Increased Cortisol Production Research Articles

Cortisol Production Rate Is Similarly Elevated in Obese Women with or without the Polycystic Ovary Syndrome

The pituitary-adrenal axis in obesity and polycystic ovary syndrome (PCOS) is marked by increased urinary excretion of cortisol and its metabolites. It is not as yet clear whether the increased cortisol production in PCOS is related to obesity per se. We investigated 15 obese PCOS women with a body mass index of 30-54 kg/m(2) and 15 healthy obese controls (body mass index 31-60 kg/m(2)) with a regular menstrual cycle. Patients and control women underwent 24-h blood sampling at 20-min intervals. Cortisol concentrations were measured with a sensitive assay. Data were analyzed with a new deconvolution program, approximate entropy, and cosinor regression. Basal, pulsatile, and total cortisol production expressed per liter distribution volume, per square meter body surface, and as absolute amount per 24 h was similar in PCOS patients and matched healthy control women. In addition, the regularity of cortisol secretion and the diurnal properties were identical. Compared with 10 lean control women, mean cortisol production per liter distribution volume was similar in the three groups, but the total 24-h cortisol production was increased in obese control women and PCOS women. This study demonstrates equally increased cortisol production in PCOS women and obese healthy control women.

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol-secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post-infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up-regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate-early gene expression, since it was most evident in the early phases p.i. and UV-inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency.

Milk ejection disorders in Swiss dairy cows: a field study

A questionnaire was sent to 2099 dairy farms to investigate the occurrence of poor milkability. Based on that, the frequency of poor milkability in Swiss dairy cows was 4% and the percentage of cows treated with oxytocin (OT) was 2%. In addition, 270 dairy farms that had reported cases of animals with poor milkability were contacted for an interview to classify the disorders. Farmers suspected disturbed milk ejection in 52%, anatomical dysfunction of the teat and/or the udder in 16% and milk ejection disorder or impaired milkability caused by discernable environmental factors in 32% of the cases. Forty-eight animals from 18 farms with suspected milk ejection disorders were selected for an experimental field study which included milk flow recording and OT administration to induce milk ejection. After cessation of the spontaneous milk flow, a low dose of OT (0.2, 0.5 or 1 i.u.) was injected i.v. to test the responsiveness of the udder to OT at a physiological level. When milk flow ceased again, 10 i.u. OT was injected i.v. (supraphysiological) to ensure complete udder emptying and to determine the residual milk. Milk ejection disorder could be confirmed in 69% of the cases, i.e. if residual milk was >20% of the total milk. Because in 27% of the animals milk ejection disorder was not confirmed on the basis of elevated residual milk, an anatomical disorder of the teat and/or the udder was suspected. Milk ejection disorder could be confirmed in 69% of the cases whereas in 27% of the suspected cases an anatomical disorder of the teat and/or the udder was suspected. An increased cortisol production in cows with milk ejection disorder was not obvious because faecal concentrations of cortisol metabolites with a 5 beta-androstane-3 alpha,11 oxo-structure were not augmented in animals with disturbed milk ejection.

Cryptochrome 2 Expression Level Is Critical for Adrenocorticotropin Stimulation of Cortisol Production in the Capuchin Monkey Adrenal

Timely production of glucocorticoid hormones in response to ACTH is essential for survival by coordinating energy intake and expenditure and acting as homeostatic regulators against stress. Adrenal cortisol response to ACTH is clock time dependent, suggesting that an intrinsic circadian oscillator in the adrenal cortex contributes to modulate the response to ACTH. Circadian clock gene expression has been reported in the adrenal cortex of several species. However, there are no reports accounting for potential involvement of adrenal clock proteins on cortisol response to ACTH. Here we explored whether the clock protein cryptochrome 2 (CRY2) knockdown modifies the adrenal response to ACTH in a primate. Adrenal gland explants from adult capuchin monkey (n = 5) were preincubated for 6 h with transfection vehicle (control) or with two different Cry2 antisense and sense probes followed by 48 h incubation in medium alone (no ACTH) or with 100 nm ACTH. Under control and sense conditions, ACTH increased cortisol production, whereas CRY2 suppression inhibited ACTH-stimulated cortisol production. Expression of the steroidogenic enzymes steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase at 48 h of incubation was increased by ACTH in control explants and suppressed by Cry2 knockdown. Additionally, we found that Cry2 knockdown decreased the expression of the clock gene brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (Bmal1) at the mRNA and protein levels. Altogether these results strongly support that the clock protein CRY2 is involved in the mechanism by which ACTH increases the expression of steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase. Thus, adequate expression levels of components of the adrenal circadian clock are required for an appropriate cortisol response to ACTH.

Hair as a retrospective calendar of cortisol production—Increased cortisol incorporation into hair in the third trimester of pregnancy

Hair has long been used in toxicology, forensic science, doping control and other fields as a biological specimen for the detection of environmental agents, drugs, or toxins. Most recent evidence suggests that also hormones are incorporated and trapped inside the growing hair. This has led to the hypothesis that cortisol measurement of distinct hair segments could provide a retrospective calendar of cortisol production for the individual. In this first proof-of-concept study in humans, we analyzed cortisol in hair donated by mothers with a neonate child (n-Mothers; N=103), mothers with toddlers 3-9 months of age (t-Mothers; N=19), and control women (N=20). We cut hair strands from each women into at least three 3-cm segments, which, based on an average hair growth rate of 1cm per month, would represent hair grown over the past three, six, and nine months, respectively. Since in the third trimester of pregnancy there is a well-documented increased production of cortisol, we expected to see elevated levels of cortisol in the most proximal hair segment of women who had just given birth to a child (n-Mothers) compared with the control women. Likewise, we expected to see elevated levels in the second, third, or fourth segment of mothers of 3-month olds, 6-months olds, and 9-months olds, respectively. These hair segments, cut at 4-12 cm from the scalp, would represent hair grown throughout the third trimester of pregnancy. Results showed that there was a strong monotonic decline in cortisol concentration from the segment closest to the scalp to the most distal hair segment (p<0.0001). Cortisol levels decreased by 30-40% from one segment to the next for the most recent four hair segments. Segments from hair older than one year had similarly, low levels of cortisol. Comparisons of cortisol levels in hair between n-Mothers and control women yielded the expected results: cortisol levels in the first 3-cm hair segment (i.e., closest to the scalp) of n-Mothers were two-fold higher than in controls (p<0.0001), probably reflecting increased cortisol levels throughout the third trimester of pregnancy. No differences in cortisol content were apparent for the second or third 3-cm segments in n-Mothers (p>0.2). When hair from mothers with 6-9 months old toddlers was analyzed, the hair segment representing the third trimester period contained the same amount of cortisol as the hair grown more recently in mothers with 3-4 months old toddlers only. Age of the women, hair curvature, hair color, and frequency of hair washes per week were unrelated to cortisol levels. We conclude that cortisol measured in human hair can be a valid reflection of increased cortisol production for a period of up to six months. Due to a rapid decline of cortisol levels in human adult hair, a retrospective calendar of cortisol exposure may be limited to the past six months.

Hair as a retrospective calendar of cortisol production – increased cortisol incorporation into hair in the third trimester of pregnancy

Hair has long been used in toxicology, forensic science, doping control and other fields as a biological specimen for the detection of environmental agents, drugs, or toxins. Most recent evidence suggests that also hormones are incorporated and trapped inside the growing hair. This has led to the hypothesis that cortisol measurement of distinct hair segments could provide a retrospective calendar of cortisol production for the individual. In this first proof-of-concept study in humans, we analyzed cortisol in hair donated by mothers with a neonate child (n-Mothers; N=103), mothers with toddlers 3-9 months of age (t-Mothers; N=19), and control women (N=20). We cut hair strands from each women into at least three 3-cm segments, which, based on an average hair growth rate of 1cm per month, would represent hair grown over the past three, six, and nine months, respectively. Since in the third trimester of pregnancy there is a well-documented increased production of cortisol, we expected to see elevated levels of cortisol in the most proximal hair segment of women who had just given birth to a child (n-Mothers) compared with the control women. Likewise, we expected to see elevated levels in the second, third, or fourth segment of mothers of 3-month olds, 6-months olds, and 9-months olds, respectively. These hair segments, cut at 4-12 cm from the scalp, would represent hair grown throughout the third trimester of pregnancy. Results showed that there was a strong monotonic decline in cortisol concentration from the segment closest to the scalp to the most distal hair segment (p 0.2). When hair from mothers with 6-9 months old toddlers was analyzed, the hair segment representing the third trimester period contained the same amount of cortisol as the hair grown more recently in mothers with 3-4 months old toddlers only. Age of the women, hair curvature, hair color, and frequency of hair washes per week were unrelated to cortisol levels. We conclude that cortisol measured in human hair can be a valid reflection of increased cortisol production for a period of up to six months. Due to a rapid decline of cortisol levels in human adult hair, a retrospective calendar of cortisol exposure may be limited to the past six months.

Cortisol Is a Suppressor of Apoptosis in Bovine Corpus Luteum1

Glucocorticoid (GC) acts as a modulator of physiological functions in several organs. In the present study, we examined whether GC suppresses luteolysis in bovine corpus luteum (CL). Cortisol (an active GC) reduced the mRNA expression of caspase 8 (CASP8) and caspase 3 (CASP3) and reduced the enzymatic activity of CASP3 and cell death induced by tumor necrosis factor (TNF) and interferon gamma (IFNG) in cultured bovine luteal cells. mRNAs and proteins of GC receptor (NR3C1), 11beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), and HSD11B2 were expressed in CL throughout the estrous cycle. Moreover, the protein expression and the enzymatic activity of HSD11B1 were high at the early and the midluteal stages compared to the regressed luteal stage. These results suggest that cortisol suppresses TNF-IFNG-induced apoptosis in vitro by reducing apoptosis signals via CASP8 and CASP3 in bovine CL and that the local increase in cortisol production resulting from increased HSD11B1 at the early and midluteal stages helps to maintain CL function by suppressing apoptosis of luteal cells.

The Role of Corticosteroids in Sepsis and Acute Respiratory Distress Syndrome

The use of corticosteroids in the treatment of critically ill patients has been the subject of controversy for decades. Information from recent clinical trials has helped to more clearly define their use in the acute setting. Cortisol is produced in response to pituitary corticortopin secretion which is in turn released in response to hypothalamic corticorpinreleasing hormone. Acute illness should result in increased cortisol production, although relative corticosteroid insufficiency can occur in sepsis and appears to be associated with increased mortality. Despite this, diagnosing corticosteroid insufficiency in patients with septic shock remains difficult. Investigators have failed to find a reliable relationship between random cortisol levels and mortality. However, recent evidence demonstrated an increase of serum cortisol < 9 mcg/dL following corticotropin stimulation is associated with increased mortality from septic shock. Corticosteroid replacement therapy in these patients reduces mortality. The mortality benefit seen in this setting may be limited to septic patients with acute respiratory distress syndrome. Corticosteroids have been used in a variety of conditions causing hypoxemic respiratory failure. While empiric steroids were widely used to treat ARDS in the 1970s and 1980s, more recent evidence from the ARDS clinical trial network failed to demonstrate a mortality benefit, despite the fact that patients were able to be weaned to unassisted breathing more quickly. Tight glycemic control in ARDS patients receiving corticosteroids can help avoid neuromyopathy and may afford an opportunity to prevent a return to assisted breathing in these patients. While steroids have also been reported for use in the treatment of SARS, no trials have been performed which support their efficacy.

Evaluation of function and disorders of the adrenal gland in neonates

Majority of sick full term newborns have adequate adrenal cortical function in response to stress. Acutely ill neonates with a basal cortisol level less than 15 μg/dL (414 nmol/L) suggest adrenal insufficiency and require function testing of adrenal function. In premature infant, immaturity of hypothalamic-pituitary adrenal axis (HPA axis), may limit the ability to increase cortisol production in response to stress. The response to low dose ACTH and CRH appears to be useful as an additional test of adrenal function. CRH stimulation has been used increasingly in neonates. The ACTH and CRH stimulated cortisol response of more than 17 μg/dL (469 nmol/L) indicates a normal response.

Modulation of ACTH-induced cortisol release by polyunsaturated fatty acids in interrenal cells from gilthead seabream, Sparus aurata

Highly unsaturated fatty acids are essential components of cellular membranes of vertebrates and can modulate physiological processes, including membrane transport, receptor function and enzymatic activities. In gilthead sea bream, dietary deficiencies of essential fatty acids of marine fish raise the basal cortisol levels and alter the pattern of cortisol release after stress. The aim of the present study was to clarify the effect of different essential fatty acids on adrenocorticotropic hormone (ACTH)-induced cortisol production and release in fish, through in vitro studies of sea bream interrenal cells maintained in superfusion and incubated with different types of fatty acids and eicosanoid production inhibitors. Results showed the first evidence of the effect of certain fatty acids on cortisol production by ACTH-stimulated interrenal cells in fish. Both arachidonic acid (ARA) and particularly eicosapentaenoic acid (EPA) promoted cortisol production in sea bream interrenal cells. Moreover, incubation with indometacin (INDO) reduced the increased cortisol production induced by EPA and ARA, suggesting mediation by their cyclooxygenase-derived products. Docosahexaenoic acid stimulated cortisol production to a lesser extent than that caused by EPA or ARA, but the inhibitory effect of INDO was not as marked as it was for the other fatty acids. In contrast, supplementation with dihomogammalinolenic acid reduced cortisol production, denoting the inhibitor effect of this fatty acid in cortisol secretion.

Metyrapone Improves Endothelial Dysfunction in Patients With Treated Depression

This study sought to examine the effect of metyrapone on endothelial dysfunction in patients with treated recurrent major depression. Depression is an independent risk factor for the development of coronary heart disease, and patients with depression have endothelial dysfunction, an atherogenic abnormality. This abnormality may be attributable to abnormal hypothalamic-pituitary-adrenal (HPA) axis function, a feature of depression, resulting in increased exposure to cortisol. Cortisol administration produces endothelial dysfunction in healthy subjects. We measured endothelial function using flow-mediated dilation (FMD) of the brachial artery in 30 patients with depression and in 36 matched control subjects. Patients were randomized (double blind) to metyrapone (an inhibitor of cortisol synthesis) or placebo, and FMD was remeasured 6 h later. At baseline, FMD was impaired in patients versus control subjects (mean [standard error]), -1.27% [0.91%] vs. 4.37% [0.59%] (p < 0.001). The FMD was similar in the placebo and the metyrapone patient groups at baseline (0.17% [1.04%] vs. -2.72% [1.30%], p = 0.11). Metyrapone significantly reduced plasma cortisol levels. There was a significant improvement in FMD in the metyrapone group from -2.72% [1.30%] to 3.82% [0.99%] (p < 0.001), whereas the change in the placebo group, from 0.17% [1.04%] to 1.15% [1.14%], was not significant. Analysis of covariation showed that the effect of metyrapone was significant (p = 0.034). Inhibition of cortisol production by metyrapone ameliorates the endothelial dysfunction seen in depression, suggesting that the mechanism of the endothelial dysfunction may involve cortisol.

Intra-adrenal interactions in fish: Catecholamine stimulated cortisol release in sea bass ( Dicentrarchus labrax L.)

The effect of the catecholamines, adrenaline and noradrenaline, on sea bass ( Dicentrarchus labrax) and sea bream ( Sparus auratus) interrenal cortisol production was studied in vitro using a dynamic superfusion system technique. Increasing concentrations of catecholamines (10 − 6 , 10 − 8 and 10 − 10 M) stimulated cortisol production in a dose-dependent manner, in sea bass only. The increase in cortisol production stimulated by adrenaline (10 − 6 M) and noradrenaline (10 − 6 M) was inhibited by sotalol (2 × 10 − 5 M), but not by prazosin suggesting that catecholamines stimulate cortisol release through the β-receptor subtype. To evaluate catecholamine-induced signal transduction in head kidney cells, measurements of cAMP production and [H 3]myo-inositol incorporation were determined in head kidney cell suspensions. Adrenaline and noradrenaline (10 − 6 M) increased cAMP production, but had no effect on total inositol phosphate accumulation. These results indicate that catecholamines released from the chromaffin cells within the interrenal tissue may act as a paracrine factor to stimulate interrenal steroidogenesis in the sea bass.

A Case of Adrenocortical Carcinoma showing Variable Cortisol Production during the Clinical Course

Patients with adrenocortical carcinoma (ACC) present with evidence of excessive adrenal steroid hormone in approximately 60% of cases, in which rapidly progressing Cushing's syndrome with or without virilization is the most frequent presentation. Some patients experience an increase or a decline in cortisol production through the progression of their ACC. We report on an unusual case of a cortisol-producing ACC, and the patient presented with a decline in cortisol production, followed by an increase in cortisol production, through the progression of the tumor. A 65-year-old woman who manifested with facial edema and weight gain was diagnosed with Cushing's syndrome, caused by cortisol producing ACC. The patient was treated with adrenalectomy. However, 8 months later, a metastatic hepatic tumor of recurred ACC was detected. At that time, the hormonal evaluation revealed that the liver mass did not produce any hormones. The patient was treated with metastatectomy. Four months later, a relapsed tumor was detected. Increased cortisol production was observed at that time. We speculate there was a change in the clonal dominance within the ACC and this change might cause such a difference. This is the first case report of ACC that showed variable hormone production during progression. (J Kor Endocrinol Soc 21:419~423, 2006)

Effects of ACTH and cAMP on steroidogenic acute regulatory protein and P450 11β-hydroxylase messenger RNAs in rainbow trout interrenal cells: Relationship with in vitro cortisol production

Steroidogenic acute regulatory protein (StAR) transfers cholesterol over the inner mitochondrial membrane, thereby making the molecule available for cholesterol side-chain cleavage enzyme, which carries out the first conversion in the steroidogenic pathway. In mammals, StAR controls this rate limiting step in steroidogenesis, and both StAR protein and StAR mRNA levels become rapidly elevated in response to tropic hormone stimulation. The relationship between StAR gene expression and steroid production in fish has not yet been well explored. We investigated the relationship between adrenocorticotropic hormone (ACTH)- and cAMP-stimulated cortisol production in vitro and levels of StAR transcripts in interrenal cells of rainbow trout. To assess the effect of ACTH on mRNA levels of a downstream steroidogenic enzyme, we also investigated the effects of ACTH on transcripts encoding 11β hydroxylase (P450 11β). In a series of experiments, juvenile rainbow trout head kidney tissue containing interrenal cells was incubated with either ACTH or dibutyryl cyclic AMP (dbcAMP). Cortisol in incubation media were measured by radioimmunoassay and total RNA was isolated from the tissue for Northern analysis or for quantitative real-time PCR. Incubation of tissue with 150 ng/mL ACTH for 1–18 h induced a progressive increase in cortisol accumulation in media, but StAR mRNA levels increased modestly and mostly insignificantly over 18 h, irrespective of treatment. Exposure of tissue for 18 h to 5, 150, 500 or 1500 ng ACTH/mL resulted in a strong increase in cortisol production, with a peak response (15-fold increase over controls) achieved with 150 ng/mL ACTH. Although there was a trend towards a dose–response effect, mean StAR mRNA levels were only significantly affected by the highest concentration of ACTH used (1500 ng/mL), which induced a less than 2-fold increase in StAR transcripts. However, there was a significant linear relationship between StAR mRNA levels and ACTH-induced cortisol accumulation in media ( p < 0.001, r 2 = 0.55). Incubation of tissue with 5 mM dbcAMP for 6 or 18 h induced large increases in cortisol accumulation in media over controls, but had no significant effect on StAR mRNA levels. By contrast, ACTH induced a clear dose-dependent increase in P450 11β transcripts, with 150 ng/mL ACTH inducing an 8-fold increase in levels compared to control; nonetheless, only a weak correlation existed between transcript levels and ACTH-induced cortisol secretion ( p < 0.003, r 2 = 0.26). Thus, despite the relatively high degree of conservation of StAR proteins in vertebrates, we have been unable to demonstrate that a rapid, acute increase in transcription of the StAR gene is the dominant mechanism supporting flow of cholesterol to the mitochondria during acute increases in cortisol production in rainbow trout. The strong stimulation of P450 11β gene transcription by ACTH probably enhances biosynthetic capacity during longer term chronic ACTH stimulation.

Biopsychological markers of distress in informal caregivers.

Thirty caregiving wives participated in a study of caregiving distress and negative mood (depressive symptoms) by making diary entries on stressful caregiving situations and collecting saliva samples 4 times a day. At the end of the 7-day study period, caregivers' salivary cortisol production was compared with their diary entries and correlated with pencil and paper self-report scores of caregiver distress and depressive symptoms. Despite the inability to control a number of factors thought to confound cortisol production (exercise, smoking, alcohol ingestion, and prescription medications), there was a statistically significant difference between No Caregiving and Caregiving cortisol, F(1,739) = 7.67, P = 0.006, with cortisol production higher when caregiving events occurred. However, efforts to code specific types of caregiving situations (e.g., 1 = indirect care; 4 = AD problem behavior care) did not further differentiate cortisol production. Although caregivers' self-reports for the same 7-day period indicated they were depressed, pencil-and-paper measures of distress and negative affect were not significantly correlated with cortisol production. The finding that this caregiving group was significantly stressed by caregiving, as evidenced by increased cortisol production during caregiving episodes, verifies the importance of further exploration of specific caregiving situations as contributory factors in caregiver health and well-being. In that saliva is a relatively economical and comparatively noninvasive biological data source for community-based stress studies, methodological limitations of the study are identified and 5 recommendations are made for future biological marker studies of caregiver distress in community-based settings.

The Orphan Nuclear Receptor NGFIB Regulates Transcription of 3β-Hydroxysteroid Dehydrogenase: IMPLICATIONS FOR THE CONTROL OF ADRENAL FUNCTIONAL ZONATION

3beta-Hydroxysteroid dehydrogenase type 2 (HSD3B2) is a steroid-metabolizing enzyme that is essential for adrenal production of mineralocorticoids and glucocorticoids. Thus, HSD3B2 is expressed at high levels in the glomerulosa and fasciculata, where these steroids are produced. In contrast, the production of dehydroepiandrosterone (DHEA) and DHEA sulfate in the adrenal reticularis is inversely correlated with the expression of HSD3B2. The reasons for the zonal expression of HSD3B2 are not known but represent an important aspect in the biochemical zonation of the adrenal. Using microarray, real time reverse transcriptase-PCR, immunohistochemistry, and HSD3B2 promoter analysis, we demonstrate that the NGFIB family of nuclear hormone receptors plays a critical part in the regulation of HSD3B2 transcription and may play an important role in the functional zonation of the adrenal gland. Microarray analysis of cortisol- versus DHEA sulfate-producing adrenal tissue demonstrated that NGIFB paralleled expression of HSD3B2 with expression much higher in cortisol-producing adrenal tissue; this observation was also demonstrated using real time reverse transcriptase-PCR analysis. In addition, immunohistochemistry confirmed that within adult and fetal adrenal gland NGFIB expression paralleled expression of HSD3B2. Transient transfections into H295R adrenal cells demonstrated that NGFIB family members enhanced HSD3B2 reporter activity but had no effect on a 17alpha-hydroxylase (CYP17) promoter construct. Deletion and mutational analyses of the 5'-flanking region of the HSD3B2 gene identified a consensus NGFIB response element that bound NGFIB in mobility shift assays. Infection of cultured human adrenal cells with adenovirus-containing NGFIB increased cortisol production by 8-fold and increased expression of HSD3B2 mRNA 26-fold over that observed in mock-infected cells. In primary cultures of adrenal cells, ACTH, an activator of HSD3B2, rapidly induced expression of NGFIB. These results suggest that NGFIB plays a crucial role in adrenal zonation by regulating HSD3B2 gene transcription.

Acid–base balance may influence risk for insulin resistance syndrome by modulating cortisol output

Frank metabolic acidosis is known to promote renal excretion of hydrogen ion by induction of glutaminase and other enzymes in the renal tubules. This induction, at least in part, reflects an increase in pituitary output of ACTH and a consequent increased production of cortisol and aldosterone; these latter hormones act on the renal tubules to promote generation of ammonia, which expedites renal acid excretion. Recent evidence suggests that the moderate metabolic acidosis associated with a protein-rich diet low in organic potassium salts – quantifiable by net acid output in daily urine – can likewise evoke a modest increase in cortisol production. Since cortisol promotes development of visceral obesity, and has a direct negative impact on insulin function throughout the body, even a modest sustained up-regulation of cortisol production may have the potential to increase risk for insulin resistance syndrome and type 2 diabetes. This thesis appears to be consistent with previous epidemiological reports correlating high potassium consumption, or a high intake of fruits and vegetables, with reduced risk for diabetes and coronary disease. Future prospective epidemiology should assess whether the estimated acid–base balance of habitual diets – calculated from the ratio of dietary protein and potassium – correlates with risk for insulin resistance syndrome and diabetes.

Spontaneous Remission of Cushing's Disease after Disappearance of a Microadenoma Attached to the Pituitary Stalk

Cushing's disease caused by a microadenoma located near the pituitary stalk is infrequent and spontaneous remission caused by necrosis of a corticotropinoma in such location has not been reported. A 42-year-old woman with ACTH-dependent Cushing's syndrome presented on magnetic resonance imaging (MRI) a 3-mm microadenoma attached to the pituitary stalk. Treatment with ketoconazole normalized urinary free cortisol (UFC) from 433.0 to 66.0 microg/day, although it failed to reduce elevated serum androgen levels (DHEAS 4770 ng/ml). After one year, treatment was stopped and UFC rose again to 936.0 microg/day but one month later the patient presented acute headache and signs of steroid withdrawal syndrome. Endocrine evaluation showed glucocorticoid and androgen deficiency (UFC 5.0 microg/day; DHEAS < 300 ng/ml); control MRI revealed disappearance of the microadenoma. Cushingoid signs subsided and steroid replacement was initiated, proving still necessary over two years after the episode. Infarction or hemorrhage of a corticotrope adenoma could be a probable underlying mechanism although its precipitating factor is unclear. Ketoconazole withdrawal, through abrupt increase in cortisol production and/or the interruption of a hypothetical inhibitory action on cell replication followed by tumor growth and compromise of vascular supply, may be considered as possible triggering factors. To the best of our knowledge, this is the first report of spontaneous remission of Cushing's disease caused by presumed infarction of a microadenoma, unusually located in the superior rim of the pituitary, attached to the stalk.

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Mutant K-ras oncogene regulates steroidogenesis of normal human adrenocortical cells by the RAF-MEK-MAPK pathway.

The result of our previous study has shown that the K-ras mutant (pK568MRSV) transfected human adrenocortical cells can significantly increase cortisol production and independently cause cell transformation. The aim of this study is to investigate the effect of the active K-ras oncogene on the cortisol production in normal human adrenocortical cells. First we used isopropyl thiogalactoside to induce the inducible mutant K-ras expression plasmid, pK568MRSV, in the stable transfected human adrenocortical cells. The result showed that the increase of RasGTP levels in transfected cells was time-dependent after isopropyl thiogalactoside induction. Additionally, results from Western blot analysis revealed significant elevation in phosphorylation of c-Raf-1 and Mitogen-activated protein kinase. We also detected the levels of mRNA encoding Cholesterol side-chain cleavage enzyme (P450SCC), 17α-Hydroxylase/17,20-lyase (P450c17) and 3β-Hydroxysteroid dehydrogenase (3βHSD) were increased in human adrenocortical cells transfected with mutant K-ras after IPTG treatment. The increase of mRNA amount in P450scc P450c17 and 3βHSD and the elevation of cortisol level were inhibited with a pretreatment of PD098059, a specific extracellular signal-regulated kinase inhibitor. In our previous report, we proved that lovastatin, a pharmacological inhibitor of p21ras function, also reversed the increase of cortisol level in mutant K-ras stably transfected human adrenocortical cells. Taken together, these findings proved that the active mutant Ras enhanced not only cell proliferation but also steroidogenesis in steroidogenic phenotype cells by activating Raf-MEK-MAPK related signal transduction pathway. Therefore, we believe that K-ras mutants influence regulation of steroidogenesis in adrenocortical cells through RAF-MEK-MAPK pathway.British Journal of Cancer (2002) 87, 1000–1005. doi:10.1038/sj.bjc.6600589 www.bjcancer.com© 2002 Cancer Research UK