Role of pituitary POMC-peptides and insulin-like growth factor II in the developmental biology of the adrenal gland

During fetal life, it is critical that there is coordinate regulation of the growth, zonation and differentiation of the fetal adrenal cortex to ensure that cells in key tissues and organs are exposed in a programmed temporal sequence to the actions of glucocorticoids. Glucocorticoids are essential for maturation of key target organs before birth, including the lung, brain, liver, gut, kidney and adrenal, and the prepartum increase in glucocorticoid synthesis and secretion by the fetal adrenal gland is critical for the successful transition to postnatal life. It is also evident that premature or abnormal exposure of embryonic or fetal tissues to glucocorticoids during critical windows of development can irreversibly alter the programmed development of organ systems. Premature or abnormal exposure of the fetus to excess glucocorticoids may occur either as a consequence of endogenous stimulation of the fetal hypothalamo-pituitary-adrenal axis (HPAA) or as a consequence of exposure to exogenous glucocorticoids in a therapeutic context. Administration of synthetic glucocorticoids to women at risk of preterm labour, for example, is a routine clinical practice designed to improve respiratory function and neonatal outcome. It is clearly important to understand what endogenous factors regulate the growth and functional maturation of the adrenal cortex during development and the consequent likelihood of exposure of developing tissues to excess corticosteroids. To date, investigations have centred on the role of ACTH 1-39 in the stimulation of adrenal growth and steroidogenesis in long gestation species, such as the primate and sheep, where maturation and differentiation of organ systems occurs predominantly before birth. In this review, we will focus on the evidence that in addition to ACTH 1-39, other pro-opio-melanocortin (POMC) derived peptides, which are synthesized, processed and secreted by the fetal pituitary, play a role in the coordinate regulation of the specific phases of growth and functional development of the fetal adrenal gland in vivo. We will discuss our recent findings on the direct in vivo actions of N-POMC 1-77 and separately, insulin like growth factor II (IGF-II), as adrenal growth factors. These studies provide an understanding of the separate regulatory mechanisms which control activation of adrenal growth and stimulation of adrenal steroidogenesis in the late gestation fetus.     

Corticosteroids, receptors, and the organ-specific functions of 11 beta-hydroxysteroid dehydrogenase.

Reversible oxidation of the biologically active corticosteroids to the inactive 11-dehydrocorticosteroids is catalyzed by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD). The properties of the enzyme based on clinical observations of individuals with defective 11 beta HSD expression, and laboratory studies of the properties and behavior of the enzyme, are consistent with separate 11 beta-dehydrogenase and 11-oxoreductase species. However, recombinant enzyme expressed in mammalian cells retain both activities, leading to the conclusion that 11 beta HSD is a unique, reversible enzyme. 11 beta HSD is present in most tissues, but its specific functions in most tissues are unknown. How the enzyme may mediate corticosteroid-receptor interaction is illustrated by studies using kidney, testis, and brain. In kidney, 11 beta HSD prevents glucocorticoids from competing inappropriately with aldosterone for mineralocorticoid receptor (MR). Lack of enzyme in humans due to natural causes or inhibition by pharmacological agents results in maximum activation of MR by glucocorticoids, leading to the clinical symptoms of apparent mineralocorticoid excess. Leydig cells of the testes synthesize testosterone, a process that is suppressed by events initiated by the binding of corticosteroid to glucocorticoid receptors (GR). Depletion of active steroid mediated by 11 beta HSD may initiate testosterone production at puberty and affect testosterone production during adult life, as for example during periods of stress. The heterogeneous distribution of MR and GR in the brain reflects the specific regional effects of glucocorticoids and mineralocorticoids on neural function. Colocalization of 11 beta HSD and corticosteroid receptors in brain may be important in controlling the specificity of corticosteroid interaction with GR and MR. The patterns of 11 beta HSD-steroid-receptor interaction illustrated with these three tissues may provide models applicable to other tissues in which corticosteroid receptors and 11 beta HSD coexist.     

Studies on arterial mineralocorticoid and glucocorticoid receptors: evidence for the translocation of steroid-cytoplasmic receptor complexes to cell nuclei

The high affinity binders for mineralocorticoids and glucocorticoids, previously reported by us as present in arterial cytosol have been further characterized. The results of this study demonstrate that these binders translocate under appropriate conditions to cell nuclei as complexes with mineralocorticoids and glucocorticoids, respectively. Thus, they exhibit a fundamental property of steroid receptors. This provides evidence for the presence in the arterial wall of a molecular mechanism(s) for the in-situ action of both mineralocorticoids and glucocorticoids.     

Identification of oxytocin and vasopressin in the testis and in adrenal tissue

Oxytocin, vasopressin and neurophysin-like immunoreactivity have been identified and measured by radioimmunoassay in extracts of human and rat testis and human fetal adrenal tissue. The authenticity of these polypeptides has been confirmed by their behaviour on high performance liquid chromatography. The concentrations of the hormone were too great to be explained by known circulating levels of the polypeptides, and their presence in steroid secreting organs suggests a possible role for them in steroidogenesis. The peptides may be taken up and concentrated by the tissues but the co-localisation of neurophysins with the hormones points towards local synthesis.     

Differences in Production of Adrenal Steroid Hormones in Pubertal Rats Exposed to Low Doses of the Endocrine Disruptor DDT during Prenatal and Postnatal Development

Production of adrenal steroid hormones in pubertal male Wistar rats exposed to low doses of DDT during both prenatal and postnatal and only postnatal development has been investigated. Rats exposed to the disruptor prenatally and postnatally, and only postnatally were characterized by opposite changes in production of mineralocorticoids, glucocorticoids, male and female sex hormones. The study revealed that daily exposure to low doses of DDT enhanced conversion of progesterone to 17-OH-progesterone and did not exert selective antiandrogenic or proestrogenic action typical for the effect of toxic and subtoxic doses. In rats, exposed to DDT during their prenatal and postnatal development, impaired morphogenesis of the adrenal cortex and circulatory disorders in zona glomerulosa contributed to reduced aldosterone and sex steroid hormones production.     

Induction of corticosteroid biosynthetic pathway by ACTH and high-density lipoprotein in newborn rat adrenocortical cells cultured in serum-free medium

In order to investigate the role of rat high-density lipoprotein (HDL) on adrenal cholesterol accumulation and steroidogenic pathways (corticosteroid, i.e., 21-hydroxysteroid biosynthesis and reductive metabolism of progesterone), newborn rat adrenal cells cultured in serum-free medium were used. Incubation of [4-14C]cholesterol-HDL in serum-free medium compared to those in medium with lipoprotein-deficient serum, in serum-free medium with ACTH compared to those without ACTH, both showed an increase of labelled cholesterol in cells and of labelled 21-hydroxysteroids excreted in medium. Substitution of serum-supplemented medium by serum-free and cholesterol-free medium led to a deep decrease of ACTH-induced steroid biosynthesis with a predominance of 20 alpha-reduced steroids; addition of HDL restored the corticosteroid biosynthesis and decreased the reductive metabolism. Addition of increased concentrations of HDL (7-150 micrograms cholesterol/ml) enhanced, in a saturable fashion, the total cholesterol uptake and the corticosteroid biosynthesis. The total cholesterol accumulation in cells exceeded by 4-fold the steroid production at saturation. The ratio between the two steroidogenic pathways increased up to 40 at saturation in favor of corticosteroids. These results suggest that HDL is at least partly internalized and that probably its constituents contribute greatly to the control of the two different steroidogenic pathways.     

Extraglandular hormonal steroidogenesis in aged rats

We have examined the metabolism in vitro of [4-¹⁴C]pregnenolone by the following organs of 2.4-year-old rats: submandibular gland, stomach, duodenum, liver, lung, heart, spleen, kidney, skin, prostate, testis and adrenal. All tissues converted pregnenolone to progesterone, the highest yields being observed with adrenal, testis and skin. Androgen formation was intense in the testis and absent in the adrenal. Moreover, 17-hydroxylation of pregnenolone occurred moderately in kidney, skin and submandibular gland and markedly in duodenum and stomach, which also produced high amounts of dehydroepiandrosterone and/or 5-androstene-3β,17β-diol. Extratesticular synthesis of androstenedione and testosterone was very low. A significant formation of 20-dihydropregnenolone was observed in all tissues but stomach, duodenum and steroidogenic endocrines. Corticosteroids were not synthesized extraadrenally, except a small amount of 11-deoxycorticosterone in the testis. These results indicate that key steroid-biosynthetic enzymes, such as 3β-hydroxysteroid dehydrogenase/Δ⁵′Δ⁴ isomerase, 17β- and 20-hydroxysteroid dehydrogenases and steroid 17-monooxygenase/17,20-lyase are also expressed extraglandularly in the rat.     

Liver X receptors regulate adrenal steroidogenesis and hypothalamic-pituitary-adrenal feedback

The nuclear hormone receptors liver X receptor alpha (LXRalpha) (NR1H3) and LXRbeta (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11beta-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.     

New Roles for Corticosteroid Binding Globulin and Opposite Expression Profiles in Lung and Liver

Corticosteroid-binding globulin (CBG) is the specific plasma transport glycoprotein for glucocorticoids. Circulating CBG is mainly synthesized in liver but, its synthesis has been located also in other organs as placenta, kidney and adipose tissue with unknown role. Using an experimental model of acute pancreatitis in cbg^-/- mice we investigated whether changes in CBG affect the progression of the disease as well as the metabolism of glucocorticoids in the lung. Lack of CBG does not modify the progression of inflammation associated to pancreatitis but resulted in the loss of gender differences in corticosterone serum levels. In the lung, CBG expression and protein level were detected, and it is noteworthy that these showed a sexual dimorphism opposite to the liver, i.e. with higher levels in males. Reduced expression of 11β-HSD2, the enzyme involved in the deactivation of corticosterone, was also observed. Our results indicate that, in addition to glucocorticoids transporter, CBG is involved in the gender differences observed in corticosteroids circulating levels and plays a role in the local regulation of corticosteroids availability in organs like lung.     

Oleoyl-estrone increases adrenal corticosteroid synthesis gene expression in overweight male rats

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10 nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5α-reductase was 3× larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands’ corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.     

Design and synthesis of functionalized piperazin-1yl-(E)-stilbenes as inhibitors of 17α-hydroxylase-C17,20-lyase (Cyp17)

The synthesis of steroid hormones is critical to human physiology and improper regulation of either the synthesis of these key molecules or activation of the associated receptors can lead to disease states. This has led to intense interest in developing compounds capable of modulating the synthesis of steroid hormones. Compounds capable of inhibiting Cyp19 (Aromatase), a key enzyme in the synthesis of estrogens, have been successfully employed as breast cancer therapies, while inhibitors of Cyp17 (17α-hydroxylase-17,20-lyase), a key enzyme in the synthesis of glucocorticoids, mineralocorticoids and steroidal sex hormones, are a key component of prostate cancer therapy. Inhibition of CYP17 has also been suggested as a possible target for the treatment of Cushing Syndrome and Metabolic Syndrome. We have identified two novel series of stilbene based CYP17 inhibitors and demonstrated that exemplary compounds in these series have pharmacokinetic properties consistent with orally delivered drugs. These findings suggest that compounds in these classes may be useful for the treatment of diseases and conditions associated with improper regulation of glucocorticoids synthesis and glucocorticoids receptor activation.     

Apolipoprotein-E messenger RNA in rat ovary is expressed in theca and interstitial cells and presumptive macrophage, but not in granulosa cells.

Apolipoprotein-E (apoE) is a constituent of various lipoproteins and is a ligand for cellular lipoprotein receptors. Unlike most apolipoproteins, apoE is synthesized in peripheral tissues, including those engaged in steroidogenesis. ApoE expression in adrenal cells inhibits cholesterol utilization for steroid synthesis and blocks signal transduction via the protein kinase-A pathway. In cultured ovarian thecal/interstitial cells, exogenous apoE has been shown to inhibit LH-induced androgen synthesis. These findings support a role for apoE as an autocrine or paracrine factor involved in regulating steroidogenesis. In the present study in situ hybridization was used to identify cell types that express apoE mRNA in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with PMSG to stimulate follicular development, and from PMSG-treated rats that were subsequently administered hCG to stimulate ovulation and luteinization. ApoE mRNA was localized to theca and interstitial cells of follicles in animals at all stages of the estrous cycle as well as in immature rats treated with PMSG. ApoE mRNA was not detected in oocytes, cumulus cells, or granulosa cells. High levels of apoE mRNA also were expressed by localized clusters of presumptive macrophages in atretic follicles and degenerating corpora lutea. This complex pattern of expression may indicate that apoE has multiple functions in the rat ovary. ApoE made by theca and interstitial cells may act locally as an autocrine factor to regulate androgen production. ApoE made in atretic follicles and regressing corpora lutea may serve to facilitate local transport and reutilization of lipid released as these structures degenerate.     

Effect of 3-acetylpyridine on the functional activity of the adrenal cortex]

A reduction of blood corticosteroid content was observed in rats blood after the administration of 3-acetylpyridine. The rats given ACTH after 3-acetylpyridine showed a lesser elevation of corticosteroids in the blood and adrenal gland tissue than the intact animals; 3-acetylpyridine diminished the activity of dehydrogenase glucose-6-phosphate in the adrenal glands. The authors suggested that the action of acetylpyridine was realized at the adrenal gland level and consisted in inhibition of the NADP-H2 generation in the dehydrogenase systems.     

Orexins (hypocretins) and adrenal function.

The recently discovered neuropeptides orexin A and B regulate feeding behavior, neuroendocrine and autonomic functions, and sleep-wakefulness by central mechanisms. The expression of orexins and orexin receptors in various peripheral organs and the presence of orexin A in blood indicate the existence of a peripheral orexin system. In rat and human adrenal glands, both OX (1) and OX (2) receptor subtypes have been described with a predominant expression of OX (2) receptors in the adrenal cortex. In male rats, adrenocortical OX (2) receptors are much higher expressed than in female rats. Various experimental data demonstrate a stimulatory effect of orexins on the secretion of adrenocortical steroids, mainly on glucocorticoids. Some results also suggest the regulation of catecholamine synthesis and release by orexins. Whether the gender-dependent expression of adrenocortical OX (2) receptors has functional correlates awaits future clarification. As plasma orexin appears to rise during hunger and hypoglycemia, orexins may link adrenal functions with energy homeostasis.     

Pluripotent steroidogenesis and ultrastructure in adrenocortical adenomas causing Cushing's syndrome

Adrenal vein catheterization data from 2 patients with adrenocortical adenomas causing Cushing's syndrome are presented and the electron-microscopic features of one of the tumors are described. Based on the catheterization data both tumors produced all three classes of adrenal steroids (mineralocorticoids, glucocorticoids and sex steroids). Electron-microscopic examination of the tumor cells suggested an origin from the zona fasciculata. If one accepts the theory of a common cellular origin of adrenal tumors, then the pattern of steroidogenesis would indicate that the postulated original 'stem' cell retains the potential of secreting all classes of adrenocortical steroids. The clinical presentation of such tumors would thus reflect the hypersecretion of one of the steroid classes relative to the others.     

11-Oxa and 17alpha-hydroxymethyl analogues of steroid hormones and their derivatives.

The syntheses of 11-oxa and 17alpha-hydroxymethyl analogues of steroid hormones and their derivatives are reported and some of their biological activities are discussed. Generally, the replacement of the 11-methylene group by oxygen results in a diminution of the progestational, androgenic-anabolic, and estrogenic activities. This effect is least pronounced in the case of the progestational activity of 11-oxa-ethisterone and particularly strong in the case of the uterotropic activity of 11-oxa-estradiol. 17alpha-acetoxymethylprogesterone and 17alpha-hydroxymethylprogesterone were synthesized by 2 pathways, 1 of which can be advantageously applied also to the synthesis of 17alpha-acyloxymethyl and 17alpha-hydroxymethyl glucocorticoids. 17alpha-acetoxymethylprogesterone was inactive in the Clauberg test even at high doses.     

Glucocorticoids coordinately regulate procollagens type I and type III synthesis

Antibodies to type I and type III procollagens were raised in rabbits and were made monospecific by chromatography on collagen and procollagen affinity columns. The antibodies were determined to be monospecific by the direct enzyme-linked immunosorbent assay and the enzyme-linked immunosorbent assay inhibition assay. Rats were treated with various doses of triamcinolone diacetate, pulse-labeled with radioactive proline for 20 min, and the procollagens were precipitated with procollagen antibodies. The degree of inhibition of procollagen type I and type III synthesis to corticosteroid treatment was the same. This coordinate effect of glucocorticoids on the synthesis of the two procollagens was reversible, dose-dependent, time-dependent, and observed in lung as well as in skin. These data indicate that glucocorticoids coordinately regulate the synthesis of type I and type III procollagen in skin and lung to the same extent.