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1.	Abadpour, Shadab, et al. (author) Interleukin-22 reverses human islet dysfunction and apoptosis triggered by hyperglycemia and LIGHT 2018 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 60:3, s. 171-183 Journal article (peer-reviewed)abstract Interleukin (IL)-22 has recently been suggested as an anti-inflammatory cytokine that could protect the islet cells from inflammation- and glucose-induced toxicity. We have previously shown that the tumor necrosis factor family member, LIGHT can impair human islet function at least partly via pro-apoptotic effects. Herein, we aimed to investigate the protective role of IL-22 on human islets exposed to the combination of hyperglycemia and LIGHT. First, we found up-regulation of LIGHT receptors (LTβR and HVEM) in engrafted human islets exposed to hyperglycemia (>11 mM) for 17 days post transplantation by using a double islet transplantation mouse model as well as in human islets cultured with high glucose (HG) (20mM glucose) + LIGHT in vitro and this latter effect was attenuated by IL-22. The effect of HG + LIGHT impairing glucose stimulated insulin secretion was reversed by IL-22. The harmful effect of HG + LIGHT on human islet function seemed to involve enhanced endoplasmic reticulum stress evidenced by up-regulation of p-IRE1α and BiP, elevated secretion of pro-inflammatory cytokines (IL-6, IL-8, IP-10 and MCP-1) and the pro-coagulant mediator tissue factor (TF) release and apoptosis in human islets, whereas all these effects were at least partly reversed by IL-22. Our findings suggest that IL-22 could counteract the harmful effects of LIGHT/hyperglycemia on human islet cells and potentially support the strong protective effect of IL-22 on impaired islet function and survival.
2.	Alheim, Katarina, et al. (author) Identification of a functional glucocorticoid response element in the promoter of the cylcin-dependant kinase inhibitor p57(Kip2) 2003 In: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 30:3, s. 359-368 Journal article (peer-reviewed)abstract Glucocorticoids are known regulators of the cell cycle, normally exerting an anti-proliferative effect. We have previously shown that glucocorticoids stimulate expression of p57(Kip2), a member of the Cip/Kip family of cyclin-dependent kinase inhibitors which, in some cell types, may account for the anti-proliferative responses seen after glucocorticoid treatment. The induction of p57(Kip2) involves primary transcriptional effects where no de novo protein synthesis is necessary, suggesting a direct interaction of the glucocorticoid receptor with the p57(Kip2) gene. In this study we have identified a functional glucocorticoid response element (GRE), located 5 kilo bases (kb) upstream of the transcription start site in the human P57(Kip2) promoter. This GRE was functional also when isolated, suggesting a direct transcriptional effect of the glucocorticoid receptor. Furthermore, mutation of this GRE abolished glucocorticoid induction of the reporter gene, whereas mutation of a nearby Sp1 site did not. Using electrophoretic mobility shift assays, we have shown that the -5 kb p57(Kip2) promoter GRE was able to compete with a well-known GRE for glucocorticoid receptor binding. Sequence comparisons with the mouse genome showed that this GRE is highly conserved, further strengthening the biological importance of this site. All these data emphasize the involvement of this GRE in the glucocorticoid-mediated induction of p57(Kip2) expression.
3.	Baryshev, M, et al. (author) Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism 2004 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 32:3, s. 903-920 Journal article (peer-reviewed)abstract The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the protein folding and processing capacity of the endoplasmic reticulum (ER). The UPR is induced by the pharmacological agents that perturb ER functions but is also activated upon excessive accumulation of the mutant secretory proteins that are unable to attain correct three-dimensional structure and are thus retained in the ER. Such defects in intracellular protein transport underlie the development of a number of phenotypically diverse inherited pathologies, termed endoplasmic reticulum storage diseases (ERSD). We have studied UPR development in two similar ERSDs, human congenital goiter caused by the C1264R and C1996S mutations in the thyroglobulin (Tg) gene and non-goitrous congenital hypothyroidism in rdw dwarf rats determined by the G2320R Tg mutation. In both cases, these mutations rendered Tg incapable of leaving the ER. A major ER chaperone immunoglobulin-binding protein (BiP), and a novel putative escort chaperone endoplasmic reticulum protein 29 KDa (ERp29) were found to be associated with Tg, which might be interpreted as the contribution of the quality control machinery to the previously shown retention of Tg in the ER. We have extended our earlier observations of ER chaperone induction with the identification of the additional ER (ERp29, ERp72, calreticulin, protein disulfide isomerase (PDI)), cytoplasmic (heat shock protein (HSP)70, HSP90) and mitochondrial (mtHSP70) upregulated chaperones and folding enzymes. Activation of the transcriptional arm of UPR, as judged by the appearance of the spliced (active) form of X-box binding protein (XBP1) and processed activating transcription factor 6 (ATF6) transcription factors was suggested to contribute to the overexpression of the ER chaperones. The processing of ATF6 was observed in both human and rat tissues with Tg mutations. Whereas, in human tissues, weak splicing of XBP1 mRNA was detected only in the C1264R mutant, all rat thyroids including wild-type contained significant amounts of the spliced form of XBP1 as opposed to human liver and rat brain tissues, implying the existence of a previously unknown tissue-specific regulation of XBP1 processing.
4.	Bjornstrom, L, et al. (author) Cross-talk between Stat5b and estrogen receptor-alpha and -beta in mammary epithelial cells 2001 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 27:1, s. 93-106 Journal article (peer-reviewed)abstract Both 17beta-estradiol and prolactin play important roles in the mammary gland, raising the possibility of functional cross-talk between the two signaling pathways. Here, we demonstrate that estrogen receptor-alpha (ERalpha) and -beta (ERbeta) are both able to potentiate transcription from a Stat5-responsive promoter when activated by prolactin. Potentiation was observed not only in the presence of 17beta-estradiol, but also in the presence of anti-estrogens such as tamoxifen and ICI 182,780. The magnitude of the response was dependent on cell-type: in the HC11 mouse mammary epithelial cell line ERbeta potentiates transcription efficiently whereas ERalpha showed low activity. Conversely, in COS-7 cells, both estrogen receptors were active. We show that activation domains in the N-terminus (AF-1) and the C-terminus (AF-2) of the ERs are dispensable for potentiation. The effects are dependent on the presence of an intact DNA-binding/hinge domain, which we show is capable of interacting with Stat5b in vitro and in HC11 cell extracts. We conclude that ERalpha and ERbeta act as coactivators for Stat5b through a mechanism which is independent of AF-1 and AF-2.
5.	Cardoso, Joao C. R., et al. (author) Corticotropin-releasing hormone family evolution : five ancestral genes remain in some lineages 2016 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 57:1, s. 73-86 Journal article (peer-reviewed)abstract The evolution of the peptide family consisting of corticotropin-releasing hormone ( CRH) and the three urocortins ( UCN1-3) has been puzzling due to uneven evolutionary rates. Distinct gene duplication scenarios have been proposed in relation to the two basal rounds of vertebrate genome doubling ( 2R) and the teleost fish-specific genome doubling ( 3R). By analyses of sequences and chromosomal regions, including many neighboring gene families, we show here that the vertebrate progenitor had two peptide genes that served as the founders of separate subfamilies. Then, 2R resulted in a total of five members: one subfamily consists of CRH1, CRH2, and UCN1. The other subfamily contains UCN2 and UCN3. All five peptide genes are present in the slowly evolving genomes of the coelacanth Latimeria chalumnae ( a lobe-finned fish), the spotted gar Lepisosteus oculatus ( a basal ray-finned fish), and the elephant shark Callorhinchus milii ( a cartilaginous fish). The CRH2 gene has been lost independently in placental mammals and in teleost fish, but is present in birds ( except chicken), anole lizard, and the nonplacental mammals platypus and opossum. Teleost 3R resulted in an additional surviving duplicate only for crh1 in some teleosts including zebrafish ( crh1a and crh1b). We have previously reported that the two vertebrate CRH/UCN receptors arose in 2R and that CRHR1 was duplicated in 3R. Thus, we can now conclude that this peptide-receptor system was quite complex in the ancestor of the jawed vertebrates with five CRH/UCN peptides and two receptors, and that crh and crhr1 were duplicated in the teleost fish tetraploidization.
6.	Cen, Jing, et al. (author) Mechanisms of beneficial effects of metformin on fatty acid-treated human islets 2018 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 61:3, s. 91-99 Journal article (peer-reviewed)abstract Elevated levels of palmitate accentuate glucose-stimulated insulin secretion (GSIS) after short-term and cause beta-cell dysfunction after prolonged exposure. We investigated whether metformin, the first-line oral drug for treatment of T2DM, has beneficial effects on FFA-treated human islets and the potential mechanisms behind the effects. Insulin secretion, oxygen consumption rate (OCR), AMPK activation, endoplasmic reticulum (ER) stress and apoptosis were examined in isolated human islets after exposure to elevated levels of palmitate in the absence or presence of metformin. Palmitate exposure doubled GSIS after 2 days but halved after 7 days compared with control. Inclusion of metformin during palmitate exposure normalized insulin secretion both after 2 and 7 days. After 2-day exposure to palmitate, OCR and the marker of the adaptive arm of ER stress response (sorcin) were significantly raised, whereas AMPK phosphorylation, markers of pro-apoptotic arm of ER stress response (p-EIF2α and CHOP) and apoptosis (cleaved caspase 3) were not affected. Presence of metformin during 2-day palmitate exposure normalized OCR and sorcin levels. After 7-day exposure to palmitate, OCR and sorcin were not significantly different from control level, p-AMPK was reduced and p-EIF2α, CHOP and cleaved caspase 3 were strongly upregulated. Presence of metformin during 7-day culture with palmitate normalized the level of p-AMPK, p-EIF2α, CHOP and cleaved caspase 3 but significantly increased the level of sorcin. Our study demonstrates that metformin prevents early insulin hypersecretion and later decrease in insulin secretion from palmitate-treated human islets by utilizing different mechanisms.
7.	Chisalita, Ioana Simona, et al. (author) Human aortic smooth muscle cells are insulin resistant at the receptor level but sensitive to IGF1 and IGF2 2009 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 43:5-6, s. 231-239 Journal article (peer-reviewed)abstract Whether insulin, in physiological concentrations, has direct effects on vascular smooth muscle cells (VSMC) remains controversial. Our aim was to characterize the mechanism for insulin resistance in VSMCs. For comparison, effects of insulin-like growth factor (IGF)-I and IGF-II were also studied. Cultured human aortic smooth muscle cells (HASMC) were used. Receptor mRNA was analysed by quantitative RT-PCR and receptor protein by ELISA and Western Blot. The biological effects were studied by thymidine incorporation and glucose accumulation. In HASMC both mRNA and protein expression of IGF-I receptors (IGF-IR) were 5 fold higher compared to insulin receptor (IR). IR isoform A mRNA was 13 times more expressed than IR isoform B. Immunoprecipitation and Western blot showed co precipitation of IR and IGF-IR indicating the presence of hybrid IR/IGF-IR. Phosphorylation of the IGF-IR β-subunit was obtained by IGF-I 10-9-10-8mol l-1 and IGF-II 10-8mol l-1. IR β-subunit was phosphorylated by IGF-I 10-8mol l-1 but not by insulin. IGF-I stimulated IRS-I at 10-8mol l-1, Akt and Erk 1/2 at 10-9-10-8mol l-1, respectively. IGF-II stimulated Akt at 10-8mol l-1 whereas insulin had no effect. IGF-I and IGF-II at a concentration of 10-8-10-7mol l-1 significantly stimulated 3H-thymidine incorporation, whereas insulin did not. 14C-Glucose accumulation was stimulated by IGF-I or IGF-II 10-8-10-7mol l-1, and also by insulin 10-7mol l-1. Our results suggest that IGF-IR and hybrid IR/IGF-IR are activated by physiological concentrations of IGF-I and IGF-II in HASMC and this causes downstream signaling and biological effects, while insulin has no effect on its receptor or downstream signaling probably due to a preponderance of IGF-IR and incorporation of IR into hybrid IR/IGF-IR.
8.	Dare, E, et al. (author) Characterization of the phosphatidylinositol-specific phospholipase C isozymes present in the bovine parathyroid and in human kidney HEK293 cells stably transfected with the human parathyroid Ca2+-sensing receptor 1998 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 21:1, s. 7-17 Journal article (peer-reviewed)abstract The regulation of parathyroid hormone secretion by the chief cells of the parathyroid is mediated by a 7-transmembrane (7-TM) Ca2+-sensing receptor (CaR), which signals via activation of pertussis toxin-insensitive G proteins, causing stimulation of phosphatidylinositol-specific phospholipase C (PI-PLC). We have identified the PI-PLC isoforms expressed in two model systems utilized for studying CaR signal transduction, i.e. dispersed bovine parathyroid cells and a human embryonic kidney cell line (HEK 293) stably transfected with the human parathyroid CaR-cDNA. All of the eight PI-PLC isozymes examined in this study were found to be expressed to varying extents in the bovine parathyroid gland and in the CaR-transfected HEK cells as assessed by immunoblotting. We localized the expression of the more abundant isozymes (beta1, beta2, beta3, gamma1, gamma2, delta2) to the chief cells of the bovine parathyroid by immunocytochemistry, while the two less abundant isozymes (delta1, beta4) were not detectable in parathyroid sections. G proteins activated by 7-TM receptors are known to activate mainly PI-PLC of the beta class. Therefore, beta1, beta2, beta3 and beta4, all expressed in the bovine parathyroid, are candidate isozymes for coupling to the CaR. A comparison of the levels of expression of PI-PLC isozymes between CaR-transfected HEK cells and non-transfected HEK cells suggested that the expression of the CaR in this human cell line does not cause a significant up-regulation of any of the PLCbeta and PLCgamma isozymes. PLCdelta2, showing predominantly nuclear localization in the parathyroid, was the sole PI-PLC isozyme with higher levels of expression in CaR-transfected HEK cells.
9.	Essand, Magnus, et al. (author) Identification and characterization of a novel splicing variant of vesicular monoamine transporter 1 2005 In: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 35:3, s. 489-501 Journal article (peer-reviewed)abstract Vesicular monoamine transporter 1 (VMAT1) is an integral protein in the membrane of secretory vesicles of neuroendocrine and endocrine cells that allows the transport of biogenic monoamines, such as serotonin, from the cytoplasm into the secretory vesicles. The full-length VMAT1 transcript is produced from 16 exons. We have identified and characterized an alternatively spliced form of VMAT1 that lacks exon 15, the next to last exon of VMAT1. The new form was therefore denoted VMAT1Delta15. Exon 15 does not contain an even multiple of three nucleotides. As a consequence, there is a shift of reading frame, and exon 16 is translated in an alternative reading frame, yielding a novel protein with a shorter and unrelated C-terminus compared with the native VMAT1 protein. VMAT1 and VMAT1Delta15 mRNAs are simultaneously expressed in normal and neoplastic neuroendocrine cells of the GI tract. However, VMAT1 expression is always higher than VMAT1Delta15 expression. We prove that VMAT1Delta15 is not localized in large, dense core vesicles as the native form but in the endoplasmic reticulum. Furthermore, while VMAT1 can take up serotonin, VMAT1Delta15 cannot, indicating different functions for the two forms of VMAT1.
10.	Farman, Helen H., 1983, et al. (author) Extra-nuclear effects of estrogen on cortical bone in males require ERαAF-1 2017 In: Journal of Molecular Endocrinology. - 0952-5041. ; 58:2, s. 105-111 Journal article (peer-reviewed)abstract Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-10) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-10 males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-10 mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.
11.	Fridmanis, Davids, et al. (author) Replacement of short segments within transmembrane domains of MC2R disrupts retention signal 2014 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 53:2, s. 201-215 Journal article (peer-reviewed)abstract The proteolysis of the pro-opiomelanocortin precursor results in the formation of melanocortins (MCs), a group of peptides that share the conserved -H-F-R-W- sequence, which acts as a pharmacophore for five subtypes of MC receptors (MCRs). MC type 2 receptor (MC2R; also known as ACTHR) is the most specialized of all the MCRs. It is predominantly expressed in the adrenal cortex and specifically binds ACTH. Unlike other MCRs, it requires melanocortin receptor accessory protein 1 (MRAP) for formation of active receptor and for its transport to the cell membrane. The molecular mechanisms underlying this specificity remain poorly understood. In this study, we used directed mutagenesis to investigate the role of various short MC2R sequence segments in receptor membrane trafficking and specific activation upon stimulation with ligands. The strategy of the study was to replace two to five amino acid residues within one MC2R segment with the corresponding residues of MC4R. In total, 20 recombinant receptors C-terminally fused to enhanced green fluorescent protein were generated and their membrane trafficking efficiencies and cAMP response upon stimulation with α-MSH and ACTH(1-24) were estimated during their stand-alone expression and coexpression with MRAP. Our results indicate that both the motif that determines the ligand-recognition specificity and the intracellular retention signal are formed by a specific extracellular structure, which is supported by the correct alignment of the transmembrane domains. Our results also indicate that the aromatic-residue-rich segment of the second extracellular loop is involved in the effects mediated by the second ACTH pharmacophore (-K-K-R-R-).
12.	Gardmo, C, et al. (author) In vivo transfection of rat liver discloses binding sites conveying GH-dependent and female-specific gene expression 2006 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 37:3, s. 433-441 Journal article (peer-reviewed)abstract The sexually dimorphic mode of GH secretion leads to a sex-differentiated expression of many hepatic target genes. Expression of the a1bg gene in rat liver is specifically induced by the female pattern of GH secretion. In this study, we have used the a1bg promoter in in vivo transfection experiments to investigate molecular mechanisms of GH-mediated female-specific hepatic gene regulation. Rat liver transfection was achieved by rapid tail vein injection of large volumes of plasmid solution. Expression of reporter constructs showed that the 160 bp proximal part of the a1bg promoter contained elements directing sex-specific expression. In vitro footprinting analysis and electromobility shift assays identified binding of hepatic nuclear factor 6 (HNF6), signal transducer and activator of transcriptions (Stat5) and nuclear factor 1 (NF1) in liver nuclear extracts to the 160 bp proximal promoter. Transfection of mutated and/or deletion constructs showed that HNF6 and NF1 binding markedly enhanced expression in female livers, whereas Stat5 reduces the sex difference by enhancing expression more strongly in male than in female rat liver. Based on our present results, we propose that adjacent binding sites for NF1 and HNF6 constitute a gene regulatory unit of importance for transducing the female-specific effect of GH in rat liver.
13.	Giandomenico, Valeria, et al. (author) Olfactory Receptor 51E1 as a Novel Target for Diagnosis in Somatostatin Receptor Negative Lung Carcinoids 2013 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 51, s. 277-286 Journal article (peer-reviewed)abstract Somatostatin receptors (SSTRs) may be used in lung carcinoids (LCs) for diagnosis and therapy, although additional targets are clearly warranted. This study aimed to investigate whether olfactory receptor 51E1 (OR51E1) may be a potential target for LCs. OR51E1 coding sequence was analyzed in LC cell lines, NCI-H727 and NCI-H720. OR51E1 transcript expression was investigated in LC cell lines and frozen specimens by quantitative real-time PCR. OR51E1, SSTR2, SSTR3, and SSTR5 expression was evaluated by immunohistochemistry on paraffin-embedded sections of 73 typical carcinoids (TCs), 14 atypical carcinoids (ACs) and 11 regional/distant metastases, and compared to OctreoScan data. Immunohistochemistry results were rendered semiquantitatively on a scale from 0 to 3+, taking into account the cellular compartmentalization (membrane vs. cytoplasm) and the percentage of tumor cells (<50% vs. >50%). Our results showed that wild-type OR51E1 transcript was expressed in both LC cell lines. OR51E1 mRNA was expressed in 9/12 TCs and 7/9 ACs (p=NS). Immunohistochemically, OR51E1, SSTR2, SSTR3 and SSTR5 were detected in 85%, 71%, 25% and 39% of TCs, and in 86%, 79%, 43% and 36% of ACs, respectively. OR51E1 immunohistochemical scores were higher or equal compared to SSTRs in 79% of TCs and 86% of ACs. Furthermore, in the LC cases where all SSTR subtypes were lacking, membrane OR51E1 expression was detected in 10/17 TCs and 1/2 ACs. Moreover, higher OR51E1 immunohistochemical scores were detected in 5/6 OctreoScan-negative LC lesions. Therefore, the high expression of OR51E1 in LCs makes it a potential novel diagnostic target in SSTR-negative tumors.
14.	Gong, Ningping, 1979, et al. (author) Acute anorexigenic action of leptin in rainbow trout is mediated by the hypothalamic Pi3k pathway 2016 In: Journal of Molecular Endocrinology. - 0952-5041. ; 56:3, s. 227-238 Journal article (peer-reviewed)abstract Leptin (Lep) is an anorexigenic hormone and regulates appetite-related neuropeptides in mammals. A number of neuropeptides have also been linked to appetite regulation in teleost fish, but Lep signaling activation and effects on appetite-regulating neurons are poorly elucidated in early vertebrates. This study uses cellular, tissue and organismal approaches to elucidate the acute, central Lep action in rainbow trout. The results demonstrate that Lep activates phosphorylation of protein kinase B (Akt) and signal transducer and activator of transcription 3 in rainbow trout hypothalamus-derived cells, and that the phosphatidylinositol-3-kinase (Pi3k) inhibitor LY294002 can suppress the Lep-induced Akt phosphorylation. Intracerebroventricular (ICV) Lep administration strongly suppresses food intake at the doses of 0.05 and 0.5 μg Lep fish-1. At low dose, Lep stimulates hypothalamic transcription of anorexigenic cocaine- and amphetamineregulated transcript (Cart) and orexigenic neuropeptide Y. At high dose, Lep stimulates hypothalamic transcription of anorexigenic proopiomelanocortin (Pomc) A1, A2, and B, while coinjection with LY294002 reverses this upregulation. The data suggest that the anorexigenic action of Lep in rainbow trout is mediated through stimulation of the anorexigenic neuropeptides Pomc and Cart. Furthermore, ICV Lep treatment increases phosphor-Akt-immunoreactive cells in the nucleus lateralis tuberis, periventricular zone along infundibulum, and lateral recess surrounded by nucleus anterior tuberis, while LY294002 inhibits this effect. Lep receptor-immunoreactive cells are also predominant in these regions. These results demonstrate that Lep activates the Pi3k-Akt pathway in the lateral tuberal hypothalamus of rainbow trout for acute appetite regulation, indicating the conservation of anorexigenic Lep action in the mediobasal hypothalamus. © 2016 Society for Endocrinology.
15.	Guilbaud, cecile, et al. (author) Cloning and sequence analysis of a cDNA encoding an androgen-dependent mouse seminal vesicle secretory protein. 1993 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 10:3, s. 279-288 Journal article (peer-reviewed)
16.	Hansson, Björn, et al. (author) Intact glucose uptake despite deteriorating signaling in adipocytes with high-fat feeding 2018 In: Journal of Molecular Endocrinology. - 0952-5041. ; 60:3, s. 199-211 Journal article (peer-reviewed)abstract To capture immediate cellular changes during diet-induced expansion of adipocyte cell volume and number, we characterized mature adipocytes during a short-term high-fat diet (HFD) intervention. Male C57BL6/J mice were fed chow diet, and then switched to HFD for 2, 4, 6 or 14 days. Systemic glucose clearance was assessed by glucose tolerance test. Adipose tissue was dissected for RNA-seq and cell size distribution analysis using coulter counting. Insulin response in isolated adipocytes was monitored by glucose uptake assay and Western blotting, and confocal microscopy was used to assess autophagic activity. Switching to HFD was accompanied by an immediate adipocyte size expansion and onset of systemic insulin resistance already after two days, followed by recruitment of new adipocytes. Despite an initially increased non-stimulated and preserved insulin-stimulated glucose uptake, we observed a decreased phosphorylation of insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB). After 14 days of HFD, both the insulin-stimulated phosphorylation of Akt substrate of 160 kDa (AS160) and glucose uptake was blunted. RNA-seq analysis of adipose tissue revealed transient changes in gene expression at day four, including highly significant upregulation of Trp53inp, previously demonstrated to be involved in autophagy. We confirmed increased autophagy, measured as an increased density of LC3-positive puncta and decreased p62 expression after 14 days of HFD. In conclusion, HFD rapidly induced systemic insulin resistance, whereas insulin-stimulated glucose uptake remained intact throughout 6 days of HFD feeding. We also identified autophagy as an early cellular process that potentially influences adipocyte function upon switching to HFD.
17.	Kasper, S, et al. (author) Selective activation of the probasin androgen-responsive region by steroid hormones 1999 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 22:3, s. 313-325 Journal article (peer-reviewed)abstract Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.
18.	Larsson, Dennis, et al. (author) Antagonistic effects of 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 on L-type Ca2+ channels and Na+/Ca2+ exchange in enterocytes from Atlantic cod (Gadus morhua). 2002 In: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 28:1, s. 53-68 Journal article (peer-reviewed)abstract There is mounting evidence that vitamin D and its metabolites play important roles in regulating plasma calcium concentrations in teleost fish as in other vertebrates. The aims of the present study were to elucidate the possible cellular target mechanisms for the rapid actions of 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in Atlantic cod enterocytes at physiological doses, and to establish the concentration and thus the physiological range of circulating 24R,25(OH)(2)D(3), 25(OH)D(3) and 1,25(OH)(2)D(3) in the Atlantic cod. The plasma concentrations of 25(OH)D(3), 1,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) were 15.3 +/- 2.7nM, 125.1 +/- 12.3pM and 10.1 +/- 23.5nM respectively. Exposure of enterocytes to 10mM calcium (Ca(2+)) evoked an increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)). This increase was suppressed by 24R,25(OH)(2)D(3) dose-dependently, with an EC(50) of 4.9nM and a maximal inhibition of 60%. 24R,25(OH)(2)D(3) (20nM) abolished an increase in [Ca(2+)](i) (approximately 252%) in the control enterocytes exposed to 10microM S(-)-BAYK-8644, suggesting that the hormone acts by inhibiting Ca(2+) entry through L-type voltage-gated Ca(2+) channels. Administration of 20nM 24R,25(OH)(2)D(3) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 20%, indicating a release of Ca(2+) from intracellular stores. Administration of 25(OH)D(3) (20nM) resulted in a biphasic change in the enterocyte [Ca(2+)](i): within 1--5s, it decreased to 87 +/- 12nM below its mean basal [Ca(2+)](i) (334 +/- 13nM), followed by a rapid recovery of [Ca(2+)](i) to a new level, 10% lower than the initial [Ca(2+)](i). The rapid decrease, the recovery rate and the final [Ca(2+)](i) were all affected dose-dependently by 25(OH)D(3), with EC(50) values of 8.5, 17.0 and 18.9nM respectively. Furthermore, the effects of 25(OH)D(3) were sensitive to sodium (Na(+)), bepridil (10microM) and nifedipine (5 microM), suggesting that 25(OH)D(3) regulates the activity of both basolateral membrane-associated Na(+)/Ca(2+) exchangers and brush border membrane-associated L-type Ca(2+) channels. Administration of 25(OH)D(3) (10nM) to enterocytes in the absence of extracellular Ca(2+) increased [Ca(2+)](i) by approximately 18%, indicating a release of Ca(2+) from intracellular stores. 1,25(OH)(2)D(3) also affected enterocyte [Ca(2+)](i) in a biphasic manner: the rapid decrease, the recovery rate, and the mean final [Ca(2+)](i) were all affected dose-dependently, with EC(50) values of 8.3, 24.5 and 7.7nM respectively. The high EC(50) values for 1,25(OH)(2)D(3) compared with circulating concentrations of 1,25(OH)(2)D(3) (130pM) suggest that this effect is pharmacological, rather than of physiological relevance in enterocyte Ca(2+) homeostasis of the Atlantic cod. It is concluded that 24R,25(OH)(2)D(3) has a physiological role in decreasing intestinal Ca(2+) uptake via inactivation of L-type Ca(2+) channels, whereas the physiological role of 25(OH)D(3) is to increase enterocyte Ca(2+) transport via activation of Na(+)/Ca(2+) exchangers, concurrent with activation of L-type Ca(2+) channels.
19.	Liu, Lian, et al. (author) Phosphorylation and inactivation of glycogen synthase kinase-3 by soluble kit ligand in mouse oocytes during early follicular development. 2007 In: Journal of Molecular Endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 38:1-2, s. 137-146 Journal article (peer-reviewed)abstract Communication between mammalian oocytes and their surrounding granulosa cells through the Kit-Kit ligand (KL, or stem cell factor, SCF) system has been shown to be crucial for follicular development. Our previous studies (Reddy et al. 2005, Liu et al. 2006) have indicated that the intra-oocyte KL-Kit-PI3 kinase (PI3K)-Akt-Foxo3a cascade may play an important role in follicular activation and early development. In the present study, using in situ hybridization and in vitro culture of growing oocytes from 8-day-old postnatal mice, we have demonstrated that another Akt substrate, glycogen synthase kinase-3 (GSK-3), is expressed in growing oocytes. Also, treatment of cultured mouse oocytes with soluble KL not only leads to increased Akt kinase activity in the oocytes, which can phosphorylate recombinant GSK-3 in vitro, but also leads to phosphorylation of oocyte GSK-3alpha and GSK-3beta, which can result in the inactivation of GSK-3 function in oocytes. In addition, we have shown that the regulation of GSK-3alpha and GSK-3beta in cultured oocytes by soluble KL is accomplished through PI3K, since the PI3K-specific inhibitor LY294002 completely abolished the KL-induced phosphorylation of GSK-3alpha and GSK-3beta. Moreover, blockage of the Kit signaling pathway by a Kit function-blocking antibody, ACK2, resulted in reduced phosphorylation of GSK-3. Taken together, our data suggest that the cascade from granulosa cell-derived KL to Kit-PI3K-Akt-GSK-3 in oocytes may take part in regulation of oocyte growth and early ovarian follicular development.
20.	Lu, Ming, et al. (author) Expression and association of TRPC subtypes with Orai1 and STIM1 in human parathyroid 2010 In: Journal of Molecular Endocrinology. - 0952-5041 .- 1479-6813. ; 44:5, s. 285-294 Journal article (peer-reviewed)abstract The mechanism behind Ca2+ entry into the parathyroid cells has been widely debated, and the molecular identities of the responsible ion channels have not been established yet. In this study, we show that the parathyroid cells lack voltage-operated Ca2+ channels. Passive store depletion by thapsigargin, on the other hand, induces a large non-voltage-activated non-selective cation current. The increase in intracellular Ca2+ caused by thapsigargin is attenuated by 2-aminoethoxydiphenyl borate, a blocker of store-operated Ca2+ entry (SOCE). Candidate molecules for non-voltage-operated Ca2+ signaling were investigated. These included members of the transient receptor potential canonical (TRPC) ion channel family, as well as Ca2+ release-activated Ca2+ modulator 1 (Orai1) and stromal interaction molecule 1 (STIM1) that are key proteins in the SOCE pathway. Using RT-PCR screening, quantitative real-time PCR, and western blot, we showed expression of TRPC1, TRPC4, and TRPC6; Orai1; and STIM1 genes and proteins in normal and adenomatous human parathyroid tissues. Furthermore, co-immunoprecipitation experiments demonstrated a ternary complex of TRPC1-Orai1-STIM1, supporting a physical interaction between these molecules in human parathyroid.
21.	Lundholm, Lovisa, et al. (author) Gene expression profiling identifies liver X receptor alpha as an estrogen-regulated gene in mouse adipose tissue. 2004 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 32:3, s. 879-92 Journal article (peer-reviewed)abstract Estrogens reduce adipose tissue mass in both humans and animals. The molecular mechanisms for this effect are, however, not well characterized. We took a gene expression profiling approach to study the direct effects of estrogen on mouse white adipose tissue (WAT). Female ovariectomized mice were treated for 10, 24 and 48 h with 17beta-estradiol or vehicle. RNA was extracted from gonadal fat and hybridized to Affymetrix MG-U74Av2 arrays. 17beta-Estradiol was shown to decrease mRNA expression of liver X receptor (LXR) alpha after 10 h of treatment compared with the vehicle control. The expression of several LXRalpha target genes, such as sterol regulatory element-binding protein 1c, apolipoprotein E, phospholipid transfer protein, ATP-binding cassette A1 and ATP-binding cassette G1, was similarly decreased. We furthermore identified a 1.5 kb LXRalpha promoter fragment that is negatively regulated by estrogen. Several genes involved in lipogenesis and lipolysis were identified as novel targets that could mediate estrogenic effects on adipose tissue. Finally, we show that ERalpha is the main estrogen receptor expressed in mouse white adipose tissue (WAT) with mRNA levels several hundred times higher than those of ERbeta mRNA.
22.	Nicholson, L B, et al. (author) Monoclonal antibodies to the human TSH receptor: epitope mapping and binding to the native receptor on the basolateral plasma membrane of thyroid follicular cells. 1996 In: Journal of molecular endocrinology. - 0952-5041. ; 16:2, s. 159-70 Journal article (peer-reviewed)abstract We have characterized four murine monoclonal antibodies (mAbs) to the extracellular domain of the human TSH receptor (TSH-R.E), the target autoantigen of Graves' disease. Recombinant TSH-R.E used as immunogen, was produced in E. coli as a fusion protein with glutathione-S-transferase or in a baculovirus-insect cell system, as a non-fusion glycoprotein. To increase the epitope specificity of the mAbs, two different strains of mice (H-2(b) and H-2(d)) were immunized. The epitopes recognized by the mAbs were characterized by immunoblotting with various recombinant constructs of TSH-R.E and by binding to overlapping synthetic peptides of the receptor. The four IgG mAbs characterized recognized epitopes localized to different regions on the TSH-R.E; amino acids 22-35 (A1O and A11, both IgG2b from H-2(b) animals), amino acids 402-415 (A7, IgG2b from H-2(b) animals) and amino acids 147-228 (A9, IgG1 from H-2(d) animals). Immunolocalization studies showed that mAb A9 recognized TSH-R.E on unfixed cryostat sections, where binding was localized to the basolateral plasma membrane of thyroid follicular cells, suggesting that this antibody reacts with the native receptor on thyroid cells. The binding of the mAbs A7, A10 and A11 was also restricted to the basal surface of thyroid cells, but only after acetone fixation of the sections, implying that the epitopes recognized on the amino and carboxyl terminus of the extracellular region of the receptor are not accessible on the native molecule. None of the mAbs stimulated cyclic AMP responses in COS-7 cells transiently transfected with full-length functioning TSH-R.E, whilst weak inhibition of binding of radiolabelled TSH to porcine membranes in a radioreceptor assay was apparent with mAb A10 and A11, but only at high concentrations of IgG. The ability of mAb A9 to bind to the native receptor without stimulating activity or inhibition of TSH binding suggests that antibody can bind to the central region of the TSH-R.E without perturbing receptor function. The availability of mAbs that recognize epitopes on different regions of the extracellular domain of TSH-R will lead to a better understanding of the autoantigenic regions on TSH-R implicated in disease activity.
23.	Nicol, L, et al. (author) Differential secretion of gonadotrophins : investigation of the role of secretogranin II and chromogranin A in the release of LH and FSH in LbetaT2 cells. 2004 In: J Mol Endocrinol. - 0952-5041. ; 32:2, s. 467-80 Journal article (other academic/artistic)
24.	Nygard, M, et al. (author) Thyroid hormone-mediated negative transcriptional regulation of Necdin expression 2006 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 36:3, s. 517-530 Journal article (peer-reviewed)abstract Unliganded thyroid hormone receptors (apoTRs) repress transcription of hormone-activated genes by recruiting corepressors to the promoters. In contrast, on promoters containing so-called negative thyroid hormone response elements (nTREs), apoTRs activate transcription. A number of different molecular mechanisms have been described as to how apoTRs activate transcription varying with the target gene of the study. Here we demonstrate that thyroid hormone regulates the transcription of the Necdin gene, a developmentally regulated candidate gene for the genomic imprinting-associated neurobehavioural disorder, Prader–Willi syndrome. ApoTRs activate Necdin expression through an nTRE in its promoter, downstream of the transcription start site. The nTRE of the Necdin gene resembles the nTREs of the TSHβ genes of the hypothalamus–pituitary–thyroid axis in the sequence, position in the promoter, and mode of activation. We show that this group of nTRE-driven genes shares the requirements for binding of the retinoic X receptor and nuclear receptor corepressor/silencing mediator of retinoid and thyroid hormone receptors (NCoR/SMRT) for full ligand-independent activation, whereas there is no need for association of the p160 family of coactivators. In accordance with the requirement for corepressors, Necdin expression is influenced by deacetylase activity, suggesting that histone deacetylases and corepressors as well could function as activators of transcription, depending on the promoter context.
25.	Palmieri, C, et al. (author) The expression of oestrogen receptor (ER)-beta and its variants, but not ERalpha, in adult human mammary fibroblasts 2004 In: Journal of molecular endocrinology. - : Bioscientifica. - 0952-5041 .- 1479-6813. ; 33:1, s. 35-50 Journal article (peer-reviewed)abstract Whilst oestrogen receptor (ER)-alpha and ERbeta have been shown to be important in the development of the mammary gland, the cell-specific expression pattern of these two receptors within the human breast is not clear. Although it is well established that in the developing rodent mammary gland stromal ERalpha mediates the secretion of growth factors which stimulate the proliferation of the ductal epithelium, the expression of ERalpha in human adult breast stromal fibroblasts is controversial, and the expression of ERbeta has not been properly defined. In the present study, we have evaluated the expression of ERalpha and ERbeta by immunohistochemistry in normal tissue samples, and in purified human breast fibroblasts by Western blotting, RT-PCR analysis and ligand-binding sucrose gradient assay. Our data clearly demonstrated that ERbeta variants, including ERbeta1, ERbeta2, ERbeta5, ERbetadelta and ERbetains, but not ERalpha, are expressed in human adult mammary fibroblasts. These results are supported by the findings that an ERbeta-selective ligand, BAG, but not the ERalpha high-affinity ligand oestradiol, can induce fibroblast growth factor-7 release and activate transcription from an oestrogen-responsive element promoter in these adult human mammary fibroblasts. Together, these observations revealed that, in the adult breast and in breast cancer, the proliferative signals derived from the stroma of adult mammary glands in response to oestrogen are not mediated by ERalpha and provide new insights into the nature of stromal-epithelial interactions in the adult mammary gland. In addition, the expression of these ERbeta variants in cells where there is no ERalpha suggested that these ERbeta splice forms may have functions other than that of modulating ERalpha activity.

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