James F. Hyde

Targeted Overexpression of Galanin in Lactotrophs of Transgenic Mice Induces Hyperprolactinemia and Pituitary Hyperplasia

We generated transgenic mice that carry 4.6 kb of the mouse galanin gene fused to 2.5 kb of the rat PRL promoter. In all transgenic lines that carried and transmitted the transgene, there were significant increases in galanin messenger RNA and peptide levels in the anterior pituitary in both male and female transgenic mice, and the elevation of galanin was restricted to the anterior lobe. Furthermore, galanin release from pituitary cells in vitro of both male and female transgenic mice was dramatically increased compared with that in control mice. At 2–4 months of age, pituitary PRL contents in female transgenic mice were increased compared with those in normal controls. Moreover, PRL messenger RNA levels were increased in female transgenic mice. However, plasma levels of PRL in female transgenic mice were not significantly higher until 6 months of age. By 11 months of age, cell numbers in the anterior pituitary were increased in female, but not male, transgenic mice. The percentage of lactotrophs in fema...

Estradiol Attenuates the Forskolin-Induced Increase in HypothalamicTyrosine Hydroxylase Activity

The purpose of this study was to evaluate interactions between estradiol and the 3′,5′ cyclic adenosine monophosphate (cAMP) signaling pathway to regulate tyrosine hydroxylase (TH) activity in hypothalamic dopaminergic neurons. The first experiment examined the ability of forskolin to activate TH in the tuberoinfundibular dopaminergic neurons of adult ovariectomized rats with or without estradiol treatment. Estradiol treatment reduced both basal and forskolin-stimulated TH activity in the median eminence. The second group of experiments examined the effect of estradiol on the forskolin-induced activation of TH in fetal hypothalamic cells cultures. Estradiol decreased basal TH activity in the hypothalamic cell cultures to 80% of control levels. Forskolin treatment for 1 h increased TH activity in a concentration-dependent manner in control and estradiol-treated cells, but estradiol attenuated the stimulatory response to 0.01–10 µM forskolin. The suppressive effect of estradiol on cAMP-dependent activation of TH was evident with 1–12 h of forskolin treatment. The responses to other activators of the cAMP- protein kinase A pathway, including dibutyryl cAMP and 8-bromo-cAMP, and to a depolarizing stimulus were blunted in estradiol-treated cultures. Forskolin treatment for 1 h increased radiolabeled phosphate incorporation into TH protein in control but not estradiol-treated cells, suggesting that estradiol interferes with the ability of the cAMP pathway to phosphorylate TH. Forskolin caused a time-dependent increase in TH mRNA signal levels in control cultures. The magnitude of the forskolin-induced increase in TH mRNA levels was less in the estradiol-treated cells after 6 h of forskolin treatment, indicating that estradiol hinders cAMP-regulated TH gene expression. These data indicate that estradiol attenuates the ability of hypothalamic dopaminergic neurons to respond to cAMP-dependent stimulation by interfering with phosphorylation mechanisms in the short term and control of TH mRNA levels in the long term.

Regulation of galanin secretion from pituitary cells in vitro by estradiol and GHRH

The effects of estradiol and growth hormone-releasing hormone (GHRH) on galanin release from anterior pituitary cells were examined in vitro. 17-beta-Estradiol (0.001-10 nM) increased galanin secretion from anterior pituitary cells in a concentration-dependent manner. Estradiol (10 nM) increased galanin release 300 and 600% from pituitary cells of ovariectomized and male rats, respectively. Immunocytochemical studies demonstrated that estradiol (10 nM) increased the number of galanin-containing cells twofold after 4 days in culture. Growth hormone-releasing hormone (1 and 10 nM) increased and SRIF (1 and 10 nM) decreased galanin release from pituitary cells of ovariectomized and male rats. We conclude that estradiol increases galanin release by a direct effect on pituitary cells, in part by increasing the number of pituitary cells synthesizing galanin. In addition, GHRH stimulates galanin release when estradiol levels are low.

PRL, Placental Lactogen, and GH Induce Na+/Taurocholate-Cotransporting Polypeptide Gene Expression by Activating Signal Transducer and Activator of Transcription-5 in Liver Cells

We investigated the transcriptional regulation of the Na+/taurocholate cotransporting polypeptide gene by PRL, placental lactogen, and GH. In primary hepatocytes, ovine PRL induced a dose-dependent phosphorylation and nuclear translocation of signal transducers and activators of transcription-5a and -5b, but not -1 or -3, whereas mouse placental lactogen I and rat GH activated -5a, -5b, and -1. In EMSAs, ovine PRL, mouse placental lactogen I, and rat GH increased the specific DNA binding of nuclear signal transducer and activator of transcription-5 to its consensus element in both transfected HepG2 cells and primary hepatocytes. PRL, placental lactogen I, and GH also increased Na+/taurocholate cotransporting polypeptide mRNA expression in hepatocytes from control and pregnant (mouse placental lactogen I) rats. Genistein, a phosphotyrosine kinase inhibitor, inhibited PRL-induced signal transducer and activator of transcription-5 activation and Na+/taurocholate-cotransporting polypeptide mRNA. In HepG2 cell...

Dopaminergic Regulation of Galanin Gene Expression in the Rat Anterior Pituitary Gland

Estrogen dramatically increases galanin mRNA and peptide levels in the rat anterior pituitary gland. We recently reported that galanin secretion in vitro from estrogen‐exposed anterior pituitary cells is regulated by hypothalamic factors; dopamine and somatostatin inhibit galanin secretion, and thyrotrophin‐releasing hormone stimulates galanin release. To determine whether galanin is regulated by a dopaminergic mechanism in vivo, we used ovariectomized Fischer 344 rats treated with 17ß‐estradiol‐containing or empty Silastic capsules. Rats were also administered bromocriptine, a dopamine receptor agonist, haloperidol, a dopamine receptor antagonist, or placebo for 2 weeks. Galanin peptide levels were measured in the anterior pituitary, neurointermediate lobe, medial basal hypothalamus, and plasma by radioimmunoassay. Plasma and pituitary prolactin levels were also determined. Bromocriptine decreased gaianin peptide levels in the anterior pituitary gland of ovariectomized rats by 30%, but had no effect on galanin in the neurointermediate lobe or medial basal hypothalamus. In contrast, haloperidol had no effect on galanin in the anterior pituitary or medial basal hypothalamus of ovariectomized rats, but decreased galanin peptide levels in the neurointermediate lobe. In the anterior pituitary gland of estrogen‐treated rats, bromocriptine increased and haloperidol decreased both galanin and prolactin levels. Galanin mRNA levels were quantified in the anterior pituitary gland by solution hybridization. Bromocriptine increased galanin mRNA levels 3‐fold in the anterior pituitary, whereas haloperidol had no effect. Galanin mRNA levels in the anterior pituitary were elevated 10‐fold by estrogen. Bromocriptine reduced galanin mRNA levels in the pituitary by 50% in estrogen‐treated rats, where again haloperidol had no effect. Estrogen increased plasma galanin levels 4‐fold compared to ovariectomized rats and this effect was reduced 60% by bromocriptine and increased 20% by haloperidol.

Galanin in Normal and Hyperplastic Anterior Pituitary Cells: From Pituitary Tumor Cell Lines to Transgenic Mice

Abstract: Studies on the regulation of galanin expression in the epithelial cells of the anterior pituitary gland have provided a wealth of insight into the cellular and molecular biology of this unique peptide. Galanin is localized within subpopulations of specific pituitary cell types, and hypothalamic as well as gonadal factors including dopamine, somatostatin, thyrotropin‐releasing hormone, growth hormone‐releasing hormone (GHRH), estrogen, and progesterone dynamically regulate its expression and release. Galanin gene expression and peptide secretion are markedly increased in estrogen‐induced prolactinomas, wherein galanin serves as both an autocrine and paracrine hormone regulating prolactin secretion. Galanin mRNA and peptide levels are also dramatically elevated in somatotroph adenomas of human GHRH transgenic mice. Moreover, galanin secretion is increased from the hyperplastic somatotrophs of hGHRH transgenic mice. However, not all pituitary adenomas are associated with increased galanin gene expression; galanin synthesis is repressed in 131I‐induced thyrotroph adenomas. Thus, galanin acts locally to regulate pituitary hormone secretion and appears to act as a mitogenic factor to increase the proliferation of pituitary cells in a cell‐type specific manner.

Effects of phencyclidine on 5-hydroxytryptophan- and suckling-induced prolactin release

UNLABELLED The effects of acute and chronic exposure to phencyclidine (PCP) on the regulation of prolactin (PRL) secretion were examined. Female Sprague-Dawley rats were injected with PCP (10 mg/kg) or saline for 14 days, or just prior to the administration of the serotonin precursor, 5-hydroxytryptophan (5-HTP). A single injection of PCP had no effect on the 5-HTP-induced rise of plasma PRL levels. In contrast, chronic administration of PCP facilitated the release of PRL induced by 5-HTP. Peak plasma PRL levels were more than 3-fold higher after chronic PCP. The acute effect of PCP on suckling-induced PRL release was also examined. PCP delayed the rise of plasma PRL levels by suckling. The magnitude and profile of PRL, however, were similar to saline controls. The pups of PCP-treated dams failed to obtain milk during the suckling episode. Exogenous oxytocin restored the milk ejection reflex in PCP-treated dams. PCP had no effect on basal PRL release from anterior pituitary cells in vitro, and failed to alter the effects of TRH or dopamine. CONCLUSIONS (1) chronic, but not acute, administration of PCP facilitates the 5-HTP-induced release of PRL, (2) acute exposure to PCP delays the suckling-induced rise in PRL and appears to inhibit oxytocin release. These data demonstrate that both acute and chronic PCP may alter the regulation of PRL release, likely through an indirect central mechanism.

Vasoactive intestinal polypeptide mRNA and peptide levels are decreased in the anterior pituitary of the human growth hormone-releasing hormone transgenic mouse.

We recently reported that galanin gene expression is markedly increased in the hyperplastic anterior pituitary gland of the human growth hormone‐releasing hormone (hGHRH) transgenic mouse. To determine if another pituitary peptide hormone with putative growth‐promoting activity is similarly affected, or if this effect is specific to the peptide galanin, we examined vasoactive intestinal polypeptide (VIP) gene expression in the hypothalamic‐pituitary axis of male hGHRH transgenic and non‐transgenic mice. The objectives were to: 1) assess VIP peptide concentrations, 2) estimate relative differences in VIP mRNA levels, 3) determine the effects of acute treatment with 17(3‐estradiol on VIP peptide and mRNA levels, and 4) quantify the density of immunoreactive VIP pituitary cells by immunohistochemistry. Four to five month old male hGHRH transgenic mice and their non‐transgenic siblings were identified by PCR. Immunoreactive VIP concentrations were decreased by 50% in the anterior pituitary glands of hGHRH transgenic mice as compared to non‐transgenic siblings. In contrast, no differences in immunoreactive VIP concentrations were observed in the hypothalamus or frontal cerebral cortex of transgenic and non‐transgenic mice. Treatment with 17β‐estradiol significantly increased VIP concentrations in the anterior pituitary gland of both transgenic and non‐transgenic mice; however, VIP peptide concentrations in the anterior pituitary glands of hGHRH transgenic mice remained 50% lower. Relative differences in VIP mRNA levels were estimated by RT‐PCR, and were found to be 2.5‐fold higher in the anterior pituitary glands of non‐transgenic mice. In contrast, no differences in VIP mRNA levels in the cerebral cortex were detected between transgenic and non‐transgenic mice. Treatment with 17(3‐estradiol increased VIP mRNA levels in the anterior pituitary, but not in the cerebral cortex. In concert with the changes in VIP peptide and mRNA, the density of immunoreactive VIP pituitary cells was decreased approximately 50% in hGHRH transgenic mice. In conclusion, unlike galanin gene expression, VIP peptide and mRNA levels are significantly decreased in the anterior pituitary gland of hGHRH transgenic mice. Moreover, these changes appear to be tissue‐specific and are likely due, in part, to the decrease in the density of VIP‐containing pituitary cells in the hyperplastic pituitary. Although the pituitary cell type(s) synthesizing VIP remains unclear, these data suggest that VIP in the anterior pituitary is not stimulating pituitary tumor development in hGHRH transgenic mice.

The effects of co‐therapy with recombinant human insulin‐like growth factor I and insulin on serum leptin levels in adolescents with type 1 diabetes mellitus

Abstract: We previously demonstrated that in patients with type 1 diabetes mellitus (DM), co‐therapy with subcutaneous (sc) recombinant human insulin‐like growth factor I (rhIGF‐I) and insulin improves glycemic control and reduces daily insulin requirements without inducing a significant change in body weight. However, it has been postulated that treatment with IGF‐I may promote beneficial changes in body composition. Consequently, we assayed serum leptin, a peptide highly correlated with total fat mass, before and during chronic rhIGF‐I administration. We studied 14 adolescents with type 1 DM (age range 12–19 yr). All patients were treated for 12 weeks with twice daily (BID) sc rhIGF‐I in combination with standard BID split‐mix insulin. At baseline, leptin concentrations were positively correlated with body mass index (BMI) (r2 = 0.52, p = 0.004), as previously described for non‐diabetic individuals. Leptin levels in diabetic females were higher than in diabetic males, and more than two times higher than in non‐diabetic female controls. Baseline leptin levels did not correlate with patient age, duration of DM or hemoglobin A1c (HbA1c) measurements. The relationship between leptin concentrations and gender was maintained throughout treatment; however, average leptin levels did not change during 12 weeks of IGF‐I + insulin co‐therapy. These data suggest that despite treatment‐induced improvements in HbA1c and serum IGF‐I levels, serum leptin concentrations are unchanged by co‐therapy with IGF‐I + insulin. Moreover, these results suggest that improved metabolic control with IGF‐I therapy is not obtained at the expense of increasing adiposity, a complication seen frequently with intensive insulin therapy.

Pituitary Hormone Gene Expression and Secretion in Human Growth Hormone-Releasing Hormone Transgenic Mice: Focus on Lactotroph Function1

The human GH-releasing hormone (hGHRH) transgenic mouse has a hyperplastic anterior pituitary gland that eventually develops into an adenoma. We showed previously that the number of lactotrophs in the male hGHRH transgenic mouse is increased 2-fold, yet there is no concomitant increase in plasma levels of PRL. To further elucidate underlying changes in lactotroph function in the hGHRH transgenic mouse, the objectives of this study were to 1) examine the relative differences in PRL gene expression in transgenic mice and their siblings, 2) quantify PRL secretion at the level of the individual cell, 3) determine whether tyrosine hydroxylase gene expression and/or activity are altered in the hypothalamus of transgenic mice, and 4) assess dopamine receptor gene expression and functional sensitivity in lactotrophs of transgenic mice. Total PRL messenger RNA (mRNA) levels were increased nearly 5-fold in the hGHRH transgenic mouse, whereas the concentrations of PRL mRNA (PRL mRNA per μg total RNA) were unchanged....