bluuebunny/crossref_metadata_2025 huggingface dataset:
Long-term in vitro exposure to high glucose increases proinsulin-like-molecules release by isolated human islets
Authors: Bertuzzi F , Saccomanno K , Socci C , Davalli AM , Taglietti MV , Berra C , Dalcin E , Monti LD , Pozza G , Pontiroli AE
Published: December 2004

The aim of this study was to determine the effect of long-term in vitro exposure to high glucose on the release and content of proinsulin and insulin in human islets. After 48 h culture in CMRL medium at 5.5 mM (control islets) and 16.7 mM glucose (experimental islets), islets were perifused and acutely stimulated with 16.7 mM glucose, followed by 3.3 mM glucose. Compared with control islets, experimental islets showed a higher basal release of true insulin and proinsulin-like-molecules (PLM), with no increase of true insulin and PLM release in response to 16.7 mM glucose, and a paradoxical true insulin release in response to 3.3 mM glucose; the PLM/total insulin ratio increased significantly after 16.7 mM glucose. Moreover these islets showed a decreased true insulin content and an increased PLM/total insulin ratio. Quantitative ultrastructural analysis of granules, supported by double gold immunostaining with monoclonal antibodies against proinsulin and insulin, showed an increased proinsulin to insulin ratio in beta-cells from experimental islets. These data support in vitro what was recently shown in vivo, and further confirm that culture in high glucose is a useful tool to mimic the effect of in vivo chronic hyperglycemia on human beta-cell function.
Differential distribution of gelatinases and tissue inhibitor of metalloproteinase-1 in the rat ovary
Authors: Bagavandoss P
Published: December 2004

The distribution of gelatinases/matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in neonatal and gonadotropin-primed immature rat ovaries was studied by immunofluorescent microscopy. Immature female Long-Evans rats were primed with 15 IU pregnant mare's serum gonadotropin (PMSG) in 100 microliters PBS. Two days later, to induce ovulation, the rats were injected with human chorionic gonadotropin (hCG, 5 IU/100 microliters PBS). The animals were killed at appropriate times and the ovaries removed and processed for cryostat or paraffin sectioning. Ovaries were also obtained from 7-day-old neonatal rats and processed as above. In the neonatal rat ovary, MMP-2 was present in the follicle and in the ovarian surface epithelium. MMP-9 was not detectable in the neonatal ovary. TIMP-1 was present in the oocyte and in the surface epithelium. In the PMSG-primed ovary, MMP-2 was present in the granulosa and thecal cells of the ovary. MMP-9 distribution, however, was restricted to the interstitial and thecal cells. TIMP-1 was mainly present in the blood vessels and thecal cells, with minor staining in the granulosa cells. In the developing corpus luteum, luteal and endothelial cells were positive for MMP-2. MMP-9 localization was restricted to the plasma membrane of the luteal and interstitial cells. TIMP-1 was clearly observed in the luteal capillaries and, to a lesser extent, in the luteal cell plasma membrane. This distribution of MMP-2, MMP-9, and TIMP-1 in the corpus luteum persisted throughout the life span of the corpus luteum. The spatial and temporal distribution of the gelatinases and TIMP-1 suggests unique roles for these proteins in the rat ovary.
Tissue-specific induction of 17 beta-hydroxysteroid dehydrogenase type IV by peroxisome proliferator chemicals is dependent on the peroxisome proliferator-activated receptor alpha
Authors: Fan LQ , Cattley RC , Corton JC
Published: December 2004

The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) family of proteins regulates the levels of the active 17 beta-hydroxy forms of sex steroids. The expression of 17 beta-HSD type IV is induced by peroxisome proliferator chemicals (PPC) in rat liver. In order to characterize more generally the impact of PPC on 17 beta-HSD expression, we determined (1) if expression of other members of the 17 beta-HSD family was coordinately induced by PPC exposure, (2) the tissues in which 17 beta-HSD was induced by PPC, and (3) whether the induction of 17 beta-HSD by PPC was dependent on the peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of PPC effects in the mouse liver. The mRNA levels of 17 beta-HSD I, II, and III were not altered in the liver, kidney, and testis or uterus of rats treated with PPC. The mRNA or 80 kDa a full-length protein levels of 17 beta-HSD IV were strongly induced in liver and kidney, but not induced in adrenals, brown fat, heart, testis, and uterus of rats treated with diverse PPC. In liver and kidneys from treated rats, additional proteins of 66 kDa, 56 kDa, and 32 kDa were also induced which reacted with the anti-17 beta-HSD IV antibodies and were most likely proteolytic fragments of 17 bega-HSD IV. Treatment of mice which lack a functional form of PPAR alpha with PPC, demonstrated that PPC-inducibility of 17 beta-HSD IV mRNA or the 80 kDa protein was dependent on PPAR alpha expression in liver and kidney. Our results demonstrate that 17 beta-HSD IV is induced by PPC through a PPAR alpha-dependent mechanism and support the hypothesis that exposure to PPC leads to alterations in sex steroid metabolism.
Altered postprandial hormone and metabolic responses in a simulated shift work environment
Authors: Ribeiro DC , Hampton SM , Morgan L , Deacon S , Arendt J
Published: December 2004

The circadian rhythms of most night shift workers do not adapt fully to the imposed behavioural schedule, and this factor is considered to be responsible for many of the reported health problems. One way in which such disturbances might be mediated is through inappropriate hormonal and metabolic responses to meals, on the night shift. Twelve healthy subjects (four males and eight females) were studied on three occasions at the same clock time (1330 h), but at different body clock times, after consuming test meals, first in their normal environment, secondly after a forced 9 h phase advance (body clock time approximately 2230 h) and then again 2 days later in the normal environment. They were given a low-fat pre-meal at 0800 h, then a test meal at 1330 h with blood sampling for the following 9 h. Parameters measured included plasma glucose, non-esterified fatty acids (NEFAs), triacylglycerol (TAG), insulin, C-peptide, proinsulin and glucose-dependent insulinotropic polypeptide, and urinary 6-sulphatoxymelatonin. In contrast with a previous study with a high-fat pre-meal, postprandial glucose and insulin responses were not affected by the phase shift. However, basal plasma NEFAs were lower immediately after the phase shift (P < 0.05). Incremental (difference from basal) TAG responses were significantly higher (P < 0.05) immediately after the phase shift compared with before. Two-day post-phase shift responses showed partial reversion to baseline values. This study suggests that it takes at least 2 days to adapt to eating meals on a simulated night shift, and that the nutritional content of the pre-meals consumed can have a marked effect on postprandial responses during a simulated phase shift. Such findings may provide a partial explanation for the increased occurrence of cardiovascular disease reported in shift workers.
Several environmental oestrogens are also anti-androgens
Authors: Sohoni P , Sumpter JP
Published: December 2004

There is presently considerable interest in endocrine disruption which is a new area of endocrinology concerned with chemicals that mimic hormones, in particular sex steroids. It has been hypothesised that exposure to such chemicals may be responsible for adverse effects in both humans and wildlife. Until now, chemicals that mimic oestrogens (so-called xenoestrogens) have been the main focus of endocrine disruption research. However, recent evidence suggests that many abnormalities in the male reproductive system may be mediated via the androgen receptor. By blocking androgen action, exposure to an anti-androgen may cause changes similar to those associated with oestrogen exposure. We have used in vitro yeast-based assays to detect oestrogenic, anti-oestrogenic, androgenic and anti-androgenic activities in a variety of chemicals of current interest. We show that many of the so-called 'environmental oestrogens' also possess anti-androgenic activity. The previously reported anti-androgenic activities of vinclozolin and p,p'-1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (DDE) were confirmed. We also found that o,p'-1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), bisphenol A and butyl benzyl phthalate were anti-androgenic. However, not all xenoestrogens are also anti-androgenic, because nonylphenol was found to be a weak androgen agonist. Our results demonstrate that hormone-mimicking chemicals can have multiple hormonal activities, which may make it difficult to interpret their mechanisms of action in vivo. Although not a specific objective of this study, our results also demonstrate that yeast-based assays are powerful tools with which to investigate both agonist and antagonistic hormonal activities of chemicals.
The sodium iodide symporter gene and its regulation by cytokines found in autoimmunity
Authors: Ajjan RA , Watson PF , Findlay C , Metcalfe RA , Crisp M , Ludgate M , Weetman AP
Published: December 2004

Iodide concentration by the thyroid gland, an essential step for thyroid hormone synthesis, is mediated by the Na+/I- symporter (NIS). To identify factors that may regulate this process, we have studied NIS gene expression in the Fisher rat thyroid cell line (FRTL-5) by a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. Increasing concentrations of bovine TSH (0.1, 1, 10, 50 and 100 mU/l), with or without tumour necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma) or interleukin-1 alpha (IL-1 alpha) were added to FRTL-5 cells previously deprived of TSH for a minimum of 5 days. RNA was extracted and samples were studied for NIS expression. TSH enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.1 mU/l, reaching a peak at 50 mU/l, an effect detected after 6 h of stimulation, but not in the first 2 h. Both TNF alpha and, to a lesser extent, IL-1 alpha inhibited basal and TSH-induced NIS expression. High concentrations of IFN gamma also downregulated TSH-stimulated NIS mRNA expression. Using the same technique, we also investigated NIS mRNA tissue distribution in two male and one female Wistar rats. High levels of NIS expression were detected in the thyroid, stomach, and mammary gland, lower levels were found in the intestine, adipose tissue and liver, borderline levels were expressed in the salivary gland, and no expression was detected in the kidneys. In summary, we have shown that TSH upregulates rat NIS gene expression in vitro, and this induction can be modulated by cytokines. Analysis of the distribution of rat NIS mRNA ex vivo demonstrated variable levels of NIS transcription in different tissue samples.
Upregulation of the angiogenic factor heparin affin regulatory peptide by progesterone in rat uterus
Authors: Milhiet PE , Vacherot F , Caruelle JP , Barritault D , Caruelle D , Courty J
Published: December 2004

Heparin affin regulatory peptide (HARP), also named pleiotropin, is a secreted polypeptide that belongs to a new family of heparin-binding growth/differentiation factors. In this study, we investigated the expression and distribution of HARP mRNA and protein in rat uterus. Semi-quantitative reverse transcriptase PCR experiments showed variations in HARP mRNA levels throughout the estrous cycle, with a maximum during diestrus, pointing to hormonal regulation of HARP mRNA expression. Uterine expression of HARP mRNA was studied in ovariectomized animals treated with 17 beta-estradiol, progesterone alone or progesterone and RU486. In these experiments, progesterone upregulated HARP mRNA expression. Induction was observed 6 h after progesterone injection and was inhibited by RU486 treatment. In contrast, after 17 beta-estradiol injection, a slight decrease in HARP mRNA expression was observed. In situ hybridization studies with digoxigenin-labeled DNA probe revealed that HARP mRNA was present in smooth muscle cells of both myometrium and blood vessels and also in endothelial cells from endometrium. Immunohistochemical studies showed that HARP expression was not limited to cells that expressed HARP mRNA, but also occurred in both the luminal and glandular epithelium even though its transcript was never detected. We conclude that HARP may mediate the effects of progesterone on the homeostasis and vascularization of uterine tissue.
Progesterone inhibits glucocorticoid-dependent aromatase induction in human adipose fibroblasts
Authors: Schmidt M , Renner C , Loffler G
Published: December 2004

In fibroblasts derived from human adipose tissue, aromatase induction is observed after exposure to 1 microM cortisol in the presence of serum or platelet-derived growth factor (PDGF). Progesterone suppresses this induction in a dose-dependent manner, 10 microM resulting in complete inhibition. A reduced cortisol concentration (0.1 microM) concomitantly reduces the progesterone concentration required for effective inhibition (10-100 nM). This effect of progesterone is specific, as neither the release of cellular enzymes nor aromatase induction by dibutyryl-cAMP, which acts independently from cortisol, are affected. However, the inhibitory effect of progesterone requires its presence throughout the induction period. Kinetic studies in intact cells reveal a reduced number of aromatase active sites upon progesterone treatment, whereas progesterone at near-physiological concentration (100 nM) does not inhibit aromatase activity in isolated microsomes. Semi-quantitative reverse transcriptase PCR analysis shows reduced amounts of aromatase mRNA in progesterone-treated cells, indicating specific inhibition of the glucocorticoid-dependent pathway of aromatase induction. The inhibitory effect of progesterone is not blocked by the anti-progestin ZK114043, excluding action via progesterone receptors and indicating competition for the glucocorticoid receptor. Progesterone must be considered a potential physiological inhibitor of glucocorticoid-dependent aromatase induction in adipose tissue. It is proposed that it is a suppressor of aromatase induction in adipose tissue in premenopausal women.
Differential activation by xenoestrogens of ER alpha and ER beta when linked to different response elements
Authors: Pennie WD , Aldridge TC , Brooks AN
Published: December 2004

The discovery of a second estrogen receptor (ER beta) has significant implications for our understanding of the molecular basis for the diverse actions of estrogen. Here we report the differential activation by natural and xenobiotic estrogens of ER alpha and ER beta when linked to different response elements. Receptor mediated activation of reporter constructs containing either the estrogen response element (ERE) from the vitellogenin (Vit) gene or from the luteinizing hormone beta (LH) gene were examined in transiently transfected Cos-1 cells. ER beta preferentially activated the consensus Vit ERE whereas ER alpha showed greater activation at the divergent LH ERE. This differential activation was observed for a number of ligands including estradiol, estrone, bisphenol A, octylphenol and diethystilbestrol. These findings show that the nature of the ERE, as well as the ratio of ER subtypes in a particular cell/tissue, will influence whether particular estrogen responsive genes are activated in the presence of natural or xenobiotic estrogens.
Mechanism of hyperthyroidism-induced renal hypertrophy in rats
Authors: Kobori H , Ichihara A , Miyashita Y , Hayashi M , Saruta T
Published: December 2004

It is well known that renal hypertrophy is induced by hyperthyroidism; however, the mechanism is not fully understood. We recently reported that cardiac hypertrophy in hyperthyroidism is mediated by enhanced cardiac expression of renin mRNA. The present study addresses the hypothesis that renal hypertrophy in hyperthyroidism is mediated by amplification of renal expression of renin mRNA. Twenty Sprague-Dawley rats were divided into control (n=5) and hyperthyroid groups by daily intraperitoneal injections of saline vehicle or thyroxine. The hyperthyroid group was subdivided further into hyperthyroid-vehicle (n=5), hyperthyroid-losartan (n=5), and hyperthyroid-nicardipine (n=5) groups by daily intraperitoneal injections of saline vehicle, losartan, or nicardipine. All rats were killed at 4 weeks, and the blood and kidneys were collected. The kidney-to-body weight ratio increased in the hyperthyroid groups (+34%). Radioimmunoassays and reverse transcriptase-polymerase chain reaction revealed increased renal renin (+91%) and angiotensin II (+65%) levels and enhanced renal renin mRNA expression (+113%) in the hyperthyroid groups. Losartan and nicardipine decreased systolic blood pressure to the same extent, but only losartan caused regression of thyroxine-induced renal hypertrophy. These results suggest that thyroid hormone activates the intrarenal renin-angiotensin system via enhancement of renal renin mRNA expression, which then leads to renal hypertrophy.
Pituitary growth hormone release and gene expression in cafeteria-diet-induced obese rats
Authors: Zhou X , De Schepper J , De Craemer D , Delhase M , Gys G , Smitz J , Hooghe-Peters EL
Published: December 2004

In human obesity as well as in rat obesity models a decrease in spontaneous and stimulated GH secretion has been a constant finding. The presence of a decreased pituitary GH synthesis in diet-induced obese male rats was investigated and its possible relationship with obesity-related changes in peripheral hormones was analyzed. Cafeteria-diet-overfed obese male Wistar rats with body fat percentage above 30% had a significantly decreased pituitary GH mRNA transcript level assessed by both Northern blot and in situ hybridization, and a lower pituitary GH protein level as demonstrated by immunocytochemistry. The GH transcript level correlated negatively with the serum leptin and positively with the IGF-I concentration. No differences in circulating tri-iodothyronine, non-fasting insulin and corticosterone levels were found between overfed and control rats. GH release by cultured pituitary cells from overfed rats was comparable to that by cells prepared from control rats. In contrast, incubation of normal pituitary cells with serum from overfed rats for 3 days gave a significantly lower GH release than after incubation with serum from non-obese rats. In conclusion, cafeteria-diet-induced obese male Wistar rats have a decreased pituitary GH gene expression and a modifiable GH release in in vitro experiments. A possible role for peripheral circulating factors, like leptin and IGF-I, in decreasing the pituitary GH synthesis and release in obese rats is discussed.
Elevated plasma cortisol reduces permeability of mammary tight junctions in the lactating bovine mammary epithelium
Authors: Stelwagen K , van Espen DC , Verkerk GA , McFadden HA , Farr VC
Published: December 2004

Induction of tight junction permeability in the mammary epithelium decreases milk secretion, and in cows tight junctions become leaky after 17 h of milk accumulation.In vitro studies demonstrate the importance of glucocorticoids for the formation and maintenance of tight junctions. In this study we examined whether cortisol can prevent mammary tight junction permeability in the lactating gland in vivo, and inhibit the associated milk loss, using our milk-accumulation model to challenge tight junction patency. Following a 4-day control period Jersey cows were subjected to a 24-h period in which they were milked twice at 0700 and 1500 h (TM;n=6), once at 0700 h (OM;n=7), or once and treated with ACTH (40 IU per 2 h, starting after 14 h of milk accumulation) to increase endogenous cortisol levels (OM+ACTH;n=7). Frequent blood samples for cortisol, lactose and glucose analyses were taken via indwelling jugular catheters. ACTH treatment resulted in a sustained elevation of systemic cortisol concentrations. Plasma lactose, an indicator of tight junction leakiness, was not changed in TM cows, but began to increase rapidly at 17 h of milk accumulation in OM cows. Treatment with ACTH prevented the increase in plasma lactose, although levels were slightly, but not significantly, higher than in TM cows, indicating that elevated plasma cortisol reduced mammary tight junction leakiness. Milk yield was reduced by 12% in both once-milked groups, despite cortisol preventing tight junction leakiness. However, the milk loss in the latter group may not be related to leaky tight junctions, but be due to a reduction in milk precursor uptake by the mammary gland. Consistent with this notion was a 34% increase in plasma glucose levels in OM+ACTH cows only.
Fetal expression of GnRH and GnRH receptor genes in rat testis and ovary
Authors: Botte MC , Chamagne AM , Carre MC , Counis R , Kottler ML
Published: December 2004

The identification of gonadal gonadotropin-releasing hormone receptors (GnRH-R) and evidence of direct inhibitory effects of GnRH agonists upon steroidogenesis in adult rat gonads, lend credence to a putative intragonadal role of a locally secreted GnRH or GnRH-like peptide. Using reverse transcription-polymerase chain reaction followed by Southern blot hybridization and sequencing, we identified, both in the ovary and in the testis of fetal and adult rats, a fully processed GnRH messenger RNA (mRNA), the sequence of which, in adult testis, was identical to that found in the hypothalamus. We also detected in the testis, but not in the ovary, a transcript containing the first intron. The ontogeny of GnRH and GnRH-R gene expression was studied in rat gonads from 14.5 to 21.5 days post-coitum (dpc), using dot blot hybridization of total RNA. During this period, the levels of cyclophilin mRNA normalized to total RNA remained unchanged. Thus, we used cyclophilin as an internal standard. GnRH mRNA was detected in the ovary at 18.5 dpc, four days later than in the testis, and similar levels were found in both sexes at birth. GnRH-R mRNA was present at 14.5 dpc in the testis and at 15.5 dpc in the ovary, with the levels at 21.5 dpc being 2.4 times higher in the testis than in the ovary. GnRH and GnRH-R mRNA levels increased in both sexes in late fetal development, but this increase appeared two days sooner in the ovary compared with the testis, thus supporting the hypothesis that expression of the GnRH and GnRH-R genes is regulated in a sex-dependent manner during fetal development. In all cases, expression of GnRH and GnRH-R preceded gonadotropin receptors in the gonads and initiation of gonadotropin secretion by the pituitary.
Assessment of the mechanism by which prolactin stimulates progesterone production by early corpora lutea of pigs
Authors: Ciereszko RE , Petroff BK , Ottobre AC , Guan Z , Stokes BT , Ottobre JS
Published: December 2004

Previously, we reported that administration of prolactin (PRL) during the early luteal phase in sows increases plasma progesterone concentrations. In the current study, we searched for the mechanisms by which PRL exerts this luteotrophic effect. The objectives of the study were (1) to examine the effect of PRL and/or low-density lipoproteins (LDL) on progesterone production by porcine luteal cells derived from early corpora lutea, and (2) to assess the ability of PRL to activate phosphoinositide-specific phospholipase C (PI-PLC) and protein kinase C (PKC) in these luteal cells. Ovaries with early corpora lutea (day 1-2 of the oestrous cycle) were obtained from the slaughterhouse. Progesterone production by dispersed luteal cells was measured after treatment with PRL, phorbol 12-myristate 13-acetate or inhibitors of PKC in the presence or absence of LDL. LDL increased progesterone concentration in the incubation medium (304.5 vs 178.6 ng/ml in control, P<0.05). PRL augmented LDL-stimulated progesterone secretion by luteal cells (to 416 ng/ml, P<0.05), but PRL alone did not affect progesterone production (209.6 ng/ml, P>0.05). Staurosporine, a PKC inhibitor, inhibited progesterone secretion stimulated by the combined action of LDL and PRL; however, such inhibition was not demonstrated when cells were treated with the PKC inhibitor, H-7. PKC activation was assessed by measuring the specific association of [H]phorbol dibutyrate (H-PDBu) with luteal cells after treatment with PRL or ionomycin (a positive control). PRL and ionomycin increased H-PDBu-specific binding in early luteal cells by 28+/-5.5% (within 5 min) and 70.2+/-19.3% (within 2 min) over control binding respectively (P<0.05). In addition, PRL did not augment the LDL-stimulated progesterone production in PKC-deficient cells. In contrast with PKC, total inositol phosphate accumulation, as well as intracellular free calcium concentrations, were not affected by PRL in the current study. We conclude that PRL, in the presence of LDL, stimulates progesterone production by early corpora lutea in vitro. Moreover, PRL appears to activate PKC, but not PI-PLC, in these cells. Thus intracellular transduction of the PRL signal may involve activation of PKC that is not dependent on PI-PLC.
Pre- and posthatch developmental changes in hypothalamic thyrotropin-releasing hormone and somatostatin concentrations and in circulating growth hormone and thyrotropin levels in the chicken
Authors: Geris KL , Berghman LR , Kuhn ER , Darras VM
Published: December 2004

Thyrotropin-releasing hormone (TRH) and somatostatin (SRIH) concentrations were determined by RIA during both embryonic development and posthatch growth of the chicken. Both TRH and SRIH were already detectable in hypothalami of 14-day-old embryos (E14). Towards the end of incubation, hypothalamic TRH levels increased progressively, followed by a further increase in newly hatched fowl. SRIH concentrations remained stable from E14 to E17 and doubled between E17 and E18 to a concentration which was observed up to hatching. Plasma GH levels remained low during embryonic development, ending in a steep increase at hatching. Plasma TSH levels on the other hand decreased during the last week of the incubation. During growth, TRH concentrations further increased, whereas SRIH concentrations fell progressively towards those of adult animals. Plasma TSH levels increased threefold up to adulthood; the rise in plasma GH levels during growth was followed by a drop in adults. In conclusion, the present report shows that important changes occur in the hypothalamic TRH and SRIH concentration during both embryonic development and posthatch growth of the chicken. Since TRH and SRIH control GH and TSH release in the chicken, the hypothalamic data are compared with plasma GH and TSH fluctuations.
11beta-hydroxysteroid dehydrogenase is a predominant reductase in intact rat Leydig cells
Authors: Leckie CM , Welberg LA ,   Seckl
Published: December 2004

11beta-Hydroxysteroid dehydrogenases (11beta-HSDs) interconvert active corticosterone and inert 11-dehydrocorticosterone. In tissue homogenates, 11beta-HSD type 1 (11beta-HSD-1) exhibits both 11beta-dehydrogenase (corticosterone inactivating) and 11beta-reductase (corticosterone regenerating) activities, whereas 11beta-HSD type 2 (11beta-HSD-2) is an exclusive dehydrogenase. In the rat testis, 11beta-HSD has been proposed to reduce glucocorticoid inhibition of testosterone production, promoting puberty and fertility. This hypothesis presupposes dehydrogenation predominates. 11beta-HSD-1 immunoreactivity has been localised to Leydig cells. However, recent studies suggest that 11beta-HSD-1 is predominantly an 11beta-reductase in many intact cells. We therefore examined the expression and reaction direction of 11beta-HSD isozymes in cultures of intact rat Leydig cells. Reverse transcriptase PCR demonstrated expression of 11beta-HSD-1, but not 11beta-HSD-2 mRNA in rat testis. Primary cultures of intact rat Leydig cells showed predominant 11beta-reductase activity, activating 50-70% of 11-dehydrocorticosterone to corticosterone over 3 h, whereas 11beta-dehydrogenation was <5%. Although both dexamethasone (10 nM) and corticosterone (1 microM) modestly inhibited LH-stimulated testosterone production by Leydig cells, inert 11-dehydrocorticosterone (1 microM) had similar effects, suggesting 11beta-reductase is functionally important. Carbenoxolone (10(-5) M) inhibited 11beta-reduction in intact Leydig cells. However, although carbenoxolone reduced Leydig cell testosterone production, this also occurred in the absence of glucocorticoids, suggesting effects distinct from modulation of corticosteroid access to Leydig cells. In conclusion, rat Leydig cell 11beta-HSD-1 is unlikely to reduce glucocorticoid access to testicular receptors. More likely, 11beta-reductase amplifies glucocorticoid action, perhaps to maintain Leydig cell metabolic and endocrine functions.
Gestational age-dependent changes in the levels of mRNAs encoding cortisol biosynthetic enzymes and IGF-II in the adrenal gland of fetal sheep during prolonged hypoxemia
Authors: Braems GA , Han VK ,   Challis
Published: December 2004

Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.
Regulation of insulin-like growth factor-binding protein-3 ternary complex formation in pregnancy
Authors: Lewitt MS , Scott FP , Clarke NM , Wu T , Sinosich MJ , Baxter RC
Published: December 2004

The IGFs are believed to be important in pregnancy and are implicated in the pathophysiology of pre-eclampsia. In adults the IGFs circulate primarily with IGF-binding protein-3 (IGFBP-3) and an acid-labile glycoprotein (ALS) in a 140 kDa complex which limits IGF bioavailability. Less than 10% of IGFBP-3 is in lower molecular weight forms. We have investigated the developmental regulation of the IGF/IGFBP system in normal and pre-eclamptic pregnancies with particular emphasis on the IGFBP-3 ternary complex. Circulating levels of IGF-I, IGFBP-3 and ALS, and their degree of association in the ternary complex in the fetus increased with gestational age. In neonatal serum from deliveries <35 weeks' gestation IGFBP-3 was predominantly in 30-50 kDa form(s) and ALS was a limiting factor for ternary complex formation. In serum from deliveries >35 weeks both ALS and IGFs were limiting but approximately 25% of IGFBP-3 was unable to form the ternary complex even in the presence of excess ALS and IGF-I. Serum IGFBP-1, -2 and -6 concentrations tended to decrease with increasing gestational age. In pre-eclamptic pregnancies, amniotic fluid IGFBP-2, -3 and -6 levels decreased with gestational age while IGFBP-1 levels did not show the normal decline. We speculate that the endocrine IGF system develops in the fetus during the third trimester of pregnancy when ALS levels increase.
Dynamics of proopiomelanocortin and prohormone convertase 2 gene expression in Xenopus melanotrope cells during long-term background adaptation
Authors: Dotman CH , van Herp F , Martens GJ , Jenks BG , Roubos EW
Published: December 2004

The toad Xenopus laevis is able to adapt its skin color to background light intensity. In this neuroendocrine reflex, the proopiomelanocortin (POMC)-derived peptide alpha-melanophore-stimulating hormone (alphaMSH) is a key regulatory factor. In animals adapting to a black background, release of alphaMSH from the pituitary pars intermedia causes dispersal of melanin in skin melanophores. To investigate the long-term in vivo dynamics of alphaMSH production during black background adaptation, the biosynthetic rate of POMC and the contents of POMC, alphaMSH and the POMC processing enzyme precursor convertase 2 (PC2) have been studied in the pars intermedia using pulse-labeling, Western blot and radioimmunoassay. In control animals, adapted to a white background, the rate of POMC biosynthesis and the POMC content were low, while high alphaMSH and PC2 contents were found. After 1 week of adaptation to a black background, the rate of POMC biosynthesis and the POMC protein content had increased 19- and 3.7-fold respectively. These parameters attained a maximum level (28- and 5. 8-fold higher than control) after 3 weeks and remained at these elevated levels for at least 12 weeks. After 1 week, the pars intermedia content of alphaMSH was only 30% of the control level, but after 6 and 12 weeks, the alphaMSH level had increased to the control level. The PC2 content decreased to 52% of control after 1 week and stabilized after 3 weeks at a level slightly lower than the control value. The results show that during long-term background adaptation a steady-state situation is reached, with a balance between the biosynthesis, enzymatic processing and release of alphaMSH. The in vivo dynamics of the processing enzyme PC2 suggest a parallel storage and release of alphaMSH and mature PC2 in the Xenopus pituitary pars intermedia.
Central administration of Y5 receptor antisense decreases spontaneous food intake and attenuates feeding in response to exogenous neuropeptide Y
Authors: Tang-Christensen M , Kristensen P , Stidsen CE , Brand CL , Larsen PJ
Published: December 2004

A number of neuropeptide Y (NPY) receptor subtypes, including the recently cloned Y5 receptor, have been implicated in the stimulation of food intake. In the present study, Y5 receptor antisense oligodeoxynucleotides (ODNs) were used to assess the potential involvement of the Y5 receptor in the regulation of spontaneous as well as NPY-induced food intake. Repeated central administration of Y5 antisense ODN significantly decreased spontaneous food intake and subsequently resulted in a significant weight loss. Furthermore, Y5 antisense ODN pre-treatment significantly inhibited the robust feeding response elicited by central administration of NPY (5.3+/-0. 8 vs 1.08+/-0.28 g, vehicle+/-s.e.m. vs Y5 ODN+/-s.e.m.). The present results provide evidence that central Y5 receptors are involved in both spontaneous as well as NPY-induced food intake, which may prove to be a new therapeutic route in the treatment of obesity and other disorders of appetite.
Crosstalk between the interleukin-6 (IL-6)-JAK-STAT and the glucocorticoid-nuclear receptor pathway: synergistic activation of IL-6 response element by IL-6 and glucocorticoid
Authors: Takeda T , Kurachi H , Yamamoto T , Nishio Y , Nakatsuji Y , Morishige K , Miyake A , Murata Y
Published: December 2004

Cytokines and steroid hormones use different sets of signal transduction pathways, which seem to be unrelated. Interleukin-6 (IL-6) uses JAK tyrosine kinase and STAT (signal transducer and activator of transcription) transcription factor. Glucocorticoid binds glucocorticoid receptor (GR), which is a member of the steroid receptor superfamily. We have studied the crosstalk between the IL-6-JAK-STAT and glucocorticoid-nuclear receptor pathways. IL-6 and glucocorticoid synergistically activated the IL-6 response element on the rat alpha2-macroglobulin promoter (APRE)-driven luciferase gene. The exogenous expression of GR enhanced the synergism. The exogenous expression of dominant negative STAT3 completely abolished the IL-6 plus glucocorticoid-induced activation of the APRE-luciferase gene. Tyrosine phosphorylation of STAT3 stimulated by IL-6 alone was not different from that by IL-6 plus glucocorticoid. The protein level of STAT3 was also not increased by glucocorticoid stimulation. The time course of STAT3 tyrosine phosphorylation by IL-6 plus glucocorticoid was not different from that by IL-6 alone. The synergism was studied on the two other IL-6 response elements, the junB promoter (JRE-IL-6) and the interferon regulatory factor-1 (IRF-1) promoter (IRF-GAS) which could be activated by STAT3. The synergistic activation by glucocorticoid on the IL-6-activated JRE-IL-6 and the IRF-GAS-driven luciferase gene was not detected. Glucocorticoid did not change the mobility of IL-6-induced APRE-binding proteins in a gel shift assay. These results suggest that the synergism was through the GR and STAT3, and the coactivation pathway which was specific for APRE was the target of glucocorticoid.
The hormonal status modulates the effect of neurokinin A on prolactin secretion in female rats
Authors: Pisera D , Theas S , De Laurentiis A , Lasaga M , Duvilanski B , Seilicovich A
Published: December 2004

We have previously reported that neurokinin A (NKA), a tachykinin closely related to substance P, increases the release of prolactin (PRL) from the anterior pituitary gland of male rats, but not from pituitaries of ovariectomized (OVX) female rats. In this study, we evaluated the influence of estrogens in the action of NKA on PRL secretion in female rats. NKA stimulated the in vitro release of PRL from pituitary glands of OVX-chronically estrogenized rats, and of proestrus and estrus rats, but had no effect in anterior pituitaries of diestrus rats. In addition, we observed that cultured anterior pituitary cells of OVX rats responded to NKA only when they were incubated for 3 days in the presence of estradiol 10(-9) M. This effect was blocked by L-659,877, an NK-2 receptor antagonist. We also studied the action of NKA on PRL release during lactation. The response of anterior pituitary cells to NKA was variable over this period. The maximal sensitivity to NKA was observed at day 10 of lactation. Furthermore, the blockade of endogenous NKA by the administration of an anti-NKA serum to lactating rats reduced the PRL surge induced by the suckling stimulus. These results show that the responsiveness of the anterior pituitary gland of female rats to NKA is modulated by the endocrine environment, and suggest that NKA may participate in the control of PRL secretion during the estrus cycle and lactation.
Gonadotrophin secretion patterns in testicular cancer patients with greatly increased human chorionic gonadotrophin serum concentrations
Authors: Madersbacher S , Gerth R , Mann K , Dirnhofer S , Berger P
Published: December 2004

Despite the fact that a number of alterations of the hypothalamic-pituitary-gonadal hormone axis have been identified in patients with testicular cancer, little is known about the gonadotrophin secretion pattern in such patients who have greatly increased human chorionic gonadotrophin (hCG) serum concentrations. The aim of this study was to assess this issue in detail using a longitudinal study design and a panel of highly sensitive and specific immunoassays. Eleven patients with non-seminomatous (n=11), and one with seminomatous testicular cancer with pretreatment hCG serum concentrations exceeding 10(5) pg/ml (>1000 mIU/ml) were selected and followed for a mean of 166 days (mean of 14 serum samples/patient) after initial diagnosis. Serum concentrations of hCG, its free alpha- (hCGalpha) and beta- (hCGbeta) subunits, human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) were determined by highly sensitive and specific enzymometric immunoassays based on a panel of monoclonal antibodies (MCA) established in our laboratory. A potential FSH-like activity (FSA) of hCG in the respective sera was determined by radioreceptor assays (RRA) for LH/CG and FSH. Specificity of FSA at the level of the receptor was assessed by MCA-based immunoabsorption studies. At diagnosis, hCG (9.8x10(7)+/-4.84x10(7) pg/ml; range 1.1x10(5)-5x10(8) pg/ml) was greatly increased and serum hFSH was undetectable (<9 pg/ml) in 11 patients, and one patient had very low, albeit detectable (approximately 30 pg/ml) hFSH concentrations. hLH was below the limit of detection (<2 pg/ml) in five individuals. During successful chemotherapy, hCG rapidly declined to physiological concentrations and hFSH/hLH returned to normal or even reached supraphysiological values. There was a highly significant negative correlation between hCG and hFSH (P=0.0001) and, to a lesser extent, hLH (P=0.0265). The ability of serum hCG to block the binding of [125I]rFSH (rat FSH) to its receptor was found to be 0.01-0.1% compared with the FSH standard; this could be reversed by an anti-hCG MCA. Addition of a specific MCA against hFSH blocked 3 microg/ml of the hFSH standard, but had no effect on the FSA of serum hCG in the FSH RRA. As observed during pregnancy, secretion of gonadotrophin -- particularly that of FSH -- is substantially or completely suppressed in patients with testicular cancer when serum hCG concentrations exceed 10(5)-10(6) pg/ml (approximately 10(3)-10(4) mIU/ml). As determined by RRA, the intrinsic FSA of tumour-derived hCG is most probably responsible for the suppression of hFSH in this group of patients with testicular cancer.
Expression and localization of the renin-angiotensin system in the rat pancreas
Authors: Leung PS , Chan WP , Wong TP , Sernia C
Published: December 2004

The possibility of an intrinsic renin-angiotensin system (RAS) in the pancreas has been raised by previous studies in which immunohistochemical examination showed the presence of angiotensin II and its receptor subtypes, type 1 (AT1) and type 2 (AT2). In the present study, gene expression of several key RAS components was investigated by reverse-transcription PCR. mRNA expression for angiotensinogen, renin and angiotensin II receptor subtypes, AT1a, AT1b and AT2 was shown. The presence of angiotensinogen protein, the mandatory component for an intrinsic RAS, was demonstrated by Western blotting and localized by immunohistochemistry to the epithelia and endothelia of pancreatic ducts and blood vessels respectively. Immunoblot analysis detected a predominant protein band of about 60 kDa in the pancreas. This was consistent with the predicted value for angiotensinogen as reported in other tissues. Together with previous findings, the present study shows that the rat pancreas expresses the major RAS component genes, notably angiotensinogen and renin, required for intracellular formation of angiotensin II. The data support the notion of an intrinsic RAS in the rat pancreas which may play a role in the regulation of pancreatic functions.
The effect of pregnancy on the expression of uterine oxytocin, oestrogen and progesterone receptors during early pregnancy in the cow
Authors: Robinson RS , Mann GE , Lamming GE , Wathes DC
Published: December 2004

The expression of oxytocin receptor (OTR) in the uterine endometrium plays an important role in the initiation of luteolysis. During early pregnancy, the conceptus secretes interferon tau (IFN|gt) which inhibits OTR up-regulation and luteolysis. In this study, uterine horn cross sections were collected on day 16 from 15 pregnant cows (PREG), 9 uninseminated controls and 5 inseminated cows with no embryo present. The latter two groups had similar results and were combined to form a single non-pregnant (NP) group. The animals were given an oxytocin challenge shortly before tissue collection to assess prostaglandin F2alpha (PGF2alpha) release through the measurement of the metabolite 13,14-dihydro-15-keto PGF2alpha (PGFM). The mRNAs for OTR, oestrogen receptor (ER) and progesterone receptor (PR) were localised by in situ hybridisation. The results were quantified by optical density (OD) measurements from autoradiographs using image analysis. OTR protein was measured by autoradiography with iodinated oxytocin antagonist and ER and PR protein was detected by immunocytochemistry. The release of PGFM after the oxytocin challenge was significantly higher in the 14 NP cows (187%+/-15%) compared with the PREG group (131%+/-11%) (P<0.01). Low concentrations of OTR mRNA were localised to the luminal epithelium (LE) in 6 out of the 14 NP cows, of which 2 also expressed OTR protein, while OTR mRNA and protein were undetectable in all the pregnant animals. These results indicated that the sampling time coincided with the onset of the luteolytic mechanism in the NP cows. On day 16 ER mRNA was detectable in both the LE and glands of both PREG and NP animals. There were no differences in either ER mRNA or protein between NP and PREG samples. PR mRNA was moderately expressed in the caruncular stroma, with lower levels in the dense caruncular-like stroma and glands. There were no differences between PREG and NP animals. The expression of PR mRNA and protein in the deep glands was variable between animals. These results suggested that, in cows, the presence of an embryo suppressed the expression of OTR, but had no effect on the expression of the transcriptionally regulated ER on day 16.
Indirect assessment of pulsatile gonadotropin-releasing hormone release in agonadal prepubertal rhesus monkeys (Macaca mulatta)
Authors: Suter KJ , Pohl CR , Plant TM
Published: December 2004

The major purpose of this study was to characterize the open-loop frequency of pulsatile GnRH release in the female rhesus monkey at an age (15-20 months) when the prepubertal restraint on the hypothalamic-pituitary axis is maximally imposed. Additionally, evidence for pulsatile GnRH release in agonadal males of comparable age was also sought. Episodic LH secretion from the pituitary was used as an indirect index of GnRH discharges. In order to maximize the sensitivity of this in situ bioassay, the responsiveness of the pituitary gonadotrophs was usually first heightened by an i.v. intermittent infusion of the synthetic peptide. Monkeys (five females, three males) were castrated between 9 and 14 months of age, implanted with indwelling venous catheters, fitted with nylon jackets and housed in specialized cages that permitted remote access to the venous circulation with minimal restraint and without interruption of the light-darkness cycle. In females, LH secretion was generally assessed at 20-day intervals during alternate nighttime (1900-0200 h) and daytime (0700-1400 h) windows. In males, LH was assessed less frequently and only at night. The mean frequency of pulsatile LH release in agonadal prepubertal females was 4 pulses/7 h during the night and 2 pulses/7 h during the day. These findings indicate that, prior to puberty in the female monkey, the GnRH pulse generator operates at a relatively slow frequency and is subjected to diurnal modulation. In males, evidence for robust pulsatile GnRH release was not observed. The striking difference in activity of the GnRH pulse generator in agonadal prepubertal male and female monkeys reinforces the view that the ontogeny of the hypothalamic drive to the pituitary-gonadal axis in higher primates, including man, is sexually differentiated.