Tetrahydrogestrinone is an androgenic steroid that stimulates androgen receptor-mediated, myogenic differentiation in C3H10T1/2 multipotent mesenchymal cells and promotes muscle accretion in orchidectomized male rats

The discovery of tetrahydrogestrinone (THG) abuse by several elite athletes led the U.S. Congress to declare it a controlled substance, although conclusive evidence of its anabolic/androgenic activity is lacking. We determined whether THG affects myogenic differentiation and androgen receptor (AR)-mediated signaling, whether it binds to AR, and whether it has androgenic and anabolic effects in vivo. Accordingly, we measured the dissociation constant for THG with a fluorescence anisotropy assay using recombinant AR-ligand binding domain. The AR nuclear translocation and myogenic activity of androstenedione were evaluated in mesenchymal, multipotent C3H10T1/2 cells. We performed molecular modeling of the THG:AR interaction. The androgenic/anabolic activity was evaluated in orchidectomized rats. THG bound to AR with an affinity similar to that of dihydrotestosterone. In multipotent C3H10T1/2 cells, THG upregulated AR expression, induced AR nuclear translocation, dose dependently increased the area of myosin heavy chain type II-positive myotubes, and up-regulated myogenic determination and myosin heavy chain type II protein expression. The interaction between AR and the A ring of THG was similar to that between AR and the A ring of dihydrotestosterone, but the C17 and C18 substituents in THG had a unique stabilizing interaction with AR. THG administration prevented the castration-induced atrophy of levator ani, prostate gland, and seminal vesicles and loss of fat-free mass in orchidectomized rats. We conclude that THG is an anabolic steroid that binds to AR, activates AR-mediated signaling, promotes myogenesis in mesenchymal multipotent cells, and has anabolic and androgenic activity in vivo. This mechanism-based approach should be useful for rapid screening of anabolic/androgenic agents.     

Androgen receptor gene expression in leucocytes is hormonally regulated: implications for gender differences in disease pathogenesis

OBJECTIVE: There is evidence that male sex hormones influence the rate of progression of inflammatory and cardiovascular diseases. We have previously shown that human leucocytes and arterial cells isolated from male donors express more androgen receptor (AR) than those from female cells, with potentially pro-atherogenic effects. We now investigate whether the gender difference in AR expression is due to genetic or hormonal regulation. DESIGN AND PATIENTS: The influence of hormones on AR expression were studied in hpg mice (a mouse model of androgen deficiency) treated with testosterone, oestradiol or dihydrotestosterone (DHT). Blood samples were obtained for leucocyte AR expression and hormone levels from 53 subjects, grouped into: 12 male [six young adult (27-45 years), six elderly (71-79 years)] and six female (young adult 25-45 years) healthy controls; six male-to-female transsexuals (M2F; 20-50 years) receiving stable pharmacological oral oestrogen treatment; six female-to-male transsexuals (F2M; 31-51 years) receiving stable androgen replacement therapy; five younger men (18-56 years) who had been receiving long-term androgen replacement therapy for hypogonadal disease; six elderly men (72-88 years) who had undergone medical castration for prostate cancer treatment; and 12 male bone marrow transplant recipients (BMT; 23-65 years) from either male or female donors. MEASUREMENTS: Serum testosterone and oestradiol concentrations were measured by established immunoflurometric assays from unextracted human serum. AR mRNA levels were measured by RT-PCR and AR protein levels by western blot (cell culture) or immunohistochemistry (mouse arteries). RESULTS: We found that AR mRNA levels were significantly down-regulated in the leucocytes of hpg mice that were treated with exogenous testosterone, oestradiol or DHT. AR protein levels were also lower in aortic tissue from the same mice. In humans, we found AR expression was significantly down-regulated by exogenous treatment with testosterone in F2M (31 +/- 13%, compared with control) or oestradiol in M2F (22 +/- 5%) but was significantly up-regulated by endogenous testosterone in BMT (128 +/- 17%). Low androgen levels measured in castrated older men were associated with markedly increased AR expression (207 +/- 26%, P < 0.05) compared with age-matched older male controls (100 +/- 2%). CONCLUSIONS: Our results indicate a regulated ability of vascular cells to respond to sex hormones, with the effects of exogenous therapies differing markedly from those due to endogenous sex hormones. We conclude that the gender difference in AR expression in vascular cells is hormonally, rather than genetically, controlled.     

A double-blind, placebo-controlled, randomized clinical trial of transdermal dihydrotestosterone gel on muscular strength, mobility, and quality of life in older men with partial androgen deficiency.

The efficacy and safety of androgen supplementation in older men remains controversial. Despite biochemical evidence of partial androgen deficiency in older men, controlled studies using T demonstrate equivocal benefits. Furthermore, the importance of aromatization and 5alpha reduction in androgen actions among older men remains unclear. Dihydrotestosterone is the highest potency natural androgen with the additional features that it is neither aromatizable nor susceptible to potency amplification by 5alpha reduction. Therefore, the effects of dihydrotestosterone may differ from those of T in older men. This study evaluated the efficacy and safety of 3 months treatment with transdermal dihydrotestosterone gel on muscle strength, mobility, and quality of life in ambulant, community-dwelling men aged 60 yr or older. Eligible men (plasma T < or =15 nmol/liter) were randomized to undergo daily dermal application of 70 mg dihydrotestosterone gel (n = 18) or vehicle (n = 19) and were studied before, monthly during, and 1 month after treatment. Among 33 (17 dihydrotestosterone, 16 placebo) men completing the study with a high degree of compliance, dihydrotestosterone had significant effects on circulating hormones (increased dihydrotestosterone; decreased total and free testosterone, LH, and FSH; unchanged SHBG and estradiol), lipid profiles (decreased total and low-density lipoprotein cholesterols; unchanged high-density lipoprotein cholesterol and triglycerides), hematopoiesis (increased hemoglobin, hematocrit, and red cell counts), and body composition (decreased skinfold thickness and fat mass; unchanged lean mass and waist to hip ratio). Muscle strength measured by isokinetic peak torque was increased in flexion of the dominant knee but not in knee extension or shoulder contraction, nor was there any significant change in gait, balance, or mobility tests, in cognitive function, or in quality of life scales. Dihydrotestosterone treatment had no adverse effects on prostate (unchanged prostate volumes and prostate-specific antigen) and cardiovascular (no adverse change in vascular reactivity or lipids) safety markers. We conclude that 3 months treatment with transdermal dihydrotestosterone gel demonstrates expected androgenic effects, short-term safety, and limited improvement in lower limb muscle strength but no change in physical functioning or cognitive function.     

Recombinant human growth hormone and recombinant human insulin-like growth factor I diminish the catabolic effects of hypogonadism in man: metabolic and molecular effects

Severe gonadal androgen deficiency can have profound catabolic effects in man. Hypogonadal men develop a loss of lean body mass, increased adiposity, and decreased muscle strength despite normal GH and insulin-like growth factor I (IGF-I) concentrations. We designed these studies to investigate whether GH or IGF-I administration to male subjects with profound hypogonadism can diminish or abolish the catabolic effects of testosterone deficiency. Moreover, we also examined the nature of the interactions among GH, IGF-I, and androgens in specific genes of the im system. A group of 13 healthy subjects (mean age, 22 +/- 1 yr) was studied at baseline (D1) and 10 weeks after being made hypogonadal using a GnRH analog (GnRHa; D2). At 6 weeks from baseline they were started on either recombinant human (rh) IGF-I (60 microg/kg, sc, twice daily) or rhGH (12.5 microg/kg, sc, daily) for 4 weeks. On each study day subjects had infusions of L-[(13)C]leucine; indirect calorimetry; isokinetic dynamometry of the knee extensors; determination of body composition (dual energy x-ray absortiometry) and hormone and growth factor concentrations, as well as percutaneous muscle biopsies. Their data were compared with those of previously studied male subjects who received only GNRHA: Administration of rhIGF-I and rhGH to the hypogonadal men had similar effects on whole body metabolism, with maintenance of protein synthesis rates, fat oxidation rates, and fat-free mass compared with the eugonadal state, preventing the decline observed with hypogonadism alone. This was further amplified by the molecular assessment of important genes in muscle function. During rhIGF-I treatment, im expression of IGF-I declined, and IGF-binding protein-4 increased, similar to the changes during GnRHa alone. However, rhGH administration was associated with a marked increase in IGF-I and androgen receptor messenger ribonucleic acid concentrations in skeletal muscle with a reciprocal decline in IGF-binding protein-4 expression in the hypogonadal men. The gene expression for myostatin did not change. These effects were accompanied by a much greater increase in plasma IGF-I concentrations after rhIGF-I (225 +/- 32 vs. 768 +/- 117 microg/L) compared with the concentrations achieved during rhGH (217 +/- 20 vs. 450 +/- 19 microg/L). We conclude that 1) rhGH and rhIGF-I both may be beneficial in preserving lean body mass and sustaining rates of protein synthesis during states of severe androgen deficiency in man; 2) GH may affect the im IGF system via an a paracrine, local production of IGF-I; 3) androgens may be necessary for the full anabolic effect of GH/IGF-I in man. These hormones, particularly GH, may play a role in the treatment of hypogonadal men rendered hypogonadal pharmacologically or those unable to take full testosterone replacement. The latter requires further study.     

Androgen deficiency: effects on body composition

Published data on the effects of androgen deficiency and testosterone administration on body composition in men and women are reviewed. In experimental paradigms, androgen deprivation decreases lean body mass and increases fat mass in men, and physiologic replacement reverses these abnormalities. The anabolic effects of testosterone administration on muscle in men are well-established, and current understanding of the underlying mechanisms are discussed. Randomized, placebo-controlled studies have been performed to investigate the effects of testosterone administration on body composition in a number of male hypogonadal states, including HIV-associated weight loss, supraphysiologic glucocorticoid administration, aging and obesity, with variable outcomes, and the results are reviewed. There are few data investigating the effects of hypoandrogenemia or androgen replacement on body composition in women, in whom endogenous testosterone levels are a fraction of those in men. A recent randomized, placebo-controlled study of physiologic testosterone replacement therapy in women with profound hypoandrogenemia due to hypopituitarism demonstrated an increase in skeletal muscle mass but no change in body fat. Further research is needed to establish the effects of endogenous androgens on the regulation of body composition in women.     

Testosterone replacement therapy for older men

Despite intensive research on testosterone therapy for older men, important questions remain unanswered. The evidence clearly indicates that many older men display a partial androgen deficiency. In older men, low circulating testosterone is correlated with low muscle strength, with high adiposity, with insulin resistance and with poor cognitive performance. Testosterone replacement in older men has produced benefits, but not consistently so. The inconsistency may arise from differences in the dose and duration of testosterone treatment, as well as selection of the target population. Generally, studies reporting anabolic responses to testosterone have employed higher doses of testosterone for longer treatment periods and have targeted older men whose baseline circulating bioavailable testosterone levels were low. Most studies of testosterone replacement have reported anabolic that are modest compared to what can be achieved with resistance exercise training. However, several strategies currently under evaluation have the potential to produce greater anabolic effects and to do so in a safe manner. At this time, testosterone therapy can not be recommended for the general population of older men. Older men who are hypogonadal are at greater risk for the catabolic effects associated with a number of acute and chronic medical conditions. Future research is likely to reveal benefits of testosterone therapy for some of these special populations. Testosterone therapy produces a number of adverse effects, including worsening of sleep apnea, gynecomastia, polycythemia and elevation of PSA. Efficacy and adverse effects should be assessed frequently throughout the course of therapy.     

An orally active selective androgen receptor modulator is efficacious on bone, muscle, and sex function with reduced impact on prostate

A number of conditions, including osteoporosis, frailty, and sexual dysfunction in both men and women have been improved using androgens. However, androgens are not widely used for these indications because of the side effects associated with these drugs. We describe an androgen receptor (AR) ligand that maintains expected anabolic activities with substantially diminished activity in the prostate. LGD2226 is a nonsteroidal, nonaromatizable, highly selective ligand for the AR, exhibiting virtually no affinity for the other intracellular receptors. We determined that AR bound to LGD2226 exhibits a unique pattern of protein-protein interactions compared with testosterone, fluoxymesterone (an orally available steroidal androgen), and other steroids, suggesting that LGD2226 alters the conformation of the ligand-binding domain. We demonstrated that LGD2226 is fully active in cell-based models of bone and muscle. LGD2226 exhibited anabolic activity on muscle and bone with reduced impact on prostate growth in rodent models. Biomechanical testing of bones from animals treated with LGD2226 showed strong enhancement of bone strength above sham levels. LGD2226 was also efficacious in a sex-behavior model in male rats measuring mounts, intromissions, ejaculations, and copulation efficiency. These results with an orally available, nonaromatizable androgen demonstrate the important role of the AR and androgens in mediating a number of beneficial effects in bone, muscle, and sexual function independent from the conversion of androgens into estrogenic ligands. Taken together, these results suggest that orally active, nonsteroidal selective androgen receptor modulators may be useful therapeutics for enhancing muscle, bone, and sexual function.     

Androgen receptor gene CAG repeat length and body mass index modulate the safety of long-term intramuscular testosterone undecanoate therapy in hypogonadal men

CONTEXT: A reliable form of androgen substitution therapy regarding kinetics, tolerance, and restoration of androgenicity is paramount in hypogonadal men. Intramuscular injection of the long-acting ester testosterone undecanoate (TU) offers a new modality. OBJECTIVE: The objective of the study was to assess the safety of TU regarding metabolic and pharmacogenetic confounders. DESIGN: This was a longitudinal one-arm open observation trial. A minimum of five individual assessments was a prerequisite. Putative modulators of safety parameters entering regression models were nadir and/or delta total testosterone concentrations, body mass index, androgen receptor (AR) gene CAG repeat length, and age. SETTING: The study was conducted at an andrological outpatient clinic. PATIENTS: Patients included 66 hypogonadal men (mean age 38 +/- 9.9 yr). MAIN OUTCOME MEASURES: A total of 515 data time points each related to prostate, erythropoiesis, lipoproteins, and circulation during 118 treatment-years with 1000 mg TU at 10- to 14-wk intervals. RESULTS: Testosterone substitution resulted in significant decrements of serum levels of low-density lipoprotein-cholesterol, resting diastolic and systolic blood pressure, and heart rate. Erythropoiesis was stimulated and concentrations of high-density lipoproteincholesterol increased. Parameters remained stable after four injections. No adverse effects regarding the prostate were observed. Significantly increased hematocrit greater than 50% was predicted by enhanced androgen action (shorter AR CAG repeats per higher testosterone levels). However, insufficient androgen action (longer AR CAG repeats per lower testosterone levels) caused pathological safety parameters (high blood pressure, adverse lipid profiles). In addition, a body mass index 30 kg/m(2) or greater represents a clinically relevant factor for the occurrence of all pathological safety parameters. Risk calculations for obese patients and nonlinear pharmacogenetic models to tailor androgen substitution are presented. CONCLUSIONS: Testosterone substitution with im TU is generally well tolerated. Modifications of androgen action are due to both AR CAG repeats and testosterone levels. Adverse observations are mostly seen in obese patients.     

Physiological testosterone replacement and arterial endothelial function in men

OBJECTIVE: The vascular effects of fluctuations in testosterone levels within the physiological range in otherwise healthy men are not known. We therefore aimed to study arterial function in hypogonadal men receiving long-term physiological androgen replacement therapy, at trough and peak testosterone levels. PATIENTS AND DESIGN: We recruited nine hypogonadal men (aged 35 +/- 4 years) receiving androgen replacement therapy, each treated with 800 mg testosterone (T) depot preparations every 6 months. MEASUREMENTS: Serum lipid and hormone levels and arterial reactivity were measured, prior to (trough T) and 2-4 weeks following testosterone administration (peak T). Each subject therefore served as their own control. Vessel diameter was measured by ultrasound at rest, during reactive hyperaemia [leading to flow-mediated dilatation (FMD), an endothelium-dependent response] and after sublingual nitroglycerin (GTN, an endothelium-independent dilator). RESULTS: Serum T (13 +/- 2 nmvs. 27 +/- 3 nm for trough and peak serum T, respectively, P < 0.001; normal adult male range 11-35 nm), and free T (195 +/- 23 pmvs. 510 +/- 93 pm, P < 0.005) significantly increased following subcutaneous depot T administration, as did serum oestradiol (100 +/- 10 pmvs. 175 +/- 9 pm, P = 0.001; normal adult male range < 250 pm). There was a significant decrease in FMD (3.6 +/- 1.1%vs. 3.0 +/- 0.8%, P < 0.01), but GTN responses were similar (9.5 +/- 0.8%vs. 10.4 +/- 1.0%, P > 0.2). Lipid, blood pressure and vessel diameter measurements were also similar before and after testosterone administration. CONCLUSION: Physiological replacement of testosterone is associated with decreased endothelium-dependent dilatation, in hypogonadal men.     

Testosterone inhibits adipogenic differentiation in 3T3-L1 cells: nuclear translocation of androgen receptor complex with beta-catenin and T-cell factor 4 may bypass canonical Wnt signaling to down-regulate adipogenic transcription factors

Testosterone supplementation in men decreases fat mass; however, the mechanisms by which it inhibits fat mass are unknown. We hypothesized that testosterone inhibits adipogenic differentiation of preadipocytes by activation of androgen receptor (AR)/beta-catenin interaction and subsequent translocation of this complex to the nucleus thereby bypassing canonical Wnt signaling. We tested this hypothesis in 3T3-L1 cells that differentiate to form fat cells in adipogenic medium. We found that these cells express AR and that testosterone and dihydrotestosterone dose-dependently inhibited adipogenic differentiation as analyzed by Oil Red O staining and down-regulation of CCAAT/enhancer binding protein-alpha and -delta and peroxisome proliferator-activated receptor-gamma2 protein and mRNA. These inhibitory effects of androgens were partially blocked by flutamide or bicalutamide. Androgen treatment was associated with nuclear translocation of beta-catenin and AR. Immunoprecipitation studies demonstrated association of beta-catenin with AR and T-cell factor 4 (TCF4) in the presence of androgens. Transfection of TCF4 cDNA inhibited adipogenic differentiation, whereas a dominant negative TCF4 cDNA construct induced adipogenesis and blocked testosterone's inhibitory effects. Our gene array analysis indicates that testosterone treatment led to activation of some Wnt target genes. Expression of constitutively activated AR fused with VP-16 did not inhibit the expression of CCAAT/enhancer binding protein-alpha in the absence of androgens. Testosterone and dihydrotestosterone inhibit adipocyte differentiation in vitro through an AR-mediated nuclear translocation of beta-catenin and activation of downstream Wnt signaling. These data provide evidence for a regulatory role for androgens in inhibiting adipogenic differentiation and a mechanistic explanation consistent with the observed reduction in fat mass in men treated with androgens.     

Tissue selectivity of the anabolic steroid, 19-nor-4-androstenediol-3beta,17beta-diol in male Sprague Dawley rats: selective stimulation of muscle mass and bone mineral density relative to prostate mass

Stimulation of prostate growth is a major concern with testosterone therapy in older hypogonadal men. As a result, nonsteroidal selective androgen receptor modulators with anabolic activity but less prostate stimulation are being developed. Anabolic steroids might exhibit similar tissue selectivity. We hypothesized the anabolic steroid 19-nor-4-androstenediol-3beta,17beta-diol (3beta,19-NA) would increase muscle, lean body mass (LBM), and bone mineral density (BMD) with little stimulation of prostate growth. Male Sprague Dawley rats were implanted with SILASTIC brand (Dow Corning, Midland, MI) capsules containing 3beta,19-NA (4, 8, or 16 cm), dihydrotestosterone (DHT) (8 cm), 19-nortestosterone (16 cm), or four empty capsules after undergoing either a sham operation (intact) or orchidectomy (ORX). Serum gonadotropins, measured after 4, 8, or 24 wk of treatment, were significantly lower in 3beta,19-NA-treated vs. untreated, intact, and ORX rats (P < 0.05), and testosterone was lowered by 3beta,19-NA-treatment of intact animals. LBM and BMD were assessed after 20 wk, and 4 wk later, rats were killed for levator ani muscle and prostate weights. Compared with ORX rats, 3beta,19-NA-treated rats had dose-dependent higher levator ani muscle weights, LBM, and BMD, which were similar to intact and DHT-treated rats at the highest 3beta,19-NA dose. In contrast, prostate weights in all 3beta,19-NA-treated groups were similar to ORX rats and lower than intact and DHT- and 19-nortestosterone-treated rats even at the highest 3beta,19-NA dose. In summary, 3beta,19-NA increases muscle and bone mass without significant stimulation of prostate growth, suggesting it may have some properties of a steroidal selective androgen receptor modulator. Anabolic steroids such as 3beta,19-NA should be studied further to determine their mechanisms of tissue selectivity and effects in men.     

Drug insight: Testosterone and selective androgen receptor modulators as anabolic therapies for chronic illness and aging

Several regulatory concerns have hindered development of androgens as anabolic therapies, despite unequivocal evidence that testosterone supplementation increases muscle mass and strength in men; it induces hypertrophy of type I and II muscle fibers, and increases myonuclear and satellite cell number. Androgens promote differentiation of mesenchymal multipotent cells into the myogenic lineage and inhibit their adipogenic differentiation, by facilitating association of androgen receptors with beta-catenin and activating T-cell factor 4. Meta-analyses indicate that testosterone supplementation increases fat-free mass and muscle strength in HIV-positive men with weight loss, glucocorticoid-treated men, and older men with low or low-normal testosterone levels. The effects of testosterone on physical function and outcomes important to patients have not, however, been studied. In older men, increased hematocrit and increased risk of prostate biopsy and detection of prostate events are the most frequent, testosterone-related adverse events. Concerns about long-term risks have restrained enthusiasm for testosterone use as anabolic therapy. Selective androgen-receptor modulators that are preferentially anabolic and that spare the prostate hold promise as anabolic therapies. We need more studies to determine whether testosterone or selective androgen-receptor modulators can induce meaningful improvements in physical function and patient-important outcomes in patients with physical dysfunction associated with chronic illness or aging.     

Pharmacokinetics of a transdermal testosterone system in men with end stage renal disease receiving maintenance hemodialysis and healthy hypogonadal men

Androgen deficiency is common in men with end stage renal disease (ESRD) on maintenance hemodialysis. Pharmacokinetics of transdermal testosterone in men receiving maintenance hemodialysis have not been studied. Our objective was to compare the pharmacokinetics of a transdermal testosterone system in healthy hypogonadal men and in men with ESRD on maintenance hemodialysis. We recruited 10 healthy hypogonadal men and 8 medically stable men on maintenance hemodialysis, 18--70 yr old, who had serum testosterone less than 300 ng/dL. After baseline sampling during a 24-h control period, two testosterone patches were applied daily for 28 days, to achieve a nominal delivery of 10-mg testosterone daily. In addition to single, pooled samples on days 7, 14, and 21, blood was drawn at 0, 2, 4, 6, 8, and 24 h on day 28 in healthy hypogonadal men and on an interdialytic day (day 21 or 28) as well as a dialysis day (day 21 or 28) in men on hemodialysis. On the dialysis day (day 21 or 28), serum free and total testosterone levels were measured hourly for 4 h before hemodialysis and for 4 h during hemodialysis. The dialysate was sampled for testosterone measurement. Baseline mean + SD total (92 +/- 82 vs. 222 +/- 50 ng/dL) and free (11 +/- 9 vs. 27 +/- 6 pg/mL) testosterone concentrations were lower in healthy hypogonadal men than in men with ESRD. After application of two testosterone patches, serum total and free testosterone concentrations rose into the midnormal range in both groups of men. Time-average, steady state (total testosterone, 506 +/- 88 vs. 516 +/- 86 ng/dL; free testosterone, 55 +/- 9 vs. 67 +/- 11 pg/mL), minimum, and maximum total and free testosterone concentrations were not significantly different between the two groups of men during treatment. Increments in total and free testosterone concentrations above baseline, baseline-subtracted areas under the total and free testosterone curves, and half-life of testosterone elimination (t(1/2), 2.1 +/- 0.1 vs. 2.1 +/- 0.2 h, P = not significant) were not significantly different between the two groups. In men receiving hemodialysis, time-average, steady state, and maximal total and free testosterone concentrations and baseline-subtracted areas under the total and free testosterone curves were higher on dialysis day than on an interdialytic day. On the day of hemodialysis, time-average total and free testosterone concentrations were not significantly different during the 4 h before or during hemodialysis. The amount of testosterone removed in the dialysate (8.4 +/- 1.6 microg during 4 h of hemodialysis) was small compared with the daily testosterone production rates in healthy young men. Serum dihydrotestosterone and estradiol concentrations increased into the normal male range and were not significantly different between the two groups. Percent suppression of LH was greater in men with ESRD than in healthy hypogonadal men. A regimen of two Testoderm TTS testosterone patches (Alza Corp., Mountain View, CA) daily can maintain serum concentrations of total and free testosterone and its metabolites dihydrotestosterone and estradiol in the midnormal range in healthy hypogonadal men and men on hemodialysis. The amount of testosterone cleared by hemodialysis is small, and hemodialysis does not significantly affect serum total and free testosterone concentrations in men treated with the testosterone patch.     

Androstenedione metabolism in cultured human osteoblast-like cells.

Bone is a target organ of androgens. The mechanism by which these steroids exert their action within bone cells is still poorly understood. The metabolism of androstenedione, the major circulating androgen in women, was, therefore, assessed in osteoblast-like bone cells cultured from bone of 16 postmenopausal women (mean age, 69 yr; range, 56-80) and 3 elderly men (mean age, 71 yr; range, 69-73) undergoing total hip replacement. Each cell strain was incubated under standardized conditions with varying concentrations of [1,2,6,7-3H]androstenedione (0.05-5 microM). In every instance 5 alpha-reduced metabolites and 17 beta-hydroxysteroids were formed. There was no correlation between the volumetric density of the resected bone and androstenedione metabolism of the corresponding cultured bone cell strains. The apparent Km for the 5 alpha-reductase activity (sum of androstanedione and dihydrotestosterone) of all 19 cell strains was 0.7 +/- 0.1 microM (mean +/- SEM), and the apparent Km for 17 beta-hydroxysteroid dehydrogenase (sum of testosterone and dihydrotestosterone) was 2.3 +/- 0.8 microM (mean +/- SEM), values similar to those reported for other androgen target organs. Our results demonstrate that human osteoblast-like cells have the capacity to transform androstenedione into the more potent biological androgens testosterone and dihydrotestosterone. Since the Km values of both 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase exceed the serum androstenedione concentration, the formation of testosterone and dihydrotestosterone appears to be mainly a function of substrate availability.     

Levels of Dihydrotestosterone, Testosterone, Androstenedione, and Estradiol in Canalicular, Saccular, and Alveolar Mouse Lungs

Androgens and estrogens are known regulators of fetal and postnatal lung development, but their levels in the developing lung have never been determined. We present here, for the first time, a gas chromatography-mass spectrometry (GC/MS) quantification of dihydrotestosterone, testosterone, androstenedione, and estradiol in canalicular, saccular, and alveolar stage lungs of both sexes. Testosterone, androstenedione, and estradiol were observed in all the analyzed lung samples from gestation day (GD) 16.5 to postnatal day (PN) 30, totalizing 383 individual mice. Levels of these three steroids decreased between birth and PN 5. In contrast, dihydrotestosterone was detected only in male samples on GD 19.5, PN 0, and PN 30. A significant sex difference was observed for testosterone and androstenedione but not for estradiol. Steroid levels were also determined in skinned hind legs for comparison. Three-way analysis of variance revealed that tissue (lung or leg) had a significant effect on testosterone levels for both sexes, but not on androstenedione and estradiol levels. Low but significant testosterone and androstenedione levels were observed in all the females and in prepubertal male samples. These levels must be sufficient to induce androgen receptor activation, as suggested by our recent report showing the presence of androgen receptor in the nucleus of several lung cells in corresponding developmental ages and sexes.     

Androgens and the ageing male

Serum testosterone levels peak in early adulthood in men and fall progressively with age. Since sex hormone binding globulin increases with age, the unbound forms of testosterone (free and bioavailable testosterone) fall more steeply than total testosterone levels. Serum testosterone levels below the normal range for young healthy adult males provide chemical evidence of androgen deficiency independent of the age of the patient. When accompanied by signs or symptoms that are compatible with androgen deficiency, treatment with testosterone should be considered in older men without evidence of prostate or breast cancer. While such therapy for younger hypogonadal men has shown benefit on libido, mood, muscle mass, muscle strength, bone mineral density and haematocrit, similar benefits in older men have not been as adequately assessed. While there is no convincing evidence that testosterone treatment in older men will increase the risk of cardiovascular or prostate cancer, long-term, well-controlled studies are lacking and needed. Treatment options for older men include injectable, transdermal and transbuccal testosterone preparations.     

Estrogen, but not androgens, regulates androgen receptor messenger ribonucleic acid expression in the developing male rat forebrain.

Testosterone is the principal gonadal hormone responsible for the masculinization of the rat nervous system. Sex differences in both the ligand and receptor availability may play a role in the process of sexual differentiation. In some brain regions, males express more androgen receptor (AR) messenger RNA (mRNA) than females by postnatal day (PND) 10. Gonadectomy on the day of birth (PND-0) eliminated the sex differences in AR mRNA expression at PND-10, and exogenous testosterone replacement restored this sex difference. Because testosterone can be converted to both androgenic and estrogenic metabolites in the brain, the present experiments were performed to determine whether androgenic or estrogenic metabolites of testosterone are responsible for region-specific regulation of AR mRNA content in the developing rat forebrain. We used a 35S-labeled riboprobe and in situ hybridization to assess relative steady-state levels of AR mRNA in animals killed on PND-10. In the principal portion of the bed nucleus of the stria terminalis (BSTpr) and medial preoptic area (MPO), males gonadectomized on PND-0 and treated daily with dihydrotestosterone propionate (DHTP), a nonaromatizable androgen, had low levels of AR mRNA that were not significantly different from AR mRNA levels in intact females. In contrast, males gonadectomized on PND-0 and treated daily with diethylstilbestrol (DES), a synthetic estrogen, maintained high, male-typical levels of AR mRNA in the BSTpr and the MPO. AR mRNA content in the VMH was not sexually differentiated in PND-10 rats and was unaffected by gonadectomy or hormone replacement. To further assess whether AR mRNA was autologously regulated, neonatal male rats were treated with the androgen receptor antagonist, flutamide. Flutamide at a dose of either 40 microg/day or 300 microg/day had no effect on AR mRNA expression in any area examined. Thus, AR mRNA is up-regulated by estrogen but is not regulated by androgen during the early postnatal period.