At the eighth week of human gestation, the fetal adrenal cortex comprises two morphologically distinct layers: an outer definitive zone (Dz) and an inner fetal zone (Fz) [6]. The Dz is thin and contains small basophilic cells, whereas the Fz is thick and contains large eosinophilic cells (Fig. 2.3). The Dz does not synthesize significant amounts of steroid hormones, but the Fz produces large quantities of DHEA and its sulfated counterpart DHEA-S. Cells of the Fz express CYP17A1, a dual function enzyme that catalyzes both a 17α-hydroxylation reaction and a 17,20-lyase reaction required for C₁₉ steroid production [1]. The lyase reaction is selectively enhanced through allosteric interactions with cytochrome b₅ (CYB₅), a protein that is abundant in the Fz [1]. A third cortical zone, the transitional zone (Tz), develops shortly after the appearance of the Fz and Dz. The Tz secretes cortisol, a hormone that promotes maturation of the lungs and other organs [8].

C₁₉ steroids secreted by the Fz are converted into estrogens through the actions of enzymes in the liver and/or placenta. The fetal pituitary, adrenal, liver, and placenta constitute a functional entity known the feto-placental unit [9] (Fig. 2.4). The concentration of estrogens in maternal plasma increases abruptly mid-gestation, reflecting production by this unit [10]. Estrogens support pregnancy by promoting maternal breast development, blood volume expansion, and uterine growth/contractility, although intact fetal adrenocortical function is not a prerequisite for term gestation or birth [11].

Adrenocorticotropic hormone (ACTH), a peptide secreted by the anterior pituitary gland, is a major regulator of fetal adrenal growth and function. ACTH promotes the production of both C₁₉ steroids and cortisol in the fetal adrenal. Disruption of hypothalamic/pituitary function (e.g., in the anencephalic fetus) impairs Fz growth and decreases estrogen levels in the maternal circulation [8].

Another important regulator of steroidogenesis in the fetus is placenta-derived corticotropin-releasing hormone (CRH), a peptide that both directly and indirectly stimulates fetal adrenal cells to produce cortisol and androgens [12, 13] (Fig. 2.4). Unlike hypothalamic CRH secretion, which is subject to feedback inhibition by glucocorticoids (see later), placental CRH release is enhanced by cortisol. At birth the placenta separates from the body, and this CRH feed-forward loop is interrupted. The newborn adrenal gland, which is almost as large as the kidney, shrinks dramatically over the first 2 weeks of life owing to apoptotic involution of the Fz . Regression of the Fz is accompanied by a reduction in C₁₉ steroid production [1]. Postnatally, the Dz differentiates into the anatomically and functionally distinct zones of the adult cortex.

The adult adrenal cortex of humans contains three layers: the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR) (Fig. 2.5) [4, 14]. The cortex is enveloped by a capsule that provides structural support and houses stem/progenitor cells of the cortex. A complex network of blood vessels within the adrenal gland ensures the efficient delivery of stimulants and the export of corticoids into the circulation [15, 16]. Blood flows centripetally in the gland, so inner cortical zones and the adrenal medulla are exposed to high concentrations of locally-produced steroids.

Rodents are used as experimental models for the study of adrenal physiology, but there are noteworthy differences between the adrenocortical zonation patterns of rodents and humans [30]. The zR is absent from the adrenal gland of the mouse. The adrenal cortex of the young mouse contains an ephemeral juxtamedullary layer, the X-zone, that regresses during puberty in males and the first pregnancy in females [31]. Lineage tracing studies have shown that the X-zone is a derivative of the fetal cortex [31], but the physiological function of the X-zone remains unclear [32]. The rat adrenal cortex has an additional layer, the undifferentiated zone (zU), located between the zG and zF. The zU contains a transitional population of cells [33].

Species also differ in the complement of steroidogenic enzymes and cofactors expressed in the adrenal cortex, and these differences have biological ramifications. For example, cells in the zF and zR of humans express CYP17A1, so cortisol is the principal glucocorticoid secreted by the adrenal gland of this species [34]. Cells in the zF of adult mice and rats lack CYP17A1, so corticosterone is the main glucocorticoid produced, and adrenal androgens are not made. A newly recognized rodent-like model is the spiny mouse (genus Acomys) [35]. In contrast to the laboratory mouse (genus Mus), the adrenal cortex of the spiny mouse has a zR. Cells in the zF and zR of the spiny mouse produce cortisol and DHEA, respectively. Thus, the adrenal physiology of the spiny mouse recapitulates that of humans [9].

Cholesterol can be derived from a combination of sources: (1) de novo biosynthesis, (2) import of lipoprotein-associated cholesteryl esters (CEs) via endocytosis of the low-density lipoprotein receptor (LDLR), (3) uptake of esterified cholesterol through the high-density lipoprotein (HDL) scavenger receptor 1 (SR-B1), and (4) hydrolysis of CEs stored with lipid droplets. These redundant sources ensure that adequate cholesterol is available for steroidogenesis.

Excess-free choleste rol, which is toxic to cells, may be esterified by the ER enzyme sterol O-acyltransferase 1 (SOAT1) [also called acyl-CoA:cholesterol O-acyltransferase 1 (ACAT1)]. The resultant CEs may be incorporated into lipid droplets for storage. Drugs that inhibit SOAT1 activity, such as mitotane and ATR-101, induce the accumulation of cholesterol and free fatty acids in the ER of adrenocortical cells (Fig. 2.9) [78–80]. This increase in cholesterol limits SREBP activation, resulting in downregulation of target genes that mediate cholesterol synthesis and uptake. The accumulation of cholesterol and free fatty acids induces ER stress, which triggers transcription of unfolded protein response (UPR) genes [81]. Persistent ER stress leads to upregulation of pro-apoptotic BAX and repression anti-apoptotic BCL2, thus inducing cell death [79]. Targeted deletion of SOAT1 in an adrenocortical cell line recapitulates the effects of pharmacological inhibitors of SOAT1 [78]. Inhibition of cholesterol esterification is thought to be the mechanism of action of mitotane, the only drug approved for the treatment of patients with adrenocortical carcinoma (ACC) (Fig. 2.9). ATR-101 is undergoing preclinical and clinical testing as a new drug for the treatment of ACC [78].

Mitochondria contain a limited amount of cholesterol, the majority of which resides in the OMM [82, 83]. Therefore, cholesterol consumed during steroidogenesis must be continually replenished. Mitochondrial stores cannot be restocked with cholesterol via simple diffusion across aqueous cytoplasmic spaces, because this compound is relatively insoluble in water. Instead, “free” cholesterol is trafficked to mitochondria via two general mechanisms: (1) vesicular transport, an energy-dependent process entailing budding and fusion of membrane vesicles, and (2) non-vesicular transport via soluble cholesterol-binding proteins [61, 71]. Close physical interactions between mitochondria and other subcellular compartments (e.g., ER, lipid droplets) promote cholesterol transfer.

Once cholesterol reaches the OMM, a group of proteins led by steroidogenic acute regulatory protein (StAR) facilitate the transport of cholesterol to the IMM, the site of pregnenolone synthesis by CYP11A1. The delivery of cholesterol from the OMM to the IMM is the rate-limiting step in steroid production in adrenocortical cells. Hormones, such as ACTH and Ang II, rapidly stimulate this process. Transport occurs preferentially at sites of close contact between the OMM and IMM, and such sites are more plentiful in tubulovesicular than lamelliform mitochondria. The preponderance of tubulovesicular mitochondria in the zF compared to the zG reflects higher levels of steroidogenesis in the former [23].

ACTH , a 39-amino acid peptide secreted by the anterior pituitary, is the principal stimulus for cortisol production [24]. When the HPA axis is activated by stress or other stimuli, neurons in the hypothalamic paraventricular nucleus secrete CRH and arginine vasopressin (AVP) into the hypophyseal portal circulation [145, 146]. CRH is the main stimulant of ACTH release. AVP acts synergistically with CRH to enhance ACTH secretion, although AVP is ineffective in the absence of CRH [147].

ACTH is derived from pro-opiomelanocortin (POMC), a 214-amino acid precursor synthesized in the anterior pituitary and limited other sites [148]. POMC can be proteolytically processed to generate ACTH and several other peptides, including α-melanocyte-stimulating hormone (α-MSH) and β-endorphin [149]. The N-terminal 18 amino acids of ACTH confer its biological activity, so shorter synthetic peptides [ACTH (1–24) and ACTH (1–18)] are used clinically in lieu of full-length ACTH. The sequence of α-MSH, which is contained within the ACTH peptide, stimulates the production of melanin by melanocytes, resulting in skin hyperpigmentation when secreted in excess, as in the ACTH-dependent Cushing syndrome.

ACTH promotes steroidogenesis at three levels: (1) acute ACTH exposure rapidly (within minutes) stimulates the transport of cholesterol from the OMM to the IMM, (2) longer term exposure to ACTH (hours) promotes transcription of genes encoding steroidogenic enzymes, notably CYP11A1, and (3) prolonged exposure to ACTH (weeks to months) promotes zonal expansion and adrenal growth [126, 150].

Cortisol is the main negative regulator of the HPA axis (Fig. 2.6). Feedback inhibition by cortisol is mediated via binding to GRs in the pituitary and hypothalamus. Cortisol also acts on suprahypothalamic centers (e.g., hippocampus) to further limit the secretion of CRH [151]. Feedback inhibition in suprahypothalamic centers is mediated by cortisol binding to GRs and MRs [152].

ACTH is secreted in regular pulses of varying amplitude. Peak ACTH secretion occurs in the early morning, coinciding with a rise in cortisol secretion [153]. The diurnal production of cortisol, which anticipates times of increased activity and energy demand, reflects an interplay between environmental cues (e.g., fluctuations in light intensity) and an internal circadian timekeeping system [154]. Disruption of this interplay contributes to the phenomenon of jetlag [155].

The circadian timekeeping system is composed of a central clock [the hypothalamic suprachiasmatic nucleus (SCN)] and subsidiary peripheral clocks in nearly every cell type, including adrenocortical cells [154]. The SCN is entrained to the light-dark cycle [156]. Highlighting the importance of the central clock, the diurnal glucocorticoid rhythm is altered by lesions of the SCN [157] or constant light exposure [158]. Signals from the SCN are relayed via the sympathetic nervous system to peripheral clocks in the adrenal cortex (Fig. 2.12). The rhythmic secretion of glucocorticoids from the adrenal gland is thought to synchronize peripheral circadian rhythms of tissues downstream of the SCN [155, 159].

The central and peripheral circadian clocks are composed of interlocking positive and negative transcriptional-translational feedback loops that oscillate with a periodicity of approximately 24 hours [154]. Genetically-engineered mice lacking of key components of the molecular clock exhibit either chronic elevation or suppression of glucocorticoid levels; rhythmic expression of certain steroidogenic genes, including Star , is altered in the adrenocortical cells of these animals [160–165]. Peripheral clocks in the adrenal cortex are thought to modulate sensitivity to ACTH, although the mechanistic basis for this effect is unclear.

The trophic effects of ACTH are mediated through its plasma transmembrane G-protein-coupled receptor MC2R (Fig. 2.13). Melanocortin 2 receptor accessory proteins (MRAPs) are small membrane proteins that are essential for ACTH signaling. MRAP deficiency is one of the causes of hereditary unresponsiveness to ACTH [166]. Two types of MRAPs have been identified: MRAP1 and MRAP2. In humans, there are two distinct isoforms of MRAP1, termed MRAPα and MRAPβ, which share the same N-terminus and transmembrane domain but differ in their C-terminal domains. MRAP1 isoforms are involved in intracellular trafficking and signaling of MC2R. MRAPα has been implicated in targeting of MC2R to the plasma membrane, whereas MRAPβ appears to enhance cAMP productio n by ACTH-bound MC2R [153]. MRAP2 also is involved in trafficking of MC2R to the plasma membrane.

Binding of ACTH to MC2R causes the α-subunit of the stimulatory heterotrimeric G-protein G_s to associate with adenylate cyclase (AC), resulting in cAMP production [167]. This rise in cAMP enhances lipid synthesis, protein synthesis, and protein phosphorylation, all of which promote cholesterol trafficking to mitochondria and the production of steroid hormones [167]. Most of these effects are mediated by PKA. cAMP binds to the regulatory subunits of PKA, allowing the catalytic subunits of PKA to phosphorylate downstream effectors. For example, PKA-induced phosphorylation and activation of HSL enhances free cholesterol formation, while phosphorylation of StAR promotes transport of cholesterol from the OMM to the IMM [111]. PKA-dependent phosphorylation enhances CE uptake by enhancing the transcription and stability of SR-B1 [70]. PKA also phosphorylates transcription factors, leading to enhanced expression of steroidogenic genes such as CYP11A1 [167]. The transcription factors phosphorylated include steroidogenic factor 1 (SF1), cAMP response element-binding protein (CREB), and activator protein 1 (AP1) [150]. PKA-mediated phosphorylation of L-type Ca²⁺ channels activates a slow but sustained influx of Ca²⁺ that triggers additional signaling pathways.

Loss-of-function mutations in the protein kinase A regulatory subunit gene (PRKAR1A) cause excessive cAMP production. Such mutations underlie Carney complex, a syndrome associated with the pituitary-independent Cushing syndrome and adrenocortical neoplasia. Conditional deletion of Prkar1a in the adrenal cortex of mice leads to not only excess glucocorticoid production but also impaired apoptosis [168], mediated in part by crosstalk between the PKA and mammalian target of rapamycin (mTOR) pathways [169].

Somatic gain-of-function mutations in the PRKACA gene, which encodes the catalytic subunit of PKA, cause the ACTH-independent Cushing syndrome due to cortisol-producing adenomas [170, 171]. Germline duplications of this gene can cause bilateral adrenal hyperplasia [170].

Although the initial and most significant actions of ACTH are mediated through cAMP and the subsequent activation of PKA, there are also PKA-independent effects of cAMP, such as those mediated by Epac (the Exchange protein directly activated by cAMP) [172, 173]. One Epac isoform, Epac2, is highly expressed in the adrenal cortex and functions to activate Rap GTPases [174]. Epac proteins regulate various cellular processes via mechanisms that include modulation of gene expression and cytoskeletal rearrangements [153, 173, 174]. Treatment with an Epac-selective analogue of cAMP is sufficient to stimulate the expression of steroidogenic enzymes and cortisol secretion [175].

The level of intracellular cAMP is determined not only by its rate of synthesis by AC but also its rate of degradation by phosphodiesterases (PDEs) [176]. In mammals there are 11 families of PDEs, each with distinctive properties [177]. In mice, treatment with a PDE-selective inhibitor increases phosphorylation of HSL and basal corticosterone secretion [178]. Loss-of-function mutations in PDE8B or PDE11A have been linked to bilateral adrenal hyperplasia and Cushing syndrome in humans [176, 178].

Hydrolysis of cAMP by PDEs produces AMP, which in turn stimulates the AMP-activated protein kinase (AMPK) [179]. Activated AMPK represses the expression of transcription factors known to stimulate steroidogenesis (e.g., NUR77 and cJUN) and activates the expression of repressors of steroidogenesis (e.g., DAX1 and cFOS) [180] (Fig. 2.14).

ACTH stimulation induces the release of arachidonic acid (AA) from phospholipid stores, and this fatty acid and its metabolites have been shown to enhance steroidogenesis [181, 182]. Intracellular levels of free AA are tightly regulated by not only phospholipases, such as phospholipase A₂ (PLA₂), but also by acyl-CoA synthetases and thioesterases. Most acyl-CoA synthetases exhibit a broad specificity with respect to fatty acid substrate, but acyl-CoA synthetase 4 (ACSL4) prefers AA as a substrate [183, 184]. Originally characterized as an activity in platelets [185], ACSL4 is highly expressed in steroidogenic tissues including the adrenal gland and gonads [183, 186]. The expression of Acsl4 mRNA is upregulated through a cAMP-dependent pathway , and pharmacological inhibition of ACSL4 reduces steroidogenesis in adrenocortical cell lines [187]. The hormonal regulation of steroid synthesis requires the concerted action of ACS4 and a second enzyme, mitochondrial acyl-CoA thioesterase 2 (ACOT2), which cleaves arachidonoyl-CoA into its component parts. These two enzymes constitute an AA generation/export system, which releases AA in mitochondria in response to ACTH stimulation [188]. AA is then metabolized into lipoxygenated or epoxygenated products that induce the expression of StAR [182, 187, 188].

ACTH triggers a transient increase in total protein tyrosine phosphatase (PTP) activity in zF cells and a concomitant decrease in the phosphotyrosine level of several proteins [189]. Treatment with PTP inhibitors reduces hormone-induced stimulation of steroidogenesis [153], and PTPs have been implicated in the regulation of StAR induction and cholesterol transport to the IMM [189]. One particular PTP, PTPN11, has been shown to regulate the expression of ACSL4 [190].

MAPKs have also been implicated in the action of ACTH [24]. This kinase family comprises the extracellular signal-related kinases ERK1/2 (p42/p44^mapk), the p38 MAPKs, and the p54 stress-activated JNK protein kinases [191]. MAPK targets in steroidogenic cells include HSL [76] and CYP17A1 (see later).

JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling is also activated by ACTH. Pharmacological or siRNA-mediated inhibition of JAK2 impairs ACTH-induced steroidogenesis [192]. Nuclear JAK2 regulates the amount of active CREB through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional upregulation of StAR [192].

Although ACTH is the principal stimulus for glucocorticoid secretion , there are several ACTH-independent mechanisms that regulate cortisol release from the adrenal cortex. A variety of neuropeptides, neurotransmitters, and cytokines bind receptors on the surface of zF cells and modulate glucocorticoid secretion [193]. Additionally, factors secreted by endothelial cells and adipocytes influence the secretion of adrenal steroids [194, 195]. The close anatomical relationship among adrenocortical cells, medullary chromaffin cells, and nerve endings facilitates paracrine modulation of adrenal secretion of glucocorticoids [193].

In addition to regulating cortisol secretion in zF cells, ACTH can induce aldosterone production in the zG. As in the zF, ACTH acts on zG cells by binding to its receptor, MCR2, and activating AC via G_s [17]. This leads to increased cAMP/PKA signaling, which facilitates the movement of cholesterol from the OMM to the IMM and activates transcription of key steroidogenic genes. Binding of ACTH to its receptor also impacts the electrical properties of zG cells [24].

The zG controls extracellular fluid volume and salt balance as part of the renin-angiotensin-aldosterone system [17, 23]. Renin is a protease secreted by the juxtaglomerular apparatus of the kidney in response to extracellular fluid depletion, low sodium concentrations, or hypotension. Renin cleaves angiotensinogen, a glycoprotein constitutively secreted into the serum by the liver, yielding the decapeptide angiotensin I (Ang I). Angiotensin-converting enzyme (ACE), a protein expressed on the surface of pulmonary and renal endothelial cells, subsequently converts Ang I into to the vasoactive octapeptide Ang II.