Search

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3β-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Stimulation of prostate growth is a major concern with testosterone therapy in older hypogonadal men. As a result, nonsteroidal selective androgen receptor modulators with anabolic activity but less prostate stimulation are being developed. Anabolic steroids might exhibit similar tissue selectivity. We hypothesized the anabolic steroid 19-nor-4-androstenediol-3beta,17beta-diol (3beta,19-NA) would increase muscle, lean body mass (LBM), and bone mineral density (BMD) with little stimulation of prostate growth. Male Sprague Dawley rats were implanted with SILASTIC brand (Dow Corning, Midland, MI) capsules containing 3beta,19-NA (4, 8, or 16 cm), dihydrotestosterone (DHT) (8 cm), 19-nortestosterone (16 cm), or four empty capsules after undergoing either a sham operation (intact) or orchidectomy (ORX). Serum gonadotropins, measured after 4, 8, or 24 wk of treatment, were significantly lower in 3beta,19-NA-treated vs. untreated, intact, and ORX rats (P < 0.05), and testosterone was lowered by 3beta,19-NA-treatment of intact animals. LBM and BMD were assessed after 20 wk, and 4 wk later, rats were killed for levator ani muscle and prostate weights. Compared with ORX rats, 3beta,19-NA-treated rats had dose-dependent higher levator ani muscle weights, LBM, and BMD, which were similar to intact and DHT-treated rats at the highest 3beta,19-NA dose. In contrast, prostate weights in all 3beta,19-NA-treated groups were similar to ORX rats and lower than intact and DHT- and 19-nortestosterone-treated rats even at the highest 3beta,19-NA dose. In summary, 3beta,19-NA increases muscle and bone mass without significant stimulation of prostate growth, suggesting it may have some properties of a steroidal selective androgen receptor modulator. Anabolic steroids such as 3beta,19-NA should be studied further to determine their mechanisms of tissue selectivity and effects in men.

The age-related decrease in serum T levels is associated with impairments in food intake and weight regulation and alterations in brain peptides that regulate energy balance. To test the hypothesis that reduced T levels contribute to altered hypothalamic cocaine-amphetamine-regulated transcript (CART) and NPY gene expression, the mRNA content of these neuropeptides was measured by in situ hybridization in sham-operated (intact), castrated, and T-replaced castrated young and old male Brown Norway rats. T levels in T-replaced young and old rats were similar to those in intact young animals. Compared with castrated rats, arcuate nucleus CART mRNA was lower and NPY mRNA was higher in both young and old T-replaced castrated animals, suggesting reciprocal regulation of these peptides by T; these T-induced changes were localized primarily in the rostral arcuate and were markedly attenuated in old animals. Compared with intact animals, paraventricular nucleus CART mRNA was lower in castrated animals and similar in T-replaced young and old rats. We conclude that hypothalamic CART and NPY neurons remain responsive to T regulation in old rats, albeit less so than in young animals, suggesting that the age-related reduction of T contributes in part to altered brain neuropeptide gene expression favoring anorexia and wasting with aging.

Pineal melatonin secretion declines with aging, whereas visceral fat, plasma insulin, and plasma leptin tend to increase. We have previously demonstrated that daily melatonin administration at middle age suppressed male rat intraabdominal visceral fat, plasma leptin, and plasma insulin to youthful levels; the current study was designed to begin investigating mechanisms that mediate these responses. Melatonin (0.4 microg/ml) or vehicle was administered in the drinking water of 10-month-old male Sprague Dawley rats (18/treatment) for 12 weeks. Half (9/treatment) were then killed, and the other half were submitted to cross-over treatment for an additional 12 weeks. Twelve weeks of melatonin treatment decreased (P<0.05) body weight (BW; by 7% relative to controls), relative intraabdominal adiposity (by 16%), plasma leptin (by 33%), and plasma insulin (by 25%) while increasing (P<0.05) locomotor activity (by 19%), core body temperature (by 0.5 C), and morning plasma corticosterone (by 154%), restoring each of these parameters toward more youthful levels. Food intake and total body fat were not changed by melatonin treatment. Melatonin-treated rats that were then crossed over to control treatment for a further 12 weeks gained BW, whereas control rats that were crossed to melatonin treatment lost BW, but food intake did not change in either group. Feed efficiency (grams of BW change per g cumulative food intake), a measure of metabolic function, was negative in melatonin-treated rats and positive in control rats before cross-over (P<0.001); this relationship was reversed after cross-over (P<0.001). Thus, melatonin treatment in middle age decreased BW, intraabdominal adiposity, plasma insulin, and plasma leptin, without altering food intake or total adiposity. These results suggest that the decrease in endogenous melatonin with aging may alter metabolism and physical activity, resulting in increased BW, visceral adiposity, and associated detrimental metabolic consequences.

Human and rat pineal melatonin secretion decline with aging, whereas visceral fat and plasma insulin levels increase. Melatonin modulates fat metabolism in some mammalian species, so these aging-associated melatonin, fat and insulin changes could be functionally related. Accordingly, we investigated the effects of daily melatonin supplementation to male Sprague-Dawley rats, starting at middle age (10 months) and continuing into old age (22 months). Melatonin was added to the drinking water (92% of which was consumed at night) at a dosage (4 microg/ml) previously reported to attenuate the aging-associated decrease in survival rate in male rats, as well as at a 10-fold lower dosage. The higher dosage produced nocturnal plasma melatonin levels in middle-aged rats which were 15-fold higher than in young (4 months) rats; nocturnal plasma melatonin levels in middle-aged rats receiving the lower dosage were not significantly different from young or middle-aged controls. Relative (% of body wt) retroperitoneal and epididymal fat, as well as plasma insulin and leptin levels, were all significantly increased at middle age when compared to young rats. All were restored within 10 weeks to youthful (4 month) levels in response to both dosages of melatonin. Continued treatment until old age maintained suppression of visceral (retroperitoneal + epididymal) fat levels. Plasma corticosterone and total thyroxine (T4) levels were not significantly altered by aging or melatonin treatment. Plasma testosterone, insulin-like growth factor I (IGF-I) and total triiodothyronine (T3) decreased by middle age; these aging-associated decreases were not significantly altered by melatonin treatment. Thus, visceral fat, insulin and leptin responses to melatonin administration may be independent of marked changes in gonadal, thyroid, adrenal or somatotropin regulation. Since increased visceral fat is associated with increased insulin resistance, diabetes, and cardiovascular disease, these results suggest that appropriate melatonin supplementation may potentially provide prophylaxis or therapy for some prominent pathologies associated with aging.

Aging in man is associated with alterations in food intake (FI) and body weight (BW). To establish a model of age-related alterations in FI and BW regulation, FI and BW were determined in young (3-month-old), middle-aged (12-month-old), and old (24-month-old) male Brown Norway rats during ad libitum feeding and after 72 h of fasting. FI was reduced with aging both during ad libitum feeding and after fasting. With fasting, young rats lost more BW than older rats, but regained BW more rapidly during refeeding. To determine whether age-related impairments in FI and BW regulation are mediated by neuropeptide Y (NPY), a potent stimulator of FI, we compared arcuate nucleus prepro-NPY (ppNPY) messenger RNA (mRNA) by in situ hybridization in fasted and ad libitum-fed (fed) young, middle-aged, and old rats. ppNPY mRNA declined with aging in both fed and fasted rats. Although ppNPY mRNA increased with fasting in all age groups, this response was attenuated with aging. In conclusion, impaired FI and BW recovery after fasting is associated with reduced NPY responsiveness to fasting in aging rats. Impaired activation of the hypothalamic NPY pathway may, therefore, contribute to age-related alterations in FI and BW regulation.

Neuropeptide-Y (NPY) is thought to modulate reproductive function and food intake. NPY neuronal activity is modulated by sex steroids, and NPY secretion declines with aging. We hypothesized that reduced NPY secretion with aging is due to decreased NPY gene expression, and that this decrease is independent of testicular feedback. To test this hypothesis, arcuate nucleus (ARC) prepro-NPY (ppNPY) mRNA levels, determined by in situ hybridization, and serum testosterone levels, determined by RIA, were compared in sham-operated and orchidectomized young (3 months old), middle-aged (13 months old), and old (23 months old) male Brown Norway (BN) rats. Hybridization area and average optical density (OD) were used as indices of ARC ppNPY mRNA content. In sham-operated rats, both ppNPY mRNA hybridization area and OD decreased progressively with aging, whereas serum testosterone levels were decreased only in old, not in middle-aged or young, rats. In orchidectomized rats, ppNPY mRNA hybridization area also decreased significantly with aging, although OD did not change significantly. The ppNPY mRNA hybridization area was lower in orchidectomized than in intact young and middle-aged rats, whereas OD was unchanged by orchidectomy. Furthermore, the effects of aging and orchidectomy on ppNPY mRNA levels were not localized to a specific region of the ARC. We conclude that in the male BN rat, ARC NPY gene expression is decreased with aging independently of the effects of testicular feedback. This reduction in NPY synthetic capacity may contribute to altered reproductive function and food intake with aging.

Fasting causes a decrease in serum gonadotropin and testosterone (T) levels in the male rat. We hypothesized that this fasting-induced decrease in serum gonadotropins is due to reduced GnRH gene expression. PreproGnRH mRNA (GnRH mRNA) and serum gonadotropin and T levels by RIA were compared in 90 day old male Wistar rats fed ad lib or fasted for 60 hours (n = 8/group). GnRH mRNA was quantitated by in situ hybridization and computerized image analysis in 18 anatomically matched 20 microns coronal sections from the medial preoptic area (MPOA) and diagonal band of Broca (DBB), using a 35S-labeled 48 base oligodeoxynucleotide probe complementary to rat GnRH mRNA. LH and FSH levels were lower in fasted vs fed rats (LH: 0.17 +/- 0.03 vs 0.36 +/- 0.06 ng/ml, p < 0.01; FSH: 6.7 +/- 0.5 vs 8.7 +/- 0.6 ng/ml, p < 0.05). T levels were also decreased in fasted (1.34 +/- 0.36 ng/ml) vs fed (2.21 +/- 0.30 ng/ml) rats, although this was not statistically significant (p = 0.08). In both the MPOA and DBB, the number of neurons expressing GnRH was lower in fasted vs fed rats (10.6 +/- 0.4 vs 15.8 +/- 1.2 cells/section, p < 0.001), while cellular GnRH mRNA content was unchanged with fasting (83 +/- 1 vs 80 +/- 2 grains/cell). These data support the hypothesis that in the male Wistar rat, decreased gonadotropin secretion with fasting is due, at least in part, to a reduction in GnRH gene expression.

In the male rat, age-associated reproductive decline is thought to be due in part to diminished GnRH secretion. We tested the hypothesis that the age-related decrease in GnRH secretion is due to decreased GnRH gene expression by comparing GnRH mRNA and peptide content in the anterior forebrain of intact young and old male rats. Since sex steroids modulate GnRH secretion, we also determined hypothalamic-pituitary responsiveness to removal of testicular feedback by comparing GnRH mRNA and gonadotropin levels in intact and orchidectomized young and old rats. In an initial study, 10 20-micron coronal sections from the medial preoptic area (MPOA) were anatomically matched and compared in intact young (3-month-old) and old (24-month-old) male F344 rats (n = 5/group). In another group of young and old male rats (n = 8-12/group), animals were randomly assigned to be either orchidectomized or sham operated. Rats were killed 21 days after surgery, and comparisons were made in 12 anatomically matched sections of MPOA and 4 matched sections of diagonal band of Broca. In both studies, GnRH mRNA was quantitated by in situ hybridization, using a 35S-labeled oligodeoxynucleotide probe complementary to rat prepro-GnRH mRNA and a computerized image analysis system. In a third study, GnRH content was measured by RIA in microdissected regions of the arcuate nucleus and median eminence in intact 3- and 24-month-old male rats (n = 10 and 8, respectively). Serum LH, FSH, and testosterone (T) levels were measured by RIA in trunk blood of all animals. The number of neurons expressing the GnRH gene in the MPOA was significantly lower in sham-operated old rats (mean +/- SEM, 10.5 +/- 0.5 cells/section) than in young rats (13.7 +/- 0.7 cells/section; P less than 0.01), while cellular GnRH mRNA content was unchanged with age (103 +/- 1 vs. 103 +/- 2 grains/cell). Similar results were obtained in intact old and young rats. GnRH peptide content was significantly decreased in the arcuate nucleus of intact old (0.5 +/- 0.08 ng/mg protein) compared to young animals (2.3 +/- 0.7 ng/mg protein; P less than 0.05), with a trend toward a decrease in the median eminence of old (53 +/- 2 ng/mg protein) vs. young rats (69 +/- 7 ng/mg protein; P = 0.06).(ABSTRACT TRUNCATED AT 400 WORDS)

Orchidectomy results in increased LH and FSH levels by removal of negative feedback at the hypothalamus and pituitary gland. However, the precise central nervous system mechanisms involved in elevation of gonadotropins after castration are unclear. We tested the hypothesis that catecholamine neuronal activity mediates the rise in serum LH that occurs after withdrawal of testosterone (T) negative feedback. The effects of acute and selective T withdrawal on brain catecholamine and LHRH activity and serum LH levels were determined in adult male rats. At the time of orchidectomy, rats were given sc implants of both T-containing and empty Silastic capsules. After recovery from surgery, the T-containing capsule was atraumatically removed from half of the animals (T-withdrawn), while the empty capsule was removed from the remaining rats (T-replaced). Rats were killed before and 6, 12, and 24 h after capsule removal. Serum T and LH levels were determined by RIA. Catecholamine content in microdissected nuclei of the LHRH neuronal system [medial preoptic nucleus, suprachiasmatic nucleus, retrochiasmatic area, arcuate nucleus (ARC), and median eminence (ME)] was measured by HPLC with electrochemical detection. Norepinephrine turnover rate was also determined in these areas by measuring the rate of decline of NE levels after blockade of synthesis with diethyldithiocarbamate. Additionally, LHRH content was measured by RIA within the ARC and ME. In T-replaced rats, the T capsules maintained serum T and LH levels within the normal range for intact male rats. In T-withdrawn rats, T levels fell into castrate range by 6 h after removal of the T capsule [0.12 +/- 0.04 ng/ml (mean +/- SEM); P less than 0.01 vs. T-replaced], and LH levels increased significantly from 0.23 +/- 0.04 ng/ml before capsule removal to 1.31 +/- 0.14 and 2.80 +/- 0.20 ng/ml 12 and 24 after T withdrawal, respectively (both P less than 0.01 vs. T-replaced). Despite a marked increase in serum LH levels, no significant changes in catecholamine content or NE turnover rate were observed in any of the hypothalamic nuclei of the LHRH neuronal system at any time after T withdrawal. ARC and ME LHRH levels also did not change significantly at any point after T withdrawal. These results suggest that activation of hypothalamic catecholamine neuronal activity is not required for the rise in serum LH levels after acute withdrawal of T negative feedback.

During sexual development in the male rat, serum testosterone (T) levels increase markedly at 45-60 days of age. At the time of the pubertal rise in T levels, activation of the hypothalamic-pituitary axis is difficult to demonstrate, since there is little change in serum LH levels and a decrease in serum FSH levels. We determined whether experimental maintenance of stable pubertal T levels in these animals as they passed through the normal age of puberty would allow demonstration of a major increase in serum gonadotropin levels. At 14-15 days of age, male rats were castrated and outfitted with either T-containing or empty Silastic capsules. Another group of rats was left intact and outfitted with empty capsules. At various times between 29 and 58 days of age, blood was drawn for measurement of serum LH, FSH, and T levels. In the T-implanted castrated rats, serum T levels were comparable to those in midpubertal intact rats, without significant differences among age groups. In this setting of stable T levels, serum LH and FSH were suppressed to levels at or below those in pubertal intact rats until 51 days of age, when they increased significantly into the untreated castrate range. In contrast, untreated castrate animals demonstrated markedly reduced serum T and elevated LH and FSH levels that did not change significantly throughout the entire study. In intact rats, serum T levels were stable until 58 days of age, when they increased over 2-fold; serum LH levels did not change significantly with age, and serum FSH levels decreased significantly by 54 days of age. A separate group of rats was castrated and outfitted with T-containing Silastic capsules at 21 days of age. In these animals, there were significant increases in hypothalamic LHRH, norepinephrine (NE), and dopamine levels and NE turnover rate at 56 compared to 36 days of age. We conclude that stable pubertal levels of T are able to suppress gonadotropin levels in castrated rats until the normal age of puberty, at which time LH and FSH levels increase markedly. This decrease in sensitivity of the hypothalamic-pituitary axis to T negative feedback at puberty is accompanied by increases in hypothalamic LHRH, NE, and dopamine levels and NE turnover rate. These results provide direct evidence for activation of the central nervous system-pituitary mechanism regulating gonadotropin secretion at puberty in the male rat.(ABSTRACT TRUNCATED AT 400 WORDS)