In humans, visceral obesity may result from local excessive tissue formation of cortisol, produced from cortisone by the action of 11 beta HSD1. 11 beta HSD1 is highly upregulated during adipocyte differentiation in parallel with other genes such as liver X receptor (LXR). Oxysterols are ligands for LXR alpha, the predominant receptor subtype in adipose tissue. 11 beta HSD1 activity is also increased by TNF alpha and attenuated by insulin. This study examines the interaction of oxysterols and retinoic acid with cytokines and insulin to regulate 11 beta HSD1 activity in human adipose tissue. Adipose stromal cells from breast and abdominal subcutaneous tissue were incubated with 22R hydroxycholesterol (22ROHC) and TNF alpha, IL-6, 9-cis retinoic acid (9 cis RA), or insulin.11 beta HSD1 activity in breast and abdominal stromal cells was increased approximately 4 and 2 fold respectively by TNF alpha. 22ROHC reduced basal and TNF alpha stimulated activity by 24% and 44% respectively in breast tissue, but was more effective in reducing basal 11 beta HSD1 activity in abdominal tissue. 22ROHC and insulin reversed TNFa stimulated 11PHSD1 activity in both tissues. 22ROHC and 9 cis RA also reduced enzyme activity, compared to oxysterol. Oxysterols can therefore modulate intracellular glucocorticoid levels by reducing basal and hormonally stimulated 11 beta HSD1 activity.

Studies in vitro and in vivo have shown that glucocorticoids and sex steroids play an important role in bone physiology and pathophysiology. In this study we investigated glucocorticoid and sex steroid conversion in osteoblasts derived from lumbar vertebrae of adult male and female rats. Progesterone was converted to inactive 20α-OH-progesterone and the conversion at day 5 was 16-fold greater than that at day 13 in both sexes (male/female, 2.7/1.7 and 0.16/0.10 nM/105cells/24 h, respectively). The conversion of inactive androstenedione to active androgen testosterone in males and females was 1.2- and 2.4-fold greater at day 5 than at day 13, respectively (male/female, 0.40/0.70 and 0.34/0.30 nM/105cells/24 h, respectively). These results suggest that osteoblasts possess 20α-hydroxysteroid dehydrogenase (HSD) and 17β-HSD and that their activities are dependent on the stage of cell differentiation. At day 5, dehydroepiandrosterone was converted to androstenedione (male/female, 0.25/0.098 nM/105cells/24 h), to 7α-OH-dehydroepiandrosterone (male/female, 0.49/0.39 nM/105cells/24 h) and to 5-androstene-3β,17β-diol (male/female, 0.18/0.37 nM/105cells/24 h), indicating the presence of 3β-HSD, 7α-hydroxylase and 17β-HSD, respectively. Both 3β-HSD and 7α-hydroxylase activities declined with cell differentiation. Hormonally inactive cortisone was converted to active cortisol (male/female, 0.34/0.29 mM/106cells/6 h) while conversion of cortisol to cortisone was not detectable, suggesting the presence of oxoreductase activity of 11β-HSD-1. These results show, for the first time, the presence of 7α-hydroxylase and 20α-HSD in osteoblasts, and provide further evidence that osteoblasts metabolize a variety of steroid hormones and can thus regulate tissue responsiveness to them.

Obesity is frequently associated with insulin-resistance and abnormal glucose homeostasis. Recent evidence indicates that TNFα may play a role in mediating the insulin-resistance of obesity through its overexpression in adipose tissue. Previously, we have shown that human adipose stromal cells contain 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mRNA and activity. The present study was designed to examine the effects of insulin on 11β-HSD1 expression in human adipose stromal cells under basal and TNFα-stimulated conditions. The cells were obtained from breast adipose tissue by collagenase digestion, and grown to confluence under replicating conditions in 10% fetal bovine serum. The cells were transferred to serum-free medium for 24 h prior to treatment with either TNFα, insulin or both for a further 24 h. The level of 11β-HSD1 reductase activity was determined by measuring the conversion of [3H]-cortisone to [3H]-cortisol at a substrate concentration of 10 nM. Treatment with TNFα at concentrations of 0.1–10 ng/ml resulted in a dose dependent increase in 11β-HSD1 reductase activity from 1.5 to 10-fold. Insulin (0.1–100 nM) had no effect under basal conditions, but inhibited the stimulatory effects of TNFα (5 ng/ml) on 11β-HSD1 reductase activity in a dose dependent fashion (8–66%) inhibition). Northern blot analysis revealed corresponding changes in the level of 11β-HSD1 mRNA, suggesting that the effects of TNFα and insulin on 11β-HSD1 activity are mediated at the level of gene transcription. The interaction between insulin and TNFα suggests that local and systemic factors may act in a concerted fashion to modulate glucocorticoid activity in adipose and other peripheral tissues.

The biological activity of glucocorticoids in target tissues can be influenced by locally produced 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), the enzyme responsible for the interconversion of cortisol and its inactive metabolite cortisone. In human adipose stromal cells, glucocorticoids are potent stimulators of the conversion of androgens to estrogens (aromatase activity). The present study was designed to determine whether 11 beta-HSD activity was present in human adipose stromal cells, and if changes in the activity of this enzyme could influence aromatase activity. 11 beta-HSD activity was determined by a radiometric conversion assay in breast adipose tissue from six patients. It was found that both dehydrogenase (cortisol to cortisone) and reductase (cortisone to cortisol) activities were present in all six subjects, and the reductase activity was always predominant. Carbenoxolone (CBX), a potent inhibitor of 11 beta-HSD, added to the culture medium at 50 and 200 microM, resulted in 39 +/- 4% and 85 +/- 1% inhibition, respectively, of both reductase and dehydrogenase activity of 11 beta-HSD. To determine whether alterations in 11 beta-HSD could influence aromatase activity, the effect of CBX (200 microM) on cortisol- and cortisone-induced changes in the conversion of androstenedione to estrone was examined. CBX prevented the stimulatory effect of cortisone and minimally potentiated the stimulatory effect of cortisol on aromatase activity, reflecting an inhibition of the local activation of cortisone and the local metabolism of cortisol, respectively. In order to determine whether the product of the 11 beta-HSD 1 gene was responsible for the observed 11 beta-HSD activity, total RNA extracts from these cells were subjected to Northern blot analysis using human 11 beta-HSD 1 cDNA as the probe. A single 1.8 11 beta-HSD 1 transcript was detected, and its abundance was reduced by CBX. No 11 beta-HSD 2 mRNA was detected. The present results demonstrate that the 11 beta-HSD 1 gene is expressed and functional in human breast adipose stromal cells and that changes in 11 beta-HSD 1 activity result in alterations in aromatase activity.

The clinical behavior of growth hormone (GH)-producing pituitary tumors is known to vary greatly; however, the events underlying this variability remain poorly understood, Herein we demonstrate that tumor overexpression of the GH-releasing hormone (GHRH) gene is one prognostically informative event associated with the clinical aggressiveness of somatotroph pituitary tumors. Accumulation of GHRH mRNA transcripts was demonstrated in 91 of a consecutive series of 100 somatotroph tumors by in situ hybridization; these findings were corroborated by Northern analysis and reverse transcriptase polymerase chain reaction, and protein translation was confirmed by Western blotting, By comparison, transcript accumulation was absent or negligibly low in 30 normal pituitary glands, GHRH transcripts were found to preferentially accumulate among clinically aggressive tumors, Specifically, GHRH mRNA signal intensity was 1) linearly correlated with Ki-67 tumor growth fractions (r = 0.71; P < 0.001), 2) linearly correlated with preoperative serum GH levels (r = 0.56; P = 0.01), 3) higher among invasive tumors (P < 0.001), and 4) highest in those tumors in which post-operative remission was not achieved (P < 0.001). Using multivariate logistic regression, a model of postoperative remission likelihood was derived wherein remission was defined by the single criterion of suppressibility of GH levels to less than 2 ng/ml during an oral glucose tolerance test, In this outcome model, GHRH mRNA signal intensity proved to be the most important explanatory variable overall, eclipsing any and all conventional clinicopathological predictors as the single most significant predictor of postoperative remission; increases in GHRH mRNA signal were associated with marked declines in remission likelihood, The generalizability of this outcome model was further validated by the model's significant performance in predicting postoperative remission in a random sample of 30 somatotroph tumors treated at another institution. These data indicate that overexpression of GHRH gene is an event associated with the neoplastic progression and clinical aggressiveness of somatotroph adenomas. More generally, these data merge essential elements of the hypothalamic and pituitary hypotheses of pituitary tumorigenesis, providing for a more unified concept of neoplastic progression in the pituitary.

Human adipose stromal cells provide an excellent model for studying a variety of metabolic processes in an in vitro system These are normal cells derived from subcutaneous or omental adipose tissue. Under specific culture condition, they will differentiate without replication into cells resembling mature adipocytes or will replicate, become confluent, and grow in subculture. The initial observation that the stromal vascular fraction of human omental adipose tissue contained a fibroblast-like cell that was a possible adipocyte precursor was made by Poznanski et al (1). Subsequent studies demonstrated enzymological and morphological properties that developed during differentiation (2-4). The primary interest has focused on the conditions required to stimulate these cells to differentiate into adipocytes, which can accumulate lipid and possess the enzymes involved in lipogenesis, including glycerol-3-phosphate dehydrogenase (GPDH) and lipoprotein lipase (LPL).

Annals of the New York Academy of SciencesVolume 774, Issue 1 p. 316-318 7α-Hydroxylation of the Adrenal Androgens Dehydroepiandrosterone and Androst-5-ene-3β, 17β-Diol Predominates in Differentiating Human Adipose Stromal Cells M. W. KHALIL, M. W. KHALIL Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this authorB. STRUTT, B. STRUTT Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this authorD. W. KILLINGER, D. W. KILLINGER Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this author M. W. KHALIL, M. W. KHALIL Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this authorB. STRUTT, B. STRUTT Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this authorD. W. KILLINGER, D. W. KILLINGER Department of Medicine and Lawson Research Institute St. Joseph's Health Centre and University of Western Ontario London, Ontario, Canada N6A 4V2Search for more papers by this author First published: December 1995 https://doi.org/10.1111/j.1749-6632.1995.tb17396.x-i1Citations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume774, Issue1Dehydroepiandrosterone (DHEA) and AgingDecember 1995Pages 316-318 RelatedInformation

The metabolism of dehydroepiandrosterone (DHA) and androstenedione (A-dione) was studied in cultured human adipose stromal cells obtained from breast tissue of six premenopausal patients undergoing reduction mammoplasty. Cells were maintained in culture in the presence of 10% fetal bovine serum. Studies were carried out during the proliferative and confluent phases of culture with radiolabelled substrates (2 μCi, 10 nM). During the early phases of replication 7α-hydroxydehydroepiandrosterone (7α-OHDHA) was formed from DHA. As the cells reached confluence, the major metabolite of DHA in cells from all patients was A-dione indicating the presence of 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD). The conversion of DHA to A-dione was variable among patients when cells were confluent with 30–80% of substrate being metabolized to this product. Adipose stromal cells synthesized estrone (E1) from DHA once A-dione formation was established. Under basal conditions E1 was obtained in cells from three of the six patients examined with up to 36% substrate converted to this product. Dexamethasone (Dex 10−7 M) stimulated E1 formation in cells from all subjects with up to 50% of substrate being converted. Parallel studies comparing the conversion of DHA with A-dione to E1 revealed that as the cells became confluent, E1 formation from both substrates was similar. The pattern of steroid metabolism was also examined in primary culture and in subculture. Passage 1 cells continued to form A-dione as a major metabolite of DHA, and did not revert to the pattern of metabolism found in primary cells during the early stages of replication, when 7α-hydroxylation predominated. Human adipose stromal cells actively metabolize DHA, producing 7α-OHDHA, A-dione and E1 as principal metabolites. Changes in the circulating levels of DHA may directly influence the formation of E1 in peripheral tissues. This source of E1 will be modulated by factors controlling 3β-HSD and aromatase activities.

We aim to correlate point mutations in the androgen receptor gene with receptor phenotypes and with clinical phenotypes of androgen resistance. In two families, the external genitalia were predominantly female at birth, and sex-of-rearing has been female. Their androgen receptor mutation changed arginine-839 to histidine. In a third family, the external genitalia were predominantly male at birth, and sex-of-rearing has been male: their codon 839 has mutated to cysteine. In genital skin fibroblasts, both mutant receptors have a normal androgen-binding capacity, but they differ in selected indices of decreased affinity for 5 alpha-dihydrotestosterone or two synthetic androgens. In transiently cotransfected androgen-treated COS-1 cells, both mutant receptors transactivate a reporter gene subnormally. The His-839 mutant is less active than its partner, primarily because its androgen-binding activity is more unstable during prolonged exposure to androgen. Adoption of a nonbinding state explains a part of this instability. In four other steroid receptors, another dibasic amino acid, lysine, occupies the position of arginine-839 in the androgen receptor. Androgen receptors with histidine or cysteine at position 839 are distinctively dysfunctional and appear to cause different clinical degrees of androgen resistance.

Lawson Research Institute, St. Joseph's Health Centre, University of Western Ontario, London, Ontario, Canada.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with <30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Studies of the metabolism of dehydroepiandrosterone (DHA) by cultured human adipose stromal cells revealed that the most abundant metabolite detected by HPLC was a polar compound accounting for up to 45% of total radioactivity. This metabolite was isolated by chromatography on Lipidex 5000 from the culture medium of breast adipose stromal cells cultured with unlabelled DHA (5 μM) and identified by combined capillary gas chromatography and mass spectrometry as 7α-hydroxydehydroepiandrosterone (7α-OHDHA). In breast adipose stromal cells, the conversion of DHA to 7α-OHDHA was linear from a substrate concentration of 10 nM to 1 μM. At 1 μM substrate concentration, the formation of 7α-OHDHA in four patients ranged from 6.1 to 22.5 ng/105 cells/24 h. Incubations carried out in primary culture and up to the fifth subculture revealed continued formation of 7α-OHDHA. Adipose stromal cells from abdomen, flank and perinephric fat also produced 7α-OHDHA from DHA. These studies have shown that 7α-OHDHA is a major metabolite of DHA in human adipose stromal cells. The variability from patient to patient and the magnitude of this conversion suggests that this pathway may play an important role in the peripheral metabolism of DHA.

The case of a 35-year-old man with pituitary macroadenoma who was complaining of reduced sexual activity is presented. Histologic examination showed a chromophobic adenoma corresponding mainly to a null cell adenoma at the ultrastructural level. Focal plurihormonality and plurimorphous differentiation of adenoma cells were demonstrated by immunohistochemical and electron-microscopic studies. It is suggested that adenomatous null cells represent pluripotent progenitor cells capable of transforming to different hormone-producing cell types. The factors accounting for differentiating to various cell populations have yet to be elucidated.

To establish whether the conversion of androstenedione (A) to estrogens and 5 alpha-reduced metabolites in human adipose tissue was determined by the site of origin of the tissue, studies were carried out on adipose stromal cells from different body sites. Adipose tissue was obtained from the breast, omentum, abdomen, lower thigh, upper thigh, buttock, and flank from patients undergoing liposuction for cosmetic reasons or at surgery. Stromal cells were isolated after incubation of the adipose tissue with collagenase and were grown in culture using alpha-minimal essential medium (MEM) + 15% fetal calf serum. Studies of A metabolism were carried out when the cells were between days 4 and 12 in culture. After an 8-hour incubation with (3H)-A as substrate, estrone (E1), testosterone (T), 5 alpha-androstanedione (5 alpha-A-dione), androsterone (AND), and dihydrotestosterone (DHT) were isolated using thin layer and paper chromatography. The conversion per 1 x 10(6) cells of A of E1 was more than 10-fold greater in the upper thigh, buttock, and flank than in the breast, lower thigh, abdomen, or omentum (0.13-3.0 vs 0.01-0.09%). The formation of 5 alpha-reduced androgens varied from 0.86-10% and was similar in tissue from different body sites. Cortisol (10(-7) M) stimulated E1 formation 3- to 10-fold in cells from all sites, whereas 5 alpha-reductase activity was either unchanged or increased moderately (up to twofold). In cells from the abdomen, omentum, and lower thigh, the formation of 5 alpha-reduced androgens was more than 10-fold greater than the formation of E1. In cells from the upper thigh, buttock, and flank, E1 formation was comparable to 5 alpha-reduced androgen formation. These studies show marked differences in the relative conversion of A to estrogens and 5 alpha-reduced androgens in adipose stromal cells depending on their site of origin, and they suggest that the distribution of body fat may be a major factor in determining the biologic effects of secreted androgens.