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Abstract
The aim of the present investigation was to evaluate the putative involvement of oxygen free radicals in interleukin-1β (IL-1β)-induced suppression of islet glucose oxidation. Isolated adult rat pancreatic islets were exposed for 1 h to liposomally encapsulated superoxide dismutase (SOD; 10 mg/ml), catalase (CAT; 10 mg/ml) and glutathione peroxidase (GPX; 5 mg/ml), after which IL-1β (25 U/ml) or hydrogen peroxide (H2O2; 0·1 mm) was added, and the incubation was continued overnight. The following day, samples were taken from the incubation media for nitrite determinations, and islet glucose oxidation rates were measured. The CAT activity increased fourfold after addition of CAT-containing liposomes. It was found that IL-1β induced a marked increase in islet nitrite production, as an index of nitric oxide formation, and that this was paralleled by a decrease in islet glucose oxidation rates. H2O2-treated islets exhibited a modest decrease in glucose oxidation rates and a minor increase in the release of nitrite to the media. Treatment of islets with liposomes containing the antioxidant enzymes SOD, CAT and GPX, either alone or in combination, did not decrease the effect of IL-1β. However, the H2O2-induced decrease in glucose oxidation rates was counteracted by the combination of the antioxidants. It was concluded that, provided the intracellular delivery of the antioxidant enzymes to the islet cells was effective, oxygen free radicals probably do not play a decisive role in IL-1β suppression of islet glucose metabolism.
Journal of Endocrinology (1994) 143, 151–156
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The capacity of the adrenal to produce cortisol is controlled in part by 21-hydroxylase (CYP21) and the production of androgens by 17-hydroxylase/17-20-lyase (CYP17), in response to secretagogues including ACTH, angiotensin-II (A-II) and insulin. In this study we examined the capacity of human adrenocortical cells to produce cortisol and androgens in response to these secretagogues and their ability to regulate the expression of CYP21 and CYP17. In H-295 cells, forskolin and A-II were found to stimulate production of cortisol relative to androstenedione and a similar pattern of steroid production was noted in primary human adrenocortical cells. Both mRNA and protein expression of CYP21 was upregulated with forskolin and A-II alone and in combination, as detected by Northern and Western blotting. Whereas expression of CYP17 mRNA and protein was up regulated in the presence of forskolin and forskolin in combination with insulin. The ability of steroidogenic factor-1 (SF-1) and nur77 to regulate transcription of these enzymes was examined. Forskolin, A-II and insulin increased the protein expression of SF-1. Increased binding of SF-1 to its response element in the presence of forskolin, A-II and insulin was observed. Nur77 was expressed primarily in the zona glomerulosa and fasciculata. Increased protein expression of nur77 and the greatest binding of nur77 to its response element was seen when cells were stimulated with A-II in combination with forskolin. These data indicate that nur77 may preferentially regulate steroid enzyme genes relevant to cortisol production and thereby regulate differential cortisol and adrenal androgen production.
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enzyme aromatase cytochrome P450 ( Means et al . 1989 ). Disruption of the aromatase gene prevents the biosynthesis of endogenous oestrogens and allows investigation into whether the absence of oestrogen alters prostate contractility in mature mice. It
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ABSTRACT
An enzyme-linked immunoadsorbent assay (ELISA) for measuring human pituitary glycoprotein free α-subunit is described. Pituitary tumours may secrete glycoprotein hormones, their free subunits (α, β) or a combination of these. Non-functioning adenomas often secrete free α-subunit. Assays for free α-subunit have previously used radioimmunoassay or immunoradiometric principles. Some of these methods are time-consuming and lack specificity and sensitivity. In order to overcome these problems, we have developed a two-site ELISA for α-subunit which uses a monoclonal antibody to α-subunit as the capture antibody to provide greater specificity. An affinity-purified polyclonal anti α-subunit conjugated to alkaline phosphatase was the signal antibody. Detection and quantification were based on phenolphthalein monophosphate conversion to phenolphthalein. The ELISA specifically measured glycoprotein free α-subunit in serum or plasma, discriminating between α-subunit and the intact glycoprotein hormones. The assay can be completed in 4 h. The assay sensitivity was 0·03 μg/l, and a normal range of 0·05 to 0·22 μg/l was established. In a retrospective study, elevated circulating glycoprotein α-subunit was detected in 22% of patients with non-functioning pituitary tumours previously treated with surgery and/or radiotherapy.
Journal of Endocrinology (1993) 136, 511–516
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Animal Reproduction Teaching and Research Center, The Ohio State University, Columbus, Ohio 43210, U.S.A.
(Received 26 September 1974)
While it is widely recognized that androgens appear to be essential for maintenance of the spermatogenic process in a variety of scrotal mammals (Steinberger, 1971) and that ambient temperatures exceeding the thermoregulatory capabilities of the scrotum and pampiniform plexus are detrimental to sperm production, surprisingly little information is available concerning the effects of heat on the enzymology of testicular steroidogenesis (VanDemark & Free, 1970). Hall (1965) has demonstrated that incorporation of [1-14C]acetate into [14C]test-osterone by rabbit testis slices in vitro is maximal at the scrotal temperature and depressed at the abdominal temperature. The data of Le Vier & Spaziani (1968), using non-replicate experiments, suggest that testicular conversion of [4-14C] cholesterol to [14C]pregnenolone (cholesterol side-chain cleavage enzyme, CSCCE) in the rat exhibits a positive in-vitro temperature coefficient over a range of 28–36 °C
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ABSTRACT
In the present study the effects of insulin, glucocorticoids and thyroid hormones on macrophage metabolism and function were investigated. The maximum activities of hexokinase, glucose-6-phosphate dehydrogenase, glutaminase and citrate synthase were determined in macrophages obtained from hormonetreated rats and those cultured for a period of 48 h in the presence of hormones. Macrophage phagocytosis was markedly inhibited by dexamethasone and thyroid hormones, remaining unchanged when insulin was added to the culture medium, however. The changes in the enzyme activities caused by hormone treatments of the rats were very similar to those found in culture. Insulin enhanced citrate synthase and hexokinase activities and diminished those of glutaminase and glucose-6-phosphate dehydrogenase. Dexamethasone had a similar effect except on glucose6-phosphate dehydrogenase. The addition of thyroid hormones to the culture medium raised the activities of glutaminase and hexokinase and reduced that of citrate synthase. The results presented support the suggestion that the effects of insulin, glucocorticoids and thyroid hormones on immune and inflammatory responses could well be mediated through changes in macrophage metabolism..
Journal of Endocrinology (1992) 135, 213–219
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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.
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The effects of prostaglandins on testicular synthesis and hydrolysis of cholesteryl esters, the activity of the enzyme involved in cleavage of the cholesterol side-chain and on serum levels of testosterone and LH have been studied. Subcutaneous administration of prostaglandins to male rats caused an increase in the concentration of cholesteryl esters in the testes, a decrease in testicular synthesis and hydrolysis of cholesteryl esters but no change in the activity of the cholesterol side-chain cleavage enzyme. There was also a significant decrease in the serum level of testosterone, but the level of LH was raised. Prostaglandins also affected the fatty acid composition of lipids in rat testicular tissue; cholesteryl esters were found to contain greater amounts of arachidonic (C20:4) and docosapentaenoic (C22:5) acids. These findings suggest that prostaglandins are involved in the turnover of cholesteryl esters in rat testicular tissue and regulate the production of androgens.
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Abstract
The performance of existing immunoassays and bioassays for activins is compromised by the presence of activin-binding proteins such as follistatin and α2 macroglobulin (α2M) in biological fluids. To overcome this problem we have developed a novel two-site enzyme immunoassay procedure for activin-A which incorporates an analyte denaturation and oxidation step. The optimized assay is sensitive (detection limit ∼10 pg/well), precise (mean within- and between-plate coefficients of variation 4·9 and 9·1% respectively) and accurate (activin-A recovery values of 102 ± 3 and 96 ± 5% for bovine follicular fluid (FF) and human serum respectively). In specificity tests, high concentrations of follistatin (500 ng/ml) and α2M (100 μg/ml) did not interfere with the response signal to activin-A. In addition, no significant cross-reactivity was observed with a range of related molecules including inhibin-A, inhibin-B, activin-B (all <0·5%), bovine pro-αC and follistatin (both <0·1%). Response curves parallel to the activin-A standard curve were obtained for a variety of test samples including bovine, human, ovine and porcine FF, human sera and conditioned medium from cultured bovine and human granulosa cells. Fractionation of bovine FF by SDS-PAGE confirmed assay specificity since only one peak of activin-A immunoreactivity was detected (M r ∼25 k) in eluted gel slices. However, gel-permeation chromatography showed that under physiological conditions all of the detectable activin-A in bovine FF eluted with apparent M r values of >700 and 60–200 k reflecting its association with binding protein(s). Analysis of bovine FF samples (n=76) from morphologically dominant follicles during the luteal phase showed that activin-A levels were positively correlated with inhibin-A (r=+0·54; P<0·001) and total β subunit immunoreactivity (r=+0·32; P<0·005) but not with total α subunit immunoreactivity (r= −0·09). Classification of these follicles according to oestrogenic status showed that activin-A, inhibin-A and total β subunit levels were highest in oestrogen-inactive follicles (P<0·01) whereas total α subunit levels were lowest in these follicles (P<0·001). Activin-A levels were measurable in all human serum samples analysed, ranging from 128 pg/ml during the normal menstrual cycle, 210 pg/ml in women undergoing ovarian hyperstimulation and ∼ 500 pg/ml in postmenopausal women to over 4000 pg/ml during pregnancy.
In conclusion, the present assay provides a reliable method for quantitating total (i.e. bound+free) activin-A concentrations in a variety of biological samples and should prove useful for further in vivo and in vitro studies in a range of species including man.
Journal of Endocrinology (1996) 148, 267–279
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Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) alpha plus TGF beta caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF alpha or TGF beta alone the cells remained healthy. In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Bcl-2, a cell survival gene, has been implicated in regulating the balance between cell proliferation and cell death in physiological processes. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA encoding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were amplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatment of T/I cells with TGF alpha plus TGF beta significantly decreased the levels of bcl-2 mRNA expression. TGF alpha plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T/I cells, followed by a progressive decline to 10% of control levels after 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha plus TGF beta caused a progressive increase in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragmentation, indicative of apoptosis, occurred after 10 h of treatment with TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression of bcl-2 and ICE prior to the time at which DNA fragmentation was observed. We propose that the gene products of bcl-2 and ICE are involved in the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.