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INTRODUCTION

The adrenal androgens (AAs), normally secreted by the fetal adrenal zone or the zona reticularis of the adrenal cortex are steroid hormones with weak androgenic activity. Although AAs do not appear to play a major role in the fully androgenized adult man, they seem to play a role in the adult woman and in both sexes before puberty. Girls, women, and prepubertal boys may be negatively affected by AA hypersecretion in contrast to adult men. This chapter reviews AA biosynthesis, regulation, physiology, and biological action.

ADRENAL ANATOMY AND ANDROGEN BIOSYNTHESIS

Adrenal gland anatomy

The adrenal glands, consisting of cortex and medulla, have a roughly pyramidal shape and lie above the upper poles of the kidneys. In keeping with their essential function, they are well supplied with arterial blood from branches of the phrenic arteries, the aorta, and the renal arteries, giving rise to the superior, middle, and inferior adrenal arteries, respectively. Arterial blood enters from the outer cortex, flows through fenestrated capillaries between the cords of cells, and drains into venules in the medulla. On the right, the adrenal vein directly enters the inferior vena cava; on the left, it usually drains into the left renal vein. The adrenal cortex is divided into three histologic and functional zones: the outer, aldosterone-secreting, called zona glomerulosa; the intermediate, cortisol-secreting, zona fasciculata; and the inner, androgen-secreting zona reticularis. Whereas the zona glomerulosa is primarily regulated by angiotensin II, both the zona fasciculata and the zona reticularis are regulated by corticotropin (ACTH) (1). Both these zones become hypofunction and atrophic when corticotropin is deficient; on the other hand, they become hypertrophic and hyperplastic when corticotropin is secreted in excess.

Fetal adrenal and development

The fetal adrenal cortex arises from mesodermal cells migrating from the coelomic epithelium very early in the embryonic period. It consists of the two adult adrenal zonae glomerulosa and fasciculata and an inner large adrenal zone, which degenerates rapidly after birth (2). The active secretion of steroids occurs by week 6 from the provisional zone, which represents the functional cortex in the fetal period. Remaining cell foci from the fetal adrenal zone presumably give rise to the adrenal zona reticularis starting at the age of 4 to 5 years in both sexes (3). The growth of this zone continues until young adulthood (20 to 25 years), remains at a plateau for 5 to 10 years, and regresses gradually after the age of 35 years. Aging results in alterations within the adrenal cortex with no significant difference in the total width of the cortex. More specifically, there is a reduction in the size of the zona reticularis and a relative increase in the outer cortical zones (4).

Biosynthesis

The anatomical alterations of the adrenal cortex resulting from aging are followed by a marked decline in circulating adrenal C19 steroids and their resulting androgen metabolites, which takes place mainly between the age groups of 20-30 and 50-60 yr, with smaller changes observed after the age of 60 yr (5). The major androgens secreted by the adrenals are dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), and androstenedione (D⁴-A). Production of testosterone (T) by these glands is minimal (6). DHEA and DHEA-S are mainly products of zona reticularis, D⁴-A and T are secreted by both zona reticularis and zona fasciculata (7; 8). All human steroid hormones derive from cholesterol. Cholesterol can be synthesized within the adrenal from acetylCoA, but about 80% derives from circulating plasma lipoproteins (LDL). The enzymes responsible for the synthesis are hydroxylases, dehydrogenases, isomerases, and desmolases, most of which require NADPH or NAD+ as cofactors (Fig.1).

Figure 1. Reaction mechanism of hydroxylations catalyzed by cytochrome P-450s of adrenal cortex mitochondria. Abbreviations: XH = substrate; XOH = product; Fp = flavoprotein; ISp = iron-sulfur protein.

ADRENAL ANDROGEN REGULATION AND PHYSIOLOGY

Regulation

Adrenal androgens are secreted by the adrenal glands in response to ACTH, a 39-amino acid peptide synthesized and secreted by the anterior pituitary (Fig.2). It is derived from Proopiomelanocortin (POMC), a large precursor molecule from which b-lipotropic hormone and/or b-endorphin are also derived (9;10). Corticotropin (1-39) is the predominant form of corticotropin in plasma and has a half- life of approximately 10 minutes (11). Its synthesis and secretion are primarily regulated by corticotropin-releasing hormone (CRH) and arginine-vasopressin (AVP), both of which are produced by parvocellular neurons of the paraventricular nucleus of the hypothalamus and act in synergy with each other (Fig.2) (12;13). Under ACTH regulation, adrenal androgens are secreted synchronously with cortisol, in both secretory episodes and circadian pattern. The circadian rhythm of the adrenal androgens is typical of the different physiologic and pathologic conditions. Patients with nonclassic 21-hydroxylase deficiency, for example, have a distinct pattern of adrenal steroid secretion characterized by a high-frequency 17-hydroxyprogesterone release accompanied by a relative nocturnal cortisol deficiency (14;15). Corticotropin-stimulated cortisol exerts major feedback inhibitory influences at the level of both the hypothalamus and the anterior pituitary by suppressing CRH, AVP, and corticotropin synthesis and secretion. Plasma DHEA, D⁴-A, and T concentrations closely parallel the circadian rhythm in plasma cortisol. Plasma DHEA-S levels do not exhibit a circadian rhythm because of the much longer circulating half-life of this sulfated steroid (16;17).

Figure 2. Schematic representation of the adrenal androgen regulation.

Numerous other endocrine signals (18) were proposed as coregulators of adrenal androgen secretion. Among these are prolactin (PRL) (19), estrogen (20-24), epidermal growth factor (25), prostaglandins (26), angiotensin (27), GH (28), gonadotropins (29;30), b-lipotropin, and b-endorphin. Glasow et al. reported the presence of PRL receptors in the human adrenal gland and suggested a direct effect of PRL on adrenal steroidogenesis that may be of particular relevance in clinical disorders characterized by hyperprolactinemia (31). Interestingly, adults with hyperprolactinemia have increased secretion of AAs by the zona reticularis, which is corrected by reduction of PRL secretion with bromocriptine (32). In women with PRL-secreting tumors there is a correlation between PRL levels and DHEA-S (33).

Interleukin-6 (IL-6) also is known to activate the HPA axis by stimulating both the CRH - and AVP -secreting neurons of the paraventricular nucleus of the hypothalamus, and their terminals at the median eminence, the cotricotrophs of the anterior pituitary, and cortisol-secreting adrenal cells in rats. In the latter it acts through specific receptors expressed mainly in the zonae fasciculata and reticularis, but also with lower density in the zona glomerulosa (34;35). The ability of IL-6 to stimulate glucocorticoids, mineralocorticoids, and androgens suggests that this cytokine might have a role in coordinating the response of all adrenocortical zones. Its secretion is regulated by different substances, such as CRH, ACTH, angiotensin II, or immune products such as IL-1a/b indicating that IL-6 may play a major role in the interaction of the adrenal function with the immune system (36).

Both corticotropin and PRL stimulate AA secretion by the fetal adrenal zone. In addition, placental CRH appears to play a major role in sustaining this zone and stimulating androgen secretion together with corticotropin and/or PRL (37).

Physiology

AAs are secreted in small amounts during infancy and early childhood, and their secretion gradually increases with age, paralleling the growth of the zona reticularis. The appearance of public hair (pubarche) results from a rise in adrenal androgen levels called adrenarche (38). The mechanism(s) by which the zona reticularis develops with age and the regulation of adrenarche onset are notunderstood. Children with premature pubarche have hormonal responses to CRH stimulation test similar in magnitude to those of prepubertal children of comparable age, ruling out a prominent role of CRH in premature pubarche (39). Gell et al. suggested that as children mature, a decrease of 3b-hydroxysteroid dehydrogenase activity in the adrenal reticularis occurs, leading to an increased production of DHEA and DHEA-S, as seen during adrenarche, by shifting pregnenolone through the 17a-hydroxylase /17, 20 lyase pathway (40). Activation of the type I Insulin-like Growth Factor (IGF) receptor was shown to enhance steroidogenic responsiveness of the fetal zone cells to ACTH by modulating the ACTH signal transduction pathway at some point downstream from ACTH receptor binding (41). Also, locally produced IGF-II modulates fetal adrenocortical cells function by increasing responsiveness to ACTH via activation of the type I IGF receptor and increases the capacity of those cells for androgen synthesis by directly augmenting the expression of P450c17 (41). Thus, IGF-II may play a pivotal role in AA production, both physiologically in utero and at adrenarche, as well as in conditions of hyperandrogenemia (41). All together, these data indicate that the IGF system is important in the regulation of the differential function of adult human adrenocortical cells (42).

The rise in plasma concentrations of the AAs at adrenarche occurs in the presence of constant levels of cortisol, suggesting that factors other than corticotropin are involved. Recent data suggest that the influences of sex and age are minor in the modulation of adrenal steroidogenesis and support the concept that extra-adrenal factors dominate in the differential modulation of AAs and cortisol (43). These may include POMC-derived peptides and an elusive adrenal androgen-stimulating factor (AASF) (Fig. 2), the existence of which has not been confirmed (44-49).

Recently, experiments by Miller and co-workers (50; 51) suggested that an increased serine phosphorylation of human P450c17 might have a role in the development of both the excessive adrenarche and hyperandrogenism of patients with the polycystic ovary syndrome resulting in a substantial increase in 17-20-lyase activity (52). P450c17 is the key enzyme that regulates androgen synthesis (53). It is the only enzyme known to be able to convert C21-precursors to the androgen pro-hormones, the 17-ketosteroids. It is a single enzyme with two activities, 17 a-hydroxylase and 17,20-lyase and serine phosphorylation appears to modulate the activity of P450c17. In particular, it promotes the 17,20-lyase activity, and at the same time it inhibits the activity of the insulin receptor (51;54-57). It was postulated that a single abnormal serine kinase might hyperphosphorylate both P450c17 and the insulin receptor, accounting for the hyperandrogenism and hyperinsulinism responsible for both premature pubarche and later in life for PCOS (58). In vitro studies, however, failed to find evidence for hyperphosphorylation of the insulin receptor-b chain and P450c17 in PCOs (58). The reason for this might be related to the many different factors needed for P450c17 optimal activity and not normally expressed in the cell line used for that study (59).

BIOCHEMISTRY

The steroid hormones produced by the adrenal cortex are members of a large family of compounds derived from the cyclopentanoperhydrophenanthrene ring structure that comprises three cyclohexane rings and one cyclopentane ring. Three 19-carbon compounds are the principal androgens secreted by the adrenals: DHEA, DHEA-S, and D⁴-A (Fig. 3). Production of testosterone by these glands is minimal. Although quantitatively DHEA-S is the major product of the adrenal, this steroid per se has extremely weak androgenic activity.

Figure 3. Adrenal androgen biosynthetic pathway. Abbreviations: CYP11A1, cholesterolmside-chain cleavage enzyme; desmolase; CYP17, 17a-hydroxylase/17,20-lyase; 3b-HSD, 3b-hydroxysteroid dehydrogenase; CYP21A2, 21-hydroxylase; CYP11B1, 11b-hydroxylase; CYP11B2, aldosterone synthase, corticosterone 18-methylcorticosterone oxidase/lyase.

CIRCULATION

Circulating steroid hormones are largely bound to plasma proteins (binding globulins and albumin). About 90% of DHEA, DHEA-S, and D⁴-A is bound to albumin and 3% is bound to sex hormone-binding globulin (SHBG). The binding globulins have high affinity and low capacity, whereas, albumin has low affinity and high capacity for steroids.

ANDROGEN RECEPTOR

The inactive androgen precursors secreted by the adrenal glands are converted to T and 5a-dihydrotestosterone (DHT) and exert their effects in most peripheral tissues by interacting with high-affinity receptor proteins. The androgen receptors (AR), members of the steroid receptor superfamily, are encoded by the AR gene located on the X-chromosome (60). This gene contains a polymorphic CAG microsatellite repeat within exon 1, encoding for a variable length of polyglutamine chain at the aminoterminal, the transactivation domain of the AR protein. Triplet-repeat DNA sequences can be sites of genetic instability, and their expansion in a variety of genes has been associated with human genetic diseases, such as fragile X-syndrome (61;62) and myotonic dystrophy (63). In the case of the AR gene, an inverse correlation of the number of CAG repeats with the risk for prostate cancer was described (64-67), and its expansion was documented in Kennedy's disease (spinal and bulbar muscular atrophy), a disorder associated with primary hypogonadism (68). In vitro studies showed that progressive expansion of the repeat length in the AR was associated with a linear decrease in its transactivation function (69). These observations support the idea that there is an optimal number of repeats, which varies in the population from 11 to 31 (average size: 21 + 2) (64).

Methylation of deoxycytosine residues is another process involved in the modulation of gene expression. Belmont et al. (70) showed that the methylation of HpaII and HhaI sites near the polymorphic CAG repeats in the first exon of the human AR (HUMARA) locus correlated with X-inactivation.

Patients with idiopathic hirsutism were shown to have a normal number of CAG repeats but with a preponderance of the shortest and most active alleles (71). These patients had also a preferential methylation of the longer AR allele compared to normal subjects, leading to inactivation of the functionally weaker gene. This skewing could allow the shorter, more active AR allele (65; 69) to be preferentially expressed explaining the peripheral hypersensitivity to androgens in hirsute patients.

Multiple "coactivators" were identified enhancing transcription of the AR gene (72), including AP-1 (73), Smad3 (74;75), nuclear factor kB (NF-kB) (76;77), sex-determining region Y (SRY) (78), and the Ets family of transcription factors (79). The relative importance of these molecules for any particular cell type remains unclear, since the ability of a putative coregulator to alter the transcriptional activity is typically examined in transient transfection experiments. Although AR is normally thought to function as a homodimer, it was also shown to heterodimerize with other nuclear receptors including the estrogen receptor (ER) (80), glucocorticoid receptor (GR) (81) and testicular orphan receptor 4 (TR4) (82). One of the major mechanisms through which coregulators might function is by forming a bridge between the DNA-bound nuclear receptor and the basal transcriptional machinery (type I regulators) (83). Coactivators may also facilitate ligand binding, promote receptor nuclear translocation, or mediate signal transduction (type II coregulators).

The role of "corepressors" in AR function is poorly defined. Three corepressors of androgen-bound AR have been identified to date, cyclin D1, calreticulin, and HBO1. However, relatively little is known about the mechanism of their repressive effect.

PERIPHERAL CONVERSION AND METABOLISM

DHEA, DHEA-S, and D⁴-A are converted to the potent androgens T and DHT in peripheral tissues. (Fig. 4). Major conversions are those of D⁴-A to T and T to DHT by the enzymes 17beta -hydroxysteroid dehydrogenase (17b-HSD) and 5a-reductase, respectively. Major peripheral sites of androgen conversion are the hair follicles, the sebaceous glands (Fig.4A), the prostate, and the external genitalia (84;85).

Figure 4. Metabolism of adrenal androgens in the pilosebaceous unit (A) and adipocyte tissue (B). Abbreviations: A, D4-androstenedione; T, testosterone; DHT, dihydrotestosterone; R, receptor; 3b-HSD, 3b-hydroxysteroid dehydrogenase; 17b-HSD, 17b-hydroxysteroid dehydrogenase; 5a-r, 5a-reductase; 3a-, 3b-diol, 3a-androstenediol, 3b-androstenediol; 3a-, 3b-diol-G, 3a-diol-glucuronide, 3b-diol-glucuronide; androsterone-G, androsterone glucorunide; ar, aromatase; E1, estrone; E2, estradiol-17b.

Active uptake of androgens and in situ estrogen synthesis occur in peripheral adipose tissue (Fig. 4B). through the enzymes 17b-HSD and aromatase, respectively (86-88). Peripheral conversion contributes significantly to circulating T levels in women, but not in men, in whom T is largely produced by the testis.

The AAs and their metabolites are inactivated or degraded in various tissues, including the liver and kidneys (89). Major biochemical routes for inactivation and excretion are conjugation of androgens to glucuronate or sulfate residues to produce hydrophilic glucuronides or sulfates that are excreted in the urine (Fig. 4A).

BIOLOGIC EFFECTS

In adult men, the conversion of adrenal D⁴-A to testosterone accounts for less than 5% of the production rate of the latter, making its participation in the physiologic androgenization of the male negligible. Excessive AA secretion appears to have no major clinical consequences in the adult man, although this may be debated. AA hypersecretion in prepubertal boys, on the other hand has clearly been associated with isosexual precocious puberty.

In adult women, adrenal D⁴-A and D⁴-A generated from peripheral conversion of DHEA contribute substantially to total androgen production and effect. In the follicular phase of the menstrual cycle, adrenal precursors account for two thirds of testosterone production and half of dihydrotestosterone production. In the midcycle, the ovarian contribution increases, and the adrenal precursors account for 40% of testosterone production. In women, increased AA production may be manifested as cystic acne, hirsutism, male type baldness, menstrual irregularities, oligoovulation or anovulation, infertility, and/or frank virilization. Excessive adrenal androgen secretion in prepubertal or pubertal girls can cause heterosexual precocious puberty.

Both gonadal and AAs contribute to the positive impact of androgenic steroids on bone cell metabolism in vitro (90). Interestingly, a recent study found that the potential anabolic effect of androgens on bone might not be mediated at the level of the mature osteoblast but at the level of fetal, less differentiated, osteoblastic cell lines (91).