bluuebunny/crossref_metadata_2025 huggingface dataset:
The Metabolic Phenotype of Prader-Willi Syndrome (PWS) in Childhood: Heightened Insulin Sensitivity Relative to Body Mass Index
Authors: Haqq Andrea M. , Muehlbauer Michael J. , Newgard Christopher B. , Grambow Steven , Freemark Michael
Published: January 2014

Context

Insulin sensitivity is higher in patients with Prader-Willi syndrome (PWS) than in body mass index-matched obese controls (OCs). Factors contributing to the heightened insulin sensitivity of PWS remain obscure. We compared the fasting levels of various hormones, cytokines, lipids, and liver function tests in 14 PWS patients and 14 OCs with those in 14 age- and gender-matched lean children (LC). We hypothesized that metabolic profiles of children with PWS are comparable with those of LC but different from those of OCs.

Results

Leptin levels were comparable in PWS patients and OCs, suggesting comparable degrees of adiposity. Glucose levels were comparable among groups. However, fasting insulin concentrations and homeostasis model assessment insulin resistance index were lower in PWS patients than in OCs (P < 0.05) and similar to LC. Moreover, high-density lipoprotein levels were lower and triglycerides higher in OCs (P < 0.05) but not PWS patients. Total adiponectin, high-molecular-weight (HMW) adiponectin and the HMW to total adiponectin ratio were higher in PWS patients (P < 0.05) than in OCs and similar to LC. High-sensitivity C-reactive protein and IL-6 levels were higher in OCs than in PWS patients or LC (P < 0.05). Nevertheless, PAI-1 levels were elevated in both OC and PWS patients. There were no group differences in glucagon-like peptide-1-(7-36)-amide, macrophage chemoattractant protein-1, TNFα, IL-2, IL-8, IL-10, IL-12p40, IL-18, resistin, total or low-density lipoprotein cholesterol, aspartate aminotransferase, or aminotransferase.

Conclusions

The heightened insulin sensitivity of PWS patients relative to OCs is associated with higher levels of adiponectin and lower levels of high-sensitivity C-reactive protein and IL-6. Future studies will determine whether PWS children are protected from obesity comorbidities such as type 2 diabetes, hyperlipidemia, and nonalcoholic fatty liver disease.
Dynamic Chromatin Modifications Control GnRH Gene Expression during Neuronal Differentiation and Protein Kinase C Signal Transduction
Authors: Iyer Anita K. , Brayman Melissa J. , Mellon Pamela L.
Published: January 2014

GnRH, a neuropeptide produced by rare, specialized hypothalamic secretory neurons, is critical for reproduction. During development, GnRH gene expression increases as neurons migrate from the olfactory placode to the hypothalamus, with highest levels in the mature, postmitotic state. While neuronal differentiation is known to be controlled by chromatin modulations, the role of chromatin dynamics in GnRH gene regulation has not been studied. Here, we use mature and immature GnRH neuronal cell models to show that both neuron-specific and protein kinase C regulation of GnRH expression are mediated by chromatin structure and histone modifications. Only in GT1-7 mature GnRH neuronal cells did GnRH regulatory elements display high sensitivity to DNase and enrichment of active histone markers histone-H3 acetylation and H3 lysine 4 trimethylation (H3K4-Me3), as well as RNA Polymerase II (RNAPII) binding and enhancer RNA transcription. In contrast, H3K9-Me2, a marker of inactive chromatin, was highest in nonneuronal cells, low in GT1-7 cells, and intermediate in immature GnRH neuronal cells. The chromatin of the GnRH gene was therefore active in mature GnRH neuronal cells, inactive in nonneuronal cells, but not fully inactive in immature GnRH neuronal cells. Activation of protein kinase C (PKC) potently represses GnRH expression. PKC activation caused closing of the chromatin and decreased RNAPII occupancy at the GnRH minimal promoter (−278/−97). At GnRH-Enhancer-1 (−2404/−2100), PKC activation decreased phosphorylated-RNAPII binding, enhancer RNA transcription, and H3 acetylation, and reciprocally increased H3K9-Me2. Chromatin modifications therefore participate in the dynamic regulation and specification of GnRH expression to differentiated hypothalamic neurons.
Progesterone Regulates Osteopontin Expression in Human Trophoblasts: A Model of Paracrine Control in the Placenta?*
Authors: Omigbodun Akinyinka , Ziolkiewicz Piotr , Tessler Cheryl , Hoyer John R. , Coutifaris Christos
Published: January 2014

Osteopontin (OPN), a matrix glycosylated phosphoprotein, has been proposed to play a role(s) in basic cellular processes, such as neovascularization and tissue remodeling, which are essential to placental morphogenesis and embryo implantation. We have shown OPN to be expressed by cytotrophoblasts of the chorionic villus, and a putative progesterone regulatory element in the OPN promoter suggests hormonal regulatory control. This led us to test the hypothesis that progesterone regulates OPN expression in human cytotrophoblasts. Cytotrophoblasts isolated from human placentas were treated with combinations of progesterone, RU486, and/or aminoglutethimide, and their expression of OPN was assessed by Northern hybridization and immunocytochemistry. The expression of OPN messenger RNA (mRNA) declined as trophoblasts aggregated, but rebounded at later times when syncytia and mononuclear cytotrophoblasts coexisted in culture. Progesterone increased OPN mRNA expression by aggregating mononuclear cytotrophoblasts. Aminoglutethimide suppression of endogenous steroidogenesis by syncytiotrophoblasts inhibited OPN expression, whereas the addition of exogenous progesterone to cells treated with aminoglutethimide reversed this inhibitory effect. These observations were confirmed at the protein level by immunocytochemistry. Treatment of cytotrophoblasts with both progesterone and RU486 inhibited the up-regulatory effect on OPN mRNA associated with exposure to progesterone alone, further confirming a direct effect of progesterone. We conclude that progesterone up-regulates OPN expression in human cytotrophoblasts, and we propose that in vivo, progesterone secretion by syncytiotrophoblasts regulates the expression of OPN by the underlying cytotrophoblasts. As the receptors for OPN,α v integrins, are expressed by syncytiotrophoblasts, we postulate that these paracrine regulatory mechanisms contribute to the adhesive and/or signaling events between the two trophoblast cell types of the chorionic villus.
Transforming Growth Factor-β1 Inhibits Membrane Association of Protein Kinase Cα in a Human Prostate Cancer Cell Line, PC3*
Authors: Lamm Marilyn L. G. , Long Denise D. , Goodwin Shannon M. , Lee Chung
Published: January 2014

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-β1 (TGF-β1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-β1 in PC3, a human prostate cancer cell line. PKCα, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKCα were decreased by a growth-inhibitory dose of TGF-β1. The response to TGF-β1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKCα protein. TGF-β1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKCα’s membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKCα, is an early event associated with TGF-β1 postreceptor signaling that might mediate suppression of cell proliferation.
Estradiol Requirements for Induction and Maintenance of the Gonadotropin-Releasing Hormone Surge: Implications for Neuroendocrine Processing of the Estradiol Signal*
Authors: Evans N. P. , Dahl G. E. , Padmanabhan V. , Thrun L. A. , Karsch F. J.
Published: January 2014

Two experiments were performed to examine the temporal requirements of the estradiol signal for the GnRH and LH surges in the ewe. Hypophyseal portal and jugular blood (to measure GnRH and LH, respectively) were sampled from ewes set up in an artificial follicular phase model. After progesterone withdrawal to simulate luteolysis, circulating estradiol was raised to a preovulatory level by inserting estradiol implants, which then were removed at different times to vary estradiol signal duration. The objective of the first experiment was to assess the effect of withdrawing estradiol at surge onset on development and maintenance of the GnRH/LH surges. Removal of estradiol, before surge onset, neither altered the LH surge in relation to that induced when the estradiol stimulus was maintained nor affected stimulation of a massive and sustained GnRH surge that outlasted the LH surge by many hours. Continued estradiol treatment, however, did prolong the GnRH surge. In the second experiment, the estradiol stimulus was shortened to test the hypothesis that estradiol need not be present for the whole presurge period to induce GnRH/LH surges. Ewes received estradiol either up to the time of surge onset (21 h) or for periods equivalent to the last 14 h, the last 7 h, or the earliest 7 h of the 21-h signal. Shortening the signal to 14 h did not reduce its ability to stimulate a full GnRH surge, but it did reduce the amplitude of the resultant LH surge. Further shortening of the signal to 7 h, however, produced a mixed response. Most animals (8 of 10 combining the two 7-h groups) did not express GnRH surges. In the two ewes that did, GnRH surge amplitude and duration were again within the range observed with the 21-h estradiol signal, but the LH response was greatly reduced.

These results indicate that, once the GnRH/LH surges of the ewe have begun, elevated estradiol is not required for surge maintenance. Development of a full GnRH surge requires elevated estradiol for only a portion of the presurge period. More prolonged exposure to estradiol, however, is needed to maximize pituitary responsiveness to GnRH. Since the estradiol signal for the GnRH surge is relatively short (7–14 h) and temporally located well in advance of the surge itself, these results are consistent with the hypothesis that estradiol is required only to activate the steroid-responsive neuronal elements and not for progression of the signal from these elements to the actual surge process of GnRH release.
Maternal Nonthyroidal Illness and Fetal Thyroid Hormone Status, as Studied in the Streptozotocin-Induced Diabetes Mellitus Rat Model*
Authors: Calvo Rosa , Morreale de Escobar Gabriella , Escobar del Rey Francisco , Obregón Maria-Jesús
Published: January 2014

We have used the streptozotocin-induced diabetes mellitus pregnant rat as a model of maternal nonthyroidal illness. We measured the effects of different degrees of diabetes mellitus on maternal body weight, the outcome of pregnancy, circulating glucose, insulin, T4, T3, rT3, and TSH in mother and fetus, T4 and T3 in maternal and fetal tissues, and iodothyronine deiodinases in liver, lung, and brain.

All of the changes in thyroid hormone status typical of nonthyroidal illnesses were observed in the mothers and were related to the degree of the metabolic imbalances. Most were controlled with a daily insulin dose of 0.5 U/100 g BW. Normalization of maternal placental T4, however, required higher insulin doses than in other maternal tissues.

The number and body weight of the fetuses, their pituitary GH contents, and their thyroid hormone status were severely affected. The total extrathyroidal T4 and T3 pools decreased to one third of normal fetal values. T4 and T3 concentrations in the fetal brain were lower than normal, and the expected increase in type II 5′deiodinase activity was not observed. The low cerebral T3 only improved with adequate insulin treatment of the dams.

It is concluded that maternal diabetes mellitus, and possibly other nonthyroidal illnesses that impair the availability of intracellular energy stores, may affect fetal brain T3 when thyroid hormones are essential for normal development.
Retinoic Acids Increase 17β-Hydroxysteroid Dehydrogenase Type 1 Expression in JEG-3 and T47D Cells, but the Stimulation Is Potentiated by Epidermal Growth Factor, 12-O-Tetradecanoylphorbol-13-Acetate, and Cyclic Adenosine 3′,5′-Monophosphate Only in JEG-3 Cells*
Authors: Piao Yun-shang , Peltoketo Hellevi , Jouppila Annukka , Vihko Reijo
Published: January 2014

Human 17β-hydroxysteroid dehydrogenase type 1 (17HSD type 1) primarily catalyzes the reduction of low activity estrone to high activity estradiol in ovarian granulosa cells and placental trophoblasts. 17HSD type 1 is also present in certain peripheral tissues, such as breast tissue. In the present study we investigated the effects of retinoic acids (RAs) together with other stimuli known to modulate estradiol production and/or cell growth on expression of 17HSD type 1 in JEG-3 choriocarcinoma cells and estrogen-responsive T47D breast cancer cells. Treatment of cultured JEG-3 and T47D cells with all-trans-RA and 9-cis-RA increased reductive 17HSD activity and 17HSD type 1 messenger RNA expression severalfold in both cell lines. On the other hand, epidermal growth factor (EGF), Ca ionophore, the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA), and cAMP elevated 17HSD type 1 expression only in JEG-3 cells. Correspondingly, the effects of RAs were potentiated by EGF, TPA, and cAMP in JEG-3 cells, whereas no such phenomenon was observed in T47D cells. In JEG-3 cells, simultaneous administration of RAs with TPA and EGF maximally resulted in approximately 40- and 20-fold increases in 17HSD type 1 messenger RNA expression, respectively. The present data indicate that RAs may stimulate estradiol biosynthesis by regulating 17HSD type 1 expression in certain breast cancer and choriocarcinoma cells. The results suggest that interaction of multiple regulatory pathways is involved in maintaining high 17HSD type 1 expression in the placenta. In addition, regulation of 17HSD type 1 expression may be different in trophoblast cells from that in breast epithelial cells.
Adrenocorticotropin Response and Nicotine-Induced Norepinephrine Secretion in the Rat Paraventricular Nucleus Are Mediated through Brainstem Receptors*
Authors: Fu Yitong , Matta Shannon G. , Valentine James D. , Sharp Burt M.
Published: January 2014

Nicotine is a potent stimulus for the secretion of ACTH, and norepinephrinergic neurons originating in the brainstem are involved. Prior reports using in vivo microdialysis in alert rats have shown that nicotine, administered ip or into the fourth ventricle, stimulated the release of norepinephrine (NE) into the hypothalamic paraventricular nucleus (PVN), the site of neurons containing CRH. In the present studies, rats received an iv infusion of nicotine into the jugular vein on alternate days during their active (dark) phase; therefore, direct correlations between the levels of NE microdialyzed from the PVN and plasma ACTH could be made in each animal. Nicotine administered iv (0.045–0.135 mg/kg) elicited dose-dependent increases in both NE and ACTH (P < 0.01). A significant correlation was found between nicotine-stimulated NE release in the PVN and ACTH secretion (r = 0.91, P < 0.01). To address whether the site(s) of action of nicotine was on presynaptic receptors on NE terminals in the PVN or on receptors on neurons in brainstem regions accessible from the fourth ventricle, the nicotinic cholinergic antagonist, mecamylamine (0.1–4.8 μg), was microinjected directly into the PVN or into the fourth ventricle before nicotine infusion. Fourth-ventricular administration of mecamylamine (1.6 μg) or higher, before iv nicotine (0.09 mg/kg), completely blocked both NE release in the PVN (IC50 = 0.64 μg) and ACTH secretion (IC50 = 0.40 μg) (P < 0.01, compared with vehicle before nicotine), whereas it was ineffective when injected directly into the PVN. The results demonstrate that the nicotinic cholinergic receptors in the brainstem, rather than presynaptic receptors within the PVN itself, mediate nicotine-stimulated PVN NE release and ACTH secretion.
Contrasted Impact of Maternal Rat Food Restriction on the Fetal Endocrine Pancreas*
Authors: Alvarez C. , Martín M. A. , Goya L. , Bertin E. , Portha B. , Pascual-Leone A. M.
Published: January 2014

The effects of food restriction of the mother (65% restriction of ad libitum food intake) on fetal and maternal insulin secretion and islet function were studied at 21 days gestation in three different rat populations: 1) undernourished from 0–7 days gestation, 2) undernourished from 7–14 days gestation, and 3) undernourished from 14–21 days gestation. The body weights of mothers were decreased in groups 2 and 3 vs. those in control fed pregnant animals, and no changes in basal parameters were found in any group. A glucose tolerance test in mothers from group 3 showed a mild intolerance to glucose and a decreased islet insulin content, although islet stimulation in vitro with glucose alone or plus arginine showed a normal insulin secretory response. Body weight was decreased in fetuses from the three groups (P < 0.01), and pancreas weight was reduced only in group 3. Insulinemia was increased in groups 2 and 3, and pancreatic insulin content increased only in group 3. However, fetuses from mothers of group 3 showed increased islet insulin content, increased response of insulin in vitro to glucose or glucose plus arginine, and hypertrophy of β-cell mass. These results indicate, first, that the development of the fetal pancreas depends on a balanced maternal glucose homeostasis and, second, that adaptive maternal changes to undernutrition seem to induce alterations on the fetal endocrine pancreas, especially when food restriction is applied during the last week of gestation.
Leptin Signaling in the Hypothalamus of Normal Rats in Vivo**This work was supported in part by a Boston Obesity Nutrition Research Center Grant (to J.C.C.), NIH Grant DK-50411 (to R.J.S.), and NIH Diabetes and Endocrinology Center Grant DK-36836 (to R.J.S.).
Authors: McCowen Karen C. , Chow Jesse C. , Smith Robert J.
Published: January 2014

Leptin has been shown to activate multiple signaling molecules in cultured cells, including Janus kinase-2, STAT (signal transducer and activator of transcription) proteins, and mitogen-activated protein kinase, and to stimulate the DNA-binding activity of STAT3 in mouse hypothalamus. In this study, the activation of candidate leptin signaling molecules in the hypothalamus of normal rats in vivo was investigated. Fasted male Sprague-Dawley rats were injected iv with recombinant murine leptin or vehicle. Plasma leptin concentrations were determined at defined time points, and the phosphorylation of signaling proteins was assessed in hypothalamic lysates. There was a marked increase in plasma leptin concentration at 2 min and a gradual decline by 45 min after leptin injection. Immunoblotting analysis of hypothalamic lysates with a phosphospecific STAT3 antibody demonstrated a time-dependent stimulation of STAT3 tyrosine phosphorylation. STAT3 phosphorylation was first evident at 5 min and was maximal at 30 min after leptin injection. By contrast, leptin did not increase the phosphorylation of Janus kinase proteins, mitogen-activated protein kinase, or STAT1 and -5 despite abundant expression of these signaling molecules in the hypothalamus. These results differ from findings in cultured cells and in vitro systems. It remains unclear how signaling is propagated downstream from the leptin receptor to STAT3, but this may involve novel signaling intermediates.
Importance of the Gonadotropin-Releasing Hormone (GnRH) Surge for Induction of the Preovulatory Luteinizing Hormone Surge of the Ewe: Dose-Response Relationship and Excess of GnRH*
Authors: Bowen Jennifer M. , Dahl Geoffrey E. , Evans Neil P. , Thrun Lori A. , Wang Yuedong , Brown Morton B. , Karsch Fred J.
Published: January 2014

The preovulatory LH surge in the ewe is stimulated by a large sustained surge of GnRH. We have previously demonstrated that the duration of this GnRH signal exceeds that necessary to initiate and sustain the LH surge. The objective of the present study was to determine whether a similar excess exists for amplitude of the GnRH surge. Experiments were performed using an animal model in which GnRH secretion was blocked by progesterone, which in itself does not block the pituitary response to GnRH. To assess the amplitude of the GnRH surge needed to induce the LH surge, we introduced artificial GnRH surges of normal contour and duration but varying amplitudes. Twelve ewes were run through 3 successive artificial follicular phases (total of 36). Six of these artificial follicular phases were positive controls, in which progesterone was removed, the estradiol stimulus was provided, and vehicle was infused. In these control cycles, animals generated endogenous LH surges. In the remaining artificial follicular phases, progesterone was not withdrawn, the estradiol stimulus was provided, and either vehicle (negative control) or GnRH solutions of varying concentrations (experimental) were infused. The circulating GnRH concentrations achieved by infusion were monitored. No LH surges were observed in negative controls, whereas LH surges were induced in experimental cycles provided a sufficient dose of GnRH was infused. A highly significant dose-response relationship was observed between the amplitude of the GnRH surge and both the amplitude of the LH surge and the area under the curve describing the LH response, but no such relationship existed between the amplitude of the GnRH surge and the duration of the LH response. In numerous cases, LH surges similar to those in the positive control animals resulted from infusion of amounts of GnRH estimated to be considerably less than those delivered to the pituitary during the endogenously generated GnRH/LH surge. These findings indicate that, in the ewe, increased GnRH secretion drives the preovulatory LH surge in a dose-dependent fashion, and they provide evidence that the amplitude of the GnRH surge may exceed that needed to generate the LH surge.
Identification of a Novel cis-Element in the 3′-Untranslated Region of Mammalian Peptidylglycine α-Amidating Monooxygenase Messenger Ribonucleic Acid
Authors: Fraboulet Sandrine , Boudouresque Françoise , Delfino Christine , Ouafik L’Houcine
Published: January 2014

Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. Growing evidence suggests that the metabolism of PAM messenger RNAs (mRNAs) can be regulated within the cytoplasm. To understand the mechanisms controlling the metabolism of PAM mRNAs, we sought to identify cis elements of the 3′-untranslated region (3′-UTR) of PAM mRNA that are recognized by cytoplasmic factors. From gel retardation assays, one sequence element is shown to form a specific RNA-protein complex. The protein-binding site of the complex was determined by ribonuclease T1 mapping, by blocking the putative binding site with antisense oligonucleotide, and by competition assays. Using 3′-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3′-UTR a novel 20-nucleotide cis element that interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) to form a 60-kDa PAM mRNA-binding protein complex. The binding activity was redox sensitive. Tissue distribution of the protein in the rat showed a marked tissue-specific expression, with ovary, testis, lung, heart septum, anterior pituitary and hypothalamus containing large amounts compared with liver, ventricle, atrium, and neurointermediate lobe. No binding activity was detectable in pancreas, intestine, or kidney extracts. Northwestern blot analysis of AtT-20 (mouse corticotrope tumor cell line) cytoplasmic extracts revealed a protein of 46 kDa. Thus, we have identified a widely distributed cellular protein that binds to a conserved domain within the 3′-UTR of PAM mRNA from many animal species. Although these data suggest that cis element-binding activity could be a cytoplasmic regulator of PAM mRNA metabolism, the functional consequences of this binding remain to be determined.
Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor*
Authors: Camarillo Ignacio G. , Thordarson Gudmundur , Ilkbahar Yonca N. , Talamantes Frank
Published: January 2014

A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide corresponding to the carboxyl-terminal 14 amino acids of the mGHR (GHR-2 peptide) was used as the antigen for antiserum production. The synthetic peptide was also used as the standard and radioligand in the RIA. The ability of the antiserum to recognize the mGHR was demonstrated by quantitating receptor concentrations in liver and mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of these samples yielded displacement curves parallel to the synthetic peptide. No significant cross-reactivity was seen with serum from virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein (mGHBP), a synthetic peptide identical to the hydrophilic tail of mGHBP, or a 14-amino acid synthetic peptide corresponding to amino acids 338–351 of mGHR (GHR-1 peptide). The concentration range of the mGHR RIA was 0.5–200 nm, and the intra- and interassay coefficients of variation were 6.5% and 6.1%, respectively. The concentration of liver GHR increased significantly during pregnancy compared with that in virgin mice, from 0.246 ± 0.045 pmol/mg protein (mean ± sem; n = 5) in the virgin animals to 1.015 ± 0.159 pmol/mg protein (n = 5) in pregnant mice. In contrast, the mGHR concentration in the mammary gland decreased significantly during pregnancy from 0.606 ± 0.201 pmol/mg protein (mean ± sem; n = 5) to 0.299 ± 0.027 pmol/mg protein (n = 5). Comparison of the total number of binding sites in livers from virgin and pregnant mice using the GH RRA and the combined results of the mGHR and mGHBP RIAs showed that the two methods gave almost identical results for livers from virgin animals, or 0.363 ± 0.063 pmol/mg protein (mean± sem; n = 3) and 0.371 ± 0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively. However, in livers from pregnant animals, the combined results from the mGHR and mGHBP RIAs were approximately 1.8 times higher than those obtained by the GH RRA, or 6.732 ± 0.612 pmol/mg protein (mean ± sem; n = 3) and 3.693 ± 0.67 pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the GH RRA, respectively. The increase in the total GH binding capacity in livers from pregnant mice compared with those from virgin animals was largely due to an increase in the GHBP content. The increase in GHR was only 2.4-fold, or from 0.153 ± 0.01 pmol/mg protein (mean ± sem; n = 3) in virgin mice to 0.364 ± 0.03 pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP increased almost 30-fold during pregnancy, or from 0.218 ± 0.003 pmol/mg protein (mean ± sem; n = 3) in virgin animals to 6.369 ± 0.607 pmol/mg protein (n = 3) in pregnant mice.
Glucocorticoid-Induced Apoptosis and Regulation of NF-κB Activity in Human Leukemic T Cells*
Authors: Ramdas Jyoti , Harmon Jeffrey M.
Published: January 2014

Glucocorticoid-induced apoptosis was investigated in glucocorticoid-sensitive 6TG1.1 and resistant ICR27TK.3 human leukemic T cells. Following glucocorticoid treatment of 6TG1.1 cells, chromatin fragmentation was observed after a delay of 24 h. Fragmentation was not observed in ICR27TK.3 cells containing mutant glucocorticoid receptors (L753F) that are activation-deficient but retain the ability to repress AP-1 activity. Nor was fragmentation observed after treatment with RU38486, indicating that repression of AP-1 activity is not involved. As described in other systems, fragmentation required ongoing protein synthesis. However, inhibition of protein synthesis with cycloheximide anytime during the first 18 h of steroid treatment was as effective in blocking chromatin fragmentation as inhibition for the entire period, suggesting that synthesis of a component with a rapid turnover rate is required. Dexamethasone treatment completely blocked 12-O-tetradecanoylphorbol 13-acetate induction of nuclear factor-κB (NF-κB) activity and elicited an increase in the amount of immunoreactive IκBα in sensitive 6TG1.1 cells but not in resistant ICR27TK.3 cells. In addition, mild detergent treatment of cell extracts indicated that a substantial amount of cytoplasmic NF-κB is complexed with IκBα or some other inhibitory factor. These results suggest that induction of a labile inhibitory factor such as IκBα may contribute to glucocorticoid-induced apoptosis.
Pituitary Lactotroph Adenomas Develop after Prolonged Lactotroph Hyperplasia in Dopamine D2 Receptor-Deficient Mice1
Authors: Asa Sylvia L. , Kelly Michele A. , Grandy David K. , Low Malcolm J.
Published: January 2014

Tuberoinfundibular dopamine tonically inhibits PRL expression and secretion from the pituitary gland by the activation of dopamine D2 receptors (D2R) localized on lactotrophs. Mutant female mice that lack D2Rs have persistent hyperprolactinemia but also develop extensive hyperplasia of pituitary lactotrophs and peliosis of the adenohypophysis at 9 to 12 months of age, while age-matched male D2R-deficient mice have no morphologic adenohypophysial lesion. We now report that both female and male D2R-deficient mice 17 to 20 months of age develop pituitary lactotroph adenomas. Of 12 aged female mice examined, all developed monohormonal PRL-immunoreactive neoplasms that had a characteristic juxtanuclear Golgi pattern of PRL staining and loss of the reticulin fiber network. Several of these adenomas were 50-fold larger than normal glands with marked suprasellar extension and invasion of brain but no gross evidence of distant metastases. They also had striking peliosis that was more marked than the lesion seen in the hyperplastic pituitaries of the younger females. These findings demonstrate that a chronic loss of neurohormonal dopamine inhibition promotes the hyperplasia-neoplasia sequence in adenohypophysial lactotrophs. Our results are analogous to previous data indicating that protracted stimulation of adenohypophysial cells by hormones or growth factors results in proliferation with initial hyperplasia followed by the development of neoplasia. Six aged male D2R-deficient mice had slightly enlarged anterior pituitaries similar in size to normal female glands. However, each case exhibited multifocal, microscopic lactotroph adenomas with strong nuclear immunoreactivity for estrogen receptors and Pit-1 transcription factor. The unexpected development of adenomas in males without preexisting or concomitant hyperplasia suggests that prolonged loss of dopamine inhibition may also cause neoplasia by distinct cellular mechanisms in male and female animals.
Pituitary Adenylate Cyclase-Activating Polypeptide Is an Auto/Paracrine Stimulator of Acute Progesterone Accumulation and Subsequent Luteinization in Cultured Periovulatory Granulosa/Lutein Cells*
Authors: Gräs Søren , Hannibal Jens , Fahrenkrug Jan
Published: January 2014

Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4–6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10−7m induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 × 10−9m), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 μg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P < 0.05), 41.2% (P < 0.02), and 50% (P < 0.002), respectively], compared with nonimmune IgG (100 μg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P < 0.02), 69.2% (P < 0.05), 43.8% (P < 0.02), and 52.2% (P < 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.
A Naturally Occurring Genetic Variant in the Human Chorionic Gonadotropin-β Gene 5 Is Assembly Inefficient*
Authors: Miller-Lindholm Amanda K. , Bedows Elliott , Bartels Cynthia F. , Ramey Jacques , Maclin Victoria , Ruddon Raymond W.
Published: January 2014

The hCGβ gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGβ gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGβ gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGβ gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in β gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether βV79M hCG had biological properties that differed from those of wild-type hCG, a β-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone α-subunit in Chinese hamster ovary cells and 293T cells. When we examined βV79M hCG biosynthesis, we detected atypical βV79M hCG folding intermediates, including a βV79M conformational variant that resulted in aβ -subunit with impaired ability to assemble with the α-subunit. The inefficient assembly of βV79M hCG appeared to be independent ofβ -subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the βV79M subunit to fold correctly. The majority of the V79M β-subunit synthesized was secreted as unassembled free β. Although the amount of αβ hCG heterodimer formed and secreted by βV79M-producing cells was less than that by wild-type β-producing cells, the hCG that was secreted as αβ V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.
Parathyroid Hormone-Related Protein in Rat Penis: Expression, Localization, and Effect on Cavernosal Pressure1
Authors: Lang Hervé , Endlich Nicole , Lindner Véronique , Endlich Karlhans , Massfelder Thierry , Stewart Andrew F. , Saussine Christian , Helwig Jean-Jacques
Published: January 2014

Although PTH-related protein-(1–36) [PTHrP-(1–36)] is known to be expressed in smooth muscle and to exert potent myorelaxant effects, its tonic effects on cavernosal smooth muscle has not yet been explored. Using the RT-PCR technique, the present study establishes that PTHrP messenger RNA is present in microdissected corpus cavernosa in the rat. In immunohistochemical studies using affinity-purified antibodies to middle regions of PTHrP, immunostaining was localized throughout the penile structures, including vessels, cavernosal smooth muscle, and trabecular fibroblasts. Strong immunostaining for PTHrP was also detected in the dorsal nerve bundles. In anesthetized rats, intracavernosally injected boluses of increasing doses of PTHrP-(1–36) (0.3–30 pmol in 100 μl saline) had little effect on intracavernosal pressure. However, they markedly potentiated the dilatory response to papaverine (8–800 nmol), increasing the papaverine-induced intracavernous pressure by 2.5-fold, close to the mean arterial pressure. In conclusion, the cavernosal expression of PTHrP messenger RNA, the distribution of immunoreactive PTHrP throughout the structuro-functional components of the erectile apparatus and its strong potentiating action on papaverine-induced cavernosal relaxation, collectively suggest that PTHrP participates in the control of cavernosal tone.
In Situ Estrogen Synthesized by Aromatase P450 in Uterine Leiomyoma Cells Promotes Cell Growth Probably Via an Autocrine/Intracrine Mechanism*
Authors: Sumitani Hiroshi , Shozu Makio , Segawa Tomoya , Murakami Kouichi , Yang Hui-Juan , Shimada Keiko , Inoue Masaki
Published: January 2014

In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1β and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5α-reductase (finasteride and flutamide). The apparent Km of androstenedione was 3 nm in the presence of dexamethasone and interleukin-1β, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10−8 and 10−7m) and androstenedione (10−8 and 10−7m) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10−10–10−7m), whereas dihydrotestosterone (10−11–10−8m) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10−8m)-treated smooth muscle-like cells was 10−11m, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10−10m). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism.
Multiple Cadherin Superfamily Members with Unique Expression Profiles Are Produced in Rat Testis1
Authors: Johnson Kamin J. , Patel Sutchin R. , Boekelheide Kim
Published: January 2014

Adhesion between germ and Sertoli cells is thought to be crucial for spermatogenesis. Cadherin superfamily proteins, including classic cadherins and protocadherins, are important mediators of cell-cell adhesion. Using a degenerate PCR cloning strategy, we surveyed the expression of cadherin superfamily members in rat testis. Similar to brain, testis expressed a large number of cadherin superfamily members: 7 classic cadherins of both types I and II, 14 protocadherins, 2 protocadherin-related cadherins, and 1 cadherin-related receptor-like protein. All three protocadherin families (α, β, and γ) were found in testis. Using a semiquantitative RT-PCR assay, messenger RNA expression was determined for each cadherin superfamily member during a postnatal developmental time-course and following ablation of specific testis cell types by ethanedimethanesulfonate, methoxyacetic acid, and 2,5-hexanedione. Diverse expression patterns were observed among the cadherins, suggesting that cadherin expression is cell type-specific in testis. The large number and variety of cadherin superfamily members found in testis supports a critical function for cadherin-mediated cell-cell adhesion in spermatogenesis.
Influence of the in Vivo Calcium Status on Cellular Calcium Homeostasis and the Level of the Calcium-Binding Protein Calreticulin in Rat Hepatocytes*
Authors: Mailhot Geneviève , Petit Jean-Luc , Demers Christian , Gascon-Barré Marielle
Published: January 2014

Little attention has been given to the consequences of the in vivo calcium status on intracellular calcium homeostasis despite several pathological states induced by perturbations of the in vivo calcium balance. The aim of these studies was to probe the influence of an in vivo calcium deficiency on the resting cytoplasmic Ca2+ concentration and the inositol-1,4,5-trisphosphate-sensitive Ca2+ pools. Studies were conducted in hepatocytes (a cell type well characterized for its cellular Ca2+ response) isolated from normal and calcium-deficient rats secondary to vitamin D depletion. Both resting cytoplasmic Ca2+ concentration and Ca2+ mobilization from inositol-1,4,5-trisphosphate -sensitive cellular pools were significantly lowered by calcium depletion. In addition, Ca deficiency was shown to significantly reduce calreticulin messenger RNA and protein levels but calcium entry through store-operated calcium channels remained unaffected, indicating that the Ca2+ entry mechanisms are still fully operational in calcium deficiency. The effects of calcium deficiency on cellular calcium homeostasis were reversible by repletion with oral calcium feeding alone or by the administration of the calcium-regulating hormone 1,25-dihydroxyvitamin D3, further strengthening the tight link between extra- and intracellular calcium. These data, therefore, challenge the currently prevailing hypothesis that extracellular Ca2+ has no significant impact on cellular Ca2+ by demonstrating that despite the large Ca2+ gradient between extra- and intracellular Ca2+ concentrations, calcium deficiency in vivo significantly alters the hormone-sensitive cellular calcium homeostasis.
Expression of Estrogen Receptor α and β in the Rhesus Monkey Corpus Luteum during the Menstrual Cycle: Regulation by Luteinizing Hormone and Progesterone*
Authors: Duffy Diane M. , Chaffin Charles L. , Stouffer Richard L.
Published: January 2014

There are conflicting reports on the presence or absence of estrogen receptor (ER) in the primate corpus luteum, and the discovery of a second type of estrogen receptor, ERβ, adds an additional level of complexity. To reevaluate ER expression in the primate luteal tissue, we used semiquantitative RT-PCR based assays and Western blotting to assess ERα and β messenger RNA (mRNA) and protein levels in corpora lutea (n = 3/stage) obtained from adult female rhesus monkeys at early (days 3–5), mid (days 6–8), mid-late (days 10–12), and late (days 14–16) luteal phase of the natural menstrual cycle. ERα mRNA levels did not vary across the stages of the luteal phase, and ERα protein was not consistently detected in luteal tissues. However, ERβ mRNA and protein levels were detectable in early and mid luteal phases and increased (P < 0.05) to peak levels at mid-late luteal phase before declining by late luteal phase. To determine if ERβ mRNA expression in the corpus luteum is regulated by LH, monkeys received the GnRH antagonist antide either alone or with 3 daily injections of LH to simulate pulsatile LH release. Treatment with antide alone or concomitant LH administration did not alter luteal ERβ mRNA levels. When monkeys also received the 3β-hydroxysteroid dehydrogenase inhibitor trilostane to reduce luteal progesterone production, luteal ERβ mRNA levels were 3-fold higher (P < 0.05) than in monkeys receiving antide + LH only. Replacement of progestin activity with R5020 reduced luteal ERβ mRNA levels to those seen in animals receiving antide + LH. Thus, there is dynamic ERβ expression in the primate corpus luteum during the menstrual cycle, consistent with a role for estrogen in the regulation of primate luteal function and life span via a receptor (ERβ)-mediated pathway. Increased ERβ expression in the progestin-depleted corpus luteum during LH exposure suggests that the relative progestin deprivation experienced by the corpus luteum between LH pulses may enhance luteal sensitivity to estrogens during the late luteal phase of the menstrual cycle.
Dissociation of Locomotor Activation and Suppression of Food Intake Induced by CRF in CRFR1-Deficient Mice
Authors: Contarino Angelo , Dellu Françoise , Koob George F. , Smith George W. , Lee Kuofen , Vale Wylie W. , Gold Lisa H.
Published: January 2014

ABSTRACT

Corticotropin-releasing factor (CRF) systems are involved in locomotor and feeding behaviors. Two distinct CRF receptor subtypes, CRFR1 and CRFR2, are thought to mediate CRF actions in the central nervous system. However, the role for each receptor in locomotor activity and feeding remains to be determined. Using CRFR1 null mutant mice, the present study examined the functional significance of this receptor in ambulation and feeding. CRF treatment of wild-type mice resulted in increased levels of locomotion whereas no change was observed in CRFR1-deficient mice as compared to vehicle-treated mutant mice. In contrast, CRF decreased food-water intake in both wild type and CRFR1-deficient mice equally. These results support an important role for CRFR1 in mediating CRF-induced locomotor activation, whereas other receptor subtypes, likely CRFR2, may mediate the appetite-suppressing effects of CRF-like peptides.