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  • 1.
    Barbu, Andreea R
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Adenoviral-mediated transduction of human pancreatic islets: importance of adenoviral genome for cell viability and association with a deficient antiviral response2005In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 146, no 5, p. 2406-2414Article in journal (Refereed)
    Abstract [en]

    As adenoviral vectors are extensively used for genetic manipulation of insulin-producing cells in vitro, there is an increasing need to evaluate their effects on the function, morphology, and viability of transduced pancreatic islets. In the present study we observed that specific adenoviral genotypes, carrying E4 and E1/E3 deletions, correlate with differential induction of necrosis in pancreatic islet cells. In particular, the adenovirus death protein encoded from the E3 region of the adenoviral genome was able to modulate the changes induced in the morphology and viability of the transduced cells. We also propose a putative role for the transcriptional regulator pIX. Although human islet cells showed an increased resistance in terms of viral concentrations required for the induction of cell toxicity, our results showed that they were unable to build up an efficient antiviral response after transduction and that their survival was dependent on the exogenous addition of alpha-interferon. An intact and fully functional beta-cell is crucial for the successful application of gene therapy approaches in type 1 diabetes, and therefore, the implications of our findings need to be considered when designing vectors for gene transfer into pancreatic beta-cells.

  • 2.
    Berg, Håkan
    et al.
    Örebro University, School of Science and Technology. Marine Science Institute, University of Texas, Austin, United States .
    Rice, Charles
    Department of Biology, Clemson University, United States .
    Rahman, Saydur
    Marine Science Institute, University of Texas, Austin, United States .
    Dong, Jing
    Marine Science Institute, University of Texas, Austin, United States .
    Thomas, Peter
    Marine Science Institute, University of Texas, Austin, United States .
    Identification and characterization of membrane androgen receptors in the ZIP9 zinc transporter subfamily: I. Discovery in female Atlantic croaker and evidence ZIP9 mediates testosterone-induced apoptosis of ovarian follicle cells2014In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 155, no 11, p. 4237-4249Article in journal (Refereed)
    Abstract [en]

    Rapid, cell surface-initiated, pregenomic androgen actions have been described in various vertebrate cells, but the receptors mediating these actions remain unidentified.We report here cloning and expression of a cDNA from Atlantic croaker (Micropogonias undulatus) ovaries encoding a 33kDa, 7-transmembrane protein with binding and signaling characteristics of a membrane androgen receptor (mAR) that is unrelated to any previously described steroid receptor. Instead croaker mAR has 81–93 % amino acid sequence identity with zinc transporter ZIP9 (SLC39A9) subfamily members, indicating it is a ZIP9 protein. Croaker ZIP9 is expressed in gonadal tissues and in brain, and is upregulated in the ovary by reproductive hormones. ZIP9 protein is localized to plasma membranes of croaker granulosa cells and human breast cancer (SKBR-3) cells stably transfected with ZIP9. Recombinant croaker ZIP9 has a high affinity (Kd 12.7 nM), limited capacity (Bmax 2.8nM/mgprotein), displaceable, single binding site specific for androgens, characteristic of steroid receptors. Testosterone activates a stimulatory G protein coupled to ZIP9, resulting in increased cAMP production. Testosterone promotes serum starvation-induced cell death and apoptosis in transfected cells and in croaker ovarian follicle cells that is associated with rapid increases in intracellular free zinc concentrations, suggesting an involvement of zinc in this nonclassical androgen action to promote apoptosis. These responses to testosterone are abrogated by treatment with ZIP9 siRNA. The results provide the first evidence that zinc transporter proteins can function as specific steroid membrane receptors and indicate a previously unrecognized signaling pathway mediated by steroid receptors involving alterations in intracellular zinc.

  • 3.
    Bergman, Julia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Botling, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Fagerberg, Linn
    KTH Royal Inst Technol, Sci Life Lab, SE-17121 Stockholm, Sweden.
    Hallström, Björn M.
    KTH Royal Inst Technol, Sci Life Lab, SE-17121 Stockholm, Sweden.
    Djureinovic, Dijana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH Royal Inst Technol, Sci Life Lab, SE-17121 Stockholm, Sweden.
    Ponten, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The human adrenal gland proteome defined by transcriptomics and antibody-based profiling2017In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 158, no 2, p. 239-251Article in journal (Refereed)
    Abstract [en]

    The adrenal gland is a composite endocrine organ with vital functions that include the synthesis and release of glucocorticoids and catecholamines. To define the molecular landscape that underlies the specific functions of the adrenal gland, we combined a genome-wide transcriptomics approach based on mRNA sequencing of human tissues with immunohistochemistry-based protein profiling on tissue microarrays. Approximately two-thirds of all putative protein coding genes were expressed in the adrenal gland and the analysis identified 253 genes with an elevated pattern of expression in the adrenal gland, with only 37 genes showing a markedly higher expression level (>5-fold) in the adrenal gland compared to 31 other normal human tissue types analyzed. The analyses allowed for an assessment of the relative expression levels for well-known proteins involved in adrenal gland function, but also identified previously poorly characterized proteins in the adrenal cortex, such as FERM domain containing 5 (FRMD5) and protein NOV homolog (NOV). In summary, we provide a global analysis of the adrenal gland transcriptome and proteome, with a comprehensive list of genes with elevated expression in the adrenal gland and spatial information with examples of protein expression patterns for corresponding proteins. These genes and proteins constitute important starting points for an improved understanding of the normal function and pathophysiology of the adrenal glands.

  • 4.
    Bergman, Julia
    et al.
    Uppsala University.
    Botling, Johan
    Uppsala University.
    Fagerberg, Linn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    M Hallström, Björn
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Djureinovic, Dijana
    Uppsala University.
    Pontén, Fredrik
    Uppsala University.
    Mathias, Uhlén
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    The human adrenal gland proteome defined by transcriptomics and antibody-based profiling.2017In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 158, no 2, p. 239-251Article in journal (Refereed)
    Abstract [en]

    The adrenal gland is a composite endocrine organ with vital functions that include the synthesis and release of glucocorticoids and catecholamines. To define the molecular landscape that underlies the specific functions of the adrenal gland, we combined a genome-wide transcriptomics approach using messenger RNA sequencing of human tissues with immunohistochemistry-based protein profiling on tissue microarrays. Approximately two-thirds of all putative protein coding genes were expressed in the adrenal gland, and the analysis identified 253 genes with an elevated pattern of expression in the adrenal gland, with only 37 genes showing a markedly greater expression level (more than fivefold) in the adrenal gland compared with 31 other normal human tissue types analyzed. The analyses allowed for an assessment of the relative expression levels for well-known proteins involved in adrenal gland function but also identified previously poorly characterized proteins in the adrenal cortex, such as the FERM (4.1 protein, ezrin, radixin, moesin) domain containing 5 and the nephroblastoma overexpressed (NOV) protein homolog. We have provided a global analysis of the adrenal gland transcriptome and proteome, with a comprehensive list of genes with elevated expression in the adrenal gland and spatial information with examples of protein expression patterns for corresponding proteins. These genes and proteins constitute important starting points for an improved understanding of the normal function and pathophysiology of the adrenal glands.

  • 5. Bicsak, T A
    et al.
    Cajander, S B
    Peng, X R
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    LaPolt, P S
    Lu, J K
    Kristensen, P
    Tsafriri, A
    Hsueh, A J
    Tissue-type plasminogen activator in rat oocytes: expression during the periovulatory period, after fertilization, and during follicular atresia.1989In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 124, no 1, p. 187-94Article in journal (Refereed)
    Abstract [en]

    The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.

  • 6. Boucher, Marie-Josée
    et al.
    Simoneau, Mélanie
    Edlund, Helena
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    The homeodomain-interacting protein kinase 2 regulates insulin promoter factor-1/pancreatic duodenal homeobox-1 transcriptional activity2009In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 150, no 1, p. 87-97Article in journal (Refereed)
    Abstract [en]

    The homeodomain transcription factor insulin promoter factor (IPF)-1/pancreatic duodenal homeobox (PDX)-1 plays a crucial role in both pancreas development and maintenance of beta-cell function. Targeted disruption of the Ipf1/Pdx1 gene in beta-cells of mice leads to overt diabetes and reduced Ipf1/Pdx1 gene expression results in decreased insulin expression and secretion. In humans, mutations in the IPF1 gene have been linked to diabetes. Hence, the identification of molecular mechanisms regulating the transcriptional activity of this key transcription factor is of great interest. Herein we analyzed homeodomain-interacting protein kinase (Hipk) 2 expression in the embryonic and adult pancreas by in situ hybridization and RT-PCR. Moreover, we functionally characterized the role of HIPK2 in regulating IPF1/PDX1 transcriptional activity by performing transient transfection experiments and RNA interference. We show that Hipk2 is expressed in the developing pancreatic epithelium from embryonic d 12-15 but that the expression becomes preferentially confined to pancreatic endocrine cells at later developmental stages. Moreover, we show that HIPK2 positively influences IPF1/PDX1 transcriptional activity and that the kinase activity of HIPK2 is required for this effect. We also demonstrate that HIPK2 directly phosphorylates the C-terminal portion of IPF1/PDX1. Taken together, our data provide evidence for a new mechanism by which IPF1/PDX1 transcriptional activity, and thus possibly pancreas development and/or beta-cell function, is regulated.

  • 7.
    Bourghardt, Johan
    et al.
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.
    Bergström, Göran
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.
    Krettek, Alexandra
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.
    Sjöberg, Sara
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.
    Borén, Jan
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden.
    Tivesten, Åsa
    Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sahlgrenska University Hospital, Göteborg, Sweden / Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, Göteborg, Sweden.
    The endogenous estradiol metabolite 2-methoxyestradiol reduces atherosclerotic lesion formation in female apolipoprotein E-deficient mice2007In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 148, no 9, p. 4128-4132Article in journal (Refereed)
    Abstract [en]

    Estradiol, the major endogenous estrogen, reduces experimental atherosclerosis and metabolizes to 2-methoxyestradiol in vascular cells. Currently undergoing evaluation in clinical cancer trials, 2-methoxyestradiol potently inhibits cell proliferation independently of the classical estrogen receptors. This study examined whether 2-methoxyestradiol affects atherosclerosis development in female mice. Apolipoprotein E-deficient mice, a well-established mouse model of atherosclerosis, were ovariectomized and treated through slow-release pellets with placebo, 17beta-estradiol (6 microg/d), or 2-methoxyestradiol [6.66 microg/d (low-dose) or 66.6 microg/d (high-dose)]. After 90 d, body weight gain decreased and uterine weight increased in the high-dose but not low-dose 2-methoxyestradiol group. En face analysis showed that the fractional area of the aorta covered by atherosclerotic lesions decreased in the high-dose 2-methoxyestradiol (52%) but not in the low-dose 2-methoxyestradiol group. Total serum cholesterol levels decreased in the high- and low-dose 2-methoxyestradiol groups (19%, P < 0.05 and 21%, P = 0.062, respectively). Estradiol treatment reduced the fractional atherosclerotic lesion area (85%) and decreased cholesterol levels (42%). In conclusion, our study shows for the first time that 2-methoxyestradiol reduces atherosclerotic lesion formation in vivo. The antiatherogenic activity of an estradiol metabolite lacking estrogen receptor activating capacity may argue that trials on cardiovascular effects of hormone replacement therapy should use estradiol rather than other estrogens. Future research should define the role of 2-methoxyestradiol as a mediator of the antiatherosclerotic actions of estradiol. Furthermore, evaluation of the effects of 2-methoxyestradiol on cardiovascular disease endpoints in ongoing clinical trials is of great interest.

  • 8.
    Bourghardt, Johan
    et al.
    Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
    Wilhelmson, Anna S. K.
    Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
    Alexanderson, Camilla
    Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
    De Gendt, Karel
    Laboratory for Experimental Medicine and Endocrinology, Department of Experimental Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
    Verhoeven, Guido
    Laboratory for Experimental Medicine and Endocrinology, Department of Experimental Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.
    Krettek, Alexandra
    Nordic School of Public Health NHV, Gothenburg, Sweden.
    Ohlsson, Claes
    Center for Bone Research, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Tivesten, Åsa
    Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, University of Gothenburg, Gothenburg, Sweden.
    Androgen receptor-dependent and independent atheroprotection by testosterone in male mice2010In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 151, no 11, p. 5428-5437Article in journal (Refereed)
    Abstract [en]

    The atheroprotective effect of testosterone is thought to require aromatization of testosterone to estradiol, but no study has adequately addressed the role of the androgen receptor (AR), the major pathway for the physiological effects of testosterone. We used AR knockout (ARKO) mice on apolipoprotein E-deficient background to study the role of the AR in testosterone atheroprotection in male mice. Because ARKO mice are testosterone deficient, we sham operated or orchiectomized (Orx) the mice before puberty, and Orx mice were supplemented with placebo or a physiological testosterone dose. From 8 to 16 wk of age, the mice consumed a high-fat diet. In the aortic root, ARKO mice showed increased atherosclerotic lesion area (+80%, P < 0.05). Compared with placebo, testosterone reduced lesion area both in Orx wild-type (WT) mice (by 50%, P < 0.001) and ARKO mice (by 24%, P < 0.05). However, lesion area was larger in testosterone-supplemented ARKO compared with testosterone-supplemented WT mice (+57%, P < 0.05). In WT mice, testosterone reduced the presence of a necrotic core in the plaque (80% among placebo-treated vs. 12% among testosterone-treated mice; P < 0.05), whereas there was no significant effect in ARKO mice (P = 0.20). In conclusion, ARKO mice on apolipoprotein E-deficient background display accelerated atherosclerosis. Testosterone treatment reduced atherosclerosis in both WT and ARKO mice. However, the effect on lesion area and complexity was more pronounced in WT than in ARKO mice, and lesion area was larger in ARKO mice even after testosterone supplementation. These results are consistent with an AR-dependent as well as an AR-independent component of testosterone atheroprotection in male mice.

  • 9.
    Brolinson, Annelie
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Fourcade, S
    Jakobsson, A
    Pujol, A
    Jacobsson, A
    Steroid hormones control circadian Elovl3 expression in mouse liver2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 6, p. 3158-3166Article in journal (Refereed)
    Abstract [en]

    The Elovl3 gene belongs to the Elovl gene family, which encodes for enzymes involved in the elongation of very long chain fatty acids. The recognized role for the enzyme is to control the elongation of saturated and monounsaturated fatty acids up to 24 carbons in length. Elovl3 was originally identified as a highly expressed gene in brown adipose tissue on cold exposure. Here we show that hepatic Elovl3 mRNA expression follows a distinct diurnal rhythm exclusively in mature male mice, with a sharp increase early in the morning Zeitgeber time (ZT) 20, peaks around ZT2, and is back to basal level at the end of the light period at ZT10. In female mice and sexually immature male mice, the Elovl3 expression was constantly low. Fasting and refeeding mice with chow or high-fat diet did not alter the Elovl3 mRNA levels. However, animals that were exclusively fed during the day for 9 d displayed an inverted expression profile. In addition, we show that Elovl3 expression is transcriptionally controlled and significantly induced by the exposure of the synthetic glucocorticoid dexamethasone. Taken together, these data suggest that Elovl3 expression in mouse liver is under strict diurnal control by circulating steroid hormones such as glucocorticoids and androgens. Finally, Elovl3 expression was found to be elevated in peroxisomal transporter ATP-binding cassette, subfamily D(ALD), member 2 ablated mice and suppressed in ATP-binding cassette subfamily D(ALD) member 2 overexpressing mice, implying a tight cross talk between very long chain fatty acid synthesis and peroxisomal fatty acid oxidation

  • 10. Brönnegård, M.
    et al.
    Arner, P.
    Hellström, L.
    Akner, Gunnar
    Örebro University, School of Health and Medical Sciences.
    Gustafsson, J. A.
    Glucocorticoid receptor messenger ribonucleic acid in different regions of human adipose tissue1990In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 127, no 4, p. 1689-1696Article in journal (Refereed)
    Abstract [en]

    The expression of glucocorticoid receptor (GR) messenger RNA (mRNA) was investigated in sc adipose tissue and isolated adipocytes from the abdominal and gluteal regions in men and women using a human GR complementary RNA probe. GR mRNA levels were 2-fold higher in female than in male abdominal tissue or adipocytes, whereas in gluteal tissue or adipocytes no sex differences were observed. GR mRNA levels in female abdominal adipocytes were 50% higher than in corresponding female gluteal adipocytes; the opposite was observed corresponding in males. Northern blot analysis of total cellular RNA isolated from abdominal and gluteal adipocytes showed hybridization of the human GR probe to an RNA species of approximately 7.1 kilobases in both regions. No sex or regional differences in GR mRNA stability were observed. The human metallothionein II (hMTII) mRNA, which is regulated by glucocorticoids at the transcriptional level, showed an opposite sex and regional pattern as GR mRNA. However, in gluteal adipose tissue no sex differences were observed in hMTII mRNA levels. The expression of beta-actin mRNA, which is not regulated by glucocorticoids, showed no sex or regional variation. By immunocytochemistry, using an anti-GR-monoclonal antibody, cytoplasmic as well as nuclear staining for GR was demonstrated in both sexes and both regions. In conclusion, variations in GR mRNA levels between sexes and body regions may explain the well known sex and tissue differences in effects of glucocorticoids on human adipose tissue.

  • 11.
    Carlsson, Annika
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hallgren, Ing-Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Johansson, Hanna
    Sandler, Stellan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Concomitant Enzyme-Linked Immunosorbent Assay Measurements of Rat Insulin, Rat C-Peptide, and Rat Proinsulin from Rat Pancreatic Islets: Effects of Prolonged Exposure to Different Glucose Concentrations2010In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 151, no 10, p. 5048-5052Article in journal (Refereed)
    Abstract [en]

    Until now, there have been few assays to measure C-peptide and proinsulin in the rat. We used a well-established rat insulin ELISA and validated two novel ELISAs for rat C-peptide and rat/mouse proinsulin to examine secretion and content of insulin, proinsulin, and C-peptide from rat islets cultured for 72 h at different glucose concentrations in culture medium. To examine long-term effects in vitro rather than short-term effects of exposure to low, normal, and high glucose, the exposure time to the different glucose concentrations was set to 72 h. The measurement uncertainty of the values obtainable from the ELISAs was determined by calculation of the variation pattern from the intraassay variation generated by unknown samples, and repeatability was determined by analysis of controls. The precision study and the analysis of controls confirm that the validated ELISAs for rat C-peptide and proinsulin would be useful for further studies on the effects of preculture in different glucose concentrations. The higher the glucose concentration used during the 72-h culture period of rat islets, the higher insulin, C-peptide and proinsulin values were obtained in a subsequent short-term glucose-challenge experiment. The proportion of proinsulin to insulin secreted increased, as did islet content, with increasing glucose concentration during preculture. We also observed a nonequimolar, glucose-dependent secretion and content of rat insulin over rat C-peptide after culture at 11.1 and 28 mM glucose.

  • 12. Caruso, Carla
    et al.
    Durand, Daniela
    Schiöth, Helgi B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Rey, Rodolfo
    Seilicovich, Adriana
    Lasaga, Mercedes
    Activation of melanocortin 4 receptors reduces the inflammatory response and prevents apoptosis induced by lipopolysaccharide and interferon-gamma in astrocytes2007In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 148, no 10, p. 4918-4926Article in journal (Refereed)
    Abstract [en]

    Alpha-MSH exerts an immunomodulatory action in the brain and may play a neuroprotective role acting through melanocortin 4 receptors (MC4Rs). In the present study, we show that MC4Rs are constitutively expressed in astrocytes as determined by immunocytochemistry, RT-PCR, and Western blot analysis. alpha-MSH (5 microm) reduced the nitric oxide production and the expression of inducible nitric oxide synthase (iNOS) induced by bacterial lipopolysaccharide (LPS, 1 microg/ml) plus interferon-gamma (IFN-gamma, 50 ng/ml) in cultured astrocytes after 24 h. alpha-MSH also attenuated the stimulatory effect of LPS/IFN-gamma on prostaglandin E(2) release and cyclooxygenase-2 (COX-2) expression. Treatment with HS024, a selective MC4R antagonist, blocked the antiinflammatory effects of alpha-MSH, suggesting a MC4R-mediated mechanism in the action of this melanocortin. In astrocytes, LPS/IFN-gamma treatment reduced cell viability, increased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells and activated caspase-3. alpha-MSH prevented these apoptotic events, and this cytoprotective effect was abolished by HS024. LPS/IFN-gamma decreased Bcl-2, whereas it increased Bax protein expression in astrocytes, thus increasing the Bax/Bcl-2 ratio. Alpha-MSH produced a shift in Bax/Bcl-2 ratio toward astrocyte survival because it increased Bcl-2 expression and also prevented the effect of LPS/IFN-gamma on Bax and Bcl-2 expression. In summary, these findings suggest that alpha-MSH, through MC4R activation, attenuates LPS/IFN-gamma-induced inflammation by decreasing iNOS and COX-2 expression and prevents LPS/IFN-gamma-induced apoptosis of astrocytes by modulating the expression of proteins of the Bcl-2 family.

  • 13.
    Dahlfors, Gunilla
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Arnqvist, Hans J.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Vascular Endothelial Growth Factor and Transforming Growth Factor-β1 Regulate the Expression of Insulin-Like Growth Factor-Binding Protein-3, -4, and -5 in Large Vessel Endothelial Cells2000In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 141, no 6, p. 2062-2067Article in journal (Refereed)
    Abstract [en]

    We investigated the effect of diabetes-associated growth factors on the expression of insulin-like growth factor-I (IGF-I) and IGF-binding proteins (IGFBPs) in cultured endothelial cells from bovine aorta. Gene expression was measured by solution hybridization, and proteins were measured by enzyme-linked immunosorbent assay, RIA, or Western blot. The cells expressed messenger RNA (mRNA) for IGFBP-2 through -6 and IGFBP-2 through -5 proteins were detected in conditioned medium. Vascular endothelial growth factor inhibited IGFBP-3 mRNA (P < 0.01) and protein expression and increased IGFBP-5 mRNA (P < 0.001) and protein. Transforming growth factor-β1 inhibited IGFBP-3 (P < 0.01), IGFBP-4 (P < 0.01), and IGF-I mRNA expression, whereas at the protein level only IGFBP-3 was significantly decreased. IGF-I, insulin, or angiotensin II did not affect IGF-I or IGFBP mRNA expression. At the protein level, IGF-I clearly increased IGFBP-5 levels in conditioned medium. In conclusion, vascular endothelial growth factor and transforming growth factor-β1 regulate IGFBP expression in bovine aortic endothelial cells. These observations provide a new aspect of regulation for the IGF-system in macrovascular endothelium, with possible implications for subendothelial smooth muscle cells and development of diabetic angiopathy.

  • 14.
    Dallner, Olof S.
    et al.
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Chernogubova, Ekaterina
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Brolinson, Annelie
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    Bengtsson, T
    Stockholm University, Faculty of Science, The Wenner-Gren Institute .
    β3-adrenergic receptors stimulate glucose uptake in brown adipocytes by two mechanisms independently of GLUT4 translocation2006In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 147, no 12, p. 5730-5739Article in journal (Refereed)
    Abstract [en]

    To identify the mechanisms whereby norepinephrine induces glucose uptake in brown adipose tissue, we used mouse brown adipocytes in culture. Proliferating brown adipocytes had high levels of glucose transporter (GLUT) 1 mRNA and low levels of GLUT4 mRNA. The ratio of GLUT4/GLUT1 mRNA expression increased during differentiation, and mature brown adipocytes had high levels of GLUT4 mRNA. The endogenous adrenergic neurotransmitter norepinephrine induced a potent increase in GLUT1 mRNA and a decrease of GLUT4 mRNA in mature brown adipocytes. The norepinephrine effect was mimicked by isoprenaline and CL 316243 and was thus mediated by beta(3)-adrenergic receptors. The cAMP analog 8-bromoadenosine-cAMP partly mimicked the response on GLUT1 mRNA increase and fully mimicked the GLUT4 mRNA decrease. We found no involvement of alpha(1) or alpha(2)-adrenergic receptors on GLUT1 or GLUT4 mRNA transcription. Norepinephrine treatment led to a large increase of GLUT1 protein amount in brown adipocytes as visualized with immunocytochemical staining and subcellular fractionation. A large part of the newly synthesized GLUT1 was found in the plasma membrane (PM). The potent transcriptional inhibitor actinomycin D fully abolished this increase of GLUT1 protein at all time points examined. Norepinephrine treatment shifted GLUT4 from the PM to an intracellular vesicular compartment. Norepinephrine increased 2-deoxy-D-glucose uptake 2-fold at an early time point ( 1 h) and 4-fold at later time point ( 5 h). Addition of actinomycin D did not block the early phase but blocked a large part of the later phase of 2-deoxy-D-glucose uptake. These results imply that adrenergic stimulation through beta(3)-adrenergic receptors induces glucose uptake in brown adipocytes via two mechanisms: 1) a mechanism not dependent on GLUT1 and GLUT4 translocation, 2) a mechanism that is dependent on de novo synthesis of GLUT1 protein and increase of GLUT1 protein at the PM.

  • 15.
    Damdimopoulos, Anastasios E.
    et al.
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden.
    Spyrou, Giannis
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden / Foundation for Biomedical Research, Academy of Athens, Athens, Greece.
    Gustafsson, Jan-Åke
    Department of Biosciences and Nutrition at Novum, Karolinska Institutet, Huddinge, Stockholm, Sweden.
    Ligands differentially modify the nuclear mobility of estrogen receptors alpha and beta2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 1, p. 339-345Article in journal (Refereed)
    Abstract [en]

    Signaling of nuclear receptors depends on the structure of their ligands, with different ligands eliciting different responses. In this study using a comparative analysis, an array of ligands was examined for effects on estrogen receptor alpha (ERalpha) and ERbeta mobility. Our results indicated that these two receptors share similarities in response to some ligands but differ significantly in response to others. Our results suggest that for ERalpha, ligands can be classified into three distinct groups: 1) ligands that do not affect the mobility of the receptor, 2) ligands that cause a moderate effect, and 3) ligands that strongly impact mobility of ERalpha. Interestingly, we found that for ERbeta such a classification was not possible because ERbeta ligands caused a wider spectrum of responses. One of the main differences between the two receptors was the response toward the antiestrogens ICI and raloxifene, which was not attributable to differential subnuclear localization or different conformations of helix 12 in the C-terminal domain. We showed that both of these ligands caused a robust phenotype, leading to an almost total immobilization of ERalpha, whereas ERbeta retained its mobility; we provide evidence that the mobility of the two receptors depends upon the function of the proteasome machinery. This novel finding that ERbeta retains its mobility in the presence of antiestrogens could be important for its ability to regulate genes that do not contain classic estrogen response element sites and do not require DNA binding and could be used in the investigation of ligands that show ER subtype specificity.

  • 16. Derde, Sarah
    et al.
    Vanhorebeek, Ilse
    Guiza, Fabian
    Derese, Inge
    Gunst, Jan
    Fahrenkrog, Birthe
    Martinet, Wim
    Vervenne, Hilke
    Ververs, Eric-Jan
    Larsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Clinical Neurophysiology.
    Van den Berghe, Greet
    Early Parenteral Nutrition Evokes a Phenotype of Autophagy Deficiency in Liver and Skeletal Muscle of Critically Ill Rabbits2012In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 153, no 5, p. 2267-2276Article in journal (Refereed)
    Abstract [en]

    Muscular and hepatic abnormalities observed in artificially fed critically ill patients strikingly resemble the phenotype of autophagy-deficient mice. Autophagy is the only pathway to clear damaged organelles and large ubiquitinated proteins and aggregates. Fasting is its strongest physiological trigger. Severity of autophagy deficiency in critically ill patients correlated with the amount of infused amino acids. We hypothesized that impaired autophagy in critically ill patients could partly be evoked by early provision of parenteral nutrition enriched with amino acids in clinically used amounts. In a randomized laboratory investigation, we compared the effect of isocaloric moderate-dose iv feeding with fasting during illness on the previously studied markers of autophagy deficiency in skeletal muscle and liver. Critically ill rabbits were allocated to fasting or to iv nutrition (220 kcal/d, 921 kJ/d) supplemented with 50 kcal/d (209 kJ/d) of either glucose, amino acids, or lipids, while maintaining normoglycemia, and were compared with healthy controls. Fasted critically ill rabbits revealed weight loss and activation of autophagy. Feeding abolished these responses, with most impact of amino acid-enriched nutrition. Accumulation of p62 and ubiquitinated proteins in muscle and liver, indicative of insufficient autophagy, occurred with parenteral feeding enriched with amino acids and lipids. In liver, this was accompanied by fewer autophagosomes, fewer intact mitochondria, suppressed respiratory chain activity, and an increase in markers of liver damage. In muscle, early parenteral nutrition enriched with amino acids or lipids aggravated vacuolization of myofibers. In conclusion, early parenteral nutrition during critical illness evoked a phenotype of autophagy deficiency in liver and skeletal muscle.

  • 17. Engdahl, Cecilia
    et al.
    Lagerquist, Marie K
    Stubelius, Alexandra
    Andersson, Annica
    Studer, Erik
    Ohlsson, Claes
    Westberg, Lars
    Carlsten, Hans
    Forsblad-d'Elia, Helena
    Department of Rheumatology and Inflammation Research, Sahlgrenska Academy at University of Gothenburg.
    Role of androgen and estrogen receptors for the action of dehydroepiandrosterone (DHEA).2014In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 155, no 3, p. 889-96Article in journal (Refereed)
    Abstract [en]

    Dehydroepiandrosterone (DHEA) is an abundant steroid hormone, and its mechanism of action is yet to be determined. The aim of this study was to elucidate the importance of androgen receptors (ARs) and estrogen receptors (ERs) for DHEA function. Orchidectomized C57BL/6 mice were treated with DHEA, DHT, 17β-estradiol-3-benzoate (E2), or vehicle. Orchidectomized AR-deficient (ARKO) mice and wild-type (WT) littermates were treated with DHEA or vehicle for 2.5 weeks. At termination, bone mineral density (BMD) was evaluated, thymus and seminal vesicles were weighted, and submandibular glands (SMGs) were histologically examined. To evaluate the in vivo ER activation of the classical estrogen signaling pathway, estrogen response element reporter mice were treated with DHEA, DHT, E2, or vehicle, and a reporter gene was investigated in different sex steroid-sensitive organs after 24 hours. DHEA treatment increased trabecular BMD and thymic atrophy in both WT and ARKO mice. In WT mice, DHEA induced enlargement of glands in the SMGs, whereas this effect was absent in ARKO mice. Furthermore, DHEA was able to induce activation of classical estrogen signaling in bone, thymus, and seminal vesicles but not in the SMGs. In summary, the DHEA effects on trabecular BMD and thymus do not require signaling via AR and DHEA can activate the classical estrogen signaling in these organs. In contrast, DHEA induction of gland size in the SMGs is dependent on AR and does not involve classical estrogen signaling. Thus, both ERs and ARs are involved in mediating the effects of DHEA in an organ-dependent manner.

  • 18.
    Engström, Linda
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Rosén, Khadijah
    Angel, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Developmental Biology-IKE . Linköping University, Faculty of Health Sciences.
    Fyrberg, Anna
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology . Linköping University, Faculty of Health Sciences.
    Mackerlova, Ludmila
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Konsman, Jan Pieter
    Engblom, David
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Systemic immune challenge activates an intrinsically regulated local inflammatory circuit in the adrenal gland2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 4, p. 1436-1450Article in journal (Refereed)
    Abstract [en]

    There is evidence from in vitro studies that inflammatory messengers influence the release of stress hormone via direct effects on the adrenal gland; however, the mechanisms underlying these effects in the intact organism are unknown. Here we demonstrate that systemic inflammation in rats elicited by iv injection of lipopolysaccharide results in dynamic changes in the adrenal immune cell population, implying a rapid depletion of dendritic cells in the inner cortical layer and the recruitment of immature cells to the outer layers. These changes are accompanied by an induced production of IL-1β and IL-1 receptor type 1 as well as cyclooxygenase-2 and microsomal prostaglandin E synthase-1 in these cells, implying local cytokine-mediated prostaglandin E2 production in the adrenals, which also displayed prostaglandin E2 receptors of subtypes 1 and 3 in the cortex and medulla. The IL-1β expression was also induced by systemically administrated IL-1β and was in both cases attenuated by IL-1 receptor antagonist, consistent with an autocrine signaling loop. IL-1β similarly induced expression of cyclooxygenase-2, but the cyclooxygenase-2 expression was, in contrast, further enhanced by IL-1 receptor antagonist. These data demonstrate a mechanism by which systemic inflammatory agents activate an intrinsically regulated local signaling circuit that may influence the adrenals’ response to immune stress and may help explain the dissociation between plasma levels of ACTH and corticosteroids during chronic immune perturbations.

  • 19.
    Engström, Linda
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Ruud, Johan
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Eskilsson, Anna
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Larsson, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Health Sciences.
    Mackerlova, Ludmila
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Kugelberg, Unn
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Qian, Hong
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Vasilache, Ana Maria
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Larsson, Peter
    Linköping University, Department of Medical and Health Sciences, Radiation Physics. Linköping University, Faculty of Health Sciences.
    Engblom, David
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Sigvardsson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Jönsson, Jan-Ingvar
    Linköping University, Department of Clinical and Experimental Medicine, Experimental Hematology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Lipopolysaccharide-Induced Fever Depends on Prostaglandin E2 Production Specifically in Brain Endothelial Cells2012In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 153, no 10, p. 4849-4861Article in journal (Refereed)
    Abstract [en]

    Immune-induced prostaglandin E2 (PGE2) synthesis is critical for fever and other centrally elicited disease symptoms. The production of PGE2 depends on cyclooxygenase-2 and microsomal prostaglandin E synthase-1 (mPGES-1), but the identity of the cells involved has been a matter of controversy. We generated mice expressing mPGES-1 either in cells of hematopoietic or nonhematopoietic origin. Mice lacking mPGES-1 in hematopoietic cells displayed an intact febrile response to lipopolysaccharide, associated with elevated levels of PGE2 in the cerebrospinal fluid. In contrast, mice that expressed mPGES-1 only in hematopoietic cells, although displaying elevated PGE2 levels in plasma but not in the cerebrospinal fluid, showed no febrile response to lipopolysaccharide, thus pointing to the critical role of brain-derived PGE2 for fever. Immunohistochemical stainings showed that induced cyclooxygenase-2 expression in the brain exclusively occurred in endothelial cells, and quantitative PCR analysis on brain cells isolated by flow cytometry demonstrated that mPGES-1 is induced in endothelial cells and not in vascular wall macrophages. Similar analysis on liver cells showed induced expression in macrophages and not in endothelial cells, pointing at the distinct role for brain endothelial cells in PGE2 synthesis. These results identify the brain endothelial cells as the PGE2-producing cells critical for immune-induced fever.

  • 20.
    Eriksson, Jan W
    et al.
    Lundberg Laboratory for Diabetes Research, Department of Medicine, Sahlgrenska University Hospital, Goteborg, Sweden.
    Lönnroth, P
    Wesslau, C
    Smith, U
    Insulin promotes and cyclic adenosine 3',5'-monophosphate impairs functional insertion of insulin receptors in the plasma membrane of rat adipocytes: evidence for opposing effects of tyrosine and serine/threonine phosphorylation1997In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 138, no 2, p. 607-612Article in journal (Refereed)
    Abstract [en]

    The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes. [125I]insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin. Subsequently prepared PM displayed a ∼3-fold increase in bound [125I]insulin when cells had been pretreated with 6 nm insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptorα-subunit. The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin. In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of [125I]insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors. Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM. The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors. Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity. In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP. The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered.                  

    Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM. It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g. produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

  • 21. Flores-Morales, A.
    et al.
    Stahlberg, N.
    Tollet-Egnell, P.
    Lundeberg, Joakim
    KTH, Superseded Departments, Biotechnology.
    Malek, R. L.
    Quackenbush, J.
    Lee, N. H.
    Norstedt, G.
    Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat2001In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 142, no 7, p. 3163-3176Article in journal (Refereed)
    Abstract [en]

    Complementary DNA microarrays containing 3000 different rat genes were used to study the consequences of severe hormonal deficiency (hypophysectomy) on the gene expression patterns in heart, liver, and kidney. Hybridization signals were seen from a majority of the arrayed complementary DNAs; nonetheless, tissue-specific expression patterns could be delineated. Hypophysectomy affected the expression of genes involved in a variety of cellular functions. Between 16-29% of the detected transcripts from each tissue changed expression level as a reaction to this condition. Chronic treatment of hypophysectomized animals with human GH also caused significant changes in gene expression patterns. The study confirms previous knowledge concerning certain gene expression changes in the above-mentioned situations and provides new information regarding hypophysectomy and chronic human GH effects in the rat. Furthermore, we have identified several new genes that respond to GH treatment. Our results represent a first step toward a more global understanding of gene expression changes in states of hormonal deficiency.

  • 22.
    Franck-Lissbrant, I
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Häggström, S
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Urology and Andrology.
    Damber, J E
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Urology and Andrology.
    Bergh, A
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Testosterone stimulates angiogenesis and vascular regrowth in the ventral prostate in castrated adult rats.1998In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 139, no 2, p. 451-6Article in journal (Refereed)
    Abstract [en]

    The castration-induced regression and testosterone stimulated regrowth of the vasculature in the rat ventral prostate lobe were studied using stereological techniques. Seven days after castration, the endothelial cell proliferation rate (bromodeoxyuridine labeling index); the total weights of blood vessel walls, blood vessel lumina, endothelial cells, glandular epithelial cells; and total organ weight were all decreased. Within 2 days after sc treatment with testosterone, the total weights of blood vessel walls, endothelial cells, and vascular lumina, as well as the endothelial cell proliferation rate, were all normalized. In contrast to the rapid response of the vasculature, the total weight of glandular epithelium and total organ weight were not normalized during the 4 days of testosterone treatment. Growth of the vasculature apparently precedes growth of the glandular epithelium. The testosterone- dependent factors stimulating the vasculature are unknown, but factors derived from epithelial cells, mast cells (which accumulate in the prostate during the first day of testosterone treatment), and tissue macrophages could all be involved. Castration-induced regression and testosterone-stimulated regrowth of the prostatic vasculature can be used as an experimental model to study factors regulating angiogenesis and organ growth in the prostate.

  • 23.
    Fredriksson, Robert
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Hägglund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Olszewski, Pawel K
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Stephansson, Olga
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Jacobsson, Josefin A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Olszewska, Agnieszka M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Levine, Allen S
    Lindblom, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Schiöth, Helgi B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    The obesity gene, FTO, is of ancient origin, up-regulated during food deprivation and expressed in neurons of feeding-related nuclei of the brain2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 5, p. 2062-71Article in journal (Refereed)
    Abstract [en]

    Gene variants of the FTO (fatso) gene have recently been strongly associated with body mass index and obesity. The FTO gene is well conserved and found in a single copy in vertebrate species including fish and chicken, suggesting that the ancestor of this gene was present 450 million years ago. Surprisingly, the FTO gene is present in two species of algae but not in any other invertebrate species. This could indicate that this gene has undergone a horizontal gene transfer. Quantitative real-time PCR showed that the gene is expressed in many peripheral and central rat tissues. Detailed in situ hybridization analysis in the mouse brain showed abundant expression in feeding-related nuclei of the brainstem and hypothalamus, such as the nucleus of the solitary tract, area postrema, and arcuate, paraventricular, and supraoptic nuclei as well as in the bed nucleus of the stria terminalis. Colabeling showed that the FTO gene is predominantly expressed in neurons, whereas it was virtually not found in astrocytes or glia cells. The FTO was significantly up-regulated (41%) in the hypothalamus of rats after 48-h food deprivation. We also found a strong negative correlation of the FTO expression level with the expression of orexigenic galanin-like peptide, which is mainly synthesized in the arcuate nucleus. These results are consistent with the hypothesis that FTO could participate in the central control of energy homeostasis.

  • 24. Galway, A B
    et al.
    Oikawa, M
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hsueh, A J
    Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C.1989In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 125, no 1, p. 126-35Article in journal (Refereed)
    Abstract [en]

    Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.

  • 25. Gianoukakis, Andrew G.
    et al.
    Jennings, Timothy A.
    King, Chris S.
    Sheehan, Christine E.
    Hoa, Neil
    Heldin, Paraskevi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm , Ludwig Institute for Cancer Research.
    Smith, Terry J.
    Hyaluronan accumulation in thyroid tissue: evidence for contributions from epithelial cells and fibroblasts2007In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 148, no 1, p. 54-62Article in journal (Refereed)
    Abstract [en]

    Graves' disease (GD) and Hashimoto's thyroiditis (HT) are autoimmune processes often associated with hyperthyroidism and hypothyroidism, respectively. Despite their diverging clinical presentations, immune activation drives both diseases and results in connective tissue accumulation of the nonsulfated glycosaminoglycan, hyaluronan. The hydrophilic property of hyaluronan contributes to the pathogenesis of thyroid-associated ophthalmopathy, dermopathy and hypothyroid myxedema. Whether hyaluronan accumulates in the thyroid and plays a role in goiter formation in GD and HT remains unknown. We report here that levels of hyaluronan are increased in thyroid tissue from individuals with both diseases compared with glands uninvolved with autoimmune disorders. The transcript encoding hyaluronan synthase (HAS)-3, one of three mammalian HAS isoforms, was detected in thyroid tissue. Isolated thyrocytes in primary culture express all three HAS isoforms when treated with IL-1beta. Thyrocytes and thyroid fibroblasts produce hyaluronan under basal culture conditions and IL-1beta enhances levels of this molecule in both cell types. On a per-cell basis, fibroblasts produce more hyaluronan than do thyrocytes under basal conditions and after cytokine treatment. Synthesis in thyrocytes can also be altered by increasing serum concentration in the medium and by modifying culture density. Our findings suggest that hyaluronan accumulation in thyroid tissue might derive from thyrocytes and fibroblasts. Moreover, this glycosaminoglycan becomes more abundant as a consequence of autoimmune disease. It may therefore contribute to increased thyroid volume in GD and HT. Coupled with the newly identified influence exerted by hyaluronan on immunocompetent cells, our findings represent potentially important insights into the pathogenesis of autoimmune thyroid diseases.

  • 26. Gougeon, Alain
    et al.
    Delangle, Aurélien
    Arouche, Nassim
    Stridsberg, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Gotteland, Jean Pierre
    Loumaye, Ernest
    Kit ligand and the somatostatin receptor antagonist, BIM-23627, stimulate in vitro resting follicle growth in the neonatal mouse ovary2010In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 151, no 3, p. 1299-1309Article in journal (Refereed)
    Abstract [en]

    In the mammalian ovary, kit ligand (KL), coded by a cAMP-stimulatable gene, is a protein that promotes initiation of follicle growth. The neuropeptide somatostatin (SST) is a small peptide that inhibits cAMP generation in many cell types. Consequently, SST receptor agonists might alter KL production and subsequent follicle growth. The present study was undertaken to look for the existence of a functional SST system in the mouse ovary, to test the effects of the SST receptor 2 (SSTR-2) antagonist BIM-23627 on in vitro folliculogenesis, and to compare them with those of KL, which was demonstrated to stimulate follicle growth in the neonatal rat ovary. Pairs of ovaries from 5-d-old mice were incubated in vitro during 15 d in the presence of either KL or BIM-23627. For every mouse, one ovary was cultured in culture medium (control), and the other ovary was cultured in the presence of either KL or BIM-23627. After 5, 10, and 15 d culture, the ovaries were histologically assessed for the content of primordial, primary, and secondary follicles. The SSTR-2 and -5, but not SST, were identified at the transcriptional and translational (mainly in granulosa cells) levels. Both KL and BIM-23627 triggered a reduction of the percentages of primordial follicles and an increase of the percentages of primary and secondary follicles when compared with control ovaries from the same animal. In conclusion, extraovarian SST, acting through its receptors 2 and 5 present on granulosa cells, may be involved in mouse folliculogenesis by reducing recruitment of resting follicles.

  • 27.
    Hellman, Bo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Salehi, Albert
    Lunds universitet.
    Gylfe, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dansk, Heléne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Grapengiesser, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Glucose generates coincident insulin and somatostatin pulses and anti-synchronous glucagon pulses from human pancreatic islets2009In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 150, no 12, p. 5334-5340Article in journal (Refereed)
    Abstract [en]

    The kinetics of insulin, glucagon and somatostatin release was studied   in human pancreatic islets. Batches of 10-15 islets were perifused and   the hormones measured with RIA in 30-sec fractions. Increase of glucose   from 3 to 20 mM resulted in a brief pulse of glucagon coinciding with   suppression of basal insulin and somatostatin release. There was a   subsequent drop of glucagon release concomitant with the appearance of   a pronounced pulse of insulin and a slightly delayed pulse of   somatostatin. Continued exposure to 20 mM glucose generated pulsatile   release of the three hormones with 7- to 8-min periods accounting for   60-70% of the secreted amounts. Glucose caused pronounced stimulation   of average insulin and somatostatin release. However, the nadirs   between the glucagon pulses were lower than the secretion at 3 mM   glucose, resulting in 18% suppression of average release. The   repetitive glucagon pulses were antisynchronous to coincident pulses of   insulin and somatostatin. The resulting greater than 20-fold variations   of the insulin to glucagon ratio might be essential for   minute-to-minute regulation of the hepatic glucose production.

  • 28.
    Hogh, K-Lynn N.
    et al.
    Northern Medical Program, University of Northern British Columbia, Prince George BC, Canada.
    Craig, Michael N.
    Northern Medical Program, University of Northern British Columbia, Prince George BC, Canada.
    Uy, Christopher E.
    Northern Medical Program, University of Northern British Columbia, Prince George BC, Canada.
    Nygren, Heli
    VTT Technical Research Centre of Finland, Espoo, Finland; Steno Diabetes Center A/S, Gentofte, Denmark.
    Asadi, Ali
    Department of Cellular and Physiological Sciences and Faculty of Medicine, University of British Columbia, Vancouver BC, Canada.
    Speck, Madeline
    Child and Family Research Institute, Vancouver BC, Canada.
    Fraser, Jordie D.
    Rudecki, Alexander P.
    Northern Medical Program, University of Northern British Columbia, Prince George BC, Canada.
    Baker, Robert K.
    Department of Cellular and Physiological Sciences and Faculty of Medicine, University of British Columbia, Vancouver BC, Canada.
    Oresic, Matej
    Örebro University, School of Medical Sciences. VTT Technical Research Centre of Finland, Espoo, Finland; Steno Diabetes Center A/S, Gentofte, Denmark.
    Gray, Sarah L.
    Northern Medical Program, University of Northern British Columbia, Prince George BC, Canada.
    Overexpression of PPARγ specifically in pancreatic β-cells exacerbates obesity-induced glucose intolerance, reduces β-cell mass, and alters islet lipid metabolism in male mice2014In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 155, no 10, p. 3843-3852Article in journal (Refereed)
    Abstract [en]

    The contribution of peroxisomal proliferator-activated receptor (PPAR)-γ agonism in pancreatic β-cells to the antidiabetic actions of thiazolidinediones has not been clearly elucidated. Genetic models of pancreatic β-cell PPARγ ablation have revealed a potential role for PPARγ in β-cell expansion in obesity but a limited role in normal β-cell physiology. Here we overexpressed PPARγ1 or PPARγ2 specifically in pancreatic β-cells of mice subjected to high-fat feeding using an associated adenovirus (β-PPARγ1-HFD and β-PPARγ2-HFD mice). We show β-cell-specific PPARγ1 or PPARγ2 overexpression in diet-induced obese mice exacerbated obesity-induced glucose intolerance with decreased β-cell mass, increased islet cell apoptosis, and decreased plasma insulin compared with obese control mice (β-eGFP-HFD mice). Analysis of islet lipid composition in β-PPARγ2-HFD mice revealed no significant changes in islet triglyceride content and an increase in only one of eight ceramide species measured. Interestingly β-PPARγ2-HFD islets had significantly lower levels of lysophosphatidylcholines, lipid species shown to enhance insulin secretion in β-cells. Gene expression profiling revealed increased expression of uncoupling protein 2 and genes involved in fatty acid transport and β-oxidation. In summary, transgenic overexpression of PPARγ in β-cells in diet-induced obesity negatively impacts whole-animal carbohydrate metabolism associated with altered islet lipid content, increased expression of β-oxidative genes, and reduced β-cell mass.

  • 29. Hsueh, A J
    et al.
    Liu, Y X
    Cajander, S
    Peng, X R
    Dahl, K
    Kristensen, P
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization.1988In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 122, no 4, p. 1486-95Article in journal (Refereed)
    Abstract [en]

    GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.

  • 30. Hägglund, A C
    et al.
    Ny, A
    Leonardsson, G
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Regulation and localization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse ovary during gonadotropin-induced ovulation.1999In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 140, no 9, p. 4351-8Article in journal (Refereed)
    Abstract [en]

    At the time of ovulation, proteolytic degradation of the follicular wall is required to release the mature oocyte. Extracellular proteases, such as serine proteases and matrix metalloproteinases (MMPs), are thought to play important roles in this process. In this study we have examined the regulation of 11 MMPs and 3 tissue inhibitors of metalloproteinases (TIMPs) during gonadotropin-induced ovulation in the mouse. Northern blot hybridization showed that messenger RNA for several MMPs and TIMPs, including gelatinase A, MT1-MMP, stromelysin-3, MMP-19, TIMP-1, TIMP-2, and TIMP-3, were present at detectable levels in the mouse ovary. In addition, ovarian extracts contained gelatinolytic activities corresponding to the inactive proforms of gelatinase A and gelatinase B. Most of the MMPs and TIMPs were expressed at a constitutive level throughout the periovulatory period. However, MMP-19 and TIMP-1 revealed a different expression pattern; they were both induced 5-10 times by hCG and reached their maximum levels at 12 h after hCG treatment, corresponding to the time of ovulation. At this time point, MMP-19 and TIMP-1 messenger RNA were localized to the granulosa and thecal-interstitial cells of large preovulatory and ovulating follicles. This temporal and spatial regulation pattern suggests that MMP-19 might be involved in the tissue degradation that occurs during follicular rupture and that TIMP-1 could have a role in terminating MMP activity after ovulation.

  • 31. Hägglund, A C
    et al.
    Ny, A
    Liu, K
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Coordinated and cell-specific induction of both physiological plasminogen activators creates functionally redundant mechanisms for plasmin formation during ovulation.1996In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 137, no 12, p. 5671-7Article in journal (Refereed)
    Abstract [en]

    Several lines of indirect evidence indicate that plasmin-mediated proteolysis plays a role in the breakdown of the follicle wall during ovulation. Consistent with this, the ovulation efficiency of mice lacking the two known physiological plasminogen activators (PAs), tissue-type PA (tPA) and urokinase-type PA (uPA), is reduced by 26%. Surprisingly, mice with a single deficiency of either tPA or uPA gene function were normal in their capacity to ovulate. In this study we used in situ hybridization and casein in situ zymography to localize the expression of messenger RNAs (mRNAs) encoding PAs and PA inhibitors and to examine the net PA activity in the mouse ovary at the time of ovulation. Although uPA mRNA expressed by granulosa cells is the most abundant and dramatically up-regulated PA before ovulation, a previously unnoticed coordinated induction oftPA mRNA was found in thecal-interstitial tissue. The existence of redundant mechanisms for plasmin production in the ovary may be the cause of the normal ovulation efficiency in single deficient mice lacking tPA or uPA. The expression of mRNAs for PA inhibitors, types 1 and 2, was low in the ovary, with minor inductions at restricted time points. In contrast, expression of protease nexin-1 (PN-1) by granulosa cells was high during the entire periovulatory period. Among subpopulations of granulosa cells, the expression of PN-1 and uPA was heterogeneous and complementary. Cumulus cells expressed high levels of PN-1 mRNA and low levels of uPA mRNA, thereby providing an inhibitory activity that may protect the mucified matrix of the cumulus oocyte complex from proteolytic degradation.

  • 32.
    Jee, Youn Hee
    et al.
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD, United States.
    Wang, Jinhee
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD, United States.
    Yue, Shanna
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD, United States.
    Jennings, Melissa
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, United States.
    Clokie, Samuel J. H.
    Birmingham Women's and Children's NHS Foundation Trust, Birmingham, United Kingdom.
    Nilsson, Ola
    Örebro University, School of Medical Sciences. Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden.
    Lui, Julian
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD, United States.
    Baron, Jeffrey
    Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda MD, United States.
    Mir-374-5p, mir-379-5p, and mir-503-5p regulate proliferation and hypertrophic differentiation of growth plate chondrocytes in male rats2018In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 159, no 3, p. 1469-1478Article in journal (Refereed)
    Abstract [en]

    Growth plate chondrocytes undergo sequential differentiation to form the resting (RZ), proliferative (PZ), and hypertrophic zones (HZ). The important role of microRNAs (miRNAs) in the growth plate was previously revealed by cartilage-specific ablation of Dicer, an enzyme essential for biogenesis of many miRNAs. To identify specific miRNAs that regulate differentiation of PZ chondrocytes to HZ chondrocytes, we microdissected individual growth plate zones from juvenile rats and performed miRNA profiling using a solution hybridization method and also miRNA-seq. Thirty-four miRNAs were differentially expressed between PZ and HZ and we hypothesized that some of the miRNAs that are preferentially expressed in PZ may serve to promote proliferation and inhibit hypertrophic differentiation. Consistent with this hypothesis, transfection of inhibitors for four of these miRNAs (mir-369-3p, mir-374-5p, mir-379-5p, mir-503-5p) decreased proliferation in primary epiphyseal chondrocytes. The inhibitors for three of these miRNAs (mir-374-5p, mir-379-5p, mir-503-5p) also increased expression of multiple genes that are associated with chondrocyte hypertrophic differentiation. We next hypothesized that preferential expression of these miRNAs in PZ is driven by the PTHrP concentration gradient across the growth plate. Consistent with this hypothesis, treatment of primary chondrocytes with a PTH/PTHrP receptor agonist, PTH1-34, increased expression of mir-374-5p, mir-379-5p, and mir-503-5p. Taken together, our findings suggest that the PTHrP concentration gradient across the growth plate induces differential expression of mir-374-5p, mir-379-5p and mir-503-5p between PZ and HZ. In PZ, the higher expression levels of these miRNAs promote proliferation and inhibit hypertrophic differentiation. In HZ, downregulation of these miRNAs inhibits proliferation and promotes hypertrophic differentiation.

  • 33.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Mattsson, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Andersson, Arne
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jansson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Islet endothelial cells and pancreatic beta-cell proliferation: studies in vitro and during pregnancy in adult rats2006In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 147, no 5, p. 2315-2324Article in journal (Refereed)
    Abstract [en]

    The growth of both tumors and nonneoplastic tissues may be influenced by signals from the vascular endothelium. In the present investigation we show that purified proliferating endothelial cells from pancreatic islets can stimulate beta-cell proliferation through secretion of hepatocyte growth factor (HGF). This secretion could be induced by soluble signals from the islets, such as vascular endothelial growth factor-A (VEGF-A) and insulin. During pregnancy, the pancreatic beta-cells display a highly reproducible physiological proliferation. We show that islet endothelial cell proliferation precedes beta-cell proliferation in pregnant animals. Vascular growth was closely associated with endocrine cell proliferation, and prominent expression of HGF was observed in islet endothelium on d 15 of pregnancy, i.e. coinciding with the peak of beta-cell proliferation. In summary, our results suggest the existence of an endothelial-endocrine axis within adult pancreatic islets, which is of importance for adult beta-cell proliferation.

  • 34.
    Johansson, Magnus
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Olerud, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Jansson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Carlsson, Per-Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Internal Medicine.
    Prolactin treatment improves engraftment and function of transplanted pancreatic islets2009In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 150, no 4, p. 1646-1653Article in journal (Refereed)
    Abstract [en]

    Transplantation of pancreatic islets is clinically used to treat type 1 diabetes, but requires multiple donors. Previous experimental studies have demonstrated that transplanted islets have a low blood vessel density, which leads to a hypoxic microenvironment. The present study tested the hypothesis that experimental prolactin pretreatment, a substance which seems to stimulate angiogenesis in endogenous islets, would increase graft blood vessel density, thereby improving transplantation outcome. Pancreatic islets from C57BL/6 mice were incubated with prolactin (500 ng/ml) or vehicle during the last 24 h of culture before syngeneic transplantation beneath the renal capsule, or recipients were injected with prolactin or vehicle for the first 7 days after transplantation. One month post-transplantation, graft vascular density, blood flow, oxygen tension, endocrine volume and function was evaluated. Also human islets were incubated with prolactin or vehicle before experimental transplantation and investigated for vascular engraftment. Vascular engraftment of syngeneically transplanted mouse islets was improved by both in vivo and in vitro prolactin pretreatment. Moreover, prolactin pretreatment in vitro of islets used for transplantation improved recovery from diabetes in a minimal islet mass model. Interestingly, also human islets subjected to prolactin treatment before experimental transplantation demonstrated improved revascularization, blood perfusion and oxygen tension when evaluated one month post-transplantation. We conclude that prolactin may improve engraftment of transplanted pancreatic islets. The protocol with pretreatment of islets ex vivo could minimize the risk of side effects when used in the clinical setting.

  • 35.
    Kawai, Mayumi
    et al.
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Naruse, Mitsuhide
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Yoshimoto, Takanobu
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Naruse, Kiyoko
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Shionoya, Kiseko
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Tanaka, Masami
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Morishita, Yoshikazu
    Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. Tokyo 194, Japan.
    Matsuda, Yuzuru
    Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd. Tokyo 194, Japan.
    Demura, Reiko
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    Demura, Hiroshi
    Department of Medicine, Znstitute of Clinical Endocrinology, Tokyo Women’s Medical College, Tokyo 162, Japan.
    C-type natriuretic peptide as a possible local modulator of aldosterone secretion in bovine adrenal zona glomerulosa1996In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 137, no 1, p. 42-46Article in journal (Refereed)
    Abstract [en]

    Although atrial and brain natriuretic peptides are well known to be involved in the regulation of cardiovascular and endocrine functions as circulating hormones, the roles of the C-type natriuretic peptide (CNP) remain unknown. We examined the effects of CNP on the secretion of aldosterone and cyclic nucleotides from bovine adrenal zona glomerulosa cells in culture. CNP produced a dose-dependent increase in the basal secretion of cGMP, with an EC50 of 3.8 x 10(-10)M. CNP significantly inhibited the ACTH-induced increase in aldosterone and cAMP in a dose-related manner, with an IC50 of 3.6 x 10(-10)M. Although ACTH itself did not increase cGMP secretion, the addition of CNP elicited a significant increase in cGMP secretion. The effects of CNP on the basal secretion of cGMP and the ACTH-induced secretion of aldosterone were significantly reversed by a nonpeptide natriuretic peptide receptor antagonist, HS-142-1. CNP immunoreactivity was localized in the zona glomerulosa by immunohistochemical staining. In addition, expression of CNP messenger RNA and natriuretic peptide B receptor messenger RNA was demonstrated by RT-PCR in the zona glomerulosa tissue and cells in culture. These findings suggest that CNP is a local factor regulating ACTH-induced aldosterone secretion through a guanylyl cyclase-cGMP pathway.

  • 36.
    Kristinsson, Hjalti
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Smith, David M.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sargsyan, Ernest
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    FFAR1 Is Involved in Both the Acute and Chronic Effects of Palmitate on Insulin Secretion2013In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 154, no 11, p. 4078-4088Article in journal (Refereed)
    Abstract [en]

    Free fatty acids (FFAs) have pleiotropic effects on the pancreatic beta-cell. Although acute exposure to FFAs stimulates glucose-stimulated insulin secretion (GSIS), prolonged exposure impairs GSIS and causes apoptosis. FFAs exert their effects both via intracellular metabolism and interaction with the FFA receptor 1 (FFAR1/GPR40). Here we studied the role of FFAR1 in acute and long-term effects of palmitate on GSIS and insulin content in isolated human islets by using the FFAR1 agonist TAK-875 and the antagonist ANT203. Acute palmitate exposure potentiated GSIS approximately 3-fold, whereas addition of the antagonist decreased this potentiation to approximately 2-fold. In the absence of palmitate, the agonist caused a 40% increase in GSIS. Treatment with palmitate for 7 days decreased GSIS to 70% and insulin content to 25% of control level. These negative effects of long-term exposure to palmitate were ameliorated by FFAR1 inhibition and further aggravated by additional stimulation of the receptor. In the absence of extracellularly applied palmitate, long-term treatment with the agonist caused a modest increase in GSIS. The protective effect of FFAR1 inhibition was verified by using FFAR1-deficient MIN6 cells. Improved beta-cell function by the antagonist was paralleled by the decreased apoptosis and lowered oxidation of palmitate, which may represent the potential mechanisms of protection. We conclude that FFAR1 in the pancreatic beta-cell plays a substantial role not only in acute potentiation of GSIS by palmitate but also in the negative long-term effects of palmitate on GSIS and insulin content.

  • 37. LaPolt, P S
    et al.
    Yamoto, M
    Veljkovic, M
    Sincich, C
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Tsafriri, A
    Hsueh, A J
    Basic fibroblast growth factor induction of granulosa cell tissue-type plasminogen activator expression and oocyte maturation: potential role as a paracrine ovarian hormone.1990In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 127, no 5, p. 2357-63Article in journal (Refereed)
    Abstract [en]

    Gonadotropin-induced ovulation is associated with oocyte maturation and preovulatory increases of tissue plasminogen activator (tPA) expression. Basic fibroblast growth factor (bFGF), an angiogenic factor found in many organs including the ovary, modulates steroidogenesis in granulosa cells and increases PA activity in endothelial cells. Here studies were performed to examine the possible roles of bFGF as an intragonadal regulator of tPA expression and oocyte maturation. In cultured granulosa cells, bFGF caused a time-dependent (onset at 24 h) and dose-dependent (ED50 = 0.6 nM) increase (up to 5-fold) in tPA enzyme activity as measured by the fibrin overlay technique. Northern blot hybridization also revealed that treatment of cells with bFGF (2 nM) increased the level of the 22S tPA messenger RNA. Slot blot analysis indicated that the effects of bFGF were time dependent and dose dependent; tPA message levels increase before tPA activity levels. bFGF (0.6 nM) also significantly increased granulosa cell cAMP production in both the absence and presence of a phosphodiesterase inhibitor. In follicle-enclosed oocytes incubated for 24 h in media with or without increasing concentrations of LH or bFGF, germinal vesicle breakdown was observed in only 1.6% of controls, but 85% of LH (1 microgram/ml)-treated oocytes underwent maturation. Likewise, bFGF induced germinal vesicle breakdown (10-80%) over a dose range of 0.6 to 333 nM. In the same follicles, bFGF, like LH, also stimulated prostaglandin E production. These results, coupled with the identification of bFGF in growing follicles, suggest that bFGF acts as an intraovarian inducer of granulosa cell tPA gene expression and oocyte maturation.

  • 38.
    Larsson, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Marsell, Richard
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Schipani, Ernestina
    Ohlsson, Claes
    Ljunggren, Östen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrinology and mineral metabolism.
    Tenenhouse, Harriet S
    Juppner, Harald
    Jonsson, Kenneth B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Orthopaedics.
    Transgenic mice expressing Fibroblast Growth Factor-23 under the control of the α1 (I) collagen promoter exhibit growth retardation, Osteomalacia and disturbed phosphate homeostasis2004In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 145, no 7, p. 3087-3094Article in journal (Refereed)
    Abstract [en]

    Mutations in the fibroblast growth factor 23 gene, FGF23, cause autosomal dominant hypophosphatemic rickets (ADHR). The gene product, FGF-23, is produced by tumors from patients with oncogenic osteomalacia (OOM), circulates at increased levels in most patients with X-linked hypophosphatemia (XLH) and is phosphaturic when injected into rats or mice, suggesting involvement in the regulation of phosphate (Pi) homeostasis. To better define the precise role of FGF-23 in maintaining Pi balance and bone mineralization, we generated transgenic mice that express wild-type human FGF-23, under the control of the alpha1(I) collagen promoter, in cells of the osteoblastic lineage. At 8 wk of age, transgenic mice were smaller (body weight = 17.5 +/- 0.57 vs. 24.3 +/- 0.37 g), exhibited decreased serum Pi concentrations (1.91 +/- 0.27 vs. 2.75 +/- 0.22 mmol/liter) and increased urinary Pi excretion when compared with wild-type littermates. The serum concentrations of human FGF-23 (undetectable in wild-type mice) was markedly elevated in transgenic mice (>7800 reference units/ml). Serum PTH levels were increased in transgenic mice (231 +/- 62 vs. 139 +/- 44 pg/ml), whereas differences in calcium and 1,25-dihydroxyvitamin D were not apparent. Expression of Npt2a, the major renal Na(+)/Pi cotransporter, as well as Npt1 and Npt2c mRNAs, was significantly decreased in the kidneys of transgenic mice. Histology of tibiae displayed a disorganized and widened growth plate and peripheral quantitative computerized tomography analysis revealed reduced bone mineral density in transgenic mice. The data indicate that FGF-23 induces phenotypic changes in mice resembling those of patients with ADHR, OOM, and XLH and that FGF-23 is an important determinant of Pi homeostasis and bone mineralization.

  • 39. Leblanc, Samuel
    et al.
    Battista, Marie-Claude
    Noll, Christophe
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Gallo-Payet, Nicole
    Carpentier, Andre C.
    Vine, Donna F.
    Baillargeon, Jean-Patrice
    Angiotensin II Type 2 Receptor Stimulation Improves Fatty Acid Ovarian Uptake and Hyperandrogenemia in an Obese Rat Model of Polycystic Ovary Syndrome2014In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 155, no 9, p. 3684-3693Article in journal (Refereed)
    Abstract [en]

    Polycystic ovary syndrome (PCOS) is mainly defined by hyperandrogenism but is also characterized by insulin resistance (IR). Studies showed that overexposure of nonadipose tissues to nonesterified fatty acids (NEFA) may explain both IR and hyperandrogenism. Recent studies indicate that treatment with an angiotensin II type 2 receptor (AT2R)-selective agonist improves diet-induced IR. We thus hypothesized that PCOS hyperandrogenism is triggered by ovarian NEFA overexposure and is improved after treatment with an AT2R agonist. Experiments were conducted in 12-week-old female JCR:LA-cp/cp rats, which are characterized by visceral obesity, IR, hyperandrogenism, and polycystic ovaries. Control JCR:LA +/? rats have a normal phenotype. Rats were treated for 8 days with saline or the selective AT2R agonist C21/M24 and then assessed for: 1) fasting testosterone, NEFA, and insulin levels; and 2) an iv 14(R,S)-[F-18]fluoro-6-thia-heptadecanoic acid test to determine NEFA ovarian tissue uptake (Km). Compared with controls, saline-treated PCOS/cp rats displayed higher insulin (100 vs 5.6 mu U/mL), testosterone (0.12 vs 0.04 nmol/L), NEFA (0.98 vs 0.48 mmol/L), and Km (20.7 vs 12.9 nmol/g.min) (all P < .0001). In PCOS/cp rats, C21/M24 did not significantly improve insulin or NEFA but normalized testosterone (P = .004) and Km(P = .009), which were strongly correlated together in all PCOS/cp rats (rho = 0.74, P = .009). In conclusion, in an obese PCOS rat model, ovarian NEFA uptake and testosterone levels are strongly associated and are both significantly reduced after short-term C21/M24 therapy. These findings provide new information on the role of NEFA in PCOS hyperandrogenemia and suggest a potential role for AT2R agonists in the treatment of PCOS.

  • 40.
    Lejonklou, Margareta H
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Barbu, Andreea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stålberg, Peter
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery.
    Skogseid, Britt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Accelerated Proliferation and Differential Global Gene Expression in Pancreatic Islets of Five-Week-Old Heterozygous Men1 Mice: Men1 Is a Haploinsufficient Suppressor2012In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 153, no 6, p. 2588-2598Article in journal (Refereed)
    Abstract [en]

    Individuals carrying heterozygous (hz) MEN1 (Multiple Endocrine Neoplasia Syndrome Type 1) germ line mutations develop endocrine tumors as a result of somatic loss of the wild-type (wt) allele. However, endocrine cell proliferation has been observed despite wt allele retention, indicating haploinsufficiency. To study downstream molecular effects of the hz haplotype, a germ line Men1 hz mouse model was used to explore differences in global endocrine pancreatic gene expression. Because islet cells of 5-wk-old hz mice express Menin from the retained wt Men1 allele, these were isolated after collagenase digestion of the pancreas, and used for global gene expression array. Wild-type littermates were used for comparison. Array findings were corroborated by quantitative PCR, Western blotting, in situ proximity ligation assay, and immunohistochemistry. The hz islets show increased proliferation: the Ki-67 index was twice as high as in wt islets (3.48 vs. 1.74%; P = 0.024). The microarray results demonstrated that several genes were differentially expressed. Some selected genes were studied on the protein level, e.g. the cytoskeletal regulator myristoylated alanine-rich protein kinase C substrate (Marcks) was significantly less expressed in hz islets, using in situ proximity ligation assay and Western blotting (P < 0.001 and P < 0.01, respectively). Further, gene ontology analysis showed that genes with higher mRNA expression in the hz endocrine pancreas were associated with e.g. chromatin maintenance and apoptosis. Lower mRNA was observed for genes involved in growth factor binding. In conclusion, despite retained Menin expression, proliferation was accelerated, and numerous genes were differentially expressed in the endocrine pancreas of 5-wk-old hz Men1 mice, corroborating the hypothesis that MEN1 is a haploinsufficient suppressor.

  • 41. Lekva, Tove
    et al.
    Berg, Jens Petter
    Lyle, Robert
    Heck, Ansgar
    Ringstad, Geir
    Olstad, Ole Kristoffer
    Michelsen, Annika Elisabet
    Casar-Borota, Olivera
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular and Morphological Pathology.
    Bollerslev, Jens
    Ueland, Thor
    Epithelial Splicing Regulator Protein 1 and Alternative Splicing in Somatotroph Adenomas2013In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 154, no 9, p. 3331-3343Article in journal (Refereed)
    Abstract [en]

    Somatotroph adenomas secrete supraphysiological amounts of GH, causing acromegaly. We have previously hypothesized that epithelial mesenchymal transition (EMT) may play a central role in the progression of these adenomas and that epithelial splicing regulator 1 (ESRP1) may function prominently as a master regulator of the EMT process in pituitary adenomas causing acromegaly. To further elucidate the role of ESRP1 in somatotroph adenomas and in EMT progression, we used RNA sequencing (RNAseq) to sequence somatotroph adenomas characterized by high and low ESRP1 levels. Transcripts identified by RNAseq were analyzed in 65 somatotroph adenomas and in GH-producing pituitary rat cells with a specific knockdown of Esrp1. The clinical importance of the transcripts was further investigated by correlating mRNA expression levels with clinical indices of disease activity and treatment response. Many of the transcripts and isoforms identified by RNAseq and verified by quantitative PCR were involved in vesicle transport and calcium signaling and were associated with clinical outcomes. Silencing Esrp1 in GH3 cells resulted in changes of gene expression overlapping the data observed in human somatotroph adenomas and revealed a decreased granulation pattern and attenuated GH release. We observed an alternative splicing pattern for F-box and leucine-rich repeat protein 20, depending on the ESPR1 levels and on changes in circulating IGF-I levels after somatostatin analog treatment. Our study indicates that ESRP1 in somatotroph adenomas regulates transcripts that may be essential in the EMT progression and in the response to somatostatin analog treatment.

  • 42. Ling, Charlotte
    et al.
    Hellgren, Gunnel
    Gebre-Medhin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Women's and Children's Health.
    Dillner, Karin
    Wennbo, Håkan
    Carlsson, Björn
    Billig, Håkan
    Prolactin (PRL) receptor gene expression in mouse adipose tissue: increases during lactation and in PRL-transgenic mice2000In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 141, no 10, p. 3564-3572Article in journal (Refereed)
    Abstract [en]

    There are indications that PRL may exert important metabolic actions on adipose tissue in different species. However, with the exception of birds, the receptor has not been identified in white adipose tissue. The present study was designed to examine the possible expression and regulation of the PRL receptor (PRLR) in mouse adipose tissue. The long PRLR messenger RNA (mRNA) splice form (L-PRLR) and two short splice forms (S2- and S3-PRLR) were detected in mouse adipose tissue by RT-PCR. Furthermore, L-PRLR mRNA was detected by ribonuclease protection assay. Immunoreactive PRLR with a relative molecular mass of 95,000 was revealed by immunoblotting. Furthermore, L-PRLR mRNA expression was demonstrated in primary isolated adipocytes. In mouse adipose tissue, the level of L-PRLR mRNA expression increased 2.3-fold during lactation compared with those in virgin and pregnant mice. In contrast, in the liver the expression of L-PRLR increased 3.4-fold during pregnancy compared with those in virgin and lactating mice. When comparing the levels of L-PRLR expression in virgin female and male mice, no difference was detected in adipose tissue. However, in virgin female liver the expression was 4.5-fold higher than that in male liver. As PRL up-regulates its own receptor in some tissues, we analyzed L-PRLR expression in PRL-transgenic female and male mice. In PRL-transgenic mice L-PRLR expression was significantly increased in both adipose tissue (1.4-fold in females and 2.4-fold in males) and liver (1.9-fold in females and 2.7-fold in males) compared with that in control mice. Furthermore, in female PRL-transgenic mice retroperitoneal adipose tissue was decreased in weight compared with that in control mice. However, no difference was detected when comparing the masses of parametrial adipose tissue. Our results suggest a direct role for PRL, mediated by PRLR, in modulating physiological events in adipose tissue.

  • 43. Liu, K
    et al.
    Brändström, A
    Liu, Y X
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Selstam, G
    Coordinated expression of tissue-type plasminogen activator and plasminogen activator inhibitor type 1 during corpus luteum formation and luteolysis in the adult pseudopregnant rat.1996In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 137, no 5, p. 2126-32Article in journal (Refereed)
    Abstract [en]

    Proteolytic activity generated by the plasminogen activator (PA) system is associated with many biological processes. Using an adult pseudopregnant rat model, we have studied how two components of the PA system, tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1), are expressed temporally and spatially during different developmental stages of the corpus luteum (CL). Northern blot analysis, in situ hybridization, in situ zymography, and fibrin overlay were used to analyze the expression and distribution of tPA and PAI-1 messenger RNA (mRNA) as well as PA activity in CL of different ages. We demonstrated that during the luteinization period (approximately days 1-2), tPA mRNA was highly and evenly expressed in newly formed CL, whereas PAI-1 mRNA was mainly detected in the central part of the same CL. In accordance with these findings, proteolytic activity generated by tPA was detected in the outer region of newly formed CL by in situ zymography. During the luteotropic period (approximately days 3-10), tPA mRNA expression was very low. PAI-1 mRNA expression was also low, but increased on day 10. As expected, proteolytic activity was very low during this period. During functional luteolysis (days 13-14) and subsequent structural luteolysis, tPA mRNA was elevated. PAI-1 mRNA was also expressed during this period. Moreover, the net PA activity, as determined by fibrin overlay, was relatively high during this period. Our studies indicate that tPA and PAI-1 are coordinately expressed in the CL, resulting in increased proteolytic activities during the luteinization and luteolytic periods. PA-mediated proteolysis may, therefore, play a role in both CL formation and luteolysis in rats.

  • 44.
    Liu, Kui
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Olofsson, Jan
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Wahlberg, Patrik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Distinct expression ofgelatinase A (MMP-2), collagenase-3 (MMP-13), membrane-type MMP 1 (MT1-MMP), and tissue inhibitor of MMPs type 1 (TIMP-1) mediated by physiological signals during formation and regression of the rat corpus luteum1999In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 140, no 11, p. 5330-5338Article in journal (Refereed)
    Abstract [en]

    The corpus luteum (CL) is a transient endocrine organ that secretes progesterone to support pregnancy. The CL is formed from an ovulated follicle in a process that involves extensive angiogenesis and tissue remodeling. If fertilization does not occur or implantation is unsuccessful, the CL will undergo regression, which involves extensive tissue degradation. Extracellular proteases, such as serine proteases and matrix metalloproteinases (MMPs), are thought to play important roles in both the formation and regression of the CL. In this study, we have examined the physiological regulation pattern and cellular distribution of messenger RNAs coding for gelatinase A (MMP-2), collagenase-3 (MMP-13), membrane type MMP 1 (MT1-MMP, MMP-14), and the major MMP inhibitor, tissue inhibitor of MMPs type 1 (TIMP-1) in the CL of adult pseudopregnant (psp) rat. Northern blot and in situ hybridization analyses revealed that gelatinase A messenger RNA was mainly expressed during luteal development, indicating that gelatinase A may be associated with the neovascularization and tissue remodeling that takes place during CL formation. Collagenase-3 had a separate expression pattern and was only expressed in the regressing CL, suggesting that this MMP may be related with luteal regression. MT1-MMP that in vitro can activate progelatinase A and procollagenase-3 was constitutively expressed during the formation, function, and regression of the CL and may therefore be involved in the activation of these MMPs. TIMP-1 was induced during both the formation and regression of the CL, suggesting that this inhibitor modulates MMP activity during these processes. To test whether the induction of collagenase-3 and TIMP-1 is coupled with luteal regression, we prolonged the luteal phase by performing hysterectomies, and induced premature luteal regression by treating the pseudopregnant rats with a PGF2alpha analog, cloprostenol. In both treatments, collagenase-3 and TIMP-1 were induced only after the serum level of progesterone had decreased, suggesting that collagenase-3 and TIMP-1 are induced by physiological signals, which initiate functional luteolysis to play a role in tissue degradation during structural luteolysis. In conclusion, our data suggest that gelatinase A, collagenase-3, and MT1-MMP may have separate functions during the CL life span: gelatinase A mainly takes part in CL formation, whereas collagenase-3 mainly takes part in luteal regression; MT1-MMP is constitutively expressed during the CL life span and may therefore serve as an in vivo activator of both gelatinase A and collagenase-3. TIMP-1 is up-regulated both during the formation and regression of the CL and may therefore regulate MMP activity during both processes.

  • 45.
    Liu, Kui
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Wahlberg, Patrik
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ny, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Coordinated and cell-specific regulation of membrane-type matrix metalloproteinase 1 (MT1-MMP) and its substrate matrix metalloproteinase 2 (MMP-2) by physiological signals during follicular development and ovulation1998In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 139, no 11, p. 4735-4738Article in journal (Refereed)
    Abstract [en]

    In the ovary, extensive tissue remodeling is required during both follicular development and the break down of the follicular wall at the time of ovulation. Extracellular proteases such as serine proteases and matrix metalloproteinases (MMPs) are thought to play pivotal roles in these processes. In this paper, we have used in situ hybridization to study the regulation and distribution of mRNA coding for MMP-2 (gelatinase A) and its cell surface activator membrane-type MMP1 (MT1-MMP) during gonadotropin induced ovulation in the rat. In ovaries of untreated immature (23 day old) rats, the levels of MT1-MMP and MMP-2 mRNA were low. MMP-2 mRNA was found in theca-interstitial cells while MT1-MMP mRNA was found in both granulosa and theca-interstitial cells and both messages were induced after stimulation with PMSG. After an ovulatory dose of hCG, the expression of MT1-MMP was dramatically down regulated in the granulosa cell layers of large preovulatory follicles but the expression remained and appeared to be up regulated together with MMP-2 in the theca-interstitial cells surrounding the large preovulatory follicles. The expression kinetics and tissue distribution supports the notion that MT1-MMP may have dual functions in the ovary. Initially MT1-MMP may act as a matrix degrading protease inside the follicle during follicular development and later, just prior to ovulation, as an activator of proMMP-2 in theca-interstitial cells surrounding preovulatory follicles.

  • 46. Liu, Y X
    et al.
    NY, Tor
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sarkar, D
    Loskutoff, D
    Hsueh, A J
    Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes.1986In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 119, no 4, p. 1578-87Article in journal (Refereed)
    Abstract [en]

    Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.

  • 47.
    Makeeva, Natalia
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Roomans, Godfried M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Myers, Jason W
    Stanford University School of Medicine, Stanford, California.
    Welsh, Nils
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Transforming growth factor-beta-activated protein kinase 1-binding protein (TAB)-1alpha, but not TAB1beta, mediates cytokine-induced p38 mitogen-activated protein kinase phosphorylation and cell death in insulin-producing cells2008In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 149, no 1, p. 302-309Article in journal (Refereed)
    Abstract [en]

    Previous studies have indicated that the p38 MAPK participates in signaling events that lead to the death of the insulin-producing beta-cell. The aim of the present study was to elucidate the role of the TGF-beta-activated protein kinase 1-binding protein 1 (TAB1) in the cytokine-induced activation of p38. Levels of TAB1 mRNA and protein were analyzed by real-time PCR and immunoblotting, and TAB1 expression in mouse and human islet cells was down-regulated using lipofection of diced-small interfering RNA. TAB1 overexpression in beta-TC6 cells was achieved by transient transfections followed by fluorescence activated cell sorting. Phosphorylation of p38, c-Jun N-terminal kinase, and ERK was assessed by immunoblotting, and viability was determined using vital staining with bisbenzimide and propidium iodide. We observed that TAB1 is expressed in insulin-producing cells. Cytokine (IL-1beta + interferon-gamma)-stimulated p38 phosphorylation was significantly increased by TAB1alpha overexpression, but not TAB1beta overexpression, in beta-TC6 cells. The TAB1alpha-augmented p38 phosphorylation was paralleled by an increased cell death rate. Treatment of islet cells with diced-small interfering RNA specific for TAB1, but not for TGF-beta-activated kinase 1, resulted in lowered cytokine-induced p38 phosphorylation and protection against cell death. The cytokine-induced phosphorylation of c-Jun N-terminal kinase and ERK was not affected by changes in TAB1 levels. Finally, TAB1 phosphorylation was decreased by the p38 inhibitor SB203580. We conclude that TAB1alpha, but not TAB1beta, plays an important role in the activation of p38 in insulin-producing cells and therefore also in cytokine-induced beta-cell death.

  • 48. Mannerås, Louise
    et al.
    Cajander, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Holmäng, Agneta
    Seleskovic, Zamira
    Lystig, Theodore
    Lönn, Malin
    Stener-Victorin, Elisabet
    A new rat model exhibiting both ovarian and metabolic characteristics of polycystic ovary syndrome2007In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 148, no 8, p. 3781-3791Article in journal (Refereed)
    Abstract [en]

    Polycystic ovary syndrome (PCOS) is a complex endocrine and metabolic disorder associated with ovulatory dysfunction, hyperandrogenism, abdominal obesity, and insulin resistance. However, its etiology is unclear, and its management is often unsatisfactory or requires a diversified approach. Here, we describe a new rat PCOS model, the first to exhibit both ovarian and metabolic characteristics of the syndrome. Female rats received the nonaromatizable androgen dihydrotestosterone (DHT) or the aromatase inhibitor letrozole by continuous administration, beginning before puberty, to activate androgen receptors. Adult DHT rats had irregular cycles, polycystic ovaries characterized by cysts formed from atretic follicles, and a diminished granulosa layer. They also displayed metabolic features, including increased body weight, increased body fat, and enlarged mesenteric adipocytes, as well as elevated leptin levels and insulin resistance. All letrozole rats were anovulatory and developed polycystic ovaries with structural changes strikingly similar to those in human PCOS. Our findings suggest that the formation of a "hyperplastic" theca interna reflects the inclusion of luteinized granulosa cells in the cyst wall rather than true hyperplasia. We conclude that the letrozole model is suitable for studies of the ovarian features of human PCOS, while the DHT model is suitable for studies of both ovarian and metabolic features of the syndrome.

  • 49.
    Manukyan, Levon
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Ubhayasekera, Sarojini J.K.A.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergquist,, Jonas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Analytical Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sargsyan, Ernest
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Bergsten, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Palmitate-induced impairments of beta-cell function are linked with generation of specific ceramide species via acylation of sphingosine2015In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 156, no 3, p. 802-812Article in journal (Refereed)
    Abstract [en]

    Prolonged exposure to palmitate impairs beta-cell function and mass. One of the proposed mechanisms is alteration in ceramide generation. In the present study, exposure to palmitate induced the level of palmitoyl transferase and ceramide synthases, enzymes of the ceramide de novo and salvage pathways, and doubled total ceramide levels, which was associated with decreased insulin secretion and augmented apoptosis in MIN6 cells and human islets. By inhibiting enzymes of the pathways pharmacologically with ISP-1 or fumonisin B1 or by siRNA we showed that Cer(14:0), Cer(16:0), Cer(20:1) and Cer(24:0) species, generated by the salvage pathway, are linked to the harmful effect of palmitate on beta-cells. Oleate attenuates negative effects of palmitate on beta-cells. When oleate was included during culture of MIN6 cells with palmitate the palmitate-induced up-regulation of the enzymes of the de novo and salvage pathways was prevented resulting in normalized levels of all ceramide species except Cer(20:1). Our data suggest that enhanced ceramide generation in response to elevated palmitate levels involves both de novo and salvage pathways. However, the negative effects of palmitate on beta-cells are attributed to generation of ceramide species Cer(14:0), Cer(16:0) and Cer(24:0) via acylation of sphingosine.

  • 50.
    McCulloch, Laura J
    et al.
    University of Exeter Medical School, Exeter, UK.
    Rawling, Tom J
    University of Exeter Medical School, Exeter, UK.
    Sjöholm, Kajsa
    The Sahlgrenska Academy at University of Gothenburg, Sweden.
    Franck, Niclas
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences.
    Dankel, Simon N
    University of Bergen and Hormone Laboratory, Haukeland University Hospital, Norway.
    Price, Emily J
    University of Exeter Medical School, Exeter, UK.
    Knight, Bridget
    University of Exeter Medical School, UK.
    Liversedge, Neil H
    Royal Devon and Exeter NHS Foundation Trust, UK.
    Mellgren, Gunnar
    University of Bergen and Hormone Laboratory, Haukeland University Hospital, Norway.
    Nyström, Fredrik
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Endocrinology.
    Carlsson, Lena M
    The Sahlgrenska Academy at University of Gothenburg, Sweden.
    Kos, Katarina
    University of Exeter Medical School, Exeter, UK.
    COL6A3 is regulated by leptin in human adipose tissue and reduced in obesity2015In: Endocrinology, ISSN 0013-7227, E-ISSN 1945-7170, Vol. 156, no 1, p. 134-146Article in journal (Refereed)
    Abstract [en]

    Fibrosis of adipose tissue (AT) increases AT rigidity, reduces its expandability and contributes to metabolic dysfunction. Collagen type VI, alpha3 (COL6A3) encodes one subunit of a fibrotic extracellular matrix (ECM) protein highly expressed in rodent AT. Knock-out of collagen VI in rodent AT led to a significant improvement in metabolic health in obese, diabetic (ob/ob) mice however, it is unknown whether this collagen has the same metabolic significance in human AT. We therefore aimed to undertake a comprehensive assessment of COL6A3 in relation to human AT and obesity. Characterisation of COL6A3 in human AT showed 5 fold higher expression in the stromalvascular fraction compared with adipocyte expression and significantly higher expression in subcutaneous than omental AT. In both depots COL6A3 expression appeared to be lowered in obesity, whilst diet and surgery-induced weight loss increased COL6A3 expression in subcutaneous AT. Leptin treatment caused a dose dependent decrease in COL6A3 expression although no effect was seen with insulin or glucose treatment and no difference observed in subjects with diabetes. In addition, we found that the collagen expression profile in humans differs significantly from rodents as COL6A3 does not appear to be the predominant collagen in adipose, muscle or liver. Our findings oppose those initially seen in rodent studies and most importantly, demonstrate a direct regulation of COL6A3 by leptin. This highlights the importance of a paracrine leptin signalling pathway in human AT and suggests an additional mechanism by which leptin can regulate ECM composition and with it AT expandability.

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