When extracting steroids from the adrenal Kendall and Reichstein identified 28 separate steroids and today we classify the steroids produced by the adrenal glands, the corticosteroids, in three major classes— glucocorticoids (cortisol, corticosterone), mineralocorticoids (aldosterone, deoxycorticosterone), and adrenal sex steroid precursors (dehydroepiandrosterone (DHEA), androstenedione).
Cholesterol is the precursor for all adrenal steroidogenesis. The principal source of cholesterol is provided from the circulation in the form of low- density lipoprotein (LDL) cholesterol. Uptake is by specific cell- surface LDL receptors present on adrenal tissue; LDL is then internalized via receptor- mediated endocytosis, the resulting vesicles fuse with lysozymes, and free cholesterol is produced following hydrolysis. However, it is clear that this cannot be the sole source of adrenal cholesterol as patients with abetalipoproteinaemia, who have undetectable circulating LDL, and patients with defective LDL receptors in the setting of familial hypercholesterolaemia still have normal basal adrenal steroidogenesis. Cholesterol can be generated de novo within the adrenal cortex from acetyl coenzyme A. In addition, there is evidence that the adrenal can utilize high- density lipoprotein (HDL) cholesterol following uptake through the HDL receptor, scavenger receptor.
The biochemical pathways involved in adrenal steroidogenesis start with the rate- limiting step of the transport of intracellular cholesterol from the outer to the inner mitochondrial mem brane. Within the mitochondrion cholesterol is then converted to pregnenolone by the cholesterol side chain cleavage enzyme, cytochrome P450scc (CYP11A1). The rapid transport of cholesterol into the mitochondria is importantly facilitated by steroidogenic acute regulatory protein, which is induced by an increase in intra cellular cAMP following binding of adrenocorticotrophic hormone (ACTH) to its receptor.
Steroidogenesis involves the concerted action of several en zymes, including a series of cytochrome P450 (CYP) enzymes (for schematic overview). CYP11A1 and the CYP11B1 and CYP11B2 enzymes are localized to the mitochondria and require an electron shuttle system— provided through adrenodoxin/ adrenodoxin reductase— for functional activity. Other CYP enzymes involved in steroidogenesis, namely 17α- hydroxylase (CYP17A1) and 21- hydroxylase (CYP21A2), are localized to the microsomal/ endoplasmic reticulum fraction and depend on electron transfer from nicotinamide adenine dinucleotide phosphate (NADPH) via the electron donor enzyme P450 oxidoreductase (POR).
After the uptake of cholesterol to the mitochondrion, side chain cleavage of cholesterol by CYP11A1 forms pregnenolone, which is converted in the cytoplasm to progesterone by the type 2 isoenzyme of 3β- hydroxysteroid dehydrogenase. Progesterone is hydroxylated to 17- hydroxyprogesterone (17OHP) through the activity of 17α- hydroxylase. 17- hydroxylation is an essential prerequisite for glucocorticoid synthesis. CYP17A1 also possesses 17,20 lyase activity, which crucially facilitates the synthesis of the sex steroid pre cursor DHEA, a reaction that also requires allosteric interaction of the flavoprotein cytochrome b5 with both CYP17A1 and POR. In humans, 17OHP is not an efficient substrate for CYP17A1, and there is negligible conversion of 17- OH progesterone to androstenedione. Adrenal androstenedione secretion is dependent upon the conversion of dehydroepiandrosterone to androstenedione by 3β- hydroxysteroid dehydrogenase (3β- HSD). 21- hydroxylation of either progesterone (zona glomerulosa) or 17OHP (zona fasciculata) is carried out by 21- hydroxylase (CYP21A2) to yield deoxycorticosterone or 11- deoxycortisol, respectively. The final step in cortisol biosynthesis takes place in the mitochondria and involves the con version of 11- deoxycortisol to cortisol by 11β- hydroxylase activity of the enzyme CYP11B1. In the zona glomerulosa, 11β- hydroxylase may also convert deoxycorticosterone to corticosterone. However, the enzyme CYP11B2, or aldosterone synthase, may also carry out this reaction and, in addition, is required for the conversion of corticosterone to aldosterone via the intermediate 18- OH corticosterone. Thus, CYP11B2 can carry out 11β- hydroxylation, 18- hydroxylation, and 18- methyl oxidation to yield the characteristic C11– 18 hemiacetal structure of aldosterone.
Cortisol is inactivated to cortisone by action of the enzyme 11β- hydroxysteroid dehydrogenase type 2 (HSD11B2) mainly in the kidney, while the opposite reaction, activation of cortisone to cortisol, is carried out by the type 1 isoenzyme, HSD3B1, mainly in the liver (Figure 1). Recent years have highlighted the important role of this system in the tissue- specific activation and inactivation of glucocorticoids. Without the action of hepatic HSD11B1, Kendall would have observed no activity of his ‘Compound E’, as cortisone does not bind the glucocorticoid receptor and conversion to cortisol by HSD11B1 is a mandatory requirement for its biological activity.
Fig1. Schematic representation of adrenal zonation and steroidogenesis, depicting histology of the three adrenocortical and the major corticosteroids and the receptors mediating their action. While cortisol and aldosterone can bind and activate the glucocorticoid and mineralocorticoid receptor, respectively, DHEA requires conversion to active androgens and further aromatization to oestrogens prior to exerting sex steroid action. (See also Plate 29.)
Glucocorticoids are secreted in relatively high amounts (cortisol 10– 20 mg/ day) from the zona fasciculata, while mineralocorticoids are secreted in low amounts (aldosterone 100– 150 µg/ day) from the zona glomerulosa. The adrenal androgen precursors DHEA, its sulphate ester dehydroepiandrosterone sulphate (DHEAS), and androstenedione are produced in the adrenal zona reticularis and represent the most abundant steroids secreted by the adult adrenal gland (>20 mg/ day). Recent work has shown that androstenedione is effectively converted to 11- hydroxyandrostenedione (11OHA4) by CYP11B1 and 11OHA4 is a major product of adrenal steroidogenesis, as measured in adrenal vein blood. Adrenal zonal- specific steroidogenesis of the three different corticosteroid classes is facilitated by tissue- specific expression of the required steroidogenic enzymes. The zona glomerulosa generally does not synthesize cortisol because it does not express 17α- hydroxylase. In contrast, aldosterone secretion is largely confined to the outer zona glomerulosa through the restricted expression of CYP11B2. Although CYP11B1 and CYP11B2 share 95% homology, the 5′ promoter sequences differ and permit regulation of the final steps in glucocorticoid and mineralocorticoid biosynthesis by ACTH and angiotensin II, respectively. DHEA is sulphated in the zona reticularis by the DHEA sulphotransferase (SULT2A1) to form DHEAS.
Regulation of Adrenal corticosteroid Synthesis
Classical endocrine feedback loops are in place to control the secretion of both cortisol and aldosterone. Cortisol inhibits the secretion of both hypothalamic corticotrophin- releasing factor and pituitary ACTH, and the aldosterone- mediated increase in sodium retention inhibits renin secretion by the juxta- glomerular cells of the kidney (Figure 2).
Fig2. Negative feedback regulation of cortisol and aldosterone secretion. (a) Glucocorticoid feedback regulation by the hypothalamic– pituitary– adrenal (HPA) axis. CRH, corticotropin- releasing hormone; ACTH, adrenocorticotropic hormone; ADH, antidiuretic hormone. (b) Mineralocorticoid regulation by the renin– angiotensin– aldosterone system (RAAS). The extracellular fraction (ECF) of potassium has an important direct influence on aldosterone secretion. ACE, angiotensin- converting enzyme; ANP, atrial natriuretic peptide. Schematic graph: Dr Nils Krone, Birmingham
Glucocorticoid synthesis is under negative feedback control of the hypothalamic– pituitary– adrenal (HPA) axis (Figure 2a). Adrenocorticotropic hormone (ACTH) secretion from the anterior pituitary is stimulated by hypothalamic corticotrophin- releasing hormone (CRH) following a circadian rhythm, with a peak around 03.00h to 04.00h. Other major effectors on CRH secretions are various forms of stress, including hypoglycaemia, hypo tension, fever, trauma, and surgery. ACTH binds to its receptor (melanocortin receptor 2, MC2R) on the adrenocortical cell sur face and stimulates import of cholesterol into the mitochondrion by steroidogenic acute regulatory protein. In parallel, transcription of genes encoding steroidogenic enzymes and proteins of the electron transfer shuttle is increased.
Mineralocorticoid synthesis is mainly under the control of the renin– angiotensin– aldosterone system (RAAS) and a potassium feedback loop (Figure 2b). A variety of factors stimulate renin secretion from renal juxtaglomerular cells, with renal perfusion being the most important regulator. Several other stimulators (β- adrenergic stimulation, prostaglandins) and inhibitors (α- adrenergic stimulation, dopamine, atrial natriuretic peptides, angiotensin II) are known. Angiotensinogen is an α2- globulin synthesized within the liver which is cleaved by renin to form angiotensin I. Angiotensin I is converted to angiotensin II by angiotensin- converting enzyme in the lung and many other peripheral tissues. Angiotensin I has no apparent biological activity but angiotensin II is a potent stimulator of aldosterone secretion. In addition, angiotensin II acts as a potent vasoconstrictor. The rate- limiting step in the RAAS is the secretion of renin, which is also controlled through a negative feedback loop. Renin is secreted from juxtaglomerular epithelial cells within the macula densa of the renal tubule in response to underlying renal arteriolar pressure, oncotic pressure, and sympathetic drive. Thus, low perfusion pressure and/ or low tubular fluid sodium content, as seen in haemorrhage, renal artery stenosis, dehydration, or salt loss, increase renin secretion. Conversely, secretion is suppressed following a high salt diet and by factors that increase blood pressure. Hypokalaemia increases and hyperkalaemia decreases renin secretion; in addition, potassium exerts a direct effect upon the adrenal cortex to increase aldosterone secretion. Angiotensin II and potassium stimulate aldosterone secretion principally by increasing the transcription of CYP11B2 through common intracellular signalling pathways. The potassium effect is mediated through membrane de polarization and opening of calcium channels, and the angiotensin II effect following binding of angiotensin II to the surface AT1 receptor and activation of phospholipase C.
The separate control of glucocorticoid biosynthesis through the HPA axis and mineralocorticoid synthesis via the renin– angiotensin system has important clinical consequences. The overwhelming majority of patients with primary adrenal failure have both cortisol and aldosterone deficiency, whereas patients with ACTH deficiency due to pituitary disease have glucocorticoid deficiency only, as the intact renin– angiotensin system maintains normal aldosterone levels.
corticosteroid Hormone Action
Both cortisol and aldosterone exert their effects following uptake of free hormone from the circulation and binding to intracellular receptors, termed the glucocorticoid and mineralocorticoid receptors (GR, MR). These are both members of the thyroid/ steroid hormone– receptor superfamily of transcription factors, comprising a C- terminal ligand binding domain, a central DNA binding domain, interacting with specific DNA sequences on target genes, and an N- terminal hypervariable region. In both cases, although there is only a single gene encoding the GR and MR, splice variants have been described resulting in α and β variants.
The binding of glucocorticoid to the GR- α in the cytosol results in activation of the steroid– receptor complex through a process which involves the dissociation of heat- shock proteins HSP 90 and HSP 70. Following translocation to the nucleus, gene transcription is stimulated or repressed following binding of dimerized GR– ligand complexes to specific DNA sequences (glucocorticoid- response element) in the promoter regions of target genes. The GR- β variant may act as a dominant negative regulator of GR- α transactivation.
In contrast to the diverse actions of glucocorticoids, mineralocorticoids have a more restricted role, principally to stimulate epithelial sodium transport in the distal nephron, distal colon, and salivary glands. This is mediated through the induction of the apical sodium channel (comprising three subunits α, β, and γ) and the α1 and β1 subunits of the basolateral Na+K+- ATPase through transcriptional regulation of a specific aldosterone- induced gene that encodes serum and glucocorticoid- induced kinase. Aldosterone binds to the MR, principally in the cytosol (though there is evidence for expression of the unoccupied MR in the nucleus) followed by translocation of the hormone– receptor complex to the nucleus.
The MR and GR share considerable homology— 57% in the steroid binding domain and 94% in the DNA binding domain. Therefore, it is perhaps not surprising therefore that there is promiscuity of ligand binding with aldosterone binding to the GR and cortisol binding to the MR. For the MR this is particularly impressive— in vitro the MR has the same inherent affinity for aldosterone, corticosterone, and cortisol. Specificity upon the MR is conferred through the ‘prereceptor’ metabolism of cortisol via the enzyme HSD11B2 in the kidney, which inactivates cortisol and corticosterone to 11- keto metabolites that do not activate the MR, thereby enabling aldosterone to bind to the MR.
For both glucocorticoids and mineralocorticoids, there is accumulating evidence for so- called non- genomic effects involving hormone response obviating the genomic GR or MR effects. A series of responses have been reported within seconds/ minutes of exposure to corticosteroids and are thought to be mediated by, as yet uncharacterized, membrane- coupled receptors.
cortisol- Binding Globulin and corticosteroid Hormone Metabolism
Over 90% of circulating cortisol is bound, predominantly to the α2- globulin cortisol- binding globulin (CBG). This 383- amino acid protein is synthesized in the liver and binds cortisol with high affinity. Affinity for synthetic corticosteroids (except prednisolone, which has an affinity for CBG of approximately 50% of that of cortisol) is negligible. Circulating CBG concentrations are approximately 700 nmol/ L; levels are increased by oestrogens and in some patients with chronic active hepatitis but generally reduced in patients with cirrhosis, nephrosis, and hyperthyroidism. The oestrogen effect can be marked, with levels increasing two- to threefold across pregnancy, and this should also be taken into account when measuring plasma ‘total’ cortisol in pregnancy and in women taking oestrogens. Inherited abnormalities in CBG synthesis are much rarer than those described for thyroid- binding globulin but include patients with elevated CBG, partial and complete deficiency of CBG, or CBG variants with reduced affinity for cortisol. In each case, alterations in CBG concentrations change total circulating cortisol concentrations accordingly but ‘free’ cortisol concentrations are normal. Only this free circulating fraction is available for transport into tissues for biological activity. The excretion of ‘free’ cortisol through the kidneys is termed urinary- free cortisol and represents only 1% of the total cortisol secretion rate. Approximately 50% of secreted cortisol appears in the urine as tetrahydrocortisol (THF), 5alpha- tetrahydrocortisol (allo- THF), and tetrahydrocortisone (THE), 25% as cortols/ cortolones, 10% as C19 steroids, and 10% as cortolic/ cortolonic acids.
Aldosterone is also metabolized in the liver and kidneys. In the liver it undergoes tetrahydro reduction, followed by its excretion in the urine as a 3- glucuronide tetrahydroaldosterone derivative. However, glucuronide conjugation at the 18 position occurs directly in the kidney, as does 3α and 5α/ 5β metabolism of the free steroid. Because of the aldehyde group at the C18 position, aldosterone is not metabolized by HSD11B2. Hepatic aldosterone clearance is reduced in patients with cirrhosis, ascites, and severe congestive heart failure.
