Transplantation of porcine adrenal spheroids for the treatment of adrenal insufficiency.

Primary adrenal insufficiency is a life-threatening disorder, which requires lifelong hormone replacement therapy. Transplantation of xenogeneic adrenal cells is a potential alternative approach for the treatment of adrenal insufficiency. For a successful outcome of this replacement therapy, transplanted cells should provide adequate hormone secretion and respond to adrenal physiological stimuli. Here, we describe the generation and characterization of primary porcine adrenal spheroids capable of replacing the function of adrenal glands in vivo. Cells within the spheroids morphologically resembled adult adrenocortical cells and synthesized and secreted adrenal steroid hormones in a regulated manner. Moreover, the embedding of the spheroids in alginate led to the formation of cellular elongations of steroidogenic cells migrating centripetally towards the inner part of the slab, similar to zona Fasciculata cells in the intact organ. Finally, transplantation of adrenal spheroids in adrenalectomized SCID mice reversed the adrenal insufficiency phenotype, which significantly improved animals' survival. Overall, such adrenal models could be employed for disease modeling and drug testing, and represent the first step toward potential clinical trials in the future.

Development and function of the fetal adrenal.

The adrenal cortex undergoes multiple structural and functional rearrangements to satisfy the systemic needs for steroids during fetal life, postnatal development, and adulthood. A fully functional adrenal cortex relies on the proper subdivision in regions or 'zones' with distinct but interconnected functions, which evolve from the early embryonic stages to adulthood, and rely on a fine-tuned gene network. In particular, the steroidogenic activity of the fetal adrenal is instrumental in maintaining normal fetal development and growth. Here, we review and discuss the most recent advances in our understanding of embryonic and fetal adrenal development, including the known causes for adrenal dys-/agenesis, and the steroidogenic pathways that link the fetal adrenal with the hormone system of the mother through the fetal-placental unit. Finally, we discuss what we think are the major open questions in the field, including, among others, the impact of osteocalcin, thyroid hormone, and other hormone systems on adrenal development and function, and the reliability of rodents as models of adrenal pathophysiology.

Latent Adrenal Insufficiency: From Concept to Diagnosis.

Primary adrenal insufficiency (PAI) is a rare disease and potentially fatal if unrecognized. It is characterized by destruction of the adrenal cortex, most frequently of autoimmune origin, resulting in glucocorticoid, mineralocorticoid, and adrenal androgen deficiencies. Initial signs and symptoms can be nonspecific, contributing to late diagnosis. Loss of zona glomerulosa function may precede zona fasciculata and reticularis deficiencies. Patients present with hallmark manifestations including fatigue, weight loss, abdominal pain, melanoderma, hypotension, salt craving, hyponatremia, hyperkalemia, or acute adrenal crisis. Diagnosis is established by unequivocally low morning serum cortisol/aldosterone and elevated ACTH and renin concentrations. A standard dose (250 µg) Cosyntropin stimulation test may be needed to confirm adrenal insufficiency (AI) in partial deficiencies. Glucocorticoid and mineralocorticoid substitution is the hallmark of treatment, alongside patient education regarding dose adjustments in periods of stress and prevention of acute adrenal crisis. Recent studies identified partial residual adrenocortical function in patients with AI and rare cases have recuperated normal hormonal function. Modulating therapies using rituximab or ACTH injections are in early stages of investigation hoping it could maintain glucocorticoid residual function and delay complete destruction of adrenal cortex.

The Role of the Hippocampus in the Neuroendocrine Response to Neurobiological Stimuli in Experiment.

We compared the levels of functional activity of cells in each adrenal zone with blood levels of corticosterone, testosterone, and neuropeptide Y in control and hippocampectomized F1(C57BL/6×DBA/2) mice during modeling of metabolic, motivational, and cognitive tension. The morphofunctional state of the adrenal glands was studied using a new morphometric approach. It was found that hippocampectomy changed the testosterone response to neurobiological stimuli; similar changes were observed in the zona reticularis of the adrenal cortex producing dehydroepiandrosterone that is involved in the regulation of testosterone secretion. At the same time, hippocampectomy enhanced the response of the peptide hormone; the index of functional activity of chromaffin cells producing this hormone also increased. These findings allow us to put forward a hypothesis that the hippocampus is involved in the regulation of mutual influences of the studied hormones and that it modulates the sensitivity of testosterone and NPY to metabolic and cognitive factors.

Adrenal Cortex Development and Maintenance: Knowledge Acquired From Mouse Models.

The adrenal cortex is an endocrine organ organized into concentric zones that are specialized to produce specific steroid hormones essential for life. The development and maintenance of the adrenal cortex are complex, as a fetal adrenal is first formed from a common primordium with the gonads, followed by its separation in a distinct primordium, the invasion of the adrenal primordium by neural crest-derived cells to form the medulla, and finally its encapsulation. The fetal cortex is then replaced by a definitive cortex, which will establish zonation and be maintained throughout life by regeneration relying on the proliferation, centripetal migration, and differentiation of several stem/progenitor cell populations whose activities are sex-specific. Here, we highlight the advances made, using transgenic mouse models, to delineate the molecular mechanisms regulating these processes.

The Age-Dependent Changes of the Human Adrenal Cortical Zones Are Not Congruent.

BACKGROUND: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF). OBJECTIVES: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms. METHODS: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3ß-hydroxysteroid dehydrogenase type II (HSD3B2), 11ß-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years. RESULTS: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007). CONCLUSION: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk.

Relationship between exploratory activity and adrenocortical activity in the black rat (Rattus rattus).

The relationship between physiological and behavioral stress markers is documented in several rodent species. However, there is no information regarding the role of adrenocortical activity in behavior of the black rat (Rattus rattus). Therefore, we hypothesize that the adrenocortical activity of black rats varies between individuals and is related to some of the behaviors in a novel environment. To test this hypothesis, we (i) validated a method for quantifying glucocorticoid metabolites from feces (fGCMs) with an enzyme immunoassay (EIA); (ii) examined variation and diurnal rhythms of feces and GCM production; and (iii) examined the relationship between GCM levels and exploratory behavioral traits. We fulfilled the first aim (i) by successfully performing an ACTH challenge test to validate the use of a 5α-pregnane-3ß,11ß,21-triol-20-one EIA for measuring fGCMs. Second (ii) we detected considerable consistent interindividual variability in production of both feces and glucocorticoids. The peak production of feces occurred in the first hour of the dark cycle, the peak of fGCMs occurred approximately 3 h later. Lastly, (iii) there was no clear relationship between behavior in the hole board test and GCMs. Grooming, a typical behavioral stress marker, was negatively associated with stress reactivity, while head-dipping in the hole-board test (traditionally considered an exploratory behavior independent of stress) was not correlated with the GCMs. This study offers a first look at GCMs in the black rat, successfully validates a method for their measurement and opens possibilities for future research of the relationship between glucocorticoids and exploratory behavior in this species.

Stem cell function and plasticity in the normal physiology of the adrenal cortex.

The adrenal cortex functions to produce steroid hormones necessary for life. To maintain its functional capacity throughout life, the adrenal cortex must be continually replenished and rapidly repaired following injury. Moreover, the adrenal responds to endocrine-mediated organismal needs, which are highly dynamic and necessitate a precise steroidogenic response. To meet these diverse needs, the adrenal employs multiple cell populations with stem cell function. Here, we discuss the literature on adrenocortical stem cells using hematopoietic stem cells as a benchmark to examine the functional capacity of particular cell populations, including those located in the capsule and peripheral cortex. These populations are coordinately regulated by paracrine and endocrine signaling mechanisms, and display remarkable plasticity to adapt to different physiological and pathological conditions. Some populations also exhibit sex-specific activity, which contributes to highly divergent proliferation rates between sexes. Understanding mechanisms that govern adrenocortical renewal has broad implications for both regenerative medicine and cancer.

A phosphodiesterase 11 (Pde11a) knockout mouse expressed functional but reduced Pde11a: Phenotype and impact on adrenocortical function.

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cyclic adenosine and guanosine mono-phosphate (cAMP and cGMP, respectively). Increased cAMP signaling has been associated with adrenocortical tumors and Cushing syndrome. Genetic defects in phosphodiesterase 11A (PDE11A) may lead to increased cAMP signaling and have been found to predispose to the development of adrenocortical, prostate, and testicular tumors. A previously reported Pde11a knockout (Pde11a-/-) mouse line was studied and found to express PDE11A mRNA and protein still, albeit at reduced levels; functional studies in various tissues showed increased cAMP levels and reduced PDE11A activity. Since patients with PDE11A defects and Cushing syndrome have PDE11A haploinsufficiency, it was particularly pertinent to study this hypomorphic mouse line. Indeed, Pde11a-/- mice failed to suppress corticosterone secretion in response to low dose dexamethasone, and in addition exhibited adrenal subcapsular hyperplasia with predominant fetal-like features in the inner adrenal cortex, mimicking other mouse models of increased cAMP signaling in the adrenal cortex. We conclude that a previously reported Pde11a-/- mouse showed continuing expression and function of PDE11A in most tissues. Nevertheless, Pde11a partial inactivation in mice led to an adrenocortical phenotype that was consistent with what we see in patients with PDE11A haploinsufficiency.

Annual regulation of adrenocortical function in migrant and resident subspecies of white-crowned sparrow.

Corticosterone affects physiology and behavior both during normal daily processes but also in response to environmental challenges and is known to mediate life history trade-offs. Many studies have investigated patterns of corticosterone production at targeted times of year, while ignoring underlying annual profiles. We aimed to understand the annual regulation of hypothalamic-pituitary-adrenal (HPA) axis function of both migrant (Zonotrichia leucophrys gambelii; n = 926) and resident (Z. l. nutalli; n = 688) subspecies of white-crowned sparrow and how it is influenced by environmental conditions - wind, precipitation, and temperature. We predicted that more dramatic seasonal changes in baseline and stress-induced corticosterone would occur in migrants to precisely time the onset of breeding and cope with environmental extremes on their arctic breeding grounds, while changes in residents would be muted as they experience a more forgiving breeding schedule and comparatively benign environmental conditions in coastal California. During the course of a year, the harshest conditions were experienced the summer breeding grounds for migrants, at which point they had higher corticosterone levels compared to residents. For residents, the winter months coincided with harshest conditions at which point they had higher corticosterone levels than migrants. For both subspecies, corticosterone tended to rise as environmental conditions became colder and windier. We found that the annual maxima in stress-induced corticosterone occurred prior to egg lay for all birds except resident females. Migrants had much higher baseline and acute stress-induced corticosterone during breeding compared to residents; where in a harsher environment the timing of the onset of reproduction is more critical because the breeding season is shorter. Interestingly, molt was the only stage within the annual cycle in which subspecies differences were absent suggesting that a requisite reduction in corticosterone may have to be met for feather growth. These data suggest that modulation of the HPA axis is largely driven by environmental factors, social cues, and their potential interactions with a genetic program.

Functional imaging of adrenal cortex. / Estudios de imagen funcional de la corteza adrenal.

The rising number of high-resolution imaging scans has increased the adrenal lesions detection, which require a differential diagnosis. Currently, the most commonly used scans are CT and MRI, but these are sometimes not very specific. In these cases, nuclear medicine scans with 131I-norcolesterol, 11C-metomidate and 18F-fludeoxyglucose help to differentiate benign vs. malignant lesions, to lateralize the involvement in hypersecretion disease, as well as to guide the therapeutic strategy in both unilateral and bilateral lesions.

Impacts of the season and reproductive status on fecal reproductive and adrenocortical steroid metabolites in zoo Cuban crocodiles (Crocodylus rhombifer).

Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.

[EVALUATION OF ADRENAL CORTICAL FUNCTION USING SALIVARY CORTISOL MEASUREMENTS IN CHILDREN WITH BRONCHIAL ASTHMA].

BACKGROUND: When corticosteroids are used for a long time in patients with bronchial asthma, a decrease in adrenal cortex function occurs. We investigated the use of salivary cortisol measurements as a simple, noninvasive method for the evaluation of the adrenal cortex function in pediatric asthmatic patients. METHODS: Plasma and salivary cortisol levels were measured from 8:00 am to 10:00 am in 248 pediatric asthmatic patients aged 0-18 years that were under long-term care management at the National Hospital Organization Fukuoka National Hospital in 2011-2013. We determined the correlation between plasma and salivary cortisol levels and calculated a salivary cortisol cut-off value for screening adrenocortical function. RESULTS: There was a significant positive correlation between salivary cortisol levels and plasma cortisol levels in all patients, patients under age 3, and patients over age 3 (r=0.759, r=0.563, r=0.827, respectively). Salivary cortisol cut-off values were 0.015µg/dL for plasma cortisol levels below 3µg/dL (sensitivity 83%, specificity 82%), and 0.045µg/dL those below 5µg/dL (sensitivity 83%, specificity 75%). 5µg/dL or less is an abnormal value, and 3µg/dL or less is considered to be adrenal insufficiency. CONCLUSION: Salivary cortisol levels were positively correlated with plasma cortisol levels in pediatric asthmatic patients. This is a useful method for frequently monitoring young children with adrenal dysfunction or severe asthma that are treated with high doses of inhaled corticosteroids.

Coping with extremes: Remarkably blunt adrenocortical responses to acute stress in two sympatric snow finches on the Qinghai-Tibet Plateau during winter relative to other seasons.

The extreme climatic conditions (ECCs) of the Qinghai-Tibet Plateau impose strong selective pressures on the evolution of phenotypic traits in free-living animals. It is not well understood how animals on the Qinghai-Tibet Plateau modify their adrenocortical functions in response to both predictable and unpredictable events of ECCs, especially when the available resources are lowest during the wintering life-history stage. To uncover potential physiological mechanisms, we studied the life history stage dependent features of morphology, the plasma corticosterone response to acute stress and brain glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) mRNA expression in two sympatric snow finches: the white-rumped snow finch (Onychostruthus taczanowskii, WRSF); and the rufous-necked snow finch, Pyrgilauda ruficollis, RNSF) in Qinghai Province, China. Our results showed that (a) baseline corticosterone and stressor-induced corticosterone levels significantly varied with life history stage, but not between the species; (b) in WRSF, GR mRNA expression in the paraventricular nucleus was higher in the wintering stage compared to the pre-basic molt stage. There were no differences in hippocampus MR mRNA expression between stages in either species; (c) in the wintering stage, the suppression of corticosterone secretion in both species was an unexpected strategy in free-living animals. Both convergent and divergent phenotypic traits of adrenocortical responses to acute stress in two sympatric snow finches contribute to our understanding of the coping mechanisms of closely related species in the severe winter on the Qinghai-Tibet Plateau.

GATA4/6 regulate DHH transcription in rat adrenocortical autografts.

Adrenal cortex autotransplantation with ACTH stimulation may be an alternative therapy for patients with bilateral adrenalectomy to avoid adrenal crisis, but its underlying mechanism has not been elucidated. Previously, we detected Dhh upregulation in rat adrenocortical autografts after transplantation. Here, we investigated potential regulators such as Gata4, Gata6, Sry and Sox9 which affect Dhh transcription in adrenocortical autografts with or without ACTH stimulation. In ACTH-stimulated autografts, Gata4 and Gata6 were downregulated compared to control autografts. This response was linked to rDhh repression. A reporter assay using the upstream region of rDhh and a GATA binding motif revealed that rDhh promoters were significantly upregulated by co-transfection with Gata4 or Gata6 or both. Sry and Sox9 expression in autografts with or without ACTH stimulation were verified by PCR and RNAscope analyses. The ovarian differentiation factors Foxl2 and Rspo1 were also upregulated in the autografts. Gata4 and Gata6 were found to be significant factors in the regulation of rDhh expression and could be associated with adrenocortical autograft maintenance. Gonadal primordia with bipotential testicular and ovarian functions may also be present in these autografts.

Chemogenetic activation of adrenocortical Gq signaling causes hyperaldosteronism and disrupts functional zonation.

The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin-angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.

The Adult Adrenal Cortex Undergoes Rapid Tissue Renewal in a Sex-Specific Manner.

Evolution has resulted in profound differences between males and females that extend to non-reproductive organs and are reflected in the susceptibility and progression of diseases. However, the cellular and molecular basis for these differences remains largely unknown. Here we report that adrenal gland tissue renewal is highly active and sexually dimorphic, with female mice showing a 3-fold higher turnover than males. Moreover, in males, homeostasis relies on proliferation of cells within the steroidogenic zone, but females employ an additional stem and/or progenitor compartment situated in the adrenal capsule. Using lineage tracing, sex reversal models, gonadectomy, and dihydrotestosterone treatments, we further show that sex-specific stem cell activity is driven by male hormones that repress recruitment of Gli1+ stem cells from the capsule and cell proliferation. Taken together, our findings provide a molecular and cellular basis for adrenal sex dimorphism that may contribute to the increased incidence of adrenal diseases in females.

Regulation of Aldosterone Secretion.

Secretion of the major mineralocorticoid aldosterone from the adrenal cortex is a tightly-regulated process enabling this hormone to regulate sodium homeostasis and thereby contribute to blood pressure control. The circulating level of aldosterone is the result of various regulatory mechanisms, the most significant being those controlled by the renin-angiotensin system and plasma potassium levels. The importance of maintaining tight control over aldosterone secretion is demonstrated by cases of dysregulation, which can result in severe hypertension and significantly increased cardiovascular risk. In this article we summarize current knowledge of the major regulatory mechanisms, focusing particularly on the systems operating within the adrenocortical zona glomerulosa cells; we also describe some of the other factors that influence aldosterone production to a lesser but still significant extent. Finally, we discuss the influence of common genetic polymorphisms on aldosterone secretion in large sections of the population and also the emerging role of microRNA as significant regulators of this system.

The Origin of a New Progenitor Stem Cell Group in Human Development.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

Isolation and characterization of adrenocortical progenitors involved in the adaptation to stress.

The adrenal gland is a master regulator of the human body during response to stress. This organ shows constant replacement of senescent cells by newly differentiated cells. A high degree of plasticity is critical to sustain homeostasis under different physiological demands. This is achieved in part through proliferation and differentiation of adult adrenal progenitors. Here, we report the isolation and characterization of a Nestin+ population of adrenocortical progenitors located under the adrenal capsule and scattered throughout the cortex. These cells are interconnected with progenitors in the medulla. In vivo lineage tracing revealed that, under basal conditions, this population is noncommitted and slowly migrates centripetally. Under stress, this migration is greatly enhanced, and the cells differentiate into steroidogenic cells. Nestin+ cells cultured in vitro also show multipotency, as they differentiate into mineralocorticoid and glucocorticoid-producing cells, which can be further influenced by the exposure to Angiotensin II, adrenocorticotropic hormone, and the agonist of luteinizing hormone-releasing hormone, triptorelin. Taken together, Nestin+ cells in the adult adrenal cortex exhibit the features of adrenocortical progenitor cells. Our study provides evidence for a role of Nestin+ cells in organ homeostasis and emphasizes their role under stress. This cell population might be a potential source of cell replacement for the treatment of adrenal insufficiency.