plasma testosterone level was associated with endothelial dysfunction in men independent of other risk factors, suggesting a protective effect of endogenous testosterone on the endothelium.

Silver-haired bat rabies virus (SHBRV) infection induces a strong virus-specific immune response in the periphery of the host, but death is common due to the failure to open the blood-brain barrier (BBB) and deliver immune effectors to central nervous system (CNS) tissues. Mice with an SJL background are less susceptible to lethal infection with rabies viruses. In addition, these animals are known to have reduced hypothalamus-pituitary-adrenal (HPA) axis activity and an elevated capacity to mediate CNS inflammatory responses. We show here that approximately one-half of PLSJL mice survive an SHBRV infection that is invariably lethal for 129/SvEv mice. This difference is associated with the elevated capacity of PLSJL mice to mediate BBB permeability changes in response to the infection. The induction of more extensive BBB permeability and CNS inflammation in these animals results in greater virus clearance and improved survival. On the other hand, treatment of SHBRV-infected PLSJL mice with the steroid hormone dehydroepiandrosterone reduced BBB permeability changes and caused greater mortality. We conclude that the infiltration of immune effectors across the BBB is critical to surviving a rabies virus infection and that HPA axis activity may influence this process.

Serum levels of the adrenal androgen dehydroepiandrosterone (DHEA) peak in men and women in the third decade of life and decrease progressively with age. Increasing numbers of middle-aged and older individuals consume over-the-counter preparations of DHEA, hoping it will retard aging by increasing muscle and bone mass and strength, decreasing fat, and improving immunologic and neurobehavioral functions. Because DHEA can serve as a precursor to more potent androgens and estrogens, like testosterone (T), dihydrotestosterone (DHT), and 17beta-estradiol (E2), supplemental DHEA use may pose a cancer risk in patients with nascent or occult prostate cancer. The steroid-responsive human LNCaP prostate cancer cells, containing a functional but mutated androgen receptor (AR), were used to compare effects of DHEA with those of T, DHT, and E2 on cell proliferation and protein and/or gene expression of AR, prostate-specific antigen (PSA), IGF-I, IGF-I receptor (IGF-IR), IGF-II, IGF-binding proteins-2, -3, and -5, (IGFBPs-2, -3, and -5), and estrogen receptor-beta (ERbeta). Cell proliferation assays revealed significant stimulation by all four steroids. DHEA- and E2-induced responses were similar but delayed and reduced compared with that of T and DHT. All four hormones increased gene and/or protein expression of PSA, IGF-IR, IGF-I, and IGFBP-2 and decreased that of AR, ERbeta, IGF-II, and IGFBP-3. There were no significant effects of hormone treatment on IGFBP-5 mRNA. DHEA and E2 responses were similar, and distinct from those of DHT and T, in time- and dose-dependent studies. Further studies of the mechanisms of DHEA effects on prostate cancer epithelial cells of varying AR status, as well as on prostate stromal cells, will be required to discern the implications of DHEA supplementation on prostatic health.

Dietary caloric restriction (CR) slows aging, extends lifespan, and reduces the occurrence of age-related diseases in short-lived species. However, it is unclear whether CR can exert similar beneficial effects in long-lived species, like primates. Our objective was to determine if CR could attenuate purported age-related changes in the 24-h release of adrenal steroids. To this end, we examined 24-h plasma profiles of cortisol, and dehydroepiandrosterone sulfate (DHEAS) in young and old, male and female rhesus macaques (Macaca mulatta) subjected to either ad libitum (AL)-feeding or CR (70% of AL) for 2-4 years. Hormone profiles from young monkeys showed pronounced 24-h rhythms. Cortisol concentrations were higher in old males but not females, whereas DHEAS rhythms were dampened with age in both sexes. The cortisol rhythms of old CR males resembled those of young control males. However, CR failed to prevent age-related declines in DHEAS and further dampened DHEAS rhythms in both sexes. Apart from the partial attenuation of the age-related cortisol elevation in the old males, 24-h adrenal steroid rhythms did not benefit from late-onset CR.

Post-traumatic stress disorder (PTSD) has been associated with dysregulation of the neuroendocrine system. In this study we examine the effects of psychotherapy in 21 PTSD patients, with and without coexisting depression, on the levels of six stress-related hormones: cortisol, dehydroepiandrosterone (DHEA), and dehydroepiandrosterone-sulfate (DHEA-S), prolactin, thyrotropin (TSH) and free thyroxin (fT4). The results show that after brief eclectic psychotherapy (BEP) significant changes occurred in levels of cortisol and DHEA. Responders showed an increase in cortisol and DHEA levels, while in non-responders both hormone levels decreased. Differences were only found after controlling for depressive symptoms. In conclusion, effective psychotherapy for PTSD may alter dysregulations in the Hypothalamus-pituitary-adrenal (HPA)-axis, but comorbid depressive symptoms should be taken into account.

Excitatory amino acids such as glutamate play important roles in the central nervous system. We previously demonstrated that a neurosteroid, dehydroepiandrosterone (DHEA), has powerful effects on the cell proliferation of human neural progenitor cells (hNPC) derived from the fetal cortex, and this effect is modulated through NMDA receptor signaling. Here, we show that glutamate can significantly increase the proliferation rates of hNPC. The increased proliferation could be blocked by specific NMDA receptor antagonists, but not other glutamate antagonists for kainate-AMPA or metabotropic receptors. The NR1 subunit of the NMDA receptor was detectable in elongated bipolar or unipolar cells with small cell bodies. These NR1-positive cells were colocalized with GFAP immunoreactivity. Detection of the phosphorylation of cAMP response element-binding protein (pCREB) revealed that a subset of NR1-positive hNPC could respond to glutamate. Furthermore, we hypothesized that glutamate treatment may affect mainly the hNPC with a radial morphology and found that glutamate as well as DHEA selectively affected elongated hNPC; these elongated cells may be a type of radial glial cell. Finally we asked whether the glutamate-responsive hNPC had an increased potential for neurogenesis and found that glutamate-treated hNPC produced significantly more neurons following differentiation. Together these data suggest that glutamate stimulates the division of human progenitor cells with neurogenic potential.

Dehydroepiandrosterone (DHEA) is a C19 steroid of adrenal origin. Notably, its secretion declines with age, a phenomenon referred to as the "adrenopause". For many years, the physiological significance of DHEA remained elusive. However, many studies have now shown that DHEA has significant immune modulatory function, exhibiting both immune stimulatory and anti-glucocorticoid effects. Although several of these studies are limited by the fact that they were carried out in rodents, who are incapable of adrenal DHEA production, and therefore have very low circulating levels of this steroid, evidence from the study of immune cells is now accumulating to suggest a role for DHEA in regulating human immunity. This ability to regulate immune function has raised interest in the therapeutic potential of DHEA as a treatment for the immunological abnormalities that arise in subjects with low circulating levels of this hormone. This has included attempts at reversing the impaired immune response of older individuals to vaccination and restoring immune regulation in patients with chronic autoimmune disease. This review summarises the reported effects of DHEA on immune function and discusses the therapeutic potential of this steroid in geriatric medicine and particularly in age-related disease with an immune component.

Adipose tissue is a site of uptake, storage, action, and metabolism of sex steroids. After menopause aromatization of androgens to estrogens in adipose tissue is one of the most important sources of estrogen in the circulation and for peripheral tissues. The aim of this study was to estimate local sex steroid concentrations in breast and abdominal subcutaneous (s.c.) adipose tissue, to compare them with plasma concentrations and to investigate possible correlations with body mass index (BMI). The patients were postmenopausal women undergoing surgery for non-oncological reasons (Group A; n = 35) and breast cancer patients (group B; n = 19). The concentrations of estrone, 17 beta-estradiol, estrone sulfate, 17 beta-estradiol sulfate, androstenedione, androstenediol (androst-5-ene-3 beta, 17 beta-diol), testosterone and dehydroepiandrosterone were measured. The method was based on frozen tissue homogenization, extraction with ethanol: acetone, delipidation, extraction of estrogens with ether, and of androgens with iso-octane in toluene, followed by RIA. The mean levels of steroids were higher in fat than in plasma, apart from testosterone. Levels of sulfates of estrogens and androstenediol were higher in breast than abdominal adipose tissue, and levels of estradiol lower. Positive correlations were found between BMI and tissue and plasma concentration of both estrone and androstenedione.

Epidemiological studies have shown that chronic work stress or unfavourable psychosocial work conditions are prospectively associated with different adverse health outcomes. The aim of the present cross-sectional study was to investigate the relationship between work-related chronic stress as well as exhaustion and a cumulative measure of physiological wear-and-tear called allostastic load (AL). AL could be a possible biological pathway for how chronic work stress and exhaustion lead to health impairments in the long run. As the teaching profession has been proposed to be a potentially high stressful occupation, chronic work stress (effort-reward-imbalance) and exhaustion were assessed in 104 female school teachers. AL was first analyzed according to McEwen's classical model comprised of ten parameters including cortisol, epinephrine and norepinephrine, dehydroepiandrosterone-sulphate (DHEA-S), waist/hip-ratio (WHR), glycosylated haemoglobin (HbA1c), high-density lipoprotein (HDL), total cholesterol/HDL-ratio, and systolic and diastolic blood pressure. Additionally it was extended to include tumor-necrosis-factor-alpha (TNF-alpha), C-reactive protein (CRP), fibrinogen, D-dimer, percent-body-fat, triglycerides, and glucose levels. A substantial proportion of our sample was highly exhausted whereas relatively few teachers showed high effort-reward-imbalance. AL scores were significantly higher in women high on effort-reward-imbalance or suffering from exhaustion. Although all teachers had been in a good health status, chronic work stress as well as exhaustion appears to be associated with changes in a multi-system summary indicator of physiological risk.

While salivary assays for some hormones are widely used, the availability of assays for salivary DHEA is limited. By adapting a commercially available radioimmunoassay serum kit, we developed a reliable, efficient and sensitive measure of DHEA in saliva that does not require separation or extraction. The minimum detection limit was 4.0 pg/ml. Intra-assay coefficients of variation (CV%) were on average 4.05, and inter-assay CVs averaged 9.70. Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, averaged 99.55 and 92.03%. Levels in matched serum and saliva samples showed strong linear relationships for adult males and females. Specific guidelines are developed for sample collection, storage, and preparation procedures. Reference ranges for salivary DHEA levels are provided for 64 children ages 8-11, 96 adolescents ages 12-17 and 48 adults ages 30-45. Salivary DHEA levels are shown to reflect developmental, gender and diurnal differences.

Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) are involved in the absorption and elimination of a wide variety of structurally unrelated amphipathic organic compounds. Several members of this protein family mediate the uptake of substrates across the basolateral membrane of hepatocytes as the first step in hepatic elimination. In contrast to the well-characterized Oatp1 and Oatp2, the localization and substrate specificity of the recently cloned Oatp4 have not been investigated in detail. Therefore, we raised an antibody against the C-terminal end of Oatp4 and localized this 85-kDa protein to the basolateral membrane of rat hepatocytes. Similar to Oatp1 and Oatp2, Oatp4 is a multispecific transporter with high affinities for bromosulfophthalein, dehydroepiandrosterone sulfate, leukotriene C4, and anionic peptides. In addition, we compared the substrate specificity of Oatp4 to that of Oatp3, which so far has mainly been shown to mediate intestinal bile acid transport. Oatp3 had a similar broad substrate specificity, but in general much lower affinities than Oatp4. Thus, while Oatp4 seems to work in concert with Oatp1 and Oatp2 in the basolateral membrane of rat hepatocytes, Oatp3 is a multispecific transport system in the small intestine.

OBJECTIVE

Hyperandrogenia and insulin resistance are heritable family traits, likely to cluster in children of polycystic ovary syndrome (PCOS) mothers.

METHODS

We performed a case control study of PCOS children (n = 32) compared with children from control women (n = 38) for reproductive and metabolic abnormalities, stratifying results by three Tanner stage groupings. The children underwent history and physical examinations, a 3-h timed urine collection, a 2-h oral glucose tolerance test, and abdominal ultrasound examination (females only). Serum was obtained in older children (age>> 8 yr) who consented.

RESULTS

Urine LH levels were significantly lower in the Tanner IV-V PCOS girls compared with controls (P = 0.04). Urine testosterone levels were significantly elevated in Tanner II-III PCOS boys compared with controls (P = 0.007). There were no significant differences in dehydroepiandrosterone levels. We validated the correlation between salivary and serum levels of insulin (insulin areas under the curve) in an adult population [n =30, Pearson correlation coefficient (r) = 0.67; P < 0.0001], which also replicated in the children (2-h insulin r = 0.57; P = 0.0004). Mean area under the curve salivary insulin levels were significantly higher in the Tanner IV-V PCOS girls in the later stages of puberty when compared with controls (3625 +/- 1372 vs. 1766 +/- 621 min x muU/ml, 95% confidence interval 475-3242; P < 0.02).

CONCLUSIONS

Hyperinsulinism may be a familial characteristic of PCOS children (or at least girls) but does not appear until the later stages of puberty. Other reproductive abnormalities that characterize PCOS may develop later.

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are metabolic intermediates in the production of potent androgens, estrogens and other less well-characterized steroids. DHEA(S) and closely related steroid hormones have a variety of immunological effects both in vitro and in vivo in experimental animals and humans. Many of these effects have been demonstrated in animal models where there is little circulating DHEA(S), and the demonstrated effects are generally seen at concentrations of DHEA(S) which are supra-physiological in man. The physiological role of DHEA(S) in the immunological system is unknown. Furthermore, the molecular mechanism of action of DHEA(S) is unclear. In this review, I focus on studies of the immunological effects of DHEA(S) and closely related steroid metabolites and analogs, mainly derived from literature published in the last five years. My purpose is to describe the demonstrated effects and to highlight some of the remaining major research issues in this field. These issues include defining the molecular mechanism of DHEA(S) action; determining whether the effect of DHEA(S) is related to the steroid itself or to a metabolic product of DHEA; determining the relationship of physiological function to the pharmacological effects; and determining the molecular basis for species-specific differences in effects.

This study examined the effects of neurosteroids on the behavior of mice in the mirrored chamber test of anxiety, and determined the potential mechanisms by which neurosteroids alter the behavior in animal models of anxiety. Allopregnanolone (AP) (0.5 and 1 mg/kg) and pregnenolone sulfate (PS) (0.5 and 2 mg/kg) significantly reduced the latency to enter the chamber, and increased both number of entries and total time spent in the chamber in a dose-dependent manner, without affecting the spontaneous locomotor activity. In contrast, dehydroepiandrosterone sulfate (DHEAS) (1 and 2 mg/kg) increased motor activity and caused an anxiogenic response, i.e., an increase in latency to enter the mirrored chamber, and a decrease in the number of entries and time spent in the chamber. Progesterone (PROG) (1-10 mg/kg), a neurosteroid precursor, and 4'-chlordiazepam (4'-CD) (0.25-1 mg/kg), a specific ligand for the mitochondrial diazepam binding inhibitor (DBI) receptor (MDR), produced a clear dose-dependent anxiolytic response in the mirrored chamber. The AP-, PROG- and 4'-CD-elicited anxiolytic behavior was blocked by picrotoxin (1 mg/kg), a GABA-A chloride channel antagonist, but not by flumazenil (2 mg/kg), a selective benzodiazepine (BZD) antagonist. In contrast, the anxiolytic effect of PS was not blocked by picrotoxin. The 4'-CD-induced anxiolytic effect was prevented by pretreatment with PK11195 (2 mg/kg), a selective partial MDR antagonist. Nifedipine (2 and 5 mg/kg), a dihydropyridine-type Ca2+ channel blocker, produced a flumazenil-resistant anxiolytic effect. Combined administration of nifedipine (2 and 5 mg/kg) and PS (0.5 and 2 mg/kg) exerted a significant additive effect in the mirrored chamber test. The potent anxiolytic effect of dizocilpine (0.5 and 1 mg/kg), an NMDA receptor antagonist, was blocked by pretreatment with DHEAS (2 mg/kg). Neurosteroids evoked changes in mirrored chamber activities resembling those elicited by triazolam (0.25 and 0.5 mg/kg). However, these effects were seen at doses that did not markedly affect locomotor activity, thereby suggesting these changes in behavior represent anxiolytic actions. Together, these results provide evidence for differential behavioral actions of the neurosteroids AP, PS and DHEAS in the mirrored chamber test of anxiety. The anxiolytic effect of PROG may be imputed to its metabolism to neurosteroid AP, while the 4'-CD-induced anxiolytic response is related to its MDR-stimulated neurosteroidogenesis and subsequent modulation of GABA-A receptor. Further, these differential effects reaffirm the contention that neurosteroids could be involved in the homeostasis of stress response.

In humans, the biotransformation of cholesterol and its hydroxylated metabolites (oxysterols) by sulfonation is a fundamental process of great importance. Nevertheless, the sulfotransferase enzyme(s) that carries out this function has never been clearly identified. Cholesterol is a relatively poor substrate for the previously cloned hydroxysteroid sulfotransferase (HST), i.e. dehydroepiandrosterone (DHEA) sulfotransferase (HST1). Recently, cloning of a single human gene that encodes for two proteins related to HST1 was reported. These newly cloned sulfotransferases (HST2a and HST2b), while exhibiting sequence similarity to other members of the soluble sulfotransferase superfamily, also contain unique structural features. This latter aspect prompted an examination of their substrate specificity for comparison with HST1. Thus, HST1, HST2a, and HST2b were overexpressed as fusion proteins and purified. Furthermore, a novel procedure for the isolation of cholesterol and oxysterol sulfonates was developed that was used in association with HPLC to resolve specific sterol sulfonates. HST1 preferentially sulfonated DHEA and, to a lesser extent, oxysterols; whereas cholesterol was a negligible substrate. The reverse, however, was the case for the HST2 isoforms, particularly HST2b, which preferentially sulfonated cholesterol and oxysterols, in contrast to DHEA, which served as a poor substrate for this enzyme. RT-PCR analysis revealed distinct patterns of HST1, HST2a, and HST2b expression. It was particularly notable that both HST2 isoforms, but not HST1, were expressed in skin, a tissue where cholesterol sulfonation plays an important role in normal development of the skin barrier. In conclusion, substrate specificity and tissue distribution studies strongly suggest that HST2a and HST2b, in contrast to HST1, represent normal human cholesterol and oxysterol sulfotransferases. Furthermore, this study represents the first example of the sulfonation of oxysterols by a specific human HST.

Dehydroepiandrosterone (DHEA) is a neurosteroid with potential effects on neurogenesis and neuronal survival in humans. However, most studies on DHEA have been performed in rodents, and there is little direct evidence for biological effects on the human nervous system. Furthermore, the mechanism of its action is unknown. Here, we show that DHEA significantly increased the growth rates of human neural stem cells derived from the fetal cortex and grown with both epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). However, it had no effect on cultures grown in either factor alone, suggesting a specific action on the EGF/LIF-responsive cell. Precursors of DHEA such as pregnenolone or six of its major metabolites, had no significant effect on proliferation rates. DHEA did not alter the small number (<3%) of newly formed neuroblasts or the large number (>95%) of nestin-positive precursors. However, the number of glial fibrillary acidic protein-positive cells, its mRNA, and protein were significantly increased by DHEA. We found both N-methyl-d-aspartate and sigma 1 antagonists, but not GABA antagonists, could completely eliminate the effects of DHEA on stem cell proliferation. Finally we asked whether the EGF/LIF/DHEA-responsive stem cells had an increased potential for neurogenesis and found a 29% increase in neuronal production when compared to cultures grown in EGF/LIF alone. Together these data suggest that DHEA is involved in the maintenance and division of human neural stem cells. Given the wide availability of this neurosteroid, this finding has important implications for future use.

A method designed for the analysis of sulfated neurosteroids and unconjugated ketonic neurosteroids in rat brain using nanoscale liquid chromatography-electrospray (nano-LC-ES) mass spectrometry is described. Neurosteroids in rat brain tissue were extracted, purified, and separated into two groups, neutral unconjugated steroids and steroid sulfates, by employing solid-phase partition, cation- and anion-exchange chromatography. The steroid sulfate fraction was analyzed by nano-LC-ES mass spectrometry. Contrary to expectations, the sulfates of pregnenolone and dehydroepiandrosterone (DHEA) were not detected. Internal standards, including pregnenolone sulfate, were recovered and the detection limit of the method was 0.3 ng/g of wet brain. Cholesterol sulfate was detected at a level of 1.2 microg/g of wet brain. The neutral unconjugated steroid fraction was derivatized with hydroxylamine hydrochloride to convert oxosteroids into their oximes. The oximes were isolated using cation-exchange chromatography and were analyzed by nano-LC-ES tandem mass spectrometry. The analyses of the neutral unconjugated steroid fraction confirmed the presence in rat brain of pregnenolone, pregnanolone isomers, progesterone, testosterone, and DHEA, which were characterized by their retention times, the mass of the protonated molecules, and characteristic fragment ions. The levels were estimated by addition of [3,4-(13)C(2)]-progesterone as an internal standard and found to be in a range of 0.04-20 ng/g.

To determine whether hyperandrogenism caused by an inborn error of adrenal steroidogenesis could produce insulin resistance, we examined insulin sensitivity in females with 21-hydroxylase deficiency. Minimal modelling was used to analyze the results of tolbutamide-modified, frequently sampled, iv glucose tolerance testing. Insulin sensitivity [Si; (min-1) (microU/mL)-1] was plotted against body mass index (BMI; defined as kilograms per m2). Six patients with nonclassical 21-hydroxylase deficiency (mean age, 27 yr; mean BMI, 23.2) underwent testing. None of these patients was in active puberty, nor was any patient being treated with glucocorticoids at the time of the study. Twelve eumenorrheic nonhyperandrogenic young adult female control subjects (mean age, 27 yr; mean BMI, 22.4) were also tested. The basal 17-hydroxyprogesterone concentration, but not the total serum testosterone level, was significantly different in the two groups (mean +/- SEM, 11,987 +/- 2,761 vs. 4,059 +/- 802 pmol/L; P < 0.05). As a group the patients' Si values were significantly lower than those of the controls (mean +/- SEM, 4.1 +/- 0.6 vs. 9.7 +/- 1.2; P < 0.05). There was no correlation between Si and basal serum 17-hydroxyprogesterone, testosterone, delta 4-androstenedione, or dehydroepiandrosterone. We conclude that chronic hypersecretion of androgen precursors due to an inborn error of metabolism can induce a reduction in insulin sensitivity.