Bruce S. Keenan

Androgen receptor in human skin fibroblasts characterization of a specific 17β-hydroxy-5α-androstan-3-one-protein complex in cell sonicates and nuclei☆

Cultured human skin fibroblasts were shown to contain an androgen binding activity (receptor) which was heat-labile and destroyed by trypsin. Specific binding was seen after incubations of these cells with 1,2-3-H-testosterone, 1,2-3-H17beta-hydroxy-5alpha-androstan-3-one (dihydrotestosterone, DHT) and 1,2-3-H-5alpha-androstane-3alpha, 17beta-diol. This receptor had a high affinity (Kd=0,2-1.6 nM) and a high degree of specificity for DHT. It was measured as a 3-H-DHT-protein complex by gel filtration chromatography using a method which distinguishes specific from nonspecific binding. Receptor activity was distributed about equally between nuclear and extranuclear components at all times studied and was present in both compartments when cell incubations were carried out at 4 degrees and 37 degrees. Saturation analysis indicated that there were 1250-18,600 binding sites per whole cell. By sucrose gradient centrifugation the receptor had a sedimentation coefficient (S20,w) of about 4. Cells grown for 8 days without serum in the medium maintained the same levels of 3-H-DHT binding. Within 15 hours puromycin (20 mug/ml) in serum-free medium caused a 40-60 percent decrease in binding for the same cell lines. Although the highest levels of 3-H-DHT binding were observed in fibroblasts from newborn foreskin, appreciable cytosol and nuclear binding were seen in cells from forearm, neck and abdominal skin. Receptor activity was stable during prolonged culture. Fibroblasts from several skin sites from patients with the androgen insensitivity syndrome (testicular feminization) had no detectable specific DHT binding. In this study it was demonstrated that skin fibroblasts can rapidly convert testosterone to its active form, DHT, bind DHT to a specific receptor protein and transport this complex to their nuclei. Therefore this may prove to be a convenient system for studying androgen action in vitro.

Treatment of persistent pubertal gynecomastia with dihydrotestosterone heptanoate

Four boys with persistent pubertal gynecomastia were given intramuscular dihydrotestosterone heptanoate (DHT-hp) at 2 to 4-week intervals for 16 weeks. By the end of treatment, breast size in all four boys had decreased 67% to 78%. Initial plasma levels of gonadotropins, estradiol, testosterone, and dihydrotestosterone (DHT) were normal. Mean plasma DHT concentration rose with the injections of DHT-hp, and remained elevated throughout the treatment period. Estradiol, LH, FSH, and testosterone decreased during treatment, as did 24-hour urinary LH and FSH. No regrowth of breast tissue was observed 6 to 15 months after treatment, although hormone concentrations had returned to near pretreatment values by 2 months after the last injection. DHT-hp has potential to be an effective medical therapy for persistent pubertal gynecomastia.

Abnormality of intracellular 5 alpha-dihydrotestosterone binding in simple hypospadias: studies on equilibrium steroid binding in sonicates of genital skin fibroblasts.

Summary: A method was developed for the measurement of the binding of [3H]5α-dihydrotestosterone, [3H]DHT and other steroids at equilibrium with intracellular androgen receptor of genital skin fibroblasts. This method utilized 0.2 M Na2MoO4 to stabilize the receptor and Sephadex G-25 chromatography to eliminate steroid metabolism. This binding protein showed the expected limited capacity, high affinity, and specificity of an androgen receptor.Using this method, penile skin cultures from 26 infants with simple hypospadias (HS) were compared with 18 controls. The [3H)DHT binding capacity (Bmax) was 10.1 ± 1.3 (±SE) fmol/mg protein for controls and 6.1 ± 1.7 for HS. The two populations were significantly different by Mann-Whitney test (P < 0.001). Equilibrium dissociation constant was similar for both groups. Surprisingly, there was no correlation between Bmax and the severity of the anatomic defect. Bmax was below the values seen in HS for two of three infants with male pseudohermaphroditism. In complete androgen insensitivity, DHT binding was unmeasurable.A subgroup exists in HS with an abnormality of intracellular androgen receptors. The lack of correlation between severity of hypospadias and Bmax suggests that additional factors, such as differences in physicochemical properties of the receptor or factors present in utero, contribute to the development of HS.

High-density lipoprotein response to 5-α-dihydrotestosterone and testosterone in Macaca fascicularis: A hormone-responsive primate model for the study of atherosclerosis☆☆☆

A decrease in high-density lipoprotein cholesterol (HDL-C), a major risk factor for coronary artery disease, occurs during puberty in males. Previous studies have shown this decrease with testosterone (T) therapy for adolescent males, but the mechanism of this effect is unknown and has not been studied in a non-human primate. Two adult male monkeys (Macaca fascicularis) were studied to determine simultaneous changes in plasma androgens and HDL-C during the phases precastration (Ci); postcastration (Cx); Cx and T therapy; Cx and dihydrotestosterone (DHT) therapy; and T and 5-alpha-reductase inhibitor therapy (4-MA). After castration, the HDL-C concentrations increased significantly in both animals (monkey A, 57.0 +/- 1.8 mg/dL SE to 66.6 +/- 2.2, P less than .005; monkey B, 62.9 +/- 1.6 to 80.2 +/- 1.7, P less than .001). T-propionate treatment produced a significant decrease in HDL-C (monkey A, 48.0 +/- 5.0, P less than .01; monkey B, 43.5 +/- 0.5, P less than .001), which was similar to HDL-C reductions seen when treated with a nonaromatizeable androgen, DHT-propionate (monkey A, 47.5 +/- 1.5, P less than .005; monkey B, 44.5 +/- 3.5, P less than .001). T and the 5-alpha-reductase inhibitor therapy did not increase HDL-C from the levels with T therapy alone (monkey A, 55.7 +/- 1.9, NS; monkey B, 57.3 +/- 0.3, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosol androgen receptor (AR) in human skin fibroblasts: Characterization of the binding reaction and differentiation from androgen binding molecules of lower affinity

Androgen binding was studied in cytosol of human fibroblasts at 4 degrees C. When 5 alpha-dihydrotestosterone (DHT) was the ligand, a curvilinear Scatchard plot was seen, which was resolved into two components: I the androgen receptor (AR), Kd = 0.12-0.44 nM, and II a low affinity species, Kd = 6.3-28 nM. The same cytosol demonstrated only type I binding for 3H-methyltrienolone (MTr), Kd = 0.10-0.40 nM. The AR, i.e., 3H-MTr binding activity, eluted at 440,000 d by gel filtration chromatography in pre-labeling and post-labeling experiments. When the ligand was 3H-DHT, binding activity in the 10,000-45,000 d range was seen in addition to AR. Thus, saturable nonreceptor steroid binding was seen for DHT but not for MTr. The latter is the preferred ligand for the study of the AR in this system.

Cessation of growth hormone secretion associated with acute elevation of the serum-free fatty acid concentration☆

Abstract Following the intravenous injection of fat (Intralipid) with heparin into five unanesthetized adult male baboons, the serum FFA level rose to a mean (±SE) of 11.30 ± 3.58 meq/liter at 5 min and the serum GH concentration fell rapidly from a mean base-line value of 8.1 ± 2.0 ng/ml to 3.4 ± 1.4 ng/ml 20 min after injection (T12 = 19 min. This fall in serum GH was more consistent and of greater magnitude than that following the injection of either fat or heparin alone. A late rise in GH was not observed through 2 hr. The depression of serum GH was not explicable on the basis of hyperglycemia. The rapid fall in the serum GH concentration after the injection of fat with heparin, with a serum halftime approximating the disappearance rate of GH in man, the consistent temporal relationship between fat with heparin injection and the fall in GH concentration, and the failure of either fat or heparin alone to consistently produce similar serum GH patterns suggest that the injection of fat with heparin with acute elevation of serum FFA concentrations to supraphysiologic levels caused virtual cessation of GH secretion. Thus, free fatty acids, in addition to glucose and amino acids, may be involved in the regulation of GH secretion.

ACTH RECEPTOR DEFECT IN ADRENOLEUKODYSTROPHY (ALD)

ACTH insensitivity is a cardinal feature of ALD, but the mechanism is unknown. We studied a 10 year old hyperpigmented boy with glucocorticoid insufficiency of 8 years duration and a history of two generalized tonic clonic seizures associated with febrile episodes with normal blood glucose. His neurological exam was normal, EEG showed non-specific slowing with normal auditory evoked responses, and Magnetic Resonance Imaging showed areas of increased signal intensity in the cerebral peduncles and the internal capsule. ALD was documented by increased plasma very long chain fatty acids, i.e., C26/C22 = 0.055 (control = 0.01 ± 0.01); C26= 1.572 μg/ml (control = 0.33 ± 0.18 pg/ml). Basal ACTH was elevated at 1840 pg/ml (normal = <100 pg/ml). Cortisol was 7.0 μg/dl with no response to exogenous ACTH. Basal and stimulated renin and aldosterone were normal. The childs leukocytes had no detectable ACTH binding sites in a radioligand binding study. In contrast, normal mononuclear leukocytes possess high and low affinity receptors for ACTH that appear identical to the prototype adrenal receptors. These studies suggest that the adrenal failure associated with ALD is secondary to an ACTH receptor defect. Whether the ACTH receptor defect is primary or secondary to the long chain lipid abnormality is under investigation.

Plasma 17α-hydroxyprogesterone and aldosterone concentrations in infants and children with congenital adrenal hyperplasia—the role of salt-losing hormones in salt wasting

I N F A N X S with congenital adrenal hyperplasia caused by a 21-hydroxylation defect in steroidogenesis often present with salt-losing crisis and hyponatremia. A severe defect in aldosterone synthesis has been demonstrated in some patients. 1 However, in some subjects with salt-losing CAH, normal aldosterone secretion rates have been demonstrated. 2 The observation that plasma 17a-hydroxyprogesterone is elevated in patients with untreated CAH, 3 together with the finding that 17-OHP and other ACTH-dependent steroids which are elevated in CAH can antagonize endogenous mineralocorticoids, ~ suggest that mineralocorticoid antagonists may play a significant role in the salt-wasting in these infants. In an attempt to clarify this relationship, we have studied plasma 17-OHP levels in 20 infants and children of different ages with salt-losing and simpie virilizing CAH. Plasma aldosterone levels also were measured on selected subjects.

Inappropriate adrenal androgen secretion with once-a-day corticosteroid therapy for congenital adrenal hyperplasia

To determine whether cortisone acetate given as a single daily dose would be as effective as the same amount divided into three doses in suppressing adrenal androgen secretion in congenital adrenal hyperplasia, we studied three patients with the salt-losing variety, who were judged to have been adequately treated during the previous year. Each patient was receiving cortisone acetate, 20.6 to 22.6 mg/m2 body surface area per day, divided into three doses, and 9 alpha-fluorohydrocortisone, 0.05 to 0.1 mg/day. Their spontaneous and adrenocorticotropic hormone-stimulated blood concentrations of 17 alpha-hydroxyprogesterone, androstenedione, and testosterone were measured initially and were normal. Cortisone acetate was then given once a day in the same total daily dose. After 3 months, plasma adrenocorticotropic hormone concentration was increased in all three patients, and in two of them the plasma levels of 17 alpha-hydroxyprogesterone and androstenedione were also increased. In the third patient, after 7 months of once-daily therapy, plasma levels of 17 alpha-hydroxyprogesterone and androstenedione were increased. We conclude that a physiologic replacement dosage of cortisone acetate given once daily is not effective in controlling excessive adrenal androgen secretion in congenital adrenal hyperplasia.

Plasma Renin Activity and the Response to Sodium Depletion in Salt-Losing Congenital Adrenal Hyperplasia

Summary: Mineralocorticoid therapy was discontinued in 14 subjects, ages 7–17 years, with salt-losing congenital adrenal hyperplasia. The level of plasma renin activity (PRA) was determined in relation to the cumulative loss of sodium (intake-urinary output) over 4–12 days. Although the magnitude and rate of response varied, PRA increased in all subjects. There was a significant positive correlation between cumulative sodium loss and PRA (r = 0.585, P < 0.001, n = 76), and a significant negative correlation between serum sodium concentration and PRA (r = - 0.736, P < 0.001, n = 76). Significant sodium loss and elevation of PRA were seen prior to the development of hyponatremia. Thus, elevated PRA appeared to be a more sensitive indicator of sodium loss than serum sodium concentration.Fifteen infants, ages 1–33 months with salt-losing congenital adrenal hyperplasia, who were treated with subcutaneous pellets of 11-deoxycorticosterone acetate (DOCA), were also studied. PRA decreased following implantation of the DOCA pellets over 1–6 months. In most instances there was a subsequent increase in PRA, beginning at 1–8 months post implantation. There was a significant negative correlation between serum sodium and PRA (r = −0.669, P < 0.001, n = 78) in these infants. However, there were four instances in which elevated PRA was associated with normal serum sodium.Sodium balance studies were also performed on 14 of these infants during 29 admissions. All balance studies were performed at approximately 3 or 6 months after DOCA pellet implantation. On a diet containing 2 mEq Na/kg body weight per day, sodium balance was positive and there was no significant change in sodium excretion over the 3 days of observation. When a 1 mEq Na/kg/day diet was given, sodium balance was neutral on day 3 of the study, and fractional urinary sodium excretion (FeNa) decreased from 0.49 ± 0.33, S.D. on day 1 to 0.23 ± .09% on day 3 (P < 0.001, n = 19). This change was statistically significant. Net sodium balance on either diet was independent of PRA. Elevated PRA was not necessarily associated with adverse symptomatology.Thus in infants treated with DOCA pellets, elevated PRA was associated with indices of sodium depletion and decreased availability of DOCA. The dietary sodium requirement for infants treated with DOCA appeared to be 1–2 mEq/kg body weight/day.Speculation: Although increased PRA indicated the subjects response to sodium depletion, it did not predict the ability to maintain sodium balance. There must be factors other than mineralocorticoid therapy, some possibly renin-dependent, contributing to the ability to compensate for sodium depletion. Nevertheless, increased PRA may be taken as an indication to either increase mineralocorticoid dosage or sodium intake.