| 1. |
- Al-Khalili, L, et al.
(författare)
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Signaling specificity of interleukin-6 action on glucose and lipid metabolism in skeletal muscle
- 2006
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:12, s. 3364-3375
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Tidskriftsartikel (refereegranskat)abstract
- We identified signaling pathways by which IL-6 regulates skeletal muscle differentiation and metabolism. Primary human skeletal muscle cells were exposed to IL-6 (25 ng/ml either acutely or for several days), and small interfering RNA gene silencing was applied to measure glucose and fat metabolism. Chronic IL-6 exposure increased myotube fusion and formation and the mRNA expression of glucose transporter 4, peroxisome proliferator activated receptor (PPAR)α, PPARδ, PPARγ, PPARγ coactivator 1, glycogen synthase, myocyte enhancer factor 2D, uncoupling protein 2, fatty acid transporter 4, and IL-6 (P < 0.05), whereas glucose transporter 1, CCAAT/enhancer-binding protein-α, and uncoupling protein 3 were decreased. IL-6 increased glucose incorporation into glycogen, glucose uptake, lactate production, and fatty acid uptake and oxidation, concomitant with increased phosphorylation of AMP-activated protein kinase (AMPK), signal transducer and activator of transcription 3, and ERK1/2. IL-6 also increased phosphatidylinositol (PI) 3-kinase activity (450%; P < 0.05), which was blunted by subsequent insulin-stimulation (P < 0.05). IL-6-mediated glucose metabolism was suppressed, but lipid metabolism was unaltered, by inhibition of PI3-kinase with LY294002. The small interfering RNA-directed depletion of AMPK reduced IL-6-mediated fatty acid oxidation and palmitate uptake but did not reduce glycogen synthesis. In summary, IL-6 increases glycogen synthesis via a PI3-kinase-dependent mechanism and enhances lipid oxidation via an AMPK-dependent mechanism in skeletal muscle. Thus, IL-6 directly promotes skeletal muscle differentiation and regulates muscle substrate utilization, promoting glycogen storage and lipid oxidation.
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| 2. |
- Ammoun, Sylwia, et al.
(författare)
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OX1 orexin receptors activate extracellular signal-regulated kinase in Chinese hamster ovary cells via multiple mechanisms : the role of Ca2+ influx in OX1 receptor signaling
- 2006
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:1, s. 80-99
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Tidskriftsartikel (refereegranskat)abstract
- Activation of OX1 orexin receptors heterologously expressedin Chinese hamster ovary (CHO) cells led to a rapid, strong,and long-lasting increase in ERK phosphorylation (activation).Dissection of the signal pathways to ERK using multiple inhibitorsand dominant-negative constructs indicated involvement of Ras,protein kinase C, phosphoinositide-3-kinase, and Src. Most interestingly,Ca2+ influx appeared central for the ERK response in CHO cells,and the same was indicated in recombinant neuro-2a cells andcultured rat striatal neurons. Detailed investigations in CHOcells showed that inhibition of the receptor- and store-operatedCa2+ influx pathways could fully attenuate the response, whereasinhibition of the store-operated Ca2+ influx pathway alone orthe Ca2+ release was ineffective. If the receptor-operated pathwaywas blocked, an exogenously activated store-operated pathwaycould take its place and restore the coupling of OX1 receptorsto ERK. Further experiments suggested that Ca2+ influx, as such,may not be required for ERK phosphorylation, but that Ca2+,elevated via influx, acts as a switch enabling OX1 receptorsto couple to cascades leading to ERK phosphorylation, cAMP elevation,and phospholipase C activation. In conclusion, the data suggestthat the primary coupling of orexin receptors to Ca2+ influxallows them to couple to other signal pathways; in the absenceof coupling to Ca2+ influx, orexin receptors can act as signalintegrators by taking advantage of other Ca2+ influx pathways.
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| 3. |
- Archer, Amena, et al.
(författare)
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Intestinal Apolipoprotein A-IV Gene Transcription Is Controlled by Two Hormone-Responsive Elements : A Role for Hepatic Nuclear Factor-4 Isoforms
- 2005
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:9, s. 2320-2334
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Tidskriftsartikel (refereegranskat)abstract
- In the small intestine, the expression of the apolipoprotein (apo) C-III and A-IV genes is restricted to the enterocytes of the villi. We have previously shown that, in transgenic mice, specific expression of the human apo C-III requires a hormone-responsive element (HRE) located in the distal region of the human apoA-IV promoter. This HRE binds the hepatic nuclear factors (HNF)-4α and γ. Here, intraduodenal injections in mice and infections of human enterocytic Caco-2/TC7 cells with an adenovirus expressing a dominant-negative form of HNF-4α repress the expression of the apoA-IV gene, demonstrating that HNF-4 controls the apoA-IV gene expression in enterocytes. We show that HNF-4α and γ functionally interact with a second HRE present in the proximal region of the human apoA-IV promoter. New sets of transgenic mice expressing mutated forms of the promoter, combined with the human apo C-III enhancer, demonstrate that, whereas a single HRE is sufficient to reproduce the physiological cephalo-caudal gradient of apoA-IV gene expression, both HREs are required for expression that is restricted to villi. The combination of multiple HREs may specifically recruit regulatory complexes associating HNF-4 and either coactivators in villi or corepressors in crypts.
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| 4. |
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| 5. |
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| 6. |
- Bladh, LG, et al.
(författare)
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Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression
- 2005
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:3, s. 632-643
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Tidskriftsartikel (refereegranskat)abstract
- Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-κB (NF-κB) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-κB signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-κB-inhibitory protein IκBα, in line with repression of NF-κB signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of IκBα was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of IκBα in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-κB is an important mechanism in the antiproliferative response to GCs.
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| 7. |
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| 8. |
- Charles, Michael A, et al.
(författare)
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PITX genes are required for cell survival and Lhx3 activation.
- 2005
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:7, s. 1893-1903
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Tidskriftsartikel (refereegranskat)abstract
- The PITX family of transcription factors regulate the development of many organs. Pitx1 mutants have a mild pituitary phenotype, but Pitx2 is necessary for the development of Rathke's pouch, expression of essential transcription factors in gonadotropes, and expansion of the Pit1 lineage. We report that lack of Pitx2 causes the pouch to undergo excessive cell death, resulting in severe pituitary hypoplasia. Transgenic overexpression of PITX2 in the pituitary can increase the gonadotrope population, suggesting that the absolute concentration of PITX2 is important for normal pituitary cell lineage expansion. We show that PITX1 and PITX2 proteins are present in similar expression patterns throughout pituitary development and in the mature pituitary. Both transcription factors are preferentially expressed in adult gonadotropes and thyrotropes, suggesting the possibility of overlap in maintenance of adult pituitary functions within these cell types. Double knockouts of Pitx1 and Pitx2 exhibit severe pituitary hypoplasia and fail to express the transcription factor LHX3. This indicates that these PITX genes are upstream of Lhx3 and have compensatory roles during development. Thus, the combined dosage of these PITX family members is vital for pituitary development, and their persistent coexpression in the adult pituitary suggests a continued role in maintenance of pituitary function.
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| 9. |
- Davies, JS, et al.
(författare)
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Ghrelin induces abdominal obesity via GHS-R-dependent lipid retention
- 2009
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 1944-9917 .- 0888-8809. ; 23:6, s. 914-924
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Tidskriftsartikel (refereegranskat)abstract
- Circulating ghrelin elevates abdominal adiposity by a mechanism independent of its central orexigenic activity. In this study we tested the hypothesis that peripheral ghrelin induces a depot-specific increase in white adipose tissue (WAT) mass in vivo by GH secretagogue receptor (GHS-R1a)-mediated lipolysis. Chronic iv infusion of acylated ghrelin increased retroperitoneal and inguinal WAT volume in rats without elevating superficial sc fat, food intake, or circulating lipids and glucose. Increased retroperitoneal WAT mass resulted from adipocyte enlargement probably due to reduced lipid export (ATP-binding cassette transporter G1 mRNA expression and circulating free fatty acids were halved by ghrelin infusion). In contrast, ghrelin treatment did not up-regulate biomarkers of adipogenesis (peroxisome proliferator-activated receptor-γ2 or CCAAT/enhancer binding protein-α) or substrate uptake (glucose transporter 4, lipoprotein lipase, or CD36) and although ghrelin elevated sterol-regulatory element-binding protein 1c expression, WAT-specific mediators of lipogenesis (liver X receptor-α and fatty acid synthase) were unchanged. Adiposity was unaffected by infusion of unacylated ghrelin, and the effects of acylated ghrelin were abolished by transcriptional blockade of GHS-R1a, but GHS-R1a mRNA expression was similar in responsive and unresponsive WAT. Microarray analysis suggested that depot-specific sensitivity to ghrelin may arise from differential fine tuning of signal transduction and/or lipid-handling mechanisms. Acylated ghrelin also induced hepatic steatosis, increasing lipid droplet number and triacylglycerol content by a GHS-R1a-dependent mechanism. Our data imply that, during periods of energy insufficiency, exposure to acylated ghrelin may limit energy utilization in specific WAT depots by GHS-R1a-dependent lipid retention.
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| 10. |
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| 11. |
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| 12. |
- Faber, Kirsten, et al.
(författare)
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Megalin is a receptor for apolipoprotein M and kidney-specific megalin-deficiency confers urinary excretion of apolipoprotein M.
- 2006
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:1, s. 212-218
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein ( apo) M is a novel apolipoprotein belonging to the lipocalin protein superfamily, i.e. proteins binding small lipophilic compounds. Like other apolipoproteins, it is expressed in hepatocytes and secreted into plasma where it associates with high-density lipoprotein particles. In addition, apoM is expressed at high levels in the kidney tubule cells. In this study, we show that the multiligand receptor megalin, which is expressed in kidney proximal tubule cells, is a receptor for apoM and mediates its uptake in the kidney. To examine apoM binding to megalin, a recombinant apoM was expressed in Escherichia coli and used in surface plasmon resonance and cell culture studies. The results showed apoM binding to immobilized megalin [ dissociation constant ( K-d) similar to 0.3-1 mu M] and that the apoM was endocytosed by cultured rat yolk sac cells in a megalin-dependent manner. To examine the importance of apoM binding by megalin in vivo, we analyzed mice with a tissue-specific deficiency of megalin in the kidney. Megalin deficiency was associated with pronounced urinary excretion of apoM, whereas apoM was not detected in normal mouse, human, or rat urine. Gel filtration analysis showed that the urinary apoM-containing particles were small and devoid of apoA-1. The results suggest that apoM binds to megalin and that megalin-mediated endocytosis in kidney proximal tubules prevents apoM excretion in the urine.
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| 13. |
- Flores-Morales, A, et al.
(författare)
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Negative regulation of growth hormone receptor signaling
- 2006
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:2, s. 241-253
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Tidskriftsartikel (refereegranskat)abstract
- GH has been of significant scientific interest for decades because of its capacity to dramatically change physiological growth parameters. Furthermore, GH interacts with a range of other hormonal pathways and is an established pharmacological agent for which novel therapeutical applications can be foreseen. It is easy to see the requirement for a number of postreceptor mechanisms to regulate and control target tissue sensitivity to this versatile hormone. In recent years, some of the components that take part in the down-regulatory mechanism targeting the activated GH receptor (GHR) have been defined, and the physiological significance of some of these key components has begun to be characterized. Down-regulation of the GHR is achieved through a complex mechanism that involves rapid ubiquitin-dependent endocytosis of the receptor, the action of tyrosine phosphatases, and the degradation by the proteasome. The suppressors of cytokine signaling (SOCS) protein family, particularly SOCS2, plays an important role in regulating GH actions. The aim of this review is to summarize collected knowledge, including very recent findings, regarding the intracellular mechanisms responsible for the GHR signaling down-regulation. Insights into these mechanisms can be of relevance to several aspects of GH research. It can help to understand growth-related disease conditions, to explain GH resistance, and may be used to develop pharmaceuticals that enhance some the beneficial actions of endogenously secreted GH in a tissue-specific manner.
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| 14. |
- Frobose, H, et al.
(författare)
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Suppressor of cytokine Signaling-3 inhibits interleukin-1 signaling by targeting the TRAF-6/TAK1 complex
- 2006
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 20:7, s. 1587-1596
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Tidskriftsartikel (refereegranskat)abstract
- IL-1 plays a major role in inflammation and autoimmunity through activation of nuclear factor κ B (NFκB) and MAPKs. Although a great deal is known about the mechanism of activation of NFκB and MAPKs by IL-1, much less is known about the down-regulation of this pathway. Suppressor of cytokine signaling (SOCS)-3 was shown to inhibit IL-1-induced transcription and activation of NFκB and the MAPKs JNK and p38, but the mechanism is unknown. We show here that SOCS-3 inhibits NFκB-dependent transcription induced by overexpression of the upstream IL-1 signaling molecules MyD88, IL-1R-activated kinase 1, TNF receptor-associated factor (TRAF)6, and TGFβ-activated kinase (TAK)1, but not when the MAP3K MAPK/ERK kinase kinase-1 is used instead of TAK1, indicating that the target for SOCS-3 is the TRAF6/TAK1 signaling complex. By coimmunoprecipitation, it was shown that SOCS-3 inhibited the association between TRAF6 and TAK1 and that SOCS-3 coimmunoprecipitated with TAK1 and TRAF6. Furthermore, SOCS-3 inhibited the IL-1-induced catalytic activity of TAK1. Because ubiquitination of TRAF6 is required for activation of TAK1, we analyzed the role of SOCS-3 on TRAF6 ubiquitination and found that SOCS-3 inhibited ubiquitin modification of TRAF6. These results indicate that SOCS-3 inhibits IL-1 signal transduction by inhibiting ubiquitination of TRAF6, thus preventing association and activation of TAK1.
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| 15. |
- Gabbi, C, et al.
(författare)
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Minireview: liver X receptor beta: emerging roles in physiology and diseases
- 2009
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 23:2, s. 129-136
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Tidskriftsartikel (refereegranskat)abstract
- Liver X receptors, LXRα and LXRβ, are nuclear receptors belonging to the large family of transcription factors. After activation by oxysterols, LXRs play a central role in the control of lipid and carbohydrate metabolism as well as inflammation. The role of LXRα has been extensively studied, particularly in the liver and macrophages. In the liver it prevents cholesterol accumulation by increasing bile acid synthesis and secretion into the bile through ATP-binding cassette G5/G8 transporters, whereas in macrophages it increases cholesterol reverse transport. The function of LXRβ is still under investigation with most of the current knowledge coming from the study of phenotypes of LXRβ−/− mice. With these mice new emerging roles for LXRβ have been demonstrated in the pathogenesis of diseases such as amyotrophic lateral sclerosis and chronic pancreatitis. The present review will focus on the abnormalities described so far in LXRβ−/− mice and the insight gained into the possible roles of LXRβ in human diseases.
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| 16. |
- Gao, H, et al.
(författare)
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Genome-wide identification of estrogen receptor alpha-binding sites in mouse liver
- 2008
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 22:1, s. 10-22
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Tidskriftsartikel (refereegranskat)abstract
- We report the genome-wide identification of estrogen receptor α (ERα)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERα-binding regions. In agreement with what has previously been reported for human cell lines, many ERα-binding regions are located far away from transcription start sites; approximately 40% of ERα-binding regions are located within 10 kb of annotated transcription start sites. Almost 50% of ERα-binding regions overlap genes. The majority of ERα-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS proteins, and Forkhead proteins as the most common motifs present in identified ERα-binding regions. To correlate ERα binding to the promoter of specific genes, with changes in expression levels of the corresponding mRNAs, expression levels of selected mRNAs were assayed in livers 2, 4, and 6 h after treatment with ERα-selective agonist propyl pyrazole triol. Five of these eight selected genes, Shp, Stat3, Pdgds, Pck1, and Pdk4, all responded to propyl pyrazole triol after 4 h treatment. These results extend our previous studies using gene expression profiling to characterize estrogen signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERα to DNA in intact chromatin.
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| 17. |
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| 18. |
- Gustafsson, JA
(författare)
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Steroids and the scientist
- 2005
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:6, s. 1412-1417
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Tidskriftsartikel (refereegranskat)abstract
- Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor α); 2) liver X receptor β (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor β, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor β agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.
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| 19. |
- Johansson, Eva M., 1900, et al.
(författare)
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Nuclear factor 1-C2 is regulated by prolactin and shows a distinct expression pattern in the mouse mammary epithelial cells during development
- 2005
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:4, s. 992-1003
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated that the transcription factor nuclear factor (NF)1-C2 plays an important role in the mammary gland for the activation of the tumor suppressor gene p53. It also activates the milk genes carboxyl ester lipase and whey acidic protein, implying that NF1-C2 participates both in the establishment of a functional gland and in protection of the gland against tumorigenesis during proliferation. In this study, we have developed a new sensitive NF1-C2-specific antiserum for immunohistochemical analyses of the NF1-C2 distribution during mammary gland development. We show that the NF1-C2 protein is present in the epithelial compartment at the virgin stage and throughout mammary gland development. However, in the lactation stage the NF1-C2 protein levels strongly decreased, and many epithelial nuclei stained negative. In situ hybridization shows that NF1-C2 transcripts are expressed in the whole epithelium at pregnancy as well as the lactation stage, indicating that the reduction in protein levels is posttranscriptionally regulated. At involution, the NF1-C2 proteins are back to high levels. Based on studies using NMuMG cells and mammary tissue from heterozygous prolactin receptor knockout mice, we also demonstrate that prolactin has a direct effect in the maintenance of the NF1-C2 protein levels in the mammary epithelial nuclei at the virgin stage and during pregnancy. Hence, we have identified another transcription factor in the mammary gland, besides signal transducer and activator of transcription 5, through which prolactin may control mammary gland development. Furthermore, our data suggest a link between prolactin and p53 in the mammary gland, through NF1-C2.
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| 20. |
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| 21. |
- Lewis, AE, et al.
(författare)
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Phosphorylation of steroidogenic factor 1 is mediated by cyclin-dependent kinase 7
- 2008
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 22:1, s. 91-104
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear receptor steroidogenic factor-1 (SF1) is critical for development and function of steroidogenic tissues. Posttranslational modifications are known to influence the transcriptional capacity of SF1, and it was previously demonstrated that serine 203 is phosphorylated. In this paper we report that serine 203 is phosphorylated by a cyclin-dependent kinase 7 (CDK7)-mediated process. As part of the CDK-activating kinase complex, CDK7 is a component of the basal transcription factor TFIIH, and phosphorylation of SF1 as well as SF1-dependent transcription was clearly reduced in cells carrying a mutation that renders the CDK-activating kinase complex unable to interact with the TFIIH core. Coimmunoprecipitation analyses revealed that SF1 and CDK7 reside in the same complex, and kinase assays demonstrated that immunoprecipitated CDK7 and purified TFIIH phosphorylate SF1 in vitro. The CDK inhibitor roscovitine blocked phosphorylation of SF1, and an inactive form of CDK7 repressed the phosphorylation level and the transactivation capacity of SF1. Structural studies have identified phosphoinositides as potential ligands for SF1. Interestingly, we found that mutations designed to block phospholipid binding dramatically decreased the level of SF1 phosphorylation. Together our results suggest a connection between ligand occupation and phosphorylation and association with the basic transcriptional machinery, indicating an intricate regulation of SF1 transactivation.
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| 22. |
- Ma, Xiaosong, et al.
(författare)
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Glucagon stimulates exocytosis in mouse and rat pancreatic {alpha} cells by binding to glucagon receptors.
- 2005
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:1, s. 198-212
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Tidskriftsartikel (refereegranskat)abstract
- Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.
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| 23. |
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| 24. |
- Nilsson, M, et al.
(författare)
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Liver X receptors regulate adrenal steroidogenesis and hypothalamic-pituitary-adrenal feedback
- 2007
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 21:1, s. 126-137
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear hormone receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11β-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.
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| 25. |
- Ono, M, et al.
(författare)
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Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone
- 2007
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 21:6, s. 1443-1457
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Tidskriftsartikel (refereegranskat)abstract
- GH plays a central role in controlling somatic growth, tissue regeneration, and intermediary metabolism in most vertebrate species through mechanisms dependent on the regulation of gene expression. Recent studies using transcript profiling have identified large cohorts of genes whose expression is induced by GH. Other results have demonstrated that signal transducer and activator of transcription (Stat) 5b, a latent transcription factor activated by the GH receptor-associated protein kinase, Jak2, is a key agent in the GH-stimulated gene activation that leads to somatic growth. By contrast, little is known about the steps through which GH-initiated signaling pathways reduce gene expression. Here we show that Stat5b plays a critical role in the GH-regulated inhibition of IGF binding protein-1 gene transcription by impairing the actions of the FoxO1 transcription factor on the IGF binding protein-1 promoter. Additional observations using transcript profiling in the liver indicate that Stat5b may be a general mediator of GH-initiated gene repression. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.
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| 26. |
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| 27. |
- Rajareddy, Singareddy, et al.
(författare)
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p27kip1 (cyclin-dependent kinase inhibitor 1B) controls ovarian development by suppressing follicle endowment and activation and promoting follicle atresia in mice
- 2007
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 21:9, s. 2189-2202
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Tidskriftsartikel (refereegranskat)abstract
- In humans, the molecular mechanisms underlying ovarian follicle endowment and activation, which are closely related to the control of female reproduction, occurrence of menopause, and related diseases such as premature ovarian failure, are poorly understood. In the current study, we provide several lines of genetic evidence that the cyclin-dependent kinase (Cdk) inhibitor 1B (commonly known as p27kip1 or p27) controls ovarian development in mice by suppressing follicle endowment and activation, and by promoting follicle death. In p27-deficient (p27−/−) mice, postnatal follicle assembly was accelerated, and the number of endowed follicles was doubled as compared with p27+/+ mice. Moreover, in p27−/− ovaries the primordial follicle pool was prematurely activated once it was endowed, and at the same time the massive follicular death that occurs before sexual maturity was rescued by loss of p27. In early adulthood, however, the overactivated follicular pool in p27−/− ovaries was largely depleted, causing premature ovarian failure. Furthermore, we have extensively studied the molecular mechanisms underlying the above-mentioned phenotypes seen in p27−/− ovaries and have found that p27 controls follicular development by several distinct mechanisms at different stages of development of the ovary. For example, p27 controls oocyte growth by suppressing the functions of Cdk2/Cdc2-cyclin A/E1 in oocytes that are arrested at the diplotene stage of meiosis I. This function of p27 is distinct from its well-known role as a suppressor of cell cycle progression. In addition, we have found that p27 activates the caspase-9-caspase-3-caspase-7-poly (ADP-ribose) polymeraseapoptotic cascade by inhibiting Cdk2/Cdc2-cyclin A/B1 kinase activities in follicles, thereby inducing follicle atresia. Our results suggest that the p27 gene is important in determining mammalian ovarian development. This study therefore provides insight into ovary-borne genetic aberrations that cause defects in folliculogenesis and infertility in humans.
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| 28. |
- Reddy, Pradeep, et al.
(författare)
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Formation of E-cadherin-mediated cell-cell adhesion activates AKT and mitogen activated protein kinase via phosphatidylinositol 3 kinase and ligand-independent activation of epidermal growth factor receptor in ovarian cancer cells
- 2005
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Ingår i: Mol Endocrinol. - : The Endocrine Society. - 0888-8809. ; 19:10, s. 2564-2578
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Tidskriftsartikel (refereegranskat)abstract
- E-cadherin is a well characterized adhesion molecule that plays a major role in epithelial cell adhesion. Based on findings that expression of E-cadherin is frequently lost in human epithelial cancers, it has been implicated as a tumor suppressor in carcinogenesis of most human epithelial cancers. However, in ovarian cancer development, our data from the current study showed that E-cadherin expression is uniquely elevated in 86.5% of benign, borderline, and malignant ovarian carcinomas irrespective of the degree of differentiation, whereas normal ovarian samples do not express E-cadherin. Thus, we hypothesize that E-cadherin may play a distinct role in the development of ovarian epithelial cancers. Using an E-cadherin-expressing ovarian cancer cell line OVCAR-3, we have demonstrated for the first time that the establishment of E-cadherin mediated cell-cell adhesions leads to the activation of Akt and MAPK. Akt activation is mediated through the activation of phosphatidylinositol 3 kinase, and both Akt and MAPK activation are mediated by an E-cadherin adhesion-induced ligand-independent activation of epidermal growth factor receptor. We have also demonstrated that suppression of E-cadherin function leads to retarded cell proliferation and reduced viability. We therefore suggest that the concurrent formation of E-cadherin adhesion and activation of downstream proliferation signals may enhance the proliferation and survival of ovarian cancer cells. Our data partly explain why E-cadherin is always expressed during ovarian tumor development and progression.
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| 29. |
- Reinbothe, Thomas, 1981, et al.
(författare)
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Glutaredoxin-1 mediates NADPH-dependent stimulation of calcium-dependent insulin secretion
- 2009
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Ingår i: Mol Endocrinol. - : The Endocrine Society. - 1944-9917 .- 0888-8809. ; 23:6, s. 893-900
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Tidskriftsartikel (refereegranskat)abstract
- Nicotinamide adenine dinucleotide phosphate (NADPH) enhances Ca(2+)-induced exocytosis in pancreatic beta-cells, an effect suggested to involve the cytosolic redox protein glutaredoxin-1 (GRX-1). We here detail the role of GRX-1 in NADPH-stimulated beta-cell exocytosis and glucose-stimulated insulin secretion. Silencing of GRX-1 by RNA interference reduced glucose-stimulated insulin secretion in both clonal INS-1 832/13 cells and primary rat islets. GRX-1 silencing did not affect cell viability or the intracellular redox environment, suggesting that GRX-1 regulates the exocytotic machinery by a local action. By contrast, knockdown of the related protein thioredoxin-1 (TRX-1) was ineffective. Confocal immunocytochemistry revealed that GRX-1 locates to the cell periphery, whereas TRX-1 expression is uniform. These data suggest that the distinct subcellular localizations of TRX-1 and GRX-1 result in differences in substrate specificities and actions on insulin secretion. Single-cell exocytosis was likewise suppressed by GRX-1 knockdown in both rat beta-cells and clonal 832/13 cells, whereas after overexpression exocytosis increased by approximately 40%. Intracellular addition of NADPH (0.1 mm) stimulated Ca(2+)-evoked exocytosis in both cell types. Interestingly, the stimulatory action of NADPH on the exocytotic machinery coincided with an approximately 30% inhibition in whole-cell Ca(2+) currents. After GRX-1 silencing, NADPH failed to amplify insulin release but still inhibited Ca(2+) currents in 832/13 cells. In conclusion, NADPH stimulates the exocytotic machinery in pancreatic beta-cells. This effect is mediated by the NADPH acceptor protein GRX-1 by a local redox reaction that accelerates beta-cell exocytosis and, in turn, insulin secretion.
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| 30. |
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| 31. |
- Robins, Tiina, et al.
(författare)
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Molecular Model of Human CYP21 Based onMammalian CYP2C5: Structural Features Correlatewith Clinical Severity of Mutations CausingCongenital Adrenal Hyperplasia
- 2006
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Ingår i: Molecular Endocrinology. - Stanford : The endocrin society. - 0888-8809 .- 1944-9917. ; 20:11, s. 2946-2964
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Tidskriftsartikel (refereegranskat)abstract
- Enhanced understanding of structure-function relationshipsof human 21-hydroxylase, CYP21, is requiredto better understand the molecular causesof congenital adrenal hyperplasia. To this end, astructural model of human CYP21 was calculatedbased on the crystal structure of rabbit CYP2C5.All but two known allelic variants of missense type,a total of 60 disease-causing mutations and sixnormal variants, were analyzed using this model. Astructural explanation for the corresponding phenotypewas found for all but two mutants for whichavailable clinical data are also discrepant with invitro enzyme activity. Calculations of protein stabilityof modeled mutants were found to correlateinversely with the corresponding clinical severity.Putative structurally important residues were identifiedto be involved in heme and substrate binding,redox partner interaction, and enzyme catalysisusing docking calculations and analysis of structurallydetermined homologous cytochrome P450s(CYPs). Functional and structural consequences ofseven novel mutations, V139E, C147R, R233G,T295N, L308F, R366C, and M473I, detected inScandinavian patients with suspected congenitaladrenal hyperplasia of different severity, were predictedusing molecular modeling. Structural featuresdeduced from the models are in good correlationwith clinical severity of CYP21 mutants,which shows the applicability of a modeling approachin assessment of new CYP21 mutations.
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| 32. |
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| 33. |
- Sandelin, A, et al.
(författare)
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Prediction of nuclear hormone receptor response elements
- 2005
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 19:3, s. 595-606
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Tidskriftsartikel (refereegranskat)abstract
- The nuclear receptor (NR) class of transcription factors controls critical regulatory events in key developmental processes, homeostasis maintenance, and medically important diseases and conditions. Identification of the members of a regulon controlled by a NR could provide an accelerated understanding of development and disease. New bioinformatics methods for the analysis of regulatory sequences are required to address the complex properties associated with known regulatory elements targeted by the receptors because the standard methods for binding site prediction fail to reflect the diverse target site configurations. We have constructed a flexible Hidden Markov Model framework capable of predicting NHR binding sites. The model allows for variable spacing and orientation of half-sites. In a genome-scale analysis enabled by the model, we show that NRs in Fugu rubripes have a significant cross-regulatory potential. The model is implemented in a web interface, freely available for academic researchers, available at http://mordor.cgb.ki.se/NHR-scan.
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| 34. |
- Shima, Yuichi, et al.
(författare)
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Pituitary Homeobox 2 Regulates Adrenal4 Binding Protein/Steroidogenic Factor-1 Gene Transcription in the Pituitary Gonadotrope through Interaction with the Intronic Enhancer
- 2008
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Ingår i: Molecular Endocrinology. - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 22:7, s. 1633-1646
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Tidskriftsartikel (refereegranskat)abstract
- Ad4BP/SF-1 (Adrenal4 Binding Protein/Steroidogenic Factor-1 (NR5A1)) is a factor important for animal reproduction and endocrine regulation, and its expression is tightly regulated in the gonad, adrenal gland, ventromedial hypothalamic nucleus (VMH), and pituitary gonadotrope. Despite its functional significance in the pituitary, the mechanisms underlying pituitary-specific expression of the gene remain to be uncovered. In this study, we demonstrate by transgenic mouse assays that the pituitary gonadotrope-specific enhancer is localized within the 6th intron of the gene. Functionally, the enhancer recapitulates endogenous Ad4BP/SF-1 expression in the fetal Rathke's pouch to the adult pituitary gonadotrope. Structurally, the enhancer consists of several elements conserved among animal species. Mutational analyses confirmed the significance of these elements for the enhancer function. One of these elements was able to interact both in vitro and in vivo with Pitx2, demonstrating that Pitx2 regulates Ad4BP/SF-1 gene transcription in the pituitary gonadotrope via interaction with the gonadotrope-specific enhancer.
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| 35. |
- Soriano, S, et al.
(författare)
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Rapid regulation of K(ATP) channel activity by 17{beta}-estradiol in pancreatic {beta}-cells involves the estrogen receptor {beta} and the atrial natriuretic peptide receptor
- 2009
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 1944-9917 .- 0888-8809. ; 23:12, s. 1973-1982
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Tidskriftsartikel (refereegranskat)abstract
- The ATP-sensitive potassium (KATP) channel is a key molecule involved in glucose-stimulated insulin secretion. The activity of this channel regulates β-cell membrane potential, glucose- induced [Ca2+]i signals, and insulin release. In this study, the rapid effect of physiological concentrations of 17β-estradiol (E2) on KATP channel activity was studied in intact β-cells by use of the patch-clamp technique. When cells from wild-type (WT) mice were used, 1 nm E2 rapidly reduced KATP channel activity by 60%. The action of E2 on KATP channel was not modified in β-cells from ERα−/− mice, yet it was significantly reduced in cells from ERβ−/− mice. The effect of E2 was mimicked by the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). Activation of ERβ by DPN enhanced glucose-induced Ca2+ signals and insulin release. Previous evidence indicated that the acute inhibitory effects of E2 on KATP channel activity involve cyclic GMP and cyclic GMP-dependent protein kinase. In this study, we used β-cells from mice with genetic ablation of the membrane guanylate cyclase A receptor for atrial natriuretic peptide (also called the atrial natriuretic peptide receptor) (GC-A KO mice) to demonstrate the involvement of this membrane receptor in the rapid E2 actions triggered in β-cells. E2 rapidly inhibited KATP channel activity and enhanced insulin release in islets from WT mice but not in islets from GC-A KO mice. In addition, DPN reduced KATP channel activity in β-cells from WT mice, but not in β-cells from GC-A KO mice. This work unveils a new role for ERβ as an insulinotropic molecule that may have important physiological and pharmacological implications.
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| 36. |
- Vidal, OM, et al.
(författare)
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In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver
- 2007
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Ingår i: Molecular endocrinology (Baltimore, Md.). - : The Endocrine Society. - 0888-8809 .- 1944-9917. ; 21:1, s. 293-311
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Tidskriftsartikel (refereegranskat)abstract
- The GH-activated signal transducer and activator of transcription 5b (STAT5b) is an essential regulator of somatic growth. The transcriptional response to STAT5b in liver is poorly understood. We have combined microarray-based expression profiling and phylogenetic analysis of gene regulatory regions to study the interplay between STAT5b and GH in the regulation of hepatic gene expression. The acute transcriptional response to GH in vivo after a single pulse of GH was studied in the liver of hypophysectomized rats in the presence of either constitutively active or a dominant-negative STAT5b delivered by adenoviral gene transfer. Genes showing differential expression in these two situations were analyzed for the presence of STAT5b binding sites in promoter and intronic regions that are phylogenetically conserved between rats and humans. Using this approach, we showed that most rapid transcriptional effects of GH in the liver are not results of direct actions of STAT5b. In addition, we identified novel STAT5b cis regulatory elements in genes such as Frizzled-4, epithelial membrane protein-1, and the suppressor of cytokine signaling 2 (SOCS2). Detailed analysis of SOCS2 promoter demonstrated its direct transcriptional regulation by STAT5b upon GH stimulation. A novel response element was identified within the first intron of the human SOCS2 gene composed of an E-box followed by tandem STAT5b binding sites, both of which are required for full GH responsiveness. In summary, we demonstrate the power of combining transcript profiling with phylogenetic sequence analysis to define novel regulatory paradigms.
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| 37. |
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| 38. |
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