Background: Estradiol 17-beta (E2) enhances the function and survival of pancreatic beta-cells but its clinical use has been questioned due to concerns regarding oncogenic potential and feminizing effects in males. The G-protein coupled estrogen receptor (GPER), expressed in pancreatic islets, exhibits estrogenic beta-cell protective effects, without the feminizing effects of the nuclear ERs. Here, we examine the outcome of selective activation of the three estrogen receptors ERα, ERβ and GPER on replication, in human pancreatic islets and the INS1-E rodent β-cell line, under hyperglycemic conditions such as occur in diabetes mellitus. Methods: Pancreatic islets from nine human donors and INS1-E cells were grown at glucose concentrations of 11mM and 25mM and examined for DNA synthesis using 3[H]-thymidine incorporation, after 24 hour treatment with E2 and specific agonists for ERα, ERβ and GPER (PPT 10nM, DPN 10nM and G1 100nM, respectively). Expression of the three ERs was examined by qRT-PCR. Results: In human islets, under glucose 11mM, agonists to ERα and GPER induced a significant ~2 folds increase in 3[H]-thymidine incorporation (p

Estrogen exerts its effects through genomic and rapid extra-nuclear signaling pathways. The classic estrogen receptors, ERα and ERβ have demonstrated favorable effects on beta-cell function and survival but their clinical utility has been questioned owing to oncogenic potential and concerns regarding feminizing effects in men. Recent work indicates, however, that the G-protein coupled estrogen receptor (GPER), expressed in pancreatic islet cells retains the estrogenic beta-cell protective effects, without the shortcomings of the classic ERs. GPER activation enhances glucose stimulated insulin secretion, reduces islet apoptosis upon insult and has been implicated in beta-cell mass expansion during pregnancy in rodent islets. Since GPER can thus comprise an attractive treatment target in hyperglycemic states we examined the effects of the three ERs on proliferation of human pancreatic islets in the presence of hyperglycemia. Islets from eight non diabetic human donors (6 males and 2 females) were grown at glucose concentrations of either 11mM or 25mM for 24 hours and examined for DNA synthesis after treatment with E2 (at increasing concentrations) and with specific agonists for ERα, ERβ and GPER (PPT 10nM, DPN 10nM and G1 100nM, respectively). At a glucose concentration of 11mM, E2 and all three ER agonists induced a significant ~2 folds increase in 3[H]-thymidine incorporation. However, under still higher glucose conditions (HG; 25mM) only the GPER agonist G1 increased proliferation equipotently (~2.5 folds; p<0.001), while stimulation of ERα and ERβ enhanced proliferation by ~1.4 folds only (p<0.05 for ERα). In search for an explanation for the differential effects, we examined expression of the three ERs at glucose concentrations of 11mM and 25mM, by qRT-PCR. High glucose reduced expression of ERα and ERβ (by 15%; p<0.05) but not the expression of GPER. Although beta-cells comprise the majority (~70%) of cells in human islets, expression of GPER has also been demonstrated in alpha and delta cells and thus the full effect of hyperglycemia on GPER expression in beta-cells may be masked. Hence, extending previous reports, our results suggest that GPER may comprise an attractive target in the therapy of human diabetes and point to the phenomenon of species specificity regarding effects of glucose on estrogen receptor subtype expression and activity. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

BACKGROUND Women of reproductive age with differentiated thyroid cancer (DTC) often need radioactive iodine (RAI) treatment after surgery. In contrast to the well-documented effect of RAI on testicular function, the potential negative effects of this treatment on ovarian reserve have been largely dismissed. The objective of this pilot study was to examine the possibility that RAI treatment is deleterious to the ovarian reserve by prospectively measuring the concentration of anti-Müllerian hormone (AMH) after RAI treatment. METHODS Thirty premenopausal women (Mage = 34 years; range 20-45 years) with a new diagnosis of DTC scheduled to undergo RAI ablation were recruited for this study. All of them had TNM stage 1 disease (T1-3, N0, or N1, M0), and were scheduled to receive RAI activities ranging from 30 to 150 mCi. AMH was measured at baseline and at 3, 6, 9, and 12 months after the administration of RAI. RESULTS Of the 30 women, only 24 returned after the baseline assessment. RAI treatment resulted in a significant decrease in AMH concentrations at three months, from 3.25 ± 2.75 to 1.9 ± 1.74 ng/mL (p < 0.0001). Only partial recovery was subsequently documented. Eighty-two percent of subjects had final values below baseline levels, such that at one year, serum AMH was still 32% lower than prior to treatment (2.36 ± 1.88 ng/mL; p < 0.005). The only two continuous variables that correlated with the extent of AMH reduction at three months were the woman's age (r = 0.51; p = 0.02) and the age at menarche (r = 0.48; p = 0.03). Importantly, the RAI dose was not associated with the extent of AMH reduction and neither were smoking or the use of birth control pills. Older subjects (≥35 years) were significantly more likely to experience a marked AMH reduction at three months (63.7 ± 18.5% vs. 33.1 ± 29.2%; p = 0.01). The only predictor of recovery after one year was the extent of AMH decrease at three months: the lower the decline, the higher the chances for recovery. CONCLUSIONS RAI in DTC has a rapid and profound effect on ovarian reserve, with only a partial recovery potential. In an era of declining human fertility, it is of relevance to recognize the potentially adverse effect of RAI in women of reproductive age. AMH measurement may be useful as a tool in this decision-making process.

Estradiol-17β (E2) and the Foxo1 transcription factor have each been implicated in the regulation of β-cell proliferation. Interaction between Foxo1and estrogen receptor alpha (ERα), effecting cell cycle, has been demonstrated in breast cancer cells, but has not been studied thus far in β-cells. Using human islets and the INS1-E β-cell line, this study investigated the contribution of Foxo1 to E2-mediated β-cell replication. Foxo1 expression was knocked down in INS1-E cells using siRNA and Foxo1 activity was inhibited in human islets with a specific Foxo1 inhibitor (AS1842856). Cells were treated with E2 and the ERα agonist PPT and evaluated for proliferation by 3[H]-thymidine incorporation and for transcriptional activity through the estrogen response element by the luciferase assay. As Foxo1 activity is regulated by post-translational modifications, the effect of E2 on phosphorylation was also assessed. In INS1-E cells, knock down of Foxo1 abrogated the proliferative response to E2 and PPT. In human islets, inhibition of Foxo1 abrogated E2-mediated proliferation and attenuated the response to PPT. Foxo1 knock down and inhibition reduced activity through the estrogen response element by 25% (p<0.05) and 50% (p<0.01) respectively, in INS1-E cells. E2 increased Foxo1 phosphorylation in a time dependent manner in INS1-E and human islets (p<0.01, p<0.05, respectively). These findings suggest that Foxo1 is involved in E2-mediated proliferation in INS1-E cells and human islets. This may have implications vis-à-vis variations in circulating endogenous E2 concentrations in diabetes.

Background: Sorafenib improves progression-free survival in patients with progressive radioactive iodine-refractory differentiated thyroid carcinoma, but causes severe side effects. Estrogens may accelerate thyroid carcinoma cell growth. Our group recently reported that isoflavone derivative 7-(0)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine (cD-tboc), a novel anti-estrogenic compound, retards the growth of both thyroid carcinoma cell lines and cultured human carcinoma cells. Vitamin D receptor (VDR) is expressed in malignant cells and responds to 1,25 dihydroxyvitamin D3 (1.25D) by decreased proliferative activity in vitro. The purpose of this study was to examine the effects of vitamin D metabolites (VDM) on the expression of estrogen receptors (ERs), VDR, and l0Hase mRNA, and to evaluate the inhibitory effect of low doses of sorafenib in combination with cDtboc and VDM on cell proliferation in cultured human papillary thyroid carcinoma (PTC). Methods: In 19 cultured PTC specimens and 19 normal thyroid specimens, harvested during thyroidectomies from the same patients, expression levels of ER alpha, ER beta, VDR, and 1 alpha-hydroxylase (l0Hase) mRNA (by quantitative real-time PCR) were determined at baseline and after treatment with VMD. Cell proliferation was determined by measurement of (3)[H] thymidine incorporation after treatment with sorafenib alone, sorafenib with added 1.25D or cD-tboc, and sorafenib with both 1.25D and cD-tboc added. Results: 1,25D increased mRNA expression of all tested genes in the malignant and normal thyroid cells, while the ERa mRNA of the normal cells was unaffected. 1.25D dose-dependently inhibited cell proliferation in the malignant cells. The inhibitory effect of sorafenib on cell proliferation in the malignant cells was amplified after the addition of cDtboc and 1.25D, such that the maximal inhibition was not only greater, but also had been attained at a 10-fold lower concentration of sorafenib (20 mu/ml). This inhibition was similar to that of the generally used concentration of sorafenib (200 mu/ml) alone. Conclusions: The demonstration that low concentrations of cDtboc and 1.25D markedly amplify the inhibitory effect of sorafenib on the growth of human PTC supports the use of a 10-fold lower concentration of sorafenib. The findings may promote a new combination treatment for progressive radioactive iodine-refractory PTC.

To further investigate the interaction between vitamin D system and estrogen-mimetic compounds in the human vasculature we studied the effect of the “less- calcemic” analog of 1,25(OH)2D3 (1,25D); JK 1624F2-2 (JKF) in the presence of selective estrogen modulator femarelle (F), the phytoestrogen daidzein (D) and estradiol-17b (E2) on 3[H] thymidine incorporation (DNA synthesis) and creatine kinase specific activity (CK) in human umbilical artery vascular smooth muscle cells (VSMC). F, D and E2, stimulated DNA synthesis at low concentrations, and inhibited it at high concentrations. All estrogen-related compounds increased CK dose- dependently. Daily treatment with JKF (1 nM for 3 days) resulted in decreased DNA synthesis, increased CK and up- regulation of the stimulation of DNA synthesis by low estrogen-related hormones whereas D- and E2- mediated inhibition of cell proliferation was abolished by JKF. In contrast, inhibition of cell proliferation by F could not be blocked by JKF. JKF also up-regulated the stimulatory effects on CK by F, E2 and D. VSMC expressed Estrogen Receptor (ER)a and ERb mRNA at a relative ratio of 2.7:1.0, respectively. JKF pretreatment increased ERa (∼50%) and decreased ERb (∼25%) expression. E2 did not affect ERs whereas both D and F up-regulated ERb (∼100%) and ERa (∼50%). Additionally, JKF increased the intracellular competitive binding of F (from ∼70 to ∼310%), of D (from ∼60 to ∼250%) and of E2 from (from ∼ 70 to ∼320%). F reciprocally modulated the vitamin D system by up-regulating VDR- and 25 hydroxyy vitamin D 1-a hydroxylase (1OHase) mRNA expression (∼120%). F also stimulated 1OHase activity as indicated by an increase in the production of 1, 25D (∼250%). A similar increase was elicited by D (∼90%) but not by E2. In conclusion, F has unique effects on human VSMC in that it can sustain inhibition of cell growth even in the presence of the vitamin D analog JKF. That JKF increases ER expression and F increased the endogenous production of 1, 25D and VDR expression offer new opportunities to modulate VSMC growth. Whether or not these mutual effects of F and JKF can be exploited to promote vascular health, particularly in estrogen-deficient states (e.g., menopause) is under investigation.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE.

ABSTRACT Background: Anticoagulant therapy is a mainstay of treatment subsequent to major orthopedic surgeries. Evidence linking anticoagulant therapy, osteoporosis, and delayed fracture healing is not conclusive. We have previously reported that rivaroxaban significantly inhibited cell growth and energy metabolism in a human osteoblastic cell line. This study analyzed the response of primary female osteoblast cells to rivaroxaban in combination with various bone-modulating hormones. Methods: Bone samples were taken from both premenopausal (pre-Ob) and postmenopausal (post-Ob) women. Cells were isolated from each sample and cultured to sub-confluence. Each sample was then treated with Rivaroxaban (10 µg/ml) in combination with the following hormones or with the hormones alone for 24 hours: 30nM estradiol-17β (E2), 390nM estrogen receptor α (ERα) agonist PPT, 420nM estrogen receptor β (ERβ) agonist DPN, 50nM parathyroid hormone (PTH), and 1nM of vitamin D analog JKF. Results: No effects were observed after exposure to rivaroxaban alone. When pre-Ob and post-Ob cells were exposed to the bone-modulating hormones as a control experiment, DNA synthesis and creatine kinase (CK)-specific activity was significantly stimulated with a greater response in the pre-Ob cells. When the cells were exposed to rivaroxaban in combination with bone-modulating hormones, the increased DNA synthesis and CK-specific activity previously observed were completely attenuated. Conclusions: Rivaroxaban significantly inhibited the stimulatory effects of bone-modulating hormones in both pre-Ob and post-Ob primary human cell lines. This finding may have clinical relevance for patients at high risk of osteoporosis managed with rivaroxaban or other factor Xa inhibitors.

Genes regulated cell-cell and cell-matrix adhesion and degradation of the extracellular matrix (ECM) have been screened as potential markers of malignant thyroid nodules. The mRNA expression levels of two of them, the ECM protein-1 (ECM1) and the type II transmembrane serine protease-4 (TMPRSS4), were shown to be an independent predictor of an existing thyroid carcinoma. The vitamin D receptor (VDR) is expressed in epithelial cells of the normal thyroid gland, as well as in malignant dividing cells, which respond to the active metabolite of vitamin D by decreased proliferative activity in vitro. We evaluated the relationship between mRNA gene expressions of TMPRSS4, ECM1 and VDR in 21 papillary thyroid carcinoma samples and compared it to 21 normal thyroid tissues from the same patients. Gene expression was considered as up-or down-regulated if it varied by more or less than 2-fold in the cancer tissue relative to the normal thyroid tissue (Ca/N) from the same patient. We found an overall significant adjusted correlation between the mRNA expression ratio (ExR) of VDR and that of ECM1 in Ca/N thyroid tissue (R= 0.648, P < 0.001). There was a high ExR of VDR between Ca/N thyroid tissue from the same patient (3.06 +/- 2.9), which also exhibited a high Ca/N ExR of ECMI and/or of TMPRSS4 (>2, P= 0.05).The finding that increased VDR expression in human thyroid cancer cells is often linked to increased ECM1 and/or TPMRSS4 expression warrants further investigation Into the potential role of vitamin D analogs in thyroid carcinoma. (C) 2016 Elsevier Ltd. All rights reserved.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

BACKGROUND:Human cultured osteoblasts from pre-Ob, post-Ob or m-Ob express different mRNAs and respond to different hormones.AIMS:To test expression and hormonal modulation of VDR and 1OHase and 1,25D production in hObs.METHODS:hObs obtained from bone explants were prepared, treated and analyzed as before.RESULTS:(i) VDR and 1OHase were expressed in all hObs. (ii) 1,25D was produced similarly. (iii) Treatment with E2, DPN (ER agonist), but not PPT (ER agonist) increased VDR. (iv) These hormones increased 1OHase. (v) Vitamin D analog JKF and PTH 1-84 increased similarly mRNAs. (vi) Treatment with E2, DPN and PPT increased 1,25D. (vii) JKF and PTH increased similarly 1,25D. (viii) DNA synthesis and CK were stimulated by all hormones in hObs.CONCLUSIONS:Hormonal modulation of VDR and 1OHase and 1,25D production is important for bone physiology but their correlation, activity and bone physiology is not yet clear.

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor β (ERβ), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here, and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0g/L; 44mM) compared to normal glucose (NG; 4.5g/L; 22mM). HG significantly increased DNA synthesis (DNA) and creatine kinase specific activity (CK) in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4"-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT; ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist), were abolished by HG. HG Itself up regulated the expression of mRNA of 12LO and 15LO and up regulated to much less extent ERβ and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12 and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

The use of anticoagulants has been associated with systemic osteoporosis and increased risk for poor fracture healing but is inevitable following major orthopedic surgery of lower limbs. Rivaroxaban A (R) is an anticoagulant recently introduced in the clinical setting, which is a specific factor Xa inhibitor. We reported previously that R significantly inhibited cell growth, energy metabolism and alkaline phosphatase activity in human osteoblastic cell line SaOS2, with no effect on mineralization, indicating transient inhibition of bone formation. We now investigated the effects of R on SaOS2 response to osteoblast-modulating hormones. At sub-confluence cells were treated with: estradiol-17β (E2), the phytoestrogens daidzein (D) and biochainin A (BA), the carboxy-pytoestrogenic derivative carboxy-D (cD), the estrogen receptor α (ERα) agonist PPT, the estrogen receptor β (ERβ) agonist DPN, parathyroid hormone (PTH) and several vitamin D metabolites and analogs with/without R for 24h. All hormones tested stimulated significantly DNA synthesis (DNA), creatine kinase (CK) and alkaline phosphatase (ALP) specific activities, but all these stimulations were totally inhibited when given together with R. R had no effect on mRNA expression of ERα, ERβ and 25 Hydroxy-vitamin D3-1α hydroxylase (1OHase), but inhibited hormonal modulations of mRNA expressions. In conclusion R inhibited significantly hormonal stimulation of different parameters indicating inhibition of not only the early stages of bone formation, but also the stimulatory effects of bone modulating hormones with a yet unclear mechanism. The relevance of these findings to human bone physiology is yet to be investigated.

BACKGROUNDEstrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor β. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy.METHODSIn vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and β (ERα and ERβ), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed.RESULTSFirst, all cell types thus harvested and grown in culture expressed both ERα and ERβ, with a variably higher abundance of ERβ over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17β (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERβ agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (∼-30%).CONCLUSIONThis study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.