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María Andrea Camilletti, Alejandra Abeledo-Machado, Jimena Ferraris, Pablo A Pérez, Erika Y Faraoni, Daniel Pisera, Silvina Gutierrez and Graciela Díaz-Torga

Ovarian steroids control a variety of physiological functions. They exert actions through classical nuclear steroid receptors, but rapid non-genomic actions through specific membrane steroid receptors have been also described. In this study, we demonstrate that the G-protein-coupled estrogen receptor (GPER) is expressed in the rat pituitary gland and, at a high level, in the lactotroph population. Our results revealed that ~40% of the anterior pituitary cells are GPER positive and ~35% of the lactotrophs are GPER positive. By immunocytochemical and immuno-electron-microscopy studies, we demonstrated that GPER is localized in the plasmatic membrane but is also associated to the endoplasmic reticulum in rat lactotrophs. Moreover, we found that local Gper expression is regulated negatively by 17β-estradiol (E2) and progesterone (P4) and fluctuates during the estrus cycle, being minimal in proestrus. Interestingly, lack of ovarian steroids after an ovariectomy (OVX) significantly increased pituitary GPER expression specifically in the three morphologically different subtypes of lactotrophs. We found a rapid estradiol stimulatory effect on PRL secretion mediated by GPER, both in vitro and ex vivo, using a GPER agonist G1, and this effect was prevented by the GPER antagonist G36, demonstrating a novel role for this receptor. Then, the increased pituitary GPER expression after OVX could lead to alterations in the pituitary function as all three lactotroph subtypes are target of GPER ligand and could be involved in the PRL secretion mediated by GPER. Therefore, it should be taken into consideration in the response of the gland to an eventual hormone replacement therapy.

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Te Du, Liu Yang, Xu Xu, Xiaofan Shi, Xin Xu, Jian Lu, Jianlu Lv, Xi Huang, Jing Chen, Heyao Wang, Jiming Ye, Lihong Hu and Xu Shen

Vincamine, a monoterpenoid indole alkaloid extracted from the Madagascar periwinkle, is clinically used for the treatment of cardio-cerebrovascular diseases, while also treated as a dietary supplement with nootropic function. Given the neuronal protection of vincamine and the potency of β-cell amelioration in treating type 2 diabetes mellitus (T2DM), we investigated the potential of vincamine in protecting β-cells and ameliorating glucose homeostasis in vitro and in vivo. Interestingly, we found that vincamine could protect INS-832/13 cells function by regulating G-protein-coupled receptor 40 (GPR40)/cAMP/Ca2+/IRS2/PI3K/Akt signaling pathway, while increasing glucose-stimulated insulin secretion (GSIS) by modulating GPR40/cAMP/Ca2+/CaMKII pathway, which reveals a novel mechanism underlying GPR40-mediated cell protection and GSIS in INS-832/13 cells. Moreover, administration of vincamine effectively ameliorated glucose homeostasis in either HFD/STZ or db/db type 2 diabetic mice. To our knowledge, our current work might be the first report on vincamine targeting GPR40 and its potential in the treatment of T2DM.

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Dieuwertje C E Spaanderman, Mark Nixon, Jacobus C Buurstede, Hetty H C M Sips, Maaike Schilperoort, Eline N Kuipers, Emma A Backer, Sander Kooijman, Patrick C N Rensen, Natalie Z M Homer, Brian R Walker, Onno C Meijer and Jan Kroon

Glucocorticoid signaling is context dependent, and in certain scenarios, glucocorticoid receptors (GRs) are able to engage with other members of the nuclear receptor subfamily. Glucocorticoid signaling can exert sexually dimorphic effects, suggesting a possible interaction with androgen sex hormones. We therefore set out to determine the crosstalk between glucocorticoids and androgens in metabolic tissues including white adipose tissue, liver and brown adipose tissue. Thereto we exposed male C57BL/6J mice to elevated levels of corticosterone in combination with an androgen receptor (AR) agonist or an AR antagonist. Systemic and local glucocorticoid levels were determined by mass spectrometry, and tissue expression of glucocorticoid-responsive genes and protein was measured by RT-qPCR and Western blot, respectively. To evaluate crosstalk in vitro, cultured white and brown adipocytes were exposed to a combination of corticosterone and an AR agonist. We found that AR agonism potentiated transcriptional response to GR in vitro in white and brown adipocytes and in vivo in white and brown adipose tissues. Conversely, AR antagonism substantially attenuated glucocorticoid signaling in white adipose tissue and liver. In white adipose tissue, this effect could partially be attributed to decreased 11B-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid regeneration upon AR antagonism. In liver, attenuated GR activity was independent of active glucocorticoid ligand levels. We conclude that androgen signaling modulates GR transcriptional output in a tissue-specific manner.

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Gen Chen, Xiangjuan Chen, Chao Niu, Xiaozhong Huang, Ning An, Jia Sun, Shuai Huang, Weijian Ye, Santie Li, Yingjie Shen, Jiaojiao Liang, Weitao Cong and Litai Jin

Baicalin is the major component found in Scutellaria baicalensis root, a widely used herb in traditional Chinese medicine, which exhibits strong anti-inflammatory, anti-viral and anti-tumor activities. The present work was devoted to elucidate the molecular and cellular mechanisms underlying the protective effects of Baicalin against diabetes-induced oxidative damage, inflammation and endothelial dysfunction. Diabetic mice, induced by streptozotocin (STZ), were treated with intraperitoneal Baicalin injections. Human umbilical vein endothelial cells (HUVECs) were cultured either in normal glucose (NG, 5.5 mM) or high glucose (HG, 33 mM) medium in the presence or absence of Baicalin for 72 h. We observed an obvious inhibition of hyperglycemia-triggered oxidative damage and inflammation in HUVECs and diabetic aortal vasculature by Baicalin, along with restoration of hyperglycemia-impaired nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway activity. However, the protective effects of Baicalin almost completely abolished in HUVECs transduced with shRNA against Nrf2, but not with nonsense shRNA. Mechanistic studies demonstrated that HG decreased Akt and GSK3B phosphorylation, restrained nuclear export of Fyn and nuclear localization of Nrf2, blunted Nrf2 downstream target genes and subsequently induced oxidative stress in HUVECs. However, those destructive cascades were well prevented by Baicalin in HUVECs. Furthermore, LY294002 and ML385 (inhibitor of PI3K and Nrf2) attenuated Baicalin-mediated Nrf2 activation and the ability of facilitates angiogenesis in vivo and ex vivo. Taken together, the endothelial protective effect of Baicalin under hyperglycemia condition could be partly attributed to its role in downregulating reactive oxygen species (ROS) and inflammation via the Akt/GSK3B/Fyn-mediated Nrf2 activation.

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Achim Lother, Lisa Deng, Michael Huck, David Fürst, Jessica Kowalski, Jennifer S Esser, Martin Moser, Christoph Bode and Lutz Hein

Aldosterone is a key factor in adverse cardiovascular remodeling by acting on the mineralocorticoid receptor (MR) in different cell types. Endothelial MR activation mediates hypertrophy, inflammation and fibrosis. Cardiovascular remodeling is often accompanied by impaired angiogenesis, which is a risk factor for the development of heart failure. In this study, we evaluated the impact of MR in endothelial cells on angiogenesis. Deoxycorticosterone acetate (DOCA)-induced hypertension was associated with capillary rarefaction in the heart of WT mice but not of mice with cell type-specific MR deletion in endothelial cells. Consistently, endothelial MR deletion prevented the inhibitory effect of aldosterone on the capillarization of subcutaneously implanted silicon tubes and on capillary sprouting from aortic ring segments. We examined MR-dependent gene expression in cultured endothelial cells by RNA-seq and identified a cluster of differentially regulated genes related to angiogenesis. We found opposing effects on gene expression when comparing activation of the mineralocorticoid receptor in ECs to treatment with vascular endothelial growth factor (VEGF), a potent activator of angiogenesis. In conclusion, we demonstrate here that activation of endothelial cell MR impaired angiogenic capacity and lead to capillary rarefaction in a mouse model of MR-driven hypertension. MR activation opposed VEGF-induced gene expression leading to the dysregulation of angiogenesis-related gene networks in endothelial cells. Our findings underscore the pivotal role of endothelial cell MR in the pathophysiology of hypertension and related heart disease.

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Viktorija Gustaityte, Martina Winkler, Ines Stölting and Walter Raasch

Based on findings that treatment with AT1 receptor blocker (ARB) prevents diet-induced obesity and that the activity of the hypothalamic–pituitary–adrenal (HPA) axis is stimulated by AngII and blocked by ARBs, we aimed to investigate whether ARB treatment can reduce stress-induced eating of cafeteria diet (CD), thus contributing to alterations in eating behavior. Sprague–Dawley rats were fed with chow or CD and treated with telmisartan (TEL, 8 mg/kg/day) or vehicle. At weeks 2 and 12, rats were stressed over five consecutive days by restraint stress (RS, 4 h) and by additional shaking at d5. Tail blood was sampled during RS to determine hormone levels. During the first period of RS, ACTH and corticosterone responses were diminished at d5 in CD- compared to chow-fed rats. Independently of feeding, TEL did not reduce stress hormones. Compared to food behavior before RS, the stress-induced CD eating increased in controls but remained unchanged in TEL-treated rats. After 12 weeks, TEL reduced weight gain and energy intake, particularly in CD-fed rats. Similar to the first RS period, corticosterone response was reduced in CD-fed rats at d5 during the second RS period. TEL did not further reduce stress hormones and did not lessen the CD eating upon RS. We conclude that CD feeding compensates for stress reactions. However, stress-induced CD eating was only reduced by TEL after short term, but not after long-term drug treatment. Thus, the potency of ARBs to lower HPA activity only plays a minor role in reducing energy intake to prevent obesity.

Free access

Christopher J Scott, Jessica L Rose, Allan J Gunn and Briony M McGrath

The control of reproductive processes involves the integration of a number of factors from the internal and external environment, with the final output signal of these processes being the pulsatile secretion of gonadotrophin-releasing hormone (GnRH) from the hypothalamus. These factors include the feedback actions of sex steroids, feed intake and nutritional status, season/photoperiod, pheromones, age and stress. Understanding these factors and how they influence GnRH secretion and hence reproduction is important for the management of farm animals. There is evidence that the RF-amide neuropeptide, kisspeptin, may be involved in relaying the effects of these factors to the GnRH neurons. This paper will review the evidence from the common domestic animals (sheep, goats, cattle, horses and pigs), that kisspeptin neurons are (i) regulated by the factors listed above, (ii) contact GnRH neurons and (iii) involved in the regulation of GnRH/gonadotrophin secretion.

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Zerui Wu, Lin Cai, Jianglong Lu, Chengde Wang, Jiaqing Guan, Xianbin Chen, Jinsen Wu, Weiming Zheng, Zhebao Wu, Qun Li and Zhipeng Su

To date, the management of dopamine agonist (DA)-resistant prolactinomas remains a major clinical problem. Previously, we determined that miRNA-93 expression increases in DA-resistant prolactinomas; however, the role of miRNA-93 in the DA resistance remains largely unexplored. Hence, this study aimed to investigate the susceptibility of tumor cells to cabergoline (CAB) and the autophagy changes in MMQ and GH3 cells after miRNA-93 overexpression or inhibition. We used bioinformatics to identify the potential target of miRNA-93. Subsequently, we analyzed the correlation between miRNA-93 and autophagy-related 7 (ATG7) using protein expression analysis and luciferase assays. Furthermore, the change in the effect of miRNA-93 was measured after ATG7 overexpression. miRNA-93 expression was elevated in DA-resistant prolactinomas, whereas the expression of its identified target, ATG7, was downregulated. miRNA-93 overexpression suppressed the cytotoxic effect of CAB in MMQ and GH3 cells. In contrast, miRNA-93 downregulation enhanced CAB efficiency and promoted cell autophagy, eventually resulting in apoptosis. These results were further confirmed in in vivo xenograft models in nude mice. ATG7 overexpression could reverse the inhibitory effect of miRNA-93 on CAB treatment. Taken together, our results suggest that miRNA-93 mediates CAB resistance via autophagy downregulation by targeting ATG7 and serves as a promising therapeutic target for prolactinoma.

Open access

K E Lines, P J Newey, C J Yates, M Stevenson, R Dyar, G V Walls, M R Bowl and R V Thakker

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1 +/ mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1 +/ mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.

Open access

Lesley A Hill, Dimitra A Vassiliadi, Ioanna Dimopoulou, Anna J Anderson, Luke D Boyle, Alixe H M Kilgour, Roland H Stimson, Yoan Machado, Christopher M Overall, Brian R Walker, John G Lewis and Geoffrey L Hammond

Corticosteroid-binding globulin (CBG) transports glucocorticoids in blood and is a serine protease inhibitor family member. Human CBG has a reactive center loop (RCL) which, when cleaved by neutrophil elastase (NE), disrupts its steroid-binding activity. Measurements of CBG levels are typically based on steroid-binding capacity or immunoassays. Discrepancies in ELISAs using monoclonal antibodies that discriminate between intact vs RCL-cleaved CBG have been interpreted as evidence that CBG with a cleaved RCL and low affinity for cortisol exists in the circulation. We examined the biochemical properties of plasma CBG in samples with discordant ELISA measurements and sought to identify RCL-cleaved CBG in human blood samples. Plasma CBG-binding capacity and ELISA values were consistent in arterial and venous blood draining skeletal muscle, liver and brain, as well as from a tissue (adipose) expected to contain activated neutrophils in obese individuals. Moreover, RCL-cleaved CBG was undetectable in plasma from critically ill patients, irrespective of whether their ELISA measurements were concordant or discordant. We found no evidence of RCL-cleaved CBG in plasma using a heat-dependent polymerization assay, and CBG that resists immunoprecipitation with a monoclonal antibody designed to specifically recognize an intact RCL, bound steroids with a high affinity. In addition, mass spectrometry confirmed the absence of NE-cleaved CBG in plasma in which ELISA values were highly discordant. Human CBG with a NE-cleaved RCL and low affinity for steroids is absent in blood samples, and CBG ELISA discrepancies likely reflect structural differences that alter epitopes recognized by specific monoclonal antibodies.