ResearchPad - advances-in-neuroendocrinology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[SUN-LB47 Extended Release Octreotide Pharmacokinetics in Healthy Subjects After Subcutaneous Injection of MTD201]]> https://www.researchpad.co/article/elastic_article_8706 An open-label, randomised, single-dose Phase I study in healthy subjects evaluated octreotide pharmacokinetics after deep-intramuscular (IM) or subcutaneous (SC) injection of MTD201 (30mg). All subjects received Sandostatin® (100μg immediate release; SIR) by deep SC injection 24h before MTD201. MTD201 is manufactured by Q-Sphera™ printing technology to minimize particle size variation and afford simpler reconstitution and less painful injection via a 21G needle. Plasma octreotide concentrations were measured over 63 days to ascertain the potential for a 6- to 8-week dosing interval. MTD201 is being developed as a next generation long-acting somatostatin analogue for maintenance management of acromegaly and neuroendocrine cancer patients. Methods: 28 healthy subjects were randomised to two groups. The reference product SIR (100μg) was injected SC, followed 24 hours later by MTD201 administered by either IM (n=14, 38mm 21G needle) or SC (n=14, 16mm 21G needle) injection. MTD201 was resuspended in WFI to give a final injection volume of 1.5mL. Plasma samples for determination of octreotide and serum samples for IGF-1 and GH levels were drawn pre-dose and over the 63-days post-dosing. Injection site reactions and AEs were recorded, scored and compared across groups. Results: MTD201 was very well tolerated by both groups with 3 mild TEAEs observed per group. Transient and mild injection site reactions were similar after all treatments. Upon MTD201 injection, a low initial burst of octreotide (<1ng/mL) was followed by a sustained period of release that extended beyond the final sampling point at Day 63. Cmax values of 5.42ng/mL (SC) and 3.68ng/mL (IM) were within the plasma exposure range reported for marketed octreotide products. Octreotide bioavailability was 47% (IM) and 62% (SC) relative to SIR. Sustained suppression of IGF-1 concentration was achieved throughout the study period to similar levels for both groups. Octreotide PK profiles and overall exposures were similar between groups, indicating that these routes may be interchangeable in clinical use.Conclusions: MTD201 (30mg) by either IM or SC injection produced continuous octreotide release over a period of at least 63 days at levels predicted to maintain efficacious plasma concentrations at steady state with a dosing interval of up to 8 weeks. Reduced plasma IGF-1 concentrations were maintained throughout the study period. Unlike marketed octreotide depot products, MTD201 can be simply and rapidly reconstituted in WFI to give a stable suspension injectable via 21G needle in a 1.5mL volume. MTD201 can be developed as an easy to inject SC or IM depot for acromegaly and NETs, with an expected dose interval of 8 weeks, and confirmed patient-centric advantages.

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<![CDATA[SUN-250 Effects of Anti-Mullerian Hormone on the Expression of Gonadotropin Subunits in Pituitary Gonadotroph Cell Models]]> https://www.researchpad.co/article/elastic_article_7145 Aim: We examined the effect of anti-Müllerian hormone (AMH) on the expression of gonadotropin subunits in pituitary gonadotrophs.Methods: The mouse pituitary gonadotroph cell line LβT2 was stimulated with AMH and the expression levels of gonadotropin subunits were determined by real-time PCR. We also examined the involvement of the Kiss-1 gene (encoding kisspeptin) and the kisspeptin receptor (Kiss-1R) in LβT2 cells. Results: A significant increase was observed in the expression level of the FSHβ subunit with AMH but not in the expression levels of gonadotropin α and LHβ subunits. A significant decrease was observed in the expression of Kiss-1 and Kiss-1R genes in LβT2 cells with AMH stimulation. Kiss-1 gene knockdown by siRNA did not alter the basal expression of gonadotropin subunits. When LβT2 cells overexpressing Kiss-1R were stimulated with kisspeptin, there was a significant increase in the gene expression levels of the gonadotropin subunits α, LHβ, and FSHβ. This inductive effect of kisspeptin was almost completely inhibited by AMH pretreatment. The GnRH-induced increase in gonadotropin subunit genes was unchanged in the presence of AMH. Conclusions: AMH can increase FSHβ subunit gene expression in pituitary gonadotroph cells. However, AMH decreases Kiss-1 and Kiss-1R gene expression within the gonadotrophs. Because AMH pretreatment abolishes kisspeptin-induced expression of gonadotropin subunit genes, AMH may control kisspeptin-regulated gonadotropin expression by inhibiting the expression and function of Kiss-1R within gonadotrophs.

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<![CDATA[SUN-244 Role of Activin, Follistatin, and Inhibin in the Regulation of KISS-1 Gene Expression in Hypothalamic Cell Models]]> https://www.researchpad.co/article/elastic_article_7103 Kisspeptin (encoded by the Kiss-1 gene) in the arcuate nucleus (ARC) of the hypothalamus governs the hypothalamic-pituitary-gonadal (HPG) axis by regulating pulsatile release of gonadotropin-releasing hormone (GnRH). Meanwhile, kisspeptin in the anteroventral periventricular nucleus (AVPV) region has been implicated in estradiol (E2)-induced GnRH surges. Kiss-1-expressing cell model mHypoA-55 exhibits characteristics of Kiss-1 neurons in the ARC region. On the other hand, Kiss-1 expressing mHypoA-50 cells originate from the AVPV region. In the mHypoA-55 ARC cells, activin significantly increased Kiss-1 gene expression. Follistatin alone reduced Kiss-1 expression within these cells. Interestingly, activin-induced Kiss-1 gene expression was completely abolished by follistatin. Inhibin A, but not inhibin B reduced Kiss-1 expression. Activin-increased Kiss-1 expression was also abolished by inhibin A. Pretreatment of the cells with follistatin or inhibin A significantly inhibited kisspeptin- or GnRH-induced Kiss-1 gene expression in mHypoA-55 cells. In contrast, in the mHypoA-50 AVPV cell model, activin, follistatin, and inhibin A did not modulate Kiss-1 gene expression. The subunits that compose activin and inhibin, as well as follistatin were expressed in both mHypoA-55 and mHypoA-50 cells. Expression of inhibin βA and βB subunits and follistatin was much higher in mHypoA-55 ARC cells. Furthermore, we found that expression of the inhibin αsubunit and follistatin genes was modulated in the presence of E2 in mHypoA-55 ARC cells. The results of this study suggest that activin, follistatin, and inhibin A within the ARC region participate in the regulation of the HPG axis under the influence of E2.

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<![CDATA[SUN-242 Transcriptome of the Amygdala in Normal Cyclic and Acyclic Gilts]]> https://www.researchpad.co/article/elastic_article_6992 Many replacement gilts fail to express estrus, which results from being nonpubertal or behavioral anestrus. Genomic studies identified neuronal and olfaction pathways associated with these reproductive phenotypes. To better understand puberty failure, the transcriptome of the amygdala in nonpubertal, behavioral anestrus and normal cyclic gilts in the early follicular and midluteal phase was evaluated (n = 8/group; mean age = 259 d). An average of 57 million reads were obtained from each of 32 TruSeq mRNA libraries and mapped to Sscrofa 11.1. Differential expression of genes (DEG; adjusted P < 0.05) was determined using DESeq2 and pathway analysis performed with iPathwayGuide. A total of 17,173 annotated genes were expressed (raw read count > 15 in ≥ 8 samples). Comparing amygdala expression in nonpubertal gilts with follicular phase control gilts revealed 6 DEG (3 unannotated) including ARRDC2, ZFAND2A, and LAMC2. The only DEG identified between behavioral anestrus gilts and luteal phase control gilts was PIK3CG. There were 88 DEG in the amygdala of follicular phase gilts compared with luteal phase gilts. Expression of 73 genes was upregulated in the amygdala of follicular phase gilts and 14 genes were more highly expressed in luteal phase gilts. Enriched pathways included TGFβ signaling and ion transport related to GABAergic and glutamatergic neuronal function. Molecular processes for chemokine binding, membrane transporters and receptor signaling through phosphatidylinositol and tyrosine kinases were upregulated in the amygdala of follicular phase gilts. Prominent cellular components included ion channels and integrins necessary for focal adhesions that promote dendritic growth and neuronal synapse. Major differences in the amygdala of prepubertal and behavioral anestrus gilts were not found, with few genes differentially expressed compared to cyclic gilts. Stage of the ovarian cycle majorly impacted gene expression related to increased neuronal activity in the amygdala of follicular phase gilts. USDA is an equal opportunity provider and employer.

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<![CDATA[SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30]]> https://www.researchpad.co/article/elastic_article_6956 The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

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<![CDATA[SUN-269 Activation of GFRAL Neurons Decreases Food Intake via Aversive Pathways]]> https://www.researchpad.co/article/elastic_article_6926 Growth and differentiation factor 15 (GDF15), an anorexigenic peptide that represents a promising candidate for anti-obesity treatment, acts via GDNF Family Receptor Alpha Like (GFRAL), which is expressed almost exclusively on a subset of neurons in the area postrema (AP). To determine the function and mechanisms of action for GFRAL neurons, we generated Gfralcre and conditional GfralCreERT mice. Although their chemogenetic (DREADD-mediated) activation promoted FOS in a variety of brainstem, hypothalamic, and limbic nuclei, GFRAL neurons projected only to the nucleus of the solitary tract (NTS) and the parabrachial nucleus (PBN), where they innervated and activated aversive/anorexigenic GCRP-expressing cells. Tetanus-toxin-mediated silencing of PBN CGRP neurons abrogated the aversive and anorexic effects of GDF15. Furthermore, while non-gastrointestinal (GI) stimuli (e.g., GDF15 and LPS, but not feeding or gut peptide mimetics) activated GFRAL neurons, chemogenetically activating these cells decreased gastric emptying, suppressed feeding, and promoted a conditioned taste aversion. These findings suggest that GFRAL neurons link non-GI anorexigenic signals to the control of gut physiology and to the aversive suppression of food intake. Additionally, because the chemogenetic activation of GFRAL neurons suppressed food intake more strongly than GDF15 in lean mice, additional modes of activating GFRAL neurons may augment the anorectic potential of GDF15.

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<![CDATA[SUN-248 Estradiol Changes Angiotensin II-Induced ERK1/2 Phosphorylation by Different Pathways in the Hypothalamus and Lamina Terminalis]]> https://www.researchpad.co/article/elastic_article_6895 Female gonadal hormones, especially 17β-estradiol (E2), are known to mediate hydromineral homeostasis and blood pressure mainly by attenuating renin-angiotensin system (RAS) actions. The RAS plays an essential role in the maintenance of hydromineral and cardiovascular homeostasis via angiotensin II (ANGII), a key component of the RAS. However, the cellular mechanisms of the interaction between E2 and ANGII and its physiological role are not fully elucidated. Recently, our group showed that ERK1/2 is involved in sodium intake and vasopressin release induced by ANGII in female rats. In addition, E2 decreases ERK1/2 phosphorylation induced by ANGII in the hypothalamus and in structures of the lamina terminalis (LT). Thus, the goal of the present study was evaluated some mechanisms that could be involved in ERK1/2 dephosphorylation induced by E2 in response to ANGII, such as MAPK phosphatase 1 (MKP-1) and GRK5. For this, Wistar female rats (~250g) were submitted to ovariectomy and on the following day they were treated with estradiol cypionate (10µg/rat, sc) or vehicle (corn oil, 0.1mL/rat, sc) for eight days. On the eighth day, the rats received an intracerebroventricular (icv, lateral ventricle) injection of angiotensin II (25ng/2μL/rat) or vehicle (0.9% saline, 2μL/rat). After five min of ANGII injection the animals were decapitated for brain collection for MKP-1 and GRK5 expression analysis by western blot. Data were analyzed using ANOVA two or three-way, followed by Newman-Keuls post-test and the level of significance was set at 5%. It was observed that E2 increased MKP-1 expression only in hypothalamus (F1,18=24.3, p<0.001) in ovariectomized rats, independent of ANGII stimulus. Because the inhibitory effect of E2 on vasopressin release induced by ANGII was reversed by PKC inhibition, it was analyzed the role of PKC on MKP-1 expression and it was observed that PKC inhibition (Chelerythrine, 100µM/2µL/rat) reversed the positive effect of E2 on MKP-1 expression (F1,30=4.7, p<0.05) in the hypothalamus. In addition, E2 decreased GRK5 expression only in the LT (F1,21=12.7, p<0.01) in response to ANGII. Taken together, these results suggest that E2 requires PKC/MKP-1 pathway to decrease ERK1/2 phosphorylation in the hypothalamus and consequent vasopressin release induced by ANGII. While in the LT, the inhibitory effect of E2 involves decreasing GRK5 expression compromising ERK1/2 phosphorylation and sodium intake induced by ANGII. A significant contribution of this work is the identification of some steps of ANGII signaling modulated by E2, which can explain, at least in part, its regulation on the central ANGII effects.

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<![CDATA[SUN-254 Angiotensin II Stimulates Microglia Cell Inflammatory Responses]]> https://www.researchpad.co/article/elastic_article_6884 Angiotensin II (AngII) is the principal effector molecule of the renin-angiotensin system (RAS). It’s effects on the cardiovascular and renal system are well-documented. AngII acts mainly via interaction with the AngII type-1 receptor (AT1R). Disordered levels of AngII lead to hypertension and cardiovascular disease. Increasing evidence suggests that AngII may also play a role in the pathophysiology of neurodegenerative diseases through unclear mechanisms. We investigated AngII, AT1R and AT2R levels in a mouse model of neurodegenerative disease, the experimentally induced autoimmune encephalomyelitis (EAE) mouse. In EAE mice, AngII and AT1R gene expression in brain tissue were significantly increased when compared to control mice (3.2 folds ±1.9, p<0.05, n=5; and 2.6 folds ±1.1, p<0.01, n=5 respectively). In addition, iNOS mRNA expression by qRT-PCR was likewise upregulated in EAE mice compared to control (3.4 ± 1.4 folds, p<0.01, n=5). We then studied the effects of AngII in human microglial cells (HMC3) -resident innate immune cells of the central nervous system (CNS). In HMC3 cells, treatment with AngII up-regulated the expression IL-6 (3.9 folds ± 1.2, p<0.01, n=4) and increased IL-6 concentration by 83% (p<0.05, n=4) by ELISA; effects that were blocked by the AT1R antagonist, Losartan. Also, AngII induced TNF-α production, increasing its concentration by 90% (p<0.05, n=4), an increase that was blocked by Losartan. We also quantified Nitric Oxide (NO) production by using Griess Reagent and reactive oxygen species (ROS) production by the MUSE Oxidative Stress assay. In these cells, NO and ROS production were significantly increased by AngII (p<0.05, n=4) and treatment with Losartan reduced their production (p<0.05, n=4). In addition, AngII treatment induced iNOS overexpression (2.5 folds ±0.8, p<0.05, n=4); results that are consistent with increases in the EAE mice. These data suggest that AngII can activate microglia cell inflammatory responses and as such may contribute to the pathophysiology of CNS inflammation and neurodegenerative diseases.

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<![CDATA[SUN-243 Deletion of KNDy Neuron-Specific KISS1 Disrupts Estrous Cyclicity and LH Pulsatility in Female Mice]]> https://www.researchpad.co/article/elastic_article_6784 Kisspeptin (encoded by Kiss1), a neuropeptide critically involved in neuroendocrine regulation of reproduction, is primarily synthesized in two discrete hypothalamic nuclei: the anteroventral periventricular area (AVPV) and arcuate nucleus (ARC). AVPV Kiss1 is important for the pre-ovulatory luteinizing hormone (LH) surge unique to females as well as estrogen-induced positive feedback control of GnRH and LH. In contrast, ARC Kiss1 neurons, which largely co-express the neuropeptides NKB and dynorphin (collectively known as KNDy neurons), are major regulators of pulsatile release of GnRH and LH, and mediate estrogen-induced negative feedback control of both GnRH and LH. Previous studies have not fully separated the specific roles for Kiss1 in the AVPV versus KNDy-ARC neurons in the downstream control of GnRH and LH release. Therefore, we generated a Pdyn-Cre/Kiss1fl/fl (KO) mouse model to target Kiss1 in the KNDy neurons to differentiate KNDy neuron-specific function from AVPV Kiss1 function in the maturation and maintenance of the reproductive axis. qRT-PCR data documented a significant reduction of Kiss1 expression in the mediobasal hypothalamus (containing ARC) compared to controls, whereas Kiss1 in the preoptic area (containing AVPV) was similar in both KO and controls. Immunofluorescent IHC confirmed a loss of kisspeptin immunoreactivity in the ARC of KO animals while expression in the AVPV remained intact. Markers of pubertal onset (day of vaginal opening and first estrus in females; day of preputial separation in males) were normal in KO mice, suggesting that AVPV Kiss1 and/or other neural signals may be sufficient for pubertal onset. In addition, body weight throughout pubertal growth was comparable between KO and control animals of both sexes. Interestingly, KO female mice had disrupted estrous cycles presenting with persistent diestrus and a small vaginal opening. In order to test our hypothesis that conditional deletion of Kiss1 in KNDy neurons disrupts or ablates episodic GnRH/LH pulsatile release, we collected serial tail blood samples from mice at diestrus and measured LH. KO female mice exhibited significantly fewer LH pulses in a 3-hour timespan compared to controls, suggesting that KNDy neurons were functionally compromised. These observations indicate the central role of KNDy neurons in the regulation of GnRH/LH pulsatility and estrous cyclicity. The functional effects of disrupted estrous cyclicity and slower LH pulses observed in KO females are currently under study to assess potential abnormalities in ovarian folliculogenesis and fertility. Future experiments will determine whether ARC Kiss1 deletion disrupts the KNDy-driven negative feedback response of LH to gonadectomy, as well as address potential sex differences in ARC Kiss1-mediated negative feedback control of LH release.

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<![CDATA[SUN-257 Evidence that Urocortin 2 Contributes to the Suppressive Effect of Metabolic Stress on LH Secretion in Female Mice]]> https://www.researchpad.co/article/elastic_article_6751 Pulsatile luteinizing hormone (LH) secretion is disrupted by numerous stimuli including metabolic stress. Insulin-induced hypoglycemia is a model of metabolic stress that suppresses LH secretion in numerous species including mice. Our recent work provides evidence that this inhibition of LH secretion occurs via suppression of neurons that contain kisspeptin (Kiss1), neurokinin B (NKB) and dynorphin (Dyn) in the arcuate (ARC) nucleus (KNDy cells). Thus, our current objective is to identify the neural components responsible for the suppression of KNDy cells during metabolic stress. Several lines of evidence support the hypothesis that the neuropeptide urocortin 2 (UCN2) has a key role in the inhibition of LH during stress in rats. First, ICV injection of UCN2 suppresses LH secretion. Second, an antagonist to the receptor for UCN2 reverses the suppression of LH during metabolic stress. Finally, restraint and osmotic stress increase UCN2 mRNA abundance in the paraventricular nucleus (PVN). To determine if UCN2 neurons in the PVN are activated during metabolic stress we performed immunohistochemistry for UCN2 and c-Fos in tissue collected 120 min after saline or insulin (0.75mU/kg) injection (n = 2/group, ovariectomized, adult, C57/BL6). Insulin significantly increased both the number of UCN2 cells (saline: 109.0 ± 8.5, insulin: 156.3 ± 10.8 cells) and the percentage of UCN2 cells that expressed c-Fos (saline: 13.1 ± 2.5%, insulin: 31.2 ± 0.8%). Next, we administered UCN2 (7.23nmol) via ICV injection to determine if this molecule suppresses LH secretion and/or mRNA abundance of KNDy genes. LH was measured in serial blood samples collected from 60 min prior to and 30-90 min following injection. Tissue was collected 3 h after ICV injection to confirm injection site and quantify mRNA abundance in ARC micropunches. In saline-treated mice (n = 5 successful injections), mean LH concentration and the number of LH pulses did not differ across sampling periods (mean: 6.4 ± 0.4 ng/mL vs. 6.0 ± 0.4 ng/mL; pulses: 2.6 ± 0.2 vs. 3.0 ± 0.3, pre vs. post). In contrast, in mice with successful UCN2 injections (n = 4) there was a significant reduction in both mean LH and the number of LH pulses following UCN2 (mean: 5.0 ± 0.3 ng/mL vs. 1.4 ± 0.2 ng/mL; pulses: 3.0 ± 0.0 vs. 0.25 ± 0.25, pre vs post). UCN2-treated animals had a significant reduction in the abundance of mRNAs encoding Kiss1 (~35%) and NKB (~40%) compared to saline-treated animals; the abundance of Dyn mRNA did not differ between treatments. These data demonstrate that PVN UCN2 cells are activated during metabolic stress and that UCN2 is sufficient to suppress LH secretion and the expression of genes involved in stimulating LH pulses. These data support the hypothesis that UCN2 released from neurons in the PVN impairs KNDy cell function and LH secretion during acute stress.

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<![CDATA[SUN-245 Sensory Neuron Metabolism Mediates Changes in Ovarian Function via LKB1]]> https://www.researchpad.co/article/elastic_article_6629 Bi-directional communication between sensory neurons to peripheral tissue to mediate physiology has become an area of interest. Previous research on peripheral neuron control of ovarian function shows that lesioning the superior ovarian nerve resulted in decreased estradiol release from the ovary; but there has been little research on how sensory neurons affect ovarian physiology. Interestingly, the metabolic activity of sensory neurons that control ovarian and follicular function may have profound effects on tissues they innervate. LKB1 (Liver Kinase B1) is a metabolic kinase that regulates cell growth and polarity. When downregulated in cortical neurons in vitro, leads to shorter axons with less branching. Female mice with LKB1 removed from their peripheral sensory neurons have litters more often and increased litter sizes compared to wild-type mice, which led to the investigation into the mechanisms into the role of LKB1 in sensory neurons in ovarian function. Nav1.8cre-LKB1fl/fl mice were used to assess the removal of LKB1 from Nav1.8-expressing neurons on the mechanisms behind reproductive viability. The estrus cycle was tracked by using vaginal lavage to collect cells from the vagina twice per day for ten days, and cytology was assessed to determine phase. Ovaries were collected from mice in all phases of the estrus cycle, sectioned at 8 micron thickness, stained with H&E. Follicle sizes and numbers were measured on 8 sections per ovary. Sensory innervation was measured by clearing whole ovary using ScaleS1 and using confocal microscopy to image through the whole tissue for sensory neurons tagged with tdTomato and DAPI. Our data indicate that Nav1.8-expressing neurons innervate the ovary from celiac ganglia, upper lumbar, and lower thoracic dorsal root ganglia. Nav1.8cre-LKB1fl/fl mice have larger litters and breed more frequently compared to cre-negative litter mates (WT). Nav1.8cre-LKB1fl/fl mice have larger ovaries, spend less time in proestrus, and have greater follicular turnover compared to WT mice. Phase-matched ovaries from Nav1.8cre-LKB1fl/fl mice in proestrus show greater numbers of antral and degenerating follicles than WT mice, but similar numbers of immature follicles; however, the size of all follicles from Nav1.8cre-LKB1fl/fl mice are smaller than follicles from WT mice. Nav1.8cre-LKB1fl/fl mice have improved reproductive viability by increased follicular turnover rate, more mature follicles, and shortened estrus cycle length.

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<![CDATA[SUN-234 Brain-Selective Estrogen Therapy to Prevent Androgen Deprivation Therapy-Related Hot Flushes]]> https://www.researchpad.co/article/elastic_article_6585 Synthetic estrogens are used in the clinic to alleviate debilitating neurological symptoms associated with androgen deprivation therapy (ADT), an effective treatment improving survival in prostate cancer patients when administered timely in the course of the cancer. However, this therapy to relieve the symptoms, most commonly hot flushes, causes feminizations that significantly diminishes patients’ compliance because of physical and psychological discomfort. Because only estrogens can provide adequate therapy of hot flushes based on current clinical practices, there is an unmet medical need for an effective, side effect-free and, consequently, compliance-gaining intervention to alleviate these vasomotor symptoms distressing prostate cancer patients on ADT. The goal with our experiments was to show that treatment with 10β,17β-dihydroxyestra-1,4-dien-3-one (DHED, a brain-selective bioprecursor prodrug of 17β-estradiol (E2) will ease ADT-associated hot flushes without feminizing side-effects.

To evaluate the effect of DHED on hot flushes the pharmacological rat hot flush model was used. Orchiectomized (ORDX) rats were treated orally with three different doses (10, 30, and 100 µg/kg) of DHED or ethynyl estradiol (EE, 200 µg/kg) for ten days. They were addicted to morphine and the tail skin temperature (TST) of saline-treated rats raised by 4.4±0.5 °C when morphine effect was withdrawn with naloxone injection. DHED and EE treatments significantly lowered such TST rise from 4.4 °C to 2.9 ±0.5°C and 1.8 ±0.5°C, respectively.

The conversion of DHED to E2 in the brain was confirmed by measuring the effect of DHED-derived E2 on the expression of progesterone receptors (PR) in the preoptic area of the hypothalamus with in situ hybridization histochemistry. Both DHED and EE treatment stimulated PR expression compared to saline-treatment in ORDX rats.

In our previous studies, we have shown the lack of conversion of DHED to E2 in the periphery in ovariectomized female rats; i.e., DHED treatment did not have uterotrophic, mammotrophic activities and did not stimulate galanin expression in the anterior pituitary. In these studies, the lack of conversion of DHED to E2 was also confirmed in male rats by measuring the expression of galanin, a highly estrogen-regulated gene, in the pituitary with quantitative RT-PCR. Contrary to EE, DHED treatment did not stimulate galanin expression in this estrogen target.

These observations support subsequent translational research focusing on DHED’s therapeutic use to remedy hot flushes and potentially other neurological symptoms in prostate cancer patients undergoing ADT to manage their malignancy. An estrogen therapy with the brain-selective DHED would provide a safe approach to prevent these neurological symptoms without causing peripheral estrogenic side effects such as gynecomastia or deep vein thrombosis.

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<![CDATA[SUN-240 Female Mice Lacking Brain Insulin Production Exhibit Learning Deficits, Anxiety, and Reduced Hippocampal Cyclin D1 Expression]]> https://www.researchpad.co/article/elastic_article_6512 Insulin dysregulation independently underlies diabetes and Alzheimer’s Disease (AD) pathology. However, the former has also been shown to be a risk factor for the latter. The ancestral insulin gene (Ins2), but not the pancreas-specific Ins1gene, is transcribed locally within the brain in mice. We confirmed that neuronal expression of Ins2 is most prominent within the hippocampus, a brain region with established roles in learning and memory, and that it was reduced by a diet known to promote neuronal dysfunction. It is not yet clear, however, how insulin produced locally within the brain influences hippocampal function, learning and memory. To eliminate brain-derived insulin, we used young and old mice with germline Ins2knockout (Ins2-/-) and their normal complement of wildtype Ins1 alleles, which had equivalent pancreatic insulin and normal glucose homeostasis. Using the Morris water maze, we found that learning and memory performance of female Ins2-/-mice was significantly impaired relative to wild-type mice, whereas the performance of male Ins2-/-and wild-type mice did not differ. During acquisition training, the swim-speed in female Ins2-/-was faster than wild-type mice, suggesting increased stress reactivity and motivation to escape from water. Indeed, anxiety-like behavior was increased in female mice as assessed by the open-field test. Using RNA sequencing to profile isolated hippocampi, we found that femaleIns2-/-mice had a significant reduction in Cyclin D1 (Ccnd1) compared with littermate controls. This observation points to a possible defect in hippocampal neurogenesis, a physiological hallmark of impaired memory and emotionality implicated in both, diabetes and AD. Together these data suggest that Ins2plays sex- and brain region-specific roles in neuronal function and perhaps adult neurogenesis.

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<![CDATA[SUN-260 Dual Role of Carboxypeptidase E in Prohormone Processing and a Novel Neurotrophic Factor Mediating Neuroprotection and Cognitive Functions in Hippocampal CA3 Neurons in Mice]]> https://www.researchpad.co/article/elastic_article_6093 Stress causes release of glucocorticoids from the adrenals which then circulate to the brain. High concentrations glucocorticoid from chronic severe stress results in pathophysiology in the brain, including neuronal degeneration, cell death and cognitive dysfunction, leading to diseases such as Alzheimer Disease and Major Depressive Disorders. Neurotrophic/growth factors such as BDNF, NGF and NT3 have been linked to these pathological conditions. Carboxypeptidase E (CPE), a proneuropeptide/prohormone processing enzyme, also named neurotrophic factor-α1(NFα1) is highly expressed in the stress-vulnerable hippocampal CA3 neurons, and was shown to have neuroprotective activity from in vitro studies. Here we investigated if CPE-NFα1 functions in vivo, independent of its enzymatic activity, and the mechanism underlying its action. We generated knock-in mice expressing a non-enzymatic form of CPE, CPE-E342Q, but not wild-type CPE. The CPE-E342Q mice showed significantly decreased neuropeptide content and exhibited obesity, diabetes and infertility due to lack of prohormone processing activity, similar to CPE-KO mice. However, they showed no hippocampal CA3 degeneration, exhibited neurogenesis in the dentate gyrus, and displayed normal spatial learning and memory, similar to CPE wild-type mice, after weaning stress; unlike CPE-KO mice which showed hippocampal CA3 neuronal degeneration and cognitive deficits. Binding studies showed that radiolabeled CPE bound hippocampal cell membrane specifically, in a saturable manner. Binding of CPE and CPE-E342Q to hippocampal neurons activated Erk signaling and pre-treatment with either of these proteins protected neurons against H2O2- or glutamate-induced neurotoxcity by increasing BCL2 expression. In vitro and in vivo inhibitor studies demonstrated that this neuroprotective effect was independent of tyrosine kinase receptor signaling. Taken together, the data provide evidence that CPE-NFα1 is a unique neurotrophic factor which acts through a non-tyrosine kinase receptor to activate Erk-BCL2 signaling to protect hippocampal CA3 neurons against stress-induced neurodegeneration and maintaining normal cognitive functions in mice.

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<![CDATA[SUN-251 The Effects of Unilateral Vagotomy in Female Rats with Blockade of the Muscarinic System of the Suprachiasmatic Nucleus on Ovulation and Estradiol Serum Levels]]> https://www.researchpad.co/article/elastic_article_5943 Several studies show that the suprachiasmatic nucleus (SCN) participates in the regulation of the functions of various endocrine organs through multisynaptic nerve pathways. Some of these pathways communicate the SCN with the dorsal motor nucleus of the vagus and the nucleus of the solitary tract, which are part of the origin of the vagus nerve (1). Previously we demonstrated that atropine (ATR) microinjection in the right SCN on the day of the proestrus, blocks ovulation, while the same treatment in the left SCN does it partially (2). In the present study we analyzed the possibility that the vagus nerve is one of the neural ways by which the SCN regulates the secretion of estradiol (E2) in the proestrus and subsequent ovulation. For this, cyclic rats were anesthetized with ketamine-xylazine at 09.00 of the day of the proestrus. The animals were randomly assigned to one of the following groups: rats with ATR (62.5 ng diluted in 0.3 µl of saline) microinjection in the right or left SCN, followed by ventral laparotomy or ipsilateral vagotomy to the microinjection side. The animals were sacrificed 5 h after surgery, and estradiol (E2) levels were measured. Other groups of animals with the same treatments were sacrificed 24 hours after surgery, and ovulation rate and number of ova shed were evaluated. The left vagus section did not modify the effects of ATR microinjection in the left SCN on ovulatory rate (2/5 vs. 4/7) and E2 secretion (46.6±9.0 vs. 51.3±9.0, pg/ml). In animals with ATR microinjection in the right SCN, the right vagus section increased the rate of ovulating animals (6/8 vs. 2/9, p <0.0001, Fisher’s exact probability test) and E2 levels (51.8±9.4 vs. 22.4 ± 4.0, p <0.05, two-way ANOVA, followed by Tukey’s multiple comparison test). Present results suggest that the right vagus nerve plays a role in the multisynaptic communication between the right SCN and the right ovary, while the left vagus does not. Reference: (1) Travagli, R. A. J. Physiol. 2007 Jul 15:582(Pt 2):471. (2) Vieyra et al., Reproductive Biology and Endocrinology. 2016 Jun 16 14(1):34, 1-11.Supported by CONACyT 236908; DGAPA-PAPIIT IN216519

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<![CDATA[SUN-241 Adult Exposure to Iodoacetic Acid Leads to Abnormal Expression of Key Genes Related to Hypothalamic and Pituitary Control of Reproductive Function]]> https://www.researchpad.co/article/N9c6abe20-50da-4cca-b018-f241ea9bf083 Water disinfection byproducts (DBPs) are formed when chemicals used to decontaminate water come into contact with natural or synthetic organic material. DBPs have been linked to a range of health concerns including reproductive disfunction. One such DBP, the monohalogenated iodoacetic acid (IAA), is formed when iodide reacts with a disinfectant, for example, chlorine. IAA is of particular health concern; not only is iodide widely present in the water supply, especially in coastal communities and those near fracking sites, but IAA has been found to be one of the most cyto- and genotoxic DBPs. Further, a previous study has indicated that in vitro IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels in ovaries. However, little is known about how IAA affects the other major components of the reproductive axis: the hypothalamus and pituitary. The reproductive axis relies on homeostatic release of hormones to communicate from one organ to another and alterations at any level may impact reproduction. So, we set out to test the hypothesis that exposure to IAA would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We continually exposed female adult CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water for approximately 35 days (postnatal day 40 (P40) to their first day in diestrus following P75.) Whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA) were collected and processed for qPCR analysis. We find that while kisspeptin (Kiss1) expression in the AVPV - the population responsible for generating the LH surge - is unchanged, 0.5 mg/L IAA exposure significantly increases Kiss1 in the ARC, which controls pulsatile GnRH release, and there is a trending increase (p=.056) at 10mg/L. We also measured ARC expression of Neurokinin B (NKB; Tac2), a neuropeptide secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We found no change in mRNA levels of Tac2. We also saw no significant changes in GnRH (Gnrh1) mRNA expression. At the level of the pituitary, there is no change in Lhb mRNA levels. Exposure to 10 mg/L IAA leads to significantly reduced Fshb expression, however FSH serum levels are not significantly changed. These data, taken together with previous findings in the ovary, indicate that IAA has the potential to disrupt each major level of the reproductive axis: ovarian follicle development and steroid synthesis, hypothalamic arcuate Kiss1 synthesis, and Fshb synthesis from the pituitary. Further research is necessary to elucidate at which levels IAA acts directly and at which it acts through action on another component of the axis. Additionally, future studies can clarify the mechanism through which IAA has these effects.

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<![CDATA[SUN-LB50 Increased In Vivo Pulsatile LH Secretion and Hypothalamic Kisspeptin, NKB, and Dynorphin RNA Levels in a PCOS-Like Mouse Model]]> https://www.researchpad.co/article/Na82b91a6-a613-4ae1-be00-f172d00ba358 <![CDATA[SUN-LB46 Differences in IGF-I Concentrations Between European and US Populations - Consequences for Reference Intervals]]> https://www.researchpad.co/article/N27c669e4-f970-4dc9-8e09-095c71c51d5f 15,000 subjects were published for the IDS iSYS IGF-I assay (J Clin Endocrinol Metab 2014). We now analyzed distribution of IGF-I results obtained in routine samples analyzed by accredited laboratories in the US and Europe, all using the IDS iSYS assay. Methods: All results from routine IGF-I measurements during the past 5 years in 4 laboratories were included (US lab n=778,173 males/710,752 females; European labs (Germany/Belgium, n=23,220 males/40,183 females). Assay performance across laboratories was confirmed through proficiency testing schemes and exchange of patient samples. We constructed RIs adjusted for age/sex from European and US cohorts separately using a modified Hoffmann approach (Am J Clin Pathol 2015), and compared to the originally published RIs (n=6697 males/8317 females, adults from Europe). A subset of US samples was used to compare IGF-I between regions with lower (Colorado) and higher (Alabama) mean body mass index (BMI). Results: Lower limits (LLs) of RIs calculated from routine results are superimposable to LLs from the original publication for all ages and sexes, regardless whether IGF-I results were from Europe or the US. For groups with sufficient n, upper limits (ULs) of RIs calculated from European routine data were also not statistically different from the originally published central 95%. However, a striking difference exists in calculated ULs from data of European and US origin: For ages 10-18 years, calculated UL on average was 149.3 ng/mL (34.6%) higher in boys and 94.9 ng/mL (19.8%) in girls from the US. In adults (19-95 years), calculated UL on average was 45 ng/mL (20.3%) higher in males and 29.7 ng/mL (13.8%) in females from the US. Within the US, mean IGF-I was significantly higher in samples from Colorado (lower mean BMI) than in Alabama (p<0.0001) across age- and sex groups, although the difference between the two states was smaller than between each of them and Europe. Conclusion: This study provides evidence that in sufficiently large datasets, both, direct sampling (as in the original publication) and the indirect Hoffmann algorithms provide statistically comparable RI limits and may be considered as accurate representation of results distribution in the disease-free populations. More importantly, we demonstrate that even with tight cross-correlation and continuous monitoring of IGF-I assay performance RIs generated in different populations can be different. Notably, in our extremely large study, the difference between Europe and the US was clinically relevant only at the UL. Although our study cannot reveal the cause of the difference, we suggest using adapted RIs for the US. ]]> <![CDATA[SUN-LB54 Nutritionally-Induced Alteration in KNDy Neuronal Expression in the Arcuate Nucleus of Ewes]]> https://www.researchpad.co/article/Nb564e6bc-4cab-4e2e-ad06-2f21c38ae6d1 <![CDATA[SUN-LB48 Increased Mortality in Acromegaly Is Mainly Due to Vascular and Respiratory Disease and Is Normalized by Control of Growth Hormone Hypersecretion]]> https://www.researchpad.co/article/Nb38e1e81-c590-44b9-a696-cf9909886c28 10yrs.1.13 (1.03-1.25) P <0.001.Lowering post-treatment GH levels lead to a significant reduction in all-cause SMR <2.5 ng/ml 1.15 (1.03-1.28) 2.5- 9.9 ng/ml 1.73 (1.47-2.02) ≥10 ng/ml 2.27 (1.72-2.95) P<0.001 and SMR in all disease categories. A similar relationship between mean GH levels and SMR was found for all disease categories and all-cause SMR < 2.5 ng/ml 1.00 (0.89-1.18) 2.5- 9.9 ng/ml 1.50 (1.33-1.69) ≥ 10 ng/ml 2.28 (1.85-2.79) P<0.001.Lowering post-treatment GH levels to less than 1 ng/ml appeared to have an additional beneficial effect for all-cause SMR, <1ng/ml 1.03 (0.89-1.18) 1-2.5 ng/ml 1.38 (1.16-1.62) P<0.001. A similar relationship was shown in cardiovascular, respiratory and malignant disease, but not cerebrovascular disease.Conclusion: Acromegaly is associated with an increased mortality from vascular, respiratory, but not malignant disease. The highest risk is within the first 5 years following diagnosis and the risk is abrogated by lowering GH levels to <1.0 ng/ml.On behalf of UK Acromegaly Register Study Group 2020 ]]>