Processes of Religious and Spiritual Influence in Adolescence: A Systematic Review of 30 Years of Research.

This is a systematic review of 30 years (1988-2017) of empirical research on processes of religious/spiritual influence in adolescence. We followed a multi-step process that resulted in 241 studies organized according to eight research questions and the corresponding methods and analyses typically used to address them. We coded these studies based on the dimensions of religiosity/spirituality and the youth outcomes involved. In some cases (quantitative studies of mediation and moderation, as well as qualitative studies) we also coded a third process variable. Results of the coding process revealed a number of interesting patterns. First, religiosity/spirituality is generally adaptive for adolescents, protecting them from negative outcomes (e.g., risk behaviors and mental illness), and promoting positive youth development and flourishing. Nevertheless, in some contexts, religiosity/spirituality may be at least partially maladaptive. Second, there is some evidence, from experimental and longitudinal studies, that relations between religiosity/spirituality and adaptive outcomes are causal. Third, there are numerous complex and dynamic processes by which religiosity/spirituality relate to youth outcomes. In terms of mediation studies, the most salient mediating processes seem to involve religiosity/spirituality dimensions, peers, values/attitudes, and social control/norms. Fourth, religiosity/spirituality is multidimensional, involving various interwoven facets at the individual and ecological levels. Private or personal aspects of religiosity/spirituality (e.g., religious/spiritual importance) tend to be more salient predictors of outcomes than public aspects of religiosity/spirituality (e.g., religious worship service attendance). The results of this systematic review point to promising directions for future research. First, more research is needed studying a broader range of dimensions of religiosity/spirituality, processes of influence, and outcomes. In terms of religiosity/spirituality, much of the prior work has focused on overall religiosity/spirituality, and religious/spiritual behaviors (e.g., worship service attendance). In terms of outcomes, the emphasis has been on religiosity/spirituality protecting against maladaptive outcomes (e.g., substance use). Second, more research is needed examining the role of culture, and using more rigorous methods (e.g., experience sampling, experimental design, longitudinal design, or mixed methods). This systematic review provides a detailed analysis of what is known regarding processes of religious/spiritual influence in the lives of adolescents, and hopefully better positions researchers to move the field forward.

Impact of Smoking, Obesity and Maternal Diabetes on SHBG Levels in Newborns.

BACKGROUND: Low levels of SHBG have become a marker for insulin resistance and diabetes. Babies born to mothers who are obese, have diabetes, or smoke during pregnancy are at greater risk of developing obesity and diabetes later in life. AIMS: To examine the impact of maternal obesity, diabetes and smoking on SHBG levels in newborns. STUDY DESIGN: This cross-sectional study is part of an ongoing multicenter, longitudinal study. SUBJECTS: 98 healthy newborns and their parents, including 16 mothers with diabetes and 31 mothers with a smoking history. OUTCOME MEASURES: Cord blood and second day venipuncture samples were collected for measurement of SHBG and insulin. RESULTS: Babies born to mothers with diabetes had lower SHBG levels in cord blood [14.0 (8.9-20.4) vs. 19.6 (14.9-25.1) nmol/L; p=0.011] and on day 2 [18.8 (12.6-21.2) vs. 22.9 (17.1-29.1) nmol/L; p=0.015] than controls. Maternal diabetes remained negatively associated with SHBG levels in cord blood (p=0.02) and on day 2 (p=0.04) when adjusted for mothers' age, smoking status, pre-pregnancy weight and weight gain during pregnancy. SHBG levels in cord blood and day 2 samples were similar in babies born to mothers who were overweight-obese but not diabetic vs. normal weight, or were smokers when compared to non-smokers. CONCLUSIONS: SHBG levels are lower in newborns born to mothers with diabetes than without diabetes, and may be a marker for babies' life-long risk for abnormal metabolic health. On the other hand, the adverse effects of tobacco smoke on the fetus do not appear to directly influence SHBG levels.

PACAP, an autocrine/paracrine regulator of gonadotrophs.

Hypothalamic-hypophysiotropic peptides are the proximate regulators of pituitary cells, but they cannot fully account for the complex functioning of these cells. Accordingly, awareness is growing that an array of peptides produced in the pituitary exert paracrine/autocrine functions. One such peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), was originally identified as a hypothalamic activator of cAMP production in pituitary cells. Gonadotrophs and folliculostellate cells are the main source of pituitary PACAP, and each pituitary cell type expresses a PACAP receptor. PACAP increases alpha-subunit (Cga) and Lhb mRNAs, and it stimulates the transcription of follistatin (Fst) that, in turn, restrains activin signaling to repress Fshb and gonadotropin-releasing hormone-receptor (Gnrhr) expression as well as other activin-responsive genes. The PACAP (Adcyap1) promoter is activated by cAMP, and pituitary cells may communicate by a feed-forward, cAMP-dependent mechanism to maintain a high level of PACAP in the fetal pituitary. At birth, pituitary PACAP declines and pituitary follistatin levels decrease, which together with increased gonadotropin-releasing hormone secretion allow Gnrhr and Fshb to increase and facilitate activation of the newborn gonads. Changes in Adcyap1 expression levels in the adult pituitary may contribute to the selective rise in follicle-stimulating hormone (FSH) from age 20-30 days to the midcycle surge and to the secondary increase in FSH that occurs before estrus. These results provide further support for the notion that PACAP is a key player in reproduction through its actions as a pituitary autocrine/paracrine hormone.

PACAP: A regulator of mammalian reproductive function.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an ancestral molecule that was isolated from sheep hypothalamic extracts based on its action to stimulate cAMP production by pituitary cell cultures. PACAP is one of a number of ligands that coordinate with GnRH to control reproduction. While initially viewed as a hypothalamic releasing factor, PACAP and its receptors are widely distributed, and there is growing evidence that PACAP functions as a paracrine/autocrine regulator in the CNS, pituitary, gonads and placenta, among other tissues. This review will summarize current knowledge concerning the expression and function of PACAP in the hypothalamic-pituitary-gonadal axis with special emphasis on its role in pituitary function in the fetus and newborn.

Signaling pathways and promoter regions that mediate pituitary adenylate cyclase activating polypeptide (PACAP) self-regulation in gonadotrophs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is thought to play a role in the development and regulation of gonadotrophs. PACAP levels are very high in the rodent fetal pituitary, and decline substantially and rapidly at birth, followed by a significant rise in FSHß and GnRH-R expression. Because there is evidence that PACAP stimulates its own transcription, we propose that this self-regulation is interrupted around the time of birth. To begin to examine the mechanisms for PACAP self-regulation, we used two well-established gonadotroph cell lines, αT3-1 cells and the more mature LßT2 cells which were transfected with a PACAP promoter-reporter construct As in vivo, the basal PACAP transcription level is significantly lower in the more mature LßT2 cells in which basal cAMP signaling is also much reduced. The PACAP promoter was stimulated by PACAP in both cell lines. Treatment with inhibitors of second messenger pathways implicated PKA, PKC and MAPK in PACAP transcription. Three regions of the PACAP promoter were found to confer inhibition or stimulation of PACAP transcription. By inhibiting cAMP response element binding (CREB) activity and mutating a proximal CREB binding site, we found that CREB is essential for promoter activation. Finally, overexpression of PACAP receptor HOP1 isoform, to increase the level in LßT2 cells to that of αT3-1 cells and simulate the E19 pituitary, increased PACAP- stimulated sensitivity and significantly altered downstream gene transcription. These results provide novel insight into the feed-forward regulation of PACAP expression that may help initiate gonadotroph function at birth.

Regulation of insulin-response element binding protein-1 in obesity and diabetes: potential role in impaired insulin-induced gene transcription.

One of the major mechanisms by which insulin modulates glucose homeostasis is through regulation of gene expression. Therefore, reduced expression of transcription factors that are required for insulin-regulated gene expression may contribute to insulin resistance. We recently identified insulin response element-binding protein-1 (IRE-BP1) as a transcription factor that binds and transactivates multiple insulin-responsive genes, but the regulation of IRE-BP1 in vivo is largely unknown. In this study, we show that IRE-BP1 interacts with the insulin response sequence of the IGF-I, IGFBP-1, and IGFBP-3 genes using chromatin immunoprecipitation assay. Furthermore, activation by IRE-BP1 is sequence specific and mimics that of the insulin effect on gene transcription. Tissue expression of IRE-BP1 is 50- to 200-fold higher in classical insulin target compared with nontarget tissues in lean animals, with a significantly reduced level of expression in the skeletal muscle and adipose tissue in obese and diabetic animals. In the liver, IRE-BP1 is localized to the nucleus in lean rats but is sequestered to the cytoplasm in obese and diabetic animals. Cytoplasmic sequestration appears to be related to inhibition of insulin-mediated phosphatidylinositol-3 kinase signaling. Therefore, in diabetes and obesity, the mechanisms involved in reducing the transactivation of the insulin response sequence by IRE-BP1 include decreased gene transcription and nuclear exclusion to prevent DNA binding. Our study supports the notion that IRE-BP1 may be relevant to the action of insulin in vivo and may play a role in the development of insulin resistance and diabetes.

Plastic versus self-expanding metallic stents for malignant hilar biliary obstruction: a prospective multicenter observational cohort study.

BACKGROUND: There are few comparative data as to whether plastic or self-expanding metallic stents are preferable for palliating malignant hilar biliary obstruction. METHODS: Thirty-day outcomes of consecutive endoscopic retrograde cholangiopancreatographies performed for malignant hilar obstruction at 6 private and 5 university centers were assessed prospectively. RESULTS: Patients receiving plastic (N=28) and metallic stents (N=34) were similar except that metallic stent recipients more often had: Bismuth III or IV tumors (16/34 vs. 5/28 P=0.043), higher Charlson comorbidity scores (P=0.003), metastatic disease (P=0.006), and management at academic centers (P=0.018). The groups had similar rates of bilateral stent placement (4/28 vs. 5/34), and similar frequency of opacified but undrained segmental ducts (7/28 vs. 5/34). Adverse outcomes including cholangitis, stent occlusion, migration, perforation, and/or the need for unplanned endoscopic retrograde cholangiopancreatography or percutaneous transhepatic cholangiography occurred in 11/28 (39.3%) patients with plastic versus 4/34 (11.8%) with metal stents (P=0.017). By logistic regression, factors associated with adverse outcomes included plastic stent placement (odds ratio 6.32; 95% confidence interval 1.23, 32.56) and serum bilirubin (1.11/mg/dL above normal: 1.01, 1.22) but not center type or Bismuth class. CONCLUSIONS: Metallic stent performance was superior to plastic for hilar tumor palliation with respect to short-term outcomes, independent of disease severity, Bismuth class, or drainage quality.

Development of cystic glandular hyperplasia of the endometrium in Mullerian inhibitory substance type II receptor-pituitary tumor transforming gene transgenic mice.

The pituitary tumor transforming gene (PTTG)/securin is an oncogene that is involved in cell cycle regulation and sister chromatid separation. PTTG is highly expressed in various tumors including ovarian tumors, suggesting that PTTG may play a role in ovarian tumorigenesis. Overexpression of PTTG resulted in induction of cellular transformation in vitro and tumor formation in nude mice. To ascertain PTTG function in ovarian tumorigenesis, we generated a transgenic mouse model of PTTG by cloning PTTG cDNA downstream of Mullerian inhibitory substance type II receptor gene promoter (MISIIR) in order to target the ovarian surface epithelium. By screening of transgenic animals, we identified five founders (four males and one female). Using the four male founders, we developed four transgenic lines. PTTG expression was increased in ovarian surface epithelium, ovarian granulosa cells, as well as in the pituitary gland. Transgenic females did not develop any visible ovarian tumors at 8-10 months of age; however, there was an overall increase in the corpus luteum mass in transgenic ovary, suggesting increased luteinization. These changes were associated with an increase in serum LH and testosterone levels. In addition, there was a generalized hypertrophy of the myometrium of MISIIR-PTTG transgenic uteri with cystic glandular and hyperplasia of the endometrium. Based on these results, we conclude that the overexpression of PTTG may be required to initiate precancerous conditions but is not sufficient to induce ovarian tumorigenesis and may require another partner to initiate cellular transformation.

Transcriptional regulation of follistatin expression by GnRH in mouse gonadotroph cell lines: evidence for a role for cAMP signaling.

GnRH applied continuously or in pulses of high frequency increases follistatin, and thereby differentially regulates FSH and LH. This study was conducted in alphaT3-1 and LbetaT2 gonadotroph cells to begin to understand the signaling pathways through which GnRH stimulates follistatin synthesis. GnRH increased follistatin expression and stimulated a follistatin-LUC reporter in LbetaT2 cells, but was inactive in alphaT3-1 cells. GnRH also increased cAMP levels and stimulated a cAMP-responsive promoter only in LbetaT2 cells. Forskolin stimulated follistatin in both cell lines. GnRH activation of follistatin was blocked by the PKA inhibitor H89 and by over-expression of a dominant-negative inhibitor of CREB (A-CREB). Activation was also suppressed by PKC depletion, and was reduced by the PKC inhibitor bisindolylmaleimide. The MEK inhibitor PD98059 blocked activation by GnRH or forskolin implying that MAPK contributes to cAMP/PKA-mediated activation of follistatin. When LbetaT2 cells were transfected with follistatin-LUC together with A-CREB, and perifused with GnRH, activation was blocked during continuous GnRH, but stimulation by hourly GnRH pulses was unaffected. These experiments provide evidence that GnRH stimulates follistatin through multiple signaling pathways, and that cAMP-CREB activation is obligatory when GnRH is applied continuously. The finding that follistatin transcription was CREB-dependent with continuous but not pulsatile GnRH implies that the mode of ligand activation of GnRH receptors modifies the transcriptional response by changing the signaling network. These results provide a mechanism linking GnRH pulsatility to the differential control of FSH-beta and LH-beta gene expression through follistatin.

Paracrine control of gonadotrophs.

There is increasing evidence for communication among pituitary cells. Hormone-producing pituitary cells may communicate with each other and with folliculostellate cells. The latter cells surround pituitary hormone-producing cells and are connected by tight junctions to form a network that allows for their coordinated function. Folliculostellate cells are targets of cytokines, peptides, and steroid hormones, and produce growth factors and cytokines, including follistatin, the dynamic regulator of follicle-stimulating hormone (FSH) production that binds activin, and limits activin signaling. Pituitary adenylate cyclase-activating peptide (PACAP) and its receptor are found in folliculostellate cells in which they stimulate transcription of the follistatin gene through cyclic adenosine monophosphate/protein kinase A (PKA) signaling. When PACAP increases, follistatin levels increase, and FSH-beta mRNA is reduced. PACAP also activates gonadotrophs to stimulate transcription of the gonadotropin alpha-subunit gene and lengthen the LH-beta mRNA, presumably to prolong it half-life, and increases responsiveness to GnRH. Accordingly, PACAP differentially regulates FSH and LH, and may prove to be a key player in reproduction through a novel paracrine mechanism.

Androgen receptors in gonadotrophs in pituitary cultures from adult male monkeys and rats.

There is substantial evidence demonstrating that the principal feedback action of androgens to decrease LH secretion in male primates, including man, is to slow the GnRH pulse generator, whereas in male rats androgens not only decrease GnRH but also suppress LH synthesis and secretion through a direct pituitary effect. Previous experiments in our laboratory revealed that testosterone (T) suppresses LH secretion and decreases alpha-subunit mRNA levels in male rat pituitary cell cultures perifused with pulses of GnRH but not in pituitary cells from adult male monkeys. In the present study, we sought to determine whether the lack of responsiveness of gonadotrophs to androgens in the primate is androgen receptor (AR) related. Primary cultures were prepared from the anterior pituitary glands of adult male monkeys and rats. Cells were identified as gonadotrophs if they were immunoreactive for LH-beta or FSH-beta. Of these cells in the monkey, 80% contained both gonadotropins, 17% contained only LH-beta, and 3% contained only FSH-beta. AR immunoreactivity (IR) was nuclear in 22% and 15%, respectively, of monkey and rat FSH-beta-positive cells in the absence of T. Following T treatment, nuclear AR IR was identified in 79% of monkey and 81% of rat gonadotrophs. T treatment similarly intensified AR IR in mouse gonadotroph alphaT3-1 and LbetaT2 cells and in monkey and rat fibroblasts. Single-cell RT-PCR confirmed coexpression of LH-beta and AR mRNA as well as LH-beta and GH mRNA in monkey gonadotrophs. Our data reveal that most monkey, as well as rat, gonadotrophs are AR-positive with nuclear localization in the presence of T. GH expression is not required for AR expression in gonadotrophs. We conclude that the failure of T to inhibit LH secretion and decrease alpha-subunit mRNA expression in the male primate is not due a disturbance in AR nuclear shuttling.

Evidence that PACAP and GnRH down-regulate follicle-stimulating hormone-beta mRNA levels by stimulating follistatin gene expression: effects on folliculostellate cells, gonadotrophs and LbetaT2 gonadotroph cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates alpha-subunit transcription and lengthens LH-beta mRNA transcripts, but reduces FSH-beta mRNA levels in rat pituitary cell cultures. PACAP also stimulates follistatin transcription, an effect which may explain the decrease in FSH-beta mRNA. To begin to investigate the cells in which PACAP activates the follistatin gene, quantitative in situ hybridization for follistatin mRNA combined with immunostaining for LHbeta and S100 protein was performed. In control cultures, follistatin mRNA was expressed in 70% of gonadotrophs and in 47% of folliculostellate cells (S-100+). PACAP increased (P<0.001) both the number of follistatin-expressing cells as well as the number of grains per cell in both gonadotrophs and folliculostellate cells, while GnRH only affected (P=0.01) gonadotrophs. Follistatin and FSH-beta gene expression in rat pituitary cultures were also measured by competitive quantitative RT-PCR and northern analysis, respectively. Both PACAP and GnRH increased (P<0.05) follistatin gene expression and suppressed (P<0.05) FSH-beta mRNA, and the effect of PACAP together with GnRH on follistatin exceeded that of GnRH alone. PACAP regulation of follistatin and FSH-beta gene expression was studied further in LbetaT2 cells that were found to express receptors for the specific PACAP receptor, PAC(1). Follistatin mRNA was undetectable in cultures exposed to control media, or stimulated with PACAP, GnRH or rh-activin-A. In contrast to the results in primary pituitary cultures, PACAP increased FSH-beta mRNA in these follistatin-deficient cells. Moreover, using transient transfection, PACAP stimulated transcription of ovine-FSH-beta-luciferase. GnRH likewise increased FSH-beta mRNA and stimulated FSH-beta gene transcription in LbetaT2 cells. Activin-A increased FSH-beta gene expression dose-dependently, and activin induction of FSH-beta mRNA was blocked completely by 3-fold excess follistatin. These results indicate that PACAP stimulates follistatin gene expression in both gonadotrophs and folliculostellate cells, and provide further evidence that follistatin is required for PACAP or continuous GnRH to down-regulate FSH-beta mRNA. These experiments suggest a mechanism by which PACAP influences FSH production selectively by an autocrine effect on gonadotrophs and by a paracrine mechanism through folliculostellate cells that involves follistatin.

Pituitary adenylate cyclase-activating polypeptide (PACAP) in fetal cord blood.

BACKGROUND: Pituitary adenylate cyclase-activating peptide (PACAP) is a centrally-acting peptide with highest concentration within the limbic area of the brain. PACAP is also expressed in and affects the functions of vascular and nervous tissues, endocrine glands, and the placenta. PACAP appears to be associated with the 'fight-or-flight' response to emergency partly through its effect on adrenal production of cortisol and catecholamines. OBJECTIVES: We sought to explore the impact of labor as a stressor on the level of PACAP in the fetus, and hypothesized that PACAP levels would be increased when labor, abnormal fetal heart tracing, or fetal growth impairment was evident. METHODS: Fetal cord venous blood samples were collected immediately after delivery from a random group of women undergoing either vaginal or Cesarean delivery. The blood was placed in chilled EDTA-aprotinin containing tubes, centrifuged, and stored at -80°C for PACAP immunoassay. Delivery data were abstracted for analysis. RESULTS: The level of PACAP in cord blood was similar in both males and females. There was a trend toward higher levels in the cord blood of fetuses delivered by Cesarean compared to those delivered vaginally. PACAP levels were unrelated to birth weight, Apgar scores, and the presence or absence of labor prior to delivery. CONCLUSIONS: While PACAP and its receptor are expressed in placenta, and PACAP protein is found in cord blood, no effect of labor stress on PACAP was found. Further research is needed to understand the role of PACAP in gestation and parturition.

Dopamine-2 receptor activation suppresses PACAP expression in gonadotrophs.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is expressed at a high level in the fetal pituitary and decreases profoundly between embryonic day 19 and postnatal day 1 (PN1), with a further decrease from PN1 to PN4. In this series of experiments, we investigated the hypothesis that dopamine 2 receptor (Drd2) activation interrupts a cAMP-dependent feed-forward loop that maintains PACAP expression at a high level in the fetal pituitary. Using single-cell RT-PCR of pituitary cell cultures from newborn rats, Drd2 mRNA was identified in gonadotrophs that were also positive for PACAP mRNA. PACAP expression in pituitary cultures from embryonic day 19 rats was suppressed by the PACAP6-38 antagonist and by the Drd2 agonist bromocriptine. Increasing concentrations of bromocriptine inhibited cAMP production as well as cAMP signaling based on cAMP response element-luciferase activity, decreased PACAP promoter activity, and decreased PACAP mRNA levels in αT3-1 gonadotroph cells. Furthermore, blockade of dopamine receptors by injecting haloperidol into newborn rat pups partially reversed the developmental decline in pituitary PACAP mRNA that occurs between PN1 and PN4. These results provide evidence that dopamine receptor signaling regulates PACAP expression under physiological conditions and lend support to the hypothesis that a rise in hypothalamic dopamine at birth abrogates cAMP signaling in fetal gonadotrophs to interrupt a feed-forward mechanism that maintains PACAP expression at a high level in the fetal pituitary. We propose that this perinatal decline in pituitary PACAP reduces pituitary follistatin which permits GnRH receptors and FSH-ß to increase to facilitate activation of the neonatal gonad.

Targeted pituitary overexpression of pituitary adenylate-cyclase activating polypeptide alters postnatal sexual maturation in male mice.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHß, but reduces FSHß mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.

Developmental changes in pituitary adenylate cyclase activating polypeptide expression during the perinatal period: possible role in fetal gonadotroph regulation.

Normal reproductive functioning may require secretion of LH independently of FSH. Variation in GnRH pulse frequency and inhibin negative feedback are mechanisms for differential gonadotropin regulation; however, the first instance of differential regulation in rats is during fetal development, prior to the establishment of GnRH connections, when LH accumulates appreciably 2-4 d prior to FSH. Pituitary adenylate cyclase activating polypeptide (PACAP) can differentially regulate the gonadotropins in vitro by stimulating alpha-subunit transcription, lengthening LHbeta transcripts and decreasing FSHbeta mRNA levels, probably through stimulation of follistatin transcription. These experiments are the first to examine whether PACAP influences gonadotroph function in perinatal pituitaries. In vivo, pituitary PACAP mRNA and peptide levels were high at embryonic d 19 and declined by 94 and 85%, respectively, after parturition. This was accompanied by a decrease of 65 and 96% in total follistatin and follistatin-288 mRNAs. These changes were temporally associated with a 20- and 6.5-fold rise in FSHbeta and GnRH receptor mRNAs, respectively, with no significant increase in LHbeta mRNA. In pituitary cell cultures from fetal and postnatal male rats, PACAP mRNA levels were likewise highest in fetal cultures in which the PACAP 6-38 antagonist decreased alpha-subunit and increased FSHbeta mRNA. PACAP 6-38 also reduced basal and GnRH-stimulated LH secretion with little effect on FSH. These data support the hypothesis that PACAP expressed at high levels in the fetal pituitary stimulates alpha-subunit expression and LH secretion and restrains FSH synthesis relative to LH and that a decline in PACAP allows for the neonatal rise in FSH and GnRH receptor because follistatin is decreased.

Transgenic expression of insulin-response element binding protein-1 in beta-cells reproduces type 2 diabetes.

Recent evidence supports the idea that insulin signaling through the insulin receptor substrate/phosphatidyl-inositol 3-kinase/Akt pathway is involved in the maintenance of beta-cell mass and function. We previously identified the insulin-response element binding protein-1 (IRE-BP1) as an effector of insulin-induced Akt signaling in the liver, and showed that the 50-kDa carboxyl fragment confers the transcriptional activity of this factor. In this investigation we found that IRE-BP1 is expressed in the alpha, beta, and delta-cells of the islets of Langerhans, and is localized to the cytoplasm in beta-cells in normal rats, but is reduced and redistributed to the islet cell nuclei in obese Zucker rats. To test whether IRE-BP1 modulates beta-cell function and insulin secretion, we used the rat insulin II promoter to drive expression of the carboxyl fragment in beta-cells. Transgenic expression of IRE-BP1 in FVB mice increases nuclear IRE-BP1 expression, and produces a phenotype similar to that of type 2 diabetes, with hyperinsulinemia, hyperglycemia, and increased body weight. IRE-BP1 increased islet type I IGF receptor expression, potentially contributing to the development of islet hypertrophy. Our findings suggest that increased gene transcription mediated through IRE-BP1 may contribute to beta-cell dysfunction in insulin resistance, and allow for the hypothesis that IRE-BP1 plays a role in the pathophysiology of type 2 diabetes.

Weaning and the developmental changes in follicle-stimulating hormone, pituitary adenylate cyclase-activating polypeptide, and inhibin B in the male rat.

Pituitary Fshb concentrations increase markedly and selectively beginning on Postnatal Day 20 in the male rat. To evaluate the factors potentially responsible for this rise in FSH, we adjusted the time of weaning, which is generally also on Day 20. Male rat pups were provided nutrients by suckling only and were weaned to laboratory chow earlier (Day 17) or later (Day 23) than normally performed in animal facilities (Day 20). Between ages 17 and 29 days, significant increases were seen in serum LH (1.4-fold) and FSH (2.4-fold) levels; pituitary expression of Lhb (5.4-fold), Fshb (21.3-fold), and inhibin beta B (Inhbb, 2.26-fold) mRNAs; and testicular expression of Inhbb (10-fold) mRNA. Concurrently, significant decreases occurred in serum inhibin B levels (1.8-fold); pituitary adenylate cyclase-activating polypeptide (Adcyap1, 4.2-fold), total follistatin (Fst, 3.5-fold), and Fst isoform 288 (5.6-fold) mRNAs; and testicular expression of inhibin beta A (8.2-fold) mRNA. Early weaning significantly increased serum FSH but not LH and increased pituitary expression of Fshb and GnRH receptor (Gnrhr) mRNAs but not Lhb. Early weaning also significantly decreased serum inhibin B but increased testicular expression of the Inhbb subunit. Early weaning also caused pituitary expression of Fst and Adcyap1 to decline earlier than in the control group. Immediately after weaning, growth accelerated substantially, and the time of weaning produced significant and differential effects on circulating leptin levels that were not related to indices of FSH production. From these observations, we propose the novel hypothesis that the increase in growth rate subsequent to weaning signals circulating inhibin B levels to fall and pituitary Adcyap1 and consequently Fst expression to decrease, and that these events together facilitate the rise in Fshb and Gnrhr expression by increasing pituitary activin signaling.

Effects of CDB-4022 on Leydig cell function in adult male rats.

CDB-4022, an indenopryridine, suppresses spermatogenesis and decreases inhibin secretion in adult male rats. In the present study, we investigated the effects of CDB-4022 on Leydig cell function. A single oral dose of CDB-4022 (2.5 mg/kg) resulted in a 2-fold decrease in serum testosterone levels after 7 days that was paralleled by a decrease in Cyp17a1 mRNA and protein levels and 17alpha hydroxylase enzymatic activity compared with vehicle-treated rats. Consistent with the lower serum testosterone levels, pituitary Lhb and Fshb mRNA levels were increased 3.2- and 2.3-fold, respectively, by CDB-4022 treatment. Ultrastructural analysis of pituitary gonadotrophs showed distended endoplasmic reticulum (ER) and fewer secretory granules in CDB-4022-treated rats, characteristic of enhanced secretory activity. Conversely, CDB-4022 increased serum progesterone levels, testicular Star mRNA and protein expression, and the number of Leydig cells per testis. Serum inhibin B levels were undetectable in CDB-4022-treated rats, while serum activin A levels were similar to controls, indicating that the CDB-4022-treated rats have an elevated activin A:inhibin B ratio. In the presence of hCG stimulation, activin A directly suppressed testosterone secretion but enhanced progesterone secretion from rat Leydig cell primary cultures. Likewise, treatment of MA-10 cells with activin A was found to enhance cAMP-stimulated progesterone secretion and STAR expression. Together, our data indicate that CDB-4022 treatment inhibits CYP17A1 and stimulates STAR expression, thereby decreasing testosterone but increasing progesterone production. We propose that unopposed actions of activin A most likely contribute to the steroid profile in rats after CDB-4022 treatment. Our findings establish CDB-4022 as a new model to examine intratesticular control mechanisms that modulate Leydig cell gene expression and function.

Pituitary adenylate cyclase activating polypeptide messenger RNA in the paraventricular nucleus and anterior pituitary during the rat estrous cycle.

The neuropeptide pituitary adenylate cyclase activating polypeptide (ADCYAP 1, or PACAP) has been demonstrated to enhance gonadotropin-releasing hormone (GnRH)-induced gonadotropin secretion and regulate gonadotropin subunit gene expression in cultures of anterior pituitary cells. In the present study, we used in situ hybridization and real-time polymerase chain reaction to examine the expression of Pacap mRNA within the paraventricular nucleus (PVN) and anterior pituitary throughout the estrous cycle of the rat. Levels of luteinizing hormone in serum and pituitary gonadotropin subunit mRNAs were evaluated and displayed cyclic fluctuations similar to those reported previously. Pacap mRNA expression in the PVN and pituitary varied significantly during the estrous cycle, with the greatest changes occurring on the day of proestrus. Pacap mRNA levels in the PVN declined significantly on the morning of diestrus. During proestrus, PVN Pacap mRNA levels significantly increased 3 h before the gonadotropin surge and then declined. Pituitary expression of Pacap mRNA also varied on the afternoon of proestrus with a moderate decline at the time of the gonadotropin surge and a significant increase later in the evening. Expression of the mRNA species encoding the 288 amino acid form of follistatin increased significantly following the rise in pituitary Pacap mRNA, at the termination of the secondary surge in follicle-stimulating hormone beta (Fshb) gene expression. These results suggest that PACAP is involved in events before and following the gonadotropin surge, perhaps through increased gonadotroph sensitivity to GnRH and suppression of Fshb subunit expression through increased follistatin, as previously observed in vitro.